Significance Intravital imaging, oxidative lipidomics, and a transplant model were used to define mechanisms that regulate neutrophil recruitment into lungs during ischemia reperfusion injury, a clinically relevant form of sterile inflammation. We found that early neutrophil-mediated damage is largely confined to the subpleural vasculature, a process that is orchestrated by a spatially restricted distribution of nonclassical monocytes that produce chemokines following necroptosis of pulmonary cells. Neutrophils disrupt the integrity of subpleural capillaries, which is associated with impaired lung function. Neutrophil-mediated vascular leakage is dependent on TLR4 expression on vascular endothelium, NOX4 signaling, and formation of neutrophil extracellular traps. Our research provides insights into mechanisms that regulate neutrophil recruitment during sterile lung inflammation and lays the foundation for developing new therapies.

OBJECTIVES Spread through air space (STAS) is recognized as a pattern of invasion in lung adenocarcinoma and has been reported to be a predictor of recurrence and survival in patients with early-stage lung adenocarcinoma. However, this parameter has not been studied well in stage III (N2) lung adenocarcinoma. In this study, we evaluated the association between STAS invasion patterns and recurrence and survival in stage III (N2) lung adenocarcinoma. METHODS We retrospectively reviewed data from 76 patients at University of Tokyo with stage III (N2) lung adenocarcinoma who underwent surgery from August 1998 to December 2013. Statistical analysis was performed to determine the impact of STAS invasion clinicopathological features and clarify the relationship between this pattern of invasion and survival. RESULTS Tumour STAS was observed in 46 of 76 patients (60.5%) and was significantly associated with the presence of lymphatic invasion (P < 0.001), papillary components (P < 0.001) and micropapillary components (P < 0.001). STAS was also significantly associated with recurrence (5-year recurrence-free probability: 19.0% vs 46.1%, P < 0.05). Univariate analyses showed that STAS was a significant risk factor for recurrence (hazard ratio 1.94, 95% confidence interval 1.07–3.51; P = 0.029). CONCLUSIONS The presence of STAS invasion pattern is a significant risk factor for recurrence in stage III (N2) lung adenocarcinoma.

| 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

Background: Cellular rejection after heart transplantation imparts significant morbidity and mortality. Current immunosuppressive strategies are imperfect, target recipient T cells, and have adverse effects. The innate immune response plays an essential role in the recruitment and activation of T cells. Targeting the donor innate immune response would represent the earliest interventional opportunity within the immune response cascade. There is limited knowledge about donor immune cell types and functions in the setting of cardiac transplantation, and no current therapeutics exist for targeting these cell populations. Methods: Using genetic lineage tracing, cell ablation, and conditional gene deletion, we examined donor mononuclear phagocyte diversity and macrophage function during acute cellular rejection of transplanted hearts in mice. We performed single-cell RNA sequencing on donor and recipient macrophages and monocytes at multiple time points after transplantation. On the basis of our imaging and single-cell RNA sequencing data, we evaluated the functional relevance of donor CCR2 + (C-C chemokine receptor 2) and CCR2 − macrophages using selective cell ablation strategies in donor grafts before transplant. Last, we performed functional validation that donor macrophages signal through MYD88 (myeloid differentiation primary response protein 88) to facilitate cellular rejection. Results: Donor macrophages persisted in the rejecting transplanted heart and coexisted with recipient monocyte-derived macrophages. Single-cell RNA sequencing identified donor CCR2 + and CCR2 − macrophage populations and revealed remarkable diversity among recipient monocytes, macrophages, and dendritic cells. Temporal analysis demonstrated that donor CCR2 + and CCR2 − macrophages were transcriptionally distinct, underwent significant morphologic changes, and displayed unique activation signatures after transplantation. Although selective depletion of donor CCR2 − macrophages reduced allograft survival, depletion of donor CCR2 + macrophages prolonged allograft survival. Pathway analysis revealed that donor CCR2 + macrophages are activated through MYD88/nuclear factor kappa light chain enhancer of activated B cells signaling. Deletion of MYD88 in donor macrophages resulted in reduced antigen-presenting cell recruitment, reduced ability of antigen-presenting cells to present antigen to T cells, decreased emergence of allograft-reactive T cells, and extended allograft survival. Conclusions: Distinct populations of donor and recipient macrophages coexist within the transplanted heart. Donor CCR2 + macrophages are key mediators of allograft rejection, and deletion of MYD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograft survival. This highlights the therapeutic potential of donor heart–based interventions.