Figure 6: ORMDL3 negatively regulates IL-2 production by CD4⁺ T cells.

(a) Experimental design used for assessing effects of knocking down genes of interest in memory CD4⁺ T cells. (b) Real-time PCR quantification of ORMDL3 and GSDMB transcript levels (relative to the housekeeping gene YWHAZ) in memory CD4⁺ T cells 48 h after knock down with control siRNA pools, ORMDL3 or GSDMB siRNA pools (n=12 donors). (c) Effects of ORMDL3 knockdown on cytokine release by memory CD4⁺ T cells activated for 48 h with antibodies to CD3 and CD28; data are expressed as fold change (FC) relative to control siRNA-treated conditions; error bars indicate mean±s.e.m. (d) Absolute values of IL-2 protein levels in culture supernatants from cells treated with the indicated siRNA pools. Data are presented as means of biological duplicates. (e) Time course of IL2 mRNA expression in activated memory CD4⁺ T cells (n=24) following treatment with ORMDL3 (open circles) or control siRNA pools (closed circle); data from each donor for different time points after stimulation is shown in the right panel. (f) Representative FACS plots showing intracellular staining of IL-2 in memory CD4⁺ T cells activated for 6 or 18 h (after knockdown with siRNA pool for ORMDL3 or control siRNA); percentage of IL-2 producing cells in each donor is shown to the right (n=18). (g) Correlation of the levels of ORMDL3 transcripts (measured at baseline) and IL2 transcripts produced following ex vivo stimulation of memory CD4⁺ T cells (n=36) with phorbol myristate acetate (PMA) and Ionomycin for 4 h. (h) Effects of GSDMB knockdown on cytokine release by memory CD4⁺ T cells, as described in c. Each dot represents data from a single donor. r, Spearman correlation coefficient; *P<0.05, **P<0.01, and ***P<0.001 by Student’s paired two-tailed t-test, and following Bonferroni correction for multiple testing in c,d and h (see Methods).