Extended Data Fig. 8: Avidities of CD4⁺ MAA/NeoAg-TCRs.

a, Avidity of de-orphanized TCRs with MAA or NeoAg specificity (n = 3 and n = 23 respectively). TCR-dependent CD137 upregulation was measured on TCR-transduced (mTRBC⁺) CD4⁺ cells upon culture with patient-derived EBV-LCLs pulsed with increasing concentrations of the cognate antigen (MAAs in the top panels, NeoAgs in bottom panels). Background activation measured on CD4⁺ T cells transduced with an irrelevant TCR was subtracted. Reactivity to DMSO-pulsed targets (0) and autologous melanoma (pdMel-CL) are reported on the left; for NeoAg-specific TCRs, the dashed lines report reactivity against wildtype peptides. A color legend depicts the different cognate antigens targeted by the deorphanized TCRs and specifies the number of TCRs able to recognize each cognate antigen and the patient the TCR belongs to. b, EC50 calculated from titration curves of MAA or NeoAg-specific TCRs (n = 3 and n = 23 respectively); note that high EC50 values correspond to low TCR avidities. Means with SD are reported, with TCR numbers corresponding to those reported in a. c, Expression levels of MAA or NeoAg transcripts (from bulk RNAseq of pdMel-CLs) from which the analyzed epitopes are generated, as a measure of cognate peptide abundance in tumor cells. Columns show means values with s.d. of values from n = 4 pdMel-CLs (symbols). d, Correlation between the avidity of antitumor CD4⁺TCRs (y axis) and target abundance (x axis), as measured by RNA expression of TCR-targeted genes detected in the autologous pdMel-CLs and expressed as transcripts per million mapped reads (TPM). Values for TCR avidity represent averages of EC50 data presented in b. The correlation is measured on de-orphanized antitumor CD4⁺TCR specificities with intratumoral T_Ex (top) or T_Reg (bottom) primary clusters, as showed in Fig. 3b, f. Significance of each linear regression is reported within each panel. Symbols: patients from whom TCRs were identified. Colors: cognate antigens of antitumor TCRs, as depicted in a.