Extended Data Fig. 3. T cell cloning using SIN3A* tumor antigen and graphical summary of TCC isolation, response of TCC88 to ps-SCL, and depiction of database screening for antigen discovery approach (see also Figs. 1 and 2 and Extended Data Fig. 4).

a, Gating strategy for isolation of TCCs from TILs responding to SIN3A* peptide. 60 replicate wells of CFSE-labeled TILs were pooled after 10 days stimulation with SIN3A* tumor antigen. Cells were stained using live-dead, CD3, CD4 and were next single-cell sorted. b, Schematic depiction of the steps for discovering target antigens for a TCC. TCCs are tested with positional scanning combinatorial peptide libraries (200 mixtures of decamer peptides) presented by BLS cells expressing a single HLA-DR molecule, which had been previously established as the restriction element for each TCC. Proliferative or cytokine responses of TCC to each of the decamer mixtures, i.e. 200 compounds are measured. Data are then used to create a scoring matrix. Antigens with predicted high stimulatory potential are identified by searches of several databases including human proteome, autologous tumor transcriptome and peptidome, pathogenic bacteria and viruses as well as human gut microbiota. Candidate peptides originating from pathogenic bacteria and gut microbiota strongly stimulated TCC88. Further, these peptides were used to stimulate other tumor-reactive TCCs as well as bulk TILs and peripheral memory CD4⁺ T cells. The schematics in panel b were created using BioRender (https://biorender.com).