N. M. Bath1, B. M. Verhoven1, N. A. Wilson1, R. R. Redfield1 1University Of Wisconsin,Transplant,Madison, WI, USA
Introduction: APRIL (A proliferation inducing ligand) and BLyS (B Lymphocyte Stimulator) are two critical survival factors for B lymphocyte maturation 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 antibody mediated rejection (AMR) in kidney transplant. Here we report our initial phenotyping and response to alloantigen in these novel rodents.
Methods: Using CRISPR/Cas9 we engineered APRIL-/- and BLyS-/- Lewis rats. Collected tissues were analyzed using flow cytometry, ELISPOT, and immunohistochemistry. APRIL-/- and BLyS-/- rats were sensitized with Brown Norway (BN) blood (complete MHC mismatch). Flow cross match and a 3 day mixed lymphocyte reaction (MLR) was performed with wild type (WT), APRIL-/-, and BLyS-/- rats to assess DSA (Donor Specific Antibody) production and cell proliferation, respectively.
Results: When challenged with alloantigen, sensitized BLyS-/- had significant decreases in DSA when compared to WT and APRIL-/-. MLR demonstrated a significant decrease in BLyS-/- cell proliferation when challenged by BN splenocytes compared to APRIL-/- and WT (p<0.02). Additionally, BLyS-/- significantly depleted antibody secreting cell production of IgM and IgG in all tissues compared to WT and APRIL-/- (p<0.04). BLyS-/- demonstrated a significant reduction of splenic marginal zone B lymphocytes detected by anti-PAX5 compared to both WT and APRIL-/- (p<0.0001).
Conclusion: BLyS-/- produced fewer alloantibodies and demonstrated a significant reduction in cell proliferation when challenged with alloantigen. Antibody secreting B lymphocytes and splenic germinal centers are also depleted in BLyS-/-, which translates into a reduction of alloantibody production. Future studies will characterize rodents deficient in both APRIL and BLyS and apply this to kidney transplant model as a method to prevent AMR.