Fig. 3. Bacteria-derived and microbiota-derived peptides can stimulate TCC88 as well as the tumour antigen.

a, Peptides with the highest predicted stimulatory scores were identified by searching various databases (see Methods). Numbers of unique 10-mer peptides within each database, synthesized peptides, TCC88 stimulatory peptides and the corresponding percentage of stimulatory peptides within each database are shown. Proliferation was measured using ³H-thymidine incorporation (see Supplementary Table 10). *The immunopeptidome is annotated using human proteins in which 11 peptides score over 80%. Hence, the rest of the synthesized peptides were chosen from 60–80% of maximum score. RNA-seq, RNA sequencing. b, TCC88 was stimulated with bacteria-derived (top) and microbiota-derived (bottom) peptides for 3 days and proliferation was measured by ³H-thymidine incorporation (data represent the mean SI value of two replicate wells). HB, UniProt bacteria database; HGM, human gut microbiota³⁴. c, Pie chart displaying the number of peptides from all databases that stimulated TCC88 equally well or better than the cognate antigen (SIN3A* tumour antigen) at a concentration of 10 µM. d, TCC88 was tested with several strongly stimulatory bacteria-derived and microbiota-derived and SIN3A* peptides at decreasing concentrations including 20, 2, 0.2, 0.02 and 0.002 µM. Irradiated BLS-DRB3*02:02 was pulsed with the peptides and used to stimulate TCC88 (data represent the mean SI value of five replicate wells ± s.e.m.). e, Supernatants were collected from TCC88 after stimulation with SIN3A*, HGM3 and HB84 peptides. Cytokine and chemokine secretion of TCC88 was examined and compared upon stimulation with these three different antigens. A specific amount of each cytokine and chemokine (pg ml⁻¹) was calculated by subtracting the cytokines in the negative control cultures (no peptide control). f, Cytotoxicity of TCC88 against the autologous tumour cell line after stimulation with 2 µM of SIN3A* and HGM3 (data represent the mean percentage of two replicate wells). E:T, effector to target. g, TCC88 was tested with HGM3 (10 µM) or SIN3A* (10 µM) peptides or both together (each 10 µM), and ³H-thymidine incorporation was measured after 3 days (data represent the mean SI value of three replicate wells ± s.e.m.). Also see Extended Data Figs. 3 and 4.

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