An age-progressive platelet differentiation path from hematopoietic stem cells causes exacerbated thrombosis.

Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.

IL7Rα, but not Flk2, is required for hematopoietic stem cell reconstitution of tissue-resident lymphoid cells.

Tissue-resident lymphoid cells (TLCs) span the spectrum of innate-to-adaptive immune function. Unlike traditional, circulating lymphocytes that are continuously generated from hematopoietic stem cells (HSCs), many TLCs are of fetal origin and poorly generated from adult HSCs. Here, we sought to further understand murine TLC development and the roles of Flk2 and IL7Rα, two cytokine receptors with known function in traditional lymphopoiesis. Using Flk2- and Il7r-Cre lineage tracing, we found that peritoneal B1a cells, splenic marginal zone B (MZB) cells, lung ILC2s and regulatory T cells (Tregs) were highly labeled. Despite high labeling, loss of Flk2 minimally affected the generation of these cells. In contrast, loss of IL7Rα, or combined deletion of Flk2 and IL7Rα, dramatically reduced the number of B1a cells, MZBs, ILC2s and Tregs, both in situ and upon transplantation, indicating an intrinsic and essential role for IL7Rα. Surprisingly, reciprocal transplants of wild-type HSCs showed that an IL7Rα-/- environment selectively impaired reconstitution of TLCs when compared with TLC numbers in situ. Taken together, our data defined Flk2- and IL7Rα-positive TLC differentiation paths, and revealed functional roles of Flk2 and IL7Rα in TLC establishment.

The lymphoid-associated interleukin 7 receptor (IL7R) regulates tissue-resident macrophage development.

The discovery of a fetal origin for tissue-resident macrophages (trMacs) has inspired an intense search for the mechanisms underlying their development. Here, we performed in vivo lineage tracing of cells with an expression history of IL7Rα, a marker exclusively associated with the lymphoid lineage in adult hematopoiesis. Surprisingly, we found that Il7r-Cre labeled fetal-derived, adult trMacs. Labeling was almost complete in some tissues and partial in others. The putative progenitors of trMacs, yolk sac (YS) erythromyeloid progenitors, did not express IL7R, and YS hematopoiesis was unperturbed in IL7R-deficient mice. In contrast, tracking of IL7Rα message levels, surface expression, and Il7r-Cre-mediated labeling across fetal development revealed dynamic regulation of Il7r mRNA expression and rapid upregulation of IL7Rα surface protein upon transition from monocyte to macrophage within fetal tissues. Fetal monocyte differentiation in vitro produced IL7R+ macrophages, supporting a direct progenitor-progeny relationship. Additionally, blockade of IL7R function during late gestation specifically impaired the establishment of fetal-derived trMacs in vivo These data provide evidence for a distinct function of IL7Rα in fetal myelopoiesis and identify IL7R as a novel regulator of trMac development.

Targeted hematopoietic stem cell depletion through SCF-blockade.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) is a curative treatment for many diverse blood and immune diseases. However, HSCT regimens currently commonly utilize genotoxic chemotherapy and/or total body irradiation (TBI) conditioning which causes significant morbidity and mortality through inducing broad tissue damage triggering infections, graft vs. host disease, infertility, and secondary cancers. We previously demonstrated that targeted monoclonal antibody (mAb)-based HSC depletion with anti(α)-CD117 mAbs could be an effective alternative conditioning approach for HSCT without toxicity in severe combined immunodeficiency (SCID) mouse models, which has prompted parallel clinical αCD117 mAbs to be developed and tested as conditioning agents in clinical trials starting with treatment of patients with SCID. Subsequent efforts have built upon this work to develop various combination approaches, though none are optimal and how any of these mAbs fully function is unknown. METHODS: To improve efficacy of mAb-based conditioning as a stand-alone conditioning approach for all HSCT settings, it is critical to understand the mechanistic action of αCD117 mAbs on HSCs. Here, we compare the antagonistic properties of αCD117 mAb clones including ACK2, 2B8, and 3C11 as well as ACK2 fragments in vitro and in vivo in both SCID and wildtype (WT) mouse models. Further, to augment efficacy, combination regimens were also explored. RESULTS: We confirm that only ACK2 inhibits SCF binding fully and prevents HSC proliferation in vitro. Further, we verify that this corresponds to HSC depletion in vivo and donor engraftment post HSCT in SCID mice. We also show that SCF-blocking αCD117 mAb fragment derivatives retain similar HSC depletion capacity with enhanced engraftment post HSCT in SCID settings, but only full αCD117 mAb ACK2 in combination with αCD47 mAb enables enhanced donor HSC engraftment in WT settings, highlighting that the Fc region is not required for single-agent efficacy in SCID settings but is required in immunocompetent settings. This combination was the only non-genotoxic conditioning approach that enabled robust donor engraftment post HSCT in WT mice. CONCLUSION: These findings shed new insights into the mechanism of αCD117 mAb-mediated HSC depletion. Further, they highlight multiple approaches for efficacy in SCID settings and optimal combinations for WT settings. This work is likely to aid in the development of clinical non-genotoxic HSCT conditioning approaches that could benefit millions of people world-wide.

Clearing the Haze: How Does Nicotine Affect Hematopoiesis before and after Birth?

Hematopoiesis is a tightly regulated process orchestrated by cell-intrinsic and cell-extrinsic cues. Over the past several decades, much effort has been focused on understanding how these cues regulate hematopoietic stem cell (HSC) function. Many endogenous key regulators of hematopoiesis have been identified and extensively characterized. Less is known about the mechanisms of long-term effects of environmental toxic compounds on hematopoietic stem and progenitor cells (HSPCs) and their mature immune cell progeny. Research over the past several decades has demonstrated that tobacco products are extremely toxic and pose huge risks to human health by causing diseases like cancer, respiratory illnesses, strokes, and more. Recently, electronic cigarettes have been promoted as a safer alternative to traditional tobacco products and have become increasingly popular among younger generations. Nicotine, the highly toxic compound found in many traditional tobacco products, is also found in most electronic cigarettes, calling into question their purported "safety". Although it is known that nicotine is toxic, the pathophysiology of disease in exposed people remains under investigation. One plausible contributor to altered disease susceptibility is altered hematopoiesis and associated immune dysfunction. In this review, we focus on research that has addressed how HSCs and mature blood cells respond to nicotine, as well as identify remaining questions.

Interleukin 7 receptor is required for myeloid cell homeostasis and reconstitution by hematopoietic stem cells.

Respiratory diseases are a leading cause of death worldwide, with vulnerability to disease varying greatly between individuals. The reasons underlying disease susceptibility are unknown, but there is often a variable immune response in lungs often. Recently, we identified a surprising novel role for the interleukin 7 receptor (IL7R), a primarily lymphoid-associated regulator, in fetal-specified, lung-resident macrophage development. Here, we report that traditional, hematopoietic stem cell-derived myeloid cells in the adult lung, peripheral blood, and bone marrow also depend on IL7R expression. Using single- and double-germline knockout models, we found that eosinophil numbers were reduced on deletion of IL7Rα. We then employed two Cre recombinase models in lineage tracing experiments to test whether these cells developed through an IL7Rα+ pathway. Despite the impact of IL7Rα deletion, IL7R-Cre labeled only a minimal fraction of eosinophils. We therefore examined the intrinsic versus extrinsic requirement for IL7R in the production of eosinophils using reciprocal hematopoietic stem cell transplantation assays. These assays revealed that extrinsic, but not eosinophil-intrinsic, IL7R is required for eosinophil reconstitution by HSCs in the adult lung. To determine which external factors may be influencing eosinophil development and survival, we performed a cytokine array analysis between wild-type and IL7Rα-deficient mice and found several differentially regulated proteins. These findings expand on our previous report that IL7R is required not only for proper lymphoid cell development and homeostasis, but also for myeloid cell homeostasis in tissues.

Chasing Mavericks: The quest for defining developmental waves of hematopoiesis.

Hematopoiesis is the process by which mature blood and immune cells are produced from hematopoietic stem and progenitor cells (HSCs and HSPCs). The last several decades of research have shed light on the origin of HSCs, as well as the heterogeneous pools of fetal progenitors that contribute to lifelong hematopoiesis. The overarching concept that hematopoiesis occurs in dynamic, overlapping waves throughout development, with each wave contributing to both continuous and developmentally limited cell types, has been solidified over the years. However, recent advances in our ability to track the production of hematopoietic cells in vivo have challenged several long-held dogmas on the origin and persistence of distinct hematopoietic cell types. In this review, we highlight emerging concepts in hematopoietic development and identify unanswered questions.