*Purpose: The formation of allogeneic antibody directed against non-self MHC, termed donor-specific antibody (DSA), is a significant barrier to successful transplant outcomes. Pre-formed DSA limits the number of compatible donors available to transplant candidates, while DSA formation after transplant causes antibody-mediated rejection. Although plasma cells are major source of DSA, the pathways by which B cells differentiate into plasma cells after encountering allogeneic antigen are poorly understood. We sought to understand which B cell populations are important for the formation of allogeneic IgG post-sensitization.

*Methods: To investigate the differentiation of allogeneic B cells, we used an in vivo model of sensitization in which H-2(b) C57Bl/6 mice are sensitized with H-2(d) Balb/c splenocytes. The formation of anti-H-2(d) IgG and IgM was assessed by flow cytometric crossmatch at days 7, 14, and 21. The phenotype of antigen-specific allogeneic B cells was assessed using H-2L(d)-specific tetramers on days 14 and 21 post sensitization with multicolor spectral flow cytometry.

*Results: Allogeneic anti-H-2(d) IgM levels peaked at day 7, while IgG was detected at day 7 and increased at days 14 and 21 following sensitization. At day 14, approximately half (55±9.5%) of H-2L(d)-specific B cells were IgM^lo. Few germinal center (GC) and post-GC CD73+ B cells were identified at day 14 and 21. However, the splenic H-2L(d)-specific B cells of sensitized mice showed an elevated frequency of CD23^loCD21^hi marginal zone (MZ) phenotype B cells at day 21 (naïve 10.4%, sensitized 23.7%, p=0.008). The MZ B cells elicited after sensitization were enriched for high PD-L2 and IRF4 expression, suggesting that they are antigen-experienced. Depletion of MZ B cells in vivo using a monoclonal antibody cocktail resulted in markedly reduced allogeneic IgG formation compared to the non-depleted group (p=0.009), demonstrating that MZ B cells are required for the majority of allogeneic IgG formation.

*Conclusions: B cells with a MZ phenotype are comprised of both innate-like and memory populations. Our results suggest that allogeneic antigen activates B cells with a marginal zone phenotype, and that this population is required for optimal formation of allogeneic IgG. Future experiments will seek to better understand the differentiation pathway that leads to the MZ phenotype post-sensitization, and to characterize phenotype of allogeneic B cells within the marginal zone.

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