Complement Enhances Foxp3+ T Regulatory Cell Induction by B Cell Lymphoma

Abstract 5176

The effect of complement on the immune system is complex and includes inhibition of aspects of the immune response. For example, we previously reported that C3b can inhibit the ability of mAb to activate NK cell-mediated antibody-dependent cellular cytotoxicity and that polymorphisms in C1q associated with lower C1q levels correlate with better therapeutic outcome. Polymorphisms in C5 and C9 complement components have also been shown to be associated with lymphoma risk. The current studies were designed to assess whether complement components may exert their inhibitory effects through the immune regulatory network, such as that mediated by T regulatory (Treg) cells.

Peripheral Blood Mononuclear Cells (PBMCs) from healthy donors were co-cultured with Raji B cell lymphoma cells for 48 hours in the presence of unmodified autologous human serum or complement-depleted serum [heat-inactivated (HI) or cobra-venom factor (CVF)-treated serum]. Cells were stained with anti-CD3-APC-Cy7, CD4-PE-Cy7, CD25-PerCP-Cy5.5 and Foxp3-APC and the percent of CD4+ T-cells that were CD25^highFoxp3⁺ (henceforth referred to as Tregs) determined. The ability of cells cultured in this manner to inhibit granzyme B expression in PHA-activated CD8+ autologous T cells was also determined.

Tregs were not induced when PBMCs were cultured without Raji cells regardless of whether the cells were cultured in serum (data not shown). However, PBMCs co-cultured with Raji B lymphoma cells showed significantly higher percentages of Tregs in the presence of unmodified human serum (*p=0.0387) when compared to HI or CVF-treated human serum (**p=0.0370 and 0.0227, respectively) (Fig. 1). Tregs enriched from PBMCs cultured with unmodified human serum and Raji cells also showed an increase in immunosuppressive function, as determined by their inhibitory effect on PHA-induced GrzB expression by autologous CD8+ T cells (*p=0.0029) (Fig. 2).

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unmodified or complement-inactivated human

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unmodified or complement-inactivated human

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We conclude that complement enhances generation of Treg cells induced by B lymphoma cells as indicated by both phenotypic and functional studies. Ongoing studies are exploring the role of individual complement components in Treg induction, and the possibility of using complement depletion as a strategy to boost immune responses against lymphoma.