Authorship note: KJL and DK are co-senior authors. Conflict of interest: DK serves on The Scientific Advisory Board of and receives research support from Compass Therapeutics. DK and KJL have a pending patent entitled "Compositions and methods for detecting CCR2 receptors" (US patent application no. 15/611,577).

No abstract

Ischemia-reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short-term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that CCR2+ classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia-reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88-dependent IL-1β production by graft-infiltrating classical monocytes, which downregulated the expression of the tight junction-associated protein ZO-2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia-reperfusion injury in the clinic.

Immunological requirements for rejection and tolerance induction differ between various organs. While memory CD8 T cells are considered a barrier to immunosuppression-mediated acceptance of most tissues and organs, tolerance induction after lung transplantation is critically dependent on central memory CD8 T lymphocytes. Here we demonstrate that costimulation blockade-mediated tolerance after lung transplantation is dependent on programmed cell death 1 (PD-1) expression on CD8 T cells. In the absence of PD-1 expression, CD8 T cells form prolonged interactions with graft-infiltrating CD11c cells; their differentiation is skewed towards an effector memory phenotype and grafts are rejected acutely. These findings extend the notion that requirements for tolerance induction after lung transplantation differ from other organs. Thus, immunosuppressive strategies for lung transplant recipients need to be tailored based on the unique immunological properties of this organ.

| INTRODUC TI ONBronchus-associated lymphoid tissue (BALT) is a pulmonary tertiary lymphoid organ (TLO) that plays a critical role in the inflammatory response to a range of lung pathology, including infection, autoimmunity, and allograft rejection. 1-3 However, recent evidence has emerged that pulmonary TLOs are not synonymous with the generation of deleterious proinflammatory responses. [4][5][6][7] To this end, the development of BALT has been associated with the downregulation of immune responses. Tumor-associated TLOs are one such example, which have been found in both human and mouse models of lung cancer. 4 Pulmonary tumor-associated TLOs are enriched in Foxp3 + regulatory T contributed equally to this manuscript and share first authorship Abbreviations: AHR, aryl hydrocarbon receptor; BALT, bronchus-associated lymphoid tissue; H&E, hematoxylin and eosin; HEV, high endothelial venule; ILC, innate lymphoid cell; NALT, nasal-associated lymphoid tissue; PNAd, peripheral nodal addressin; RORγt, retinoic acid receptor-related orphan receptor gamma t; SLO, secondary lymphoid organ; TLO, tertiary lymphoid organ; γδ, gamma-delta. Long-term survival after lung transplantation remains profoundly limited by graft rejection. Recent work has shown that bronchus-associated lymphoid tissue (BALT), characterized by the development of peripheral nodal addressin (PNAd)-expressing high endothelial venules and enriched in B and Foxp3 + T cells, is important for the maintenance of allograft tolerance. Mechanisms underlying BALT induction in tolerant pulmonary allografts, however, remain poorly understood. Here, we show that the development of PNAd-expressing high endothelial venules within intragraft lymphoid follicles and the recruitment of B cells, but not Foxp3 + cells depends on IL-22. We identify graft-infiltrating gamma-delta (γδ) T cells and Type 3 innate lymphoid cells (ILC3s) as important producers of IL-22. Reconstitution of IL-22 at late time points through retransplantation into wildtype hosts mediates B cell recruitment into lymphoid follicles within the allograft, resulting in a significant increase in their size, but does not induce PNAd expression. Our work has identified cellular and molecular requirements for the induction of BALT in pulmonary allografts during tolerance induction and may provide a platform for the development of new therapies for lung transplant patients. K E Y W O R D S animal models: murine, basic (laboratory) research/science, immunobiology, lung transplantation/pulmonology, lymph node, lymphocyte biology, tolerance: experimental