Pseudomonas Aeruginosa Infection of Lung Transplants Breaks Established Tolerance through B Cell-Intrinsic Innate Immune Signaling That Promotes Intragraft Plasma Cell Generation and Donor Specific Antibody Accumulation

Abstract

Antibody mediated rejection (AMR) has become an increasingly recognized form of lung transplant rejection. Our group recently identified Pseudomonas aeruginosa infection as a risk factor for clinical AMR but whether infection is sufficient for donor specific antibody (DSA) production and AMR remains unknown. Here we ask in a mouse model of orthotopic lung allograft transplantation whether airway infection with P. aeruginosa promotes DSA generation and AMR. Balb/c lungs were transplanted into the following recipients: B6 WT (wildtype), uMT KO (No B cells), AID/uS KO (Have B cells but are unable to secrete Ig), CD19 Cre , CD19 Cre x TLR4fl/fl and CD19 Cre x MyD88fl/fl. Lung recipients were immunosuppressed with costimulatory blockade on post-operative day (POD) 0 (CD40L Ab) and POD 2 (CTLA4Ig) to induce allograft acceptance. On POD 21 recipients received intratracheal saline or 2 x 105 pfu/g body weight of live P. aeruginosa (clinical isolate M57-15) and then treated with ciprofloxacin from POD 22 to 25. On POD 42 peripheral blood was assessed for DSA and graft tissue evaluated for C4d deposition or quantitated for plasma cells (PC) by flow cytometric analysis (FACS). P. Aeruginosa infection stimulated high amounts of DSA generation and marked C4d deposition. In contrast, C4d deposition was nearly absent in uMT and AID/uS KO recipients. Infected recipients with TLR4 or MyD88 targeted-deficiency in B cells had sharply lower DSA when compared to WT controls. P. aeruginosa infection drove a 4-fold expansion of allograft resident PCs that was largely dependent on maintaining B cell TLR4-MyD88 signaling. In contrast, the spleen and bone marrow had only modest changes in PC abundance despite infection. Surprisingly, re-transplantation of P. aeruginosa-infected lung allografts into secondary uMT KO recipients revealed that lung allograft-resident PCs are capable of producing large amounts of DSA. Collectively, these data show that P. aeruginosa infection abrogates established lung transplant tolerance through engaging innate immune signaling pathways within the B cell compartment. Additionally, we make the novel observation that the lung is a suitable environment for PC development with implications for clinical AMR.