UBXN3B is crucial for B lymphopoiesis.

BACKGROUND: The ubiquitin regulatory X (UBX) domain-containing proteins (UBXNs) are putative adaptors for ubiquitin ligases and valosin-containing protein; however, their in vivo physiological functions remain poorly characterised. We recently showed that UBXN3B is essential for activating innate immunity to DNA viruses and controlling DNA/RNA virus infection. Herein, we investigate its role in adaptive immunity. METHODS: We evaluated the antibody responses to multiple viruses and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza in tamoxifen-inducible global and constitutive B cell-specific Ubxn3b knockout mice; quantified various immune populations, B lineage progenitors/precursors, B cell receptor (BCR) signalling and apoptosis by flow cytometry, immunoblotting and immunofluorescence microscopy. We also performed bone marrow transfer, single-cell and bulk RNA sequencing. FINDINGS: Both global and B cell-specific Ubxn3b knockout mice present a marked reduction in small precursor B-II (>60%), immature (>70%) and mature B (>95%) cell numbers. Transfer of wildtype bone marrow to irradiated global Ubxn3b knockouts restores normal B lymphopoiesis, while reverse transplantation does not. The mature B population shrinks rapidly with apoptosis and higher pro and activated caspase-3 protein levels were observed following induction of Ubxn3b knockout. Mechanistically, Ubxn3b deficiency leads to impaired pre-BCR signalling and cell cycle arrest. Ubxn3b knockout mice are highly vulnerable to respiratory viruses, with increased viral loads and prolonged immunopathology in the lung, and reduced production of virus-specific IgM/IgG. INTERPRETATION: UBXN3B is essential for B lymphopoiesis by maintaining constitutive pre-BCR signalling and cell survival in a cell-intrinsic manner. FUNDING: United States National Institutes of Health grants, R01AI132526 and R21AI155820.

Interleukin-17 directly stimulates tumor infiltrating Tregs to prevent cancer development.

Background: Interleukin-17 (IL-17) family cytokines promote protective inflammation for pathogen resistance, but also facilitate autoimmunity and tumor development. A direct signal of IL-17 to regulatory T cells (Tregs) has not been reported and may help explain these dichotomous responses. Methods: We generated a conditional knockout of Il17ra in Tregs by crossing Foxp3-YFP-Cre mice to Il17ra-flox mice (Il17ra ΔTreg mice). Subsequently, we adoptively transferred bone marrow cells from Il17ra ΔTreg mice to a mouse model of sporadic colorectal cancer (Cdx2-Cre +/Apc F/+), to selectively ablate IL-17 direct signaling on Tregs in colorectal cancer. Single cell RNA sequencing and bulk RNA sequencing were performed on purified Tregs from mouse colorectal tumors, and compared to those of human tumor infiltrating Treg cells. Results: IL-17 Receptor A (IL-17RA) is expressed in Tregs that reside in mouse mesenteric lymph nodes and colon tumors. Ablation of IL-17RA, specifically in Tregs, resulted in increased Th17 cells, and exacerbated tumor development. Mechanistically, tumor-infiltrating Tregs exhibit a unique gene signature that is linked to their activation, maturation, and suppression function, and this signature is in part supported by the direct signaling of IL-17 to Tregs. To study pathways of Treg programming, we found that loss of IL-17RA in tumor Tregs resulted in reduced RNA splicing, and downregulation of several RNA binding proteins that are known to regulate alternative splicing and promote Treg function. Conclusion: IL-17 directly signals to Tregs and promotes their maturation and function. This signaling pathway constitutes a negative feedback loop that controls cancer-promoting inflammation in CRC.

IRAK4 is an immunological checkpoint in neuropsychiatric systemic lupus erythematosus.

The search for dementia treatments, including treatments for neuropsychiatric lupus (NPSLE), has not yet uncovered useful therapeutic targets that mitigate underlying inflammation. Currently, NPSLE's limited treatment options are often accompanied by severe toxicity. Blocking toll-like receptor (TLR) and IL-1 receptor signal transduction by inhibiting interleukin-1 receptor-associated kinase 4 (IRAK4) offers a new pathway for intervention. Using a pre-clinical NPSLE model, we compare lupus-like B6.MRL-Faslpr (MRL) mice with B6.MRL-Faslpr-IRAK4 kinase-dead (MRL-IRAK4-KD) mice, which are were less prone to 'general' lupus-like symptoms. We demonstrate that lupus-prone mice with a mutation in the kinase domain of IRAK4 no longer display typical lupus hallmarks such as splenomegaly, inflammation, production of hormones, and anti-double-stranded (ds)DNA antibody. water maze behavioral testing, which measures contextual associative learning, revealed that mice without functional IRAK4 displayed a recovery in memory acquisition deficits. RNA-seq approach revealed that cytokine and hormone signaling converge on the JAK/STAT pathways in the mouse hippocampus. Ultimately, the targets identified in this work may result in broad clinical value that can fill the significant scientific and therapeutic gaps precluding development of cures for dementia.

UBXN9 governs GLUT4-mediated spatial confinement of RIG-I-like receptors and signaling.

The cytoplasmic RIG-I-like receptors (RLRs) recognize viral RNA and initiate innate antiviral immunity. RLR signaling also triggers glycolytic reprogramming through glucose transporters (GLUTs), whose role in antiviral immunity is elusive. Here, we unveil that insulin-responsive GLUT4 inhibits RLR signaling independently of glucose uptake in adipose and muscle tissues. At steady state, GLUT4 is docked at the Golgi matrix by ubiquitin regulatory X domain 9 (UBXN9, TUG). Following RNA virus infection, GLUT4 is released and translocated to the cell surface where it spatially segregates a significant pool of cytosolic RLRs, preventing them from activating IFN-ß responses. UBXN9 deletion prompts constitutive GLUT4 trafficking, sequestration of RLRs, and attenuation of antiviral immunity, whereas GLUT4 deletion heightens RLR signaling. Notably, reduced GLUT4 expression is uniquely associated with human inflammatory myopathies characterized by hyperactive interferon responses. Overall, our results demonstrate a noncanonical UBXN9-GLUT4 axis that controls antiviral immunity via plasma membrane tethering of cytosolic RLRs.

UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors.

The Retinoic acid-Inducible Gene I (RIG-I) like receptors (RLRs) are the major viral RNA sensors essential for the initiation of antiviral immune responses. RLRs are subjected to stringent transcriptional and posttranslational regulations, of which ubiquitination is one of the most important. However, the role of ubiquitination in RLR transcription is unknown. Here, we screen 375 definite ubiquitin ligase knockout cell lines and identify Ubiquitin Protein Ligase E3 Component N-Recognin 5 (UBR5) as a positive regulator of RLR transcription. UBR5 deficiency reduces antiviral immune responses to RNA viruses, while increases viral replication in primary cells and mice. Ubr5 knockout mice are more susceptible to lethal RNA virus infection than wild type littermates. Mechanistically, UBR5 mediates the Lysine 63-linked ubiquitination of Tripartite Motif Protein 28 (TRIM28), an epigenetic repressor of RLRs. This modification prevents intramolecular SUMOylation of TRIM28, thus disengages the TRIM28-imposed brake on RLR transcription. In sum, UBR5 enables rapid upregulation of RLR expression to boost antiviral immune responses by ubiquitinating and de-SUMOylating TRIM28.

Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. Astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout (Timp1KO) mice do not efficiently remyelinate following a demyelinating injury. Here, we performed an unbiased proteomic analysis and identified a fibronectin-derived peptide called Anastellin (Ana) that was unique to the Timp1KO astrocyte secretome. Ana was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Ana is known to act upon the sphingosine-1-phosphate receptor 1, and we determined that Ana also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro. Administration of FTY720 to wild-type C57BL/6 mice during MOG35-55-experimental autoimmune encephalomyelitis ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 (Timp1KO) had no effect. Analysis of Timp1 and fibronectin (FN1) transcripts from primary human astrocytes from healthy and multiple sclerosis (MS) donors revealed lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Last, analyses of proteomic databases of MS samples identified Ana peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high disease activity. A role for Ana in MS as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and innate remyelination potential in the MS brain.

CD8+ T cell depletion prevents neuropathology in a mouse model of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) or Krabbe's disease is a fatal genetic demyelinating disease of the central nervous system caused by loss-of-function mutations in the galactosylceramidase (galc) gene. While the metabolic basis for disease is known, the understanding of how this results in neuropathology is not well understood. Herein, we report that the rapid and protracted elevation of CD8+ cytotoxic T lymphocytes occurs coincident with clinical disease in a mouse model of GLD. Administration of a function-blocking antibody against CD8α effectively prevented disease onset, reduced morbidity and mortality, and prevented CNS demyelination in mice. These data indicate that subsequent to the genetic cause of disease, neuropathology is driven by pathogenic CD8+ T cells, thus offering novel therapeutic potential for treatment of GLD.

Capturing the multifaceted function of adipose tissue macrophages.

Adipose tissue macrophages (ATMs) bolster obesity-induced metabolic dysfunction and represent a targetable population to lessen obesity-associated health risks. However, ATMs also facilitate adipose tissue function through multiple actions, including adipocyte clearance, lipid scavenging and metabolism, extracellular remodeling, and supporting angiogenesis and adipogenesis. Thus, high-resolution methods are needed to capture macrophages' dynamic and multifaceted functions in adipose tissue. Herein, we review current knowledge on regulatory networks critical to macrophage plasticity and their multifaceted response in the complex adipose tissue microenvironment.

Astrocytic TIMP-1 regulates production of Anastellin, a novel inhibitor of oligodendrocyte differentiation and FTY720 responses.

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. We have previously demonstrated that murine astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout ( Timp1 KO ) mice do not efficiently remyelinate following a demyelinating injury. To better understand the basis of this, we performed unbiased proteomic analyses and identified a fibronectin-derived peptide called anastellin that is unique to the murine Timp1 KO astrocyte secretome. Anastellin was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Anastellin is known to act upon the sphingosine-1-phosphate receptor 1 (S1PR1), and we determined that anastellin also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro . Further, administration of FTY720 to wild-type C57BL/6 mice during MOG 35-55 -EAE ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 in astrocytes ( Timp1 cKO ) had no effect. Analysis of human TIMP1 and fibronectin ( FN1 ) transcripts from healthy and multiple sclerosis (MS) patient brain samples revealed an inverse relationship where lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Lastly, we analyzed proteomic databases of MS samples and identified anastellin peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high versus low disease activity. The prospective role for anastellin generation in association with myelin lesions as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and the innate remyelination potential of the the MS brain. Significance Statement: Astrocytic production of TIMP-1 prevents the protein catabolism of fibronectin. In the absence of TIMP-1, fibronectin is further digested leading to a higher abundance of anastellin peptides that can bind to sphingosine-1-phosphate receptor 1. The binding of anastellin with the sphingosine-1-phosphate receptor 1 impairs the differentiation of oligodendrocytes progenitor cells into myelinating oligodendrocytes in vitro , and negates the astrocyte-mediated therapeutic effects of FTY720 in the EAE model of chronic CNS inflammation. These data indicate that TIMP-1 production by astrocytes is important in coordinating astrocytic functions during inflammation. In the absence of astrocyte produced TIMP-1, elevated expression of anastellin may represent a prospective biomarker for FTY720 therapeutic responsiveness.

Antigen-specific downregulation of miR-150 in CD4 T cells promotes cell survival.

MicroRNA-150 (miR-150) has been shown to play a general role in the immune system, but very little is known about its role on CD4+ T cell responses. During T cell responses against superantigen Staphylococcal Enterotoxin A, miR-150 expression was down-regulated in antigen-specific CD4+ T cells but up-regulated in CD8+ T cells. CD4+ and CD8+ T cell clonal expansion was greater in miR-150-KO mice than in WT mice, but miR-150 selectively repressed IL-2 production in CD4+ T cells. Transcriptome analysis of CD4+ T cells demonstrated that apoptosis and mTOR pathways were highly enriched in the absence of miR-150. Mechanistic studies confirmed that miR-150 promoted apoptosis specifically in antigen-specific CD4+ T cells, but not in bystander CD4+ nor in CD8+ T cells. Furthermore, inhibition of mTOR-linked mitochondrial superoxidedismutase-2 increased apoptosis in miR-150-/- antigen-specific CD4+ T. Thus, miR-150 impacts CD4+ T cell helper activity by attenuating IL-2 production along with clonal expansion, and suppresses superoxidedismutase to promote apoptosis.

Interleukin-17 Promotes the Infiltration of CD8+ T Cells into the Brain in a Mouse Model for Alzheimer's Disease.

BACKGROUND: Interleukin-17 (IL-17) family cytokines play critical roles in inflammation and pathogen resistance. Inflammation in the central nervous system, denoted as neuroinflammation, promotes the onset and progression of Alzheimer's disease (AD). Previous studies showed that IL-17A neutralizing antibody treatment alleviated Amyloid ß (Aß) burden in rodent models of AD, while overexpression of IL-17A in mouse lateral ventricles rescued part of the AD pathology. However, the involvement of IL-17 in AD and its mechanism of action remain largely unknown. METHODS: To investigate the role of IL-17 in AD, we crossed mice lacking the common receptor of IL-17 signaling (IL-17RA knockout mice) to the APP/PS1 mouse model of AD. We then analyzed the composition of immune cells and cytokines/chemokines during different phases of AD pathology, and interrogated the underlying mechanism by which IL-17 may regulate immune cell infiltration into AD brains. RESULTS: Ablation of IL-17RA in APP/PS1 mice decreased infiltration of CD8+ T cells and myeloid cells to mouse brain. IL-17 was able to promote the production of myeloid- and T cell-attracting chemokines CXCL1 and CXCL9/10 in primary glial cells. We also observed that IL-17 is upregulated in the late stage of AD development, and ectopic expression of IL-17 via adenoviral infection to the cortex trended towards worsened cognition in APP/PS1 mice, suggesting a pathogenic role of excessive IL-17 in AD. CONCLUSION: Our data show that IL-17 signaling promotes neuroinflammation in AD by accelerating the infiltration of CD8+ T lymphocytes and Gr1+ CD11b+ myeloid cells.

Macrophages at the Crossroad of Meta-Inflammation and Inflammaging.

Macrophages are central players in systemic inflammation associated with obesity and aging, termed meta-inflammation and inflammaging. Activities of macrophages elicited by the two chronic conditions display shared and distinct patterns mechanistically, resulting in multifaceted actions for their pathogenic roles. Drastically expanded tissue macrophage populations under obesity and aging stress attribute to both enhanced recruitment and local expansion. Importantly, molecular networks governing the multifaceted actions of macrophages are directly altered by environmental cues and subsequently contribute to metabolic reprogramming, resulting in meta-inflammation in obesity or inflammaging in aging. In this review, we will summarize how meta-inflammation and inflammaging affect macrophages and the molecular mechanisms involved in these processes.

Nexinhib20 Inhibits Neutrophil Adhesion and ß2 Integrin Activation by Antagonizing Rac-1-Guanosine 5'-Triphosphate Interaction.

Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting ß2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced ß2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and ß2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.

Splice factor polypyrimidine tract-binding protein 1 (Ptbp1) primes endothelial inflammation in atherogenic disturbed flow conditions.

NF-κB-mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through adhesion molecules Icam1 and Vcam1. The endothelium is primed for cytokine activation of NF-κB by exposure to low and disturbed blood flow (LDF)but the molecular underpinnings are not fully understood. In an experimental in vivo model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NF-κB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam1 induction by tumor necrosis factor (TNF)-α stimulation as a readout, we performed a CRISPR Cas9 knockout screen and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no effect on cell growth or response to apoptotic stimuli, but reversed LDF splicing patterns and inhibited NF-κB nuclear translocation and transcriptional activation of downstream targets, including Icam1 and Vcam1. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo, endothelial-specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice, limiting atherosclerosis. This may be mediated, in part, by allowing inclusion of a conserved alternative exon in Ripk1 leading to a reduction in Ripk1 protein. Our data show that Ptbp1, which is induced in a subset of the endothelium by platelet recruitment at regions of LDF, is required for priming of the endothelium for subsequent NF-κB activation, myeloid cell recruitment and atherosclerosis.

Evaluating the glycolytic potential of mouse costimulated effector CD8+ T cells ex vivo.

Studying the metabolic fitness of T cells is fundamental to understand how immune responses are regulated. Here, we describe a step-by-step protocol optimized to efficiently generate and isolate effector antigen-specific CD8+ T cells ex vivo using costimulation. We also detail steps to evaluate their metabolic activity using Seahorse technology. This protocol can be used to measure the glycolytic potential of effector murine T cells in response to different manipulations, such as infections, adjuvant studies, gene editing, or metabolite supplementation. For complete details on the use and execution of this protocol, please refer to Agliano et al. (2022).

Optimal CD8+ T cell effector function requires costimulation-induced RNA-binding proteins that reprogram the transcript isoform landscape.

Boosting T cell activation through costimulation directs defense against cancer and viral infections. Despite multiple studies targeting costimulation in clinical trials, the increased potency and reprogramming of T cells endowed by costimulation is poorly understood. Canonical dogma states that transcription mediates T cell activation. Here, we show that the spliceosome, controlling post-transcriptional alternative splicing and alternative polyadenylation, is the most enriched pathway in T cells after CD134/CD137 costimulation. Costimulation of CD8+ T cells significantly increases expression of 29 RNA-binding proteins while RNA-seq uncovers over 1000 differential alternative splicing and polyadenylation events. Using in vivo mouse and in vitro human models, we demonstrate that RNA-binding protein Tardbp is required for effector cytokine production, CD8+ T cell clonal expansion, and isoform regulation after costimulation. The prospect of immune response optimization through reprogramming of mRNA isoform production offered herein opens new avenues for experimentally and therapeutically tuning the activities of T cells.

Nicotinamide breaks effector CD8 T cell responses by targeting mTOR signaling.

Nicotinamide (NAM) shapes T cell responses but its precise molecular mechanism of action remains elusive. Here, we show that NAM impairs naive T cell effector transition but also effector T cells themselves. Although aerobic glycolysis is a hallmark of activated T cells, CD8+ T cells exposed to NAM displayed enhanced glycolysis, yet producing significantly less IFNγ. Mechanistically, NAM reduced mTORC1 activity independently of NAD+ metabolism, decreasing IFNγ translation and regulating T cell transcriptional factors critical to effector/memory fate. Finally, the role of NAM in a biomedically relevant model of lung injury was tested. Specifically, a NAM-supplemented diet reduced systemic IL-2, antigen-specific T cell clonal expansion, and effector function after inhalation of Staphylococcus aureus enterotoxin A. These findings identify NAM as a potential therapeutic supplement that uncouples glycolysis from effector cytokine production and may be a powerful treatment for diseases associated with T cell hyperactivation.

Concentration of vaginal and systemic cytokines obtained early in pregnancy and their impact on preterm birth.

OBJECTIVE: A number of factors can lead to a maternal pro-inflammatory response resulting in a spontaneous preterm birth. However, it remains unknown if an upregulation in the maternal immune system early in pregnancy leads to an increase in pro-inflammatory cytokines and ultimately preterm birth. Therefore, we hypothesize an increase in vaginal and systemic pro-inflammatory cytokines early pregnancy is associated with an increased risk of preterm birth. STUDY DESIGN: Patients initiating prenatal care prior to 14 weeks gestation were recruited for eligibility. A vaginal swab and serum sample was obtained at the first prenatal visit and these were then stored at -80 C. Patients were then followed for their gestational age at delivery. Five patients delivering preterm (cases) were matched with ten patients delivering at term (controls) based on age, BMI, smoking status and ethnicity. The serum and vaginal swabs from the cases and controls were then analyzed for the following cytokines using a multiplex cytokine assay: GM-CSF, IL-1b, IL-6, TNFα, and Rantes. RESULTS: A total of 116 patients were screened for eligibility and 96 of these patients had samples obtained prior to 14 weeks gestation. Of these 96, 5 had a spontaneous preterm birth and these were matched to 10 controls. There was no difference detected in the cytokine concentrations of GM-CSF, IL-1b, IL-6, TNFα, and Rantes in the serum or cervicovaginal fluid between cases and controls. CONCLUSION: This study demonstrates there is no difference in cytokine concentrations of several pro-inflammatory cytokines in the vagina or in the serum prior to 14 weeks gestation in patients delivering preterm. Therefore, the concentration of the cytokines analyzed in this study from the vagina and serum have little predictive value on the risk of preterm birth. Further research is needed to deepen our understanding of the mechanisms leading to preterm birth.

AtheroSpectrum Reveals Novel Macrophage Foam Cell Gene Signatures Associated With Atherosclerotic Cardiovascular Disease Risk.

BACKGROUND: Whereas several interventions can effectively lower lipid levels in people at risk for atherosclerotic cardiovascular disease (ASCVD), cardiovascular event risks remain, suggesting an unmet medical need to identify factors contributing to cardiovascular event risk. Monocytes and macrophages play central roles in atherosclerosis, but studies have yet to provide a detailed view of macrophage populations involved in increased ASCVD risk. METHODS: A novel macrophage foaming analytics tool, AtheroSpectrum, was developed using 2 quantitative indices depicting lipid metabolism and the inflammatory status of macrophages. A machine learning algorithm was developed to analyze gene expression patterns in the peripheral monocyte transcriptome of MESA participants (Multi-Ethnic Study of Atherosclerosis; set 1; n=911). A list of 30 genes was generated and integrated with traditional risk factors to create an ASCVD risk prediction model (30-gene cardiovascular disease risk score [CR-30]), which was subsequently validated in the remaining MESA participants (set 2; n=228); performance of CR-30 was also tested in 2 independent human atherosclerotic tissue transcriptome data sets (GTEx [Genotype-Tissue Expression] and GSE43292). RESULTS: Using single-cell transcriptomic profiles (GSE97310, GSE116240, GSE97941, and FR-FCM-Z23S), AtheroSpectrum detected 2 distinct programs in plaque macrophages-homeostatic foaming and inflammatory pathogenic foaming-the latter of which was positively associated with severity of atherosclerosis in multiple studies. A pool of 2209 pathogenic foaming genes was extracted and screened to select a subset of 30 genes correlated with cardiovascular event in MESA set 1. A cardiovascular disease risk score model (CR-30) was then developed by incorporating this gene set with traditional variables sensitive to cardiovascular event in MESA set 1 after cross-validation generalizability analysis. The performance of CR-30 was then tested in MESA set 2 (P=2.60×10-4; area under the receiver operating characteristic curve, 0.742) and 2 independent data sets (GTEx: P=7.32×10-17; area under the receiver operating characteristic curve, 0.664; GSE43292: P=7.04×10-2; area under the receiver operating characteristic curve, 0.633). Model sensitivity tests confirmed the contribution of the 30-gene panel to the prediction model (likelihood ratio test; df=31, P=0.03). CONCLUSIONS: Our novel computational program (AtheroSpectrum) identified a specific gene expression profile associated with inflammatory macrophage foam cells. A subset of 30 genes expressed in circulating monocytes jointly contributed to prediction of symptomatic atherosclerotic vascular disease. Incorporating a pathogenic foaming gene set with known risk factors can significantly strengthen the power to predict ASCVD risk. Our programs may facilitate both mechanistic investigations and development of therapeutic and prognostic strategies for ASCVD risk.

A high OXPHOS CD8 T cell subset is predictive of immunotherapy resistance in melanoma patients.

Immune checkpoint inhibitor (ICI) therapy continues to revolutionize melanoma treatment, but only a subset of patients respond. Major efforts are underway to develop minimally invasive predictive assays of ICI response. Using single-cell transcriptomics, we discovered a unique CD8 T cell blood/tumor-shared subpopulation in melanoma patients with high levels of oxidative phosphorylation (OXPHOS), the ectonucleotidases CD38 and CD39, and both exhaustion and cytotoxicity markers. We called this population with high levels of OXPHOS "CD8+ TOXPHOS cells." We validated that higher levels of OXPHOS in tumor- and peripheral blood-derived CD8+ TOXPHOS cells correlated with ICI resistance in melanoma patients. We then developed an ICI therapy response predictive model using a transcriptomic profile of CD8+ TOXPHOS cells. This model is capable of discerning responders from nonresponders using either tumor or peripheral blood CD8 T cells with high accuracy in multiple validation cohorts. In sum, CD8+ TOXPHOS cells represent a critical immune population to assess ICI response with the potential to be a new target to improve outcomes in melanoma patients.