Figure 5.

GITR ligation increases ex vivo proliferation of CD4⁺ and CD8⁺ TIL in response to tumor antigens presented by mRNA‐transfected autologous B cells. Effects of soluble GITRL (1 μg/mL crosslinked with anti‐HA antibody) or 10 μg/mL humanized agonistic anti‐GITR antibody on proliferation of (a) CD4⁺ TIL, and (b) CD8⁺ TIL upon 6 days’ culture of CFSE‐labeled tumor‐derived mononuclear cells with B cell blasts electroporated with mRNA encoding tumor antigens GPC3 or MAGEC2 (or eGFP as a negative control). Proliferation was measured by determination of percentages of CFSE^low CD4⁺ or CD8⁺ T cells at the end of the culture. Baseline proliferation (= % of CFSE^low T cells in the presence of eGFP‐electroporated B cells) was normalized to 100% for each tested patient, and is indicated by a closed green circle. The data depicted as ‘B cells + TIL’ demonstrate that TIL from all patients showed enhanced responses to MAGEC2 as compared to eGFP, while TIL of 6 patients also responded to GPC3. For those patients whose TIL responded to both tumor antigens, the average response to GPC3‐ and MAGEC2‐electroporated B cells was depicted. Each line represents one patient. An irrelevant human IgG1 antibody served as an isotype‐matched control antibody for the anti‐GITR antibody. Data show responses in TIL cultures of n = 8 patients. * = p < 0.05; ** = p < 0.01 compared to baseline (B cells + TIL). (c) Representative FACS contour plots of one patient display the proliferation of CD4⁺ and CD8⁺ TIL in response to autologous eGFP or MAGEC2‐electroporated B cells.