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. 2013 Jul 1;210(7):1433–1445. doi: 10.1084/jem.20121277

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© 2013 Obermajer et al.

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Figure 1. — Key role of NOS2 and the canonical cGMP-mediated NO signaling pathway in the MDSC-promoted Th17 differentiation of TILs from OvCa patients and human naive and memory CD4⁺ Th cells. (A) Induction of IL-17A, Rorc (encoding RORγt), FoxP3 (indicating de novo differentiation of FoxP3⁺ T reg cells from naive precursors), and T-bet gene expression in anti-CD3/CD28–expanded naive CD4⁺ T cells by tumor–isolated MDSCs (mean ± SD from six patients), as compared with control CD11b⁺ cells (mean ± SD from three healthy donors). (B) IL-17A production levels and percentages of IL-17A⁺ cells (mean ± SD from n donors), and representative intracellular staining (IL-17A vs. IFN-γ, right) in naive CD4⁺ T cells (n = 6 healthy donors) or OvCa-infiltrating CD4⁺ TILs (n = 3 patients) stimulated with anti-CD3/CD28 antibodies in the presence of cancer-isolated MDSCs or control CD11b⁺ cells. (C) IL-17A production by naive versus memory CD4⁺ T cells (mean ± SD from n = 4 healthy donors) stimulated with either anti-CD3/CD28 antibodies or TNF-matured allogeneic DCs in the presence of MDSCs or control CD11b⁺ cells. (D and E) Percentage of IL-17A⁺ or IFN-γ⁺ CD4⁺ T cells (D) and IL-17A secretion (E) in anti-CD3/CD28/MDSC–expanded naive CD4⁺ T cells (D) and CD4⁺ TILs (E) in the presence of specific inhibitors of NOS (L-NMMA), COX2 (celecoxib), IDO-, ARG-, or IL-10. The data (mean ± SD) from one representative experiment (performed in replicates: D, triplicate cultures; E, quadruplicate cultures). The results were confirmed in three independent experiments using different patients/healthy donors. (F) Representative staining of IL-17A⁺ or IFN-γ⁺ CD4⁺ T cells in co-cultures of anti-CD3/CD28–expanded CD4⁺ TILs and MDSCs in the presence of specific inhibitors of COX2 (celecoxib) and NOS (L-NMMA). The results were confirmed in three independent experiments using different patients. (G) Relative gene expression of IL-17A and Rorc, induced in anti-CD3/CD28–expanded naive CD4⁺ T cells cultured in the presence of MDSCs in the presence of a specific inhibitor of NOS (L-NMMA). The graphs present the mean ± SD from one representative experiment (triplicate cultures) of two (using different patients/healthy donors). (H) Relative gene expression of NOS2 and IL-17A in 7 d ex vivo anti-CD3/CD28–expanded cultures of OvCa ascites cells from 10 OvCa patients (n = 10; r² = 0.7692; P < 0.001). (I) IL-17A production in anti-CD3/CD28–expanded cultures of naive or memory CD4⁺ T cells in the presence of MDSCs, with or without specific inhibitors of NOS2 (1400W) or cGMP (ODQ). Data (mean ± SD) from one representative experiment (triplicate cultures). The results were confirmed in three independent experiments using different patients/healthy donors. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.