Mechanisms Mediating Asthma Tolerance Induced By Regulatory Dendritic Cells Through CD4+CD25+Foxp3+ T Regulatory Cells

IL-10-treated tolerogenic dendritic cells (DC10) have been used as a powerful tool for the treatment of experimental asthmatic responses. We explored the important role of CD4+CD25+Foxp3+ regulatory T cells (Treg) in the induction of tolerance by DC10 in three levels. We first used the siRNA approach to silence IL-10 gene expression in murine DC10 and we generated DC10 from CD80/86 or H-2Iab gene-KO mice. When used to treat asthmatic mice, the IL-10-silenced and MHCII-deficient DC10 had lost their abilities to induce tolerance, as determined by airway hyperresponsiveness (AHR), eosinophilia and Th2 responses, while the CD80-/-/86-/- DC10 had reduced tolerogenic activities. Further studies showed that the OVA-presenting DC10 up-regulated expression of the Treg-associated markers ICOS, PD-1, GITR, LAG-3 and CTLA-4 on pulmonary Treg cells in treated asthmatic mice while DC10 (CD80-/-CD86-/-), DC10 (Iab-/-) and DC10 (IL-10-/-) treatments lead to reduced Treg activation. This demonstrated that the activities of tolerogenic DCs are differentially affected by their expression of IL-10, MHC II and costimulatory molecules. DC10 induce allergen tolerance in asthmatic mice, during which the animals-lung Th2 T effector cells (Teff) are displaced by activated Treg cells. Intestinal DCs have been shown to promote oral tolerance by inducing antigen-naïve T cells to differentiate into Treg, but whether DCs can induce Teff to differentiate into Treg remains uncertain. We addressed this question in OVA-asthmatic mice that were treated with DC10. DC10 delivery maximally activated lung Treg in these animals at 3 wk post-treatment, as determined by in vitro and in vivo Treg-inhibitory assays. In parallel cultures OVA-, but not house dust mite (HDM)-, presenting DC10 induced ≈43% of CFSE-labeled CD25-/loFoxp3- Teff cells from asthmatic OVA-TCR transgenic mice to redifferentiate into Treg. We recapitulated this in vivo using OVA-asthmatic mice that were co-injected with OVA- or HDM-presenting DC10 (i.p.) and CFSE-labeled CD4+CD25 /loFoxp3- Teff cells (i.v.) from the lungs of asthmatic DO11.10 mice. From ≈7 to 21% of the activated (i.e., dividing) DO11.10 Teff that were recovered from the lungs, lung-draining lymph nodes or spleens of the OVA-DC10 recipients had redifferentiated into Treg. These data indicate that DC10 treatments induce tolerance at least in part by inducing Teff to redifferentiate into Treg. Finally, we assessed the relative efficiency with which natural Treg cells (nTreg) and induced Treg cells (iTreg) tolerize Teff cells from asthmatic mice. The iTreg were induced either by culture of Teff cells with OVA-presenting DC10 or by injecting these Teff cells into DC10-treated asthmatic mice. We purified nTreg from allergen-naïve mice and also cultured them with DC10 before use, or co-injected them into DC10-treated recipients. The iTreg were 26-41% more effective than analogous nTreg in reducing Teff cell proliferation and IL-4/IL-5 secretion in vitro. Neutralization of IL-10, but not TGF, eliminated the suppressive activities of iTreg, which also expressed higher levels of PD-1, LAG3 & CTLA-4. Transfer of 5×105 nTreg had no impact on AHR or IgE levels in asthmatic recipients, but reduced the airway eosinophil and IL-4/IL-5 responses by 5-19%, while the iTreg normalized AHR and reduced all airway responses to allergen challenge by 82-96%. Taken together, our data show that DC10 provide three signals to induce Treg differentiation directly from allergen-experienced Teff, and that iTreg and nTreg of the same antigen specificity employ distinct mechanisms to effect tolerance. They also indicate that iTreg are distinctly superior to nTreg in induction of tolerance