*Purpose: Antibody mediated rejection (AMR) is a major cause of kidney allograft failure. APRIL (A proliferation inducing ligand) and BLyS (B Lymphocyte Stimulator) are two critical survival factors for B lymphocytes and plasma cells, the main source of alloantibody. We generated rats deficient in APRIL and BLyS to characterize the effects of targeting these cytokines in our established rodent model of AMR in kidney transplant. Previously we reported the initial phenotyping of APRIL^-/- and BLyS^-/- Lewis rats. Here we describe the phenotype and response to alloantigen in novel dual knockout rats, APRIL^-/-/BLyS^-/- Lewis rats.

*Methods: Using CRISPR/Cas9 we engineered APRIL^-/- and BLyS^-/- Lewis rats. We subsequently mated APRIL^-/- and BLyS^-/- rats to create dual APRIL^-/-/BLyS^-/- Lewis rats. Genotype was confirmed using RT-PCR. APRIL^-/-/BLyS^-/- rats were sensitized with Brown Norway blood (complete MHC mismatch). Tissues were analyzed using flow cytometry and ELISPOT. Donor Specific Antibody (DSA) was measured by CD45R⁺ and CD3⁺ flow cross match (sum MFI of IgG1, IgG2a, IgG2c) against Brown Norway splenocytes from sensitized wild type (WT), APRIL^-/-, BLyS^-/-, and APRIL^-/-/BLyS^-/- rats.

*Results: Sensitized APRIL^-/-/BLyS^-/- significantly depleted spleen and lymph node IgM secreting cells and spleen and bone marrow IgG secreting cells compared to WT (P<0.03). When challenged with alloantigen, sensitized APRIL^-/-/BLyS^-/- had significantly lower DSA production compared to WT and APRIL^-/- in CD45R⁺ and CD3⁺ flow cross matches (P<0.04). APRIL^-/-/BLyS^-/- demonstrated significantly fewer marginal zone B lymphocytes (CD45RA⁺HIS57⁺) in spleen and bone marrow than WT (P<0.05). Naïve B cells (CD3^–CD45R⁺IgD⁺CD27^–) and transitional zone B cells (CD3^–CD45R⁺IgD⁺CD38⁺CD24⁺⁺) were significantly decreased in APRIL^-/-/BLyS^-/- compared to WT and APRIL^-/- (P<0.03). Plasma cells (CD3^–CD45R^–IgD^–IgM^–CD27⁺CD38⁺) were reduced in APRIL^-/-/BLyS^-/- compared to all other groups but was not significant. No significant differences in B lymphocyte subsets or DSA production were noted between BLyS^-/- and APRIL^-/-/BLyS^-/-.

*Conclusions: Sensitized APRIL^-/-/BLyS^-/- significantly depleted IgM and IgG secreting cells, which is further demonstrated by a decrease in DSA production when challenged with alloantigen. Additional phenotypic differences were seen in mature B lymphocyte subsets, which suggests an arrest in development past the immature B lymphocyte stage. Future studies will apply this model to a rodent kidney transplant model as means to prevent AMR.

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