Immunology & Cell Biology

Hematopoietic stem cell (HSC) transplantation (HST) is a curative treatment for many hematopoietic cancers and bone marrow (BM) disorders but is currently limited by numerous complications including a lengthy recovery period, prolonged neutropenia resulting in severe infections and bleeding, and a high incidence of graft vs. host disease (GVHD). While clinical studies have demonstrated that sex mismatch, notably male recipients with female donor cells, results in increased risk of GVHD (likely due to male recipient minor histocompatibility antigens targeted by donor female T-cells 1), increased non-relapse mortality, and decreased overall survival, the mechanisms underlying sex-determinants on hematopoiesis and post-transplant recovery are not clear. In this manuscript we have identified: 1) unique expression of hematopoietic niche factors in the BM and spleens of male and female mice, 2) altered kinetics of hematopoietic reconstitution following transplantation when male vs. female BM is used as the donor cell source, 3) a sex-specific role for the recipient niche in promoting post HST recovery, and 4) a dose-dependent role for exogenous sex hormones in maintaining hematopoietic stem and progenitor cells (HSPCs). Taken together, these data demonstrate that sex-specific cellular and molecular signaling occurs during hematopoietic regeneration. Further identifying novel sex-dependent determinants of regeneration following transplantation will not only enhance understanding of steady state versus regeneration hematopoiesis but may also reveal unique (and potentially sex-specific) therapeutic targets to accelerate hematologic recovery. Key PointsO_LIMale and female mice display altered kinetics of regeneration following HST due to unique niche factors in hematopoietic compartments. C_LIO_LIExogenous steroid sex hormones uniquely regulate the pool of hematopoietic stem and progenitor cells and may impact transplantation outcomes. C_LI

Severe burn injury induces long-lasting immune dysfunction, but the molecular mechanisms underlying this phenomenon remain unclear. We hypothesized that burn injury leads to epigenetic and transcriptional reprogramming of innate immune cells. Splenic F4/80 macrophages were isolated from mice at days 2, 9, and 14 days post-20% contact burn injury. Targeted transcriptomics and MAPit single-molecule chromatin profiling were used to assess immune, metabolic, and epigenetic changes. Canonical pathway analysis was performed to infer functional shifts over time. Burn injury induced a biphasic response in macrophages. Early after injury (Day 2), there was broad transcriptional suppression and epigenetic silencing of inflammatory regulators, including Stat3, Traf6, and Nfkb1. Over time (Days 9 and 14), loci associated with anti-inflammatory mediators such as Il-10 and Socs3 exhibited progressive chromatin opening and transcriptional upregulation. Metabolic gene profiles revealed persistent suppression of mitochondrial and oxidative phosphorylation programs. Canonical pathway analysis demonstrated early IL-10 signaling activation with sustained suppression of classical macrophage activation pathways. Chromatin architecture changes included nucleosome sliding and ejection events, consistent with dynamic, locus-specific regulation. This work challenges the classical notion of burn-induced immune suppression as purely a consequence of systemic inflammation. Instead, we reveal a programmed and locus-specific epigenetic architecture that may shape macrophage immune and metabolic function long after the acute phase.

Nuclear factor-{kappa}B (NF-{kappa}B) is a transcription factor with a key role in a great variety of cellular processes from embryonic development to immunity, the outcome of which depends on the fine-tuning of NF-{kappa}B activity. The development of sensitive and faithful reporter systems to accurately monitor the activation status of this transcription factor is therefore desirable. To address this need, over the years a number of different approaches have been used to generate NF-{kappa}B reporter mice, which can be broadly subdivided into bioluminescence- and fluorescence-based systems. While the former enables whole-body visualization of the activation status of NF-{kappa}B, the latter have the potential to allow the analysis of NF-{kappa}B activity at single cell level. However, fluorescence-based reporters frequently show poor sensitivity and excessive background or are incompatible with high-throughput flow cytometric analysis. In this work we describe the generation and analysis of ROSA26 knockin NF-{kappa}B reporter (KappaBle) mice containing a destabilized EGFP, which showed sensitive, dynamic, and faithful monitoring of NF-{kappa}B activity at the single-cell level of various cell types during inflammatory and infectious diseases.

Characterization of the functional effects of cDC2s in vivo requires model systems in which cDC2s are depleted. Previous literature has reported a loss of cDC2s in mice lacking the transcription factor IRF21,2. We sought to further characterize the cDC2 defect in these animals. Here, we find that the requirement for IRF2 in cDC2 development and survival is cell-extrinsic and correlated to the development of dermatitis in the Irf2-/- model system. We also find that Flt3L-mediated in vitro development of cDC1s and cDC2s, but not pDCs, is abrogated in Irf2-/- bone marrow, as well as in wild-type bone marrow cultured with IFN. Loss of interferon (IFN) signaling in Irf2-/- mice restored cDC2 development in vivo and cDC1 and cDC2 development in vitro. We therefore conclude that IRF2 is required for cDC2 development in a cell-extrinsic manner dependent on IFN signaling.

Multiple waves of hematopoietic progenitors with distinct lineage potentials are differentially regulated in time and space. We show that the first thymic seeding progenitors comprise a unique population of bipotent cells that generate lymphoid tissue inducer and invariant V{gamma}5+ T cells. Both populations are of embryonic origin and induce the maturation of medullary thymic epithelial cells. Indeed, temporal depletion of the first wave of thymocytes results in a five-fold reduction of mature medullary thymic epithelial cells, after birth. We further show that these progenitors are of hematopoietic stem cell, and not, of yolk sac origin, despite the temporal overlap between the onset of lymphopoiesis and the transient expression of lymphoid transcripts in yolk sac precursors, that does not impact their strict erythro-myeloid potential. 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.

While the Bacille-Calmette-Guerin (BCG) vaccine is used to prevent tuberculosis, it also offers protection against a diverse range of non-mycobacterial infections. However, the underlying protective mechanisms in humans are not yet fully understood. Here, we surveyed at single-cell resolution the gene expression and chromatin landscape of human bone marrow, aspirated before and 90 days after BCG vaccination or placebo administration. We show that BCG vaccination significantly alters both the gene expression and epigenetic profiles of human hematopoietic stem and progenitor cells (HSPCs). Changes in gene expression occur primarily on the most uncommitted stem cells and are reflective of a persistent myeloid bias. In contrast, BCG-induced changes in chromatin accessibility are most prevalent within differentiated progenitor cells at sites influenced by Kruppel-like factor (KLF)/SP and EGR transcription factors (TFs). These TFs are also activated in the most uncommitted stem cells, indicating that activated TFs, which drive persistent changes in HSC gene expression, likely also drive chromatin dynamics appearing within downstream progenitor cells. This perspective contests the prevailing notion that epigenetic modifications linked to innate immune memory transfer directly from stem cells to their differentiated derivatives. Finally, we show that alterations in gene expression and chromatin accessibility in HSPCs due to BCG vaccination were highly correlated (r>0.8) with the IL-1{beta} secretion capacity of paired PBMCs upon secondary immune challenge. Overall, our findings shed light on BCG vaccinations profound and lasting effects on HSPCs and its influence on innate immune responses.

The activated B-cell (ABC) to plasmablast transition is the cusp of antibody secreting cell (ASC) differentiation but is incompletely defined. We apply expression time-courses, parsimonious gene correlation network analysis, and ChIP-seq to explore this in human cells. The transition initiates with input signal loss leading within hours from cell growth dominant programs to enhanced proliferation, accompanied from 24h by ER-stress response, secretory optimization and upregulation of ASC features. Clustering of genomic occupancy for ASC transcription factors (TFs) IRF4, BLIMP1 and XBP1 with CTCF and histone marks defines distinct patterns for each factor in plasmablasts. Integrating TF-associated clusters and modular gene expression identifies a dichotomy: XBP1 and IRF4 significantly link to gene modules induced in plasmablasts, but not to modules of repressed genes, while BLIMP1 links to modules of ABC genes repressed in plasmablasts but is not significantly associated with modules induced in plasmablasts. Pharmacological inhibition of the G9A (EHMT2) histone-methytransferase, a BLIMP1 co-factor that catalyzes repressive H3K9me2 marks, leaves functional ASC differentiation intact but de-represses ABC-state genes. Thus, in human plasmablasts IRF4 and XBP1 emerge as the dominant association with ASC gene expression, while BLIMP1 links to repressed modules with particular focus in repression of the B-cell activation state.

In the avian host, comprehensively cataloging immune cell types, their transcriptome profiles, and varying molecular responses to pathogen challenges are necessary steps toward a better understanding of the interplay between genetics and disease resilience. We present a first nuclei atlas of immune cell types derived from the three main immune organs of layer chickens, including spleen, bursa, and thymus. In bursa we also present, an accounting of cell type activation with the bacterial toxin lipopolysaccharide (LPS). Our analysis includes 36,370 total nuclei and 16, 12, and 12 transcriptionally distinct clusters for spleen, bursa, and thymus, respectively. We discover nuclei molecular profiles that uniquely distinguish states of the transcriptome within cell type that could serve as new means to characterize avian immune subtypes. We further subcluster refined immune cell type classifications, specifically highlighting the transcriptomic diversity of B and T cell subtypes. In the bursa, inferred intercellular communication and signaling pathway enrichment analyses across immune and non-immune cell types demonstrate the unappreciated complexity of the B cell repertoire in a model mimicking systemic bacterial infection. This census of all cell types in both primary and one major secondary avian immune organ system, although preliminary, provides a first review of how nuclei transcribe numerous genes, known and unknown, a critical prerequisite for the study avian immunogenetics by cell type.

The thymus, a crucial organ for T cell development, undergoes transient involution following exposure to sublethal total body irradiation. The impact of sublethal irradiation on the thymus is reportedly bimodal: thymic involution recurs after the recovery of the thymus from the initial impact of acute sublethal irradiation. While the second impact of acute irradiation has been acknowledged for thymocytes, its influence on thymic epithelial cells (TECs), which are crucial for thymic T cell differentiation and selection, remains to be elucidated. In this study, we aim to elucidate this influence. Mice were subjected to acute sublethal total body irradiation, and TECs were evaluated at three distinct time points: during the initial impact, during the recovery phase post-initial impact, and during the second impact phase. Flow cytometry analysis revealed that during the second impact phase, mTECs were reduced, whereas cTECs remained unaffected. Among mTECs, the subset expressing high levels of the co-stimulatory molecule CD80 (mTEChi) experienced the most pronounced reduction. RNA sequencing analysis of mTEChi cells at early differentiation stages revealed significant alterations in gene expression profiles during the second impact phase. Notably, gene signatures of tuft-like TECs and Aire-expressing TECs were preferentially influenced in these mTEChi subpopulations. These findings suggest that acute total body irradiation disrupts mTEC frequencies and gene expression in a bimodal manner, possibly compromising thymic functions over extended periods.

Sepsis survivors frequently develop long-term immune dysfunction, but the epigenetic mechanisms underlying persistent myeloid suppression remain unclear. Myeloid-derived suppressor cells (MDSCs), whose function is shaped by host age and sex, are key contributors to post-sepsis immune dysregulation. Here, we present a high-resolution epigenetic map targeting gene promoters of MDSCs after sepsis using MAPit-FENGC, a single-molecule assay that simultaneously profiles DNA methylation and chromatin accessibility. In a clinically relevant murine model including young and older adult male and female mice, splenic MDSCs were isolated for MAPit-FENGC and single-cell RNA sequencing. Unsupervised clustering identified nine promoter classes reflecting chromatin dynamics: age- and sex-dependent sepsis-induced opening (Classes 1-4), persistent closure with varying levels of DNA methylation (Classes 5-7), and constitutive openness post-sepsis (Classes 8, 9). Transcriptomic profiling corroborated these promoter states, linking accessibility with gene expression. These findings establish how epigenetic reprogramming of MDSCs may shape age- and sex-specific immune trajectories in sepsis survivors.

Summary/AbstractB-cell development is initiated by the stepwise differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating the mature B-cells that mediate protective immunity. This highly regulated process also generates clonal immunological diversity via recombination of immunoglobulin genes. While several transcription factors that control B-cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for the earliest steps in B-cell differentiation. Erg initiates a transcriptional network involving the B-cell lineage defining genes, Ebf1 and Pax5, that directly promotes the expression of key genes involved in V(D)J recombination and formation of the B-cell receptor. Complementation of the Erg-deficiency with a productively rearranged immunoglobulin gene rescued B-cell development, demonstrating that Erg is an essential and exquisitely stage specific regulator of the gene regulatory network controlling B-lymphopoiesis.

Purpose and appropriate sample typesThis 25-parameter, 22-colour full spectrum flow cytometry panel was designed and optimised for the comprehensive enumeration and functional characterisation of innate lymphoid cell (ILC) subsets in mouse tissues (Table 1). The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR+ ILC3, NCR- ILC3, CCR6+ lymphoid tissue-inducer (LTi)-like ILC3 and mature natural killer (NK) cell populations. Further characterisation of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset-specific cytokine markers, and proliferation markers. Development and optimisation of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12-myrisate 13-acetate, ionomycin and pro-ILC activating cytokines. O_TBL View this table: org.highwire.dtl.DTLVardef@1b1415forg.highwire.dtl.DTLVardef@3adc6forg.highwire.dtl.DTLVardef@5e0aeaorg.highwire.dtl.DTLVardef@1e9e61org.highwire.dtl.DTLVardef@2ff463_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONSummary Table C_TABLECAPTION C_TBL Ethical compliance statementAll mouse experiments were performed in accordance with the recommendations in the Guide for the Use of Laboratory Animals of Imperial College London, with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All animal procedures and care conformed strictly to the UK Home Office Guidelines under the Animals (Scientific Procedures) Act 1986, and the protocols were approved by the Home Office of Great Britain.

The proliferation, differentiation and survival of cells of the macrophage lineage depends on signals from the macrophage colony-stimulating factor receptor (CSF1R). On a C57BL/6J background homozygous kinase-dead Csf1r mutation (Csf1rE631K/E631K - E631Km/m) causes perinatal lethality. Here we demonstrate that E631Km/m mice on a mixed genetic background (C57 x BALB/c F2) are osteopetrotic and growth retarded but viable as adults with no other gross developmental deficits. They lack osteoclasts, microglia and most peripheral tissue resident macrophages and exhibit perturbed hematopoiesis. Although CD169+ tissue resident macrophages in bone marrow are considered an essential component of the hematopoietic niche, CD169 is undetectable in E631Km/m marrow and F4/80+ macrophages are depleted. These changes are associated with expansion of mature and immature granulocytes and reduced B cells, whereas monocytes and stem and progenitor populations are unaffected as a proportion of total cells. Erythropoiesis in bone marrow is maintained in E631Km/m mice, associated with a residual population of CSF1R-independent CD169-ve/F4/80+ macrophages. Nevertheless, splenic extramedullary hematopoiesis in E631Km/m mice indicates a degree of bone marrow insufficiency. Red pulp macrophages are retained but CD169+ marginal metallophil macrophages are absent and CD209b+ (SIGNR1) macrophages are present but disorganized. Circulating white blood cell count is unchanged in E631Km/m mice, but the proportion of neutrophils is greatly increased whilst B cells and monocytes are reduced. This novel model reveals the essential roles of CSF1R-dependent macrophages in hematopoiesis and demonstrates that many developmental and homeostatic functions attributed to murine resident tissue macrophages are redundant and/or specific to inbred mouse strains.

The extracellular matrix (ECM) forms the scaffold in which cells reside and interact. The composition of this scaffold guides the development of local immune responses and tissue function. With the advent of multiplexed spatial imaging methodologies, investigating the intricacies of cellular spatial organisation are more accessible than ever. However, the relationship between cellular organisation and ECM composition has been broadly overlooked. Using imaging mass cytometry, we investigated the association between cellular niches and their surrounding matrix environment during allergic airway inflammation in two commonly used mouse strains. By first classifying cells according to their canonical intracellular markers and then by developing a novel analysis pipeline to independently characterise a cells ECM environment, we integrated analysis of both intracellular and extracellular data. Applying this methodology to three distinct tissue regions we reveal disparate and restricted responses. Recruited neutrophils were dispersed within the alveolar parenchyma, alongside a loss of alveolar type I cells and an expansion of alveolar type II cells. This activated parenchyma was associated with increased proximity to hyaluronan and chondroitin sulphate. In contrast, infiltrating CD11b+ and MHCII+ cells accumulated in the adventitial cuff and aligned with an expansion of the subepithelial layer. This expanded subepithelial region was enriched for closely interacting stromal and CD11b+ immune cells which overlaid regions enriched for type-I and type-III collagen. The cell-cell and cell-matrix interactions identified here will provide a greater understanding of the mechanisms and regulation of allergic disease progression across different inbred mouse strains and provide specific pathways to target aspects of remodelling during allergic pathology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/623782v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@dcaf56org.highwire.dtl.DTLVardef@7b5d7forg.highwire.dtl.DTLVardef@1376d4eorg.highwire.dtl.DTLVardef@1e95801_HPS_FORMAT_FIGEXP M_FIG C_FIG

Idiopathic pulmonary fibrosis (IPF) is a morbid fibrotic lung disease with limited treatment options. The pathophysiology of IPF remains poorly understood, and elucidation of the cellular and molecular mechanisms of IPF pathogenesis is key to the development of new therapeutics. B-1 cells are an innate B cell population which play an important role linking innate and adaptive immunity. B-1 cells spontaneously secrete natural IgM and prevent inflammation in several disease states. One class of these IgM recognize oxidation-specific epitopes (OSE), which have been shown to be generated in lung injury and to promote fibrosis. A main B-1 cell reservoir is the pleural space, adjacent to the typical distribution of fibrosis in IPF. In this study, we demonstrate that B-1 cells are recruited to the lung during injury where they secrete IgM to OSE (IgMOSE). We also show that the pleural B-1 cell reservoir responds to lung injury through regulation of the chemokine receptor CXCR4. Mechanistically we show that the transcription factor Id3 is a novel negative regulator of CXCR4 expression. Using mice with B-cell specific Id3 deficiency, a model of increased B-1b cells, we demonstrate decreased bleomycin-induced fibrosis compared to littermate controls. Furthermore, we show that mice deficient in secretory IgM (sIgM-/-) have higher mortality in response to bleomycin-induced lung injury, which is partially mitigated through airway delivery of the IgMOSE E06. Additionally, we provide insight into potential mechanisms of IgM in attenuation of fibrosis through RNA sequencing and pathway analysis, highlighting complement activation and extracellular matrix deposition as key differentially regulated pathways.

Chronic, low-grade inflammation has a widespread and significant impact on health, especially in Western society. While inflammation is beneficial for the removal of microbes, low-grade inflammation never resolves and can cause or worsen other diseases. The process by which low-grade inflammation occurs remains poorly understood. Here we exposed murine bone-marrow derived monocytes to chronic lipopolysaccharide (LPS) stimulation at low dose or high dose, as well as a PBS control. The cells were profiled for genome-wide H3K27ac modification and gene expression. The gene expression of TRAM-deficient and IRAK-M-deficient monocytes with LPS exposure was also analyzed. We discover that low-grade inflammation preferentially utilizes the TRAM/TRIF-dependent pathway of TLR4 signaling, and induces the expression of interferon response genes. In contrast, acute inflammation uniquely upregulates metabolic and proliferative pathways that also appear to be TRAM-dependent. The extensive differences in the epigenomic landscape between low-dose and high-dose conditions suggest the importance of epigenetic regulations in driving differential responses. Our data provide potential targets for future mechanistic or therapeutic studies.

Allogeneic bone marrow transplantation remains the only therapeutic option for a wide range of hematological malignancies despite the risk of possible adverse, immune-related events, such as infection and acute graft-versus-host disease (aGVHD). aGVHD is characterized by T-cell activation, defective B-cell development and osteoblastic niche destruction in bone marrow (BM) among other issues. Transplant conditioning regimens cause excessive inflammatory cytokines production and impaired regulatory T-cell control of aberrant T-cell activation. Here, we show that mesenchymal cells (MSCs) upregulated CD40 upon irradiation at the expense of mesenchymal markers, and that CD40 endows MSC of regulatory function on Treg homeostasis and fitness. Transplantation of wild type hematopoietic cells into a CD40-null recipient reduces Treg numbers allowing persistent T-cell activation and pro-inflammatory cytokines production causing, impaired B-lymphopoiesis. These evidences find correlation in aGVHD patients showing the loss of CD40+ BM-MSCs along with reduction in cells of the B-lineage. Modeling aGVHD in mice we show that the elimination of CD40+ BM-MSCs relies on their higher expression of MHC-I molecules. Indeed, aGVHD mice compared to MHC-matched controls showed the loss of MHC-I + radio-resistant host BM-MSCs. Our data point to CD40+ MHC-I+BM-MSCs as a key regulator of BM tolerogenic niches. Key pointsO_LICD40 regulates BM immunological tolerance following total body irradiation (TBI) and transplantation (BMT). C_LIO_LILoss of CD40+MHC-IhighBM-MSCs is associated to BM manifestation of aGVHD in human and murine model. C_LI

Both identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. However, a method that reliably and readily profiles DNA base modifications is still needed to finely study Th cell differentiation. Cytosine methylation (5mC) and cytosine hydroxymethylation (5hmC) are DNA modifications that identify stable cell phenotypes but their potential to characterize intermediate cell transitions has not yet been evaluated. To assess transition states in Th cells, we developed a new method to profile Th cell identity using cas9-targeted single molecule nanopore sequencing and found that 5mC and 5hmC can be used as markers of cellular identity. Targeting as few as 10 selected genomic loci, we were able to distinguish major differentiated T cell subtypes as well as intermediate phenotypes by their native DNA 5mC/5hmC patterns. Moreover, by using off-target sequences we were able to infer transcription factor activities relevant to each cell subtype. Our analysis demonstrates the importance of epigenetic regulation by 5mC and 5hmC modifications in the establishment of Th cell identity. Furthermore, our data highlight the potential to exploit this immune profiling application to elucidate the pathogenic role of Th transition states in autoimmune diseases.

Purpose and appropriate sample typesThis 19-parameter, 18-colour flow cytometry panel was designed and optimised to enable the comprehensive and simultaneous immunophenotyping of distinct T-cell and B-cell subsets within murine lymphoid tissues (Table 1). Cellular populations identified by employing this OMIP include 4 major subsets of B-cells (memory, activated, plasma cells and plasmablasts) and 7 major subsets of CD4+ T-cells (naive, central memory, effector memory, helper, regulatory, follicular helper and follicular regulatory). Staining was performed on freshly isolated splenocytes from 21-day-old neonatal BALB/c mice, however due to the omission of mouse strain-specific markers, this OMIP can be implemented across a range of murine models where in-depth immunophenotyping of the diverse repertoire of T-cell and B-cell populations localised within lymphoid tissues is required. O_TBL View this table: org.highwire.dtl.DTLVardef@1b488fdorg.highwire.dtl.DTLVardef@18dd356org.highwire.dtl.DTLVardef@2dce91org.highwire.dtl.DTLVardef@154cc27org.highwire.dtl.DTLVardef@27237c_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONSummary Table C_TABLECAPTION C_TBL

Adipocyte depots throughout the body are physiologically and molecularly distinct. With age, adipocytes increase in and around aged thymi. However, thymic adipocytes completely lack molecular characterization. We developed and optimized methods to isolate adipocyte nuclei from mouse thymi of different ages and sexes. Single-nucleus multiomic analysis of male and female mice aged 4-9 months reveals that thymic adipocytes are heterogeneous, with at least two distinct populations. One subpopulation harbors a transcription and chromatin signature consistent with beige/brown fat. A larger subpopulation more strongly resembles classic white adipose tissue and expresses genes associated with epithelial-to-mesenchymal transition (EMT) and antigen presentation. Analysis of differentially open chromatin in the white compared to beige adipose population identifies binding sites for Foxn1 and HIF-1/Arnt, consistent with a situation in which thymic white adipose cells emerge from thymic epithelial cells, possibly under hypoxic conditions. Immunofluorescence microscopy confirmed the expression of UCP1 protein in cells within the thymic parenchyma, most prominently in subcapsular cortical regions. This resource reveals a complex milieu of thymic adipocytes and identify multiple avenues for directly probing their ontogeny, dynamics and functional significance.