Fig 2: Immuno-profiling revealed an overall immune-suppressive TME in Tmem30-/- (PSout) tumors(A–G) Flow cytometry quantitative analysis of TILs at the endpoint (day 16). Shown are no infiltrating differences in CD45+CD11b-CD3+CD4+ T cells (B), CD45+CD11b-CD3+CD8+ T cells (C), CD45+CD11b-CD3+CD4+CD25+ Foxp3+ Tregs (D), and CD45+CD11b+ Ly6G-Ly6C+ Mo-MDSC (F); but more infiltrating cells in CD45+CD11b+Ly6G-Ly6C-F4/80 TAMs (E) and CD45+CD11b+Ly6C-Ly6G+ PMN-MDSC (G).(H) Representative flow plots of MHC II expression in TAMs.(I) Flow cytometry analysis of MHC II+ and MHC II- TAM distribution (n = 5).(J and K) Flow cytometry quantitative analysis of MHC II+ and MHC II- TAMs (n = 5).(L) RNA-seq analysis of sorted TAMs in WT or PSout (Tmem30-/-) TMEs. Shown is the Z score of gene expression (n = 4 for PSout and n = 3 for WT).(M and N) Geometric mean fluorescence intensity (gMFI) of CD163 (M) and CD206 (N) shows higher expression on TAMs (n = 5).(O–Q) qPCR analysis of MHC II, CD163, and CD206 in MHC II+ and MHC II- TAMs from both MC38-WT and Tmem30a-/- TMEs (n = 4).(R and S) gMFI of PD-1 (R) and Tim-3 (S) shows lower expression on CD8+T cells (n = 5).(T) Flow cytometry analysis of PD-1 and TIM-3 CD8+ T cell distribution (n = 5).(U) Representative flow plots and analysis of tetramer+ in CD8+ T cells (n = 5). Data were representative of at least two independent experiments (except RNAseq). Data represent mean ± SEM and were analyzed with unpaired t test. Ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001. Tmem30a-/- clone #1 cells were used for these figures.