Innate lymphoid cells (ILCs) are a diverse group of immune cells that possess many effector functions of T cells but lack somatically generated receptors. They have crucial roles in early immune responses and in maintaining tissue integrity. We review ILC developmental pathways and progenitors in mice, with a particular focus on adult bone marrow but also addressing fetal liver. We present recent insights into the earliest steps of ILC specification, as well as new evidence for developmental pathways that generate two functionally distinct types of natural killer cells. The anatomical locations where ILC progenitors are identified are outlined and evidence supporting ILC development in tissues examined. In addition, key transcriptional regulators that support ILC development are discussed. Although ILC and T-lineage cells use many of the same transcriptional controllers during development, we present new evidence indicating ILC transcriptional programs diverge from T-lineage programs at the earliest known steps of ILC lineage specification.

During mouse development, hematopoietic stem and progenitor cells (HSPCs) originate from hemogenic endothelial cells (ECs) through a process of endothelial-to-hematopoietic transition. These HSPCs are thought to fully sustain adult hematopoiesis. However, it remains unknown whether adult ECs retain hemogenic potential. Here, we used in vivo genetic lineage tracking at population and single-cell (sc) levels, scRNA sequencing, and bone marrow (BM) transplantation to detect hemogenic ECs in adult mice. We identify and characterize BM-resident, adult Cdh5/VE-Cadherin+ ECs that produce hematopoietic cell-progeny in vitro and in mice. These adult hemogenic ECs and their hematopoietic cell progeny give rise to hematopoietic cells following adoptive transfer into adult mice. Furthermore, blood cells generated from adult and developmental ECs comparably home to peripheral tissues, where they similarly contribute to inflammatory responses. Thus, our results identify previously unrecognized BM-derived adult hemogenic ECs that generate HSPC and functional mature blood cells.

Understanding the mechanisms that initiate innate lymphoid cell (ILC) specification may reveal how ILC functions in immunity and tissue homeostasis are programmed. Using an Il7r-lineage tracing mouse strain, we identified developmental intermediates between lymphoid progenitors and Tcf7+ ILC progenitors in the mouse bone marrow (BM). Transcriptional analysis of these intermediates identified very early expression of the transcription factor nuclear factor, interleukin 3 regulated (NFIL3). Nfil3-/- mice lacked all BM ILC-specified precursors. Forced expression of NFIL3 in lymphoid progenitors induced Tcf7+ ILC-lineage cells capable of generating all ILC subsets and conventional natural killer cells. Transient activity of NFIL3 in lymphoid precursors recapitulated BM ILC development in vitro, leading to the generation of all adult ILCs. Mechanistically, NFIL3 initiated ILC specification by inducing expression of key transcriptional regulators of ILC development-Tox, Id2, Gata3, Tcf7, and Zbtb16. Our findings indicate an apex role for NFIL3 in ILC specification and provide an in vitro approach to generate ILC-lineage cells with potential therapeutic utility.

Age-related thymic involution leads to diminished output of naïve T-cells. While this process is suggested to increase the risk of disease severity in the elderly following infection, direct evidence is lacking. We developed two mouse models that allow us to experimentally prevent or reverse thymic involution. Constitutive Myc expression in thymic epithelial cells (TEC) of middle-aged mice enhanced thymic function, and increased numbers of peripheral naïve CD4 and CD8 T-cells. Inducible Myc expression reversed age-related thymic involution and partially recovered peripheral naïve T-cell numbers. Importantly, improving thymic function in these settings preserved T-cell-dependent antibody responses and significantly reduced T-cell-associated mortality after infection with Toxoplasma gondii. Improved thymic function also rebalanced age-associated alterations in the Treg pool, and mitigated loss of the transcriptional Th1 signature in aged conventional T-cells. Our findings support the value of TEC-focused thymic regeneration strategies for enhancement of T-cell-mediated immunity in the elderly.

The thymus produces T cells, and an improved understanding of thymus biology is likely to have therapeutic potential. Here we summarize the 2025 ThymUS conference, focusing on fundamental insights and translational potential.

The maintenance of serum antibodies requires the persistence of plasma cells within the bone marrow (BM). However, little is understood about why relatively few BM plasma cells live for extended periods. We consider two opposing viewpoints. We first consider the notion that sustained antibody titers requires localization of plasma cells to specialized BM niches where they access cell extrinsic survival factors, including extracellular ATP (eATP). We then consider the alternative possibility that plasma cell survival requires optimized cell intrinsic control of antibody synthesis supported by eATP stimulation of purinergic receptors. Based on the latter view we propose that many BM plasma cells fail to achieve maximal longevity due to suboptimal protein homeostasis rather than compromised access to cell extrinsic survival cues.

Hematopoietic stem cells (HSC) with multilineage potential are critical for T cell reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT). The Kitlo HSC subset is enriched for multipotential precursors, but their T cell potential remains poorly characterized. Using a preclinical allo-HCT mouse model, we demonstrate that Kitlo HSCs provide superior thymic recovery and T cell reconstitution, resulting in improved immune responses to post-transplant infection. Kitlo HSCs with augmented bone marrow (BM) lymphopoiesis mitigate age-associated thymic alterations and enhance T cell recovery in middle-aged mice. Mechanistically, chromatin profiling reveals Kitlo HSCs exhibiting higher activity of lymphoid-specifying transcription factors, such as, ZBTB1. Zbtb1 deletion diminishes HSC engraftment and T cell potential; by contrast, reinstating Zbtb1 in megakaryocytic-biased Kithi HSCs rescues hematopoietic engraftment and T cell potential in vitro and in vivo. Furthermore, age-associated decline in Kitlo HSCs is associated with diminished T lymphopoietic potential in aged BM precursors; meanwhile, Kitlo HSCs in aged mice maintain enhanced lymphoid potential, but their per-cell capacity is diminished. Lastly, we observe an analogous human BM KITlo HSC subset with enhanced lymphoid potential. Our results thus uncover an age-related epigenetic regulation of lymphoid-competent Kitlo HSCs for T cell reconstitution.

Plasma cells produce large quantities of antibodies and so play essential roles in immune protection1. Plasma cells, including a long-lived subset, reside in the bone marrow where they depend on poorly defined microenvironment-linked survival signals1. We show that bone marrow plasma cells use the ligand-gated purinergic ion channel P2RX4 to sense extracellular ATP released by bone marrow osteoblasts through the gap-junction protein pannexin 3 (PANX3). Mutation of Panx3 or P2rx4 each caused decreased serum antibodies and selective loss of bone marrow plasma cells. Compared to their wild-type counterparts, PANX3-null osteoblasts secreted less extracellular ATP and failed to support plasma cells in vitro. The P2RX4-specific inhibitor 5-BDBD abrogated the impact of extracellular ATP on bone marrow plasma cells in vitro, depleted bone marrow plasma cells in vivo and reduced pre-induced antigen-specific serum antibody titre with little posttreatment rebound. P2RX4 blockade also reduced autoantibody titre and kidney disease in two mouse models of humoral autoimmunity. P2RX4 promotes plasma cell survival by regulating endoplasmic reticulum homeostasis, as short-term P2RX4 blockade caused accumulation of endoplasmic reticulum stress-associated regulatory proteins including ATF4 and B-lineage mutation of the pro-apoptotic ATF4 target Chop prevented bone marrow plasma cell demise on P2RX4 inhibition. Thus, generating mature protective and pathogenic plasma cells requires P2RX4 signalling controlled by PANX3-regulated extracellular ATP release from bone marrow niche cells. We demonstrate the role of the ligand-gated purinergic ion channel P2RX4 in maintaining mouse plasma cells in their bone marrow niche.

Background: Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective de novo T-cell generation and thymic recovery, restoring the adaptive immune system after Hematopoietic Cell Transplantation (HCT). Recent studies have brought new insights into functional heterogeneity within HSCs, revealing an organized and predictable framework governing the adoption of lineage-restricted fates. However, the gene-regulatory networks underlying lymphoid determination and their conservation with aging in HSCs need to be elucidated. This study aims to comprehensively investigate the molecular profile of young and old HSCs and identify HSC subsets with multilineage transcriptional programs. Results: We conducted single-cell multiomic RNA and ATAC sequencing of young and old HSCs defined by Lineage-CD34-CD48-CD150+Sca-1+Kit+. We identified two CD117 (Kit) HSC subsets, Kit lo and Kit hi, with unique transcriptional profiles. Kit hi HSCs were characterized by quiescence, platelet bias, and low-output gene signatures, while Kit lo HSCs enriched for multi-lineage and high-output gene signatures. We observed a decreased frequency of Kit lo HSCs in aged mice. Although Kit lo HSCs have been previously described to exhibit increased self-renewal (Shin et al. JEM, 2014), their T-cell potential and the molecular underpinnings governing lymphoid differentiation programs are unknown. We performed in vitro studies, S17 stromal assay, and artificial thymic organoids (ATOs), which revealed an enhanced output of lymphoid progenitors and T-cells from Kit lo HSCs, consistent across all age cohorts. We next compared Kit lo vs. Kit hi HSCs in a competitive allogeneic HCT model. Kit lo HSCs demonstrated improved thymic recovery and post-HCT T-cell reconstitution, independent of age. To orthogonally evaluate post-HCT thymic function, we assessed Recent Thymic Emigrants (RTEs) output, which further substantiated superior recovery in mice receiving Kit lo HSCs. We further observed that Kit lo HSC-derived T-cells exhibited better proliferation in response to an acute Listeria monocytogenes infection. Next, we transplanted middle-aged mice to evaluate the rejuvenation potential of different Kit HSC subsets. We observed that Kit lo HSCs partly mitigated age-related changes in the thymic microenvironment, specifically by enhancing the regeneration of thymic epithelial cells (TECs) in middle-aged mice, thereby reversing the decline in T-cell production. We found differential expression and activity of key transcription factors (TFs), including Runx3, Zbtb7a, and Ezh1, associated with lymphoid differentiation through integrative transcriptional and chromatin accessibility analyses of Kit lo HSCs. These TFs notably showed distinct patterns in Kit lo HSCs independent of age, indicating their potential role in driving their enhanced T-cell potential. To establish the existence of a comparable human subset, we interrogated a human BM CITE-Seq dataset (Sommarin et al. Biorxiv 2021) for the mouse Kit lo gene signature. In concordance with our findings in mice, we observed an enrichment of the Kit lo gene program in young HSCs and decreased frequency of Kit lo HSCs in old BM. Next, we used ATOs to validate differential lymphoid potential. We found increased T-cell output originating from Kit lo HSCs, thus underscoring their enhanced lymphoid capacity and potential clinical relevance. Conclusion: Collectively, we demonstrate a distinct HSC subset, Kit lo HSC, with an age-conserved lymphoid gene program that enhances T-cell production and facilitates thymic regeneration. Importantly, we demonstrate the presence of an analogous HSC subset in humans, revealing the relevance of these insights to human health and disease. Thus, Kit lo HSCs have therapeutic potential to counteract age-associated immune senescence and treatment-related immune suppression.

Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons. Single-cell technologies have unveiled a complex understanding of NK cells that has led to variations in nomenclature and inconsistencies across the scientific literature. Here, Vivier and colleagues used these technologies to dissect the heterogeneity of NK cells, revealing three prominent NK cell subsets in healthy human blood.

The immune system encodes information about the severity of a pathogenic threat in the quantity and type of memory cells it forms. This encoding emerges from lymphocyte decisions to maintain or lose self-renewal and memory potential during a challenge. By tracking CD8+ T cells at the single-cell and clonal lineage level using time-resolved transcriptomics, quantitative live imaging, and an acute infection model, we find that T cells will maintain or lose memory potential early after antigen recognition. However, following pathogen clearance, T cells may regain memory potential if initially lost. Mechanistically, this flexibility is implemented by a stochastic cis-epigenetic switch that tunably and reversibly silences the memory regulator, TCF1, in response to stimulation. Mathematical modeling shows how this flexibility allows memory T cell numbers to scale robustly with pathogen virulence and immune response magnitudes. We propose that flexibility and stochasticity in cellular decisions ensure optimal immune responses against diverse threats.

Natural killer (NK) cells function by eliminating virus-infected cells or tumor cells during early defenses. However, the early development of NK cells and lineage relationships between NK cells and helper innate lymphoid cells (ILCs) remain elusive. Common precursors for ILCs (ILCPs) can differentiate into both helper ILCs and NK cells. Here, we identified a NK lineage-restricted progenitor population, early NK progenitor (ENKP), which does not develop from ILCPs, thus ENKP may represent the ILCP-independent pathway of NK cell development. Competitive chimera experiment shows ENKPs generate NK cells more efficiently than ILCPs, suggesting that ENKP-dependent pathway is the major pathway for NK cell development. scRNA-seq shows ENKP-derived NK cells express Ly49 receptors and higher levels of cytotoxic genes whereas ILCP-derived NK cells have very low expression of Ly49 receptors and express higher levels of genes implicated in tissue residency such as CD69 and CD200R. Furthermore, Ly49H+ NK cells which response to MCMV infection mostly develop from ENKPs but not ILCPs. Consistently, ENKP-derived NK cells but not ILCP-derived NK cells expanded dramatically after MCMV infection. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets.

Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.

Thymic epithelial cells (TEC) control T cell development and play essential roles in establishing self-tolerance. By using Foxn1-Cre –driven ablation of Klf6 gene in TEC, we identified Klf6 as a critical factor in TEC development. Klf6 deficiency resulted in a hypoplastic thymus—evident from fetal stages into adulthood—in which a dramatic increase in the frequency of apoptotic TEC was observed. Among cortical TEC (cTEC), a previously unreported cTEC population expressing the transcription factor Sox10 was relatively expanded. Within medullary TEC (mTEC), mTEC I and Tuft-like mTEC IV were disproportionately decreased. Klf6 deficiency altered chromatin accessibility and affected TEC chromatin configuration. Consistent with these defects, naïve conventional T cells and invariant natural killer T cells were reduced in the spleen. Late stages of T cell receptor–dependent selection of thymocytes were affected, and mice exhibited autoimmunity. Thus, Klf6 has a prosurvival role and affects the development of specific TEC subsets contributing to thymic function.

Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the for-mation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor T cell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indi-rectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

Thymic epithelial cells (TEC) make up the thymic microenvironments that support the generation of a functionally competent and self-tolerant T-cell repertoire. Infections, starvation, and aging, among other factors, cause thymus involution with loss of T-cell generation and decreased frequency of naive T-cells in secondary lymphoid organs. However, the causes and consequences of thymus involution are not clearly understood. Since thymus function declines with involution, we need a better understanding how the thymus controls T cell development. We found that constitutive overexpression of Myc (cMycTg) in TEC prevents thymus involution, causing a dramatic increase in thymus size in adult mice (Cowan et al, 2019). In addition, cMycTg mice restored frequencies of CD4+ and CD8+ naive T-cell populations to the levels observed in young adult mice. In unpublished work, we have found that prevention of thymus involution rescues aging mice from lethal Toxoplasma gondii infection. During the acute infection, old mice showed a significantly decreased frequency of parasite-specific T-cells and increased serum levels of IFNγ, IL6 and TNFα. Furthermore, antibody depletion of T-cells in aging mice challenged with Toxoplasma gondii prolonged their survival. These findings identify a role for thymic involution in the age-associated mortality of Toxoplasma gondii infection. We are now investigating the mechanisms responsible for the pathogenic immune responses seen in aged individuals to understand how improved thymic function modifies immune responses. AMS is supported by the patronage of the Children’s Hospital of Mexico. SCS, JEC and AB are supported by the NIH Intramural Cancer Research Training Award to post-doctoral fellows.

T cells are critical components of the adaptive immune system; they develop from bone-marrow derived precursors that migrate to the thymus. There, precursors undergo a sequence of lineage specification and commitment steps that give rise to multiple T cell lineages serving essential functions in defenses against infection and immune homeostasis. The diversity of thymocytes, and their complex interactions with the thymic stroma, including epithelial cells and other hematopoietic compartments, have hampered our understanding of T cell development. For greater insight into intrathymic developmental trajectories and their transcriptional control, we and others have utilized single cell RNA- and ATAC-sequencing (scRNA-seq and scATAC-seq). However, single-cell approaches do not assess contextual cell-cell interactions. To address this limitation, we used in-situ transcriptomics, specifically the 10× Genomics “Visium” technology, which assays in-situ gene expression in 55-micron spots spanning whole mouse thymus lobes. We will present initial results of these experiments, including mapping of thymocyte differentiation stages, location of developmental bifurcations, and the spatial progression of lineage specific gene expression. Briefly, we use statistical models informed by scRNA-seq data to infer the location, relative to the thymic architecture, of lineage decisions. Additionally, we combine positional information of thymocytes and stromal cells to infer cell-cell interactions that direct lineage decisions. Collectively, this analysis sheds light on the interactions that guide development of the diverse T cell family from a common precursor and characterizes the journey of these cells to reach maturity.