FIGURE 2 from Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma
Authors: Lee Moon Hee , Theodoropoulos Jason , Huuhtanen Jani , Bhattacharya Dipabarna , Järvinen Petrus , Tornberg Sara , Nísen Harry , Mirtti Tuomas , Uski Ilona , Kumari Anita , Peltonen Karita , Draghi Arianna , Donia Marco , Kreutzman Anna , Mustjoki Satu 
Published: July 2023

<p>REP TIL T cells co-cultured with tumor cells show differences in IFNγ and TNFα expressions. <b>A,</b> REP TILs were cocultured with the corresponding tumor cells (<i>n</i> = 10) either for 6 or 48 hours. The cytokine secretion of CD4<sup>+</sup> and CD8<sup>+</sup> 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<sup>+</sup> (0.74% vs. 0.19%, <i>P</i> = 0.044) and CD8<sup>+</sup> T cells (0.78% vs. 0.13%, <i>P</i> = 0.025) compared with baseline REP TILs without tumor cell co-culture. No differences were observed at 6 hours co-cultures (<a href="#SMF3" target="_blank">Supplementary Fig. S3A</a>). BASELINE_48h = only REP TILs at 48h, CO-CULTURE_48h = cocultured REP TILs without any stimulation at 48 hours. <b>B,</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<sup>+</sup> (median 13.55% vs. 1.84%, <i>P</i> = 0.048) and CD8<sup>+</sup> T cells (45.55% vs. 22.94%, <i>P</i> = 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 (<a href="#SMF3" target="_blank">Supplementary Fig. S3D</a>). 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. <b>C,</b> 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<sup>+</sup> T cell IFNγ and TNFα expressions was observed between unstimulated and stimulated co-cultures (median 0.72% vs. 1.33%, <i>P</i> = 0.098). A moderate increase in CD4<sup>+</sup> T cell IFNγ and TNFα expressions was also observed (0.46% 1.089%, <i>P</i> = 0.012), but not in the CD8<sup>+</sup> 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. <b>D,</b> Prolonged co-culture conditions (48 hours) did not lead to increased IFNγ and TNFα production in the CD3<sup>+</sup>, CD4<sup>+</sup>, and CD8<sup>+</sup> 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; *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; ****, <i>P</i> < 0.0001.</p>