Regulatory T cell suppression is potentiated by target T cells in a cell contact, IL-35- and IL-10-dependent manner

Abstract

Regulatory T cells (T(reg)) are believed to suppress conventional T cell (T(conv)) proliferation in vitro in a contact-dependent, cytokine-independent manner, based in part on experiments in which T(reg) and T(conv) are separated by a permeable membrane. We show that the production of IL-35, a novel inhibitory cytokine expressed by natural T(reg), increases substantially following contact with T(conv). Surprisingly, T(reg) were able to mediate potent suppression of T(conv) across a permeable membrane when placed in direct contact with T(conv) in the upper chamber of a Transwell plate. Suppression was IL-35 and IL-10 dependent, and T(conv) activation was required for maximal potentiation of T(reg) suppression. These data suggest that it is the induction of suppression, rather than the function of T(reg) that is obligatorily contact dependent.

Figure 1. IL-35 and, to a lesser extent, IL-10 expression is potentiated by contact with T_conv

T_conv or T_reg cells from the spleens and lymph nodes of Foxp3^gfp, C57BL/6, Ebi3^−/−, or Il10^−/− mice were purified by FACS. (A) RNA was extracted and cDNA generated from Foxp3^gfp T_conv and T_reg, alone or from co-cultures (re-sorted based on GFP expression), and from T_conv:T_reg co-cultures re-sorted based on differential Thy1 markers. Relative mRNA expression was determined by quantitative real-time PCR from the populations and conditions indicated; unstimulated, stimulated for 48 h with anti-CD3/CD28, and/or cultures containing both T_conv and T_reg cells . (B) Supernatants were collected from purified T_conv or T_reg cells under indicated conditions and IL-10 secretion measured using Luminex™ technology. (C) Relative Ebi3 (left panel) and Il12a (right panel) mRNA expression of the T_conv and T_reg populations indicated using the same experimental approach described in (A). (D) Purified T_reg in the absence or presence of T_conv were cultured for 72 h with anti-CD3/CD28. Supernatant was collected for overnight IP with an anti-Il12a (p35) mAb, eluted proteins resolved on an SDS-PAGE gel and blotted with anti-Ebi3 mAb. Data represent the mean ± SEM of (A) 3–8, (B) 6 and (D) 4 independent experiments. Statistical analysis: *p< 0.05, ** p< 0.01.

Figure 2. T_reg mediated suppression is potentiated by T_conv cell contact in an IL-10- and IL-35-dependent, but not IL-2, manner

T_conv or T_reg cells from the spleens and lymph nodes of C57BL/6, Ebi3^−/−, Il12a^−/− and Il10^−/− mice were purified by FACS. (A) Cells assayed for regulatory capacity (T_conv or T_reg alone or in combination at a 4:1 T_conv: T_reg ratio) were cultured in the top chambers of a Transwell™ culture plate as indicated. Freshly purified wild-type “responder” T_conv were cultured in the bottom chamber of the 96-well flat bottom plates in medium containing anti-CD3/anti-CD28-coated latex beads. After 64 h in culture, 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 72 h assay. Cultures were harvested and cpm determined. (B) Purified T_reg cells from wild-type, Ebi3^−/− and Il10^−/− mice were cultured at the indicated ratios with purified T_conv and assayed for regulatory capacity in the top wells of the Transwell™ plate. In parallel, T_reg were assayed for regulatory capacity when T_reg were in direct contact with responder T_conv in the bottom chamber of the Transwell™ plate. Suppression of purified responder T_conv cells was measured by [³H]-thymidine incorporation. (C) RNA was extracted from cells in the bottom chamber of the Transwell™ plate. Groups analyzed were those cultured with no cells in the top chamber or WT T_conv co-cultured with T_reg (WT, Il10^−/−). IL-2 expression was determined by real-time quantitative PCR. Data represent the mean ± SEM of (A) 4, (B) 2, (C) 2 and independent experiments. Statistical analysis: *p< 0.05. Counts per minute of T_conv cells activated alone, in the absence of any suppressors, were 25,000 – 60,000.

Figure 3. Regulatory capacity of fresh and pre-activated fixed T_reg

T_conv or T_reg cells from the spleens and lymph nodes of C57BL/6, Ebi3^−/−, and Il10^−/− mice were purified by FACS. Cells were cultured fresh or pre-activated for 24h prior to culture, with or without fixation with 20% formaldehyde. Cells assayed for regulatory capacity (T_conv or T_reg alone or in combination at a 4:1 T_conv:T_reg ratio) were cultured in (A) direct contact with responder T_conv in a 96-well round bottom plate. Statistical analysis indicates fixation does not affect the ability of pre-activated T_reg to suppress while in direct contact with responder T_conv (B) top chambers of a Transwell™ culture plate as indicated. Freshly purified wild-type “responder” T_conv were activated in the bottom chamber of the 96-well plates. After 64 h in culture, 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 72 h assay. Cultures were harvested and cpm determined. Statistical analysis: *p< 0.05.

Figure 4. T_reg mediated suppression across the Transwell™ is IL-10 dependent

Purified T_reg were assayed for regulatory capacity in conventional T_reg and Transwell™ T_reg assays in the presence of an IL-10 neutralizing antibody (10µg/ml) or with recombinant IL-10 (100ng/ml) as indicated. Percent suppression was calculated in reference to the activated T_conv control under the conditions indicated. All % suppression to the left of the line was in reference to T_conv without anti-IL-10 or rIL-10 and the reference for % suppression to the right of the line was T_conv with anti-IL-10 or rIL-10, respectively. Data represent the mean ± SEM of 3 independent experiments. Statistical analysis: *p< 0.05. Counts per minute of T_conv cells activated alone, in the absence of any suppressors, were 25,000 – 60,000.

Figure 5. T_reg mediated suppression is potentiated, in part, by TCR signals derived from T_conv cell contact

(A) Purified T_conv with or without fixation with 20% formaldehyde were stimulated for 72 h with anti-CD3/CD28-coated beads and assayed for their proliferative capacity by [³H]-thymidine incorporation. (C) Purified Vβ8⁺ or Vβ8⁻ T_conv were stimulated for 72 h with anti-Vβ8-coated beads and assayed for their proliferative capacity by [³H]-thymidine incorporation. (B and D) Purified T_conv or T_reg cells were cultured under indicated conditions; unstimulated, stimulated for 72 h with anti-CD3/CD28-coated beads with or without fixation with 20% formaldehyde (B), or anti-Vβ8-coated beads (D) and assayed for their ability to suppress responder T_conv proliferation across the Transwell™ membrane. Data represent the mean ± SEM of 4–6 independent experiments. Statistical analysis: *p< 0.05. Counts per minute of T_conv cells activated alone, in the absence of any suppressors, were 25,000 – 60,000.

Figure 6. T_sup-derived IL-10 may contribute to the regulatory milieu

Wild-type T_reg were cultured with T_conv from wild-type, Ebi3^−/− and Il10^−/− mice in the top chambers of the Transwell™ plate. The ability of these co-cultures to suppress fresh responder T_conv proliferation in the bottom chamber was measured by [³H]-thymidine incorporation. Data represent the mean ± SEM of 5 independent experiments. Statistical analysis: *p< 0.05, ** p< 0.01. Counts per minute of T_conv cells activated alone, in the absence of any suppressors, were 25,000 – 60,000.