Fig 2: The expression of maturation surface markers and cytokines in the supernatants of BMDCs stimulated with the novel lipopeptides. BMDCs were treated with the synthetic lipopeptides respectively, and then cells were collected after 48h. The expression levels of CD40 and CD80 were analyzed by flow cytometry. (A, B) Representative flow cytometry dot plots. (C, D) Normalized expression level of maturation markers. (E–G) The expression of IL-1ß, TNF-a and IL-12 in the supernatants of BMDCs were detected by ELISA (n=3). Data were presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Deletion of Dhx9 in TECs compromised the development and maturation of TECs. (A–C) The representative pictures (A), weight (B) and cell numbers (C) of the thymus isolated from 4-week-old WT and Dhx9 cKO mice (n = 10 for each group). (D) Immunofluorescence staining of frozen thymic sections of WT and Dhx9 cKO mice. Red: KRT5-marked medullary regions; Green: KRT8-marked cortical regions; Scale bars: 500 μm. (E) The representative thymic H&E staining in 4-week-old WT and Dhx9 cKO mice; Scale bars: 1000 μm. (F) Flow cytometric profiles, frequencies, and cell numbers of CD45-EpCAM+ TECs in 4-week-old WT and Dhx9 cKO mice (n = 7 for each group). (G) Representative flow cytometry plots, frequencies, and cell numbers of UEA1+Ly51- mTECs and UEA1-Ly51+ cTECs in 4-week-old WT (n = 7) and Dhx9 cKO mice (n = 6). (H) Representative flow cytometry plots and frequencies showed the maturation of mTECs, as measured by the expression of CD40, CD80, and MHCII in 4-week-old WT (n = 8) and Dhx9 cKO mice (n = 6). (I) Representative flow cytometry plots and frequencies exhibited the maturation stage by the expression of CD80 and Aire in 4-week-old WT (n = 8) and Dhx9 cKO mice (n = 6). One representative histogram represented the mean ± SD. The unpaired, two-tailed student’s t-test was used. *P < 0.05, **P < 0.01, ***P < 0.001 compared with WT control mice.

Fig 4: Tr1 cells suppress immune responses via IL-10 signaling.(A) Representative histogram of surface MHC-II, CD40, CD80, and CD86 on BMDCs in each group (n = 4 for each group). (B) Quantitative data of the MFI of surface MHC-II, CD40, CD80, and CD86 on BMDCs in each group (n = 4 for each group). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (C) Representative histogram of CFSE dilution of CD8+ T cells in each group (n = 4 for each group). (D) Quantitative data of the proportion of CFSEdim proliferating CD8+ T cells in each group (n = 4 for each group). Values are shown as the mean ± SEM; ***P < 0.001 and ****P < 0.0001.

Fig 5: MEKi suppress M2-like macrophages and MDSCs.a Flow cytometric analysis of the impact of MEKi GDC-0623 and/or anti-CD40 Ab treatment on the macrophage infiltrate in MC-38 and PDA30364 tumors, as performed on four mice per treatment group of the experiments shown in Fig. 3 b–c. Representative dot plots are shown. Cells were pre-gated on living CD45+, CD11b+, F4/80+ cells. One-way ANOVA with post hoc Dunnetts’s test (treatment groups vs. control). Significance levels are indicated by asterisks (*p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001). b Polarization of mouse bone marrow-derived macrophage cultures towards M1- or M2-like phenotype, as assessed by flow cytometry marker analysis. Mean ± s.e.m, n = 6. c Cell viability of polarized macrophages after 3-day treatment with GDC-0623. Left panel: representative dose–response curves. Relative IC50 values were calculated by four-parameter logistic curve fit with a bottom constraint > 0. Mean ± s.e.m, n = 3. Right panel: Ki67/PI-based cell cycle analyses of tumor cells treated with 1 µm GDC-0623 and respective quantification. Mean ± s.e.m, n = 3. Unpaired student t test (medium vs. GDC-0623 for each cell cycle phase; FDR (Q = 1%), two-stage step-up method of Benjamini, Krieger and Yuketieli). d Representative dot plots of myeloid cell populations in MC-38 and PDA30364 tumors. e Representative dot plots of 'Gr1+', Ly6C+Ly6G+, and Ly6C+ Ly6G- myeloid cell populations in PDA30364 tumors. Cells were pre-gated on living CD45+, CD11b+ cells; quantitation normalized to all living cells. Mean ± s.e.m, n = 4. One-way ANOVA with post hoc Dunnett’s test (treatment groups vs. control). F Cell viability of bone marrow-derived MDSC cultures after 3-day treatment with GDC-0623; assay performed as described above. Mean ± s.e.m, n = 3.