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.2021 Mar 23:12:644520.
doi: 10.3389/fimmu.2021.644520. eCollection 2021.

Identification of NY-ESO-1157-165 Specific Murine T Cell Receptors With Distinct Recognition Pattern for Tumor Immunotherapy

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Identification of NY-ESO-1157-165 Specific Murine T Cell Receptors With Distinct Recognition Pattern for Tumor Immunotherapy

Helin Zhang et al. Front Immunol..

Abstract

New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a promising target for T-cell receptor-engineered T cell (TCR-T) therapy, and targeting the human leukocyte antigen (HLA)-A2 restricted NY-ESO-1157-165 epitope has yielded remarkable clinical benefits in the treatment of multiple advanced malignancies. Herein, we report the identification of two NY-ESO-1157-165 epitope-specific murine TCRs obtained from HLA-A*0201 transgenic mice. NY-ESO-1157-165 specific TCRs were isolated after vaccinating HLA-A2 transgenic mice with epitope peptides. HZ6 and HZ8 TCRs could specifically bind to NY-ESO-1157-165/HLA-A2 and were capable of cytokine secretion with engineered Jurkat T cells and primary T cells upon recognition with K562 target cells expressing the single-chain trimer (SCT) of NY-ESO-1157-165/HLA-A2. The reactivity profiles of the HZ6 and HZ8 TCRs were found to be distinct from one another when co-cultured with K562 target cells carrying alanine-substituted NY-ESO-1157-165 SCTs. The binding characterization revealed that the recognition pattern of the HZ6 TCR to NY-ESO-1157-165/HLA-A2 was substantially different from the widely used 1G4 TCR. These findings would broaden the understanding of immunogenicity of the NY-ESO-1157-165, and the two identified TCRs may serve as promising candidates for the future development of TCR-T therapy for tumors.

Keywords: New York esophageal squamous cell carcinoma 1; T cell receptor-engineered-T cell; T-cell receptor; human leukocyte antigen-A*0201; tumor.

Copyright © 2021 Zhang, Sun, Wang, Zeng, Cao, Han, Tan and Gao.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Single-cell isolation and sequencing of NY-ESO-1157−165-specific T cells from HLA-A*0201 transgenic mice.(A) Schematic of rapid cloning and functional verification strategies.(B,C) NY-ESO-1157−165/HLA-A2 tetramer-positive CD8+ T cells from HLA-A*0201 transgenic mice, Mus-2(B) and Mus-177(C) from 2 independent experiments, immunized with NY-ESO-1157−165, or phosphate-buffered saline (PBS) (Mus-PBS-1 and Mus-PBS-2) were analyzed with flow cytometry. Thirty-six and 48 NY-ESO-1157−165/HLA-A2 tetramer-positive CD8+ T cells from Mus-2 and Mus-177, respectively, were single-cell sorted.(D,E) TCR repertoire analysis of cloned NY-ESO-1157−165/HLA-A2 tetramer-positive CD8+ T cells from Mus-2(D) and Mus-177(E). The number in the brackets indicates the number and frequency of T cells expressing the same TRAV or TRBV in the total number of TCRα or TCRβ clones from single cells.
Figure 2
Figure 2
Specific binding of HZ6 and HZ8 TCRs with NY-ESO-1157−165/HLA-A2 in HEK-293T cells system.(A) Verification strategy for the detection of TCR binding specificity. Cloned TCRs and human CD3-CD8 complex were co-transfected into 293T cells. Binding of NY-ESO-1157−165/HLA-A2 tetramer-PE to TCRs expressed on 293T cells was analyzed with flow cytometry.(B) Binding of NY-ESO-1157−165/HLA-A2 tetramer with CD3-expressing 293T cells co-transfected with TCR and human CD3-CD8 complex constructs. Detection with HPV-E6/HLA-A2 tetramer-PE was enrolled as negative control to verify the binding specificity of HZ6 and HZ8 TCRs (lower flow chart).
Figure 3
Figure 3
Reactivity of HZ6 and HZ8 TCR engineered Jurkat T cells to NY-ESO-1157−165 WT-SCT-K562 or alanine-substituted pMHC-SCTs (SCT-K562 mutants) target cells.(A) Schematic of the functional evaluation assay of the TCRs in Jurkat T cells. TCR engineered Jurkat T cells co-transduced with human CD3-CD8 construct were co-cultured with K562 target cells that were transduced with NY-ESO-1157−165/HLA-A2 single-chain trimer (SCT-K562 cells). The reactivity of TCR engineered Jurkat T cells was evaluated by detection of the IL-2 secretion after co-culturing of effector cells and target cells.(B) Flow cytometry histogram comparison of NY-ESO-1157−165/HLA-A2 expression on the surface of K562 cells transduced with NY-ESO-1157−165 WT-SCT (WT-SCT-K562) and K562 cells without transduction.(C) Flow cytometry analysis of TCR expression on Jurkat T cells transduced with TCR and CD3-CD8 before and after14 daysin vitro culture in the presence of 2 μg/ml puromycin.(D) ELISA measuring secretion of IL-2 from untransduced, HZ6-CD8, and HZ8-CD8 Jurkat effector cells following 48 h co-incubation with WT-SCT-K562 target cells or K562 cells as control.(E) Alanine scanning approach for HZ6 and HZ8 TCR in Jurkat T cells. HZ6-CD8-Jurkat cells or HZ8-CD8-Jurkat cells were co-cultured with WT-SCT-K562 or alanine-substituted pMHC-SCTs K562 (SCT-K562 mutants) cells. IL-2 concentrations in the supernatant were measured by ELISA. For(D,E), the data is a representative of 3 independent experiments with two technical replicates. Means ± SD for a representative experiment are shown.
Figure 4
Figure 4
Function evaluation of HZ6 TCR-engineered primary T cells.(A) Schematic of the function evaluation assay in primary T cells for HLA-A2-restricted and NY-ESO-1157−165 specific TCRs. Primary T cells transduced TCRs were used as effector cells, and K562 cells transduced NY-ESO-1157−165 SCT were utilized as target cells. The reactivity of HZ6 TCR was evaluated by detecting IFN-γ secretion after co-culturing of effector cells and target cells.(B,C) Detection of IFN-γ-producing HZ6 TCR transduced primary T cells upon stimulating with SCT-K562 cells using ELISPOT assay. Primary T cells of 2 donors, D1 and D2, were enrolled in this experiment.(B) The spot forming cells (SFCs) were shown and the green number on the lower right of each well was SFCs number per 2 × 104 T cells.(C) Statistical analysis of IFN-γ secreting HZ6 TCR transduced primary T cells stimulated with NY-ESO-1157−165 SCT-K562 cells or with medium alone as mock based on the results in(B).(D) ELISA measuring secretion of IFN-γ from HZ6 TCR transduced or non-transduced primary T cells following 48 h co-incubation with WT-SCT-K562 or control K562 cells. Means ± SD for 2 technical replicates are shown.(E) Alanine scanning approach for HZ6 TCR in primary T cells from 2 donors (D1 and D2). HZ6-D1 T cells or HZ6-D2 T cells were co-cultured with WT-SCT-K562 or alanine-substituted pMHC-SCTs-K562 (SCT-K562 mutants) cells. IFN-γ concentrations in the supernatant were measured by ELISA. Means ± SD for 2 technical replicates are shown.
Figure 5
Figure 5
SPR characterizaiton of the binding between HZ6 TCR and WT or alanine substituted NY-ESO-1157−165/HLA-A2 pMHCs.(A) The pMHCs were immobilized on the chip and serial dilutions of HZ6 TCR were then flowed through. The figures represent measurements at equilibrium with serial 2-fold dilutions of HZ6 TCR with concentrations ranging from 50 to 0.39 μM.(B) The graph shows binding affinity as a percentage relative to the binding affinity to the WT peptide.
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