B cells, and in particular, memory B cells (Bmem) are sources of donor specific antibodies (DSA) that cause allograft injury. Understanding requirements for Bmem function/survival could guide novel developing therapies aimed at preventing/treating DSA-mediated allograft injury. Expanding upon published work in anti-viral immunity, we tested the hypothesis that autophagy is required for function/survival of donor-reactive Bmem. We observed 4-25 fold higher levels of autophagy genes (p<0.05) and more autophagosome+ cells (62.4% vs 35.1% p<0.05) in IgG+ vs IgG- B cells. To specifically analyze autophagy in memory B cells we sensitized mice expressing a tamoxifen inducible YFP reporter under the Activation-Induced Cytidine Deaminase (AID) promoter (AID-CreERT2; Rosa-YFP transgenic mice), and analyzed YFP+ B cells 5 mo later. Imaging flow analysis showed more autophagosomes in YFP+ Bmem vs YFP- B cells (p<0.05). Compared with WT B cells, B cells from conditional KO mice deficient in autophagy protein 7 (CD19-Cre ATG7KO) contained less activated autophagy protein LC3 and showed impaired processing of autophagy substrate p62, confirming that absent ATG7 impairs autophagy in the B cells. Primary alloantibody responses were similar in ATG7KO and control mice 2 weeks after immunization with BALB/c splenocytes and fell equally over time. To assess secondary Bmem dependent responses we then challenged sensitized animals 4-5 mo post-sensitization with heart transplant from BALB/c donors and 7 days later measured serum alloantibodies and quantified donor specific splenic B cells by ELISpot. Whereas DSA at all titers increased significantly (2.46 to 5.07 fold) in sensitized control animals, titers in ATG7KO mice were minimally increased (1.18 to 1.47 fold p<0.05 vs controls, n=6/group). Frequencies of donor reactive B cells were 4-fold lower in the ATG7KO vs control heart recipients (21 vs 86/2.5X10⁶ splenocytes), supporting the hypothesis that B cell autophagy is required for Bmem survival/function. Similar results were observed in control animals treated with the pharmacological autophagy inhibitor 3-methyladenine vs vehicle (n=5/grp). Together the data show for the first time that alloreactive Bmem require autophagy for function/survival and demonstrate feasibility for therapeutically targeting autophagy to inhibit alloreactive B cells and thereby prevent/remove DSA.

CITATION INFORMATION: Leventhal J, Fribourg M, Ni J, Heeger P. Allo-Specific Memory B Cell Recall Responses Are Dependent on B Cell Autophagy. Am J Transplant. 2016;16 (suppl 3).

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