Coexpression of CD71 and CD117 Identifies an Early Unipotent Neutrophil Progenitor Population in Human Bone Marrow.

Neutrophils are the most abundant peripheral immune cells and thus, are continually replenished by bone marrow-derived progenitors. Still, how newly identified neutrophil subsets fit into the bone marrow neutrophil lineage remains unclear. Here, we use mass cytometry to show that two recently defined human neutrophil progenitor populations contain a homogeneous progenitor subset we term "early neutrophil progenitors" (eNePs) (Lin-CD66b+CD117+CD71+). Surface marker- and RNA-expression analyses, together with in vitro colony formation and in vivo adoptive humanized mouse transfers, indicate that eNePs are the earliest human neutrophil progenitors. Furthermore, we identified CD71 as a marker associated with the earliest neutrophil developmental stages. Expression of CD71 marks proliferating neutrophils, which were expanded in the blood of melanoma patients and detectable in blood and tumors from lung cancer patients. In summary, we establish CD117+CD71+ eNeP as the inceptive human neutrophil progenitor and propose a refined model of the neutrophil developmental lineage in bone marrow.

Single Cell High Dimensional Analysis of Human Peripheral Blood Mononuclear Cells Reveals Unique Intermediate Monocyte Subsets Associated with Sex Differences in Coronary Artery Disease.

Monocytes are associated with human cardiovascular disease progression. Monocytes are segregated into three major subsets: classical (cMo), intermediate (iMo), and nonclassical (nMo). Recent studies have identified heterogeneity within each of these main monocyte classes, yet the extent to which these subsets contribute to heart disease progression is not known. Peripheral blood mononuclear cells (PBMC) were obtained from 61 human subjects within the Coronary Assessment of Virginia (CAVA) Cohort. Coronary atherosclerosis severity was quantified using the Gensini Score (GS). We employed high-dimensional single-cell transcriptome and protein methods to define how human monocytes differ in subjects with low to severe coronary artery disease. We analyzed 487 immune-related genes and 49 surface proteins at the single-cell level using Antibody-Seq (Ab-Seq). We identified six subsets of myeloid cells (cMo, iMo, nMo, plasmacytoid DC, classical DC, and DC3) at the single-cell level based on surface proteins, and we associated these subsets with coronary artery disease (CAD) incidence based on Gensini score (GS) in each subject. Only frequencies of iMo were associated with high CAD (GS > 32), adj.p = 0.024. Spearman correlation analysis with GS from each subject revealed a positive correlation with iMo frequencies (r = 0.314, p = 0.014) and further showed a robust sex-dependent positive correlation in female subjects (r = 0.663, p = 0.004). cMo frequencies did not correlate with CAD severity. Key gene pathways differed in iMo among low and high CAD subjects and between males and females. Further single-cell analysis of iMo revealed three iMo subsets in human PBMC, distinguished by the expression of HLA-DR, CXCR3, and CD206. We found that the frequency of immunoregulatory iMo_HLA-DR+CXCR3+CD206+ was associated with CAD severity (adj.p = 0.006). The immunoregulatory iMo subset positively correlated with GS in both females (r = 0.660, p = 0.004) and males (r = 0.315, p = 0.037). Cell interaction analyses identified strong interactions of iMo with CD4+ effector/memory T cells and Tregs from the same subjects. This study shows the importance of iMo in CAD progression and suggests that iMo may have important functional roles in modulating CAD risk, particularly among females.

Atherosclerosis Impairs Naive CD4 T-Cell Responses via Disruption of Glycolysis.

Objective: CD4 T cells are important regulators of atherosclerotic progression. The metabolic profile of CD4 T cells controls their signaling and function, but how atherosclerosis affects T-cell metabolism is unknown. Here, we sought to determine the impact of atherosclerosis on CD4 T-cell metabolism and the contribution of such metabolic alterations to atheroprogression. Approach and Results: Using PCR arrays, we profiled the expression of metabolism genes in CD4 T cells from atherosclerotic apolipoprotein-E knockout mice fed a Western diet. These cells exhibited dysregulated expression of genes critically involved in glycolysis and fatty acid degradation, compared with those from animals fed a standard laboratory diet. We examined how T-cell metabolism was changed in either Western diet­fed apolipoprotein-E knockout mice or samples from patients with cardiovascular disease by measuring glucose uptake, activation, and proliferation in CD4 T cells. We found that naive CD4 T cells from Western diet­fed apolipoprotein-E knockout mice failed to uptake glucose and displayed impaired proliferation and activation, compared with CD4 T cells from standard laboratory diet­fed animals. Similarly, we observed that naive CD4 T-cell frequencies were reduced in the circulation of human subjects with high cardiovascular disease compared with low cardiovascular disease. Naive T cells from high cardiovascular disease subjects also showed reduced proliferative capacity. Conclusions: These results highlight the dysfunction that occurs in CD4 T-cell metabolism and immune responses during atherosclerosis. Targeting metabolic pathways within naive CD4 T cells could thus yield novel therapeutic approaches for improving CD4 T-cell responses against atheroprogression.

Patrolling Monocytes Control NK Cell Expression of Activating and Stimulatory Receptors to Curtail Lung Metastases.

The role of nonclassical, patrolling monocytes in lung tumor metastasis and their functional relationships with other immune cells remain poorly defined. Contributing to these gaps in knowledge is a lack of cellular specificity in commonly used approaches for depleting nonclassical monocytes. To circumvent these limitations and study the role of patrolling monocytes in melanoma metastasis to lungs, we generated C57BL/6J mice in which the Nr4a1 superenhancer E2 subdomain is ablated (E2 -/- mice). E2 -/- mice lack nonclassical patrolling monocytes but preserve classical monocyte and macrophage numbers and functions. Interestingly, NK cell recruitment and activation were impaired, and metastatic burden was increased in E2 -/-mice. E2 -/- mice displayed unchanged "educated" (CD11b+CD27+) and "terminally differentiated" (CD11b+CD27-) NK cell frequencies. These perturbations were accompanied by reduced expression of stimulatory receptor Ly49D on educated NK cells and increased expression of inhibitory receptor NKG2A/CD94 on terminally differentiated NK cells. Thus, our work demonstrates that patrolling monocytes play a critical role in preventing lung tumor metastasis via NK cell recruitment and activation.

Naive CD8+ T Cells Expressing CD95 Increase Human Cardiovascular Disease Severity.

OBJECTIVE: Cardiovascular disease (CVD) remains a significant global health concern with a high degree of mortality. While CD4+ T cells have been extensively studied in CVD, the importance of CD8+ T cells in this disease, despite their abundance and increased activation in human atherosclerotic plaques, remains largely unknown. Thus, the objective of this study was to compare peripheral T-cell signatures between humans with a high (severe) risk of CVD (including myocardial infarction or stroke) and those with a low risk of CVD. Approach and Results: Using mass cytometry, we uncovered a naive CD8+ T (TN) cell population expressing CD95 (termed CD95+CD8+ stem cell memory T [CD8 TSCM] cells) that was enriched in patients with high compared with low CVD. This T-cell subset enrichment within individuals with high CVD was a relative increase and resulted from the loss of CD95lo cells within the TN compartment. We found that CD8 TSCM cells positively correlated with CVD risk in humans, while CD8+ TN cells were inversely correlated. Atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice also displayed respective 7- and 2-fold increases in CD8+ TSCM frequencies within the peripheral blood and aorta-draining paraaortic lymph nodes compared with C57BL/6J mice. CD8+ TSCM cells were 1.7-fold increased in aortas from western diet fed ApoE-/- mice compared with normal laboratory diet-fed ApoE-/- mice. Importantly, transfer of TSCM cells into immune-deficient Rag.Ldlr recipient mice that lacked T cells increased atherosclerosis, illustrating the importance of these cells in atherogenesis. CONCLUSIONS: CD8+ TSCM cells are increased in humans with high CVD. As these TSCM cells promote atherosclerosis, targeting them may attenuate atherosclerotic plaque progression.

Neuropilin-1 Expression on CD4 T Cells Is Atherogenic and Facilitates T Cell Migration to the Aorta in Atherosclerosis.

Neuropilin 1 (Nrp1) is a type I transmembrane protein that plays important roles in axonal guidance, neuronal development, and angiogenesis. Nrp1 also helps migrate thymus-derived regulatory T cells to vascular endothelial growth factor (VEGF)-producing tumors. However, little is known about the role of Nrp1 on CD4 T cells in atherosclerosis. In ApoE-/- mice fed a Western diet for 15 wk, we found a 2-fold increase in Nrp1+Foxp3- CD4 T cells in their spleens, periaortic lymph nodes, and aortas, compared with chow-fed mice. Nrp1+Foxp3- CD4 T cells had higher proliferation potential, expressed higher levels of the memory marker CD44, and produced more IFN-γ when compared with Nrp1- CD4 T cells. Treatment of CD4 T cells with oxLDL increased Nrp1 expression. Furthermore, atherosclerosis-susceptible mice selectively deficient for Nrp1 expression on T cells developed less atherosclerosis than their Nrp1-sufficient counterparts. Mechanistically, we found that CD4 T cells that express Nrp1 have an increased capacity to migrate to the aorta and periaortic lymph nodes compared to Nrp1- T cells, suggesting that the expression of Nrp1 facilitates the recruitment of CD4 T cells into the aorta where they can be pathogenic. Thus, we have identified a novel role of Nrp1 on CD4 T cells in atherosclerosis. These results suggest that manipulation of Nrp1 expression on T cells can affect the outcome of atherosclerosis and lower disease incidence.

Scavenger Receptor CD36 Directs Nonclassical Monocyte Patrolling Along the Endothelium During Early Atherogenesis.

OBJECTIVE: Nonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature. APPROACH AND RESULTS: To study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE-/- (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet-fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36-/- mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein. CONCLUSIONS: Our studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12. These data suggest that NCM function in an atheroprotective manner through sensing and responding to oxidized lipoprotein moieties via scavenger receptor engagement during early atherogenesis.

Cardif (MAVS) Regulates the Maturation of NK Cells.

Cardif, also known as IPS-1, VISA, and MAVS, is an intracellular adaptor protein that functions downstream of the retinoic acid-inducible gene I family of pattern recognition receptors. Cardif is required for the production of type I IFNs and other inflammatory cytokines after retinoic acid-inducible gene I-like receptors recognize intracellular antigenic RNA. Studies have recently shown that Cardif may have other roles in the immune system in addition to its role in viral immunity. In this study, we find that the absence of Cardif alters normal NK cell development and maturation. Cardif(-/-) mice have a 35% loss of mature CD27(-)CD11b(+) NK cells in the periphery. In addition, Cardif(-/-) NK cells have altered surface marker expression, lower cytotoxicity, decreased intracellular STAT1 levels, increased apoptosis, and decreased proliferation compared with wild-type NK cells. Mixed chimeric mice revealed that the defective maturation and increased apoptotic rate of peripheral Cardif(-/-) NK cells is cell intrinsic. However, Cardif(-/-) mice showed enhanced control of mouse CMV (a DNA ß-herpesvirus) by NK cells, commensurate with increased activation and IFN-γ production by these immature NK cell subsets. These results indicate that the skewed differentiation and altered STAT expression of Cardif(-/-) NK cells can result in their hyperresponsiveness in some settings and support recent findings that Cardif-dependent signaling can regulate aspects of immune cell development and/or function distinct from its well-characterized role in mediating cell-intrinsic defense to RNA viruses.

NR4A1 (Nur77) deletion polarizes macrophages toward an inflammatory phenotype and increases atherosclerosis.

RATIONALE: NR4A1 (Nur77) is a nuclear receptor that is expressed in macrophages and within atherosclerotic lesions, yet its function in atherosclerosis is unknown. OBJECTIVE: Nur77 regulates the development of monocytes, particularly patrolling Ly6C(-) monocytes that may be involved in resolution of inflammation. We sought to determine how absence of nuclear receptor subfamily 4, group A, member 1 (NR4A1) in hematopoietic cells affected atherosclerosis development. METHODS AND RESULTS: Nur77(-/-) chimeric mice on a Ldlr(-/-) background showed a 3-fold increase in atherosclerosis development when fed a Western diet for 20 weeks, despite having a drastic reduction in Ly6C(-) patrolling monocytes. In a second model, mice deficient in both Nur77 and ApoE (ApoE(-/-)Nur77(-/-)) also showed increased atherosclerosis after 11 weeks of Western diet. Atherosclerosis was associated with a significant change in macrophage polarization toward a proinflammatory phenotype, with high expression of tumor necrosis factor-α and nitric oxide and low expression of Arginase-I. Moreover, we found increased expression of toll-like receptor 4 mRNA and protein in Nur77(-/-) macrophages as well as increased phosphorylation of the p65 subunit of NFκB. Inhibition of NFκB activity blocked excess activation of Nur77(-/-) macrophages. CONCLUSIONS: We conclude that the absence of Nur77 in monocytes and macrophages results in enhanced toll-like receptor signaling and polarization of macrophages toward a proinflammatory M1 phenotype. Despite having fewer monocytes, Nur77(-/-) mice developed significant atherosclerosis when fed a Western diet. These studies indicate that Nur77 is a novel target for modulating the inflammatory phenotype of monocytes and macrophages and may be important for regulation of atherogenesis.

ATP-binding cassette transporter G1 intrinsically regulates invariant NKT cell development.

ATP-binding cassette transporter G1 (ABCG1) plays a role in the intracellular transport of cholesterol. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid Ags. In this study, we demonstrate that ABCG1 regulates iNKT cell development and functions in a cell-intrinsic manner. Abcg1(-/-) mice displayed reduced frequencies of iNKT cells in thymus and periphery. Thymic iNKT cells deficient in ABCG1 had reduced membrane lipid raft content, and showed impaired proliferation and defective maturation during the early stages of development. Moreover, we found that Abcg1(-/-) mice possess a higher frequency of Vß7(+) iNKT cells, suggesting alterations in iNKT cell thymic selection. Furthermore, in response to CD3ε/CD28 stimulation, Abcg1(-/-) thymic iNKT cells showed reduced production of IL-4 but increased production of IFN-γ. Our results demonstrate that changes in intracellular cholesterol homeostasis by ABCG1 profoundly impact iNKT cell development and function.

Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a novel subset of CD9+ monocytes associated with COVID-19 hospitalization.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9+ monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9+ monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9+ monocytes to COVID-19 progression.

Single cell transcriptomics and TCR reconstruction reveal CD4 T cell response to MHC-II-restricted APOB epitope in human cardiovascular disease.

Atherosclerosis is accompanied by a CD4 T cell response to apolipoprotein B (APOB). Major Histocompatibility Complex (MHC)-II tetramers can be used to isolate antigen-specific CD4 T cells by flow sorting. Here, we produce, validate and use an MHC-II tetramer, DRB1*07:01 APOB-p18, to sort APOB-p18-specific cells from peripheral blood mononuclear cell samples from 8 DRB1*07:01+ women with and without subclinical cardiovascular disease (sCVD). Single cell RNA sequencing showed that transcriptomes of tetramer+ cells were between regulatory and memory T cells in healthy women and moved closer to memory T cells in women with sCVD. TCR sequencing of tetramer+ cells showed clonal expansion and V and J segment usage similar to those found in regulatory T cells. These findings suggest that APOB-specific regulatory T cells may switch to a more memory-like phenotype in women with atherosclerosis. Mouse studies showed that such switched cells promote atherosclerosis.

Frontline Science: Kindlin-3 is essential for patrolling and phagocytosis functions of nonclassical monocytes during metastatic cancer surveillance.

Nonclassical monocytes maintain vascular homeostasis by patrolling the vascular endothelium, responding to inflammatory signals, and scavenging cellular debris. Nonclassical monocytes also prevent metastatic tumor cells from seeding new tissues, but whether the patrolling function of nonclassical monocytes is required for this process is unknown. To answer this question, we utilized an inducible-knockout mouse that exhibits loss of the integrin-adaptor protein Kindlin-3 specifically in nonclassical monocytes. We show that Kindlin-3-deficient nonclassical monocytes are unable to patrol the vascular endothelium in either the lungs or periphery. We also find that Kindlin-3-deficient nonclassical monocytes cannot firmly adhere to, and instead "slip" along, the vascular endothelium. Loss of patrolling activity by nonclassical monocytes was phenocopied by ablation of LFA-1, an integrin-binding partner of Kindlin-3. When B16F10 murine melanoma tumor cells were introduced into Kindlin-3-deficient mice, nonclassical monocytes showed defective patrolling towards tumor cells and failure to ingest tumor particles in vivo. Consequently, we observed a significant, 4-fold increase in lung tumor metastases in mice possessing Kindlin-3-deficient nonclassical monocytes. Thus, we conclude that the patrolling function of nonclassical monocytes is mediated by Kindlin-3 and essential for these cells to maintain vascular endothelial homeostasis and prevent tumor metastasis to the lung.

A practical guide to rodent islet isolation and assessment.

Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes.

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Identification of an Early Unipotent Neutrophil Progenitor with Pro-tumoral Activity in Mouse and Human Bone Marrow.

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however, unipotent neutrophil progenitors are not well defined. Here, we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly, we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models, including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases, including cancer.

Apolipoprotein AI prevents regulatory to follicular helper T cell switching during atherosclerosis.

Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here we show that during atherogenesis Treg cells lose Foxp3 expression and their immunosuppressive function, leading to the conversion of a fraction of these cells into T follicular helper (Tfh) cells. We show that Tfh cells are pro-atherogenic and that their depletion reduces atherosclerosis. Mechanistically, the conversion of Treg cells to Tfh cells correlates with reduced expression of IL-2Rα and pSTAT5 levels and increased expression of IL-6Rα. In vitro, incubation of naive T cells with oxLDL prevents their differentiation into Treg cells. Furthermore, injection of lipid-free Apolipoprotein AI (ApoAI) into ApoE-/- mice reduces intracellular cholesterol levels in Treg cells and prevents their conversion into Tfh cells. Together our results suggest that ApoAI, the main protein in high-density lipoprotein particles, modulates the cellular fate of Treg cells and thus influences the immune response during atherosclerosis.

Loss of ABCG1 influences regulatory T cell differentiation and atherosclerosis.

ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol accumulation and alters T cell homeostasis, which may contribute to progression of atherosclerosis. Here, we investigated how the selective loss of ABCG1 in T cells impacts atherosclerosis in LDL receptor-deficient (LDLR-deficient) mice, a model of the disease. In LDLR-deficient mice fed a high-cholesterol diet, T cell-specific ABCG1 deficiency protected against atherosclerotic lesions. Furthermore, T cell-specific ABCG1 deficiency led to a 30% increase in Treg percentages in aorta and aorta-draining lymph nodes (LNs) of these mice compared with animals with only LDLR deficiency. When Abcg1 was selectively deleted in Tregs of LDLR-deficient mice, we observed a 30% increase in Treg percentages in aorta and aorta-draining LNs and reduced atherosclerosis. In the absence of ABCG1, intracellular cholesterol accumulation led to downregulation of the mTOR pathway, which increased the differentiation of naive CD4 T cells into Tregs. The increase in Tregs resulted in reduced T cell activation and increased IL-10 production by T cells. Last, we found that higher ABCG1 expression in Tregs was associated with a higher frequency of these cells in human blood samples. Our study indicates that ABCG1 regulates T cell differentiation into Tregs, highlighting a pathway by which cholesterol accumulation can influence T cell homeostasis in atherosclerosis.

The cholesterol transporter ABCG1 links cholesterol homeostasis and tumour immunity.

ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol efflux from cells and regulates intracellular cholesterol homeostasis. Here we demonstrate a role of ABCG1 as a mediator of tumour immunity. Abcg1(-/-) mice have dramatically suppressed subcutaneous MB49-bladder carcinoma and B16-melanoma growth and prolonged survival. We show that reduced tumour growth in Abcg1(-/-) mice is myeloid cell intrinsic and is associated with a phenotypic shift of the macrophages from a tumour-promoting M2 to a tumour-fighting M1 within the tumour. Abcg1(-/-) macrophages exhibit an intrinsic bias towards M1 polarization with increased NF-κB activation and direct cytotoxicity for tumour cells in vitro. Overall, our study demonstrates that the absence of ABCG1 inhibits tumour growth through modulation of macrophage function within the tumour, and illustrates a link between cholesterol homeostasis and cancer.

The nuclear receptor nr4a1 controls CD8 T cell development through transcriptional suppression of runx3.

The NR4A nuclear receptor family member Nr4a1 is strongly induced in thymocytes undergoing selection, and has been shown to control the development of Treg cells; however the role of Nr4a1 in CD8(+) T cells remains undefined. Here we report a novel role for Nr4a1 in regulating the development and frequency of CD8(+) T cells through direct transcriptional control of Runx3. We discovered that Nr4a1 recruits the corepressor, CoREST to suppress Runx3 expression in CD8(+) T cells. Loss of Nr4a1 results in increased Runx3 expression in thymocytes which consequently causes a 2-fold increase in the frequency and total number of intrathymic and peripheral CD8(+) T cells. Our findings establish Nr4a1 as a novel and critical player in the regulation of CD8 T cell development through the direct suppression of Runx3.