Fig. 6: Inhibition of T cell steroidogenesis stimulates anti-tumor immunity.
From: Tumors induce de novo steroid biosynthesis in T cells to evade immunity
a–h Comparing Cyp11a1 cKO and Cd4-Cre control mice with B16-F10 cells injected subcutaneously. Representative of three independent experiments. a Tumor-infiltrating macrophages (Lin−CD11b+) were purified by cell sorting at day 12. Arg1 and Tgfβ1 mRNA expression was quantified by RT-qPCR, with mRNA expression level normalized by Gapdh mRNA expression. N = 6. b, c Tumor-infiltrating macrophages (Lin−CD11b+F4/80+) analyzed by flow cytometry at day 12 to examine iNOS and Arg1 expression. Representative FACS profile of the expression (b). Representative graphical representation of one experiment (c). N = 5. d Tumor-infiltrating CD3e+CD8+ T cells purified by cell sorting at day 12, reactivated ex vivo, and Ifnγ and Tnfα mRNA expression quantified by RT-qPCR, with mRNA expression level normalized by Rplp0 expression. N = 6. e Cytotoxic T-lymphocyte degranulation assay. CD107a/LAMP1 expression on tumor-infiltrating CD8+ T cells was analyzed by flow cytometry after 12 days post-inoculation of B16-F10 cells. N = 8 (ctrl), 9 (cKO) Gating: All cells>singlets>live cells>CD8+ T cell>CD107a. f NK cell degranulation assay by measuring CD107a/LAMP1 expression on tumor-infiltrating NK cells. N = 8 (ctrl), 9 (cKO). Gating: All cells>singlets>live cells>NK cells>CD107a. g CD4+ T cell degranulation assay by measuring CD107a/LAMP1 expression on CD4+ T cells. N = 8. Gating: All cells>singlets>live cells>CD4+ T cell>CD107a. h Flow cytometric analysis to show the changes in B16-F10 subcutaneous tumor-infiltrating Treg (CD4+CD3e+FoxP3+) populations upon Cyp11a1 deletion. N = 9. i–l Comparing immunophenotype in Cyp11a1 inhibitor, AG, treated and untreated mice with B16-F10 cells injected subcutaneously. All are representative of two independent experiments. i Representative FACS profile to show iNOS and Arg1 expression in tumor-infiltrating CD45+Lin−CD11b+F4/80+ macrophages (left panel). Graphical presentation the iNOS and Arg1 expression of a representative experiment (right panel). N = 5. j–l CD107a/LAMP1 expression on CD4+ T cells (j), CD8+ T cells (k) and NK cells (l) was analyzed by flow cytometry after 12 days post-inoculation of B16-F10 cells. N = 4 (ctrl), 3 (cKO). Gating: All cells>singlets>live cells>CD4+ or CD8+ T or NK cells>CD107a. In this figure, all error bars represent mean with s.d. P-value was calculated by unpaired two-tailed t-test. N represents biologically independent animals.
