Lymphoid origin of intrinsically activated plasmacytoid dendritic cells in mice.

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.

Recipient tissue microenvironment determines developmental path of intestinal innate lymphoid progenitors.

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.

Protocol for in vitro generating innate lymphoid cells from mouse α4ß7+ lymphoid progenitors.

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 α4ß7+ 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.

Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors.

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 CD84lo 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.

Human yolk sac-derived innate lymphoid-biased multipotent progenitors emerge prior to hematopoietic stem cell formation.

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.

A new model of human lymphopoiesis across development and aging.

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 CD117lo 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.

Distinct developmental pathways generate functionally distinct populations of natural killer cells.

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 CD56dim and CD56bright 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.

Circadian-clock-controlled endocrine and cytokine signals regulate multipotential innate lymphoid cell progenitors in the bone marrow.

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.

GFI1B specifies developmental potential of innate lymphoid cell progenitors in the lungs.

Tissue-resident innate lymphoid cells (ILCs) play a vital role in the frontline defense of various tissues, including the lung. The development of type 2 ILCs (ILC2s) depends on transcription factors such as GATA3, RORα, GFI1, and Bcl11b; however, the factors regulating lung-resident ILC2s remain unclear. Through fate mapping analysis of the paralog transcription factors GFI1 and GFI1B, we show that GFI1 is consistently expressed during the transition from progenitor to mature ILC2s. In contrast, GFI1B expression is limited to specific subsets of bone marrow progenitors and lung-resident ILC progenitors. We found that GFI1B+ lung ILC progenitors represent a multi-lineage subset with tissue-resident characteristics and the potential to form lung-derived ILC subsets and liver-resident ILC1s. Loss of GFI1B in bone marrow progenitors led to the selective loss of lung-resident IL-18R+ ILCs and mature ILC2, subsequently preventing the emergence of effector ILCs that could protect the lung against inflammatory or tumor challenge.

FOXO1 and FOXO3 Cooperatively Regulate Innate Lymphoid Cell Development.

Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15Rß (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment.

Contribution of Dendritic Cell Subsets to T Cell-Dependent Responses in Mice.

BATF3-deficient mice that lack CD8+ dendritic cells (DCs) showed an exacerbation of chronic graft-versus-host disease (cGVHD), including T follicular helper (Tfh) cell and autoantibody responses, whereas mice carrying the Sle2c2 lupus-suppressive locus with a mutation in the G-CSFR showed an expansion of CD8+ DCs and a poor mobilization of plasmacytoid DCs (pDCs) and responded poorly to cGVHD induction. Here, we investigated the contribution of CD8+ DCs and pDCs to the humoral response to protein immunization, where CD8neg DCs are thought to represent the major inducers. Both BATF3-/- and Sle2c2 mice had reduced humoral and germinal center (GC) responses compared with C57BL/6 (B6) controls. We showed that B6-derived CD4+ DCs are the major early producers of IL-6, followed by CD4-CD8- DCs. Surprisingly, IL-6 production and CD80 expression also increased in CD8+ DCs after immunization, and B6-derived CD8+ DCs rescued Ag-specific adaptive responses in BATF3-/- mice. In addition, inflammatory pDCs (ipDCs) produced more IL-6 than all conventional DCs combined. Interestingly, G-CSFR is highly expressed on pDCs. G-CSF expanded pDC and CD8+ DC numbers and IL-6 production by ipDCs and CD4+ DCs, and it improved the quality of Ab response, increasing the localization of Ag-specific T cells to the GC. Finally, G-CSF activated STAT3 in early G-CSFR+ common lymphoid progenitors of cDCs/pDCs but not in mature cells. In conclusion, we showed a multilayered role of DC subsets in priming Tfh cells in protein immunization, and we unveiled the importance of G-CSFR signaling in the development and function pDCs.

Temporal Gene Expression Profiles Reflect the Dynamics of Lymphoid Differentiation.

Understanding the emergence of lymphoid committed cells from multipotent progenitors (MPP) is a great challenge in hematopoiesis. To gain deeper insight into the dynamic expression changes associated with these transitions, we report the quantitative transcriptome of two MPP subsets and the common lymphoid progenitor (CLP). While the transcriptome is rather stable between MPP2 and MPP3, expression changes increase with differentiation. Among those, we found that pioneer lymphoid genes such as Rag1, Mpeg1, and Dntt are expressed continuously from MPP2. Others, such as CD93, are CLP specific, suggesting their potential use as new markers to improve purification of lymphoid populations. Notably, a six-transcription factor network orchestrates the lymphoid differentiation program. Additionally, we pinpointed 24 long intergenic-non-coding RNA (lincRNA) differentially expressed through commitment and further identified seven novel forms. Collectively, our approach provides a comprehensive landscape of coding and non-coding transcriptomes expressed during lymphoid commitment.

Modeling human yolk sac hematopoiesis with pluripotent stem cells.

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.

Transition from cMyc to L-Myc during dendritic cell development coordinated by rising levels of IRF8.

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.

Liver type 1 innate lymphoid cells develop locally via an interferon-γ-dependent loop.

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.

Ontogenic shifts in cellular fate are linked to proteotype changes in lineage-biased hematopoietic progenitor cells.

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.

Generation of High Quality Memory B Cells.

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.

A wave of bipotent T/ILC-restricted progenitors shapes the embryonic thymus microenvironment in a time-dependent manner.

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.

C/EBPα induces Ebf1 gene expression in common lymphoid progenitors.

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.