Fig 2: PKM2 Tetramerization Blocks T Cell Activation In VitroMurine CD4+CD62+ T cells were stimulated in vitro with CD3/CD28 antibodies in the presence of DMSO (CTRL), TEPP-46 50 µM, or 100 µM.(A and B) Cells were collected after 24 h of stimulation.(A) Quantification of Il2 mRNA in activated T cells by qRT-PCR (n = 9 from three independent experiments).(B) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. IL-2 production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced IL-2 production by TEPP-46-treated cells. Right, quantification of the percentage of IL-2-producing cells and IL-2 mean fluorescence intensity (MFI) in CTRL versus TEPP-46-treated cells (n = 8 from 3 independent experiments).(C–F) Cells were collected after 3 days of stimulation.(C) Top, representative flow cytometry plot displaying T cell proliferation assessed as CellTrace violet dilution. Bottom, a division index was calculated with FlowJo software to quantify T cell proliferation (n = 5 from four independent experiments).(D) Expression of surface CD62L, CD44, and CD25 was evaluated by flow cytometry. The percentage of expressing cells and the MFI are shown (n = 3 from 2 independent experiments).(E) Quantification of Tnfa mRNA levels in activated T cells by qRT-PCR (n = 6 from 6 independent experiments).(F) Cells were re-stimulated in vitro with PMA and ionomycin in the presence of brefeldin A. TNF-a production was then evaluated by flow cytometry after intracellular cytokine staining. Left, representative plot showing reduced TNF-a production by TEPP-46 treated cells. Right, quantification of the percentage of TNF-a-producing cells and TNF-a MFI in CTRL versus TEPP-46-treated cells (n = 5 from 2 independent experiments). For all panels, data are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001 compared to CTRL condition, by one-way ANOVA with Dunnett's post-hoc test.

Fig 3: 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 4: 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 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.