FIGURE 2 from Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma

REP TIL T cells co-cultured with tumor cells show differences in IFNγ and TNFα expressions. A, REP TILs were cocultured with the corresponding tumor cells (n = 10) either for 6 or 48 hours. The cytokine secretion of CD4⁺ and CD8⁺ T cells was analyzed with intracellular flow cytometry staining. The expression of IFNγ and TNFα moderately increased in REP TILs after the co-culture with tumor cells without additional T cell stimulation. After 48 hours co-culture, IFNγ and TNFα levels were increased in both CD4⁺ (0.74% vs. 0.19%, P = 0.044) and CD8⁺ T cells (0.78% vs. 0.13%, P = 0.025) compared with baseline REP TILs without tumor cell co-culture. No differences were observed at 6 hours co-cultures (Supplementary Fig. S3A). BASELINE_48h = only REP TILs at 48h, CO-CULTURE_48h = cocultured REP TILs without any stimulation at 48 hours. B, The immunophenotype of T cells after co-culture with tumor cells was analyzed with flow cytometry. The expression of LAG-3 moderately decreased in the CD4⁺ (median 13.55% vs. 1.84%, P = 0.048) and CD8⁺ T cells (45.55% vs. 22.94%, P = 0.029) compared with REP TIL baseline cells at 6 hours. The same trends were not observed when REP TILs were co-cultured for 48 hours (Supplementary Fig. S3D). Although not significant, the expression of PD-1 expression increased in half of the patients. BASELINE_6h = only REP TILs at 6h, CO-CULTURE_6h co-cultured REP TILs without any stimulation at 6 hours. C, T cell activation potential was assessed by stimulating the cells with anti-CD3 (OKT3), anti-CD28, anti-CD49d antibodies, and IFNγ and TNFα cytokine secretion was measured as described above. When REP TILs were stimulated and co-cultured for 6 hours, an increase in the CD3⁺ T cell IFNγ and TNFα expressions was observed between unstimulated and stimulated co-cultures (median 0.72% vs. 1.33%, P = 0.098). A moderate increase in CD4⁺ T cell IFNγ and TNFα expressions was also observed (0.46% 1.089%, P = 0.012), but not in the CD8⁺ T cells. CO-CULTURE_6h = cocultured REP TILs without any stimulation at 6h, TSTIM_6h = cocultured REP TILs that were T cell stimulated at 6 hours. D, Prolonged co-culture conditions (48 hours) did not lead to increased IFNγ and TNFα production in the CD3⁺, CD4⁺, and CD8⁺ T cell compartments. CO-CULTURE_48h = cocultured REP TILs without any stimulation at 48h, TSTIM_48h = cocultured REP TILs that were T cell stimulated at 48 hours. Non-parametric Wilcoxon matched-pairs signed rank test was used for all co-culture analyses. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.