T cell antigen discovery via trogocytosis

Abstract

T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide-major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide-MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy.

Fig. 1 |. Target cell trogocytosis is antigen specific.

a, Schematic of cognate TCR-antigen pairs. Red/blue coloring indicates cognate TCR-antigen pairs. F5-TCR (red) is paired with the SCT expressing melanoma antigen MARTI peptide, and 1G4-TCR (blue) is paired with the SCT expressing cancer-testis antigen NYESO1 peptide. Yellow stars represent membrane proteins. b-d, FACS plots of biotinylated F5-Jurkat T cells (b) after co-incubation with cognate MART1-K562 target cells (ZsGreen⁺) expressing SCT of HLA-A2/MART1_{26–35(A27L)} or noncognate NYEOS1-K562 target cells expressing SCT of HLA-A2/NYESO1_{157–165(C165V)}. c, Transfer of biotinylated membrane proteins from Jurkat T cells (LNGFR+) to K562 target cells (ZsGreen⁺). d, Antigen-specific transfer of LNGFR and TCR from F5-Jurkat T cells to cognate MART1-K562 target cells. e, FACS plot overlay of either NYESO1-K562 or MART1-K562 cells after co-incubation with 1G4-Jurkat or F5-Jurkat T cells. SCT-K562 cells (ZsGreen⁺) were assessed for acquisition of TCR using an anti-human TCRα-β (TCRαβ) antibody. Data are representative of three independent experiments.

Fig. 2 |. Trogocytosis is titratable and augmented by the presence of CD8.

a-c, Comparison of trogocytosis capability of (a) F5-Jurkat or 1G4-Jurkat cells with HLA-A2 expressing K562 cells (A2-K562) loaded with different doses of MART1 (ELAGIGILTV; top) or NYESO1 (SLLMWITQV; bottom) heteroclitic peptide; (b) F5-Jurkat or 1G4-Jurkat cells with A2-K562 cells loaded with different MART1 (top) or NYESO1 (bottom) peptide variants; and (c) CD8⁺ or CD8^- F5-Jurkat or 1G4-Jurkat cells with MART1-K562 or NYESO1-K562 cells (n = 3). Statistical analysis of quantification was performed using unpaired two-tailed Student’s t-test. Data are presented as mean± s.e.m. and are representative of two independent experiments.

Fig. 3 |. Target cell trogocytosis is sufficiently sensitive to identify one cognate-antigen-expressing target cell from 10,000 non-antigen-expressing cells.

a,b, Schematic and representative FACS plots for the mixture of cognate NYESO1-K562 target cells and noncognate A2-K562 cells (a) and the mixture of cognate MART1-K562 target cells to noncognate A2-K562 cells (b) co-incubated with either F5-Jurkat (top) or 1G4-Jurkat (bottom) cells. An overlay of the noncognate A2-K562 cells (gray) provides a clear comparison of trogocytosis in cognate versus noncognate K562 cells. Data are representative of three independent experiments.

Fig. 4 |. Target cell trogocytosis validates ligands for public F5- and 1G4-TCR.

a, Schematic outline of trogocytosis used in the context of an A2-retricted library containing 12,055 epitopes. b, Histograms representing the mean fluorescence of second-round-sorted trogocytosis⁺ SCT-K562 cells after a validating co-incubation with F5-Jurkat or 1G4-Jurkat. SCT-K562 cells (eGFP⁺) were assessed for acquisition of TCR with an anti-mouse TCR (muTCR) antibody. Data are representative of two independent experiments. c, Representative FACS plots of 1G4-TCR or F5-TCR dextramer binding by sorted trogocytosis⁺ SCT-K562 cells (n = 1). d, Identification of enriched peptides after two rounds of trogocytosis selection by NGS. Rank average represents the ranking of enriched peptides, calculated based on the abundance of each peptide among all peptides, from experimental triplicates or quadruplicates

Fig. 5 |. Target cell trogocytosis identifies the cognate ligand for a neoantigen-specific, subject-derived TCR.

a, Identification of enriched peptides after two rounds of trogocytosis selection by NGS. Rank average represents the ranking of enriched peptides, calculated based on the abundance of each peptide among all peptides, from experimental quadruplicates. b, Validation of TCR-neoantigen pairing via trogocytosis. Data are representative of two independent experiments. c, Cytotoxicity of neo-TCR T cells against mutUSP7-K562 or MART1-K562 cells in various effector-to-target (E/T) ratios (presented as percentage specific lysis). Data are presented as mean± s.e.m. and are representative of two independent experiments.