Fig. 4

A77 1726 reciprocally regulates Th17/Treg imbalance in vivo. Spleen tissues isolated from each group of mice on day 42 after the first A77 1726 injection were stained for CD4⁺ IL-17⁺ (Th17) cells and CD4⁺ CD25⁺ Foxp3⁺ (Treg) cells using monoclonal antibodies against CD4 (red) and IL-17 (green) or against CD4 (red) CD25 (blue), and FoxP3 (green). To determine the effects of A77 1726 on the phosphorylation of STAT3 and STAT5 in vivo, isolated spleen tissues was subjected to immunofluorescence staining with monoclonal antibodies against CD4 (red), pSTAT3^Tyr705 (green), pSTAT3^Ser727 (green), or pSTAT5^Tyr694 (green). To identify HO-1-expressing CD4⁺ T cells, spleen tissue of IL-1Ra-KO mice with or without A77 1726 treatment was stained with antibodies against CD4 (green) and HO-1 (red). Original magnification ×400 (left). CD4⁺ T cells positive for IL-17, CD25⁺ Foxp3⁺, pSTAT3^Tyr705, pSTAT3^Ser727, pSTAT5^Tyr694, and HO-1 were enumerated visually at higher magnification, as projected on a screen (with each confocal image representative of 4 fields of view), with results quantified as the mean ± SD number of positive cells in 5 mice per group (right). *P < 0.05; **P < 0.01