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Hematopoietic stem cell (HSC)-independent lymphopoiesis has been elucidated in murine embryos. However, our understanding regarding human embryonic counterparts remains limited. Here, we demonstrated the presence of human yolk sac-derived lymphoid-biased progenitors (YSLPs) expressing CD34, IL7R, LTB, and IRF8 at Carnegie stage 10, much earlier than the first HSC emergence. The number and lymphopoietic potential of these progenitors were both significantly higher in the yolk sac than the embryo proper at this early stage. Importantly, single-cell/bulk culture and CITE-seq have elucidated the tendency of YSLP to differentiate into innate lymphoid cells and dendritic cells. Notably, lymphoid progenitors in fetal liver before and after HSC seeding displayed distinct transcriptional features, with the former closely resembling those of YSLPs. Overall, our data identified the origin, potential, and migratory dynamics of innate lymphoid-biased multipotent progenitors in human yolk sac before HSC emergence, providing insights for understanding the stepwise establishment of innate immune system in humans., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Building a simple and efficient in vitro differentiation system is crucial for studying the regulatory mechanisms during the development of innate lymphoid cells (ILCs). Here, we present a protocol for generating ILC subsets from α 4 β 7 + lymphoid progenitors (αLPs). We describe steps for murine cell isolation from fetal liver and adult bone marrow, flow cytometry sorting for αLPs, and cell culture. We then detail procedures for flow cytometry analysis of ILCs. This protocol significantly simplifies the differentiation process through ILC differentiation in vitro. For complete details on the use and execution of this protocol, please refer to Wu et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a . These CLP-derived pDCs, which we refer to as 'B-pDCs', have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN-α secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid-derived pDCs, display elevated levels of the cell surface receptor tyrosine kinase AXL, mirroring human AXL + transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice., Competing Interests: AA, JD, KG, ZS, CR, ZH, BL, DO, JL, VI, LE, GG, GI, SY, HT No competing interests declared, (© 2024, Araujo, Dekker et al.)

Innate lymphoid cells (ILCs) are critical in maintaining tissue homeostasis, and during infection and inflammation. Here we identify, by using combinatorial reporter mice, a rare ILC progenitor (ILCP) population, resident to the small intestinal lamina propria (siLP) in adult mice. Transfer of siLP-ILCP into recipients generates group 1 ILCs (including ILC1 and NK cells), ILC2s and ILC3s within the intestinal microenvironment, but almost exclusively group 1 ILCs in the liver, lung and spleen. Single cell gene expression analysis and high dimensional spectral cytometry analysis of the siLP-ILCPs and ILC progeny indicate that the phenotype of the group 1 ILC progeny is also influenced by the tissue microenvironment. Thus, a local pool of siLP-ILCP can contribute to pan-ILC generation in the intestinal microenvironment but has more restricted potential in other tissues, with a greater propensity than bone marrow-derived ILCPs to favour ILC1 and ILC3 production. Therefore, ILCP potential is influenced by both tissue of origin and the microenvironment during development. This may provide additional flexibility during the tuning of immune reactions., (© 2024. The Author(s).)

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34 + progenitors prior to B/T cell emergence. While these TdT + cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84 lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis., (© 2024. The Author(s).)

Over the past decade our research has implemented a multimodal approach to human lymphopoiesis, combining clonal-scale mapping of lymphoid developmental architecture with the monitoring of dynamic changes in the pattern of lymphocyte generation across ontogeny. We propose that lymphopoiesis stems from founder populations of CD127/interleukin (IL)7R - or CD127/IL7R + early lymphoid progenitors (ELPs) polarized respectively toward the T-natural killer (NK)/innate lymphoid cell (ILC) or B lineages, arising from newly characterized CD117 lo multi-lymphoid progenitors (MLPs). Recent data on the lifelong lymphocyte dynamics of healthy donors suggest that, after birth, lymphopoiesis may become increasingly oriented toward the production of B lymphocytes. Stemming from this, we posit that there are three major developmental transitions, the first occurring during the neonatal period, the next at puberty, and the last during aging., Competing Interests: Declaration of interests The authors have no conflict of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H + NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56 dim and CD56 bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Innate lymphoid cells (ILCs), strategically positioned throughout the body, undergo population declines over time. A solution to counteract this problem is timely mobilization of multipotential progenitors from the bone marrow. It remains unknown what triggers the mobilization of bone marrow ILC progenitors (ILCPs). We report that ILCPs are regulated by the circadian clock to emigrate and generate mature ILCs in the periphery. We found that circadian-clock-defective ILCPs fail to normally emigrate and generate ILCs. We identified circadian-clock-controlled endocrine and cytokine cues that, respectively, regulate the retention and emigration of ILCPs at distinct times of each day. Activation of the stress-hormone-sensing glucocorticoid receptor upregulates CXCR4 on ILCPs for their retention in the bone marrow, while the interleukin-18 (IL-18) and RORα signals upregulate S1PR1 on ILCPs for their mobilization to the periphery. Our findings establish important roles of circadian signals for the homeostatic efflux of bone marrow ILCPs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

T cells and innate lymphoid cells (ILCs) share expression of many key transcription factors during development and at mature stage, resulting in striking functional similarities between these lineages. Taking into account ILC contribution is thus necessary to appreciate T cell functions during immune responses. Furthermore, understanding ILC development and functions helps to understand T cells. Here we provide methods and protocols to isolate pure populations of multipotent precursors to T cells and innate lymphoid cells (ILCs) from adult mouse bone marrow, using flow cytometric sorting. These include precursors to all lymphocytes (viz., LMPPs and ALPs) and multipotent precursors to ILCs that have been recently refined (viz., specified EILPs, committed EILPs, and ILCPs)., (© 2023. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Innate lymphoid cells (ILCs) play important roles in regulating tissue homeostasis and innate immune responses. Generation of ILCs after engraftment of pluripotent stem cell (PSC)-derived hematopoietic progenitors (iHPCs) has not yet been reported. Here, we document that ILCs exist in Rag2 -/- Il2rg -/- recipients engrafted with PSC-derived iHPCs guided by Runx1 and Hoxa9 expression. Upon transplantation, iHPCs immediately give rise to ILC-related progenitors containing common helper ILC progenitors in the bone marrow, followed by a more restricted population named ILC progenitors, which are able to further differentiate into mature ILCs in the primary and secondary immunodeficient recipients. The PSC-derived ILCs exhibit multiple tissue distributions and normal immunological functions. Single-cell transcriptomics illustrates the developmental trajectory of PSC-derived ILCs in vivo, which is consistent with that of natural ILCs. Our study provides insights into the generation of ILCs in animals transplanted with PSC-derived iHPCs as a cell source., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Peña-Pérez, Kharazi, Frengen, Krstic, Bouderlique, Hauenstein, He, Somuncular, Li Wang, Dahlberg, Gustafsson, Johansson, Walfridsson, Kadri, Woll, Kierczak, Qian, Westerberg, Luc and Månsson.)

In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac-derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor., Competing Interests: Disclosures: M.H. Atkins and G.M. Keller reported a patent to "compositions and methods for generating human yolk sac-like hematopoietic cells" pending. G.M. Keller reported personal fees from BlueRock Therapeutics and VistaGen Therapeutics outside the submitted work. No other disclosures were reported., (© 2021 Atkins et al.)

During dendritic cell (DC) development, Myc expression in progenitors is replaced by Mycl in mature DCs, but when and how this transition occurs is unknown. We evaluated DC development using reporters for MYC, MYCL, and cell cycle proteins Geminin and CDT1 in wild-type and various mutant mice. For classical type 1 dendritic cells (cDC1s) and plasmacytoid DCs (pDCs), the transition occurred upon their initial specification from common dendritic cell progenitors (CDPs) or common lymphoid progenitors (CLPs), respectively. This transition required high levels of IRF8 and interaction with PU.1, suggesting the use of EICEs within Mycl enhancers. In pDCs, maximal MYCL induction also required the +41kb Irf8 enhancer that controls pDC IRF8 expression. IRF8 also contributed to repression of MYC. While MYC is expressed only in rapidly dividing DC progenitors, MYCL is most highly expressed in DCs that have exited the cell cycle. Thus, IRF8 levels coordinate the Myc-Mycl transition during DC development., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Anderson et al.)

Protection against pathogen re-infection is mediated, in large part, by two humoral cellular compartments, namely, long-lived plasma cells and memory B cells. Recent data have reinforced the importance of memory B cells, particularly in response to re-infection of different viral subtypes or in response with viral escape mutants. In regard to memory B cell generation, considerable advancements have been made in recent years in elucidating its basic mechanism, which seems to well explain why the memory B cells pool can deal with variant viruses. Despite such progress, efforts to develop vaccines that induce broadly protective memory B cells to fight against rapidly mutating pathogens such as influenza virus and HIV have not yet been successful. Here, we discuss recent advances regarding the key signals and factors regulating germinal center-derived memory B cell development and activation and highlight the challenges for successful vaccine development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Inoue, Shinnakasu and Kurosaki.)

The pathways that lead to the development of tissue-resident lymphocytes, including liver type 1 innate lymphoid cells (ILC1s), remain unclear. We show here that the adult mouse liver contains Lin - Sca-1 + Mac-1 + hematopoietic stem cells derived from the fetal liver. This population includes Lin - CD122 + CD49a + progenitors that can generate liver ILC1s but not conventional natural killer cells. Interferon-γ (IFN-γ) production by the liver ILC1s themselves promotes the development of these cells in situ, through effects on their IFN-γR + liver progenitors. Thus, an IFN-γ-dependent loop drives liver ILC1 development in situ, highlighting the contribution of extramedullary hematopoiesis to regional immune composition within the liver., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The process of hematopoiesis is subject to substantial ontogenic remodeling that is accompanied by alterations in cellular fate during both development and disease. We combine state-of-the-art mass spectrometry with extensive functional assays to gain insight into ontogeny-specific proteomic mechanisms regulating hematopoiesis. Through deep coverage of the cellular proteome of fetal and adult lympho-myeloid multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs), and granulocyte-monocyte progenitors (GMPs), we establish that features traditionally attributed to adult hematopoiesis are conserved across lymphoid and myeloid lineages, whereas generic fetal features are suppressed in GMPs. We reveal molecular and functional evidence for a diminished granulocyte differentiation capacity in fetal LMPPs and GMPs relative to their adult counterparts. Our data indicate an ontogeny-specific requirement of myosin activity for myelopoiesis in LMPPs. Finally, we uncover an ontogenic shift in the monocytic differentiation capacity of GMPs, partially driven by a differential expression of Irf8 during fetal and adult life., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

During embryonic development, multiple waves of hematopoietic progenitors with distinct lineage potential are differentially regulated in time and space. Two different waves of thymic progenitors colonize the fetal thymus where they contribute to thymic organogenesis and homeostasis. The origin, the lineage differentiation potential of the first wave, and their relative contribution in shaping the thymus architecture, remained, however, unclear. Here, we show that the first wave of thymic progenitors comprises a unique population of bipotent T and innatel lymphoid cells (T/ILC), generating a lymphoid tissue inducer cells (LTi's), in addition to invariant Vγ5+ T cells. Transcriptional analysis revealed that innate lymphoid gene signatures and, more precisely, the LTi-associated transcripts were expressed in the first, but not in the second, wave of thymic progenitors. Depletion of early thymic progenitors in a temporally controlled manner showed that the progeny of the first wave is indispensable for the differentiation of autoimmune regulator-expressing medullary thymic epithelial cells (mTECs). We further show that these progenitors are of strict hematopoietic stem cell origin, despite the overlap between lymphopoiesis initiation and the transient expression of lymphoid-associated transcripts in yolk sac (YS) erythromyeloid-restricted precursors. Our work highlights the relevance of the developmental timing on the emergence of different lymphoid subsets, required for the establishment of a functionally diverse immune system., (© 2021 by The American Society of Hematology.)

C/EBPα is required for formation of granulocyte-monocyte progenitors (GMP) and also participates in B lymphopoiesis. The common lymphoid progenitor (CLP) and preproB populations but not proB cells express Cebpa, and pan-hematopoietic deletion of the +37 kb Cebpa enhancer using Mx1-Cre leads not only to reduced GMP but also to 2-fold reduced marrow preproB and >15-fold reduced proB and preB cells. We now show that IL7Rα-Cre-mediated deletion of the +37 kb Cebpa enhancer, which occurs in 89% of Ly6D+ and 65% of upstream Ly6D- CLP, leads to a 2-fold reduction of both preproB and proB cells, and a 3-fold reduction in preB cells, with no impact on GMP numbers. These data support a direct role for C/EBPα during B lineage development, with reduced enhancer deletion in Ly6D- CLP mediated by IL7Rα-Cre diminishing the effect on B lymphopoiesis compared to that seen with Mx1-Cre. Amongst mRNAs encoding key transcriptional regulators that initiate B lymphoid specification (PU.1, E2A, IKAROS, EBF1, FOXO1, and BACH2), only Ebf1 levels are altered in CLP upon Mx1-Cre-mediated Cebpa enhancer deletion, with Ebf1 reduced ~40-fold in Flt3+Sca-1intc-kitintIL7Rα+ CLP. In addition, Cebpa and Ebf1 RNAs were 4- and 14-fold higher in hCD4+ versus hCD4- CLP from Cebpa-hCD4 transgenic mice. Histone modification ChIP-Seq data for CLP indicate the presence of active, intronic Ebf1 enhancers located 270 and 280 kb upstream of the transcription start sites. We identified a cis element in this region that strongly binds C/EBPα using the electrophoretic mobility shift assay. Mutation of this C/EBPα-binding site in an Ebf1 enhancer-TK-luciferase reporter leads to a 4-fold reduction in C/EBPα-mediated trans-activation. These findings support a model of B lymphopoiesis in which induction of Ebf1 by C/EBPα in a subset of CLP contributes to initiation of B lymphopoiesis., Competing Interests: The authors have declared that no competing interests exist.

Early hematopoietic progenitors undergo sophisticated developmental processes to become committed innate lymphoid cell (ILC) progenitors and ultimately mature ILC subsets in the periphery. Basic leucine zipper ATF-like transcription factor (Batf) plays important roles in lymphocyte biology. We report here that Batf regulates the production of bone marrow ILC progenitors and maintenance of peripheral ILCs. The expression of Batf is induced during ILC development at the α-lymphoid progenitor stage in response to the cytokine IL-7. As a potential mechanism, up-regulated Batf binds and activates transcription of the Nfil3 gene to promote ILC hematopoiesis. Batf is necessary to maintain normal numbers of early and late ILC progenitors in the bone marrow and mature ILC1, ILC2, ILC3, and NK cells in most peripheral tissues. Batf deficiency causes ILC lymphopenia, leading to defective ILC responses to inflammatory cytokines and defective immunity to enteric bacterial infections. Thus, Batf plays critical roles in bone marrow hematopoiesis, peripheral homeostasis, and effector functions of ILCs., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8 + memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8 + memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8 + memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin - c-Kit + Sca-1 + Flt3 + cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP - subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

During postnatal life, thymopoiesis depends on the continuous colonization of the thymus by bone-marrow-derived hematopoietic progenitors that migrate through the bloodstream. The current understanding of the nature of thymic immigrants is largely based on data from pre-clinical models. Here, we employed single-cell RNA sequencing (scRNA-seq) to examine the immature postnatal thymocyte population in humans. Integration of bone marrow and peripheral blood precursor datasets identified two putative thymus seeding progenitors that varied in expression of CD7; CD10; and the homing receptors CCR7, CCR9, and ITGB7. Whereas both precursors supported T cell development, only one contributed to intrathymic dendritic cell (DC) differentiation, predominantly of plasmacytoid dendritic cells. Trajectory inference delineated the transcriptional dynamics underlying early human T lineage development, enabling prediction of transcription factor (TF) modules that drive stage-specific steps of human T cell development. This comprehensive dataset defines the expression signature of immature human thymocytes and provides a resource for the further study of human thymopoiesis., Competing Interests: Declaration of Interests A.R. is an Scientific Advistory Board member of Thermo Fisher Scientific, Neogene Therapeutics, Asimov, and Syros Pharmaceuticals; an equity holder of Immunitas; and a founder of and equity holder in Celsius Therapeutics. The remaining authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The caudal hematopoietic tissue in zebrafish, the equivalent to the fetal liver in mammals, is an intermediate hematopoietic niche for the maintenance and differentiation of hematopoietic stem and progenitor cells before homing to the thymus and kidney marrow. As one of the ultimate hematopoietic organs, the thymus sustains T lymphopoiesis, which is essential for adaptive immune system. However, the mechanism of prethymic T lymphoid progenitors migrating to the thymus remains elusive. In this study, we identify an Rho GTPase Rac2 as a modulator of T lymphoid progenitor homing to the thymus in zebrafish. rac2 -Deficient embryos show the inability of T lymphoid progenitors homing to the thymus because of defective cell-autonomous motility. Mechanistically, we demonstrate that Rac2 regulates homing of T lymphoid progenitor through Pak1-mediated AKT pathway. Taken together, our work reveals an important function of Rac2 in directing T lymphoid progenitor migration to the thymus during zebrafish embryogenesis., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Neonatal CD4 + and CD8 + T cells have historically been characterized as immature or defective. However, recent studies prompt a reinterpretation of the functions of neonatal T cells. Rather than a population of cells always falling short of expectations set by their adult counterparts, neonatal T cells are gaining recognition as a distinct population of lymphocytes well suited for the rapidly changing environment in early life. In this review, I will highlight new evidence indicating that neonatal T cells are not inert or less potent versions of adult T cells but instead are a broadly reactive layer of T cells poised to quickly develop into regulatory or effector cells, depending on the needs of the host. In this way, neonatal T cells are well adapted to provide fast-acting immune protection against foreign pathogens, while also sustaining tolerance to self-antigens.

Introduction: Innate lymphoid cells (ILCs) can provide early cytokine help against a variety of pathogens in the lungs and gastrointestinal tract. Type 2 ILC (ILC2) are comparable to T helper 2 cells found in the adaptive immune system, which secrete cytokines such as interleukin 5 (IL-5) and IL-13 and have been found to play roles in host defense against helminth infections and in allergic responses. Recent studies have identified that programmed cell death protein 1 (PD-1) and peroxisome proliferator activated receptor-γ (PPAR-γ) are highly expressed by ILC2. We examined whether PD-1 plays a role in ILC2 function and whether there was any connection between PD-1 and PPAR-γ METHODS: To ensure that only innate immune cells were present, ILC2 cells were examined from RAG1 -/- and PD-1 -/- xRAG1 -/- mice under steady-state or following inoculation with IL-33. We also tested ILC2 generated from bone marrow of RAG1 -/- and PD-1 -/- xRAG1 -/- mice for their production of cytokines. These in vitro-derived ILC2 were also exposed to agonist and antagonist of PPAR-γ., Results: We found that ILC2 from PD-1 -/- xRAG1 -/- mice had reduced frequencies of IL-5 and IL-13 producing cells both in vitro upon IL-33 stimulation and in vivo following intraperitoneal administration of IL-33 when compared with ILC2 from RAG1 -/- mice. However, by adding IL-2, IL-25, and thymic stromal lymphopoietin to the in vitro cultures, the frequency of IL-5 and IL-13 expressing ILC2 from PD-1 -/- xRAG1 -/- mice became similar to the frequency observed for ILC2 from RAG1 -/- mice. In addition, PPAR-γ agonists and antagonists were found to increase and decrease PD-1 expression on ILC2 respectively., Conclusions: These findings illustrate that chronic loss of PD-1 plays a role in ILC2 function and PD-1 expression can be modulated by PPAR-γ., (© 2019 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator ( rtTA3) . For selection of LMO2 -expressing ciLPs ( LMO2 -ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro . LMO2 -ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44 + CD25 - myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44 + CD25 - myeloid cell subpopulations expressed c-KIT, suggesting that LMO2 -ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2 -ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44 + CD25 - myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2 -ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

A bstract: The crosstalk between hematopoietic stem cells (HSC) and bone marrow (BM) microenvironment is critical for homeostasis and hematopoietic regeneration in response to blood formation emergencies after injury, and has been associated with leukemia transformation and progression. Intercellular signals by the BM stromal cells in the form of cell-bound or secreted factors, or by physical interaction, regulate HSC localization, maintenance, and differentiation within increasingly defined BM HSC niches. Gap junctions (GJ) are comprised of arrays of membrane embedded channels formed by connexin proteins, and control crucial signaling functions, including the transfer of ions, small metabolites, and organelles to adjacent cells which affect intracellular mechanisms of signaling and autophagy. This review will discuss the role of GJ in both normal and leukemic hematopoiesis, and highlight some of the most novel approaches that may improve the efficacy of cytotoxic drugs. Connexin GJ channels exert both cell-intrinsic and cell-extrinsic effects on HSC and BM stromal cells, involved in regenerative hematopoiesis after myelosuppression, and represent an alternative system of cell communication through a combination of electrical and metabolic coupling as well as organelle transfer in the HSC niche. GJ intercellular communication (GJIC) in the HSC niche improves cellular bioenergetics, and rejuvenates damaged recipient cells. Unfortunately, they can also support leukemia proliferation and survival by creating leukemic niches that provide GJIC dependent energy sources and facilitate chemoresistance and relapse. The emergence of new strategies to disrupt self-reinforcing malignant niches and intercellular organelle exchange in leukemic niches, while at the same time conserving normal hematopoietic GJIC function, could synergize the effect of chemotherapy drugs in eradicating minimal residual disease. An improved understanding of the molecular basis of connexin regulation in normal and leukemic hematopoiesis is warranted for the re-establishment of normal hematopoiesis after chemotherapy.

Reprogramming differentiated cells into induced pluripotent stem cells (iPSCs) consists in dedifferentiation of the cells into the pluripotent state, i.e., stem cells. Since T cells play a pivotal role in our immune system, T cell reprogramming into iPSCs and subsequent redifferentiation of iPSCs toward the original cells hold a great promise for future cell therapy and for further exploring the biology of such T cells. Mucosal-associated invariant T (MAIT) cells are an innate-like T cells linking innate immunity to adaptive immunity, and believed to be implicated in host protection to infection, in inflammation, and in immune homeostasis, which makes them an attractive target for the clinical intervention. In this chapter, we will outline the protocol for reprogramming MAIT cells to pluripotency with Sendai virus vector and redifferentiation. This technique will allow expansion of MAIT cells for cell therapy against the intractable infectious diseases such as HIV/Tuberculosis or cancer.

Understanding the origins and developmental trajectory of innate lymphoid cell (ILC) progenitors has been of substantial interest to the fields of ILC biology and immunology. While mature ILC are rare lymphocytes, ILC progenitors represent an even smaller fraction of cells, providing additional challenges in studying them. Moreover, though the approaches to studying these cells are conceptually straightforward, the technical nuances that underlie them can substantially affect the quality of the data. Herein, we provide a detailed protocol for assessing the frequency of ILC progenitors in the bone marrow, their phenotype, and their potential to develop into mature ILC. These methods make up the foundation of in vivo investigations into ILC development, and we hope these thorough protocols and associated notes facilitate additional, high-quality inquiries into this fascinating field.

Innate lymphoid cells (ILCs) are tissue-resident lymphoid cells that reside mostly at barrier surfaces and participate in the initial response against pathogens. They are classified into different types based on effector programs that are based on cytokine production and transcription factor expression. They all derive from the common lymphoid precursor, but the molecular mechanisms regulating ILC subset development is not well understood. Experiments using Id2 knockout mice have previously shown that E protein activity inhibition is an absolute requirement for the development of all ILC subsets. In this study, we use a genetic approach to demonstrate that small increases in E protein activity during ILC development selectively inhibit type 2 ILC development. Type 1 ILCs are mostly unperturbed, and type 3 ILC show only a minor inhibition. This effect is first evident at the ILC2 progenitor stage and is ILC intrinsic. Therefore, our results demonstrate that modulation of E protein activity can bias cell fate decisions in developing ILCs., (Copyright © 2019 The Authors.)

Hypoxia plays a major role in the physiology of hematopoietic and immune niches. Important clues from works in mouse have paved the way to investigate the role of low O 2 levels in hematopoiesis. However, whether hypoxia impacts the initial steps of human lymphopoiesis remains unexplored. Here, we show that hypoxia regulates cellular and metabolic profiles of umbilical cord blood (UCB)-derived hematopoietic progenitor cells. Hypoxia more specifically enhances in vitro lymphoid differentiation potentials of lymphoid-primed multipotent progenitors (LMPPs) and pro-T/natural killer (NK) cells and in vivo B cell potential of LMPPs. In accordance, hypoxia exacerbates the lymphoid gene expression profile through hypoxia-inducible factor (HIF)-1α (for LMPPs) and HIF-2α (for pro-T/NK). Moreover, loss of HIF-1/2α expression seriously impedes NK and B cell production from LMPPs and pro-T/NK. Our study describes how hypoxia contributes to the lymphoid development of human progenitors and reveals the implication of the HIF pathway in LMPPs and pro-T/NK-cell lymphoid identities., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Innate lymphoid cells (ILCs) play critical roles in defending infections and maintaining mucosal homeostasis. All ILCs arise from common lymphoid progenitors (CLPs) in bone marrow. However, how CLPs stratify and differentiate into ILC lineages remains elusive. Here, we showed that Yeats4 is highly expressed in ILCs and their progenitors. Yeats4 conditional KO in the hematopoietic system causes decreased numbers of ILCs and impairs their effector functions. Moreover, Yeats4 regulates α 4 β 7 + CLP differentiation toward common helper ILC progenitors (CHILPs). Mechanistically, Yeats4 recruits the Dot1l-RNA Pol II complex onto Lmo4 promoter through recognizing H3K27ac modification to initiate Lmo4 transcription in α 4 β 7 + CLPs. Additionally, Lmo4 deficiency also impairs ILC lineage differentiation and their effector functions. Collectively, the Yeats4-Lmo4 axis is required for ILC lineage commitment., (© 2019 Liu et al.)

NK cells are generated from hematopoietic stem cells (HSC) residing in the bone marrow (BM), similar to other blood cells. Development toward mature NK cells occurs largely outside the BM through travel of CD34+ and other progenitor intermediates toward secondary lymphoid organs. The BM harbors multipotent CD34+ common lymphoid progenitors (CLPs) that generate T, B, NK, and Dendritic Cells and are devoid of erythroid, myeloid, and megakaryocytic potential. Over recent years, there has been a quest for single-lineage progenitors predominantly with the objective of manipulation and intervention in mind, which has led to the identification of unipotent NK cell progenitors devoid of other lymphoid lineage potential. Research efforts for the study of lymphopoiesis have almost exclusively concentrated on healthy donor tissues and on repopulation/transplant models. This has led to the widely accepted assumption that lymphopoiesis during disease states reflects the findings of these models. However, compelling evidences in animal models show that inflammation plays a fundamental role in the regulation of HSC maturation and release in the BM niches through several mechanisms including modulation of the CXCL12-CXCR4 expression. Indeed, recent findings during systemic inflammation in patients provide evidence that a so-far overlooked CLP exists in the BM (Lin - CD34 + DNAM-1 bright CXCR4 + ) and that it overwhelmingly exits the BM during systemic inflammation. These "inflammatory" precursors have a developmental trajectory toward surprisingly functional NK and T cells as reviewed here and mirror the steady state maintenance of the NK cell pool by CD34 + DNAM-1 - CXCR4 - precursors. Our understanding of NK cell precursor development may benefit from including a distinct "inflammatory" progenitor modeling of lymphoid precursors, allowing rapid deployment of specialized Lin - CD34 + DNAM-1 bright CXCR4 + -derived resources from the BM.

Dendritic cells (DC) are currently classified as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Through a combination of single-cell transcriptomic analysis, mass cytometry, in vivo fate mapping and in vitro clonal assays, here we show that, at the single-cell level, the priming of mouse hematopoietic progenitor cells toward the pDC lineage occurs at the common lymphoid progenitor stage, indicative of early divergence of the pDC and cDC lineages. We found the transcriptional signature of a pDC precursor stage, defined here, in the IL-7Rα + common lymphoid progenitor population and identified Ly6D, IL-7Rα, CD81 and CD2 as key markers of pDC differentiation, which distinguish pDC precursors from cDC precursors. In conclusion, pDCs developed in the bone marrow from a Ly6D hi CD2 hi lymphoid progenitor cell and differentiated independently of the myeloid cDC lineage.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that originate in the bone marrow and are continuously replenished from hematopoietic progenitor cells. Conventional DCs (cDCs) and plasmacytoid DCs (pDCs) are distinguished by morphology and function, and can be easily discriminated by surface marker expression, both in mouse and man. Classification of DCs based on their ontology takes into account their origin as well as their requirements for transcription factor (TF) expression. cDCs and pDCs of myeloid origin differentiate from a common DC progenitor (CDP) through committed pre-DC stages. pDCs have also been shown to originate from a lymphoid progenitor derived IL-7R + FLT3 + precursor population containing cells with pDC or B cell potential. Technological advancements in recent years have allowed unprecedented resolution in the analysis of cell states, down to the single cell level, providing valuable information on the commitment, and dynamics of differentiation of all DC subsets. However, the heterogeneity and functional diversification of pDCs still raises the question whether different ontogenies generate restricted pDC subsets, or fully differentiated pDCs retain plasticity in response to challenges. The emergence of novel techniques for the integration of high-resolution data in individual cells promises interesting discoveries regarding DC development and plasticity in the near future.

Human myelopoiesis has been proposed to occur through oligopotent common myeloid progenitor (CMP) and lymphoid-primed multipotent progenitor (LMPP) populations. However, other studies have proposed direct commitment of multipotent cells to unilineage fates, without specific intermediary lineage cosegregation patterns. We here show that distinct human myeloid progenitor populations generate the neutrophil/monocyte and mast cell/basophil/eosinophil lineages as previously shown in mouse. Moreover, we find that neutrophil/monocyte potential selectively cosegregates with lymphoid lineage and mast cell/basophil/eosinophil potentials with megakaryocyte/erythroid potential early during lineage commitment. Furthermore, after this initial commitment step, mast cell/basophil/eosinophil and megakaryocyte/erythroid potentials colocalize at the single-cell level in restricted oligopotent progenitors. These results show that human myeloid lineages are generated through two distinct cellular pathways defined by complementary oligopotent cell populations., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Hematopoietic stem cells (HSCs) are maintained in a perivascular niche in bone marrow, in which leptin receptor + (LepR) stromal cells and endothelial cells synthesize factors required for HSC maintenance, including stem cell factor (SCF). An important question is why LepR + cells are one hundred times more frequent than HSCs. Here, we show that SCF from LepR + cells is also necessary to maintain many c-kit + -restricted hematopoietic progenitors. Conditional deletion of Scf from LepR + cells depleted common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythrocyte progenitors (MEPs), pre-megakaryocyte-erythrocyte progenitors (PreMegEs), and colony-forming units-erythroid (CFU-Es), as well as myeloid and erythroid blood cells. This was not caused by HSC depletion, as many other restricted progenitors were unaffected. Moreover, Scf deletion from endothelial cells depleted HSCs, but not progenitors. Early erythroid progenitors were closely associated with perisinusoidal LepR + cells. This reveals cellular specialization within the niche: SCF from LepR + cells is broadly required by HSCs and restricted progenitors while SCF from endothelial cells is required mainly by HSCs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Single-cell RNA sequencing (scRNA-seq) is a powerful tool to study immune cells, which enables an unbiased way to discover novel cell populations, biological meaningful cellular heterogeneity, and cell lineage development trajectories. Advances in scRNA-seq technologies and computational data analysis have driven a revolution in our understanding of the immune system in health and disease. Technically, the key step for scRNA-seq analysis is making a high-quality cDNA library for sequencing. Here, we describe a plate-based protocol to prepare single-cell cDNA library of bone marrow innate lymphoid precursors for next generation sequencing-based transcriptome analysis.

Rapid advances have been made to uncover the mechanisms that regulate dendritic cell (DC) development, and in turn, how models of development can be employed to define dendritic cell function. Models of DC development have been used to define the unique functions of DC subsets during immune responses to distinct pathogens. More recently, models of DC function have expanded to include their homeostatic and inflammatory physiology, modes of communication with various innate and adaptive immune lineages, and specialized functions across different lymphoid organs. New models of DC development call for revisions of previously accepted paradigms with respect to the ontogeny of plasmacytoid DC (pDC) and classical DC (cDC) subsets. By far, development of the cDC1 subset is best understood, and models have now been developed that can separate deficiencies in development from deficiencies in function. Such models are lacking for pDCs and cDC2s, limiting the depth of our understanding of their unique and essential roles during immune responses. If novel immunotherapies aim to harness the functions of human DCs, understanding of DC development will be essential to develop models DC function. Here we review emerging models of DC development and function., (© 2019 Elsevier Inc. All rights reserved.)

The caspase (Casp) family of proteases regulate both lymphocyte apoptosis and activation. Here, we show that Casp6 regulates early B-cell development. One-week-old Casp6 knockout (Casp6 KO) mice have significantly more splenic B-cell subsets than wild-type (WT) mice. Adult Casp6 KO mice have normal levels of total splenic B cells but have increased numbers of B1a B cells and CD43 + "transitional" or splenic red pulp (RP) B cells. These results suggested that Casp6 may function to control B-cell numbers under nonhomeostatic conditions and during B-cell development. Consistent with this model, reconstitution of B cells was dysregulated in Casp6 KO mice after sublethal irradiation. Furthermore, bone marrow pro-B, pre-B and immature B-cell numbers were significantly higher in 1-week-old Casp6 KO mice than in 1-week-old WT mice. Casp6 KO pro-B cells proliferated more in response to IL-7 than WT pro-B cells, suggesting that Casp6 regulates early B-cell responses to IL-7. Indeed, adult and aged Casp6 KO mice had elevated numbers of IL-7αR + Sca1 + precursors of common lymphoid progenitors, suggesting Casp6 may help regulate progenitors of B cells and early B-lineage cells. Casp6 regulates B-cell programs both during early development and after antigen stimulation in the periphery., (© 2018 Australasian Society for Immunology Inc.)

Given that FLT3 expression is highly restricted on lymphoid progenitors, it is possible that the established role of FLT3 in the regulation of B and T lymphopoiesis reflects its high expression and role in regulation of lymphoid-primed multipotent progenitors (LMPPs) or common lymphoid progenitors (CLPs). We generated a Flt3 conditional knock-out (Flt3 fl/fl ) mouse model to address the direct role of FLT3 in regulation of lymphoid-restricted progenitors, subsequent to turning on Rag1 expression, as well as potentially ontogeny-specific roles in B and T lymphopoiesis. Our studies establish a prominent and direct role of FLT3, independently of the established role of FLT3 in regulation of LMPPs and CLPs, in regulation of fetal as well as adult early B cell progenitors, and the early thymic progenitors (ETPs) in adult mice but not in the fetus. Our findings highlight the potential benefit of targeting poor prognosis acute B-cell progenitor leukaemia and ETP leukaemia with recurrent FLT3 mutations using clinical FLT3 inhibitors., (© 2018 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

The non-hematopoietic cell fraction of the bone marrow (BM) is classically identified as CD45 - Ter119 - CD31 - (herein referred to as triple-negative cells or TNCs). Although TNCs are believed to contain heterogeneous stromal cell populations, they remain poorly defined. Here we showed that the vast majority of TNCs (∼85%) have a hematopoietic rather than mesenchymal origin. Single cell RNA-sequencing revealed erythroid and lymphoid progenitor signatures among CD51 - TNCs. Ly6D + CD44 + CD51 - TNCs phenotypically and functionally resembled CD45 + pro-B lymphoid cells, whereas Ly6D - CD44 + CD51 - TNCs were enriched in previously unappreciated stromal-dependent erythroid progenitors hierarchically situated between preCFU-E and proerythroblasts. Upon adoptive transfer, CD44 + CD51 - TNCs contributed to repopulate the B-lymphoid and erythroid compartments. CD44 + CD51 - TNCs also expanded during phenylhydrazine-induced acute hemolysis or in a model of sickle cell anemia. These findings thus uncover physiologically relevant new classes of stromal-associated functional CD45 - hematopoietic progenitors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Interleukin-7 (IL-7) and Flt3-ligand (FL) are two cytokines important for the generation of B cells, as manifested by the impaired B cell development in mice deficient for either cytokine or their respective receptors and by the complete block in B cell differentiation in the absence of both cytokines. IL-7 is an important survival and proliferation factor for B cell progenitors, whereas FL acts on several early developmental stages, prior to B cell commitment. We have generated mice constitutively over-expressing both IL-7 and FL. These double transgenic mice develop splenomegaly and lymphadenopathy characterized by tremendously enlarged lymph nodes even in young animals. Lymphoid, myeloid and dendritic cell numbers are increased compared to mice over-expressing either of the two cytokines alone and the effect on their expansion is synergistic, rather than additive. B cell progenitors, early progenitors with myeloid and lymphoid potential (EPLM), common lymphoid progenitors (CLP) and lineage - , Sca1 + , kit + (LSK) cells are all increased not only in the bone marrow but also in peripheral blood, spleen and even lymph nodes. When transplanted into irradiated wild-type mice, lymph node cells show long-term multilineage reconstitution, further confirming the presence of functional hematopoietic progenitors therein. Our double transgenic mouse model shows that sustained and combined over-expression of IL-7 and FL leads to a massive expansion of most bone marrow hematopoietic progenitors and to their associated presence in peripheral lymphoid organs where they reside and potentially differentiate further, thus leading to the synergistic increase in mature lymphoid and myeloid cell numbers. The present study provides further in vivo evidence for the concerted action of IL-7 and FL on lymphopoiesis and suggests that extramedullary niches, including those in lymph nodes, can support the survival and maintenance of hematopoietic progenitors that under physiological conditions develop exclusively in the bone marrow.

Medullary thymic epithelial cells (mTECs) act as one of the major stromal components in the thymus for selection and maturation of both conventional T cells and non-conventional T cells. Extensive efforts have been spent to understand how mTEC development and function are regulated. Although RelB has been well accepted to be a critical transcriptional factor for mTEC development, the underlying mechanisms still remain largely unclear. In this study, by generating thymic epithelial cell specific RelB deficient mice, we found that epithelial intrinsic RelB is required for mTEC homeostatic proliferation. Mechanistically, RelB regulates the expression of genes involved in cell cycle. Functionally, lack of intrinsic RelB in thymic epithelial cells results in dramatically reduced population of mTECs and impaired development of thymic invariant natural killer T (iNKT) cells and intraepithelial lymphocyte precursors (IELPs). This study thus reveals an epithelial intrinsic role of RelB on mTEC development and function.

Recent evidence supports that bone marrow (BM)-derived hematopoietic progenitor cells play an important role in lung injury and fibrosis. While these cells give rise to multiple cell types, the ST2 (Il1rl1)-expressing group 2 innate lymphoid cells (ILC2s) derived from BM progenitors have been implicated in tissue repair and remodeling, including in lung fibrosis. To further investigate the precise role of BM-derived ILC2s in the pathogenesis of fibrotic lung disease, their importance in the bleomycin-induced lung fibrosis model was evaluated by analyzing the effects of selective ST2 deficiency in the BM compartment. The results showed that while ST2-sufficient control mice exhibited activation of lung IL-33/ST2 signaling, ILC2 recruitment, IL-13 induction, and fibrosis, these responses were significantly diminished in ST2-deficient-BM chimera mice, with selective loss of ST2 expression only in the BM. This diminished response to bleomycin was similar to that seen in ST2 global knockout mice, suggesting the predominant importance of ST2 from the BM compartment. In wild-type mice, ILC2 recruitment to the lung was accompanied by a concomitant decrease in ST2 + BM cells. ST2-deficient BM cells were unresponsive to IL-33-induced ILC2 maturation. Finally, lineage-negative wild-type, but not ST2-deficient BM cells from bleomycin-treated mice stimulated lung fibroblast type I collagen expression, which was associated with elevated TGFβ expression in the BM cells. Taken together, these findings suggested that the BM-derived ILC2s were recruited to fibrotic lung through the IL-33/ST2 pathway, and contributed to fibroblast activation to promote lung fibrosis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The transcription factor PU.1 is required for the development of mature myeloid and lymphoid cells. Due to this essential role and the importance of PU.1 in regulating several signature markers of lymphoid progenitors, its precise function in early lymphopoiesis has been difficult to define. Here, we demonstrate that PU.1 was required for efficient generation of lymphoid-primed multipotent progenitors (LMPPs) from hematopoietic stem cells and was essential for the subsequent formation of common lymphoid progenitors (CLPs). By contrast, further differentiation into the B-cell lineage was independent of PU.1. Examination of the transcriptional changes in conditional progenitors revealed that PU.1 activates lymphoid genes in LMPPs, while repressing genes normally expressed in neutrophils. These data identify PU.1 as a critical regulator of lymphoid priming and the transition between LMPPs and CLPs.

Group 2 innate lymphoid cells (ILC2s) are derived from common lymphoid progenitors (CLPs) via several specific precursors, and the transcription factors essential for ILC2 differentiation have been extensively studied. However, the external factors regulating commitment to the ILC lineage as well as the sites and stromal cells that constitute the optimal microenvironment for ILC2-specific differentiation are not fully defined. In this study, we demonstrate that three key external factors, the concentration of interleukin 7 (IL-7) and strength and duration of Notch signaling, coordinately determine the fate of CLP toward the T, B, or ILC lineage. Additionally, we identified three stages of ILC2 in the fetal mesentery that require STAT5 signals for maturation: ILC progenitors, CCR9 + ILC2 progenitors, and KLRG1 - immature ILC2. We further demonstrate that ILC2 development is supported by mesenteric platelet-derived growth factor receptor α (PDGFRα) + glycoprotein 38 (gp38) + mesenchymal cells. Collectively, our results suggest that early differentiation of ILC2 occurs in the fetal liver via IL-7 and Notch signaling, whereas final differentiation occurs in the periphery with the aid of PDGFRα + gp38 + cells., (© 2018 Koga et al.)

Common lymphoid progenitors (CLPs) differentiate to T and B lymphocytes, dendritic cells, natural killer cells, and innate lymphoid cells. Here, we describe culture conditions that, for the first time, allow the establishment of lymphoid-restricted, but uncommitted, long-term proliferating CLP cell lines and clones from a small pool of these cells from normal mouse bone marrow, without any genetic manipulation. Cells from more than half of the cultured CLP clones could be induced to differentiate to T, B, natural killer, dendritic, and myeloid cells in vitro. Cultured, transplanted CLPs transiently populate the host and differentiate to all lymphoid subsets, and to myeloid cells in vivo. This simple method to obtain robust numbers of cultured noncommitted CLPs will allow studies of cell-intrinsic and environmentally controlled lymphoid differentiation programs. If this method can be applied to human CLPs, it will provide new opportunities for cell therapy of patients in need of myeloid-lymphoid reconstitution., (© 2018 by The American Society of Hematology.)