Maintenance of tolerance after lung transplantation is dependent on the preservation of allograft tissue-resident Foxp3+ T cells

Mechanisms of allograft tolerance differ between organs. We have previously shown that Foxp3+ T cell (Treg)-enriched bronchus-associated lymphoid tissue is induced in tolerant murine lung allografts and that graft-resident Tregs suppress B cell activation to maintain tolerance. Here, using our previously described retransplant model, we examined whether continuous recruitment of Tregs to the allograft is required to maintain tolerance. We found that graft-infiltrating Tregs migrate to BALT where they interact with graft-resident CD11c+ and Foxp3+ cells. Graft-infiltrating and graft-resident Tregs are likely of thymic origin but have different gene expression profiles at the single cell level. Following retransplantation of a tolerized left lung graft into a non-immunosuppressed B6.Foxp3-DTR secondary recipient, we depleted graft-infiltrating Tregs by administering diphtheria toxin. When graft-infiltrating Tregs are depleted, the lung allograft has gross and histological evidence of rejection and is associated with B cell activation and development of donor-specific alloantibodies in the serum. These phenotypes are rescued when B cell-depleting anti-CD20 antibody is administered, further supporting an antibody-mediated mechanism by which graft-infiltrating Tregs maintain tolerance. Finally, we show that recombinant IL-33 administration can be used to expand the Treg population following murine lung transplant. Overall design: Lungs from a BALB/c donor transplanted into an immunosuppressed B6 (CD45.2) primary recipient, and then retransplanted into a B6 (CD45.1) secondary recipient at least 30 days later were harvested at 7 days post-retransplant. Lung tissue was digested, and single cell suspensions were prepared. Cells from two transplanted lungs were pooled together. CD45.1+, CD45.2+, and CD45- cells were sorted by flow cytometry to create three separate libraries for single cell RNA sequencing.