Fig 2: MOG35-55/I-Ab dimer modulates the expression of CD3 and TCR as well as the mTGF-ß1 and IL-10 suppressive cytokines in MOG35-55-specific T cells.(A–D) The 2D2 splenocytes were stimulated with MOG35-55 (25 µg/mL) in vitro in the presence of MOG35-55/I-Ab dimer (1.2 nM) or PBS. After 5 days culture, cells were harvest and stained with anti-CD4-FITC mAb, anti-CD3-PE-Cy7 mAb, anti-TCR-Va3.2-APC mAb, anti-mTGF-ß1-PE and/or anti-IL-10-PE mAb. (A–B) The MFIs of (A) PE-cy7-CD3 and (B) APC-TCR Va3.2 on CD4+ cells were analyzed by flow cytometry and expressed as the mean ± SD from 6 mice in each group. (C-D) MOG35-55/I-Ab dimer intervention induced a population of CD3lowTCRlowCD4+ cells with high level of (C) mTGF-ß1 and (D) IL-10 expression. The right panel represents the mean ± SD of MFI of PE-mTGF-ß1 and PE-IL-10 on CD3highTCRhighCD4+/CD3lowTCRlowCD4+ cells in dimer intervention group (n = 3). (E) Splenocytes were isolated from mice treated with PBS or MOG35-55/I-Ab dimer on day 18 post-immunization and stained with anti-CD3-APC, anti-CD4-FITC, anti-TGF-ß1-PE, and/or anti-IL-10-PE Abs. CD4+ cells were gated for analysis by flow cytometry. (a–b) The mean percentage of CD3lowCD4+ cells and the MFI of APC-CD3 on CD4+ cells from 6 mice in each group are represented as the mean ± SD. (c) The MFIs of PE-mTGF-ß1 and PE-IL-10 on CD3lowCD4+/CD3highCD4+ cells in dimer treated-group are expressed as the mean ± SD from 6 mice respectively. *, P<0.05; **, P<0.01; ***, P<0.001 and n.s., nonsignificant.

Fig 3: MOG35-55/I-Ab dimer treatment abrogates autoreactive Th1 and Th17 expansion in vivo.MOG35-55 immunized mice were treated with PBS, excess MOG35-55 peptide, Mulv env145-158/I-Ab dimer or MOG35-55/I-Ab dimer. On day 18 post-immunization, cells were isolated from spleen (SPN) and lymph nodes (LN) for IFN-?+ Th1 and IL-17+ Th17 cells detection by intracellular staining. (A) Gated for CD3+CD4+ (B) Gated for CD3+CD8+. The left panel shows one representative sample from each group. The right panel represents the mean ± SD of percentage of Th1 or Th17 cells with differences (n=4). *, P<0.05; **, P<0.01; ***, P<0.001 and n.s., nonsignificant.

Fig 4: Immune phenotype of twelve-week-old THCD11b-Cre mice. (A) CD11b+Ly6G+ neutrophils, (B) CD11b+F4/80+ macrophages, (C) CD3+CD8+ cytotoxic T lymphocytes and (D) CD3+CD4+ T helper lymphocytes in the bone marrow of twelve-week-old THflox/flox and THCD11b-Cre mice. (E) CXCL1, (F) CCL2, (G) IL6 and (H) IL10 relative mRNA expression in whole tibiae of twelve-week-old THflox/flox and THCD11b-Cre mice. n = 4-7; *P < 0.05; **P < 0.01; ****P < 0.0001.

Fig 5: Heterozygous Nr2f6+/- mice similarly show strong tumor growth benefit. Tumor growth curves of Nr2f6+/+, Nr2f6+/- and Nr2f6-/- mice that received a 1×105 B16-OVA melanoma cells (Nr2f6+/+ (n = 21), Nr2f6+/- (n = 11), and Nr2f6-/- (n = 21)) or c 5×104 MC38 tumor cells (Nr2f6+/+ (n = 16), Nr2f6+/- (n = 5), and Nr2f6-/- (n = 10)) subcutaneously and were monitored three times a week showing a significant tumor growth delay as well as a survival benefit (b-d) in Nr2f6 gene-modulated mice (p<0.0001 for both tumor cell lines, ANOVA). e Tumor growth curves of Nr2f6+/+ (IgG2b n = 5, aPD-L1 n = 10), Nr2f6-/- (IgG2b n = 7, aPD-L1 n = 12) and Nr2f6+/- (IgG2b n = 3, aPD-L1 n = 3) mice injected s.c. with 5×105 B16-OVA melanoma cells and treated with PD-L1 blockade (continuous line) or IgG2b (dashed line) (n = 3–5). Both aPD-L1 treated Nr2f6+/- (p = 0.0179, ANOVA) and Nr2f6-/- (p < 0.0001, ANOVA) showed a significant slower tumor growth when compared to treated wild-type controls. f, g In vitro flow cytometry analysis of isolated CD4+ or CD8+ T cells activated with anti-CD3 mAb (5 µg) and anti-CD28 mAb (1 µg) at d3 from Nr2f6+/+, Nr2f6+/- or Nr2f6-/- mice (n = 3). Analysis of IL-2 and IL-17-producing CD4+ Th0 and Th17 T cells, Ki67+ proliferating CD4+ and CD8+ T cells, and IFN? as well as IL-2 production of CD8+ T cells. Percentage of positive cells relative to parental gate is shown; experiments were repeated at least three times. Results shown are derived from at least two independent experiments. Error bars represent the mean ± SEM