Figure 8.

Co-culture with ⁹⁰Y-NM600-treated tumor cells results in activation of IFNγ production in CD8+ T cells and increased expression of CTLA-4 (C4) on CD4⁺ and CD8⁺ T cells. (A) Tumor cells were plated on petri dishes and radiated with a prescribed cumulative dose of 12 Gy of ⁹⁰Y-NM600 (140 µCi administered activity). Three days following addition of ⁹⁰Y-NM600 to tumor cell culture media, splenocytes were added to the co-culture or empty culture plates without tumor cells, and 1 day following addition, splenocytes were harvested for analysis. For each culture condition CD4⁺ and CD8⁺ T cells were analyzed by flow cytometry for viability, activation status using IFNγ as a marker and expression of inhibitory CTLA-4 (schematic created with Biorender.com). (B-D) Quantification of number of CD4⁺ and CD8⁺ T cells alive at the end of co-culture with tumor cells (B, C) or without tumor cells (D). (E-G) Quantification of IFNγ expression on CD4⁺ and CD8⁺ T cells in co-culture with tumor cells (E, F) or without tumor cells (G). (H-J) Quantification of CTLA-4 expression on CD4⁺ and CD8⁺ cells in co-culture with tumor cells (H, I) or without tumor cells (J). (K) Tumor cell expression of STING suppresses the induction of CTLA-4 on CD4⁺ and CD8⁺ T cells upon co-culture of splenocytes with ⁹⁰Y-NM600-treated B16 melanoma cells. (L) Addition of anti-CTLA-4 treatment increases expression of IFNγ on CD8⁺ T cells compared to ⁹⁰Y-NM600 alone in a STING dependent fashion. Number of live cells and expression quantification was compared via Student's T test (A-J) or one-way ANOVA with Tukey's HSD post hoc test (K, L).