.2014 Sep 26;345(6204):1623-1627.

doi: 10.1126/science.1255904. Epub 2014 Sep 11.

Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4

Hye Sun Kuehn^#¹, Weiming Ouyang^#², Bernice Lo^#^{3 4}, Elissa K Deenick^{5 6}, Julie E Niemela¹, Danielle T Avery⁵, Jean-Nicolas Schickel⁷, Dat Q Tran⁸, Jennifer Stoddard¹, Yu Zhang^{4 9}, David M Frucht², Bogdan Dumitriu¹⁰, Phillip Scheinberg¹⁰, Les R Folio¹¹, Cathleen A Frein¹², Susan Price^{3 4}, Christopher Koh¹³, Theo Heller¹³, Christine M Seroogy¹⁴, Anna Huttenlocher^{14 15}, V Koneti Rao^{3 4}, Helen C Su^{4 9}, David Kleiner¹⁶, Luigi D Notarangelo¹⁷, Yajesh Rampertaap¹⁸, Kenneth N Olivier¹⁸, Joshua McElwee¹⁹, Jason Hughes¹⁹, Stefania Pittaluga¹⁶, Joao B Oliveira²⁰, Eric Meffre⁷, Thomas A Fleisher¹, Steven M Holland^{4 18}, Michael J Lenardo^{3 4}, Stuart G Tangye^{5 6}, Gulbu Uzel¹⁸

Affiliations

¹ Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
² Laboratory of Cell Biology, Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892, USA.
³ Molecular Development of the Immune System Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
⁴ NIAID Clinical Genomics Program, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
⁵ Immunology and Immunodeficiency Group, Immunology Division, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia.
⁶ St. Vincent's Clinical School Faculty of Medicine, University of New South Wales, Sydney, NSW 2010, Australia.
⁷ Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06511, USA.
⁸ Department of Pediatrics, University of Texas Medical School, Houston, TX 77030, USA.
⁹ Immunological Diseases Unit, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
¹⁰ Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, MD 20892, USA.
¹¹ Radiology and Imaging and Sciences, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
¹² Clinical Research Directorate, Clinical Monitoring Research Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
¹³ Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892, USA.
¹⁴ Department of Pediatrics, University of Wisconsin, Madison, WI 53706, USA.
¹⁵ Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI 53706, USA.
¹⁶ Laboratory of Pathology, National Cancer Institute, Bethesda, MD 20892, USA.
¹⁷ Division of Immunology and Manton Center for Orphan Disease Research, Children's Hospital, Harvard Medical School, Boston, MA 10217, USA.
¹⁸ Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
¹⁹ Merck Research Laboratories, Merck & Co., Boston, MA 02130, USA.
²⁰ Instituto de Medicina Integral Prof. Fernando Figueira-IMIP, 50070 Recife-PE, Brazil.

^# Contributed equally.

PMID:25213377
PMCID: PMC4371526
DOI: 10.1126/science.1255904

Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4

Hye Sun Kuehn et al. Science.2014.

.2014 Sep 26;345(6204):1623-1627.

doi: 10.1126/science.1255904. Epub 2014 Sep 11.

Authors

Affiliations

¹ Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
² Laboratory of Cell Biology, Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892, USA.
³ Molecular Development of the Immune System Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
⁴ NIAID Clinical Genomics Program, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
⁵ Immunology and Immunodeficiency Group, Immunology Division, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia.
⁶ St. Vincent's Clinical School Faculty of Medicine, University of New South Wales, Sydney, NSW 2010, Australia.
⁷ Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06511, USA.
⁸ Department of Pediatrics, University of Texas Medical School, Houston, TX 77030, USA.
⁹ Immunological Diseases Unit, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
¹⁰ Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, MD 20892, USA.
¹¹ Radiology and Imaging and Sciences, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
¹² Clinical Research Directorate, Clinical Monitoring Research Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
¹³ Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892, USA.
¹⁴ Department of Pediatrics, University of Wisconsin, Madison, WI 53706, USA.
¹⁵ Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI 53706, USA.
¹⁶ Laboratory of Pathology, National Cancer Institute, Bethesda, MD 20892, USA.
¹⁷ Division of Immunology and Manton Center for Orphan Disease Research, Children's Hospital, Harvard Medical School, Boston, MA 10217, USA.
¹⁸ Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
¹⁹ Merck Research Laboratories, Merck & Co., Boston, MA 02130, USA.
²⁰ Instituto de Medicina Integral Prof. Fernando Figueira-IMIP, 50070 Recife-PE, Brazil.

^# Contributed equally.

PMID:25213377
PMCID: PMC4371526
DOI: 10.1126/science.1255904

Abstract

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory receptor found on immune cells. The consequences of mutations in CTLA4 in humans are unknown. We identified germline heterozygous mutations in CTLA4 in subjects with severe immune dysregulation from four unrelated families. Whereas Ctla4 heterozygous mice have no obvious phenotype, human CTLA4 haploinsufficiency caused dysregulation of FoxP3(+) regulatory T (Treg) cells, hyperactivation of effector T cells, and lymphocytic infiltration of target organs. Patients also exhibited progressive loss of circulating B cells, associated with an increase of predominantly autoreactive CD21(lo) B cells and accumulation of B cells in nonlymphoid organs. Inherited human CTLA4 haploinsufficiency demonstrates a critical quantitative role for CTLA-4 in governing T and B lymphocyte homeostasis.

PubMed Disclaimer

Figures

**Fig. 1. Clinical phenotype and pedigree of the patients**
(A) Top: Computed tomography images of lung and brain from patient A.II.1. Bottom: Histological section (magnification 20×) from a duodenal biopsy from a healthy donor (HD) and patient A.II.1 stained for CD3 (brown cells), showing an increased number of transepithelial T cells within the villi. (B) Flow cytometric analyses of CD4⁺ cells or total lymphocytes stained for the indicated surface markers from a healthy donor and patient A.I.1. Data showing decreased CD45RA⁺CD62L⁺ naïve CD4⁺ T cells are representative of three patients (A.I.1, A.II.1, and B.I.1). Programmed cell death–1 (PD1) expression data shown are representative of five patients (A.I.1, A.II.1, B.I.1, C.II.1, and D.II.1) and three healthy donors. Data showing decreased circulating B cells are representative of two patients (A.I.1 and A.II.1). (C) Mutations in patient alleles displayed on a schematic of the four exons ofCTLA4, pedigrees, and phenotype summary highlighting organs (gray) with inflammatory infiltrates and autoimmune cytopenias for affected family members.TM, transmembrane domain. (D) Protein and mRNA expression of CTLA-4 in T_reg cells. Left: Levels of CTLA-4 expression in T_reg cells (CD4⁺CD25⁺FoxP3⁺) were assessed by intracellular staining. The numbers in the upper right corner depict mean fluorescence intensity (MFI) of anti-CD152 (CTLA-4) staining. Dotted line indicates the peak of CTLA-4 expression in a healthy donor. Data shown are representative of three experiments. Right: Levels ofCTLA4 mRNA in T_reg cells (CD4⁺CD25⁺CD127^lo) sorted from seven different healthy donors and four patients were measured by real-time PCR using the probe forCTLA4 transcript variant 1 (full length) and normalized to GAPDH. Data are means of replicates from six experiments. For relative gene expression, all data were normalized to the same HD.The horizontal lines indicate mean values from healthy donors or patients.

**Fig. 2. Abnormal T_reg cell phenotype and function in patients**
(A) Flow cytometric analysis of FoxP3 and CD25 in CD4⁺ T cells from healthy donor (HD) and patients. (B) Mean fluorescence intensity of FoxP3 and CD25 in CD4⁺ FoxP3⁺ T cells from healthy donors and patients. Data are means T SEM of replicates of indicated patient [A.I.1 (N = 7), A.II.1 (N = 1), B.I.1 (N = 2), C.II.1 (N = 2), D.II.1 (N = 4)] and 10 healthy donors.TheN values represent number of replicates from each patient. ***P = 0.0007 (Mann-Whitney test). Bar graph: Percentage of CD25⁺ and CD25^– cells among CD4⁺FoxP3⁺ in 10 healthy donors and five patients. (C) Autologous and heterologous suppressive activities of T_reg cells from five healthy donors and four patients (A.I.1, B.I.1, C.II.1, and D.II.1). The horizontal lines indicate the mean values. Data are from three experiments, with each indicated patient paired with one or two healthy donors. *P = 0.0239, **P = 0.0037 (pairedt test).

**Fig. 3. CTLA-4 deficiency leads to a Tcell hyperproliferative phenotype**
(A) Proliferation of PBMCs obtained from healthy donors or patients. PBMCs were stained with CellTrace violet and stimulated with (black line) or without (gray) anti-CD3 and anti-CD28. Numbers indicate percentage of cells having undergone at least one cellular division, assessed by dye dilution. Graphs at right indicate percentages of cells divided from healthy donor and patient in pairs. Data are shown as in (A) and are from five experiments with a healthy donor and indicated patient tested in pairs. *P = 0.0107, ***P = 0.0005 (pairedt test). (B) Proliferation of PBMCs withCTLA4 knockdown. Healthy donor PBMCs were transfected with control (Con) or CTLA4 siRNA. Knockdown efficiency was evaluated using real-time PCR (left). Data are means ± SEM from three experiments, each with a different healthy donor. Cells were then assessed for proliferation and CD25 expression. Data shown are representative of three experiments with three healthy donors tested. (C) Reconstitution of wild-type CTLA-4 in patient cells. Patient D.II.1 PBMCs were transfected with control vector (pDsRed-N1) or CTLA-4–DsRed-N1 vector. Proliferation of DsRed⁺ CD4 and CD8 patient Tcells. Data shown are representative of three experiments with a total of three patients (B.I.1, C.II.1, D.II.1). (D) Effect of in vitro treatment with CTLA-4–Ig on proliferation of PBMCs. Patient D.III.1 PBMCs were stimulated with anti-CD3 in the presence (+) or absence (–) of CTLA-4–Ig. Data shown are representative of three experiments with a total of three patients (B.I.1, D.II.1, and D.III.1). Percentages of cells having undergone at least one cellular division are indicated in each histogram. Gray peak shows unstimulated cells.

**Fig. 4. Abnormal B cell phenotype in patients**
(A) Percentages of B cells for patients A.I.1, A.II.1, B.I.1, C.II.1, D.II.1, and D.III.1; horizontal lines indicate means ± SD. **P = 0.0047 (Mann-Whitney test). (B) Absolute B cell numbers in blood for the indicated patients. Dotted lines indicate lower limit of normal range from the healthy donors. (C) B cells were labeled with antibodies to CD19, CD20, CD27, and CD10. Top panel: Proportions of B cells corresponding to transitional (CD10⁺CD27^–), naïve (CD10^–CD27^–), or memory (CD10^–CD27⁺) subsets. Middle and bottom panels: Abnormal accumulation of CD19^hiCD21^lo B cells within the population of CD20⁺CD10^–CD27^– B cells (middle) and CD20⁺CD27^– B cells (bottom) in CTLA-4–deficient patients compared to a healthy donor. (D) Enriched CD19⁺ cells (HD1 and A.I.1) or total PBMCs (HD2, HD3, A.II.1, C.II.1, and D.II.1) were stimulated with F(ab′)₂ specific for anti-IgM, IL-4, and CD40 ligand. CD19⁺ cell proliferation was assessed by dye dilution.

See this image and copyright information in PMC

Comment in

Immunology. Autoimmunity by haploinsufficiency.
Rieux-Laucat F, Casanova JL.Rieux-Laucat F, et al.Science. 2014 Sep 26;345(6204):1560-1. doi: 10.1126/science.1260791.Science. 2014.PMID:25258064No abstract available.
Balance and imbalance in the immune system: life on the edge.
Topalian SL, Sharpe AH.Topalian SL, et al.Immunity. 2014 Nov 20;41(5):682-4. doi: 10.1016/j.immuni.2014.11.005. Epub 2014 Nov 20.Immunity. 2014.PMID:25517610Free PMC article.

Cited by

The ABACHAI clinical trial protocol: Safety and efficacy of abatacept (s.c.) in patients with CTLA-4 insufficiency or LRBA deficiency: A non controlled phase 2 clinical trial.
Krausz M, Uhlmann A, Rump IC, Ihorst G, Goldacker S, Sogkas G, Posadas-Cantera S, Schmidt R, Feißt M, Alsina L, Dybedal I, Recher M, Warnatz K, Grimbacher B.Krausz M, et al.Contemp Clin Trials Commun. 2022 Sep 24;30:101008. doi: 10.1016/j.conctc.2022.101008. eCollection 2022 Dec.Contemp Clin Trials Commun. 2022.PMID:36262801Free PMC article.
Clinical genetics in 2014: New monogenic diseases span the immunological disease continuum.
Savic S, McDermott MF.Savic S, et al.Nat Rev Rheumatol. 2015 Feb;11(2):67-8. doi: 10.1038/nrrheum.2014.215. Epub 2014 Dec 23.Nat Rev Rheumatol. 2015.PMID:25536483
Granulomatous interstitial nephritis with CTLA-4 haploinsufficiency: a case report.
Kohatsu K, Suzuki T, Takimoto M, Matsui K, Hashiguchi A, Koike J, Shirai S.Kohatsu K, et al.BMC Nephrol. 2022 Nov 16;23(1):367. doi: 10.1186/s12882-022-02999-x.BMC Nephrol. 2022.PMID:36384506Free PMC article.
Genetic Testing in Patients with Autoimmune Lymphoproliferative Syndrome: Experience of 802 Patients at Cincinnati Children's Hospital Medical Center.
Xu X, Denton J, Wu Y, Liu J, Guan Q, Dawson DB, Bleesing J, Zhang W.Xu X, et al.J Clin Immunol. 2024 Jul 26;44(7):166. doi: 10.1007/s10875-024-01772-z.J Clin Immunol. 2024.PMID:39060684Free PMC article.
A Rare Central Nervous System Involvement Due to CTLA-4 Gene Defect.
Rovshanov S, Göçmen R, Barişta İ, Çağdaş D, Üner A, Çilingir V, Filik İT, Tan Ç, Aytekin ES, Tezcan İ, Özen PA, Tuncer A.Rovshanov S, et al.Noro Psikiyatr Ars. 2022 Aug 15;59(3):248-252. doi: 10.29399/npa.27900. eCollection 2022.Noro Psikiyatr Ars. 2022.PMID:36160072Free PMC article.

See all "Cited by" articles

References

1. Bluestone JA, et al. Nat. Rev. Immunol. 2010;10:797–803. - PubMed
1. Goodnow CC. Cell. 2007;130:25–35. - PubMed
1. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Cell. 2008;133:775–787. - PubMed
1. Josefowicz SZ, Lu LF, Rudensky AY. Annu. Rev. Immunol. 2012;30:531–564. - PMC - PubMed
1. Benoist C, Mathis D. Cold Spring Harb. Perspect. Biol. 2012;4:a007021. - PMC - PubMed

Publication types

Actions
Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Movatterモバイル変換

Account

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Full text links

Actions

Share

Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4

Affiliations

Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4

Authors

Affiliations

Abstract

Figures

Comment in

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Associated data

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Molecular Biology Databases