Figure 5

Feline CD4⁺CD25⁺T cell-mediated iTreg conversion occurs by a TGFβ/TGFβRII dependent mechanism. A-C. To confirm blocking of TGFβ (A) or GARP (B) on CD4⁺CD25⁺ Treg cells and TGFβRII (C) on CD4⁺CD25^- Th cells, cells were pretreated with unconjugated specific or isotype control antibody, then analyzed by flow cytometry using conjugated antibody to confirm blocking of TGFβ and GARP on CD4⁺CD25⁺ and TGFβRII on CD4⁺CD25^- cells. Data represent three experiments. D-G. ConA-activated Th targets were pre-treated with anti-TGFβRII antibodies and/or Tregs were pre-treated with anti-TGFβ or anti-GARP antibodies for 30 min, washed, co-cultured for 5 days, then target cells were analyzed for induction of Treg phenotype and function. D. Dot plots demonstrating surface TGFβ expression following conversion of Th cells to iTregs and blocking TGFβ/TGFβRII or GARP prior to co-culture (upper panels). Surface expression of both (1) GARP and TGFβ were decreased by blocking (2) TGFβ, (3) TGFβRII, (4) TGFβ/TGFβRII, or (5) GARP (lower panel). Data represents one blocking study. E. Treatment of CD4⁺CD25⁺ converter cells with anti-TGFβ (lane 1) or anti-GARP (lane 2) or of CD4⁺CD25^- target cells with anti-TGFβRII (lane 3) prior to co-culture prevented induction of FoxP3 in Th targets. Lane 4: Th targets with no antibody blockade. F, G. Suppressor function of iTregs was evaluated by ³H-thymidine proliferation (F) or by IL2 suppression assay (G). Suppressor function of iTregs was reduced by anti-TGFβ treatment of converter Treg cells (F black bar, G lane 4) or by anti-TGFβRII treatment of target Th cells (F pattern bar, G lane 5) prior to conversion. Conversion was completely abrogated when TGFβ and TGFβRII were blocked simultaneously or when GARP was blocked during conversion (G lanes 6 and 7). Data in F represents mean + SEM of three experiments; G mean ± SEM of experimental triplicates.