Figure 1. Human IL-35 treatment of T_conv induces autocrine IL-35 expression and confers a regulatory phenotype.

T_conv purified by FACS from cord blood were treated with IL-35 or control at 25% of total culture volume for 9 days during activation (αCD3/CD28, and IL-2). (a) RNA was extracted, cDNA generated and qPCR performed. Relative Ebi3 (left panel) and Il12a (right panel) mRNA expression. (b) Cytokine treated cells were re-purified and stained with an isotype or a p35-specific antibody following 4h activation with PMA and ionomycin. Intracellular staining of IL-35 was determined by FACS. (c) Microscopic analysis of p35 expression was similarly determined following 4h activation with PMA and ionomycin. Anti- p35 or isotype control antibodies (shown in red), phalloidin (shown in green) and DAPI (shown in blue). (d) Proliferation of cytokine treated cells was determined by [³H]-thymidine incorporation (e) T_conv cells were mixed at indicated ratios (T_conv: suppressor) with control or IL-35 treated T_conv, hIL-2 and anti-CD3- + anti-CD28-coated latex beads for 9 days. Proliferation was determined by [³H]-thymidine incorporation. The mean of 4 representative experiments with similar cpm is shown. (f) Control or IL-35 treated T_conv were cultured in the top chambers of a Transwell^™ culture plate as indicated. Freshly purified responder T_conv were cultured in the bottom chamber of the 96-well flat bottom plates in medium containing hIL-2 and anti-CD3- + anti-CD28-coated latex beads. Top chambers were removed and [³H]-thymidine was added directly to the responder T_conv cells in the bottom chambers of the original Transwell^™ plate for the final 8 h of the 9 day assay. (g) T_conv cells were activated in the presence of IL-35 at 25% of total culture volume. Following conversion with cytokines, suppression assays were supplemented with neutralizing IL-10, TGFβ, or IL-35 to assess their requirement for indicated cytokines to mediate suppression. Counts per minute of T_conv cells activated alone, in the absence of control or IL-35, were 20,000–125,000 (f) and 25,000 – 570,000 (g). Data represent the mean ± SEM of (a)10 (b) 4, (c-g) independent experiments [* p < 0.05, ** p < 0.005, *** p < 0.001, NS = not significant].