Fig. 2
Identification of neo-epitope specific TILs. a–c Representative example of neo-epitope (ZCCHC11 P1265H )-specific TILs in patient CTE-0015. a IFNγ ELISpot showing the average of triplicate + SD (PHA: phytohemagglutinin; No Ag: no peptide) and peptide-MHC multimer staining. All other neo-epitopes are described in Supplementary Fig. 3 and Table 2. b Intracellular cytokine staining showing the frequency of viable IFNγ, TNFα, and IL-2 cytokine-producing TILs. c Proportion of patients with neo-epitope reactive TILs. d Cumulative analysis (SPICE) showing the functional composition (cytokine profiles) of neo-epitope specific TILs (mean ± SD). e Repertoire of neo-epitopes in PBLs and TILs in 19 patients. Each colored square represents one neo-epitope for which T-cell reactivity was validated either exclusively in PBLs (blue), exclusively in TILs (red), in both compartments (orange) or in none of them (white). For 5 out of 19 patients no tumor samples were available for TIL generation (black squares). f Neo-epitope-specific TILs from patient CTE-0011 were FACS sorted with HLA-A1101-SEPT9 R289H multimer. Sorted cells (left) underwent TCR sequencing, as well as autologous bulk PBLs, bulk tumor and bulk TIL cultures. The Manhattan plot reports the V/J recombination of the T-cell receptor β (TCRβ) of SEPT9 R289H -specific T cells: V and J segments are represented according to chromosomal location on the x and y-axis, respectively. On the right, the distribution of the TCR frequencies is shown for PBL, tumor and TILs. Dominant SEPT9 R289H -specific TCRβ are identified by red bars (and arrows) in tumor and TILS, whereas no SEPT9 R289H -specific TCRs was identified in PBLs. gTRIM26 G497W -specific circulating T cells from patient CTE-0010 were isolated based on CD137 upregulation and immediately processed for TCR-sequencing analysis (center). TCR repertoire analysis was performed in parallel on matched bulk PBLs, bulk tumor, and bulk TIL cultures (right). The most frequent shared neo-epitope specific TCR was ranked according to its relative frequency in PBL and tumor (red bars and arrows). The same TCR was undetectable in IL-2-expanded TIL cultures. Elements of box plots represent median (line), 25% and 75% confidence limit (box limits) and 10% and 90% confidence limit (whiskers)
