Extended Data Fig. 7. Stimulatory effect of IPdBPs on peripheral memory T cells (see also Fig. 5).

a, CD45RA⁻ PBMCs from patient 1635WI were CFSE-labeled and stimulated with a pool of IPdBPs (see Supplementary Table 13). 60 replicate wells were pooled after 10 days and stained with CD3, CD4 and CD8. Proliferating (CFSE^dim) CD4⁺ T cells were sorted and short-term expanded. b, Expanded and IPdBP-pre-stimulated peripheral CD4⁺ T cells were tested with 10 µM of autologous tumor peptides derived from over-expressed or mutated proteins (Supplementary Table 13). Proliferation was measured using ³H-thymidine incorporation (data represents mean value of 4 replicate wells ±SEM). GM-CSF ELISA was used as another read-out for T cell activation. GM-CSF was measured by ELISA and is depicted in (pg/ml) after subtraction of the negative control. The peptide pool that was used to pre-stimulate peripheral blood CD4+ T cells (IPdBP pool) served as a positive control (see material and methods section). c, Cytokine/chemokine response of peripheral memory CD4⁺ T cells that had been pre-stimulated with the pool of IPdBPs and short-term expanded in vitro, to the autologous tumor peptide (LRP6) as well as the IPdBP pool was measured. Specific amount of each cytokine/chemokine (pg/ml) was calculated by subtracting the cytokines in the negative control cultures (no peptide control). d–e, Percentage and frequency overlap of unique productive TCRVβ sequences of CFSE^dim peripheral memory CD4⁺ T cells responding to IPdBP pool with TCRVβ sequences of bulk TILs. Overlap of unique productive TCRVβ sequences of IPdBP-reactive peripheral CD4⁺ (CFSE^dim) (d) and bacteria/microbiota-reactive peripheral CD4⁺ (CFSE^dim) (e).

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