Gene expression profiles of OT-1 T cells co-cultured with MHC-I Negative Tumors and Antigen Loaded Macrophages

OT-1 T-cells were co-cultured either alone in T-cell media supplemented with IL-2 (50IU/mL IL-2), with OVA loaded macrophages, or with both OVA loaded macrophages and CT2A-TRP2-β2mKO tumor cells. Cells were cultured at a 5:1 T-cell to tumor ratio, and 2:1 T-cell to macrophage ratio. After 24 h of co-culture, CD8 T-cells were FACS sorted, and RNA extracted (RNeasy Mini Kit, Qiagen). RNA was analyzed on an nCounter MAX Analysis System (Nanostring) with the PanCancer Immune profiling panel (Nanostring) according to manufacturer instructions. Expression dat OT-1 T-cells were isolated from OT-1 mice by culturing OT-1 splenocytes in T-cell media supplemented with 50IU/mL IL-2 and 1 μM OVA SIINFEKL peptide (Anaspec) for 48 h. Cells were purified for CD8 T-cells as described above and subsequently cultured in TCM with 50IU/mL IL-2, splitting every 24h for a total of 4 days. Gene sets from OVA-activated OT-1 T cells co-cultured with OVA antigen-negative CT2A-TRP2-B2mKO tumor cells or from OVA-activated OT-1 T cells alone were both compared against naïve OT-1 T cells. OVA negative B2mKO tumor lines were used to ensure the absence of tumor-TCR interactions. OT-1 TCR activation for these experiments was accomplished by concomitant culture with OVA MΦ.