Figure 6. IL-35-producing Foxp3⁻ iT_R35 develop in vivo.

Foxp3^gfp mice were infected with Trichuris muris. CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ cells were purified from spleen, small intestine IEL and LPL, or large intestine IEL and LPL by FACS, RNA extracted and cDNA generated. Ebi3 (a) and Il12a (b) expression of the populations indicated. (c, d, g, h) Foxp3^gfp mice or Ebi3^−/− Foxp3^gfp were injected with 120,000 B16 cells i.d. on the right flank. Tumors and spleens were excised after 15–17 days, CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ cells, purified by FACS, RNA extracted and cDNA generated. Ebi3 (c) and Il12a (d) expression of the populations indicated. (e, f) Foxp3^gfp mice or Ebi3^−/− Foxp3^gfp were injected with 2×10⁶ MC38 cells subcutaneously on the right flank. Tumors and spleens were excised after 12 days, CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ cells purified by FACS, RNA extracted and cDNA generated. Ebi3 (e) and Il12a (f) expression of the populations indicated. (g) Following B16 cell inoculation, purified T cells from the spleen or tumor were cultured for 24 h to allow secretion of IL-35. Culture supernatants from indicated cultures were immunoprecipitated with anti-p35 mAb, resolved by SDS-PAGE and probed with anti-Ebi3 mAb to identify IL-35 secretion. No IL-35 secretion was seen in either the splenic or tumor-infiltrating lymphocytes from Ebi3^−/− mice (g). Purified cells were assayed for regulatory capacity by mixing populations indicated at a 4:1 ratio with fresh responder T_conv cells for 72 h. Proliferation was determined by [³H]-thymidine incorporation. Counts per minute of T_conv cells activated alone were 16,000–33,000 (h). Data represent the mean ± SEM of 8–10 mice per group from 2–3 independent experiments (B16) and 1 experiment (MC38) [* p < 0.05, ** p < 0.005, *** p < 0.001, NS = not significant].