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Introduction Platelets serve dual roles as a critical component of clot formation and as regulators of immune responses (1, 2). Activated platelets promote inflammation, while circulating resting platelets limit inflammation [...], Lung megakaryocytes (Mks) are largely extravascular with an immune phenotype (1). Because bone marrow (BM) Mks are short lived, it has been assumed that extravascular lung Mks are constantly "seeded" from the BM. To investigate lung Mk origins and how origin affects their functions, we developed methods to specifically label lung Mks using CFSE dye and biotin delivered via the oropharyngeal route. Labeled lung Mks were present for up to 4 months, while BM Mks had a lifespan of less than 1 week. In a parabiosis model, lung Mks were partially replaced over 1 month from a circulating source. Unlike tissue-resident macrophages, using [MDS1.sup.-Cre-ERT2] TdTomato mice, we found that lung Mks arose from hematopoietic stem cells. However, studies with FlkSwitch mTmG mice showed that lung Mks were derived from a Flt3-independent lineage that did not go through a multipotent progenitor. CFSE labeling to track lung Mk-derived platelets showed that approximately 10% of circulating platelets were derived from lung-resident Mks at steady state, but in sterile thrombocytopenia this was doubled (~20%). Lung-derived platelets were similarly increased in a malaria infection model (Plasmodium yoelii) typified by thrombocytopenia. These studies indicate that lung Mks arise from a Flt3- BM source, are long-lived, and contribute more platelets during thrombocytopenia.

Introduction Platelets are most commonly described as the megakaryocyte-derived (Mk-derived) cellular mediators of thrombosis. Mk differentiation and platelet production have been extensively studied in the bone marrow (BM) environment, but [...], Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow-resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term [Mk.sub.L], had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an [Mk.sub.L]-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that [Mk.sub.L] internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, [Mk.sub.L] induced [CD4.sup.+] T cell activation in an MHC II-dependent manner both in vitro and in vivo. These data indicated that [Mk.sub.L] had key immune regulatory roles dictated in part by the tissue environment.