Fuyi Liao

TGF-β-activating integrins promote solid-organ fibrosis, suggesting their use as a molecular marker of disease. Chronic lung allograft dysfunction (CLAD), a progressive fibrotic complication that limits lung transplant survival, is driven by intragraft TGF-β activation. However, the expression patterns of TGF-β-activating integrins remain undefined in lung transplants. Single-cell RNA sequencing in a mouse CLAD model revealed high levels of the TGF-β-activating integrin αvβ6, which was mainly localized to fibrosis-associated Krt8+ transitional alveolar cells (AT1/2), while tolerant transplants lacked both αvβ6 expression and Krt8+AT1/2 cells. Molecular imaging with a newly developed positron emission tomography radiotracer specific for αvβ6, [64Cu]Cu-DOTA-A20-K16R, showed significantly higher uptake in CLAD versus tolerant transplants. In contrast, [64Cu]Cu-DOTA-A20-K16R allograft uptake was reduced by treatments that lowered αvβ6 expression and CLAD severity. Finally, [64Cu]Cu-DOTA-A20-K16R autoradiographic analysis on human explanted lungs with CLAD showed elevated activity that correlated with αvβ6 expression. Collectively, these findings demonstrate the potential utility of αvβ6 molecular imaging to detect CLAD pathogenesis.

How pathogens inhibit transplant tolerance remains unclear. Here, we found that Pseudomonas aeruginosa infection, but not other common bacterial respiratory infections, increases antibody-mediated rejection (AMR) risk in recipients of lung transplants. To explore this relationship, we performed orthotopic lung transplants in mice, infected recipients with P. aeruginosa , and observed for the development of AMR. Intravital two-photon microscopy showed that P. aeruginosa rapidly invaded bronchial-associated lymphoid tissues, which resulted in acute lymphocytotoxicity, including the death of forkhead box P3 (Foxp3) ⁺ CD4 ⁺ T cells that are required to suppress AMR. P. aeruginosa –mediated AMR required expression of the type III secretion system (T3SS), which injects exotoxins into the cell cytoplasm. Through a combination of mutagenesis and epitope tagging experiments, we revealed that T3SS exotoxin T ADP ribosyl-transferase activity was sufficient for graft-resident Foxp3 ⁺ CD4 ⁺ T cell apoptosis, leading to myeloid differentiation primary response 88 (Myd88)–dependent generation of T-box expressed in T cells (T-bet)– and C-X-C motif chemokine receptor 3 (CXCR3)–positive germinal center and memory B cells with high donor antigen avidity. We also found that T-bet ⁺ and CXCR3 ⁺ B cells were elevated in biopsies from recipients of lung transplants who were diagnosed with AMR. In mice, CXCR3 deficiency restricted to B cells or CXCR3 blockade prevented AMR despite P. aeruginosa infection. Our work has identified a previously unrecognized role of bacterial virulence in lung allograft rejection and suggests potential strategies to prevent AMR for those at high risk of P. aeruginosa infection after transplant.

A greater understanding of chronic lung allograft dysfunction (CLAD) pathobiology, the primary cause of mortality after lung transplantation, is needed to improve outcomes. The complement system links innate to adaptive immune responses and is activated early post-lung transplantation to form the C3 convertase, a critical enzyme that cleaves the central complement component C3. We hypothesized that LTx recipients with a genetic predisposition to enhanced complement activation have worse CLAD-free survival mediated through increased adaptive alloimmunity. We interrogated a known functional C3 polymorphism (C3R102G) that increases complement activation through impaired C3 convertase inactivation in two independent LTx recipient cohorts. C3R102G, identified in at least one out of three LTx recipients, was associated with worse CLAD-free survival, particularly in the subset of recipients who developed donor-specific antibodies (DSA). In a mouse orthotopic lung transplantation model, impaired recipient complement regulation resulted in more severe obstructive airway lesions when compared to wildtype controls, despite only moderate differences in graft-infiltrating effector T cells. Impaired complement regulation promoted the intragraft accumulation of memory B cells and antibody-secreting cells, resulting in increased DSA levels. In summary, genetic predisposition to complement activation is associated with B cell activation and worse CLAD-free survival. BRIEF SUMMARY: Lung transplant recipients genetically predisposed to impaired complement regulation demonstrate worse chronic rejection-free survival. This phenotype is associated with intragraft B cell-activation and donor-specific antibodies.