Figure 5. CD39⁺, CXCL13⁺, high-frquency clonotype signature also identifies CD4⁺ neoantigen-reactive T cells.

(A) Cell-surface protein-based tSNE plot of TIL from patient 1. Light blue dots indicate CD4⁺ T cells, red dots indicate EGFR-reactive CD4⁺ T cells, large blue dots indicate ATG4C-reactive CD4⁺ T cells. (B) Volcano plot showing the differentially expressed genes between the orange cluster and other CD4⁺ T-cells shown in (A). (C, G, L and Q) tSNE plots showing CD4⁺ cells by blue dots and CD39 protein-, CXCL13-, TIGIT-, and FOXP3-expressing cells by red dots. Frequency of cells in total CD4⁺ cells is shown by %. (D) Cell number of the candidate clonotypes within the orange cluster shown in (A), (red bars: neoantigen reactive, blue bars: non-reactive). (E, J and O) CXCL13 expressions in T-cell clones analyzed. (F, K and P) Neoantigen reactivities of TCR-transduced autologous T-cells were examined by co-culturing these cells with autologous dendritic cells pulsed with cognate mutated or wildtype 25-mer peptides. After a 20-hour co-culture, T-cell activation was examined by measuring IFNγ in the cell culture sup by ELISA. (H and M) clonotype frequency of TCRs analyzed within CD3⁺ TIL (light blue CD39⁻, red: CD39⁺ CXCL13⁺ and neoantigen-reactive, dark blue CD39⁺ CXCL13⁺ but not neoantigen-reactive). (I and N) Frequencies of candidate clonotypes in PBL measured by TRBV deep sequencing. (R) Expression of CD39 protein and CXCL13 in the four clonotypes tested for neoantigen-reactivity. All the tSNE plots in Figure 5 are cell surface protein-based.