Objectives: Patients operationally tolerant to a kidney graft display a whole blood transcriptional B-cell signature. As these patients only rarely develop an allo-immune response, we hypothesized that they could display an abnormal B cell-response. Materials and methods: We used a controlled primary culture combining BCR signal, TLR activation, CD40 triggering, and cytokines to explore T-dependent differentiation of purified B cells into terminally differentiated plasma cells. CD20, CD24, CD27, CD38, and IgM/IgD expression was monitored by flow-cytometry. B-cell apoptosis and proliferation were measured using a dual active Caspase-3 and BrdU staining at day 0, 2, 4, and 6. Cytokine, Ig production, and markers of differentiation were followed using Flow-cytomix multiplex kits and RT-PCR. Results: Operationally tolerant recipients harbored a higher frequency of CD20+CD24hiCD38hi transitional B cells and CD20+CD38loCD24lo naive B cells compared to patients with stable graft function. These results correlated with a decreased frequency of CD20+CD27+ memory B cells and CD20-CD38+CD138+ terminally differentiated plasma cells, suggesting abnormal differentiation of B cells. When cultured in vitro, B cells from operationally tolerant patients proliferate normally in vitro but produce more IL10 than B cells from stable patients and healthy volunteers, whereas no difference was observed for TGFΒ and IL6 cytokines. Moreover, whereas they express normal level of PAX5, BACH2 and BCL6, B cells from tolerant recipients show a defective expression of IRF4, PRDM1, XBP1, three factors of the end step of differentiation into plasma cells. Finally, B cells from these patients harbored a susceptibility to cell death apoptosis. Conclusion: In culture, B cells from tolerant patients produce more IL10, display a lack of terminal differentiation and are more prone to cell death. To which extend this observation is involved in tolerance induction and/or maintenance remains to be established yet.

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