Extended Data Fig. 6: Antigen specificity of CD4⁺ tumor-reactive TCRs.

a, Antigen specificity screening of 76 CD4⁺TCRs with direct or indirect tumor recognition. Upregulation of CD137 was assessed by flow-cytometry on CD4⁺ T cells transduced with previously identified tumor-specific or APC-tumor antigen reactive TCRs. Antigen recognition was tested upon culture with autologous EBV-LCLs pulsed with pools of peptides corresponding to immunogenic neoantigens (NeoAgs) predicted to bind patients’ HLA class I or II restrictions or detected through Mass-spectrometry (MS) within the HLA class II immunopeptidome (see Supplementary Table 6), known melanoma associated antigens (MAAs) or immunogenic viral epitopes (CEF pools). Reactivity was also assessed against an irrelevant long peptide (Ova II) or in the presence of polyclonal stimulators (PMA/ionomycin) as negative and positive controls, respectively. Background, assessed using DMSO-pulsed target cells, was subtracted from each condition. Colored dots – confirmed antigen-reactive TCRs, colored based on highest reactivity against a particular antigen, as per the legend, compared to the other tested antigens; white dots –TCRs reactive against an antigen which was not the highest of the panel of antigens tested, and hence considered a cross-reactive response; grey dots - negative responses. b, Deconvolution of antigen specificity of TCRs reactive to NeoAg-peptide pools. Colored dots indicate the deconvoluted cognate antigens, with antigens corresponding to colors represented in panel c. c, Distribution of antigen specificities of tumor reactive CD4⁺TCRs per patient successfully deorphanized after screening. Colors denote the distinct peptides recognized by individual TCRs.