*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: B6 (H-2^b) mice received fully MHC mismatched BALB/c (H-2^d) cardiac allografts. The shuttling of MZ B cells between spleen marginal zone and the follicle is regulated by balanced signals through S1PR1 and CXCR5. We used S1PR1 signaling inhibitor FTY720 to prevent MZ B cells return to the MZ area. Recipient FTY720 treatment did not alter the numbers of spleen B cells with MZ phenotype (CD21/35^hiIgM^hi), however spleen histology showed very few B220⁺ cells in the perifollicular area, consistent with their retention in the follicles. As FTY720 has many other effects besides affecting MZ B cell trafficking, we tested the role of MZ B cells in DSA production by using CD19^CreNotch2^fl/fl mice with defective development of MZ B cells (MZB KO) as heart allograft recipients. To further test MZ B cell contribution to DSA production, we treated WT B6 recipients of BALB/c heart allografts with anti-mouse CD20 18B12 IgG1 mAb that depletes >95% of splenic FO B cells but only 40-60% MZ B cells. We also used Blimp1 reporter mice as recipients to determine the origin antibody secreting cells early after heart transplantation.

*Results: DSA generation against both donor class I and II was markedly impaired in FTY720-treated WT heart allograft recipients. Similarly, DSA development in MZB KO recipients was significantly delayed compared to control littermates even though the priming of donor-reactive IFNγ producing T cells was comparable in both groups. Preferential FO B cell depletion reduced serum IgG DSA levels only at later time points. In contrast, DSA levels on d. 7 posttransplant were similar in 18B12 IgG1 treated and untreated groups indicating that these early IgG alloantibodies are produced by residual MZ B cells. Blimp1 was up-regulated in CD21/CD35^hi MZ B cells by day 7 posttransplant, consistent with their differentiation to early antibody secreting cells.

*Conclusions: Our results indicate that MZ B cells are required for optimal DSA responses to vascularized heart allografts. While MZ B cells are sufficient to support early IgG DSA production, the defects in DSA are not limited to early stages suggesting that MZ B cells orchestrate subsequent immune responses by FO B cells.

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