Extended Data Fig. 5: Effect of pregnenolone sulfate on T cell proliferation.
From: Metabolome modulation of the host adaptive immunity in human malaria
a, Experimental design of the T cell proliferation assay (created with biorender.com). CFSE-labelled PBMCs (1.5 × 105) from 10 healthy donors were cultured with or without anti-CD3/anti-CD28 stimulation in the presence or absence of 400 µM pregnenolone sulfate. T cell proliferation was assessed using flow cytometry after five days of culturing. b, Bar plots showing the division (right) and expansion (left) indices in the four experimental conditions for PBMCs from the 10 individuals. Proliferation and replication indices are shown in Fig. 6. c, Flow cytometry plots of CFSE-stained PBMCs derived from gated T cells for each of the 10 individuals under the indicated unstimulated untreated (blue), unstimulated pregnenolone sulfate-treated (purple), stimulated untreated (green), and stimulated pregnenolone sulfate-treated (red) conditions. d, Bar plots showing log2 mean fluorescence intensities (log2 MFI) of IL-2 and IL-4 cytokines quantified in culture supernatants of unstimulated untreated (n = 4, blue), unstimulated pregnenolone sulfate-treated (n = 4, purple), stimulated untreated (n = 10, green), and stimulated pregnenolone sulfate-treated (n = 10, red) PBMCs using flow cytometry. Cytokines that show statistically significant differences between stimulated pregnenolone sulfate-treated/untreated conditions are shown in Fig. 6. Differences between the groups presented in b. and d. were assessed using a paired two-tailed Student’s t-test. Bar and whiskers represent mean ± SD. The grey lines in bar plots connect matched samples. (e) Flow cytometric plots showing the gating strategy used in the T cell proliferation assay shown in Fig. 7. Lymphocytes were gated according to FS-A and SS-A. CD3+ live cells were gated according to 7-AAD and PE-A, and CFSE stained CD3+ cells were detected using FITC-A.
