Increased release of IL-10 from Foxp3⁺ CD4⁺CD25⁺ Tregs isolated from the lungs of anti–IL-6R–treated mice. CD4⁺CD25⁺ T cells and CD4⁺CD25^– T cells were isolated from lung cells in the different experimental groups, after which cytokine production was analyzed. The purity of the CD4⁺CD25⁺ cell population was 95–98% as determined by FACS analysis during cell sorting. (A–C) Lung CD4⁺CD25⁺ T cells isolated from OVA-sensitized, anti–IL-6R antibody–treated mice released increased amounts of IL-10 (B) per cell as compared to CD4⁺CD25⁺ T cells from OVA-sensitized and -challenged, untreated or OVA-sensitized, IgG-treated mice. In contrast, lung CD4⁺CD25^– T cells produced little IL-10 (B) but more TGF-β (A) and some IFN-γ (C). n = 6. By contrast, CD4⁺CD25⁺ isolated from gp130-Fc–treated mice released less IL-10 (B), IFN-γ (C), and TGF-β (A), while the CD4⁺CD25^– isolated from the same mice released the same amount of IL-10 (B) and less TGF-β (A). (D) Expression of Foxp3 on CD4⁺CD25⁺ lung T cells upon anti–IL-6R antibody treatment. CD4⁺CD25⁺ and CD4⁺CD25^– lung T cells from untreated or anti–IL-6R antibody–treated, OVA-sensitized mice were separated as described above. This was followed by RNA extraction and analysis of Foxp3 or β-actin expression by RT-PCR. (E) Real-time PCR for Foxp3 in CD4⁺CD25⁺ and CD4⁺CD25^– cells is reported as the ratio of Foxp3 to HGPRT. Anti–IL-6R antibody treatment led to a significant upregulation of Foxp3 as compared to OVA treatment. This experiment was performed 3 times in duplicate. *P < 0.05; **P < 0.01.