*Purpose: Production of high affinity isotype-switched donor specific alloantibodies (DSA) after transplantation is associated with germinal center formation by follicular (FO) B cells whereas the contribution of marginal zone (MZ) B cells is poorly understood. The goal of our study was to test the role of MZ B cells during alloimmune responses.

*Methods: To evaluate MZ B cell responses after transplantation, B6.Blimp-1 (H-2^b) reporter mice were transplanted with BALB/c (H-2^d) heart allografts. To test MZ B cells contribution to DSA production we treated B6.WT recipients of BALB/c heart allografts with anti-mCD20 18B12 IgG1 mAb that depletes >95% splenic FO but only 40-60% MZ B cells. Serum levels of class I and class II-reactive IgG and IgM DSA were determined by ELISA. We used S1PR1 signaling inhibitor FTY720 to prevent MZ B cells from returning to MZ area. To specifically test the role of MZ B cells in DSA production we used CD19^CreNotch2^fl/fl mice (ΔMZB) with defective development of MZ B cells as heart allograft recipients. In addition, sera collected fromΔMZB recipients and littermate controls on d.14 posttransplant was transferred to RAG1^-/- recipients of BALB/c heart allograft

*Results: Blimp1⁺ MZ B cells in recipient spleen peaked at d. 7 and declined by d. 14 posttransplant, suggesting their differentiation into early antibody secreting cells. Preferential FO B cell depletion reduced serum IgG DSA levels only at later time points (d. 14 and d. 21), indicating that early IgG DSA are produced by residual MZ B cells. CD4 T cells depletion prior to transplantation showed that these early MZ B cell responses were critically dependent on T cell help. Treatment of heart allograft recipients with FTY720 displaced MZ B cells from spleen MZ area and markedly impaired both class I and class II DSA generation. IgG and IgM DSA generation in ΔMZB recipients was significantly impaired compared to control littermates, even though the priming of donor-reactive IFNγ producing T cells was similar between groups. Transfer of littermate but not ΔMZB sera collected on d.14 posttransplant to RAG1^-/- BALB/c heart allograft recipients induced C4d complement deposition, indicating that MZ B cells are required to generate high levels of complement-activating DSA.

*Conclusions: Our findings are the first demonstration for the role of MZ B cells in humoral alloimmune responses following solid organ transplantation and identify MZ B cells as a potential therapeutic target for minimizing de novo DSA production and antibody-mediated rejection in transplant recipients.

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