Fig 2: Notch-Driven Differentiation Yields Functional Human cDC1sDCs generated from CD34+ progenitors in cultures with OP9 or OP9-DL1 were analyzed in parallel to primary DCs from PB.(A and B) Cytokine production by DCs stimulated for 14 hr with a cocktail of TLR agonists (poly-I:C, LPS, CL075, and CpG).(A) Representative flow cytometric analysis of intracellular cytokine production (TNF, IL-12, and IFN-a) in the indicated gated DC subsets from OP9-DL1 cultures. Grey contours represent unstimulated cells; numbers represent the cytokine-positive fraction.(B) Proportion of cytokine-positive cDC1s (purple), cDC2s (red), or pDCs (blue) generated from CD34+ progenitors in culture with OP9 (n = 4) or OP9-DL1 (n = 3) cells compared with PB primary (n = 7) cells following TLR stimulation. Circles, histograms, and bars represent individual experiments, mean, and SEM, respectively; p values are indicated.(C and D) T cell stimulation by DCs cultured with sorted allogeneic blood CD3+ T cells.(C) Representative flow cytometric analysis of T cell proliferation in response to culture with DCs. The data show output of a T cell and cDC1 (generated with OP9-DL1) culture. CD11c+ CD141+ cDC1s could be identified (purple gate) and gated out. CD3+ T cells were subdivided by CD8 and CD4 expression. Cell division was indicated by CFSE dilution (turquoise gate).(D) Proportion of CD4+ or CD8+ T cells that underwent division (CFSE dilution) in culture with cDC1 (purple) or cDC2 (red) isolated from PB or generated in culture with OP9 or OP9-DL1 (DL1) cells. T cells cultured alone or with beads coated with anti-CD3 plus anti-CD28 were used as negative (Neg) and positive (Pos) controls, respectively. Responses to blood DCs were generated from 2–3 DC donors and 3 T cell donors (2–6 independent experiments). Responses to cultured DCs were generated from 2 BM donors combined with 3 T cell donors (4–6 independent experiments). Each circle represents an independent experiment (mean of 1–3 technical replicates). Histograms and bars represent mean and SEM, respectively. The p values were derived from unpaired two-tailed Student’s t test.

Fig 3: Effect of IL-10 on H295R cells. a Concentration of IL-10 secreted by BM-hMSC presented as the mean ± SD (n=4). Relative gene expression of b CYP17A1, c CYP11A1, and d DENND1A after IL-10 treatment. e Testosterone secretion and f androstenedione secretion by H295R cells after IL-10 treatment. Relative gene expression of inflammatory markers g IL-6 (IL6), h TNF-a (TNFA), and i IL-1ß (IL1B) after IL-10 treatment. *p<0.05, **p<0.005; NS, not significant. b–i All graphs are presented as the mean ± SD (n=3). Statistical significances were determined by 2-way ANOVA or nonparametric T-test (Mann-Whitney test) using GraphPad Prism 9

Fig 4: Effect of BM-hMSC secretome on H295R cells and human PCOS theca cells. After 24 hours of treatment, a percentage of Ki67-positive cells, Annexin V-positive cells, and Annexin V+/7AAD double-positive cells in the BM-hMSC secretome-treated vs. control (basal media) H295R cells. b Relative mRNA expression of CYP17A1, CYP11A1, and DENND1A in BM-hMSC secretome-treated vs. control H295R cells. c Protein expression analysis of CYP17A1, CYP11A1,and DENND1A by western blot in BM-hMSC secretome-treated vs. control H295R cells. d Quantification of western blot by analyzing band intensity. e Relative mRNA expression of CYP17A1 in two different human PCOS theca cells after BM-hMSC secretome treatment vs. control (basal media). f Average protein expression level of CYP17A1 in human PCOS theca cells after BM-hMSC secretome treatment vs. control. g Testosterone secretion by H295R cells and human PCOS theca cells; BM-hMSC secretome vs. control group. h Relative gene expression of inflammatory marker IL1B and TNFA in BM-hMSC secretome-treated vs. control basal media in H295R cells. *p<0.05, **p<0.005, ***p<0.0005; NS, not significant. All graphs are presented as the mean ± SD (n=3). Statistical significances were determined by 2-way ANOVA or nonparametric T-test (Mann-Whitney test) using GraphPad Prism 9

Fig 5: TEPP-46 Constrains Th1 Cell DevelopmentMurine CD4+CD62+ T cells were activated in vitro for 3 days with CD3/CD28 antibodies under Th1-polarizing conditions in the presence of DMSO (CTRL condition) or TEPP-46 50 µM or 100 µM.(A) Flow cytometry evaluation of IFN-? and TNF-a production by Th1 cells after intracellular cytokine staining. Left, representative plots showing reduced IFN-? and TNF-a production by TEPP-46 treated cells. Right, quantification of the percentage of IFN-?/TNF-a-producing cells and of IFN-?/TNF-a MFI in CTRL versus TEPP-46-treated cells (n = 5–6 from 4 independent experiments).(B and C) Quantification of Tnfa/Ifng (B) and Tbx21/Eomes (C) mRNA levels by qPCR (n = 5–6 from 3 independent experiments). For all panels, data are the mean ± SD. ***p < 0.001 or ****p < 0.0001 compared to CTRL condition, by one-way ANOVA with Dunnett's post-hoc test.