*Purpose: The allogeneic humoral immune response is largely composed of T cell dependent antibody responses produced after B and T cells interact within the germinal center (GC) microenvironment of lymphoid tissues. The purpose of this study was to generate an in vitromodel of the GC reaction and examine GC B cell differentiation in naive and sensitized nonhuman primates (NHPs).

*Methods: CD20+ B cells and CD3+ T cells were isolated by magnetic bead separation from the peripheral blood of naive and skin allograft sensitized MHC-mismatched pairs of NHPs. Allogeneic B and T cells were cultured in plates pre-coated with murine stromal cells engineered to express human CD40L. Cells and supernatants were harvested every 2 days for immune phenotyping by flow cytometry and for measurement of IgG levels by ELISA.

*Results: Activation of the cultured B cells was assessed by MHC II and CD86 expression, which was highest at day 4 in both naive and sensitized recipients. However, CD86 expression remained significantly elevated at day 8 in the sensitized GC culture compared to naive (Figure A, p<0.05). The proportion of naive CD27-IgD+ B cells in the cultures decreased from 55% at day 2 to 5% by day 8 in both naive and sensitized cultures. However, the sensitized cultures demonstrated a more rapid maturation of B cells with significantly lower frequencies of naive B cells as early as day 4 (13.3% vs. 25.6%, p<0.05). In addition, the differentiation of B cells into CD20-CD38+ plasmablasts was significantly higher in sensitized cultures as evidenced by greater frequencies of plasmablasts at day 8 (Figure B, p<0.05). Total IgG production in the culture supernatants significantly increased over time from 2.5 ng/mL at day 2 to 6100 ng/mL at day 8 and was similar in both naive and sensitized cultures.

*Conclusions: These results demonstrate an efficient and reliable method for modeling GC reactions in vitrowith the ability to monitor B cell activation, differentiation, and antibody production over time. This in vitro platform provides functional information that can quantify different levels of allosensitization in nonhuman primates and may serve as a useful adjunct to conventional donor specific antibody detection in evaluating sensitized recipients.

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