US20110236401A1

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Publication number: US20110236401A1
Application number: US13/005,346
Authority: US
Inventors: Kenneth M. Murphy; Theresa L. Murphy
Original assignee: Washington University in St Louis WUSTL
Current assignee: Washington University in St Louis WUSTL
Priority date: 2004-11-15
Filing date: 2011-01-12
Publication date: 2011-09-29
Also published as: WO2007001459A3; WO2007001459A8; US8188232B1; WO2007001459A2

Abstract

The invention derives from the identification of HVEM as the native ligand for BTLA. The invention provides compositions and methods for modulating BTLA-HVEM interactions and BTLA and HVEM activity, which are useful for modulating immune responses. Agonists and antagonists of the BTLA-HVEM interaction are provided, and methods of treating a variety of conditions through the modulation of immune responses are provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/719,356, filed May 15, 2007, which is a National Stage Entry of PCT/US2005/041446, filed Nov. 15, 2005, which claims the benefit of U.S. Provisional Application Ser. No. 60/628,474, filed Nov. 15, 2004, the disclosures of which are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to mechanisms of immunomodulation, as well as to compositions and methods useful for modulating an immune response for therapeutic purposes. The invention relates to the treatment of cancer, infection, autoimmune disease, inflammation, and allergy by immunomodulatory means. The invention further relates to the treatment of transplant recipients, and to the reduction of graft versus host reactions in patients by immunomodulatory means.

BACKGROUND

Positive and negative costimulatory signals play critical roles in the modulation of T cell activity. Positive costimulation, in addition to T cell receptor (TCR) engagement, is required for optimal activation of naïve T cells, whereas negative costimulation is believed to be required for the acquisition of immunologic tolerance to self, as well as the termination of effector T cell functions. Upon interaction with B7.1 or B7.2 on the surface of antigen-presenting cells (APC), CD28, the prototypic T cell costimulatory molecule, emits signals that promote T cell proliferation and differentiation in response to TCR engagement, while the CD28 homologue cytotoxic T lymphocyte antigen-4 (CTLA-4) emits signals that inhibit T cell proliferation and effector functions (Chambers et al., Ann. Rev. Immunol., 19:565-594, 2001; Egen et al., Nature Immunol., 3:611-618, 2002).

Agents capable of modulating positive and negative costimulatory signals are highly desirable for use in the modulation of adaptive immune responses. Many autoimmune disorders are known to involve autoreactive T cells and autoantibodies. Agents that are capable of inhibiting the activation of lymphocytes that are specific for self antigens are desirable. Similarly, under certain conditions it is desirable to inhibit normal immune responses to antigen. For example, the suppression of normal immune responses in a patient receiving a transplant is desirable, and agents that exhibit such immunosuppressive activity are highly desirable.

Conversely, many cancer immunotherapies, such as adoptive immunotherapy, expand tumor-specific T cell populations and direct them to attack and kill tumor cells (Dudley et al., Science 298:850-854, 2002; Pardoll, Nature Biotech., 20:1207-1208, 2002; Egen et al., Nature Immunol., 3:611-618, 2002). Agents capable of augmenting tumor attack are highly desirable.

In addition, immune responses to many different antigens (e.g., microbial antigens or tumor antigens), while detectable, are frequently of insufficient magnitude to afford protection against a disease process. Agents capable of promoting and/or prolonging the activation (delaying termination) of lymphocytes that are specific for such antigens are highly desirable.

Costimulatory signals, particularly positive costimulatory signals, also play a role in the modulation of B cell activity. For example, B cell activation and the survival of germinal center B cells require T cell-derived signals in addition to stimulation by antigen. CD40 ligand present on the surface of helper T cells interacts with CD40 on the surface of B cells and provides such a positive costimulatory signal to B cells.

Recently, a negative costimulatory receptor analogous to CTLA-4 was identified on B cells and T cells (Watanabe et al., Nat. Immunol., 4:670-679, 2003; U.S. patent application Ser. No. 10/600,997, filed 20 Jun. 2003; both of which are expressly incorporated herein in their entirety by reference). B and T lymphocyte attenuator (BTLA) is an immunoglobulin domain-containing glycoprotein with a Grb2 binding site, an immunoreceptor tyrosine-based inhibitory motif (ITIM), and an immunoreceptor tyrosine-based switch motif (ITSM). Partial BTLA sequences were disclosed previously (WO 99/40100 andWO 02/07294) though the complete sequence, distribution, and function of BTLA was not reported. Additionally, the partial BTLA sequences disclosed were asserted to correspond to secreted proteins rather than a functional receptor on the surface of lymphocytes.

BTLA acts a negative regulator of both B and T lymphocyte activity (Watanabe et al., Nat. Immunol., 4:670-679, 2003). Crosslinking BTLA with antigen receptors induces its tyrosine phosphorylation and association with the Src homology domain 2 (SH2)-containing protein tyrosine phosphatases SHP-1 and SHP-2, and attenuates production of interleukin 2 (IL-2). BTLA-deficient T cells show increased proliferation, and BTLA-deficient mice have increased specific antibody responses and enhanced sensitivity to experimental autoimmune encephalomyelitis.

Based on indirect evidence, the ligand for BTLA was previously asserted to be B7x (Watanabe et al., supra). However, as disclosed herein, B7x does not bind to BTLA. The identification of BTLA's cognate ligand thus remains highly desirable for an understanding of BTLA function, and for diagnostic and therapeutic purposes.

Herpes virus entry mediator (“HVEM”), a member of the TNF/NGF receptor family, is another positive costimulatory receptor that additionally mediates the entry of herpes simplex virus (HSV) into cells (Montgomery et al., Cell. Nov. 1, 1996; 87(3):427-36). Anti-HVEM antibodies and a soluble hybrid protein containing the HVEM ectodomain have been shown to inhibit such HVEM-dependent viral entry. HSV-1 glycoprotein D (gD), a structural component of the HSV envelope, binds to HVEM to facilitate viral entry (Whitbeck et al., J Virol. August 1997; 71(8):6083-93). HVEM binds two cellular ligands, secreted lymphotoxin alpha and LIGHT (Mauri et al., Immunity. January 1998; 8(1):21-30). HSV-1 gD inhibits the interaction of HVEM with LIGHT. Additionally, targeted disruption of LIGHT causes immunomodulatory defects (Scheu et al., J. Exp. Med., 195:1613-1624, 2002). Additionally, a phagederived peptide BP-2 reportedly binds to HVEM and can compete with HSV-1 gD (Carfi et al., Mol. Cell 8:169-179, 2001; Sarrias et al., Mol. Immunol., 37:665-673, 2000).

SUMMARY OF THE INVENTION

The present disclosure establishes that Herpes virus entry mediator (HVEM) is the cognate ligand of BTLA. HVEM belongs to the TNF receptor family of proteins and is itself a costimulatory receptor expressed on naive T cells. HVEM is also expressed to a lesser extent on dendritic cells, resting B cells, and macrophages. HVEM has four extracellular cysteine-rich domains (CRDs) and interacts with two known TNF family members, LIGHT and lymphotoxin alpha (LTa), through CRD2 and CRD3. For further discussion of HVEM, see for example Granger et al., Cytokine Growth Factor Rev., 14:289-96, 2003; and Croft, Nat. Rev. Immunol., 3:609-620, 2003. As disclosed herein, HVEM directly binds to BTLA and stimulates BTLA activity. As further disclosed herein, HVEM binding to BTLA can reduce the activation of BTLA expressing lymphocytes, as well as decrease the effector activity of BTLA expressing lymphocytes.

The present disclosure also establishes that B7x does not directly bind to BTLA and does not directly modulate BTLA activity. B7x is expressed in a wide variety of normal and cancer cells, and was previously reported to be a ligand for BTLA based on indirect evidence. It was postulated that the interaction of B7x with BTLA inhibited both B and T cell responses, and was a means by which B7x-expressing tumor tissue inhibited the activity of tumor-specific T cells. It was further posited that B7x expressed on non-tumor non-lymphoid tissue served to maintain immunological tolerance to self antigens.

Stemming from the discovery of the HVEM-BTLA interaction, in one aspect, the present invention provides BTLA antibodies, sometimes referred to herein as BTLA blocking antibodies. A BTLA antibody of the invention is capable of specifically binding to a BTLA protein and is capable of reducing the binding of the BTLA protein to an HVEM protein. Especially preferred are BTLA antibodies that specifically bind to a region of the BTLA Ig domain, which region binds to the HVEM CRD1 domain. Such a BTLA antibody is capable of binding to a fragment of the BTLA Ig domain, which fragment is capable of binding to an HVEM CRD1 domain.

In one embodiment, a BTLA antibody is capable of binding to a mouse BTLA Ig domain.

In one embodiment, a BTLA antibody is capable of binding to a mouse BTLA Ig domain in a human BTLA tetramer.

In one embodiment, a BTLA antibody is capable of binding to a human BTLA Ig domain.

In one embodiment, a BTLA antibody is capable of binding to a human BTLA Ig domain in a human BTLA tetramer.

In one embodiment, a BTLA antibody is capable of binding toward the DEBA face of the Ig fold of BTLA. The phrase “DEBA face” refers to the regions of the BTLA molecule composed of the beta strands labelled “D”, “E”, “B”, and “A” strands. See, for example, structure of BTLA ectodomain deposited at NCBI by C. A. Nelson, D. H. Fremont, Midwest Center For Structural & Genomics (Mcsg), 26 Aug. 2004. See also Compaan et al., J Biol Chem. Sep. 16, 2005, Epub manuscript M507629200.

In one embodiment, a BTLA antibody is capable of binding an epitope of BTLA that is capable of binding to an antibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA antibody is capable of competing with an antibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’ for binding to BTLA.

In one embodiment, a BTLA antibody is capable of binding to an epitope of BTLA that is homologous to an epitope capable of binding an antibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA antibody is capable of binding to an epitope comprising one or more residues selected from the group consisting of R55, Q63,Q 102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA antibody is capable of binding to an epitope comprising one or more residues selected from the group consisting of the residues in a BTLA protein corresponding to the residues V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA antibody is capable of binding to an epitope comprising one or more residues selected from the group consisting of the residues in human BTLA corresponding to the residues V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA antibody is capable of binding to an epitope comprising one or more residues selected from the group consisting of V36, Q37, L38, L49, E57, C79, K93, and S96 in the human BTLA sequence set forth at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA antibody is capable of binding to an epitope comprising one or more residues in a human BTLA corresponding to residues from the group consisting of V36, Q37, L38, L49, E57, C79, K93, and S96 in the human BTLA sequence set forth at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA antibody is capable of binding to a polypeptide having at least about 80%, more preferably 85%, more preferably 90%, more preferably 95% identity to the amino acid sequence set forth by residues 37-47, 39-49, 41-49, 50-60, 58-68, 80-90, 97-107, 50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137, 55-102, 50-107, and 41-137 of murine BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA antibody is capable of binding to a polypeptide selected from the group consisting of the amino acid sequences set forth by residues 37-47, 39-49, 41-49, 50-60, 58-68, 80-90, 97-107, 50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137, 55-102, 50107, and 41-137 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA antibody is capable of binding to a polypeptide having at least about 80%, more preferably 85%, more preferably 90%, more preferably 95% identity to the amino acid sequence set forth by residues 31-41, 32-42, 35-43, 44-54, 52-62, 74-84, 88-98, 44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 49-93, 44-98, 35-98, and 35-128 of the human BTLA isoform found at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA antibody is capable of binding to a polypeptide selected from the group consisting of the amino acid sequences set forth by residues 31-41, 32-42, 35-43, 44-54, 52-62, 74-84, 88-98, 44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 49-93, 44-98, 35-98, and 35-128 of the human BTLA isoform found at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA antibody is selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA antibody is capable of competing with CMV UL144 for binding to BTLA.

In one embodiment, the invention provides BTLA antibodies which are monoclonal antibodies.

In one embodiment, the invention provides BTLA antibodies which are human antibodies.

In one aspect, the invention provides a hybridoma that produces a BTLA antibody disclosed herein.

In one aspect, the invention provides BTLA antibodies that are capable of modulating BTLA activity.

In one embodiment, the invention provides BTLA antibodies that are antagonistic BTLA antibodies, which are capable of reducing BTLA activity. Such antibodies are capable of reducing the activation of BTLA by HVEM. Preferably, such antagonistic BTLA antibodies are also capable of reducing the activation of BTLA by another ligand which binds to the HVEM binding region of BTLA, such as UL144. The UL144 open reading frame in human cytomegalovirus (CMV) encodes a homologue of the herpesvirus entry mediator, HVEM, a member of the tumor necrosis factor receptor superfamily (Lurain et al., J Virol. December 1999; 73(12):10040-50).

In another embodiment, the invention provides BTLA antibodies that are agonistic BTLA antibodies, which are capable of increasing BTLA activity. Such antibodies are capable of increasing BTLA activity in a cell having BTLA on its surface.

In one aspect, the invention provides HVEM antibodies, sometimes referred to herein as HVEM blocking antibodies. An HVEM antibody specifically binds to an HVEM protein and is capable of reducing the binding of the HVEM protein to a BTLA protein. Especially preferred are HVEM antibodies that specifically bind to a region of the HVEM CRD1 domain that binds to the BTLA Ig domain. Such an HVEM antibody is capable of binding to a fragment of the HVEM CRD1 domain, which fragment is capable of binding to a BTLA Ig domain. Preferred HVEM antibodies do not bind to the HVEM CRD2 or HVEM CRD3 domains, though antibodies binding to the CRD2 and/or CRD3 domains in addition to the CRD1 domain may be used in the methods herein.

In one embodiment, the invention provides HVEM antibodies which are monoclonal antibodies.

In one aspect, the invention provides a hybridoma that produces a HVEM antibody disclosed herein.

In one embodiment, the invention provides HVEM antibodies which are human antibodies.

In one aspect, the invention provides HVEM antibodies that are capable of modulating BTLA activity.

In a preferred embodiment, the invention provides HVEM antibodies that are antagonistic HVEM antibodies, which are capable of reducing the ability of HVEM to activate BTLA on the surface of a cell.

In another embodiment, the invention provides HVEM antibodies that are agonistic HVEM antibodies, which are capable of binding to HVEM and stimulating HVEM activity in a cell, thereby mimicking BTLA. HVEM activity in this sense includes increased NF-kB activity and increased AP-1 activity.

In one embodiment, the invention provides HVEM antibodies that do not inhibit the binding of HVEM to LIGHT or LTa.

In one embodiment, the invention provides HVEM antibodies that additionally reduce the binding of HSV-1 glycoprotein D to HVEM.

In one aspect, the invention provides BTLA-HVEM antagonists. A BTLA-HVEM antagonist may be any of a wide variety of bioactive agents capable of reducing the activation of BTLA by HVEM. In a preferred embodiment, a BTLA-HVEM antagonist is capable of reducing the binding of an HVEM CRD1 domain to a BTLA Ig domain. While many BTLA-HVEM antagonists are capable of binding to BTLA, such a BTLA-HVEM antagonist does not increase BTLA activity in a cell expressing BTLA on its surface.

Preferred BTLA-HVEM antagonists are capable of reducing BTLA activity in a cell having BTLA on its surface. In a preferred embodiment, the cell is a lymphocyte, a T cell, a CD4⁺ T cell, a TH1 cell, a CD8⁺ T cell, a B cell, a plasma cell, a macrophage, or an NK cell.

Suitable bioactive agents include BTLA antibodies and HVEM antibodies (e.g., monoclonal, polyclonal, single chain, and/or bispecific antibodies as well as Fab and F(ab)₂fragments, variants and derivatives thereof). Suitable bioactive agents also include fragments and truncated forms of BTLA and HVEM proteins, fusion proteins, and the like, for example, soluble proteins and polypeptides comprising or consisting essentially of a BTLA Ig domain fragment capable of binding an HVEM CRD1 domain; soluble proteins and polypeptides comprising or consisting essentially of an HVEM CRD1 domain or fragment thereof capable of binding a BTLA Ig domain; a BTLA Ig domain peptide, a CRD1 domain peptide. Suitable bioactive agents also include small molecule chemical compositions.

In one embodiment, the invention provides a BTLA-HVEM antagonist capable of reducing the binding of a BTLA protein to an HVEM protein, wherein the antagonist does not comprise an HVEM CRD2 domain, an HVEM CRD3 domain, or both, and wherein the antagonist does not bind to an HVEM CRD2 domain or an HVEM CRD3 domain, with the proviso that the antagonist is not an HSV-1 glycoprotein D, a phage-derived peptide BP-2, or a soluble protein comprising a complete BTLA Ig domain capable of binding said HVEM protein.

In the methods herein, glycoprotein D and phage-derived peptide BP-2, as well as HVEM-binding fragments thereof, and fusion proteins comprising the same, may be used as BTLA-HVEM antagonists.

Preferred BTLA-HVEM antagonists are capable of binding to a BTLA Ig domain and are capable of reducing the binding of the BTLA Ig domain to an HVEM CRD1 domain. Especially preferred are BTLA-HVEM antagonists capable of binding to a region of the BTLA Ig domain that binds to the HVEM CRD1 domain. Such a BTLA-HVEM antagonist is capable of binding to a fragment of the BTLA Ig domain, which fragment is capable of binding to an HVEM CRD1 domain.

In one embodiment, a BTLA-HVEM antagonist binds an epitope of BTLA that is capable of binding to an antibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA-HVEM antagonist is capable of competing with an antibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’ for binding to BTLA.

In one embodiment, a BTLA-HVEM antagonist binds to an epitope of BTLA that is homologous to an epitope capable of binding an antibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA-HVEM antagonist is capable of binding to an epitope comprising one or more residues selected from the group consisting of V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to an epitope comprising one or more residues selected from the group consisting of the residues in a BTLA protein corresponding to the residues V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to an epitope comprising one or more residues selected from the group consisting of the residues in human BTLA corresponding to the residues V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to an epitope comprising one or more residues selected from the group consisting of V36, Q37, L38, L49, E57, C79, K93, and S96 in the human BTLA sequence set forth at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to an epitope comprising one or more residues of human BTLA corresponding to residues from the group consisting of V36, Q37, L38, L49, E57, C79, K93, and S96 in the human BTLA sequence set forth at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to a polypeptide having at least about 80%, more preferably 85%, more preferably 90%, more preferably 95% identity to the amino acid sequence set forth by residues 37-47, 39-49, 41-49, 50-60, 58-68, 80-90, 97-107, 50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137, 55-102, 50-107, and 41-137 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to a polypeptide selected from the group consisting of the amino acid sequences set forth by residues 37-47, 39-49, 41-49, 50-60, 58-68, 80-90, 97-107, 50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137, 55-102, 50-107, and 41-137 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to a polypeptide having at least about 80%, more preferably 85%, more preferably 90%, more preferably 95% identity to the amino acid sequence set forth by residues 31-41, 32-42, 35-43, 44-54, 52-62, 74-84, 88-98, 44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 49-93, 44-98, 35-98, and 35-128 of the human BTLA isoform found at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM antagonist is capable of binding to a polypeptide selected from the group consisting of the amino acid sequences set forth by residues 31-41, 32-42, 35-43, 44-54, 52-62, 74-84, 88-98, 44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 4993, 44-98, 35-98, and 35-128 of the human BTLA isoform found at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM antagonist is capable of competing with CMV UL144 for binding to BTLA.

In one embodiment, a BTLA-HVEM antagonist is capable of competing with HSV-1 glycoprotein D for binding to HVEM.

In one embodiment, a BTLA-HVEM antagonist is a BTLA antibody.

In one embodiment, a BTLA-HVEM antagonist is an HVEM antibody.

In one aspect, the invention provides BTLA-HVEM antagonists that comprise a BTLA Ig domain fragment capable of binding an HVEM CRD1 domain. In another aspect, the invention provides BTLA-HVEM antagonists that consist essentially of a BTLA Ig domain fragment capable of binding an HVEM CRD1 domain.

Accordingly, in a preferred embodiment, the invention provides BTLA-HVEM antagonists that are BTLA fusion proteins which are capable of binding to an HVEM CRD1 domain and reducing the binding of the HVEM CRD1 domain to a BTLA Ig domain. Preferred BTLA fusion proteins do not bind to the CRD2 or CRD3 domains of HVEM. Preferred BTLA fusion proteins can compete with an HVEM antibody disclosed herein for binding to an HVEM CRD1 domain. Preferred BTLA fusion proteins do not comprise an entire BTLA Ig domain.

In another preferred embodiment, the invention provides BTLA-HVEM antagonists that are BTLA protein fragments which are capable of binding to the CRD1 domain of HVEM and reducing the binding of the HVEM CRD1 domain to a BTLA Ig domain. Preferred BTLA protein fragments do not bind to the CRD2 or CRD3 domains of HVEM. In a preferred embodiment, a BTLA protein fragment consists essentially of a BTLA Ig domain fragment that is capable of binding to an HVEM CRD1 domain. Preferred BTLA protein fragments can compete with an HVEM antibody disclosed herein for binding to an HVEM CRD1 domain.

In one aspect, the invention provides BTLA-HVEM antagonists that comprise an HVEM CRD1 domain or fragment thereof capable of binding to a BTLA Ig domain. In another aspect, the invention provides BTLA-HVEM antagonists that consist essentially of an HVEM CRD1 domain or fragment thereof capable of binding to a BTLA Ig domain.

Accordingly, in a preferred embodiment, the invention provides BTLA-HVEM antagonists that are HVEM fusion proteins which are capable of binding to a BTLA Ig domain and reducing the binding of the BTLA Ig domain to an HVEM CRD1 domain. Such HVEM fusion proteins lack an HVEM CRD2 and/or CRD3 domain. Preferred HVEM fusion proteins can compete with a BTLA antibody disclosed herein for binding to a BTLA Ig domain.

In another preferred embodiment, the invention provides BTLA-HVEM antagonists that are HVEM protein fragments which are capable of binding to a BTLA Ig domain and reducing the binding of the BTLA Ig domain to an HVEM CRD1 domain. Such HVEM protein fragments lack an HVEM CRD2 and/or CRD3 domain. In a preferred embodiment, an HVEM protein fragment consists essentially of an HVEM CRD1 domain or fragment thereof which is capable of binding to a BTLA Ig domain. Preferred HVEM protein fragments can compete with a BTLA antibody disclosed herein for binding to a BTLA Ig domain.

In one embodiment, the invention provides fusion proteins that comprise an Fc region of an immunoglobulin.

In one embodiment, for use in the methods herein, HSV-1 glycoprotein D may be used as a BTLA-HVEM antagonist.

In one embodiment, a BTLA-HVEM antagonist is capable of reducing tyrosine phosphorylation on the intracellular domain of BTLA protein in a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of reducing association of BTLA protein with SHP-2, PI3K, or Grb2 in a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of increasing proliferation of a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of increasing IL-2 production by a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of increasing or prolonging antibody production by a cell having said BTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of increasing or prolonging the cytotoxicity of a cell having said BTLA protein on its surface.

In one aspect, the invention provides BTLA-HVEM agonists. A BTLA-HVEM agonist may be any of a wide variety of bioactive agents capable of activating BTLA and thereby mimicking the activity of HVEM.

Preferred BTLA-HVEM agonists are capable of increasing BTLA activity in a cell having BTLA on its surface. In a preferred embodiment, the cell is a lymphocyte, a T cell, a CD4⁺ T cell, aT_H1 cell, a CD8⁺ T cell, a B cell, a plasma cell, a macrophage, or an NK cell.

Suitable bioactive agents include BTLA antibodies (e.g., monoclonal, polyclonal, single chain, and/or bispecific antibodies as well as Fab and F(ab)₂fragments, variants and derivatives thereof). Suitable bioactive agents also include fragments and truncated forms of HVEM proteins, fusion proteins, and the like, such as soluble proteins and polypeptides comprising or consisting essentially of an HVEM CRD1 domain or fragment thereof capable of binding to a BTLA Ig domain and increasing BTLA activity, and lacking a CRD2 and/or CRD3 domain. Suitable bioactive agents also include small molecule chemical compositions.

In one embodiment, the invention provides a BTLA-HVEM agonist capable of binding to BTLA protein and increasing BTLA activity, wherein the agonist does not comprise an HVEM CRD2 domain, an HVEM CRD3 domain, or both, with the proviso that the agonist is not a human CMV UL144 protein.

In one embodiment, for use in the methods herein, CMV UL144, BTLA-binding fragments thereof, and fusion proteins comprising the same, may be used as a BTLA-HVEM agonist. Further regarding UL144, see Cheung et al., PNAS 102:13218-13223, 2005.

Preferred BTLA-HVEM agonists bind to a BTLA Ig domain and are capable of reducing the binding of the BTLA Ig domain to an HVEM CRD1 domain, and mimicking the stimulation of BTLA by HVEM. Especially preferred are BTLA-HVEM agonists capable of binding to a region of the BTLA Ig domain that binds to the HVEM CRD1 domain.

In one embodiment, a BTLA-HVEM agonist binds an epitope of BTLA that is capable of binding to an antibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA-HVEM agonist is capable of competing with an antibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’ for binding to BTLA.

In one embodiment, a BTLA-HVEM agonist binds to an epitope of BTLA that is homologous to an epitope capable of binding an antibody selected from the group consisting of ‘6A6’, ‘6F7’, ‘6G3’, ‘6H6’, ‘8F4’, and ‘3F9.D12’.

In one embodiment, a BTLA-HVEM agonist is capable of binding to an epitope comprising one or more residues selected from the group consisting of V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM agonist is capable of binding to an epitope comprising one or more residues selected from the group consisting of the residues in a BTLA protein corresponding to the residues V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM agonist is capable of binding to an epitope comprising one or more residues selected from the group consisting of the residues in human BTLA corresponding to the residues V42, Q43, L44, R55, Q63, Q102, and C85 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM agonist is capable of binding to an epitope comprising one or more residues selected from the group consisting of V36, Q37, L38, L49, E57, C79, K93, and S96 in the human BTLA sequence set forth at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM agonist is capable of binding to an epitope comprising one or more residues in human BTLA corresponding to residues from the group consisting of V36, Q37, L38, L49, E57, C79, K93, and S96 in the human BTLA sequence set forth at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM agonist is capable of binding to a polypeptide having at least about 80%, more preferably 85%, more preferably 90%, more preferably 95% identity to the amino acid sequence set forth by residues 37-47, 39-49, 41-49, 50-60, 58-68, 80-90, 97-107, 50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137, 55-102, 50-107, and 41-137 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM agonist is capable of binding to a polypeptide selected from the group consisting of the amino acid sequences set forth by residues 37-47, 39-49, 41-49, 50-60, 58-68, 80-90, 97-107, 50-90, 55-85, 58-90, 63-85, 80-107, 85-102, 127-137, 55-102, 50-107, and 41-137 of murine C57BL/6 BTLA (SEQ ID NO:1).

In one embodiment, a BTLA-HVEM agonist is capable of binding to a polypeptide having at least about 80%, more preferably 85%, more preferably 90%, more preferably 95% identity to the amino acid sequence set forth by residues 31-41, 32-42, 35-43, 44-54, 52-62, 74-84, 88-98, 44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 49-93, 44-98, 35-98, and 35-128 of the human BTLA isoform found at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM agonist is capable of binding to a polypeptide selected from the group consisting of the amino acid sequences set forth by residues 31-41, 32-42, 35-43, 44-54, 52-62, 74-84, 88-98, 44-84, 49-79, 52-84, 57-79, 74-98, 79-93, 118-128, 4993, 44-98, 35-98, and 35-128 of the human BTLA isoform found at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

In one embodiment, a BTLA-HVEM agonist is capable of competing with CMV UL144 for binding to BTLA.

In one embodiment, a BTLA-HVEM agonist is a BTLA antibody.

In one aspect, the invention provides BTLA-HVEM agonists that comprise an HVEM CRD1 domain or fragment thereof capable of binding to a BTLA Ig domain and stimulating BTLA activity. In another aspect, the invention provides BTLA-HVEM agonists that consist essentially of an HVEM CRD1 domain or fragment thereof capable of binding to a BTLA Ig domain and stimulating BTLA activity.

Accordingly, in a preferred embodiment, the invention provides BTLA-HVEM agonists that are agonistic HVEM fusion proteins which are capable of binding to a BTLA Ig domain, reducing the binding of the BTLA Ig domain to an HVEM CRD1 domain, and stimulating BTLA activity. Such agonistic HVEM fusion proteins lack an HVEM CRD2 and/or CRD3 domain. Preferred agonistic HVEM fusion proteins can compete with a BTLA antibody disclosed herein for binding to a BTLA Ig domain.

In another preferred embodiment, the invention provides BTLA-HVEM agonists that are agonistic HVEM protein fragments which are capable of binding to a BTLA Ig domain, reducing the binding of the BTLA Ig domain to an HVEM CRD1 domain, and stimulating BTLA activity. Such agonistic HVEM protein fragments lack an HVEM CRD2 and/or CRD3 domain. In a preferred embodiment, an agonistic HVEM protein fragment consists essentially of an HVEM CRD1 domain or fragment thereof which is capable of binding to a BTLA Ig domain and stimulating BTLA activity. Preferred agonistic HVEM protein fragments can compete with a BTLA antibody disclosed herein for binding to a BTLA Ig domain.

In one embodiment, a BTLA-HVEM agonist is capable of increasing tyrosine phosphorylation on the intracellular domain of BTLA protein in a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM agonist is capable of increasing association of BTLA protein with SHP-2, PI3K, or Grb2 in a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM agonist is capable of decreasing proliferation of a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM agonist is capable of decreasing IL-2 production by a cell having BTLA protein on its surface.

In one embodiment, a BTLA-HVEM agonist is capable of decreasing antibody production by a cell having said BTLA protein on its surface.

In one embodiment, a BTLA-HVEM antagonist is capable of decreasing the cytotoxicity of a cell having said BTLA protein on its surface.

In one aspect, the invention provides methods for modulating BTLA activity which involve the use of BTLA-HVEM agonists or BTLA-HVEM antagonists described herein.

In one aspect, the invention provides methods for decreasing BTLA activity, comprising contacting BTLA or HVEM with a BTLA-HVEM antagonist.

In a preferred embodiment, the method comprises contacting a cell having BTLA on its surface with a BTLA-HVEM antagonist, wherein the cell is capable of contacting HVEM protein in the absence of the BTLA-HVEM antagonist. In one embodiment, the methods involve use of a BTLA antibody.

In a preferred embodiment, the cells having BTLA on their surface are lymphocytes, NK cells, or macrophages.

In another embodiment, the method comprises contacting HVEM protein with a BTLA-HVEM antagonist, wherein the HVEM protein is capable of contacting a cell having BTLA on its surface in the absence of the BTLA-HVEM antagonist. In one embodiment, the methods involve use of an HVEM antibody.

In a preferred embodiment, the HVEM protein is on the surface of a dendritic cell or a lymphocyte.

In another embodiment, the invention provides methods for decreasing BTLA activation by a BTLA ligand that is capable of competing with HVEM for binding to BTLA, which comprise the use of a BTLA-HVEM antagonist. In one embodiment, the BTLA ligand is CMV UL144.

In one aspect, the invention provides methods for increasing BTLA activity comprising contacting a cell having BTLA on its surface with a BTLA-HVEM agonist. In one embodiment, the methods involve use of a BTLA antibody.

In one aspect, the invention provides methods for modulating lymphocyte activation which involve the use of BTLA-HVEM agonists or BTLA-HVEM antagonists described herein.

In one aspect, the invention provides methods for increasing lymphocyte activation. In one embodiment, the methods comprise contacting a lymphocyte having BTLA on its surface with a BTLA-HVEM antagonist, wherein the lymphocyte is capable of contacting HVEM protein in the absence of the BTLA-HVEM antagonist. In one embodiment, the methods involve use of a BTLA antibody.

In one embodiment, the methods involve contacting HVEM protein with a BTLA-HVEM antagonist, wherein the HVEM protein is capable of contacting a lymphocyte having BTLA on its surface in the absence of the BTLA-HVEM antagonist. In one embodiment, the methods involve use of a HVEM antibody.

In a preferred embodiment, a lymphocyte in which activation is increased is selected from the group consisting of naïve T cells, CD8⁺ T cells, CD4⁺ T cells, TH1 cells, naive B cells, and plasma cells.

In another embodiment, the invention provides methods for decreasing lymphocyte activation, comprising contacting a lymphocyte having BTLA on its surface with a BTLA-HVEM agonist. In one embodiment, the Methods involve use of a BTLA antibody.

In a preferred embodiment, a lymphocyte in which activation is decreased is selected from the group consisting of naïve T cells, CD8⁺ T cells, CD4⁺ T cells, TH1 cells, naive B cells, and plasma cells.

In one aspect, the invention provides methods for modulating lymphocyte effector activity.

In one aspect, the invention provides methods for decreasing lymphocyte effector activity, comprising contacting a lymphocyte having BTLA on its surface with a BTLA-HVEM agonist. In one embodiment, the methods involve the use of a BTLA antibody. Decreasing lymphocyte effector activity includes promoting the termination of effector activity, i.e., shortening the duration of effector activity.

In one aspect, the invention provides methods for increasing and/or prolonging lymphoctye effector activity, comprising contacting a lymphocyte having BTLA on its surface with a BTLA-HVEM antagonist. In one embodiment, the methods involve the use of a BTLA antibody. In another embodiment, the methods involve contacting an HVEM protein with a BTLA-HVEM antagonist. Prolonging effector activity includes delaying the termination of effector activity.

In another aspect, the invention provides methods for modulating an immune response to an antigen, which involve the use of BTLA-HVEM agonists or BTLA-HVEM antagonists described herein.

In one aspect, the invention provides methods for increasing an immune response to an antigen, comprising contacting a lymphocyte having BTLA on its surface with a BTLA-HVEM antagonist, wherein the lymphocyte has specificity for the antigen, and wherein the lymphocyte is capable of contacting HVEM protein in the absence of the BTLA-HVEM antagonist. In one embodiment, the methods involve use of a BTLA antibody.

In another embodiment, the methods comprise contacting HVEM protein with a BTLA-HVEM antagonist, wherein the HVEM protein is capable of contacting a lymphocyte having BTLA on its surface in the absence of the BTLA-HVEM antagonist, wherein the lymphocyte has specificity for the antigen. In one embodiment, the methods involve use of a HVEM antibody.

In a preferred embodiment, the antigen is a cancer cell antigen.

In another preferred embodiment, the antigen is a viral antigen.

In another preferred embodiment, the antigen is presented by a pathogen.

In another preferred embodiment, the antigen is provided by a vaccine.

In a preferred embodiment, the lymphocyte having BTLA on its surface and specificity for the antigen is contacted with a BTLA-HVEM antagonist in vivo.

In a preferred embodiment, the HVEM protein is contacted with a BTLA-HVEM antagonist in vivo.

In a preferred embodiment, the lymphocyte having specificity for the antigen is selected from the group consisting of naïve T cells, CD8⁺ T cells, CD4⁺ T cells,T_H1 cells, naive B cells, and plasma cells.

In one embodiment, the methods further comprise administering antigen to a patient receiving the BTLA-HVEM antagonist.

In one embodiment, the methods further comprise administering a bioactive agent that increases a positive costimulatory signal to a patient receiving the BTLA-HVEM antagonist.

In one embodiment, the methods further comprise administering a bioactive agent that decreases a negative costimulatory signal to a patient receiving the BTLA-HVEM antagonist. For example, it is contemplated that use of a BTLA-HVEM antagonist will be synergistic in combination with agents capable of providing CTLA-4 blockade as described in U.S. Pat. Nos. 5,855,887; 5,811,097;and 6,051,227, and International Publication WO 00/32231, the disclosures of which are expressly incorporated herein by reference.

In one embodiment, the invention provides methods for increasing an immune reaction against a tumor in a patient, comprising contacting a lymphocyte having BTLA on its surface with a BTLA-HVEM antagonist, wherein the lymphocyte has specificity for a cancer cell antigen associated with the tumor and is capable of contacting HVEM protein. In one embodiment, the methods involve use of a BTLA antibody.

In another embodiment, the methods comprise contacting HVEM protein with a BTLA-HVEM antagonist, wherein the HVEM protein is capable of contacting a lymphocyte having BTLA on its surface, and wherein the lymphocyte has specificity for a cancer cell antigen associated with the tumor. In one embodiment, the methods involve use of a HVEM antibody.

In a preferred embodiment, the methods further comprise administering a cancer cell antigen to the patient.

In a preferred embodiment, the methods further comprise administering a bioactive agent that increases a positive costimulatory signal.

In a preferred embodiment, the methods further comprise administering a bioactive agent that decreases a negative costimulatory signal to the cancer patient. For example, it is contemplated that use of a BTLA-HVEM antagonist will be synergistic in combination with agents capable of providing CTLA-4 blockade as described in U.S. Pat. Nos. 5,855,887; 5,811,097;and 6,051,227, and International Publication WO 00/32231.

In a preferred embodiment, the lymphocyte having BTLA on its surface and specificity for the cancer cell antigen is contacted with a BTLA-HVEM antagonist in vivo.

In a preferred embodiment, the HVEM protein is contacted with a BTLA-HVEM antagonist invivo.

In a preferred embodiment, the lymphocyte having specificity for the cancer cell antigen is selected from the group consisting of naïve T cells, CD8⁺ T cells, CD4⁺ T cells,T_H1 cells, naive B cells, and plasma cells.

In one aspect, the invention provides methods for inhibiting tumor growth, comprising administering to a patient a therapeutically effective amount of a BTLA-HVEM antagonist.

In one aspect, the invention provides methods for treating cancer, comprising administering to a patient a therapeutically effective amount of a BTLA-HVEM antagonist.

In a preferred embodiment, the methods further comprise administering a bioactive agent that decreases a negative costimulatory signal to the cancer patient. For example, it is contemplated that use of a BTLA-HVEM antagonist will be synergistic in combination with agents capable of providing CTLA-4 blockade as described in U.S. Pat. Nos. 5,855,887; 5,811,097; and 6,051,227, and International Publication WO 00/32231.

In one aspect, the invention provides methods for reducing an immune response to an antigen, comprising contacting a lymphocyte having BTLA on its surface with a BTLA-HVEM agonist, wherein the lymphocyte has specificity for the antigen. In one embodiment, the methods involve use of a BTLA antibody.

In a preferred embodiment, the antigen is a graft cell antigen.

In another preferred embodiment, the antigen is a self antigen.

In another preferred embodiment, the lymphocyte having specificity for the antigen is selected from the group consisting of naïve T cells, CD8⁺ T cells, CD4⁺ T cells,T_H1 cells, naive B cells, and plasma cells.

In one embodiment, the methods further comprise administering a bioactive agent that decreases a positive costimulatory signal to the patient.

In one embodiment, the methods further comprise administering an immunosuppressant to the patient.

In one embodiment, the methods further comprise administering a bioactive agent that increases a negative costimulatory signal to the patient.

In one embodiment, the invention provides methods for reducing an immune reaction against a graft in a patient, comprising contacting a lymphocyte having BTLA on its surface with a BTLA-HVEM agonist, wherein the lymphocyte has specificity for a graft cell antigen. In one embodiment, the BTLA-HVEM agonist is a BTLA antibody.

In another preferred embodiment, the lymphocyte having specificity for the antigen is selected from the group consisting of naïve T cells, CD8 T cells, CD4⁺ T cells,T_H1 cells, naive B cells, and plasma cells.

In one aspect, the invention provides methods for reducing rejection of a graft by a patient, comprising administering to the patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for prolonging the survival of a graft in a patient, comprising administering to the patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing a graft versus host response in a patient, comprising administering to the patient a therapeutically effective amount of a BTLA-HVEM antagonist.

In one embodiment, the methods further comprise administering a bioactive agent that increases a positive costimulatory signal to the patient.

In one embodiment, the methods further comprise administering a bioactive agent that decreases a negative costimulatory signal to the patient. For example, it is contemplated that use of a BTLA-HVEM antagonist will be synergistic in combination with agents capable of activating CTLA-4 as described in U.S. Pat. Nos. 5,855,887; 5,811,097;and 6,051,227, and International Publication WO 00/32231.

In one aspect, the invention provides methods for treating a patient having an autoimmune disease, comprising administering to the patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one embodiment, the autoimmune disease is selected from the group consisting of Rheumatoid arthritis,type 1 diabetes, autoimmune thyroiditis, and Lupus.

In one aspect, the invention provides methods for treating a patient having an allergic reaction, comprising administering to the patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for preventing a patient from having an allergic reaction, comprising administering to the patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing an allergic reaction in a patient, comprising administering to the patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing an asthmatic response in a patient, comprising administering to the patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for enhancing recovery from an asthmatic response in a patient, comprising administering to the patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for treating asthma, comprising administering to an asthma patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing an inflammatory reaction in a patient, comprising administering to the patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing the interaction of cell having BTLA on its surface and a second cell having HVEM on its surface. The methods involve the use of a BTLA-HVEM antagonist or a BTLA-HVEM agonist. In a preferred embodiment, the methods involve use of a BTLA antibody or a HVEM antibody. In a preferred embodiment, the cell having BTLA on its surface is selected from the group consisting of naïve T cells, CD8⁺ T cells, CD4⁺ T cells,T_H1 cells, naive B cells, and plasma cells.

In one aspect, the invention provides methods for modulating memory cell formation, comprising contacting a lymphocyte exposed to antigen with a BTLA-HVEM agonist or antagonist. In a preferred embodiment, the methods involve the use of a BTLA antibody.

In one aspect, the invention provides methods for modulating tolerance of self antigen, comprising contacting a lymphocyte exposed to self antigen with a BTLA-HVEM agonist or antagonist. In a preferred embodiment, the methods involve the use of a BTLA antibody.

Also provided are adjuvant compositions comprising at least one of the BTLA-HVEM antagonists described herein.

Also provided are immunosuppressant compositions comprising at least one of the BTLA-HVEM agonists described herein.

In another aspect, the present invention provides methods of screening for BTLA-HVEM agonists and BTLA-HVEM antagonists, which agonists and antagonists find therapeutic uses for the modulation of immune reactions.

The invention further contemplates the use of the aforementioned polypeptides in immunoassays.

The invention further contemplates the use of the aforementioned polypeptides as immunogens for the production of antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 BTLA recognizes a ligand on naive T cells. Splenocytes from BALB/c and C57BL/6 mice were collected and either were directly stained (None) or were activated with plate-bound 500A2 (Anti-CD3; 1:200 dilution of ascites fluid) or soluble anti-IgM (10 μg/ml) for 48 h, and then were stained with BTLA-Fc or PD-L1-Fc fusion protein (shaded histograms) followed by anti-human IgG-phycoerythrin (Anti-human IgG-PE), anti-CD4-tricolor and anti-B220-FITC. Open histograms, staining with human IgG1 isotype control in place of Fc fusion protein.

FIG. 2 BTLA tetramer staining identifies a ligand on CD4+ and CD8+ cells. (a) Splenocytes and lymph node cells from pooled C57BL/6 and BALB/c mice were stained with anti-CD8-FITC, antiCD4-CyChrome, and either streptavidin-phycoerythrin (open histograms) or BTLA tetramer-phycoerythrin (shaded histograms). Dot plots (left) show the CD4-CD8 gates used for single-color histograms of BTLA tetramer-phycoerythrin staining (right). (b) Splenocytes from pooled C57BL/6 and BALB/c mice were left untreated or were activated 48 h with anti-CD3 or anti-IgM as described inFIG. 1 or with lipopolysaccharide (1 μg/ml) for 24 h and were stained with antiB220-FITC, anti-CD4-CyChrome, and either streptavidin-phycoerythrin (open histograms) or BTLA tetramer-phycoerythrin (shaded histograms). (c) Thymocytes from pooled C57BL/6 and BALB/c mice were stained with anti-CD8-FITC, anti-CD4-CyChrome, and either streptavidin-phycoerythrin (open histograms) or BTLA tetramer-phycoerythrin (shaded histograms). The dot plot (left) shows the CD4/CD8 gates used for the single-color histograms of BTLA-tetramer staining.

FIG. 3 BTLA ligand expression is modulated during T cell activation. D011.10 splenocytes were stimulated with 0.3 μM OVA peptide inT helper type 1 conditions (T_H1; 10 U/ml of IL-12 and 10 μg/ml of anti-IL-4) orT helper type 2 conditions (T_H2; 100 U/ml of IL-4 and 3 μg/ml of anti-IL-12). Cultures were collected after activation (time, horizontal axis) and were stained with anti-CD4-FITC and BTLA tetramer-phycoerythrin. Filled circles, streptavidin-phycoerythrin (SA-PE) staining ofT helper type 1 cultures without BTLA tetramer. MFI, mean fluorescence intensity.

FIG. 4 HVEM is a ligand for BTLA. (a) NIH 3T3 cells and BJAB cells were transduced with splenocyte cDNA libraries and were directly stained with anti-Thy1.1-FITC and the C57BL/6 BTLA tetramer-phycoerythrin (Before sorting). These cells were sorted for the highest 0.5% population of BTLA tetramer staining with BTLA-phycoerythrin tetramer and Thy1.1-FITC and were subjected to an additional three rounds of similar sequential purification. After the fourth round of sorting, cell populations were expanded and cells were stained (After sorting). Numbers in each quadrant indicate the percentage of live cells in the indicated gate. (b) BJAB cells were transduced with the retroviruses mHVEM-IRES-GFP (mHVEM; mouse), hHVEM-IRES-GFP (hHVEM; human), 4-1 BB-IRES-GFP (4-1 BB; mouse) and LTBR-1 RES-GFP (LT(3R; mouse) and were stained with C57BL/6 BTLA tetramer-phycoerythrin or BALB/c BTLA tetramer-phycoerythrin. Numbers in dot plots indicate the percentage of BTLA tetramer staining in the GFP-positive population. (c,d) HVEM activates BTLA phosphorylation and SHP-2 association. EL-4 cells (EL4), BJAB cells expressing GFP (BJAB-GFP) or BJAB cells expressing mouse HVEM (BJAB-mHVEM) were added (+) or not added (−) for 4 min at 37° C. at a density of 25×10⁶cells/ml. Cells were left untreated (−) or were treated (+) with pervanadate (VO₄) for 4 min. Total cell lysates were prepared and were immunoprecipitated with 6A6 (anti-mouse BTLA), and immunoblots were probed for SHP-2 (c) or for phosphotyrosine (d) in immunoprecipitates (IP) or in lysates without immunoprecipitation. Immunoblots using the isotype control for immunoprecipitation were negative for SHP-2 association (data not shown). Data in c and d are representative of four independent experiments. (e) BJAB cells were transduced with retrovirus mHVEM-ires-GFP or hHVEM-ires-GFP and were stained with human IgG1 isotype control (hIgG1), mB7x-Fc, mBTLA-Fc or hBTLA-Fc followed by anti-human IgG-phycoerythrin. Numbers in dot plots show the percentage of fusion protein staining in the GFP-positive population.

FIG. 5 HVEM is the unique ligand for BTLA and interacts through CRD1. (a) Splenocytes from wild-type (Tnfrsf14+/+) or HVEM-deficient (Tnfrsfl4−/−) mice were stained with anti-CD4-FITC (CD4+), anti-CD8-FITC (CD8+) or anti-B220-FITC (B220+) and either C57BL/6 BTLA tetramer-phycoerythrin (shaded histograms) or streptavidin-phycoerythrin alone (open histograms). (b) Splenocytes from wild-type (BTLA+/+) and BTLA−/− mice were stained with anti-B220-FITC (top) or anti-CD11c-FITC (bottom) and with either mHVEM-Fc (shaded histograms) or isotype control human IgG1 (open histograms) followed by anti-human IgG-phycoerythrin. (c) Splenocytes from wild-type (Tnfrsfl4+/+) and Tnfrsfl4−/− mice were stained with B220-FITC (top) or CD11c-FITC (bottom) and mHVEM-Fc or isotype control human IgG1 (open histograms), followed by anti-human IgG-phycoerythrin. (d) BJAB cells were left uninfected or were transduced with retroviruses expressing mouse HVEM-GFP fusion protein (mHVEM-GFP), the HVEM deletion mutant lacking N-terminal CRD1 as a GFP fusion protein (mHVEMCRDI-GFP), intact human HVEM (hHVEM-IRES-GFP) or chimeric HVEM containing mouse CRD1 linked to human CRD2 (m/hHVEM-IRES-GFP). Left, cells stained with BTLA tetramer-phycoerythrin (shaded histograms) or streptavidin-phycoerythrin alone (open histograms); right, cells stained with either anti-hHVEM (shaded histograms) or a mouse IgG1 isotype control (9E10) followed by goat anti-mouse IgG1-phycoerythrin. Single-color histograms were gated on GFP-positive live cells. Right margin, composition of the HVEM constructs, with mouse CRDs (open ovals) and human CRDs (shaded ovals).

FIG. 6 HVEM expression on APCs inhibits T cell proliferation. (a) CD4+ cells were purified from BALB/c mice by magnetic separation and were stimulated (1×10⁶cells/ml) with plate-bound anti-CD3 (2C11; dose, horizontal axis) and increasing concentrations (wedges; 0, 0.3, 1.0, 3.0 and 10.0 μg/ml) of plate-bound LIGHT. Cultures were pulsed with [³H]thymidine at 48 h and were collected at 60 h. Data represent c.p.m. s.d. from one of three similar experiments. (b) CD4+ T cells from D011.10 mice were purified by magnetic separation, followed by cell sorting for CD4+B220-CD11ccells to more than 98% purity, and were added to cultures alone (T alone) or with (T+) CHO cells expressing I-Ad, I-Ad and B7.1, or I-Ad and BTLA, plus various concentration of OVA peptide (horizontal axis), and proliferation was measured as described in a. (c) T cells prepared as described in b were cultured alone or with CHO cells expressing I-Ad, or I-Ad and HVEM, plus various concentrations of OVA peptide, and proliferation was measured as described in a. (d) T cells prepared as described in b were cultured alone or with CHO cells expressing I-Ad, or I-Ad and B7.1, or I-Ad, B7.1 and HVEM, and were activated with various concentration of OVA peptide. Proliferation was measured as described in a.

FIG. 7 HVEM inhibits T cell proliferation in a BTLA-dependent way. (a) Highly purified D011.10 CD4+ T cells from wild-type (BTLA+/+) or BTLA−/− mice were prepared as described inFIG. 6, were labeled with CFSE and were cultured for 3 or 4 d with CHO cells expressing I-Ad, or I-Ad and BTLA, or I-Ad and HVEM, plus 0.03 or 0.3 μM OVA peptide. Cells were analyzed by flow cytometry. Data are single-color histograms of CFSE gated on CD4+T cells. Numbers indicate percentage of live cells that have divided at least once, as indicated by the gate drawn. (b) T cells prepared as described in a were cultured for 3 or 4 d with CHO cells expressing I-Ad and B7.1, I-Ad, B7.1 and BTLA, or I-Ad, B7.1 and HVEM, plus 0.03 or 0.3 μM OVA peptide, and were analyzed as described in a. Numbers indicate percentage of live cells that have divided at least once.

FIG. 8 Polymorphisms in the BTLA Ig domain. A,Exon 2 of BTLA, comprising the Ig domain, was amplified by PCR from genomic DNA of the indicated mouse strains and sequenced. The amino acid alignment of the Ig domains of BALB/c (SEQ ID NO:3), MLR/Ipr (SEQ ID NO:4), and C57BL/6 (SEQ ID NO:5) BTLA is shown, starting with the aspartic acid (D) residue that corresponds to residue 37 of the entire BTLA protein. The last line of the alignment shows a consensus sequence (bottom), with differences between BALB/c and MLR/Ipr (#) and differences between BALB/c and C57BL/6 (*) shown. B, Strains sharing identical alleles of BTLA are grouped together under the index headings of BALB/c, MLR/Ipr, and C57BL/6.

FIG. 9 Production of mAbs to allelic variants of murine BTLA. A and B, BJAB cells were stably transfected with retroviral constructs expressing the extracellular/transmembrane domains of BTLA from C57BL/6 (BJAB.B6 BTLA-GFP, solid histogram) or BALB/c (BJAB.BALB/c BTLA-GFP, dotted histogram) as GFP fusion proteins. Cells were stained with the indicated purified mAbs or postimmune serum (hamster anti-BTLA (A) serum, mouse anti-BTLA (B) serum). Secondary staining was with either anti-hamster IgG (A) or anti-mouse Ig (B). Histograms shown are gated on GFP+BJAB. B6 BTLA-GFP or BJAB.BALB/c BTLA-GFP cells stained separately. Shaded histogram for the hamster and mouse immune serum are controls using normal hamster serum or normal mouse serum to stain a mixture of BJAB. B6 BTLA-GFP and BJAB.BALB/c BTLA-GFP cells. Shaded histogram for mAb staining shows the isotype control of either hamster IgG (A) or murine IgG1 (B) staining a mixture of cells. C, Splenocytes from C57BL/6 or BALB/c wild-type mice (solid histogram) or BTLA−/− mice (dotted histogram) were stained with 6A6 (left) or 6F7 (right). BTLA−/− staining was equivalent to that of the isotype control (shaded histogram). D, Lysates from 25×10⁶cells BJAB. B6 BTLA-GFP or BJAB.BALB/c BTLA-GFP cells were immunoprecipitated (IP) with 10 μg of the indicated Ab and Western blots probed (Blot) with either 6F7, or with anti-GFP Ab, as indicated. As controls, cell lysates were immunoprecipitated with mouse or hamster IgG as indicated (lanes 7-10). E, EL4 cells were incubated in the absence (−) or presence (+) of pervanadate for 4 min at 37° C., and lysed in 1% Triton X-100 lysis buffer, immunoprecipitated (IP) with 6A6 or isotype control Ab (PIP anti-GST) and Western blots probed (Blot) with antiSHP-2 as described.

FIG. 10 Mapping epitopes recognized by BTLA Abs using Yeast Display. A panel of yeast cells expressing the indicated BTLA Ig domain Aga2 fusion proteins was analyzed for Ab staining. As a positive control, expression of the fusion protein was confirmed first for each line using staining with anti-HA Ab specific for the HA-tag incorporated into the BTLA-Aga2 fusion protein, and was positive for each line tested (data not shown). Yeast cells were stained with the anti-BTLA Ab indicated on top of each column. The amino acid substitutions (and corresponding nucleotide substitutions) in each yeast line are indicated on the left. Single-color histograms are marked (*) to indicate mutations that are not recognized by the corresponding Ab.

FIG. 11 BTLA shows broad and allelic-specific expression on lymphoid cell populations. A, Four-color FACS analysis was conducted on splenocytes from C57BL/6 (solid histogram) or BALB/c (dotted histogram). Two-color histograms (upper row) of the indicated markers used to gate cells for single-color histograms of 6A6 (middle row) or 6F7 (lower row) staining are shown. In the columns one, two, and three, cells were stained with anti-B220 allophycocyanin, anti-CD4 CyChrome, anti-CD8 FITC, and either biotinylated b-6A6 or b-6F7 followed by SA-PE secondary. In columns four, five, and six, cells were stained with anti-I-Ad PE (BALB/c cells) or anti-I-Ab PE (C57BU6 cells), and anti-CD11b FITC (fourth column), CD11c-FITC (fifth column), or anti-DX-5 FITC (sixth column), and b-6A6 or b-6F7 followed by SA-CyChrome secondary. Shaded histograms are staining of a mixture of C57BL/6 and BALB/c splenocytes using isotype controls of biotinylated hamster IgG (middle row) and mouse IgG1 (lower row). The numbers shown in top panels are the percentage of live cells within the indicated gate. The identity of the gated population is indicated in the panel. B, C57BL/6 and BALB/c splenocytes were stained with Abs to identify the following B cell populations: follicular B cells (FO), IgMlowCD21/CD35int; marginal zone (MZ), IgMhighCD21/CD35high; transitional (TR), IgMlowCD21/CD35low. Staining with the pan-BTLA-specific Ab 6F7 revealed equivalent BTLA levels between strains for all subsets.

FIG. 12 BTLA is expressed during late stages of B and T lymphocyte development. A, Thymocytes from C57BL/6 (solid histogram) or BALB/c (dotted histogram) mice were stained with a combination of markers, anti-B220 RTC, anti-CD11c FITC, anti-CD11b FITC, anti-GR-1 FITC, antiDX-5 FITC, CD4-CyChrome, CD8-PE, and either biotinylated (b)-6A7 or b-mouse IgG1, and SA-allophycocyanin. The two-color histogram (first panel) is gated on marker (FITC)-negative live cells, and the numbers indicate the percentage of cells in the indicated gates. Single-color histograms for each gate are shown for b-6F7/SA-allophycocyanin staining for CD4⁻CD8⁻ double negative (DN), CD4+CD8+ double positive (DP), CD4+ single positive (CD4 SP), or CD8+ single positive (CD8 SP) populations. Shaded histograms are staining the b-mouse IgG1 isotype control. B, Bone marrow cells were stained with anti-B220 allophycocyanin, anti-IgM PerCp Cy5.5, either b-6F7 or murine IgG1 biotin, and SA-PE. The numbers are the percentage of live gated cells within the three numbered gates. BTLA expression is shown in the single-color histograms for each gate;gate 1, Pre-B cells and Pro-B cells (IgM-B220low);gate 2, Immature B cells (IgM+B220low);gate 3, Mature B cells (IgM+B220high). Shaded regions are mouse IgG1 isotype control staining.

FIG. 13 BTLA expression during CD4+ T cell activation and Th1 polarization. A, DO11.10 transgenic T cells were purified by cell sorting and activated with 0.3 μM OVA peptide 324-336 under Th1 or Th2 conditions (see Examples). Cells were harvested either before activation (Day 0) or on the indicated day following primary activation, and stained with KJ1-26 Tricolor, b-6F7, and SA-PE. T cells were restimulated with OVA peptide onday 7 andday 14. B, BALB/c splenocytes were stimulated with 10 μg/ml anti-IgM and 5 μg/ml anti-CD40 (left) or 1 μg/ml LPS (right). Single-color histograms of B220+ cells (anti-B220-FITC) are shown for b-6F7/SA-PE staining on day 0 (dotted histogram) and day 2 (solid histogram) after activation. Shaded histograms are the biotinylated mouse IgG1 isotype control.

FIG. 14 BTLA is induced on anergic CD4+ T cells, but not CD4+CD25+ regulatory T cells. A, HA-TCR T cells were transferred into and subsequently harvested from B10.D2 mice (naive), C3-HAhigh mice (anergized) or B10.D2 mice infected with vaccinia-HA (activated) on

days

2, 3, 4, or 7 after transfer as indicated. After harvest, T cells were isolated using combined magnetic bead and fluorescence sorting, and cDNA probe prepared and hybridized to Affymetrix microarrays M174A, M174B, and M174C. Relative BTLA expression intensity was determined using a latin-squares approach in Affymetrix Microarray Suite, version 5.1. software. Expression of myosin Vila gene is shown as a control. B, CFSE-labeled HA-TCR T cells were adoptively transferred into B10.D2 mice (naive), C3-HAhigh mice (anergized), or B10.D2 mice immunized with vaccinia-HA (activated), and harvested onday 6 as in A. Cells were stained with anti-CD4 allophycocyanin, anti-Thy1.1 PerCP, and either b-6F7 or murine IgGI-biotin, and SA-PE. BTLA expression is shown as single-color histogram for CFSE+ (naive) or CFSE-(activated and anergized) for CD4+ Thy1.1+ donor cells. C, Splenocytes harvested from recipients as in A were restimulated with HA peptide and proliferation measured onday 2. D, Splenocytes and lymph node cells from BALB/c mice were enriched for CD25-negative and CD25-positive populations using anti-CD25-PE and magnetic beads as described in Materials and Methods, and stained with anti-CD4-Cy-chrome, and biotin-conjugated 6F7, or biotin-IgG1, followed by SA-allophycocyanin. Two-color dot plots are shown for CD25 and CD4 (left panels), or single-color histograms gated on CD4+ cells for 6F7 (middle panels) or anti-PD-1 (right panels) for the CD25− (top row) and CD25+ (bottom row) fractions. For BTLA staining, histograms are shown for both the freshly isolated cells (thin histogram) and 36 h antiCD3-activated cells (thick histogram). Shaded histograms are the staining of the mouse IgG1 istoype control. E, Cells isolated in D were stimulated with the indicated amount of anti-CD3 and proliferation measured after 2 days.

FIG. 15 BTLA−/− mice have modestly augmented IgG3 responses to T-independent Ag. 129SvEv wild-type mice or BTLA−/− mice (n=5) were immunized with 50 pg NP-Ficoll in alum by i.p. injection. Atday 14, relative isotype-specific anti-NP Ab titer in serum was determined by ELISA. Data are shown as the percentage of the Ab titer produced in serum of naive BTLA+/+ or BTLA−/− mice. Mean±SD is shown.

FIG. 16 BTLA−/− parental cells engraft and initially expand.

FIG. 17 BTLA−/− parental cells fail to survive following transfer.

FIG. 18 BTLA−/− cells do not persist as GHVD progresses. Until aboutday 9, the expansion of WT and BTLA KO donor T cells is similar; At later times, BTLA−/− show rapid decrease in the number of remaining donor cells.

FIG. 19 HVEM induces BTLA-phosphorylation and SHP-2 recruitment in trans.

FIG. 20 HVEM on APCs inhibits T cell proliferation through BTLA. HVEM on APCs inhibits T cell proliferation: HVEM does not inhibit BTLA−/− T cells.

FIG. 21 HVEM on APCs inhibits T cell proliferation through BTLA.

FIG. 22 HVEM inhibition is overcome by strong costimulation. HVEM inhibition of T cells is less with stronger co-stimulation. HVEM inhibition of T cells is less at highest antigen doses.

FIG. 23 6A6 binds to amino acid residues E34 and R73 of BTLA. Antibody interactions are most affected by E34Q and R73Q mutations, and slightly affected by H23Q and W56C mutations. E34 and R73 are E63 and R102 in full length protein.

FIG. 24 shows the amino acid sequence of human BTLA, also found at Genbank Accession No. AAP44003.1 (SEQ ID NO:2).

FIG. 25 PD-1 and BTLA are expressed on BAL CD4 T cells: C57BL/6 mice were sensitized and challenged with Ovalbumin. On

days

1, 3, 4, and 7 following challenge, groups of mice were euthanized and the cells recovered in the BAL analyzed for expression of CD4 and PD-1 or BTLA by 2-color flow cytometry. The percentage of cells positive for CD4 as a fraction of either the total sample or of the lymphocyte gate as well as the total number of CD4+ cells recovered is indicated in each box. Histograms of PD-1 or BTLA expression on the CD4+ cells are shown for

days

3, 4 and 7. Representative data of 3 independent experiments is presented.

FIG. 26 PD-1 and BTLA have a minor effect on acute allergic airway inflammation: C57BL/6, PD-1 −/− and BTLA −/− mice (n=5 per group) were sensitized and challenged with OVA. 3 days following challenge, the mice were euthanized and samples collected for analysis. A) Total cell counts in the BAL fluid. B) Differential analysis of the cell types present in the BAL. C) Representative fields of H and E stained sections (40× magnification). *=P<0.05 **=P<0.005 compared to C57BL/6 by 2 tailed T test. Representative data from 5 independent experiments is shown.

FIG. 27 Expression of the ligands for PD-1 and BTLA during allergic airway inflammation: Total RNA was isolated from whole lungs of allergen challenged mice on the indicated days post-challenge or from primary cultured murine tracheal epithelial cells (mTEC). RT-PCR was performed using specific primers that spanned intronic sequences of each gene. Shown is representative data from 2 independent experiments.

FIG. 28 shows that PD-1 and BTLA-deficient mice have a prolonged duration of airway inflammation: C57BL/6, PD-1 −/− and BTLA −/− mice were sensitized and challenged with OVA. On

days

10 and 15 cohorts of mice (n=5/group) were euthanized and samples collected for A) analysis of the BAL and B) histology. *=p<0.05 compared to C57BL/6 using a 2 tailed T-test.

FIG. 29 shows graphs and micrographs illustrating that BTLA and HVEM, but not PD-1, regulate the survival of partially MHC-mismatched cardiac allografts. a, The lack of BTLA or HVEM, or administration of a neutralizing anti-BTLA mAb, led to rejection of all MHC class II-mismatched cardiac allografts within 3-4 wk of transplantation, whereas wild-type (WT) recipients accepted Bm12 allografts indefinitely. Data were generated from six to 12 allografts/group; p<0.001 for BTLA−/−, HVEM−/−, or anti-BTLA mAb-treated group vs respective WT controls. Panels at the right show acute cellular rejection of Bm12 allografts harvested 2 wk after transplant from BTLA−/−, but not WT, recipients (H&E-stained paraffin sections; original magnifications, ×300). b, In contrast to BTLA and HVEM, a lack of PD-1 still allowed >80% long-term survival of MHC class II-mismatched cardiac allografts (p<0.05 compared with isotype-treated WT control), and an absence of both PD-1 and BTLA (DKO) led to only a minor acceleration of allograft rejection compared with lack of BTLA alone (p<0.05 vs BTLA−/− alone) in B6 recipients of Bm12 cardiac allografts. Data were generated from four to eight allografts per group. c, Lack of BTLA led to rejection of all MHC class I-mismatched cardiac allografts, whereas WT recipients accepted Bm1 allografts indefinitely. Data were generated from six to 12 allografts/group (p<0.001). Panels at the right show histologic evidence of developing cellular rejection of Bm1 allografts harvested 4 wk after transplant from BTLA−/−, but not WT, recipients (H&E-stained paraffin sections; original magnifications, ×300).

FIG. 30 shows graphs illustrating that BTLA suppresses T cell responses to MHC class II alloantigens. a, Intragraft mRNA expression of BTLA, PD-1, and ligands was determined by qPCR; data are expressed as the fold increase compared with naive heart and are representative of three separate experiments (Bm123B6 cardiac allografts). b, Compared with wild-type (WT) CD4⁺ T cells, CD4⁺ T cells from BTLA−/− mice had markedly enhanced proliferative responses to Bm12 APC. Data at 72 h are expressed as a percentage of live BrdU⁺ CD4 cells at each stimulator (S) to responder (R) ratio (pooled triplicate wells). c, Assessment of alloactivation induced CD4⁺ T cell proliferation at 72 h induced by irradiated Bm12 APC; the percentage of dividing CD4⁺ T cells was determined by CFSE dilution. d, Markedly increased proliferation of CFSE-labeled BTLA−/− CD4⁺ T cells 72 h after transfer into irradiated Bm12 hosts. Data are representative of two experiments with similar results. e, Marginally increased proliferation of CFSE-labeled BTLA−/− CD8⁺ T cells 72 h after transfer into irradiated Bm12 hosts. Data are representative of two experiments with similar results. f, Significantly increased responder frequency in BTLA−/− recipients of class II-mismatched cardiac allografts, as shown by harvesting ofrecipient spleens 10 days after transplant and stimulation of recipient splenocytes in vitro with irradiated Bm12 (p<0.001 at all ratios) or B6 DC (syngeneic control) for 24 h. Donor-specific responder frequency was expressed as the number of IFN-y spot-forming cells (SFC) per 1×10⁶splenocytes, and data (mean±SD) are representative of two experiments.

FIG. 31 shows graphs and photographic images demonstrating that BTLA targeting prolongs survival of fully MHC-mismatched cardiac allografts. Targeting of BTLA significantly prolonged BALB/c cardiac allograft in fully allogeneic B6 recipients, as shown using BTLA-/recipients (a) and anti-BTLA mAb in wild-type (WT) mice (b). c, In addition, a subtherapeutic course of rapamycin (RPM; 10 μg/kg/day, i.p., for 14 days) significantly prolonged cardiac allograft survival compared with either identically treated WT mice or BTLA−/− controls. Allograft survival data in a-c were obtained from six to eight transplants per group. d, BALB/c hearts transplanted to WT or BTLA−/− B6 mice were harvested 7 days after transplant for qPCR. Data from three allografts per group are expressed as the fold increase compared with native heart. e, Western blots of CXCR3 and IP-10 proteins, using extracts of three allografts per group. The effects of targeting BTLA, alone or in combination with low dose RPM, on allogeneic T cell proliferation and cytokine production were determined by adoptive transfer of CFSE-labeled splenocytes from WT or BTLA−/− mice to B6D2F1 hosts, and recipient spleens were harvested at 72 h. The responses of donor T cells were identified by gating on Kd-Dd-cells. Data are shown as an overlay of CFSE histograms (f) and analysis of intracellular cytokine production (g). The figure in each box is the percentage of the indicated population, and data are representative of two experiments with similar results.

FIG. 32 shows graphs illustrating the dominant role of PD-1 in regulating the survival of fully MHC-mismatched cardiac allografts. a, Dual PD-1/BTLA−/− (DKO) recipients rejected fully MHC-disparate allografts at the same speed as wildtype (WT) recipients. b, Neutralization of PD-1 in BTLA−/− recipients reversed the prolongation of survival seen in BTLA−/− mice (p<0.001). c, The dominant role of PD-1 was also seen by the quick rejection of allografts in DKO mice, despite therapy with rapamycin (RPM; 10 μg/kg/day, i.p., for 14 days), in marked contrast to the prolonged survival in BTLA−/− recipients treated with the same dose of RPM (p<0.001). d, The key contribution of PD-1, but not BTLA, in promoting the survival of fully MHC mismatched cardiac allografts in RPM-treated recipients was confirmed by the rapid onset of acute rejection in BTLA−/− recipients treated with anti-PD-1 mAb (p<0.001).

FIG. 33 shows plots that demonstrate increased PD-1 expression and function by alloreactive T cells of BTLA−/− recipients of fully MHC-mismatched cardiac allografts. a, Intragraft mRNA expression of BTLA, PD-1, and ligands was determined by qPCR. Data are expressed as the fold increase compared with naïve heart and are representative of three separate experiments (BALB/c3B6 cardiac allografts). b, PD-1 expression by alloreactive T cells determined by adoptive transfer of CFSE-labeled wild-type (WT) or BTLA−/− splenocytes to irradiated Bm12 or B6D2F1 hosts, with or without added rapamycin (RPM; 0.01 mg/kg, i.p., for 3 days). Figures indicate the percentages of PD-1 cells in the divided and undivided donor T cell populations. c, Increased proliferation of CFSE-labeled T cells from DKO mice or PD-1-/mice vs WT or BTLA−/− controls after adoptive transfer to F1 hosts, with or without RPM therapy. Analysis of corresponding intracellular cytokine production by the groups shown in c was undertaken, alone (d) or in conjunction with RPM therapy (e). Cells were stained with KdPE and CD4− or CD8-PerCP, and IL-2 or IFN-y APCs and donor cells were identified as the KdDd-population; the percentage of each indicated population is shown.

FIG. 34 shows graphs illustrating that as the strength of T cell signaling increases, PD-1 induction predominates over that of BTLA. Increasing T cell activation by mature fully allogeneic BALB/c bone marrow-derived DC leads to increasing expression of PD-1, rather than BTLA, by C57BL/6 CD4 and CD8 T cells, as shown by flow cytometric analysis of cells cultured at varying stimulator (S) to responder (R) ratios for 72 h. Data are representative of three such experiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term “HVEM CRD1 domain” refers to the CRD1 domain of an HVEM protein. An HVEM CRD1 domain binds to a BTLA Ig domain, and can be specifically bound by a preferred HVEM antibody disclosed herein. The HVEM CRD1 domain does not include the CRD2 or CRD3 domains of the HVEM protein. A preferred CRD1 domain is that set forth by residues 41-76 of the human HVEM protein sequence at Genbank accession no. AAB58354.1 (SEQ ID NO:6). See Montgomery et al., Cell, 87:427-436, 1996. Other preferred CRD1 domains are those having at least about 80%, 85%, 90% or 95% identity to the sequence set forth by residues 41-76 of the human HVEM protein sequence at Genbank accession no. AAB58354.1 (SEQ ID NO:6). Another preferred CRD1 domain is that set forth by residues 3980 of the murine HVEM protein sequence at Genbank accession no. AAQ08183.1 (SEQ ID NO:7). Otherpreferred CRD 1 domains are those having at least about 80%, 85%, 90% or 95% identity to the sequence set forth by residues 39-80 of the murine HVEM protein sequence at Genbank accession no. AAQ08183.1 (SEQ ID NO:7).

As used herein, the term “HVEM CRD1 domain peptide” refers to a peptide corresponding in sequence to a region of the CRD1 domain of HVEM, which peptide can bind to the Ig domain of BTLA. An HVEM CRD1 domain peptide is capable of reducing the binding of the HVEM CRD1 domain to the BTLA Ig domain, and is a BTLA-HVEM antagonist.

As used herein, the term “BTLA Ig domain” refers to the portion of a BTLA protein corresponding to the portion of BTLA that has been used to identify the HVEM-BTLA interaction. In particular, the BTLA Ig domain, as used herein, comprises an immunogloublin domain. Further, as compared to the BTLA sequence of C57BL/6 mouse, as found at Genbank accession no. NP 808252.1 (SEQ ID NO:1), the BTLA Ig domain corresponds to amino acids 30-166. Further, as compared to the human BTLA sequence found at Genbank accession no. AAP44003.1 (SEQ ID NO:2), the BTLA Ig domain corresponds to amino acids 31-149. A BTLA Ig domain binds to an HVEM CRD1 domain. Further, a fragment of a BTLA Ig domain binds to an HVEM CRD1 domain, and can be specifically bound by a preferred BTLA antibody disclosed herein. Some preferred BTLA Ig domains comprise a cysteine residue corresponding to residue C85 of the murine BL/6 BTLA isoform (SEQ ID NO:1), which corresponds to residue C79 of the human BTLA isoform found at Genbank accession no. AAP44003.1 (SEQ ID NO:2).

As used herein, the term “BTLA Ig domain peptide” refers to a peptide corresponding in sequence to a region of the Ig domain of BTLA, which peptide can bind to the CRD1 domain of HVEM and is capable of reducing the binding of the BTLA Ig domain to the HVEM CRD1 domain. Such peptides are BTLA-HVEM antagonists.

As used herein, the term “HVEM blocking antibody” refers to an antibody that specifically binds to HVEM and reduces binding of HVEM to BTLA. Preferred HVEM blocking antibodies bind to the CRD1 domain, more preferably to a segment thereof that binds to the Ig domain of BTLA.

As used herein, the term “BTLA blocking antibody” refers to an antibody that specifically binds to BTLA and reduces binding BTLA to HVEM. Preferred BTLA blocking antibodies bind to the Ig domain of BTLA, preferably to a segment thereof that binds to the HVEM CRD1 domain.

As used herein, the term “BTLA-HVEM antagonist” refers to a bioactive agent capable of reducing BTLA activity in a cell having BTLA on its surface. Preferred BTLA-HVEM antagonists are capable of reducing the binding of HVEM on the surface of a cell to BTLA on the surface of the same or a second cell. In some preferred embodiments, BTLA-HVEM antagonists are capable of binding to the BTLA Ig domain. Binding of a BTLA-HVEM antagonist to BTLA on the surface of a cell does not increase BTLA activity in the cell.

As used herein, the term “BTLA-HVEM agonist” refers to a bioactive agent capable of increasing BTLA activity in a cell having BTLA on its surface, thereby mimicking the action of HVEM on BTLA. Preferred BTLA-HVEM agonists are capable of reducing the binding of HVEM on the surface of a cell to BTLA on the surface of the same or a second cell.

Both HVEM and BTLA are synthesized and inserted into the plasma membrane as transmembrane proteins, and thereby expose respective extracellular domains. The phrase “on the surface of a cell” in respect of BTLA or HVEM refers to non-soluble BTLA and HVEM protein localized at the plasma membrane.

As used herein, the term “antagonistic HVEM antibody” refers to an antibody that specifically binds to HVEM and can reduce the ability of HVEM to increase BTLA activity in a cell having BTLA on its surface.

As used herein, the term “antagonistic BTLA antibody” refers to an antibody that specifically binds to BTLA and can reduce the ability of HVEM to increase BTLA activity in a cell having BTLA on its surface. Binding of an antagonistic BTLA antibody to BTLA on the surface of a cell does not increase BTLA activity in the cell.

As used herein, the term “agonistic BTLA antibody' refers to an antibody that specifically binds to BTLA, is capable of reducing the binding of HVEM to BTLA, and increases BTLA activity in a cell having BTLA on its surface.

By “BTLA activity” and variations thereof is meant intracellular signaling and the effects thereof, caused by the binding of BTLA on the surface of a cell by a BTLA agonist, e.g., HVEM on the surface of a second cell; CMV UL144. BTLA activity includes but is not limited to inhibition of lymphocyte activation; phosphorylation of BTLA intracellular domain tyrosine residues, particularly those in the Grb2 binding site, the immunoreceptor tyrosine-based inhibitory motif (ITIM), and/or the immunoreceptor tyrosine-based switch motif (ITSM); binding of BTLA to SHP-1 and/or SHP-2; activation of SHP-1 and/or SHP-2; binding of BTLA to Grb2; and binding of BTLA to p85 of PI3K.

By “modulating BTLA activity” is meant increasing or decreasing BTLA activity, which includes completely decreasing BTLA activity such that no BTLA activity is detectable.

As used herein, the term “lymphocyte activation” refers to the processes attendant B cell and T cell activation in primary or subsequent immune responses, which processes include but are not limited to cell proliferation, differentiation, migration, and survival, as well as effector activities exhibited by B cells and T cells such as, but not limited to, cytokine production, antibody production, Fas ligand production, chemokine production, granzyme production and release, and antigen presentation. Accordingly, as used herein, modulation of lymphocyte activation includes modulation of effector function, such as modulation of the termination of effector function, etc. Numerous assays are well known to the skilled artisan for detecting and/or monitoring such processes.

By “modulating lymphocyte activation” is meant increasing or decreasing lymphocyte activation, which includes decreasing lymphocyte activation such that no lymphocyte activation is detectable.

Decreasing”, “reducing”, “inhibiting”, and grammatical equivalents thereof are used interchangeably herein and refer to reductions in levels of binding, activity, etc., which include reductions to levels beyond detection, including complete inhibition. Reduced binding can be effected, for example, by competitive binding of an antagonist.

As used herein, the term “immune response” includes T and/or B cell responses, i.e., cellular and/or humoral immune responses.

By “inhibiting tumor growth” is meant maintaining or reducing the tumor burden of an animal having an extant tumor, which includes eradicating the tumor. Even though the tumor burden is maintained or reduced, cancer cell proliferation may be ongoing.

As used herein, “human antibodies” includes humanized antibodies.

It will be evident herein that the use of “BTLA” and “HVEM” refers to BTLA protein and HVEM protein in many instances.

In some embodiments herein, CRD1 domains, and BTLA Ig domains are identified by their percent identity to a particular CRD1 or BTLA sequence. As is known in the art, a number of different programs can be used to identify whether a protein or nucleic acid has sequence identity or similarity to a known sequence. For a detailed discussion, see D. Mount, Bioinformatics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 2001, ISBN 0-87969-608-7. Sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, “Current Methods in Sequence Comparison and Analysis,” Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by HIgG1ns & Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266: 460-480 (1996)]. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschul et al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions; charges gap lengths of k a cost of 10+k; Xu set to 16, and Xg set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to⁻22 bits. A percent amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the longer sequence in the aligned region. The longer sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the protein sequences set forth in the figures, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, the percent sequence identity of sequences shorter than those shown in the figures will be determined using the number of amino acids in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.

In one embodiment, only identities are scored positively (+1) and all forms of Sequence variation including gaps are assigned a value of 0, which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the shorter sequence in the aligned region and multiplying by 100. The longer sequence is the one having the most actual residues in the aligned region.

(a) BTLA Antibodies and HVEM AntibodiesAntibody Structure

The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site.

Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.

The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J. Immunol. 148:1547-1553 (1992). In addition, bispecific antibodies may be formed as “diabodies” (Holliger et al. “‘Diabodies’: small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448 (1993)) or “Janusins” (Traunecker et al. “Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells” EMBO J 10:3655-3659 (1991) and Traunecker et al. “Janusin: new molecular design for bispecific reagents” Int J Cancer Suppl 7:51-52 (1992)). Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies. Bispecific antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab′, and Fv).

Human Antibodies and Humanization of Antibodies

Human antibodies avoid certain of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient. In order to avoid the utilization of murine or rat derived antibodies, it has been postulated that one can develop humanized antibodies or generate fully human antibodies through the introduction of human antibody function into a rodent so that the rodent would produce antibodies having fully human sequences.

Human Antibodies

Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated provides an ideal source for production of fully human monoclonal antibodies (Mabs). Fully human antibodies are expected to minimize the immunogenic and allergic responses intrinsic to mouse or mouse-derivatized Mabs and thus to increase the efficacy and safety of the administered antibodies. The use of fully human antibodies can be expected to provide a substantial advantage in the treatment of chronic and recurring human diseases, such as cancer, which may require repeated antibody administrations.

Mouse strains have been engineered with large fragments of the human Ig loci and to produce human antibodies in the absence of mouse antibodies.

See Green et al. Nature Genetics 7:13-21 (1994). The XenoMouse™ strains were engineered with yeast artificial chromosomes (YACs) containing 245 kb and 190 kb-sized germline configuration fragments of the human heavy chain locus and kappa light chain locus, respectively, which contained core variable and constant region sequences. Further reported work involved the introduction of greater than approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and kappa light chain loci, respectively, to produce XenoMouse™ mice. See Mendez et al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures of which are hereby incorporated by reference.

Such approaches are further discussed and delineated in European Patent No.,EP 0 463 151 B1, grant published Jun. 12, 1996, International Patent Application No., WO 94/02602, published Feb. 3, 1994, International Patent Application No., WO 96/34096, published Oct. 31, 1996, and WO 98/24893, published Jun. 11, 1998. The disclosures of each of the above-cited patents, applications, and references are hereby incorporated by reference in their entirety.

In an alternative approach, others have utilized a “minilocus” approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a p constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, and 5,814,318 each to Lonberg and Kay, U.S. Pat. No. 5,591,669 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, 5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and Dunn. See also European Patent No. 0 546 073 B1, International Patent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, the disclosures of which are hereby incorporated by reference in their entirety. See further Taylor et al., Nucleic Acids Research 20:62876295 (1992); Chen et al. International Immunology 5:647-656 (1993); Tuaillon et al., Proc. Natl. Acad. Sci. USA 90:3720-3724 (1993); Choi et al., Nature Genetics 4:117-123 (1993); Lonberg et al., Nature 368:856-859 (1994); Taylor et al., International Immunology 6:579-59.1 (1994); Tuaillon et al., J. Immunol. 154:6453-6465 (1995); Fishwild et al., Nature Biotech. 14:845-851(1996); the disclosures of which are hereby incorporated by reference in their entirety.

BTLA and HVEM proteins and fragments thereof, HVEM CRD1 domain peptides, BTLA Ig domain peptides, BTLA fusion proteins, and HVEM fusion proteins may be used to generate BTLA antibodies and HVEM antibodies of the present invention.

The term “antibody” as used herein includes both monoclonal and polyclonal antibodies as well as antibody fragments, as are known in the art, including Fab, F(ab)₂, single chain antibodies (Fv for example), chimeric antibodies, humanized antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. Antibody fragments include those portions of the antibody that bind to an HVEM CRD1 domain or a BTLA Ig domain.

Preferably, when a BTLA or HVEM protein fragment is to be used as an immunogen to generate antibodies, the fragment must share at least one epitope or determinant with the full length protein, particularly in an HVEM CRD1 domain or a BTLA Ig domain. By epitope or determinant herein is meant a portion of a protein which will generate and/or bind an antibody. Thus, in most instances, antibodies made to a smaller or truncated BTLA or HVEM protein will be able to bind to the corresponding full length protein. In a preferred embodiment, the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.

In one embodiment, the invention provides antagonistic BTLA antibodies that are capable of reducing, including eliminating, one or more biological functions of the BTLA protein expressed at the surface of a cell. That is, the addition of an antagonistic BTLA antibody (polyclonal, or preferably monoclonal) to a cell expressing BTLA at its surface can reduce or eliminate at least one BTLA activity. BTLA activity includes but is not limited to the inhibition of lymphocyte activation; phosphorylation of tyrosine residues in the Grb2 binding site, the ITIM, or the ITSM; binding to SHP-1 and/or SHP-2; and activation of SHP-1 and/or SHP-2. The reduction of BTLA activity is observed in the presence of BTLA agonist (eg. HVEM on the surface of a second cell) which stimulates BTLA activity in the absence of an antagonistic BTLA antibody. In a preferred embodiment, such an antagonistic BTLA antibody interferes with the binding of HVEM on the surface of one cell to BTLA on the surface of a second cell.

Generally, at least a 25% decrease in BTLA activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred.

Such antagonistic BTLA antibodies are sometimes referred to herein as function-blocking antibodies. Such antibodies have the ability to increase B and T lymphocyte activation by decreasing BTLA activity in lymphocytes. Further, such antibodies have the ability to modulate immunoglobulin production by B cells expressing BTLA, and more particularly, to increase immunoglobulin production.

In an alternative embodiment, the invention provides agonistic BTLA antibodies that increase or potentiate one or more biological functions of the BTLA protein expressed at the surface of a cell. That is, the addition of an agonistic BTLA antibody (polyclonal, or preferably monoclonal) to a cell expressing BTLA at its surface will increase or potentiate at least one BTLA activity. BTLA activity includes but is not limited to the inhibition of lymphocyte activation; phosphorylation of tyrosine residues in the Grb2 binding site, the ITIM, or the ITSM; binding to SHP-1 and/or SHP-2; and activation of SHP-1 and/or SHP-2.

In a preferred embodiment, the agonistic BTLA antibodies are function-activating antibodies. Such antibodies have the ability to decrease B and T lymphocyte activation by increasing BTLA activity in lymphocytes. Further, such antibodies have the ability to modulate immunoglobulin production by B cells expressing BTLA, and more particularly, to decrease immunoglobulin production.

A BTLA antibody of the invention specifically binds to the BTLA Ig domain of a BTLA protein. By “specifically bind” herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10⁻⁴-10⁻⁶M⁻¹, with a preferred range being 10⁻⁷-10⁻⁹M⁻¹.

The BTLA proteins bound by BTLA antibodies may be human BTLA proteins, murine BTLA proteins, or other, preferably mammalian, BTLA proteins. In a preferred embodiment, the BTLA protein is a human BTLA protein.

The murine BTLA gene is polymorphic, and variations in sequence within the Ig domain that binds to murine HVEM are described herein in the figures. Despite their sequence variation, the murine BTLA Ig domains are each capable of binding to murine HVEM, and a number of BTLA blocking antibodies are capable of binding to multiple isoforms of murine BTLA.

The human BTLA gene is also polymorphic, as disclosed in U.S. application Ser. No. 10/600,997, expressly incorporated herein in its entirety by reference. As disclosed herein, human HVEM is capable of binding to human BTLA. It is within the skill of the artisan to determine if alternative alleles of human BTLA are capable of binding to HVEM. As used herein, the term “BTLA” includes any human isoform of BTLA that is capable of binding to HVEM.

In a preferred embodiment, the present invention provides monoclonal BTLA antibodies that specifically bind to murine and/or human BTLA proteins.

In one embodiment, the invention provides antagonistic HVEM antibodies that are capable of reducing, including eliminating, the ability of HVEM protein when expressed at the surface of a cell to increase BTLA activity in a second cell expressing BTLA at its surface. BTLA activity includes but is not limited to the inhibition of lymphocyte activation; phosphorylation of tyrosine residues in the Grb2 binding site, the ITIM, or the ITSM; binding to SHP-1 and/or SHP-2; and activation of SHP-1 and/or SHP-2. In a preferred embodiment, such an antagonistic HVEM antibody interferes with the binding of HVEM on the surface of one cell to BTLA on the surface of a second cell.

Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred.

Such antibodies have the ability to increase B and T lymphocyte activation by decreasing BTLA activity in lymphocytes. Further, such antibodies have the ability to modulate immunoglobulin production by B cells expressing BTLA, and more particularly, to increase immunoglobulin production.

The HVEM antibodies of the invention specifically bind to HVEM CRD1 domains. By “specifically bind” herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10⁻⁴-10⁻⁶M⁻¹, with a preferred range being 10⁻⁷-10⁻⁹M⁻¹.

The HVEM proteins bound by HVEM antibodies may be human HVEM proteins, murine HVEM proteins, or other, preferably mammalian, HVEM proteins.

HVEM protein sequences and encoding nucleic acid sequences are well known in the art. For example, see Montgomery et al., Cell, 87: 427-436, 1996; Kwon et al., Journal of Biological Chemistry, 272:14272-14276, 1997; Hsu et al., Journal of Biological Chemistry 272:13471-13474, 1997.

The term “antibody”, as used herein, includes immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. The phrase “complementary determining region” (CDR) includes the region of an antibody molecule which comprises the antigen binding site.

The antibody may be an IgG such as IgG1, IgG2, IgG3 or IgG4; or IgM, IgA, IgE or IgD isotype. The constant domain of the antibody heavy chain may be selected depending upon the effector function desired. The light chain constant domain may be a kappa or lambda constant domain.

The term “antibody” as used herein also encompasses antibody fragments, and in particular, fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of such binding fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antibody.” Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

Still further, an antibody or fragment thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody fragments and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof, e.g. humanized, chimeric, etc. Preferably, antibodies of the invention bind specifically or substantially specifically to an HVEM CRD1 domain or a BTLA Ig domain. The terms “monoclonal antibodies” and “monoclonal antibody composition”, as used herein, refer to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody molecules that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.

The antibodies described herein may be humanized antibodies, e.g., antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. Such humanized antibodies may also include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

(b) BTLA-binding domain peptides and HVEM-binding domain peptides

As used herein, peptide refers to at least two covalently attached amino acids, which includes proteins, polypeptides, and oligopeptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus, “amino acid” or “peptide residue” as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline, and norleucine are considered amino acids for the purposes of the invention. “Amino acids” also includes imino residues such as proline and hydroxyproline. The side chains may be either the D- or L-configuration, or combinations thereof. Although the bond between each amino acid is typically an amide or peptide bond, it is to be understood that peptide also includes analogs of peptides in which one or more peptide linkages are replaced with other than an amide or peptide linkage, such as a substituted amide linkage, an isostere of an amide linkage, or a peptide or amide mimetic linkage (See, for example, Spatola, “Peptide Backbone Modifications,” in Chemistry and Biochemistry of Amino Acids Peptides and Proteins, Weinstein, Ed., Marcel Dekker, New York (1983); Son et al., J. Med. Chem. 36:3039-3049 (1993); and Ripka and Rich, Curr. Opin. Chem. Biol. 2:441-452 (1998)).

Typically, peptides will generally be less than about 100 amino acids, less that about 50 amino acids, or less than about 20 amino acids.

A peptide herein is typically an isolated or purified peptide. As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The phrase “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. Preparations of a peptide are substantially free of precursors in preparation having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

The peptides of this invention can be made by chemical synthesis methods which are well known to the ordinarily skilled artisan. See, for example, Fields et al.,Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Peptides can be synthesized using the automated Merrifield techniques of solid phase synthesis with the a NH₂protected by either t-Boc or Fmoc chemistry using side chain protected amino acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431.

After complete assembly of the desired peptide, the resin is treated according to standard procedures to cleave the peptide from the resin and deblock the functional groups on the amino acid side chains. The free peptide is purified, for example by HPLC, and characterized biochemically, for example, by amino acid analysis, mass spectrometry, and/or by sequencing. Purification and characterization methods for peptides are well known to those of ordinary skill in the art.

Longer synthetic peptides can be synthesized by well-known recombinant DNA techniques. Many standard manuals on molecular cloning technology provide detailed protocols to produce the peptides of the invention by expression of recombinant DNA and RNA. To construct a gene encoding a peptide of this invention, the amino acid sequence is reverse translated into a nucleic acid sequence, preferably using optimized codon usage for the organism in which the gene will be expressed. Next, a gene encoding the peptide is made, typically by synthesizing overlapping oligonucleotides which encode the peptide and necessary regulatory elements. The synthetic gene is assembled and inserted into the desired expression vector. Nucleic acids which comprise sequences that encode the peptides of this invention are also provided. The synthetic gene is inserted into a suitable cloning vector and recombinants are obtained and characterized. The peptide is then expressed under conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

(c) Fusion Proteins

Variant polypeptides of the present invention may also be fused to another, heterologous polypeptide or amino acid sequence to form a chimera. In some embodiments, fusion proteins comprise fusion partners comprising labels (e.g. autofluorescent proteins, survival and/or selection proteins), stability and/or purification sequences, toxins, or any other protein sequences of use. Additional fusion partners are described below. In some instances, the fusion partner is not a protein.

In another embodiment, a polypeptide of the invention is fused with human serum albumin to improve pharmacokinetics.

In a further embodiment, a polypeptide of the invention is fused to a cytotoxic agent. In this method, the polypeptide of the invention acts to target the cytotoxic agent to cells, resulting in a reduction in the number of afflicted cells. Cytotoxic agents include, but are not limited to, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like, as well as radiochemicals.

Peptide Tags

Various tag polypeptides and their respective antibodies are well known in the art. Epitope tags may be placed at the amino-or carboxyl-terminus of a polypeptide of the invention to enable antibody detection. Also, the epitope tag enables a polypeptide of the invention to be readily purified by affinity purification. Examples of peptide tags include, but are not limited to, poly-histidine (poly-His) or poly-histidine-glycine (poly-His-Gly) tags; the flu HA tag polypeptide [Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)]; the c-myc tag [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; the Herpes Simplex virus glycoprotein D (gD) tag [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)1 the Flag-peptide [Hopp et al., BioTechnology 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem. 266:15163-15166 (1991)]; and theT7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. U.S.A. 87:6393-6397 (1990)].

Labels

In one embodiment, a polypeptide of the invention is modified by the addition of one or more labels. For example, labels that may be used are well known in the art and include but are not limited to biotin, tag and fluorescent labels (e.g. fluorescein). These labels may be used in various assays as are also well known in the art to achieve characterization.

(d) Additional BTLA-HVEM Agonists and BTLA-HVEM Antagonists

It will be appreciated that additional bioactive agents may be screened for BTLA-HVEM antagonistic activity and BTLA-HVEM agonistic activity. In a preferred embodiment, candidate bioactive agents are screened for their ability to reduce binding of BTLA to HVEM. In another preferred embodiment, candidate bioactive agents are screened for their ability to reduce BTLA activation by HVEM.

The assays preferably utilize human BTLA and human HVEM proteins, although other BTLA and HVEM proteins may also be used.

In a preferred embodiment, the methods comprise combining an Ig domain of a BTLA protein, or an HVEM binding portion thereof, with a candidate bioactive agent, and determining the binding of the candidate agent to the BTLA domain. In another preferred embodiment, the methods involve combining an HVEM CRD1 domain, or a BTLA binding portion thereof, with a candidate agent, and determining the binding of the candidate agent to the HVEM domain.

The term “candidate bioactive agent” as used herein describes any molecule, e.g., protein, small organic molecule, carbohydrates (including polysaccharides), polynucleotide, lipids, etc. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection. In addition, positive controls, i.e. the use of agents known to bind BTLA or HVEM may be used.

Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons, more preferably between 100 and 2000, more preferably between about 100 and about 1250, more preferably between about 100 and about 1000, more preferably between about 100 and about 750, more preferably between about 200 and about 500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides, e.g., peptidomimetics. Peptidomimetics can be made as described, e.g., in WO 98/56401.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.

In a preferred embodiment, the candidate bioactive agents are organic chemical moieties or small molecule chemical compositions, a wide variety of which are available in the art.

(e) Additional Therapeutic Agents

In a further embodiment, the bioactive agents disclosed herein, including BTLA-HVEM agonists and BTLA-HVEM antagonists, including antagonistic BTLA antibodies and agonistic BTLA antibodies, may be advantageously combined with one or more additional therapeutic agents.

In one aspect, the BTLA-HVEM antagonists described herein can be administered in combination with additional immune response stimulating agents such as, e.g., cytokines as well as various antigens and vaccine preparations including tumor antigens and tumor vaccines. In preferred embodiments, such cytokines stimulate antigen presenting cells, e.g., GM-CSF, M-CSF, G-CSF, IL-3, IL-12, etc. Additional proteins and/or cytokines known to enhance T cell proliferation and secretion, such as IL-2, IL-2, B7, anti-CD3 and anti-CD28 can be employed simultaneously or sequentially with the BTLA-HVEM antagonists to augment the immune response. The subject therapy may also be combined with the transfection or transduction of tumor cells with genes encoding for various cytokines or cell surface receptors, as is known in the art. See, e.g. Ogasawara et al. (1993)Cancer Res.53:3561-8 and Townsend et al. (1993)Science259:368-370.

In another aspect, the BTLA-HVEM agonists described herein can be administered in combination with immunosuppressive agents, e.g., antibodies against other lymphocyte surface markers (e.g., CD40) or against cytokines, other fusion proteins, e.g., CTLA4Ig, or other immunosuppressive drugs (e.g., cyclosporin A, FK506-like compounds, rapamycin compounds, or steroids).

It is further contemplated that methods using BTLA-HVEM antagonists and BTLA-HVEM agonists may be synergistically combined with immunotherapies based on modulation of other positive and negative costimulatory pathways, and with CTLA-4 modulation in particular. For example, BTLA-HVEM antagonists may be advantageously combined with CTLA-4 blocking agents as described in U.S. Pat. Nos. 5,855,887; 5,811,097;and 6,051,227, and International Publication WO 00/32231. Such CTLA-4 blocking agents inhibit T cell down-regulation mediated by CTLA-4 interaction with B7 family members B71 and B72 expressed on lymphoid and dendritic cells. Similarly, BTLA-HVEM agonists may be advantageously combined with CTLA-4 mimicking agents such as CTLA-4Ig, which has already found clinical use as an immunosuppressive agent.

As used herein the term “rapamycin compound” includes the neutral tricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs, and other macrolide compounds which are thought to have the same mechanism of action as rapamycin (e.g., inhibition of cytokine function). The language “rapamycin compounds” includes compounds with structural similarity to rapamycin, e.g., compounds with a similar macrocyclic structure, which have been modified to enhance their therapeutic effectiveness. Exemplary Rapamycin compounds suitable for use in the invention, as well as other methods in which Rapamycin has been administered are known in the art (See, e.g. WO 95/22972, WO 95/16691, WO 95/04738, U.S. Pat. Nos. 6,015,809; 5,989,591; U.S. Pat. Nos. 5,567,709; 5,559,112; 5,530,006; 5,484,790; 5,385,908; 5,202,332; 5,162,333; 5,780,462; 5,120,727).

The language “FK506-like compounds” includes FK506, and FK506 derivatives and analogs, e.g., compounds with structural similarity to FK506, e.g., compounds with a similar macrocyclic structure which have been modified to enhance their therapeutic effectiveness. Examples of FK506 like compounds include, for example, those described in WO 00/01385. Preferably, the language “rapamycin compound” as used herein does not include FK506-like compounds.

(f) Administration of Therapeutic Compositions

The bioactive agents of the present invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By “biologically compatible form suitable for administration in vivo” is meant a form of the agent to be administered in which any toxic effects are outweighed by the therapeutic effects of the agent. The term subject is intended to include living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Administration of a bioactive agent as described herein can be in any pharmacological form, including a therapeutically active amount of a BTLA antibody, optionally in combination with an additional therapeutic agent as described herein, and a pharmaceutically acceptable carrier. Administration of a therapeutically effective amount of the therapeutic compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. For example, a therapeutically active amount of a BTLA antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The bioactive agent (e.g., BTLA antibody) may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the bioactive agent may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.

To administer a bioactive agent comprising a protein, e.g. a BTLA antibody, by other than parenteral administration, it may be necessary to coat the protein with, or co-administer the protein with, a material to prevent its inactivation. A bioactive agent such as a BTLA antibody may be administered to an individual in an appropriate carrier, diluent or adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Exemplary adjuvants include alum, resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, dilsopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol 7:27).

The bioactive agent may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

In one embodiment, a pharmaceutical composition suitable for injectable use includes sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition will preferably be sterile and fluid to the extent that easy syringability exists. It will preferably be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating one or more bioactive agents, together or separately with additional immune response stimulating agents or immunosupressants, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the bioactive agent into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

When a bioactive agent comprising a peptide is suitably protected, as described above, the protein may be orally administered, for example, with an inert diluent or an assimilable edible carrier. As used herein “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary bioactive agents can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of bioactive agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the bioactive agent(s) and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an agent for the treatment of sensitivity in individuals.

The specific dose can be readily calculated by one of ordinary skill in the art, e.g., according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in assay preparations of target cells. Exact dosages are determined in conjunction with standard dose-response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration.

The toxicity and therapeutic efficacy of the bioactive agents described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test agent which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In one embodiment of the present invention a therapeutically effective amount of an antibody to BTLA or HVEM is administered to a subject. As defined herein, a therapeutically effective amount of antibody (i.e., an effective dosage) ranges from about 0.001 to 50 mg/kg body weight, preferably about 0.01 to 40 mg/kg body weight, more preferably about 0.1 to 30 mg/kg body weight, about 1 to 25 mg/kg, 2 to 20 mg/kg, 5 to 15 mg/kg, or 7 to 10 mg/kg body weight. The optimal dose of the antibody given may even vary in the same patient depending upon the time at which it is administered.

The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an antibody can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated with antibody in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result from the results of assays designed to monitor transplant status (e.g., whether rejection or an immune response in the subject has occurred) as known in the art or as described herein.

In one embodiment, a pharmaceutical composition for injection could be made up to contain 1 ml sterile buffered water, and 1 to 50 mg of antibody. A typical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 150 mg of antibody. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980), which is incorporated herein by reference. The compositions comprising the present antibodies can be administered for prophylactic and/or therapeutic treatments. In therapeutic application, compositions can be administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for this use will depend upon the clinical situation and the general state of the patient's own immune system. For example, doses for preventing transplant rejection may be lower than those given if the patient presents with clinical symptoms of rejection. Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of the bioactive agents described herein sufficient to effectively treat the patient.

Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used. It is also provided that certain protocols may allow for one or more agents describe herein to be administered orally. Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms. Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, olyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art. The formulations can also contain substances that diminish proteolytic degradation and/or substances which promote absorption such as, for example, surface active agents.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. Kits for practice of the instant invention are also provided. For example, such a kit comprises a bioactive agent such as, e.g., an antibody reactive with BTLA or HVEM, together with a means for administering the antibody conjugate, e.g., one or more syringes. The kit can come packaged with instructions for use.

(g) Modulation of Immune Responses

The present invention provides methods for modulating lymphocyte activity and immune responses to antigens using BTLA-HVEM antagonists and BTLA-HVEM agonists described herein. The methods are useful for modulating the activity of, for example, naïve T cells, CD8+ Tc cells, CD4+ cells,T_H1 cells, and B cells.

BTLA-HVEM antagonists are used alone or in combination with other therapeutic agents to reduce the negative costimulatory signals emitted by BTLA, and to reduce the suppression and/or attenuation of lymphocyte activity mediated by BTLA signaling.

BTLA-HVEM agonists are used alone or in combination with other therapeutic agents to increase negative costimulatory signals emitted by BTLA, thereby increasing the suppression and/or attenuation of lymphocyte activity mediated by BTLA signaling.

In a preferred embodiment, the methods comprise contacting a lymphocyte expressing BTLA on its surface, or a second cell expressing HVEM on its surface, or both, with a BTLA-HVEM antagonist, wherein the lymphocyte and second cell are able to contact each other such that BTLA on the lymphocyte can bind to HVEM on the second cell, and wherein the BTLA-HVEM antagonist reduces the activation of BTLA on the lymphocyte by HVEM on the second cell.

In another preferred embodiment, the methods comprise contacting a lymphocyte expressing BTLA on its surface with a BTLA-HVEM agonist, such that the BTLA-HVEM agonist increases BTLA activity in the lymphocyte.

(h) Antigens

In one aspect, the invention provides methods for modulating an immune response to antigen. Such antigens can be, for example, tumor-associated antigens, pathogen antigens, and autoantigens (self antigens). Antigenic stimulation may be a result of ongoing malignancy or infection, and/or may be a result of exposure to antigens delivered by vaccine.

A wide variety of antigens may find use in the invention. In particular, the adjuvant compositions provided herein may be advantageously combined with antigenic stimulation from tumor-associated antigens or pathogen antigens to increase lymphocyte activity against the corresponding tumor or pathogen. Generally, suitable antigens may be derived from proteins, peptides, polypeptides, lipids, glycolipids, carbohydrates and DNA found in the subject tumor or pathogen.

Tumor-associated antigens finding utility herein include both mutated and non-mutated molecules which may be indicative of a single tumor type, shared among several types of tumors, and/or exclusively expressed or over-expressed in tumor cells in comparison with normal cells. In addition to proteins and glycoproteins, tumor-specific patterns of expression of carbohydrates, gangliosides, glycolipids and mucins have also been documented.

Exemplary tumor-associated antigens for use in the subject cancer vaccines include protein products of oncogenes, tumor suppressor genes and other genes with mutations or rearrangements unique to tumor cells, reactivated embryonic gene products, oncofetal antigens, tissue-specific (but not tumor-specific) differentiation antigens, growth factor receptors, cell surface carbohydrate residues, foreign viral proteins and a number of other self proteins.

Specific embodiments of tumor-associated antigens include, e.g., mutated antigens such as the protein products of the Ras p21 protooncogenes, tumor suppressor p53 and HER-2/neu and BCR-abl oncogenes, as well as CDK4, MUM1,Caspase 8, and Beta catenin; overexpressed antigens such asgalectin 4,galectin 9, carbonic anhydrase, Aldolase A, PRAME, Her2/neu, ErbB-2 and KSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionic gonadotropin (hCG); self antigens such as carcinoembryonic antigen (CEA) and melanocyte differentiation antigens such asMart 1/Melan A, gp100, gp75, Tyrosinase, TRP1 and TRP2; prostate associated antigens such as PSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene products such asMAGE 1,MAGE 3,MAGE 4,GAGE 1,GAGE 2, BAGE, RAGE, and other cancer testis antigens such as NYESO1, SSX2 and SCP1; mucins such as Muc-1 and Muc-2; gangliosides such as GM2, GD2 and GD3, neutral glycolipids and glycoproteins such as Lewis (y) and globo-H; and glycoproteins such as Tn, Thompson-Freidenreich antigen (TF) and sTn. Also included as tumor-associated antigens herein are whole cell and tumor cell lysates as well as immunogenic portions thereof, as well as immunoglobulin idiotypes expressed on monoclonal proliferations of B lymphocytes for use against B cell lymphomas.

Tumor-associated antigens and their respective tumor cell targets include, e.g., cytokeratins, particularly

cytokeratin

8, 18 and 19, as antigens for carcinoma. Epithelial membrane antigen (EMA), human embryonic antigen (HEA-125), human milk fat globules, MBr1, MBr8, Ber-EP4, 17-1A, C26 and T16 are also known carcinoma antigens. Desmin and muscle-specific actin are antigens of myogenic sarcomas. Placental alkaline phosphatase, beta-human chorionic gonadotropin, and alphafetoprotein are antigens of trophoblastic and germ cell tumors. Prostate specific antigen is an antigen of prostatic carcinomas, carcinoembryonic antigen of colon adenocarcinomas. HMB-45 is an antigen of melanomas. In cervical cancer, useful antigens could be encoded by human papilloma virus. Chromagranin-A and synaptophysin are antigens of neuroendocrine and neuroectodermal tumors. Of particular interest are aggressive tumors that form solid tumor masses having necrotic areas. The lysis of such necrotic cells is a rich source of antigens for antigen-presenting cells, and thus the subject compositions and methods may find advantageous use in conjunction with conventional chemotherapy and/or radiation therapy.

Tumor-associated antigens can be prepared by methods well known in the art. For example, these antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells (e.g., as described in Cohen et al., Cancer Res., 54:1055 (1994)), by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens. The antigen may also be in the form of a nucleic acid encoding an antigenic peptide in a form suitable for expression in a subject and presentation to the immune system of the immunized subject. Further, the antigen may be a complete antigen, or it may be a fragment of a complete antigen comprising at least one epitope.

Antigens derived from pathogens known to predispose to certain cancers may also be advantageously utilized in conjunction with the compositions and methods provided herein. It is estimated that close to 16% of the worldwide incidence of cancer can be attributed to infectious pathogens, and a number of common malignancies are characterized by the expression of specific viral gene products. Thus, the inclusion of one or more antigens from pathogens implicated in causing cancer may help broaden the host immune response and enhance the prophylactic or therapeutic effect of the cancer vaccine. Pathogens of particular interest for use herein include the hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (heptomas), Epstein Barr virus (EBV) (Burkitt lymphoma, nasopharynx cancer, PTLD in immunosuppressed individuals), HTLV1 (adult T cell leukemia), oncogenic human papilloma viruses types 16, 18, 33, 45 (adult cervical cancer), and the bacterium Helicobacter pylori (B cell gastric lymphoma).

Also contemplated herein are pathogen antigens derived from infectious microbes such as virus, bacteria, parasites and fungi and fragments thereof, in order to increase lymphocyte activity in response to active infection or improve the efficacy of prophylactic vaccine therapy. Examples of infectious virus include, but are not limited to: Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astroviruses).

Also, gram negative and gram positive bacteria serve as antigens in vertebrate animals. Such gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species,andSalmonellaspecies. Specific examples of infectious bacteria include but are not limited to:Helicobacterpyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteriasps (e.g.M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group AStreptococcus),Streptococcus agalactiae(Group BStreptococcus),Streptococcus(viridans group),Streptococcusfaecalis, Streptococcus bovis, Streptococcus(anaerobicsps.),Streptococcus pneumoniae, pathogenic Campylobactersp.,Enterococcussp.,Haemophilus infuenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacteriumsp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroidessp.,Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia,andActinomyces israelli.

Examples of pathogens also include, but are not limited to, infectious fungi that infect mammals, and more particularly humans. Examples of infectious fingi include, but are not limited to:Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.Examples of infectious parasites includePlasmodiumsuch asPlasmodium falciparum, Plasmodium malariae, Plasmodium ovate,andPlasmodium vivax.Other infectious organisms (i.e. protists) includeToxoplasma gondii.

Other medically relevant microorganisms that serve as antigens in mammals and more particularly humans are described extensively in the literature, e.g., see C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entire contents of which is hereby incorporated by reference. In addition to the treatment of infectious human diseases, the compositions and methods of the present invention are useful for treating infections of nonhuman mammals. Many vaccines for the treatment of non-human mammals are disclosed in Bennett, K. Compendium of Veterinary Products, 3rd ed. North American Compendiums, Inc., 1995.

(i) Treatment of Cancer

The present invention also provides immunotherapeutic methods for treating cancer, comprising administering to a patient a therapeutically effective amount of a BTLA-HVEM antagonist, either alone or in combination with other therapeutic compositions.

In a preferred embodiment, immunization is done to promote a tumor-specific T cell immune response. In this embodiment, a BTLA-HVEM antagonist is administered in combination with a tumor-associated antigen. The combination of a tumor-associated antigen and a BTLA-HVEM antagonist promotes a tumor specific T cell response, in which T cells encounter reduced negative costimulatory signals mediated by BTLA as compared to those in the absence of the BTLA-HVEM antagonist.

In one aspect, the present invention provides a medicament for the treatment of cancer, wherein the medicament comprises a BTLA-HVEM antagonist. Also provided are methods for making a medicament useful for the treatment of cancer, which medicament comprises a BTLA-HVEM antagonist.

(j) Treatment of Autoimmune Disease, Allergy, and Asthma

The present invention also provides methods for inhibiting autoimmune responses and treating autoimmune diseases, comprising administering to a patient a therapeutically effective amount of a BTLA-HVEM agonist. Without being bound by theory, administration of a therapeutically effective amount of a BTLA-HVEM agonist inhibits the activity of autoreactive T and B cells that specifically recognize autoantigens and otherwise negatively affect the physiology of cells that bear them.

Autoimmune disease as used herein includes Rheumatoid arthritis,type 1 diabetes, autoimmune thyroiditis, and Lupus. Additional autoimmune diseases are described, for example, in Mackay et al., NEJM, 345:340-350, 2001.

In one aspect, the present invention provides a medicament for the treatment of autoimmune disease, wherein the medicament comprises a BTLA-HVEM agonist. Also provided are methods for making a medicament useful for the treatment of autoimmune disease, which medicament comprises a BTLA-HVEM agonist.

In another aspect, the invention provides methods for preventing or reducing an allergic reaction in a patient, comprising administering to a patient a therapeutically effective amount of a BTLA-HVEM agonist.

In one aspect, the present invention provides a medicament for the treatment or prevention of allergy, wherein the medicament comprises a BTLA-HVEM agonist. Also provided are methods for making a medicament useful for the treatment or prevention of allergy, which medicament comprises a BTLA-HVEM agonist.

In one aspect, the invention provides methods for reducing the severity of an asthmatic reaction in a patient, comprising administering to a patient a therapeutically effective amount of a BTLA-HVEM antagonist.

In one aspect, the invention provides methods for shortening the duration of an asthmatic reaction in a patient, comprising administering to a patient a therapeutically effective amount of a BTLA-HVEM antagonist.

In one aspect, the invention provides methods for improving recovery from an asthmatic reaction in a patient, comprising administering to a patient a therapeutically effective amount of a BTLA-HVEM antagonist.

(k) Increasing Graft Survival

The present invention also provides methods for inhibiting a host immune response to transplanted tissue, comprising administering to a patient receiving transplanted tissue a therapeutically effective amount of a BTLA-HVEM agonist. Administration of the therapeutically effective amount of the BTLA-HVEM agonist inhibits the host immune response to the tissue and prolongs its survival.

In one aspect, the present invention provides a medicament for use as an immunosuppressant, wherein the medicament comprises a BTLA-HVEM agonist. Also provided are methods for making such a medicament.

All references cited herein are expressly incorporated herein in their entirety by reference.

EXAMPLESExperimental BTLA Antibodies

Armenian hamsters and Balb/c BTLA KO mice were immunized with oxidatively refolded BI/6 BTLA tetramer protein. The ability of antibody to block binding of BTLA tetramer to HVEM was determined.


Clone	Isotype	Allelic	Blocking?	Applications

6A6	Hamster IgG	BL/6 only	Yes	FACS, IP
6F7	Mouse IgG1, kappa	Balb/c and	Yes	FACS, IP, WB
		BL/6
6G3	Mouse IgG1, kappa	Balb/c and	Yes	FACS, IP
		BL/6
6H6	Mouse IgG1, kappa	Balb/c and	Weak	FACS, IP
		BL/6
8F4	Mouse IgG1, kappa	Balb/c and	Yes	FACS, IP
		BL/6
3F9.D12	Mouse IgG1, kappa	Balb/c and	Yes	FACS, IP
		BL/6
3F9.C6.	Mouse IgG1, kappa	BL/6 only	No	FACS, IP, WB

Yeast Display Data: The ability of antibody to bind to BTLA mutants was determined.+indicates binding.


Bl/6 BTLA
Mutations	6A6	3F9.C6	6G3	6F7	3F9.D12

P41E	+	+	+	+	+
T45N	+	+	+	+	+
P41 E, T47K	+	+	+	+	+
P41 E, Q52H	+	+	+	+	+
P41 E,	+	−	+	+	+
P41 E, Q63E	−	+	+	+	+
P41 E,	+/−	+	+/−	+/−	+/−
P41 E, S91G	+	+	+	+	+
P41E,	−	+	+	+	+
P41 E,	+	+	+	+	+
WEHI.2	−	−	−	−	−

(I) BTLA Ligand Binding and BTLA Activation

B7 molecules bind to MYPPPY motif on CD28 and CTLA4 within FG loop. For example, PD-L1 and PD-L2 bind to FG loop of PD-1. The FG loop of BTLA is four amino acids shorter than FG loop in CD28/CTLA4, and epitope mapping places HVEM interaction towards ‘DEBA’ face of Ig fold.

BTLA Binds Naive T Cells but Does not Bind B7x

To test for direct interactions between BTLA and B7x, we made NIH 3T3 cell lines stably expressing the extracellular domains of B7x, BTLA, and programmed death 1 (PD-1) and its ligand (PD-L1), and stained the cells with PD-1 and BTLA tetramers and with PD-L1 and B7x Fc fusion proteins. Whereas PD-1 tetramer bound to cells expressing PD-L1, as expected, the BTLA tetramer did not bind to cells expressing PD-L1 or B7x. Furthermore, our B7x-Fc fusion protein did not bind cells expressing BTLA.

To identify potential ligands on normal lymphocytes, we next used a BTLA-Fc fusion protein and BTLA tetramer to stain splenocytes (FIGS. 1 and 2). As a control, the PD-L1Fc fusion protein showed selective binding to activated but not resting CD4+ T cells and B220+ B cells (FIG. 1), as expected and consistent with the reported inducibility of PD-1 expression. Notably, our BTLA-Fc fusion protein showed specific binding to resting CD4+ and CD8+ T cells but not to B220+ B cells (FIG. 1). In addition, this binding was greatly reduced after T cell activation by treatment with antibody to CD3 (anti-CD3) but was not affected by B cell activation. As B7x is not reported to be expressed by naive T cells, the binding of the BTLA-Fc fusion protein to T cells is not consistent with an interaction with B7x. However, we independently confirmed that B7x was not expressed by T cells by examining the expression of B7x and several other CD28-B7 family members. B7x mRNA was most highly expressed in heart and lung but was absent from spleen, lymph node and naive CD4+ T cells. In contrast, we confirmed the expected lymphoid-specific expression pattern for several CD28- B7 family members, including ICOS, PD-1 and BTLA.

To confirm and characterize the potential ligand on T cells identified by the BTLA-Fc fusion protein, we next analyzed the properties of BTLA tetramers binding to lymphocytes (FIG. 2). The BTLA tetramer showed strong binding to both CD4+ and CD8+ T cells obtained from both spleen and lymph nodes and bound weakly to non-T lymphocytes (FIG. 2a). Furthermore, binding to T cells was reduced after anti-CD3 stimulation (FIG. 2b), similar to our results obtained with BTLA-Fc fusion proteins (FIG. 1). In contrast, treatment of splenocytes with antiimmunoglobulin M (anti-IgM) or lipopolysaccharide did not reduce BTLA tetramer staining of CD4+ T cells. BTLA tetramers showed very slight binding to resting and activated B cells (FIG. 2b). We also examined BTLA tetramer staining in thymic subsets (FIG. 2c). BTLA tetramer staining was lowest in CD4+CD8+ (double-positive) thymocytes and showed more staining in mature CD4+ or CD8+ thymocytes and double- negative (CD4-CD8-) thymocytes, again indicating some physiological regulation of the BTLA ligand. As BTLA tetramer binding was modulated 48 h after anti-CD3 stimulation of T cells, we did a more detailed kinetic analysis using D011.10 T cells activated in vitro with ovalbumin (OVA) peptide (FIG. 3). Again, BTLA tetramer binding was regulated during activation, initially increasing by twofold at 24 and 48 h after antigen-specific stimulation, decreasing onday 3 andday 4, and increasing again byday 7. Expression of this BTLA ligand was similar in both T helper type 1- and T helper type 2-inducing conditions. Thus, BTLA tetramers and BTLA-Fc fusion proteins have very similar binding properties to lymphocytes, and a BTLA ligand is expressed by resting T cells and undergoes regulation during thymocyte development and T cell activation.

Cloning a BTLA-Interacting Protein

We constructed a retroviral cDNA library from lymphocytes and transduced two host cell lines, BJAB and NIH 3T3, that were negative for BTLA tetramer binding (FIG. 4a). After four successive rounds of sorting, we obtained lines uniformly positive for BTLA tetramer staining, which we used to amplify retrovirus-specific inserts. From BJAB cells, we obtained a predominant RT-PCR product that we identified as mouse HVEM. From NIH 3T3 cells we also obtained mouse HVEM as the main component of RT-PCR isolates. Among the minor retroviral inserts identified from NIH 3T3 cells, 4-1 BB was the only transmembrane receptor; it also belongs to the TNFR superfamily.

We next tested these isolates as candidates for direct interactions with BTLA tetramers. We expressed full-length cDNA clones of mouse HVEM, human HVEM, mouse 4-1 BB and mouse LTI3R, which binds the same ligands (LIGHT and LTα) as HVEM, in BJAB cells and analyzed these cells for binding to BTLA tetramers. We specifically constructed BTLA tetramers from both the C57BL/6 and BALB/c alleles to identify any potential allelic differences in binding (FIG. 4b). We found specific binding of both forms of BTLA tetramers to green fluorescent protein (GFP)-positive BJAB cells expressing mouse HVEM but not to BJAB cells expressing human HVEM, mouse 4-1 BB or mouse LTI3R or to GFP-negative uninfected BJAB cells.

HVEM Induces BTLA Phosphotylation

We next sought to determine if HVEM could induce BTLA phosphorylation (FIGS. 4c, d). We analyzed BTLA phosphorylation in EL4 cells using immunoprecipitation immunoblot analysis as described above. EL4 cells had low expression of BTLA but no detectable HVEM, as assessed by BTLA tetramer binding. We therefore examined EL4 cells for BTLA phosphorylation and SHP-2 coimmunoprecipitation after contact with mouse HVEM expressed by BJAB cells. EL4 cells alone showed neither coimmunoprecipitation of SHP-2 with BTLA (FIG. 4c) nor direct tyrosine phosphorylation of BTLA (FIG. 4d). Mixing of EL4 cells with HVEM-expressing BJAB cells induced both coimmunoprecipitation of SHP-2 with BTLA and tyrosine phosphorylation of BTLA. In contrast, mixing EL4 cells with HVEM negative BJAB cells induced neither coimmunoprecipitation of SHP-2 with BTLA nor BTLA phosphorylation. As controls, pervanadate treatment of EL-4 cells induced coimmunoprecipitation of SHP-2 and tyrosine phosphorylation of BTLA, but BJAB cells alone, either HVEM-negative or expressing HVEM, showed neither SHP-2 coimmunoprecipitation nor BTLA phosphorylation. Thus, these results show that HVEM can induce BTLA tyrosine phosphorylation and association with SHP-2.

HVEM-BTLA Interactions are Conserved in Human.

Because tetramers of mouse BTLA bound mouse HVEM but not human HVEM, we sought to determine if the BTLA-HVEM interaction was conserved in humans. Therefore, we generated a human BTLA-Fc fusion protein and characterized its interactions with mouse and human HVEM (FIG. 4e). The mouse BTLA-Fc fusion protein bound to BJAB cells expressing mouse HVEM but not cells expressing human HVEM, confirming the data obtained with mouse BTLA tetramers (FIG. 4b). In addition, the human BTLA-Fc fusion protein bound to BJAB cells expressing human HVEM (FIG. 4e). The human BTLA-Fc fusion protein also bound, although more weakly, to BJAB cells expressing mouse HVEM. These interactions were specific, as the isotype control antibody and the B7x-Fc fusion protein did not bind to BJAB cells expressing either mouse or human HVEM. Thus, the interaction between BTLA and HVEM occurs in human lymphocytes, as it does in mouse lymphocytes. Also, although cross-species interactions are noted for human BTLA and mouse HVEM (FIG. 4e), it seems that this cross-species interaction is weaker than the intraspecies interaction.

BTLA Interacts with the CRD I Region of HVEM

HVEM is a member of the TNFR superfamily and interacts with the two known TNF family members LIGHT and LTα. Because HVEM has multiple ligands, we sought to determine whether we could detect additional ligands for BTLA. Thus, we compared binding of BTLA tetramers to wild-type and HVEM-deficient lymphocytes (FIG. 5a). BTLA tetramers showed no detectable specific binding to HVEM-deficient CD4+ or CD8+ T cells but showed the expected binding to wild-type T cells. Even the low binding of BTLA tetramer to B cells was reduced to undetectable amounts in HVEM-deficient B cells (FIG. 5a). Thus, we found no evidence of additional ligands for BTLA in mice.

The interaction between HVEM and LIGHT can be detected with an HVEM-Fc fusion protein containing the four extracellular CRD regions of HVEM fused to the Fc region of human IgG1. We therefore sought to determine whether this HVEM-Fc fusion protein can also bind BTLA (FIG. 5b). Because LIGHT is expressed by CD11c+ DCs but not by B220+ B cells, we compared the binding of HVEM-Fc fusion protein to B cells and DCs from wild-type and BTLA-deficient mice (FIG. 5b). The HVEM-Fc fusion protein bound to wild-type B cells but not to Btla−/− B cells. In contrast, the HVEM-Fc fusion protein bound to wild-type DCs with only slightly reduced binding to BTLA−/− DCs. We next compared the binding of HVEM-Fc fusion protein to wild-type and LIGHT-deficient (Tnfrsfl4−/−) B cells and DCs (FIG. 5c). The HVEM-Fc fusion protein bound to wild-type and Tnfrsfl4−/− B cells and DCs with nearly equal intensity. In addition, HVEM expression was actually increased in BTLA−/− mice compared with that in wild-type mice. This result might indicate that endogenous HVEM expression is regulated by interaction with BTLA, similar to the reported regulation of HVEM expression by LIGHT. Furthermore, this result formally shows that HVEM expression does not require BTLA as a ‘chaperone’. These results might suggest that BTLA is the only ligand for HVEM on B cells, but such conclusions based solely on soluble staining reagents may be misleading, and it is possible that HVEM could also interact with other unknown molecules on B cells. For DCs, it seems that both BTLA and LIGHT are ligands for HVEM.

We sought to identify which domains of HVEM are involved in BTLA interactions. HVEM has four extracellular CRDs; it binds LIGHT and LTa through CRD2 and CRD3 and binds herpes glycoprotein D through CRD1. We constructed a series of HVEM mutants, including a mouse HVEM GFP fusion protein, an HVEM deletion mutant lacking the N-terminal CRD1 as a GFP fusion protein, an intact human HVEM, and a chimeric HVEM containing mouse CRD1 linked to human CRD2. We expressed this panel of HVEM mutants in BJAB cells and examined binding of the mouse BTLA tetramer (FIG. 5d). As expected, the BTLA tetramer did not bind uninfected BJAB cells but bound to wild-type mouse HVEM. However, the mouse BTLA tetramer did not bind to the HVEM mutant lacking CRD1. In addition, BTLA tetramer did not bind to human HVEM but did bind to the mouse-human chimeric HVEM (FIG. 5d). As a control, we assessed the amounts of human HVEM expressed by these cell lines (FIG. 5d), confirming expression of the human and chimeric HVEM molecules. These results indicate an important function for the CRD1 domain of mouse HVEM for BLTA interactions but do not exclude the possibility of a contribution by other domains.

HVEM Inhibits Antigen-Driven T Cell Proliferation

HVEM is expressed by several types of cells, including T cells, B cells and DCs, complicating the analysis of potential interactions between cells expressing LIGHT, BTLA and HVEM. Thus, we first sought to confirm the reported costimulatory effects of LIGHT on CD4+ T cells in our system. We stimulated highly purified CD4+ T cells with increasing amounts of anti-CD3 in the presence of various concentrations of plate-bound LIGHT (FIG. 6a). At suboptimal concentrations of anti-CD3 stimulation, LIGHT strongly augmented T cell proliferation in a dose-dependent way. At the highest dose of anti-CD3, the costimulatory effect of LIGHT was reduced slightly because of an increase in the LIGHT- independent proliferation. These data confirm reports that LIGHT engagement of HVEM provides positive costimulation.

We next tested whether BTLA or HVEM expression by antigen-presenting cells (APCs) inhibited or activated T cells. For this, we produced a panel of Chinese hamster ovary (CHO) cells expressing various combinations of I-Ad and B7-1 plus either BTLA or HVEM using retrovirus transduction and cell sorting. We confirmed expression of I-Ad, B7-1, BTLA and HVEM by these cell lines using flow cytometry. We sought to determine if BTLA expression by APCs costimulated D011.10 T cells (FIG. 6b). CHO cells expressing I-Ad alone supported minimal T cell proliferation, similar to that seen with T cells and peptide alone. As a positive control, CHO cells expressing I-Ad and B7-1 supported higher proliferation in response to OVA peptide. In contrast, BTLA expression by APCs did not augment T cell proliferation induced by CHO cells expressing I-Ad alone (FIG. 6b), as did expression of B7-1, suggesting that BTLA does not provide costimulation to T cells through HVEM engagement.

Whereas BTLA, unlike LIGHT, may not activate HVEM, HVEM seems to activate BTLA, as evidenced by BTLA phosphorylation and SHP-2 association (FIGS. 4c, d). Thus, we sought to determine whether HVEM expression by APCs influenced T cell proliferation (FIG. 6c). The peptide dose-dependent proliferation supported by CHO cells expressing I-Ad alone was reduced when HVEM was coexpressed on these CHO cells (FIG. 6c). Furthermore, as expected, B7-1 increased T cell proliferation induced by peptide and I-Ad (FIG. 6d), shifting the dose-response to lower concentrations of peptide. Again, coexpression of HVEM on these CHO cells reduced peptide-dependent T cell proliferation. The inhibition produced by HVEM at the highest peptide concentrations was smaller than the inhibition seen with intermediate stimulation.

We extended this analysis using T cells labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE;FIG. 7). In addition, we tested whether the inhibitory effect of HVEM on T cell proliferation required BTLA by using BTLA−/− D011.10 T cells. Using CHO cells lacking B7-1 expression, we did not note T cell proliferation at the lowest dose of OVA peptide (0.03 μM) ondays 3 and 4 (FIG. 7a). However, higher peptide concentrations (0.3 μM) induced T cell proliferation on

days

3 and 4. In these conditions, expression of BTLA on CHO cells had no effect on T cell proliferation at anytime. However, expression of HVEM on CHO cells greatly reduced T cell proliferation, which occurred only in wild-type D011.10 T cells, not BTLA−/− T cells, and was evident on

days

3 and 4 after activation.

We next examined the effects of HVEM on T cell proliferation in response to antigen presentation by CHO cells expressing B7-1 (FIG. 7b). Again, B7-1 increased T cell proliferation induced by peptide and I-Ad, shifting the dose-response to lower concentrations of peptide, as demonstrated by larger numbers of cellular divisions at lower doses of peptide; this was clearly evident onday 3 as well asday 4. In these conditions, coexpression of BTLA on CHO cells had no effect on T cell proliferation. In contrast, coexpression of HVEM on CHO cells caused a reduction in proliferation of wild-type D011.10 T cells, but this was evident only at the lowest peptide dose and was evident only onday 3, notday 4, after T cell activation. This inhibition of T cell proliferation was specific to BTLA, as we found it only in wild-type but not BTLA−/− T cells. In summary, HVEM inhibits both costimulation-independent and costimulation dependent proliferation, but is more effective in blocking activation of antigen stimulated T cells at low B7-1 expression. Furthermore, HVEM-mediated inhibition of T cell proliferation requires BTLA expression by T cells.

Methods (I)Mice

C57BL/6 and BALB/c mice (Jackson Labs) were bred in our facility. BTLA−/− mice were backcrossed to BALB/c for nine generations and were subsequently crossed onto the D011.10 T cell receptor-transgenic background. LIGHT-deficient mice were previously described and HVEM- deficient (Tnfrsfl4−/−) mice will be described elsewhere.

Plasmids and Retroviral Constructs.

The sequences of all oligonucleotides are provided in Sedy et al., Nature Immunology (2005) 6:90-98. For preparation of B7x-B7h-GFP-RV, a PCR product made withprimers 5′Bg12 mB7x and B7xB7h bottom using IMAGE cDNA clone 3709434 as the template, plus a PCR product made with primers B7xB7h top and 3′RI GFP using the B7h-GFP plasmid (a gift from W. Sha, University of California, Berkeley, Calif.) as the template, were annealed and amplified with Pfu polymerase withprimers 5′Bg12 mB7x and 3′RI GFP. This product, encoding the B7x extracellular domain, B7h transmembrane and cytoplasmic domains fused to GFP, was digested with BgIII and EcoRI and was cloned into IRES- GFP-RV that had been digested with BgIII and EcoRI.

The plasmid huHVEM-IRES-GFP-RV was produced by amplification of huHVEM withprimers 5′Bg12 huHVEM and 3′Xho1 huHVEM using IMAGE cDNA clone 5798167 (Invitrogen) as the template, followed by digestion with BgIII and XhoI and ligation into Tb-lym-IRES-GFP-RV that had been digested with BgIII and XhoI, replacing the Tb-lym cDNA with that of huHVEM. Similarly, m4-1BB-IRES-GFP-RV was prepared withprimers 5′Bgl2 m4-1BB and 3′Xho1 m4-1BB using library plasmid as the template, followed by digestion with BgIII and XhoI and ligation into Tb-lym-IRES-GFPRV. The plasmid mLT R-IRES-GFP-RV was prepared withprimers 5′Bg12 mLT R and 3′Sal1 mLT R using IMAGE cDNA clone 5293090 (Invitrogen) as the template, followed by digestion with BgIII and SalI and ligation into Tb-lym-IRES-GFP-RV. The plasmid mHVEM-FL-IRES-GFP-RV was similarly prepared withprimers 5′Bg12 mHVEM and 3′Xho1 mHVEM using, as the template, cDNA from library infected BJAB cells sorted for BTLA tetramer binding, followed by digestion with BgIII and XhoI and ligation into Tb-lym-IRES-GFP-RV. Three amino acid changes (N58S, K92R and E128G) in mouse HVEM cDNA cloned from the retrovirus library, compared with that of mouse HVEM cDNA from the 129 SvEv mouse strain, were implemented by Quick Change mutagenesis (Stratagene) to generate mHVEM(129)-IRES-GFP-RV with serial application of the primers S-N top plus S-N bot; R-K top plus R-K bot; and G-E top plus G-E bot.

The plasmid mHVEM-FL-GFP-RV was made from two PCR products, withprimers 5′Bg12 mHVEM and mHVEM/GFP bot using mHVEM-FL-IRES-GFP-RV as the template, and primers mHVEM/GFP top and 3′GFP+Sal using mHVEM-FL-IRES-GFP-RV as the template; the PCR products were annealed, amplified withprimers 5′Bg12 mHVEM and 3′GFP+Sal, digested with BgIII and SalI and ligated into IRES-GFP-RV that had been digested with BgIII and SalI. The plasmid mHVEM-FL-GFP-RV CRD1 was made by Quick Change mutagenesis from mHVEM-FL-GFP-RV with primers mHVEM dl top and mHVEM dl bot. The plasmid m/hHVEM-IRES-GFP-RV (mouse CRD1 fused to human CRD2) was made from two PCR products, withprimers 5′Bg12 mHVEM and m/hHVEM bot using mHVEM-FL-IRES-GFP-RV as the template, and primers m/hHVEM top and 3′Xho hHVEM using hHVEM-IRES-GFP-RV as the template; the PCR products were annealed, amplified withprimers 5′Bgl2 mHVEM and 3′Xho huHVEM, digested with BgIII and XhoI and ligated into Tb-lym-IRES-GFP-RV that had been digested with BgIII and XhoI. C57BL/6-BTLA-GFP-RV, a BTLA-GFP chimera, was prepared from two PCR products, with primers J1ORV1 (Bgl 2) and 3′J10+10 using C57BL/6 BTLA cDNA as the template, andprimers 5′GFP+10 and 3′GFP+Sal using GFP cDNA as the template; the PCR products were annealed, amplified with J1ORV1 (Bgl 2) and 3′GFP+Sal, digested with BgIII and SalI and ligated into Tb-lym-IRES-GFP-RV that had been digested with BgIII and XhoI. A cytoplasmic deletion of this construct, BTLA-trunc-GFP-RV, was made by site- directed mutagenesis (Stratagene) with primers mj1ltrunc top and mj1ltrunc bottom.

PD-1-GFP-RV was made by amplification of the PD-1 coding region with primers PD15′ and PD13′ using PD-1 cDNA as the template (a gift from T. Honjo, Kyoto University, Kyoto, Japan); the PCR product was digested with BgIII and BamHI and was cloned into A183-GFP MSCV that had been digested with BgIII and BamHI (a gift from W. Shay. Similarly, PD-L1-GFP-RV was made by amplification of the region encoding PD-L1 with primers PD-L1G5′ and PD-LIG3′ using PD-L1 cDNA (a gift from T. Honjo) as the template; the PCR product was digested with BgIII and BamHI and was ligated into AIB3-GFP MSCV.

PD-1 pET28 was made by amplification of the immunoglobulin domain of PD-1 with primers PD1Tet5′ and PD1Tet3′ using PD-1-GFP-RV plasmid as the template, followed by digestion with NcoI and BamHI and ligation intoMLL1-pET28 (a gift from D. Fremont, Washington University, St. Louis, Mo.) that had been digested with NcoI and BamHI. Similarly, B6-BTLA pET28 was made by amplification of the extracellular immunoglobulin domain of BTLA with primers J11TetMus5′ and J11TetB63′ using C57BL/6 BTLA-GFP-RV plasmid as the template, followed by digestion with NcoI and BamHI and ligation into MLL1- pET28. Similarly, BALB-BTLA pET28 was made with primers J11TetMus5′ and J11TetWEHI3′ using mJ11W1 as the template, and digestion with NcoI and BamHI and ligation into MML1-pET28. The immunoglobulin domain was ‘corrected’ to e authentic BALB/c allelic sequence (data not shown) by serial mutagenesis with primers Wle23k5′ and Wle23k3′ followed by primers W1h38n3B and W1h38n5C.

Fc Fusion Proteins

For the creation of CD47-Fc-aTP-ires-GFP-RV, a bicistronic retroviral vector for Fc fusion proteins, CP318 (a gift from Lewis Lanier, University of California, San Francisco, Calif.) was digested with PfIF I and NotI, treated with Vent polymerase and ligated into m1L-12R-ires-GFP-RV that had been digested with BgIII and XhoI and treated with mung bean nuclease. The plasmids mBTLA-Fc-aTP-ires-GFP-RV, mB7x-Fc-aTP-ires-GFP-RV, mPDLI-Fc-aTP-ires-GFP-RV and hBTLA-Fc-ocTP-ires-GFP-RV were made by ligation of the following XhoI-digested PCR products containing the immunoglobulin domains regions of these genes into the XhoI site of CD47-Fc-aTP- ires-GFP-RV. The product mBTLA was made withprimers 5′xho mJ11 dodecamer and 3′xho mJ11 dodecamer using as a template the C57BL/6 splenocyte phage library (Stratagene). The product mB7x was made withprimers 5′xho mB7x dodecamer and 3′xho mB7x dodecamer using IMAGE cDNA clone 3709434 (Invitrogen) as the template. PD-LI was made withprimers 5′xho mPDL2 dodecamer and 3′Xho PDL1 dodecamer using pBacPAK8-PDL1 (a gift from T. Honjo) as the template. Human BTLA was made withprimers 5′Xho hJ11 Ig and 3′Xho hJ11 Ig using hJ11(corr)ires- GFP-RV as the template.

Fc fusion proteins were produced by transfection of Phoenix E cells, were purified with Affiprep protein A columns (Biorad) and were dialyzed against PBS and stored at −70° C. For flow cytometry, cells were stained with 200 ng of purified Fc-fusion protein or, for hBTLA-Fc fusion protein, 1 ml of supernatant, followed by phycoerythrin-conjugated anti-human IgG (heavy plus light) that had been adsorbed against proteins from mouse, rat, cow and other species (Jackson Immunoresearch), and anti-mCD4-tricolor (Caltag) and anti-mB220-fluorescein isothiocyanate (FITC; BD-Pharmingen).

Production of Tetramers

Tetramers produced with plasmid PD-1 pET28, B6-BTLA pET28 or BALB-BTLA pET28 were transformed into BL21-CodonPlus (DE3) RIPL Competent Cells (Stratagene). Purified proteins were biotinylated in vitro with BirA ligase (Avidity), purified by gel filtration and concentrated. Tetramers were formed by the addition of biotinylated protein to streptavidin-phycoerythrin at a molar ratio of 1:4.

Cell Lines

BJAB and NIH 3T3 cells were from A. Chan (Washington University, St. Louis, Mo.); EL- 4 cells were from T. Ley (Washington University, St. Louis, Mo.); 293T cells were from R. Schreiber (Washington University, St. Louis, Mo.); CHO cells were from A. Sharpe (Harvard University, Boston, Mass.); and Phoenix A and E packaging cells were from American Type Culture Collection. Retrovirus constructs were packaged either in Phoenix A or E cells by calcium phosphate transfection. CHO cells were transduced by retrovirus packaged by transfection of 293T cells with pYITG plus pCGP (a gift from W. Sha) and were sorted for GFP to more than 95% purity, followed by staining with 6A6 (anti-BTLA) or BTLA-phycoerythrin tetramers.

Retrovirus Library

Purified BALB/c and C57BL/6 splenocytes were left unstimulated or were activated for 48 h with plate-bound anti-CD3 (500A.2 ascites) or soluble anti-IgM (Jackson Immunoresearch), then RNA was purified (RNeasy mini kit; Qiagen) and mRNA was made with the Nucleotrap mRNA purification kit (Clontech), full-length cDNA was made with the SMART cDNA Library Construction Kit (Clontech) and double-stranded cDNA was made by long-distance PCR with 5′PCR primer and CDS III/3′PCR primer; the PCR products were digested with SfH, size fractionated, amplified cDNA ligated into Sfi1-digested MSCV-ires-Thy1.1 retrovirus vector (a gift from W. Sha) and were transduced into XL-10 gold (Stratagene) for a library transcript complexity of 2×10⁶. The library plasmid was purified without further amplification by CsCl gradient ultracentrifugation. Infected NIH-3T3 cells (8×10⁶) and infected BJAB cells (6×10⁶) were generated from retrovirus made by calcium phosphate transfection of Phoenix E cells; the total number of infected cells was assessed by anti-Thy1.1-FITC (eBioscience) staining. Serial rounds of cell sorting used anti-Thy1.1-FITC and BALB/c and C57BL/66 BTLA tetramers. When the sorted cells were more than 80% positive for Thy1.1 and BTLA tetramer, RNA was prepared and reverse-transcribed, cDNA was amplified with Taq polymerase andprimers Sfi 5′ andSfi 3′, and PCR products were cloned into pGEM-T Easy (Promega).

T Cell Purification and Stimulation

T cells were purified (>90%) with anti-CD4 magnetic beads (Miltenyi) and, where indicated (FIGS. 6b-d,7) by subsequent sorting for populations that were negative for B220-FITC and CD11c- phycoerythrin and positive for CD4-CyChrome (>98%). For T cell stimulation with anti-CD3 and LIGHT, 2C11 (BD Pharmingen) was coated onto 96-well plates, followed by LIGHT (PeproTech) at the indicated doses (FIG. 6a). Purified T cells were plated at a density of 1×10⁶cells/ml in 100 μl media per well. CHO cells were treated in media for 16 h at 37° C. with 50 μg/ml of mitomycin C (Sigma), were washed twice in PBS and were plated at a density of 1×10⁶cells/ml in 100 μl media in 96-well plates for proliferation assays or in 1 ml media in 24-well plates for CFSE analysis. For proliferation assays, purified T cells were plated directly onto CHO cells at a density of 1×10⁶cells/ml in 100 μl media and OVA peptide. After 48 h, cells were pulsed for 12 h with 1 μCi/well of [³H]thymidine. For CFSE analysis, purified T cells were washed three times with PBS, were incubated for 8 min at 20° C. with 1 μM CFSE (Molecular Probes), were ‘quenched’ with fetal calf serum, were washed twice with media and were plated directly onto CHO cells at a density of 1×10⁶cells/ml in 1 ml media plus OVA peptide. After 3 and 4 d, cells were stained with CD4-FITC and were analyzed by flow cytometry.

Immunoblot Analysis

For cell-mixing experiments, 25×10⁶EL4 cells were mixed with 25×10⁶BJAB cells expressing GFP or 25×10⁶BJAB cells expressing mouse HVEM in 1 ml for 4 min at 37° C. and were lysed. Extracts were precleared with protein G-Sepharose (Pharmacia), followed by immunoprecipitation with 9 μg of 6A6 (anti-mBTLA) or isotype control Armenian hamster IgG (Santa Cruz) and 40 ul protein G-Sepharose (Pharmacia), then were washed and analyzed by SDS-PAGE. Immunobiot analyses for SHP-2 and phosphotyrosine were done as described in Watanabe et al, Nat. Immunol. (2003) 4:670-679 and Gavrieli et al, Biochem. Biophys. Res. Commun. 312:1236-1243 (2003).

For further details regarding Example I, including references, see Sedy et al., Nat. Immunol., 6:90-98, which is expressly incorporated herein in its entirety by reference.

(II) BTLA Polymorphism and BTLA Binding AntibodiesAllelic Polymorphisms in BTLA

We previously generated BTLA cDNA from several sources, including from the cell line WEHI 231, a commercial murine C57BL/6 splenocyte cDNA library, and 129SvEv mice, finding several polymorphisms within the BTLA Ig domain coding sequence. To determine the basis of differences, we sequenced the coding region for the BTLA Ig domain from genomic DNA of several inbred and wild mouse strains (FIG. 8). Among 23 strains, we identified three distinct alleles of BTLA, differing in their predicted amino acid sequence and potential predicted disulphide bonding pattern (FIG. 8a). The allele represented by BALB/c was present in CBA/J, SJL/J, New Zealand White (NZW), BXSB, C3H/J, New Zealand Black (NZB/BinJ), NOD, 129SvEv, and 129SWJ (FIG. 8b). A second allele, represented by the strains MLR/Ipr, AKR, SWR, CALB/RK, and DBA/2J, differed from the BALB/c allele at only one amino acid, containing histidine rather than asparagine at residue 38 of the BTLA protein. These two alleles each have five cysteine residues within the Ig domain, predicting two disulfide bonds and one unpaired cysteine. The third allele, represented by C57BL/6, was also present in B10.PL and several wild-derived inbred strains, and differed from the BALB/c and MLR/Ipr alleles at 10 and 11 amino acid residues, respectively (FIG. 8a). Notably, the C57BL/6 allele has a cysteine atamino acid residue 49, making six total cysteine residues with three predicted disulfide bonds in the BTLA Ig domain. As a control, we found no sequence polymorphisms in the PD-1 Ig domain from BALB/c, MLR/Ipr, and C57BL/6.

Generation of Allele-Specific mAbs to Murine BTLA

To generate anti-BTLA mAbs, we immunized Armenian hamsters and BTLA−/− BALB/c mice with recombinant Ig domain of the C57BL/6 BTLA allele. To allow the identification of Abs that could potentially recognize either the BALB/c or C57BL/6 allele of BTLA, hybridoma supernatants were screened for binding to BJAB cells expressing either the C57BL/6 or BALB/c allele of BTLA as a GFP fusion protein. One hamster anti-BTLA Ab, 6A6, was identified that reacted only with the C57BL/6, but not the BALB/c, allele of BTLA (FIG. 9a). The majority of the murine anti-BTLA mAbs reacted with both the C57BL/6 and BALB/c BTLA alleles, including 6F7, 6G3, 8F4, and 3F9.D12 (FIG. 9b). One murine Ab, 3F9.C6, reacted only with C57BL/6 BTLA, and not with BALB/c BTLA. Another Ab, 6H6, reacted with both alleles, but stained the C57BL/6 allele more highly than the BALB/c allele. For each of these Abs, staining was observed on wild type splenocytes, but not splenocytes of BTLA−/− mice (FIG. 9c), suggesting that these Abs in fact recognize BTLA, and react with native BTLA as well.

To further assess how these Abs interact with BTLA, we characterized their behavior in IP and Western blot analysis (FIGS. 9dande). The pan-specific Abs 6F7 and 6G3 each specifically immunoprecipitated both the C57BL/6 and BALB/c BTLA-GFP fusions proteins from BJAB cells (FIG.9d, bottom panel). Importantly, the C57BL/6-specific 6A6 Ab did immunoprecipitate the C57BL/6 BTLA allele, but not the BALB/c allele (FIG. 9d, comparelanes 3 and 6), indicating that the allelic specificity observed by FACS analysis extends to its behavior in IP Western blot analysis. Also, these interactions seen in IP Western blot analysis were specific because no BTLA was immunoprecipitated using mouse or hamster IgG1 as an isotype control (FIG. 9d, lanes 7-10).

Notably, although equivalent amounts of each BTLA allele were immunoprecipitated when assessed by immunoblotting for the GFP epitope of the fusion proteins, detection of the Ig domain by IP Western blot analysis was not equally efficient. Following immunoprecipitation, the C57BL/6 BTLA Ig domain was detected much more strongly than the BALB/c allele by 6G3 and 6F7, both pan-specific anti-BTLA Abs, (FIG. 9d, top panel,

lanes

1, 2, and 4-6). These results may indicate differential sensitivity between alleles for recognition or detection of the Ig domains, even using pan-specific Abs, which could result from differential sensitivity to denaturation of the antigenic epitope. Whatever the cause, it is necessary to consider this fact when using IP Western blot analysis in comparing BTLA from varying allelic backgrounds. Finally, certain Abs allow coimmunoprecipitation of BTLA-associated proteins. For example, IP Western blot analysis using 6A6 reproduces the known specific and inducible coassociation of SHP-2 with BTLA following pervanadate treatment (FIG. 9e).

Mapping Antigenic Epitopes Recognized by Anti-BTLA Abs

To map which of the polymorphic residues differing between BALB/c and C57BL/6 BTLA were involved in strain-specific reactivity of 6A6 and 3F9.C6, we used yeast display technology. We first expressed the BTLA Ig domain as an Aga2 fusion protein, and then generated a series of mutant BTLA Ig domains with single amino acid substitutions at the polymorphic residues, replacing BALB/c residues into the C57BL/6 allele one residue at a time (FIG. 10). This series of wild type and mutant BTLA proteins were then analyzed for reactivity with pan-specific anti-BTLA mAbs and two B6- specific Abs, 6A6 and 3F9.C6 (FIG. 10). As a positive control, we confirmed that the pan-specific anti- BTLA mAb 6F7 recognized the wild type C57BL/6 BTLA Ig domain, and also recognized each of the single residue substitutions of BTLA (FIG. 10, left column), as expected for pan-specific reactivity. In contrast, the two C57BL/6-specific Abs recognized some, but not all of BTLA mutants. Specifically, 6A6 showed a very selective loss of reactivity only with the Q27E, C49W, and Q66R substitutions, indicating that these residues are involved in the strain-specific recognition of BTLA. A distinct pattern of reactivity was observed with 3F9.C6, with a selective loss of reactivity with the R107W substitution and reduced reactivity with the Q27E substitution. Also, whereas 6A6 reactivity is sensitive to the C49W substitution, which disrupts one of three predicted disulphide bonds, 3F9.C6 reactivity remains in this substitution. These results indicate that the C57BL/6 specificity of these two Abs derive from interactions with the distinct, but polymorphic, region of the BTLA Ig domain.

In summary, at least two of the BTLA alleles can be distinguished by their antigenic structure, as shown by two C57BL/6-specific anti-BTLA Abs. Importantly, we also identified several pan-specific anti-BTLA Abs, which now allow direct comparisons of the fine specificity of tissue expression of native BTLA expression between various murine strains.

Distribution and Expression of Murine BTLA

In our previous studies, we were restricted to analyzing BTLA expression either by mRNA expression or by using epitope-tags because we lacked Abs to native BTLA. Conceivably, we failed to detect low but physiologically important levels of BTLA on certain lymphocyte subsets for this reason. Thus, we examined BTLA surface expression on various lymphoid subsets again, using both allele-specific Ab 6A6 and pan-specific Ab 6F7 (FIG. 11).

First, BTLA was expressed uniformly on B cells at levels that were similar for C57BL/6 and BALB/c mice (FIG. 11a). CD4+ and CD8+ T cells expressed lower levels of BTLA compared with B cells, but again, at levels that were similar for C57BL/6 and BALB/c mice. For 6A6, we found that a subpopulation of CD11b+ cells, CD11c+ dendritic cells, and DX5+ cells were positive for BTLA expression, and again identified only in C57BL/6 cells as expected (FIG. 11a,middle row). Using the pan-specific 6F7 Ab, we found that B cells express the highest levels of BTLA, again at levels similar between C57BL/6 and BALB/c mice, with lower levels expressed in CD4 and CD8 T cells (FIG. 11a,lower row). Interestingly, using the pan-specific reagent 6F7, we found that BTLA was expressed on CD11c+ BALB/c cells at levels similar to CD11c+ C57BL/6 cells, but that BTLA was only expressed on CD11b+ macrophages and DX5+ NK cells from C57BL/6 mice, but not in BALB/c mice (FIG. 11a, lower row). The fact that 6F7 detects BTLA expression on B cells, T cells, and CD11c+ cells from both BALB/c and C57BL/6 mice serves as a control for its ability to bind BTLA from both strains. Thus, the selective binding of 6F7 to DX5+ and CD11b+ cells only in C57BL/6, not BALB/c mice, indicates a difference between these strains for BTLA expression by these cell types. Thus, these strains appear to have a distinct difference in the cell types expressing detectable BTLA, explaining the differences between BTLA expression reported previously.

We also examined BTLA expression in splenic B cell populations (FIG. 11b). BTLA expression was detected at the highest levels on follicular B cells (1gMlowCD21/CD35int), and at reduced levels on marginal zone B cells (1gMhighCD21/CD35high) and transitional B cells (1gMlowCD21/CD35low) (FIG. 11b). Notably, because the 6F7 pan-specific Ab was used for analysis, we can also conclude that the levels on each subpopulation of B cells are similar between C57BL/6 and BALB/c mice (FIG. 11b).

We next examined BTLA expression in thymocyte and B cell development (FIG. 12). In thymus, BTLA was expressed at highest levels on mature CD4+ T cells, and at slightly reduced levels on CD8+ T cells (FIG. 12A). BTLA expression on immature CD4-CD8− T cells or CD4+ CD8+ double positive T cells was nearly undetectable (FIG. 12a). In bone marrow, BTLA was expressed at the highest levels on B220highlgM+ mature B cells (FIG. 12b), and was detected at relatively low levels on B220low/IgM+ immature B cells. BTLA expression was undetectable on B220+IgM− pro-B cells and pre-B cells. Further, we found no differences between C57BL/6 or BALB/c mice for the levels of BTLA expression on the thymocyte and bone marrow populations.

Finally, we examined the BTLA expressed on CD4+ T cells under various conditions of activation and polarization by cytokines (FIG. 13a). BTLA surface expression on resting CD4+ T cells was induced by 10-fold onday 2 following activation with Ag and APCs, decreased byday 4, and was nearly undetectable byday 7 after activation (FIG. 13a). The rapid increase in BTLA expression byday 2 on Ag-activated CD4+ T cells occurred both in Th1 inducing or Th2-inducing conditions (FIG. 13a). Upon secondary T cell activation, BTLA was again highly induced 2 days following activation, again in both Th1 and Th2 cultures (data not shown). However, tertiary activation of T cells revealed selective induction in the Th1 cultures, but not in the Th2 cultures (FIG. 13a). These results suggest that BTLA expression on CD4+ T cells is initially controlled primarily by T cell activation and not by factors governing Th1 or Th2 differentiation. The delayed loss of BTLA inducibility in Th2 cells might suggest a silencing process rather than a Th1-specific pathway for induction, which would be consistent with our initial finding that BTLA expression is not dependent on Stat4 or Stat1. Finally, the rapid modulation of BTLA expression, peaking onday 2 and extinguished byday 7, suggests that it may act in the mid-phases of T cell activation following interactions with APCs.

In contrast to the activation-dependent expression of BTLA seen in CD4+ T cells, BTLA expression on B cells was maintained at high levels throughout activation by LPS or anti-IgM stimulation (FIG. 13b). These results differ slightly from the reported 3- to 10-fold decrease in BTLA expression following LPS activation of B cells. Nonetheless, our results agree with that report in the finding of high levels of BTLA expression on B220+B cells in the periphery, and to some degree, the constitutive nature of its expression.

Selective Induction of BTLA an Anergic T Cells

Previously, a method of anergy induction for naive CD4+ T cells was developed that involves adoptive transfer of Ag-specific CD4+ T cells into recipients expressing Ag on somatic tissues. Specifically, clone 6.5 transgenic T cells, reactive to HA peptide 110-120 presented by I-Ad, become anergic when transferred into recipient mice expressing a membrane bound form of HA targeted for expression on lung and prostate tissue. We analyzed BTLA expression following T cell transfer on various days after transfer using Affymetrix gene arrays and FACS (FIGS. 14,aandb). We found that BTLA mRNA was highly induced in these anergic CD4+ T cells in this system, compared with CD4+ T cells activated by Agexpressing vaccinia virus (FIG. 14a). At 2 days after transfer, BTLA expression by T cells undergoing anergy induction was twice the level of naive T cells, and significantly higher than activated T cells. This induction was more evident byday 3 andday 4 following transfer, with BTLA expression about 3-fold higher than in naive T cells. By contrast, BTLA levels were substantially reduced in fully activated T cells compared with naive or anergic T cells at these times (FIG. 14a). As a control, myosin Vila, a constitutive “housekeeping” gene, showed essentially no change in these three conditions over these times. Thus, BTLA mRNA appears to decline more rapidly than BTLA surface protein in activated T cells because activated T cells express peak BTLA surface levels at day 2 (FIG. 13), but show reduced BTLA mRNA (FIG. 14b). These observations are consistent with the reduced BTLA surface expression byday 4 and the essentially undetectable BTLA expression byday 7.

We next measured BTLA expression by FACS under conditions of anergy induction or activation (FIG. 14b). Notably, the highest levels of BTLA surface expression coincided with induction of anergy in vivo. Specifically, 6 days after transfer, anergic T cells expressed about 10-fold higher BTLA than naive T cells, and about 3-fold higher than in vivo-activated T cells (FIG. 14b). We verified that the CD4+ T cells transferred into HA-expressing recipients did become anergic as defined by lack of proliferation (FIG. 14c), consistent with previous reports. For comparison, we also wished to evaluate BTLA expression on conventional naive CD4+ T cells (CD4+CD25−) T cells or T regulatory cells (CD4+CD25+) either as resting cells ex vivo or after in vitro activation with anti-CD3 (FIG. 14d). As expected, BTLA was expressed at low levels on naive T cells, and was induced about 10-fold 36 h after anti-CD3 treatment. Freshly isolated T regulatory cells expressed similar levels of BTLA as freshly isolated naive CD4+ T cell, but showed only a slight increase after treatment with antiCD3 (FIG. 14d). As a control, we confirmed that T regulatory cells, but not naive T cells, expressed PD-1, consistent with previous reports. As a further control, we showed that the isolated CD25+ T regulatory cells failed to proliferate in vitro, in contrast to the robust proliferation of freshly isolated naive T cells (FIG. 14e). In summary, BTLA shows a pattern of expression that is somewhat distinct from that of CTLA-4 and PD-1 in terms of its response to anergy induction and expression by T regulatory cells.

Role of BTLA in T Cell-Independent Ab Responses

Our initial analysis of BTLA was motivated by consideration of its role in T cell activation. However, the fact that B cells express the highest level of BTLA, and the constitutive nature of this expression, motivated a second examination of its effect on Ab production. In our study, we examined T cell-independent Ab responses using immunization with NP-Ficoll in wild-type mice or BTLA−/− 129SvEv mice, which express the BALB/c allele of BTLA. We immunized cohorts of mice with one injection of NP-Ficoll in alum and measured production of anti-NP Abs of specific isotypes on day 14 (FIG. 15). For the isotypes IgM, IgG1, IgA, we found no specific changes in levels of anti-NP Abs. For IgG2a or IgG2b, we found only slight increases in anti-NP Abs in the BTLA−/− compared with wild-type mice. However, for Abs of the IgG3 isotype, which is primarily associated with T-independent responses, we found an about 2-fold increased in anti-NP-specific Abs in BTLA−/− mice compared with wild-type mice. The size of this difference is consistent with the relatively modest increases in B cell and T cell proliferation responses described for BTLA−/− cells previously, and is consistent with an inhibitory rather than activating role of BTLA. However, the relatively modest magnitude of this effect could also be an indication that BTLA expression by B cells may serve a purpose other than cell-intrinsic signaling, such as perhaps delivery of a signal toward cells expressing ligands for BTLA.

Methods (II)

The following Abs used for FACS analysis were from BD Pharmingen: CD4-CyChrome (RM4-5), CD8-FITC (53-6.7), B220-allophycocyanin (RA3-6B2), CDHb-FITC (M1/70), CD11c-FITC (HL3), DX5-FITC, I-Ad-PE (AMS-32.1), I-Ab-PE (AF6-120.1), IgM-PerCP Cy5.5 (R6.60.2), CD21/CD25-FITC (7G6), CD25-allophycocyanin (PC61), CD62 ligand-FITC (MEL-14), Thy1.1-PerCP (OX-7), goat anti-mouse Ig-PE, mouse anti-Armenian/Syrian hamster IgG-PE (mixture), Streptavidin (SA)-PE, SA-CyChrome, and SA-aliophycocyanin. KJ1-26 Tricolor, hamster IgG-biotin, and murine IgGI-biotin were from Caltag Laboratories. All FACS analysis included an initial incubation with 2.4G2 (anti-CD16/CD32; BD Pharmingen) to block Fc receptor interactions.

Sequencing of BTLA and PD-1 Ig Domains

Exon 2 of BTLA or PD-1, encompassing the Ig domain, was amplified by PCR from genomic DNA from a panel of mouse strains using Easy-A High Fidelity PCR Cloning Enzyme (Stratagene) and the following intronic primers: BTLA (sense) ATGGTCCTTCTAAGAGTGAAC (SEQ ID NO: 8), (antisense) ATAGATGGTCTGGGGTAGATC (SEQ ID NO:9) and PD-1 (sense) CAGGCTCCTTCCTCACAGC (SEQ ID NO:10), (antisense) CTAAGAGGTCTCTGGGCAG3′ (SEQ ID NO:11).

PCR products were cloned into the pGEM-T Easy vector (Promega) and inserts from at least three individual subclones from each strain were sequenced using the T7 universal primer.

Generation of Soluble BTLA Ig Domain

The Ig domain of C57BL/6 BTLA was PCR amplified from cDNA using the following primers: BTLA (sense) CATGCCATGGAGAAAGCTACTAAGAGGAAT (SEQ ID NO:12) and BTLA (antisense) CGGGATCCTGAAGAGTTTTGAGTCCTTTC-3′ (SEQ ID NO:13). The product was subcloned into the pET28 vector (Novagen) that had been modified to contain a BirA biotinylation sequence (GGGLNDIFEAQKIEWHE) (SEQ ID NO:14) onto the C terminus of the BTLA Ig domain. Proteins were expressed as insoluble inclusion bodies in BL21 (DE3) Codon Plus RIL cells (Stratagene) and refolded.

Production of mAbs to BTLA

Armenian hamsters or BALB/c background BTLA−/− mice were immunized with 100 μg of refolded C57BL/6 BTLA Ig domain protein in CFA, boosted biweekly with 100 μg of protein in IFA, and received a final i.v. boost 3 days before fusion. Splenocytes were fused with the P3X63Ag8 myeloma, and hybridoma supernatants screened for binding to BJAB cells expressing either C57BL/6 or BALB/c BTLA Ig domains as GFP fusion proteins. The BTLAGFP chimera was prepared by splicing by overlap extension (SOEing). A PCR fragment containing the BTLA cDNA with a 3′ tail annealing to the 5′ end of GFP was amplified by PCR made using Vent polymerase, the primers J1ORV1-BgIII (AGCTCTGAAGATCTCTAGGGAGGAAG) (SEQ ID NO: 15) and 3′J 10+10 (CCTTGCTCACACTTCTCACACAAATGGATGC) (SEQ ID NO: 16) with DO11.10 BTLA cDNA as template. A second fragment containing GFP cDNA, without its start codon, with a 5′ tail annealing to the 3′ end of BTLA was amplified by PCR using Vent polymerase and theprimers 5′ GFP+10 (TGTGAGAAGTGTGAGCAAGGGCGAGGAGC) (SEQ ID NO: 17) and 3′ GFP+Sal (ACGCGTCGACTTACTTGTACAGCTCGTCCATG) (SEQ ID NO: 18) with the GFP cDNA as template. The chimeric BTLA-GFP fusion cDNA was amplified by PCR from a mixture of these two PCR fragments using the primers J1ORV1-BgIII and 3′ GFP+Sal, digested with BgIII and SalI, and cloned into the BgIII/SalI sites of IRES-GFP-RV to produce D011.10-BTLA-GFP-RV. A cytoplasmic deletion was made using site directed mutagenesis (Stratagene) and theprimers mj 1 Itrunc top (GTTGATATTCCAGTGAGCAAGGGCGAGGAG) (SEQ ID NO: 19) andmj 1 Itrunc bottom (CTTGCTCACTGGAATATCAACCAGGTTAGTG) (SEQ ID NO: 20) to produce D011.10-BTLA-trunc-GFP-RV. The C56BL/6 version of BTLA trunc-GFPRV was made by purifying a natural BgIII/BamHI fragment from a BTLA cDNA cloned from a mouse spleen cDNA phage library (Stratagene). This fragment was then cloned into the BgIII/BamHI digested D011.10-BTLA trunc-GFP-RV to produce C57BL/6-BTLA trunc-GFPRV.

Positive hybridomas were expanded and Abs purified using MAPS II-protein A columns. Hamster monoclonal 6A6 is of the IgG isotype, whereas all murine Abs are IgG1x. Unless otherwise stated, all Abs were biotinylated using EZ-Link Sulfo-NHS-LC-biotin (Pierce) and detected with SA- conjugated fluorochromes. This procedure eliminated secondary Ab cross-reactivity with murine cells.

Yeast Display Mapping

The Ig domain of the C57BL/6 BTLA allele was amplified from cDNA using theprimers 5′-GGAATTCCATATGCAGCCAAGTCCTGCCTG-3′ (SEQ ID NO: 21) and 5′-CATGCTAGCGAGAAAGCTACTAAGAGGAA-3′ (SEQ ID NO: 22) and subcloned into the NdeI and the NheI sites of the pCT302-AGA2d vector to create an HA-tagged fusion to the Aga2 peptide. QuickChange mutagenesis was used to introduce mutations into this construct using the following primer pairs:

	C26At,
	(SEQ ID NO: 23)
	CAGTGCAACTTAATATTACGAGGAATTCCAAACAG;

	C26Ab,
	(SEQ ID NO: 24)
	CTCGTAATATTAAGTTGCACTGGACACTCTT;

	C32At,
	(SEQ ID NO: 25)
	GCAACTTACTATTAAGAGGAATTCCAAACAGTCTGC;

	C32Ab,
	(SEQ ID NO: 26)
	AATTCCTCTTAATAGTAAGTTGCACTGGACA;

	G48Ct,
	(SEQ ID NO: 27)
	GAATCCCAAACACTCTGCCAGGACAGGAGAGT;

	G48Cb,
	(SEQ ID NO: 28)
	CTGGCAGAGTGTTTGGAATTCCTCGTAATAG;

	A55Tt,
	(SEQ ID NO: 29)
	ACAGTCTGCCTGGACAGGAGAGTTATTTAAAATT;

	A55Tb,
	(SEQ ID NO: 30)
	TCCTGTCCAGGCAGACTGTTTTGAATTCCT;

	C79Gt,
	(SEQ ID NO: 31)
	GAGTTATTTAAAATTGAATGTCCTGTGAAATACTGTGT;

	C79Gb,
	(SEQ ID NO: 32)
	AGGACATTCAATTTTAAATAACTCTCCTGTCC;

	T147Gt,
	(SEQ ID NO: 33)
	ATGGAACAATCTGGGTACCCCTTGAGGTTAGCC;

	T147Gb,
	(SEQ ID NO: 34)
	GGGTACCCAGATTGTTCCATTGTGCTTAC;

	A163G/T168Gt,
	(SEQ ID NO: 35)
	TTGAGGTTGGCCCGCAGCTATACACTAG;

	A163/T168Gb,
	(SEQ ID NO: 36)
	GCTGCGGGCCAACCTCAAGGGGTACACAGA;

	A197Gt,
	(SEQ ID NO: 37)
	TTGGGAAGAAAATCGATCAGTTCCGGTTTTTGTTCT;

	A197Gb,
	(SEQ ID NO: 38)
	AACTGATCGATTTTCTTCCCAACTAGTGTA;

	C320Gt,
	(SEQ ID NO: 39)
	ATCCATGTGAGAGAAAGGACTCAAAACTCTTCA;
	and

	C320Gb,
	(SEQ ID NO: 40)
	AGTCCTTTCTCTCACATGGATGGTTACTGAATG.

Transformation of EBY100.Agal yeast with each construct resulted in surface expression of the BTLA mutant. Expression level was confirmed by anti-HA staining. Yeast cells were stained with anti-BTLA Abs as indicated to determine mutations that abolished Ab recognition.

CD4+ T Cell Activation and Expression Analysis

DOI 1.10 TCR transgenic cells were activated with 0.3 μM OVA peptide (amino acids 323- 339) and irradiated (2000 rad) BALB/c splenic APCs. Th1 conditions consisted of heat-killed Listeria monocytogenes, IL-2 (40 U/ml; Takeda Chemical Industries), and 10 μg/ml anti-IL- 4 (11 B11). Th2 cells were differentiated in 100 U/ml IL-4, 3 pg/ml anti-IL-12 (TOSH), and IL-2. Cells were restimulated with Ag and APCs on

days

7 and 14. Th1/Th2 phenotypes were confirmed at

days

7 and 14 by intracellular cytokine staining for IFN-γ and IL-4.

Gene Microarray

Anergic T cells were isolated by adoptively transferring 2.5×10⁶Thy1.1+HA-specific T cells to recipient mice (C3-HAhigh). After 4 days in vivo, animals were sacrificed via CO₂asphyxiation. Spleens were harvested, and subjected to ACK lysis. Adoptively transferred HA-specific T cells were enriched by binding the resulting cells with Abs to CD8a (53-6.7), B220 (RA3-6B2) and Thy1.2 (30-H12), followed by incubation with SA-conjugated magnetic microbeads (Miltenyi Biotec). Unwanted cells were depleted by passage over LS columns (Miltenyi Biotec) according to the manufacturer's protocol. The remaining cells were stained with an Ab to Thy1.1 (OX-7) and further enriched using fluorescence-based cell sorting on a FACSVantage TurboSort (BD Biosciences). The resulting populations were between 95 and 99% pure. Cells were kept at 4° C. throughout the enrichment procedure. In vitro assays confirmed the anergic phenotype of the sorted cells. All Abs were purchased from BD Pharmingen. This procedure specifically avoids ligation of the TCR or CD4 during the isolation process. Activated, memory and naive clonal T cells were isolated in an analogous manner, using a specific viral construct (vaccinia-HA) to activate the cells after adoptive transfer to nontransgenic B10.d2 mice. RNA was isolated from each T cell population using the RNAeasy kit according to the manufacturer's instructions (Qiagen), and cRNA probe was prepared. Fragmented cRNA was hybridized to mouse GeneChips MU174A, MU174B, and MU174C per Affymetrix standard hybridization protocol. Each chip contained about 12,000 different genes (chip A) per expressed sequence tag (EST) with (chips B and C), for a total of about 36,000 genes per EST from the three chips. A single gene/EST was represented by a probe set defined by 16-20 perfect match oligonucleotides that span the length of the gene, as well as 16 oligonucleotides with 1 by mismatch. The intensity of a gene was determined by evaluating the perfect match and mismatch intensities, as described in Affymetrix Microarray Suite, version 5.1 software (Affymetrix). The experiment was replicated once, for a total of two replicate intensities within each condition. To identify probe sets associated with an anergic phenotype, we used the hypothesis-based analysis of microarrays algorithm with theboolean hypothesis day 4 anergy>naive ANDday 4 anergy>day 4 activation.

Assessment of Anergy by Proliferation

On indicated days following transfer of HA-TCR transgenic T cells, 20×10⁶splenocytes were incubated with increasing doses of HA peptide. Proliferation was assayed after 48 h, with a [³H]thymidine pulse in the final 12 h.

BTLA Expression by Naive, Activated, and Anergic CD4+ T Cells

HA-TCR transgenic T cells were enriched by depletion of CD8+ and B220+ cells as earlier described. Cells were CFSE-labeled before adoptive transfer of 2.5×10⁶clonotypic cells via tail vein injection. Cells were stained with anti-Thy1.1 PerCP and the anti-BTLA Ab 6F7-biotin, followed by SA-PE.

Purification and Activation of CD44-CD25+ T Regulatory Cells

Splenocytes and lymph node cells from BALB/c mice were isolated. Following erythrocyte lysis, B220+ cells were depleted by magnetic separation with anti-B220 Microbeads (Miltenyi Biotec). The negative fraction was stained with CD25-PE (BD Pharmingen) and anti-PE Microbeads (Miltenyi Biotec) and magnetically separated into CD25+ and CD25− fractions. Enrichment was assessed by FACS as shown (seeFIG. 14d). Contaminating non-CD4+ cells were mainly B220+ or CD8+ cells. To activate T cells, 1×10⁶cells/ml of each fraction were cultured on flat-bottom plates coated with 10 ug/ml 2G11 (anti-CD3; BD Pharmingen) for 48 h. Cells were pulse with 1 μCi/well [³H]thymidine for an additional 12 h.

Ab Response to NP-Ficoll

Eight-week-old BTLA+/+ and BTLA−/− littermate mice on the 129SvEv background were immunized i.p. with 50 pg of nitrophenyl (NP)-Ficoll (Biosearch Technologies) in Imject alum (Pierce). Sera were collected onday 14, and the titers of anti-NP were determined by ELISA using NP25-BSA (Biosearch Technologies) for Ab capture and the Southern Biotechnology clonotyping/HRP kit for IgG subclass-specific ELISA (Southern Biotechnology Associates).

For further details regarding Example II, including references, see Hurchla et al., J. Immunol., 174: 3377-3385, 2005, which is expressly incorporated herein in its entirety by reference.

(III) BTLA-HVEM Effects in Graft SurvivalBMA and HVEM Regulate Acceptance of Partially MHC-Mismatched Cardiac Allografts

Primarily vascularized cardiac allografts are the most frequent organ transplant undertaken in mice and may be performed across full MHC disparities, with rejection in 7-8 days, or across MHC class I or II disparities, which leads to long-term survival (>100 days). The basis for this unexpectedly long-term survival of cardiac transplants across partial MHC disparities is unknown and has received little attention. As anticipated from the literature, we indeed found that cardiac allografts performed across an MHC class II mismatch (Bm12 B6) survived long term in wild-type recipients (mean survival time (MST), >100 days; n=6). Histologic assessment of these allografts harvested at 2 wk after transplant showed preservation of myocardial architecture and generally only sparse mononuclear cell infiltration (FIG. 29a). In contrast, BTLA−/− recipients rejected Bm12 cardiac allografts by 2-3 wk after transplant (MST, 14.3±3.8 days; n=12; p<0.001), and histology showed a marked increase in leukocyte infiltration and myocardial injury (FIG. 29a). In addition, comparable abrogation of Bm12 allograft survival was seen with mAb targeting of BTLA in wild-type recipients (MST, 23.2±3.2; n=6; p<0.001) or by engraftment of recipients lacking the BTLA ligand, HVEM (MST, 17.4±4.2 days; n=8; p<0.001;FIG. 29a). Thus, BTLA and HVEM are required to allow long-term survival of partially mismatched cardiac allografts. In contrast to results obtained with BTLA−/− recipients, PD-1−/− recipients receiving Bm12 cardiac allografts exhibited an 80% long-term allograft survival (FIG. 29b), although we did observe a minor role for PD-1 in regulating responses to Bm12 cardiac allografts. Dual BTLA−/− and PD-1−/− knockout mice (DKO) mice rejected Bm12 donor hearts more rapidly (MST, 10.5+1.5 days; n=4) than singly deficient BTLA−/− recipients (p<0.05) or wild-type controls (p<0.0001;FIG. 29b).

Like MHC class II-mismatched grafts, MHC class I-mismatched (Bm1 B6) cardiac allografts survived long term when transplanted to wild-type B6 mice, but were rejected in BTLA−/− mice (FIG. 29c). Furthermore, in contrast to wild-type B6 recipients, the MHC class I-mismatched allografts in BTLA−/− recipients showed increased mononuclear cell infiltration and progressive tissue damage indicative of the development of cellular rejection (FIG. 29c). PD-1−/− recipients receiving Bm1 cardiac allografts had 100% long-term allograft survival. Collectively, these findings indicate that BTLA, in contrast to PD-1, is capable of inhibiting the generation of a functional allogeneic immune response in the context of partial MHC mismatches.

BTLA Suppresses MHC Class II-Dependent T Cell Responses

The unexpected rejection of Bm12 allografts by BTLA−/−, but not PD-1−/−, mice suggested that BTLA and PD-1, or their ligands, might be differentially expressed in partially MHC-mismatched allografts. BTLA mRNA expression within Bm12 allografts was 20-fold higher than PD-1 at 7 days after transplant, whereas no BTLA expression was detected within Bm12 hearts engrafted into BTLA−/− recipients, indicating BTLA expression primarily by infiltrating host leukocytes (FIG. 30a). Comparable BTLA expression was observed within long-surviving allografts. Unlike BTLA, only very low levels of PD-1 were detected in Bm12 allografts in either wild-type or BTLA−/− recipients (FIG. 30a). No differences in the levels of expression of HVEM, PD-L1, or PD-L2 were seen between wild-type and BTLA−/− recipients (FIG. 30a). These data suggest that in the Bm12 B6 model, BTLA is the predominant inhibitory receptor expressed by infiltrating alloreactive T cells, and that in the absence BTLA, there is no compensatory increase in expression of additional inhibitory molecules.

We next studied the in vitro and in vivo responses of T cells from wild-type and BTLA/− mice to MHC class II Ags. First, we examined the in vitro proliferation of purified wild-type or BTLA−/− CD4+ T cells cocultured with irradiated Bm12 DC. Proliferation of BTLA−/− T cells was increased compared with that of wild-type T cells, as measured by either BrdU incorporation (FIG. 30b) or CFSE dilution (FIG. 30c). To assess in vivo responses, 40 million CFSE-labeled wild-type or BTLA−/− splenocytes were adoptively transferred into irradiated Bm12 hosts, and donor CD4+ T cell proliferation was assessed. Although a large portion of wild-type CD4+ T cells remained undivided 72 h after adoptive transfer, almost all BTLA−/− CD4+ T cells had entered the cycle and proceeded through several rounds of division (FIG. 30d). Hence, BTLA regulates CD4+ T cell alloactivation and proliferative responses to MHC class II Ags.

MHC class II-restricted CD4+ T cell proliferation dominates host alloresponses in the Bm12 B6 model, although host responses are known to include stimulation of CD8+ precursor CTL by class II-restricted CD4+ T cells. We found that although proliferative responses of CD8+ T cells in irradiatedBm 12 hosts were low compared with those of CD4 cells, the alloactivation and proliferation of CD8+ T cells from BTLA−/− mice were marginally increased over control cells in this assay system (FIG. 30e). We examined recipient anti-donor responder frequencies by ELISPOT, with the readout of IFN-spot-forming cells by recipient splenocytes. BTLA−/− recipient splenocytes had significantly higher anti-donor responder frequencies when challenged with Bm12 APCs (FIG. 30f), consistent with the increased allogeneic proliferation in vitro and the accelerated graft rejection in vivo of T cells from BTLA−/− mice.

Minor Role of BTLA in Fully MHC-Mismatched Alloresponses

We next tested whether BTLA played a similar dominant role in regulating responses to fully MHC-mismatched cardiac allografts as it did for partially MHC-mismatched cardiac allografts. Wild-type recipients (B6, H-2b) rejected cardiac grafts (BALB/c, H-2d) in 7-10 days (MST, 8±1 days; n=6), whereas BTLA−/− recipients showed a small and unexpected prolongation of graft survival (MST, 12±5 days; n=6; p<0.05;FIG. 31a). In addition, wild-type mice treated with a neutralizing anti-BTLA mAb showed a similar prolongation of allograft survival (MST, 13±1 days; n=4; p<0.05) compared with control IgG treated recipients (MST, 8+1 days; n=4;FIG. 31b). Furthermore, addition of a subtherapeutic course of rapamycin prolonged graft survival in wild-type mice by a few days (MST, 11±2 days; n=6; p<0.05), but significantly prolonged graft survival in BTLA−/− mice (MST, 53±12 days; n=8; p<0.001), with 25% of the latter recipients achieving long-term acceptance (FIG. 31c). Hence, in the case of fully MHC-mismatched cardiac allografts, loss of BTLA did not accelerate allograft rejection, but, rather, caused a surprising, albeit small, increase in allograft survival. By contrast, the presence or the absence of BTLA had no effect on the tempo of rejection of B6 cardiac allografts by BALB/c recipients; all allografts were rejected within 7-10 days (n=4/group; p>0.05).

To understand the prolongation of fully MHC-mismatched graft survival, we measured the expression of cytokines and chemokine receptors important to host T cell recruitment in this model, using allografts harvested 7 days after transplant. We found decreases in IL-2 and IFN-mRNA in BTLA−/− recipients compared with wild-type recipients (FIG. 31d). We also found reduced expression of CXCR3 and CCR5 in BTLA−/− recipients compared with wild-type recipients (FIG. 31d). Therapy with rapamycin accentuated differences in cytokine and chemokine receptor mRNA expression between BTLA−/− and wild-type recipients (FIG. 31d). Given a key role for IFN-induced IFN-inducible protein 10 (IP-10) production in promoting CXCR3+ cell recruitment and allograft rejection in this model, we performed Western blotting, which confirmed that allografts in BTLA−/− recipients had reduced IP-10 and CXCR3 proteins compared with wild-type controls, with or without rapamycin therapy (FIG. 31e).

To assess whether the lack of BTLA affected the strong alloactivation and proliferation induced in T cells by 72 h in this model, we used the parent-to-F1 model involving transfer of CFSE-labeled cells across fully allogeneic barriers (FIG. 31f). In this model, the activation and cell cycle progression of CD4+ responses were similar for BTLA−/− and wild-type cells, and CD8+ T cells from BTLA−/− mice were only marginally decreased compared with those from wild-type controls (FIG. 31f). However, the evaluation of intracellular cytokine production by alloreactive T cells showed decreased IL-2 and IFN-production by alloreactive BTLA−/− CD4+ and CD8+ T cells compared with wild-type T cells (FIG. 31g). Again, a subtherapeutic rapamycin dose caused a modest decrease in proliferation of BTLA−/− T cells compared with wild-type T cells, particularly CD8+ responses (FIG. 31f), and decreased production of IL-2 and IFN- by both T cell subsets (FIG. 31g). These data indicate that T cell activation, proliferation, and production of cytokines such as IL-2 and IFN- are decreased in BTLA−/− mice, especially when recipients are treated with limited immunosuppression, and that these impaired responses are associated with modulation of chemokine/chemokine receptor effector pathways.

Involvement of PD-9 and BTLA in Fully MHC-Mismatched Alloresponses

In considering explanations for the differing effects of BTLA in the partial MHC mismatch and full MHC mismatch models, we wondered whether differential reliance on PD-1 between these models might play a role. Therefore, we examined the contributions of both PD-1 and BTLA in the fully MHC-mismatched model (FIG. 32). We found, first, that BALB/c cardiac allografts were rejected at similar rates (p>0.05) by C57BL/6 wild-type mice and DKO mice (FIG. 32a). Second, consistent with the DKO data, mAb blockade of PD-1 increased the rate of rejection of fully MHC-mismatched allografts by BTLA−/− recipients (p<0.001;FIG. 32b). Third, the duration of allograft survival in BTLA−/− recipients receiving subtherapeutic course of rapamycin (MST, 53±12 days; n=8) was markedly decreased by loss of PD-1, as seen by examining either DKO recipients (MST1 12.8±2.2 days; n=4; p<0.001;FIG. 32c) or by mAb Ab blockade of PD-1 in BTLA−/− mice (MST, 14.0±3.5 days; n=4; p<0.001;FIG. 32d). In summary, in contrast to partial MHC-mismatched allografts, the responses against fully MHC-mismatched cardiac allografts are regulated by both BTLA and PD-1.

We next asked whether PD-1 regulated the proliferation and function of T cells responding to fully MHC-mismatched allografts. Analysis by qPCR of BALB/c cardiac allografts harvested onday 7 after transplant from C57BL/6 recipients showed intragraft expression of BTLA, PD-1, and their ligands, HVEM, PD-L1, and PD-L2 (FIG. 33a). By comparison with wild-type recipients, BALB/c allografts harvested from BTLA−/− recipients had increased PD-1 expression (FIG. 33a; p<0.01). In contrast to PD-1 expression, the expression of HVEM, PD-L1, and PD-L2 was not increased in BTLA−/− recipients. These results suggest that in the absence of BTLA, host leukocytes might express more PD-1 in response to allostimulation.

To directly examine PD-1 expression by alloreactive wild-type or BTLA−/− T cells, we adoptively transferred CFSE-labeled splenocytes into irradiated Bm12 (class II-mismatched) or B6D2F1 (fully MHC-mismatched) recipients. At analysis 72 h later, we found that in the MHC class II partial mismatch, PD-1 was weakly expressed by alloreactive CD4+ T cells, but not at all by CD8+ T cells, from wild-type or BTLA−/− mice (FIG. 33b). In contrast, with a full MHC mismatch, PD-1 expression by both CD4+ and CD8+ donor T cells was markedly increased, and the extent of PD-1 expression was higher in BTLA−/− vs wild-type T cells (FIG. 33b). Moreover, treatment with rapamycin reduced PD-1 expression by wild-type T cells, but had only minor effects on PD-1 induction by T cells from BTLA−/− mice (FIG. 33b).

Lastly, we used in vivo and in vitro approaches to examine the roles of BTLA and PD-1 in regulating T cell proliferation and cytokine production in response to fully MHC-mismatched allostimulation (FIGS. 33,c-e). Compared with wild-type or BTLA−/− cells, DKO cells showed enhanced proliferation (FIGS. 33c) and Th1 cytokine production (FIG. 33d). Therapy with rapamycin decreased the alloactivation-induced proliferation (FIG. 33c) and cytokine production (FIG. 33e) of CD4+ and CD8+ T cells from wild-type and BTLA-/donors, but did not block these events in DKO CD4+ or CD8+ T cells (FIGS. 33,cande). Indeed, the production of IL-2 and IFN- was increased in DKO T cells compared with wild-type and BTLA−/− T cells (FIG. 33d), including in the presence of rapamycin therapy (FIG. 33e). Collectively, these data indicate that 1) PD-1 expression is highly induced on the surfaces of alloreactive CD4+ and CD8+ T cells upon exposure to fully MHC-disparate allografts; 2) the levels of PD-1 on alloreactive CD4+ and CD8+ T cells are still further increased in the absence of BTLA; and 3) increased PD-1 expression is associated with inhibitory effects on the alloantigen-induced production of cytokines such as IL-2 and IFN-. In associated in vitro studies, as T cell activation increased in response to allogeneic DC (FIG. 34), the induction of PD-1 was increasingly apparent compared with that of BTLA. BTLA up-regulation occurred upon T cell activation, but did not show expansion comparable with that of PD-1 with increasing T cell activation, suggesting that the strength of T cell activation determines the relative importance of these two pathways.

Methods (III)Mice

BTLA−/−, PD-1−/−, and dual BTLA−/− and PD-1−/− mice were backcrossed for more than eight generations on a C57BL/6 background; HVEM−/− mice were generated by homologous recombination and backcrossed more than five generations on a B6 background. Wild-type C57BL/6 (H-2b), BALB/c (H-2d), C57BL6/DBA F1 (H-2b/d), Bm12 (B6.C-H2bm12/KhEg), and Bm1 (B6.C-H2bml/ByJ) mice were purchased from The Jackson Laboratory, housed in specific pathogen-free conditions, and used for studies approved by the institutional animal care and use committee of Children's Hospital of Philadelphia.

An Armenian hamster anti-mBTLA neutralizing mAb, 6A6, was described previously in Sedy et al. Nature Immunol. 6:90-98 (2005), and we purchased mAbs for flow cytometry (BD Pharmingen) and Abs for Western blotting (Santa Cruz Biotechnology). Labeling of cells with CFSE (Molecular Probes) was undertaken as previously reported,

Quantitative PCR (qPCR)

We performed qPCR as previously described. Briefly, RNA was extracted with TRIzol (Invitrogen Life Technologies), RT of random hexamers was performed with an ABI PRISM 5700 unit (Applied Biosystems), and specific primer and probe sequences for target genes were used for qPCR amplification of total cDNA (TaqMan PDAR; Applied Biosystems). Relative quantitation of target cDNA was determined using a control value of 1; the sample cDNA content was expressed as the fold change from the control value. Differences in cDNA input were corrected by normalizing signals obtained with specific primers to ribosomal RNA; nonspecific amplification was excluded by performing RT-PCRs without target cDNA.

Flow Cytometry

Alloreactive T cells were generated by i.v. injection of 40×10⁶CFSE-labeled B6 spleen and lymph node cells into B6/DBA F1 recipients, a parent F1 MHC mismatch in which only donor cells respond. Splenocytes harvested after 3 days were incubated with CD4-PE, CD8-PE, CD25-PE, CD44-PE, CD62L-PE, PD-1-PE, ICOS-PE, and biotin-conjugated anti-H-2Kd and anti-H-2Dd mAb. Donor alloreactive T cells were identified by gating on H-2Kd and H-2Dd cells (FACSCalibur; BD Biosciences), and their proliferation was assessed by CFSE division profiles. For intracellular cytokine staining, splenocytes (3×10⁶/ml) were treated with Golgi-Stop (BD Pharmingen), stimulated for 4 h with PMA (3 ng/ml) and ionomycin (1 μM) in 24-well plates in complete medium (RPMI 1640, 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μM 2-ME), and stained with cell surface markers (CD4-PE or CD8-PE, biotin-conjugated H-2Kd or H-2Dd, followed by streptavidin- PerCP), fixed, and stained with IFN-allophycocyanin or IL-2-allophycocyanin after permeabilization (Perm-Wash buffer; BD Pharmingen).

In Vitro Cellular Assays

For propagation of bone marrow-derived DC, bone marrow cells harvested from the femurs and tibia were cultured for 5-7 days in 24-well plates (2×10⁶/well) in medium plus mouse GM-CSF (5 ng/ml) and IL-4 (10 ng/ml). One-way MLR cultures were performed in triplicate, using magnetic column-eluted splenic T cells (2×10⁵/well) as responders and gamma-irradiated (20 Gy) DC as stimulators. Cultures were maintained in complete medium for 72-96 h, and T cell proliferation was determined by BrdU incorporation or CFSE dilution profile. BrdU staining with a BrdU labeling kit (BD Pharmingen) was performed using the manufacturer's instructions. Cells were pulsed with BrdU, treated with FcR-blocking CD16/CD32 mAbs, stained with cell surface markers, fixed, permeabilized, treated with DNase/Triton X-100, stained with anti-BrdII mAb, and analyzed by flow cytometry.

ELISPOT

Immunospot assays for IFN- were performed by coating ELISPOT plates (BD Pharmingen) with anti-IFN-mAb, blocking, and addition of responder cells isolated from cardiac transplant recipients plus donor splenocytes or bone marrow-derived DC as stimulators; recipient splenocytes or DC were used as syngeneic controls. At 24 h, cells were discarded, and wells were washed, followed by biotinylated anti-IFN-mAb, streptavidin-HRP, and substrate. Spots were counted using an Immunospot Analyzer (Cellular Technology), and recipient anti-donor responder frequency was determined as the number of IFN-spot-forming cells per-10⁶splenocytes.

Western Blots

Grafts were sonicated in lysis buffer containing Triton X-100 and protease inhibitors, followed by centrifugation and assay of supernatant protein content. Proteins were reduced, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked, incubated with primary and HRP-linked secondary Abs, and, after the substrate reaction, analyzed using National Institutes of Health Image.

Transplantation

Intra-abdominal vascularized cardiac allografting was performed as previously described using 6- to 8-wk-old mice. Briefly, donor ascending aorta and pulmonary artery were anastomosed end-to-side to recipient infrarenal aorta and inferior vena cava, respectively. Graft survival was assessed twice daily by abdominal palpation; rejection was defined as total cessation of cardiac contraction and was confirmed by histology.

Immunopathology

Portions of harvested allografts were fixed in formalin, paraffin-embedded or snap-frozen, and analyzed by immunoperoxidase staining with mAbs and an Envision kit (DakoCytomation).

Statistics

Allograft survival was used to generate Kaplan-Meier survival curves, and comparison between groups was performed by log-rank analysis.

For further details regarding Example III, including references, see Tao et al., J. Immunol., 175:5774-5782, 2005, which is expressly incorporated herein in its entirety by reference.

(IV) BTLA-HVEM Effects in AsthmaRegulated Expression of PD-1 and BTLA During Acute Allergic Airway Inflammation.

We first determined the kinetics of lymphocyte accumulation and receptor expression in vivo by examining the cells recovered in the bronchoalveolar lavage (BAL) fluid. Mice were systemically sensitized and challenged with OVA. At 1, 3, 4, or 7 days following challenge groups of mice were euthanized and BAL performed. On 1 day following challenge, few CD4+ T cells were found in the BAL fluid. Significantly increased numbers of CD4 T cells appeared byday 3 which peaked byday 7 post-challenge (FIG. 25). We next examined the expression of PD-1 and BTLA on CD4 T cells recovered in the BAL fluid. Consistent with previous reports that PD-1 expression is induced on activated cells, we found that PD-1 expression gradually increased, being detectible onday 3 and reaching its maximum onday 7 following challenge. BTLA expression exhibited a reciprocal pattern with expression being greatest onday 3 and nearly undetectable by day 7 (FIG. 25).

Given the distinct patterns of expression of these receptors on BAL T cells, we next examined the phenotype of mice deficient in either BTLA or PD-1 in the acute allergic airway inflammation model (FIG. 26). Both BTLA-deficient and PD-1-deficient mice showed some increase in inflammatory cell recruitment compared to wild type mice (FIGS. 26aand26b). All genotypes had a mixed inflammatory cell infiltrate, although there was an increased percentage of neutrophils and eosinophils in the BTLA deficient mice (FIGS. 26aand26b). Examination of the lung tissues revealed a mild increase in the intensity of inflammatory infiltrates in PD-1 and BTLA-deficient animals compared to wild type controls. Thus, while PD-1 and BTLA have been reported as being potent inhibitory receptors, we found only a relatively mild increase in the inflammatory response following of acute allergic airway inflammation in the absence of either of these inhibitory receptors.

Delayed Expression of Ligands for BTLA and PD-1 in Acute Allergic Airway Inflammation.

Given the documented inhibitory activity of BTLA and PD-1 in vivo, we were surprised that the absence of either of these receptors did not have a greater effect on acute airway inflammation. We therefore speculated that the ligands for these receptors might not be expressed thereby not allowing this axis of regulation to be apparent. We examined the expression of mRNA for Herpes Virus Entry Mediator (HVEM), the ligand for BTLA, and PD-L1 and PD-L2, the ligands for PD-1, during an extended time course of airway inflammation (FIG. 27). Expression of HVEM message was nearly undetectable in the first four days of acute allergic airway inflammation following challenge but became apparent byday 7 and was maximal byday 10 and 15 (FIG. 25, upper panels). Likewise, the expression of PD-L1 was first detectable atday 2, but remained relatively low in expression until approximatelyday 7 today 10. Expression of PD-L2, a second ligand for PD-1, was maximum atday 4 following intranasal challenge, and declined subsequently. Interestingly, both HVEM and PD-L1 were detectable in RNA samples obtained from cultured murine tracheal epithelial cells (mTEC), suggesting that the source of ligand may be non-immune cells of the lung.

BTLA and PD-1 Limit the Duration of Acute Allergic Airway Inflammation.

Because the ligands for PD-1 and BTLA were maximally expressed in the second week following intranasal challenge, we next examined BAL cell numbers and compositions atday 10 andday 15 following intranasal challenge (FIG. 28). Wild type mice had completely resolved the inflammation, as evidenced by a low number of cells recovered in the BAL fluid and histology at

days

10 and 15 following challenge. In stark contrast, mice deficient in BTLA and PD-1 showed a persistent increase in BAL cells onday 10 following intranasal challenge. Furthermore, the composition of these cells in this fluid revealed a greater proportion of lymphocytes and eosinophils in comparison to the few cells in the wild type mice, which consisted predominantly of macrophages. Even onday 15, examination of BTLA-deficient mice revealed the continued presence increased numbers of lymphocytes and eosinophils: Direct histological examination of H and E stained sections also demonstrated persistent inflammation in the lungs of both PD-1 and BTLA-deficient mice at

days

10 and 15, whereas the wild type mice had complete resolution in this time frame. Thus, these receptors are critical for the normal resolution of airway inflammation.

T cell dependent immune responses are determined by the coordinate integration of signals derived from both cell:cell interactions and soluble mediators. We have recently described a novel role for CD28 signaling not only in the early priming phase but also in maintenance of the effector phase of allergic airway inflammation. These studies focused on an acute model, acting between

days

1 and 3. By contrast, the present results show that the inhibitory receptors BTLA and PD-1 exert a slight effect in attenuating the degree of acute inflammation but have a profound effect on the duration of inflammation, suggesting they act to terminate the immune response. We also observed a temporal regulation of expression of the ligands for these receptors during the course of the inflammatory response. Therefore, these data support that the regulated expression of inhibitory receptors on lymphocytes and their ligands in the lung are critical for the proper termination of the acute inflammatory response. In the absence of the inhibitory receptors on lymphocytes, the normally self limited acute inflammatory response progresses to a chronic infiltrate that persists for at least 15 days. We propose, based on these findings, that abnormalities in this axis could play a role in pathologic situations such as chronic persistent asthma and may represent novel targets for therapeutic intervention.

Methods (IV)Mice

BTLA-deficient mice were generated as previously described. PD-1 deficient mice were obtained from Tasuka Honjo (Kyoto University, Kyoto Japan). C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Me.). All mice were housed in specific pathogen free facilities at Washington University School of Medicine. All animal studies have been approved by the Washington University Animal Studies Committee.

Antibodies

Anti-BTLA antibody (Clone 6F7, mouse IgG1) was generated as previously described. Anti-PD-1 and anti-CD4 antibodies were purchased from Ebiosciences. Flow cytometric analysis was performed on a FacsCalibur cytometer using Cellquest software (Becton Dickinson Corporation). Analysis was performed using FloJo software.

RT-PCR

Total RNA was extracted from lung tissue of control or allergen challenged mice using Trizol (Invitrogen). Random primed cDNA was prepared using the Retroscript kit (Ambion). Specific primers for PDL1, PDL2 and HVEM were designed that spanned intronic sequences. Control primers amplify ribosomal S15 RNA and are provided with the Retroscript Kit.

Experimental Allergic Airway Inflammation

Mice were sensitized and challenged with Ovalbumin. Briefly, mice were injected i.p. with Ova adsorbed to alum on

days

0 and 7. Onday 14 they received an intranasal challenge of 50 μl of 1% Ova in the morning and afternoon. Samples were collected as previously described on the indicated days following inhaled challenge.

Preparation of murine tracheal epithelial cells: Primary mouse airway epithelial cells were cultured and differentiated using an established high fidelity model of the mouse airway. Briefly, epithelial cells were harvested from mouse tracheas of C57/BL6 strain mice (5-6 weeks old) using pronase digestion. Cells were purified by differential adherence of fibroblasts to yield a preparation composed of greater than 99% epithelial cells determined by expression of cytokeratin. Mouse tracheal epithelial cells (MTEC) were cultured in the presence of growth factor supplemented media on semi-permeable membranes (Transwell, Corning-Costar, Corning, N.Y.). Media was maintained in upper and lower chambers until the transmembrane resistance was greater than 1,000 Ohms⁻cm2 indicating tight junction formation. Media was then removed from the upper chamber to establish an air-liquid interface (ALI) condition used for epithelial cell differentiation. Cells were differentiated for at least 7 days at ALI to generate a multilayer model of the airway composed of ciliated, secretory, and basal airway epithelial cells. RNA was prepared fromday 7 ALI cultures using Trizol reagent.

(V) Effect of BTLA Loss of Function on Humoral Response

We immunized cohorts of mice with one injection of NP-Ficoll in alum and measured production of anti-NP antibodies of specific isotypes onday 14. For the isotypes IgM, IgG1, IgA, we found no specific changes in levels of anti-NP antibodies. For IgG2a or IgG2b, we found only slight increases in anti-NP antibodies in the BTLA−/− compared to wild type mice. However, for antibodies of the IgG3 isotype, which is primarily associated with T-independent responses, we found approximately a two-fold increased in anti-NP specific antibodies in BTLA−/− mice compared to wild type mice.

In addition, spontaneous germinal centers have been observed at a higher frequency than control in aging BTLA−/− mice.

(VI) BTLA Modulates Response to Viral Infection

Wildtype and BTLA knockout mice were infected with Sendai virus and monitored for three weeks. BTLA knockout mice maintained higher body weight and exhibited greater survival following infection with Sendai virus. Similar results were obtained using West Nile virus.