Supplementary Fig. 3: PlGF does not affect IFN-γ and IL-10 production, T cell proliferation, or cellular composition of splenocytes.
(a) IFN-γ and IL-10 production by splenocytes from Plgf-KO (n = 6) and WT mice (n = 10). Mouse splenocytes (5 × 105) were stimulated with anti-CD3 and anti-CD28 (α-CD3/28, 1 μg/ml) for 48 hours. The levels of IFN-γ and IL-10 in the culture supernatants were measured using ELISAs. (b) CD4+ T cells (1 × 105) of WT and Plgf-KO mice were differentiated under TH1, TH2 or TH17 polarizing conditions for 5 days (see Methods). The concentrations of IL-17 and IFN-γ in the culture supernatants were determined by an ELISA. * P < 0.05 versus TH17 cells of Plgf-KO mice. (c) Splenocyte proliferation in response to TCR stimulation. Splenocytes from Plgf-KO (n = 8) and WT mice (n = 8) were stimulated with α-CD3/28 for 48 hours. Cell proliferation was measured using the [3H] thymidine incorporation assay. (d) Immune cell compositions of Plgf-KO (n = 2) and WT splenocytes (n = 2). Splenocytes from these mice were stained with APC-labeled anti-CD4, PE-labeled anti-CD8, FITC-labeled anti-CD19, and FITC-labeled anti-CD11. Cells were analyzed using flow cytometry. (e) No effect of exogenous PlGFCM on production of IFN-γ by T cells. Sorted CD4+ T cells (1 × 105, n = 3) were differentiated under TH1, TH2, and TH17-polarizing conditions with exogenous PlGFCM (100 ng/ml) for 5 days. Concentrations of IFN-γ in the supernatants were analyzed by ELISA. Data are presented as the mean and SEM of three independent experiments performed in triplicate.
