Fig 2: Vß spectratyping of expanded TIL from first recurrence ascites. CD8+ TAL were stimulated with the HSDL1L25V peptide epitope or media alone and stained with antibodies to CD8, 4-1BB and each of 8 tubes of the Vß spectratyping kit. A. HSDL1L25V epitope stimulated CD8+ TAL were gated as 4-1BB+ and 4-1BB-, and the Vß spectratype plots of 4-1BB+ and 4-1BB- TAL are shown. The Vß of HSDL1L25V-reactive T cells found in peripheral blood are highlighted in red. B. Bars represent the percentage of all 4-1BB+ (black) or 4-1BB- (white) T cells expressing each Vß protein. Asterisks show the Vß of HSDL1L25V-specific T cell lines isolated from peripheral blood.

Fig 3: Validation of TRAPeS and comparison to other methods (A) Success rates for reconstruction of productive CDR3 in various CD8+ T cell data sets. Each line depicts the fraction of cells with a productive alpha or beta chain in a given data set with each one of the following methods—150 bp sequencing (black line), short paired-end data reconstructed using TRAPeS (red), TraCeR (turquoise), scTCRseq (gray), VDJPuzzle (dark blue) or Trinity (light blue). (B) Specificity of TRAPeS. Fraction of cells with identical CDR3 sequence between 150 bp data and the 25–30 bp data reconstructed either by TRAPeS, TraCeR, scTCRseq, VDJPuzzle or Trinity. This was calculated as the fraction out of cells with a productive chain in both 150 and 25–30 bp data. (C) Sensitivity of TRAPeS. Same as b, except the fraction of cells is calculated out of the total number of cells that had a successful reconstruction using 150 bp sequencing only. (D) Single cell RNA-sequencing captures a variety of clonal responses. Bars represent the Gini coefficient of each human CD8+ T cell data set. The Gini coefficient can range from zero (a complete heterogeneous population) to one (a complete homogenous population). Pie charts represent the distribution of clones in each population, n represents the number of cells with a successful reconstruction of both alpha and beta chains.

Fig 4: Isolation of highly enriched, mutation-reactive T cell lines. For each panel, examples of T cell mini-lines 1F3 and 4C5 are shown. A. Mini-lines identified in the screen were REP expanded and stimulated in duplicate wells with peptides encompassing each tumor-specific mutation (N = 37 mutations) in ELISPOT assays. Bars represent the number of IFN-? spot forming cells in each well. X-axis represents each individual mutation peptide pool with the reactive peptide indicated. B. FACS sorting plots for T cell line that proved reactive to a single mutation. CD8 (X-axis) and 4-1BB (Y-axis) double positive cells were FACS sorted. C. IFN-? ELISPOT assay of T cell lines responding to individual minimal peptides contained within the mutation specific peptide pool. Bars represent the number of spot forming cells per well. Amino acids sequences of each peptide are shown on the X-axis. D. Flow cytometry plots of T cell lines stained with antibodies towards Vß regions (X-axis) and 4-1BB (Y-axis). T cell lines were stained with the full complement of Vß antibodies, and only the positive Vß stains are shown (Vß 13.1 for well 1F3; and Vß 1 for well 4C5). E. IFN-? ELISPOT assay of T cell cross-reactivity to WT versions of minimal epitopes. Lines represent the number of IFN-? spot forming cells responding to 10-fold titrations of cognate mutated minimal epitope (squares) and the corresponding wild type peptide (triangles).

Fig 5: 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).