Figure 2. Tmed10 deletion enhances CD8 T cell activation by impairing PD-1 trafficking to the cell surface. (a) Quantification of mass spectrometric readout of PD-1^OST co-immunoprecipitations⁷⁶ in murine T cells. Each data point indicates the results obtained from a single immunoprecipitation. Error bars denote SD. Statistical analysis was performed with a Student’s t-test. (b) Representative flow cytometry histograms for the abundance of PD-1-PE (left) and CD137-APC (right) in OT-I/Cas9 CD8 T cells of the indicated genotypes and treatment. The dotted line indicates the level of the background signal. (c) Quantification by flow cytometry of PD-1 abundance on OT-I/Cas9 CD8 T cells carrying the indicated sgRNAs in the presence or absence of brefeldin A (200 ng/mL). Each data point indicates data obtained with CD8 T cells from an independent spleen. Error bars denote SD. Statistical analysis was performed with a one-way ANOVA, followed by a Tukey post hoc test. (d) REVIGO⁸⁰ representation of significant gene ontology terms. a.u., arbitrary units. (e) Gene Set Enrichment Analysis plot of the gene set GO_T_CELL_ACTIVATION comparing activated OT-I/Cas9 cells carrying an sgRNA targeting Tmed10, or a non-targeting control. (f) As in (d) but for the gene set GO_T_CELL_PROLIFERATION. (g) Cytokine release of IFN-γ (left), IL-2 (middle) and TNF (right) as measured by cytometric bead array of sgCtrl or sgTmed10 OT-I/Cas9 CD8 T cells, after co-culture with B16F10-OVA tumor cells in a 1:4 ratio in the presence or absence of PD-1 antibody (10 µg/mL). Each data point indicates data obtained with CD8 T cells from an independent spleen. Error bars denote SD. Statistical analysis was performed with a one-way ANOVA, followed by a Tukey post hoc test. (h) Proteomic differences between OT-I/Cas9 CD8 T cells carrying either an sgRNA targeting Tmed10 or a non-targeting control sgRNA after activation with CD3 antibody for 24 hours as measured by mass spectrometry. The data is based on three independent spleens for each genotype. Statistical analysis was performed by a Student’s t-test. (i) Western blot analysis of PD-1, TMED10, TMED9 and TMED2 in OT-I/Cas9 CD8 T cells carrying the indicated sgRNA construct after activation with CD3 antibody for 24 hours. The size markings indicate the size of the closest molecular weight marker. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; FDR, false discovery rate; IFN, interferon; IL, interleukin; NES, normalized enrichment score; PD-1, programmed cell death protein-1; TNF, tumor necrosis factor.