Abstract LB-152: Complement promotes induction of Foxp3+ T regulatory cells by B lymphoma cells and professional antigen-presenting cells

Abstract

Abstract Introduction: The complement system has complex interactions with the cellular immune system. Malignant B cells have been reported to enhance T regulatory (Treg) cell activity. However, the effect of complement on the Treg cells is not known. The objectives of the current study were to evaluate whether complement can enhance Treg induction by either malignant B cells or professional antigen-presenting cells (APCs) and to analyze its role on the development of active immune responses. Methods: Peripheral blood mononuclear cells (PBMCs) or enriched CD4+ T cells from healthy donors were co-cultured with Raji B lymphoma cells or enriched myeloid dendritic cells (mDCs). These cells were treated with either unmodified autologous human serum, as a source of complement, or serum from which complement had been inhibited by heat-inactivation (HI), cobra-venom factor (CVF) or soluble complement receptor 1 (sCR1) treatment. Co-cultures to which purified C5a protein was added were also evaluated. Following additional incubation, percent CD4+ T cells expressing the Treg phenotype (CD3+CD4+CD25highFoxp3+) was evaluated by flow cytometry. Treg function was determined by analyzing their inhibitory effect on mitogen-stimulated CD8+ T cells. To assess the in vivo effect of complement on the development of an active immune response, C57Bl/6 mice were immunized with single, subcutaneous injection of a model antigen, ovalbumin (100 μg/mice), mixed with complement-inhibiting agents and anti-ovalbumin antibody response was monitored by ELISA. Results 1. Raji B lymphoma cells induced significantly higher percentages of Tregs in co-cultured PBMCs in the presence of unmodified human serum (p=0.038) when compared to HI or CVF or sCR1-treated human serum (p=0.037, 0.022 and 0.0002, respectively). 2. Treg induction was also increased when CD4+ T cells were co-cultured with mDCs in the presence of serum complement or purified C5a protein (p=0.002, p=0.039, respectively). 3. Treg function, as indicated by suppression of granzyme B production by mitogen-treated CD8+ T cells, was reduced by complement depletion. 4. In vivo, local complement inhibition at the site of immunization resulted in significantly higher anti-ovalbumin IgM responses (inhibition vs no inhibition – p<0.05). Conclusion: Serum complement, including C5a, promotes induction of Treg cells by B lymphoma cells and APCs, such as mDCs, which can be reduced by inhibition of serum complement. In vivo complement inhibition enhances active immune responses. These studies suggest a immune-regulatory role for complement proteins in lymphoma and that local complement depletion could be used as a novel strategy to boost active immune responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-152. doi:10.1158/1538-7445.AM2011-LB-152