The Effect of Interleukin-10-differentiated Dendritic cell Therapy on B cell Tolerance in a Mouse Model of Asthma

IL-10-differentiated dendritic cells (DC10) that have been loaded with specific allergen can effectively reverse the asthma phenotype in mice, but how they suppress the asthma-phenotype B cell response is unclear. Herein we assessed the mechanism(s) by which DC10 and DC10-induced regulatory T cells (DC10-iTreg) affect B cell responses in asthma-phenotype mice. We found that the numbers of lung-resident OVA-specific IgG1-secreting B cells in asthma-phenotype mice rapidly declined after cessation of allergen sensitization, and that DC10 immunotherapy had no significant effect on this B cell population. However, DC10 treatment accelerated the decline in IgG1-secreting B cell numbers in bone marrow and spleen at ≥2 weeks post-treatment. Transtracheal delivery of allergen-loaded DC10 to asthma-phenotype mice also suppressed lung IgG1- and IgA-secreting B cell responses when assessed two days after treatment. Only DC10 that were carrying intact antigen on their surface could suppress B cell antibody production in vitro, and that effect was antigen-specific and contact-dependent. DC10 that had fully processed allergen to which they had been exposed lost their capacity to suppress IgG1-B cells, although they could still suppress asthma-phenotype Th2 responses. DC10-induced Tregs isolated from the lungs of DC10-treated asthma-phenotype mice suppressed IgG1-B cell responses in vitro but did not have a significant impact on asthma-phenotype lung B cell IgG1 secretion when assessed two days after transtracheal delivery. We also investigated whether DC10 induced B cell tolerance through regulatory B cells (Breg). DC10 upregulated CD5 and CD1d expression when co-cultured with asthma-phenotype lung B cells, but this effect was not allergen-specific. CD5+ B cells isolated from co-cultures of DC10 and asthma-phenotype lung CD19+ B cell suppressed stimulatory DC activation of cognate T cell proliferative responses, but this effect was independent of DC10 exposure to cognate allergen. This CD5+ B cell activity was also IL-10- and TGF-β-independent. In contrast to our in vitro observations, immunotherapy of asthma-phenotype mice with OVA-loaded DC10 did not amplify the inhibitory functions of CD5+ B cells isolated from lungs. OVA- but not irrelevant allergen-pulsed DC10 immunotherapy did, however, significantly increase the numbers of IL-10-producing splenic B cells at two weeks after treatment of asthma-phenotype mice. However, these IL-10-expressing B cells did not suppress stimulatory DC activation of cognate T cells in vitro. We had previously shown that treatment of asthma-phenotype mice with iTreg purified from DC10-treated mice effectively suppresses the asthma phenotype and IgG1-B cell responses in vitro. However, in the current study we observed that i.v. delivery of DC10-iTreg, that were generated in vitro, had no discernible impact on plasma OVA-specific IgG1, IgE or IgA antibody levels, or on airway IL-4, IL-5, IL-13, or IL-10 cytokine responses to recall allergen challenge. In addition, the numbers of OVA-specific IgG1-secreting B cells in the lung, bone marrow or spleen of asthma-phenotype mice did not change following this DC10-iTreg treatment, although iTreg did suppress IgA-B cell response in the lung, but not in the bone marrow or spleen. In summary, our data suggest that DC10 do not directly engage B cells to down-regulate OVA-specific IgG1 B cell responses in DC10-treated asthma-phenotype mice, although DC10 that carry intact cognate allergen on their cell surface can suppress IgG1 secretion in vitro. DC10 upregulated CD5 expression on B cells in vitro, but had no significant impact on the regulatory functions of CD5+ B cells and did not promote induction of IL-10-producing Breg in vivo. We conclude that DC10 treatment did not promote tolerance through IL-10-producing Breg induction, and that it is more likely that Treg or other regulatory cells induced by DC10 immunotherapy are important for full realization of B cell tolerance