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Background: Cardiac allograft vasculopathy (CAV) is the leading cause of late graft failure and mortality after heart transplantation (HT). Current strategies for early diagnosis and effective treatment of CAV are lacking. Using single-cell RNA-sequencing in peripheral blood mononuclear cells (PBMCs), we sought to investigate cell-specific gene expression profiles and T cell receptor repertoires in CAV that may inform novel biomarkers and pathways to interrupt CAV pathogenesis., Methods: Whole blood was collected from 22 HT recipients with angiographically-confirmed CAV and 18 HT recipients without CAV. PBMCs were isolated and subjected to single-cell RNA-sequencing using a 10X Genomics microfluidic platform. Downstream analyses focused on differential expression of genes, cell compositional changes, and T cell receptor repertoire analyses., Results: Across 40 PBMC samples, we isolated 134,984 cells spanning 31 cell types. Compositional analyses showed subtle, but significant increases in CD4+ T central memory cells, and CD14+ and CD16+ monocytes in high-grade CAV (CAV-2 and CAV-3). 745 genes were differentially expressed in a cell-specific manner in high-grade CAV, enriched for putative pathways involved in inflammation and angiogenesis. Intersection with the druggable genome prioritized 68 targets, including targets with approved drugs in cardiovascular disease (e.g., canakinumab). There were no significant differences in T cell clonality or diversity with increasing CAV severity., Conclusions: Unbiased whole transcriptomic analyses at single-cell resolution identify unique, cell-specific gene expression patterns in CAV, suggesting the potential utility of peripheral gene expression biomarkers in diagnosing CAV. Furthermore, precision targeting of these pathways may offer opportunities to mitigate CAV pathogenesis., (Copyright © 2024 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Temporal ordering of cellular events offers fundamental insights into biological phenomena. Although this is traditionally achieved through continuous direct observations 1,2 , an alternative solution leverages irreversible genetic changes, such as naturally occurring mutations, to create indelible marks that enables retrospective temporal ordering 3-5 . Using a multipurpose, single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo, with incorporation of cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during mouse embryonic development, unconventional developmental relationships between cell types and new epithelial progenitor states by their unique genetic histories. Analysis of mouse adenomas, coupled to multiomic and single-cell profiling of human precancers, with clonal analysis of 418 human polyps, demonstrated the occurrence of polyclonal initiation in 15-30% of colonic precancers, showing their origins from multiple normal founders. Our study presents a multimodal framework that lays the foundation for in vivo recording, integrating synthetic or natural indelible genetic changes with single-cell analyses, to explore the origins and timing of development and tumorigenesis in mammalian systems., (© 2024. The Author(s).)

Females have an increased prevalence of many Th17 cell-mediated diseases, including asthma. Androgen signaling decreases Th17 cell-mediated airway inflammation, and Th17 cells rely on glutaminolysis. However, it remains unclear whether androgen receptor (AR) signaling modifies glutamine metabolism to suppress Th17 cell-mediated airway inflammation. We show that Th17 cells from male humans and mice had decreased glutaminolysis compared to females, and that AR signaling attenuated Th17 cell mitochondrial respiration and glutaminolysis in mice. Using allergen-induced airway inflammation mouse models, we determined females had a selective reliance upon glutaminolysis for Th17-mediated airway inflammation, and AR signaling attenuated glutamine uptake in CD4+ T cells by reducing expression of glutamine transporters. Minimal reliance on glutamine uptake in male Th17 cells compared to female Th17 cells was also found in circulating T cells from patients with asthma. AR signaling thus attenuates glutaminolysis, demonstrating sex-specific metabolic regulation of Th17 cells with implications for Th17 or glutaminolysis targeted therapeutics.

Obesity is an established risk factor for breast cancer development and worsened prognosis; however, the mechanisms for this association - and the potential benefits of weight loss - have not been fully explored. The adipose environment surrounding breast tumors, which is inflamed in obesity, has been implicated in tumor progression. An emerging therapeutic target for cancer is TREM2, a transmembrane receptor of the immunoglobulin superfamily that is expressed on macrophages in adipose tissue and tumors. We utilized genetic loss of function ( Trem2 +,+ and Trem2 -/- ) models and dietary (lean, obese, and weight loss) intervention approaches to examine impacts on postmenopausal breast cancer. Remarkably, Trem2 deficiency ameliorated tumor growth in lean, but not obese or weight loss mice. Single-cell RNA sequencing, in conjunction with VDJ sequencing of tumor and tumor-adjacent mammary adipose tissue (mAT Tum-adj ) immune cells, revealed that tumors of lean Trem2 -/- mice exhibited a shift in clonal CD8 + T cells from an exhausted to an effector memory state, accompanied with increased clonality of CD4 + Th1 cells, that was not observed in any other diet-genotype group. Notably, identical T cell clonotypes were identified in the tumor and mAT Tum-adj of the same mouse. Finally, an immune checkpoint study demonstrated that αPD-1 therapy restricted tumor growth in lean and weight loss, but not obese mice. We conclude that weight history is relevant when considering potential efficacy of TREM2 inhibition in postmenopausal breast cancer. This work reveals immunological interactions between tumors and surrounding adipose tissue, highlighting significant differences under obese and weight loss conditions., Competing Interests: Conflict of interest disclosure statement: JCR is a founder and consultant for Sitryx Therapeutics.

Heat is a cardinal feature of inflammation, yet its impacts on immune cells remain uncertain. We show that moderate-grade fever temperatures (39°C) increased murine CD4 T cell metabolism, proliferation, and inflammatory effector activity while decreasing regulatory T cell suppressive capacity. However, heat-exposed T helper 1 (T H 1) cells selectively developed mitochondrial stress and DNA damage that activated Trp53 and stimulator of interferon genes pathways. Although many T H 1 cells subjected to such temperatures died, surviving T H 1 cells exhibited increased mitochondrial mass and enhanced activity. Electron transport chain complex 1 (ETC1) was rapidly impaired under fever-range temperatures, a phenomenon that was specifically detrimental to T H 1 cells. T H 1 cells with elevated DNA damage and ETC1 signatures were also detected in human chronic inflammation. Thus, fever-relevant temperatures disrupt ETC1 to selectively drive apoptosis or adaptation of T H 1 cells to maintain genomic integrity and enhance effector functions.

Tumors consist of cancer cells and a wide range of tissue resident and infiltrating cell types. Tumor metabolism, however, has largely been studied on whole tumors or cancer cells and the metabolism of infiltrating immune cells remains poorly understood. It is now clear from a range of analyses and metabolite rescue studies that metabolic adaptations to the tumor microenvironment (TME) directly impede T-cell and macrophage effector functions. The drivers of metabolic adaptation to the TME and metabolic immune suppression include depletion of essential nutrients, accumulation of waste products or immune suppression metabolites, and metabolic signaling through altered posttranslational modifications. Each infiltrating immune cell subset differs, however, with specific metabolic requirements and adaptations that can be maladaptive for antitumor immunity. Here, we review T-cell and macrophage adaptation and metabolic immune suppression in solid tumors. Ultimately, understanding and addressing these challenges will improve cancer immunotherapy and adoptive chimeric antigen receptor T-cell therapies., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

Accumulating evidence suggests that metabolic rewiring in malignant cells supports tumour progression not only by providing cancer cells with increased proliferative potential and an improved ability to adapt to adverse microenvironmental conditions but also by favouring the evasion of natural and therapy-driven antitumour immune responses. Here, we review cancer cell-intrinsic and cancer cell-extrinsic mechanisms through which alterations of metabolism in malignant cells interfere with innate and adaptive immune functions in support of accelerated disease progression. Further, we discuss the potential of targeting such alterations to enhance anticancer immunity for therapeutic purposes., (© 2024. Springer Nature Limited.)

Inborn errors of metabolism (IEMs) and immunity (IEIs) are Mendelian diseases in which complex phenotypes and patient rarity have limited clinical understanding. Whereas few genes have been annotated as contributing to both IEMs and IEIs, immunometabolic demands suggested greater functional overlap. Here, CRISPR screens tested IEM genes for immunologic roles and IEI genes for metabolic effects and found considerable previously unappreciated crossover. Analysis of IEMs showed that N-linked glycosylation and the hexosamine pathway enzyme Gfpt1 are critical for T cell expansion and function. Further, T helper (T H 1) cells synthesized uridine diphosphate N -acetylglucosamine more rapidly and were more impaired by Gfpt1 deficiency than T H 17 cells. Screening IEI genes found that Bcl11b promotes the CD4 T cell mitochondrial activity and Mcl1 expression necessary to prevent metabolic stress. Thus, a high degree of functional overlap exists between IEM and IEI genes, and immunometabolic mechanisms may underlie a previously underappreciated intersection of these disorders.

The transcription factor FOXN1 plays an established role in thymic epithelial development to mediate selection of maturing thymocytes. Patients with heterozygous loss-of-function FOXN1 variants are associated with T cell lymphopenia at birth and low TCR excision circles that can ultimately recover. Although CD4+ T cell reconstitution in these patients is not completely understood, a lower proportion of naive T cells in adults has suggested a role for homeostatic proliferation. In this study, we present an immunophenotyping study of fraternal twins with low TCR excision circles at birth. Targeted primary immunodeficiency testing revealed a heterozygous variant of uncertain significance in FOXN1 (c.1205del, p.Pro402Leufs*148). We present the immune phenotypes of these two patients, as well as their father who carries the same FOXN1 variant, to demonstrate an evolving immune environment over time. While FOXN1 haploinsufficiency may contribute to thymic defects and T cell lymphopenia, we characterized the transcriptional activity and DNA binding of the heterozygous FOXN1 variant in 293T cells and found the FOXN1 variant to have different effects across several target genes. These data suggest multiple mechanisms for similar FOXN1 variants pathogenicity that may be mutation specific. Increased understanding of how these variants drive transcriptional regulation to impact immune cell populations will guide the potential need for therapeutics, risk for infection or autoimmunity over time, and help inform clinical decisions for other variants that might arise., (Copyright © 2024 The Authors.)

Clear cell renal cell carcinoma (ccRCC) is an aggressive cancer driven by VHL loss and aberrant HIF-2α signaling. Identifying means to regulate HIF-2α thus has potential therapeutic benefit. Acetyl-CoA synthetase 2 (ACSS2) converts acetate to acetyl-CoA and is associated with poor patient prognosis in ccRCC. Here we tested the effects of ACSS2 on HIF-2α and cancer cell metabolism and growth in ccRCC models and clinical samples. ACSS2 inhibition reduced HIF-2α levels and suppressed ccRCC cell line growth in vitro, in vivo, and in cultures of primary ccRCC patient tumors. This treatment reduced glycolytic signaling, cholesterol metabolism, and mitochondrial integrity, all of which are consistent with loss of HIF-2α. Mechanistically, ACSS2 inhibition decreased chromatin accessibility and HIF-2α expression and stability. While HIF-2α protein levels are widely regulated through pVHL-dependent proteolytic degradation, we identify a potential pVHL-independent pathway of degradation via the E3 ligase MUL1. We show that MUL1 can directly interact with HIF-2α and that overexpression of MUL1 decreased HIF-2α levels in a manner partially dependent on ACSS2. These findings identify multiple mechanisms to regulate HIF-2α stability and ACSS2 inhibition as a strategy to complement HIF-2α-targeted therapies and deplete pathogenically stabilized HIF-2α.

PD-1 blockade has been approved for head and neck squamous cell carcinoma (HNSCC) patients. However, many HNSCC patients do not respond to this treatment, and other tumor microenvironmental factors may promote resistance to PD-1 blockade. We previously identified increased expression of the inhibitory receptor NKG2A on CD8+ T cells in HNSCC tumors compared with T cells in matching PBMC samples. Mechanisms that promote NKG2A expression and the role of NKG2A on human T cells in the tumor microenvironment, however, are uncertain. In this study, we show that tumor-conditioned media (TCM) of HNSCC cancer cell lines or ascites fluid from colorectal carcinoma patients is sufficient to induce the expression of NKG2A and other inhibitory receptors on activated CD8+ T cells isolated from PBMCs of healthy donors. Boiling or small molecular mass cutoff filtering did not eliminate the effect of TCM, suggesting that a small molecule promotes NKG2A. T cell activation in TCM decreased the basal and maximal mitochondrial respiration to metabolically restrain CD8+ T cells. Functionally, T cell activation in TCM reduced CD8+ T cell cytotoxicity as shown by lower production of cytokines, granzyme B, and perforin. Furthermore, TCM prevented CD8+ T cells from killing cancer cells in response to an anti-CD19/anti-CD3 bispecific T cell engager. Thus, a small secreted molecule from HNSCC cells can induce NKG2A expression and promote T cell dysfunction. Our findings may lead to targets for novel cancer therapies or biomarkers for NKG2A blockade response and provide a model to study T cell dysfunction and impaired metabolism., (Copyright © 2024 The Authors.)

Obesity is a leading risk factor for progression and metastasis of many cancers 1,2 , yet can in some cases enhance survival 3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells 6-8 . Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-1 9-12 . Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8 + T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Several chronic inflammatory diseases have been linked to high-salt (HS) diets. Chronic inflammation is an established causative hallmark of cancer. However, a direct role of HS diets in tumorigenesis is yet to be defined. Previous orthotopic murine breast tumor studies have shown that short-term HS diets caused inhibition of tumor growth through the activation of cytotoxic adaptive immune responses. However, there have been experimental challenges in developing a viable chronic HS-diet-based murine tumor model. To address this, we have developed a novel chronic HS diet tumor model through the sequential passaging of tumor cells in mice under HS dietary conditions. Two orthotopic murine triple-negative breast cancer models, 4T1 tumor cells injected into BALB/c mice and Py230 tumor cells injected into C57Bl/6 mice, were utilized in our study. For the HS diet cohort, prior to orthotopic injection with tumor cells, the mice were kept on a 4% NaCl diet for 2 weeks. For the regular salt (RS) diet cohort, the mice were kept on a 1% NaCl diet. Following syngeneic cancer cell injection, tumors were allowed to grow for 28 days, following which they were collected to isolate immune cell-depleted cancer cells (passage 1, P1). The tumor cells from P1 were reinjected into the next set of non-tumor-bearing mice. This procedure was repeated for three cycles (P2-P4). In P1, compared to the RS diet cohort, we observed reduced tumor kinetics in both murine tumor models on the HS diet. In contrast, by P4, there was significantly higher tumor progression in the HS diet cohort over the RS diet cohort. Flow cytometry analysis demonstrated an 8-fold increase in tumor-initiating stem cells (TISCs) from P1 to P4 of the HS diet cohort, while there were no significant change in TISC frequency with sequential passaging in the RS diet cohort. Molecular studies showed enhanced expression of TGFβR2 and CD80 on TISCs isolated from the P4 HS diet cohort. In vitro studies demonstrated that TGFβ stimulation of these TISCs increased the cellular expression of CD80 molecules. Further, the chronic HS diet selectively induced the glycolytic metabolic phenotype over the mitochondrial oxidative phosphorylation phenotype in TISCs, which is needed for the production of metabolites during tumor cell differentiation and proliferation. The infiltrating CD8 and CD4 T-lymphocytes in P4 tumors demonstrated increased expression of the immune checkpoint inhibitor (ICI) CTLA4, a known binding partner of CD80, to cause immune exhaustion and pro-tumorigenic effects. Interestingly, anti-TGFβ monoclonal antibodies (mAbs) played a synergistic role in further enhancing the anti-tumor effect of anti-CTLA4 mAb. In summary, our findings demonstrated that chronic HS diet increased the frequency of TISCs in tumors leading to blunting of cytotoxic adaptive immune responses causing tumor proliferation. Furthermore, a combination of anti-TGFβ with current ICI-based immunotherapies could exert more favorable anti-cancer clinical outcomes.

Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

Impairing the BET family coactivator BRD4 with small-molecule inhibitors (BETi) showed encouraging preclinical activity in treating acute myeloid leukemia (AML). However, dose-limiting toxicities and limited clinical activity dampened the enthusiasm for BETi as a single agent. BETi resistance in AML myeloblasts was found to correlate with maintaining mitochondrial respiration, suggesting that identifying the metabolic pathway sustaining mitochondrial integrity could help develop approaches to improve BETi efficacy. Herein, we demonstrated that mitochondria-associated lactate dehydrogenase allows AML myeloblasts to utilize lactate as a metabolic bypass to fuel mitochondrial respiration and maintain cellular viability. Pharmacologically and genetically impairing lactate utilization rendered resistant myeloblasts susceptible to BET inhibition. Low-dose combinations of BETi and oxamate, a lactate dehydrogenase inhibitor, reduced in vivo expansion of BETi-resistant AML in cell line and patient-derived murine models. These results elucidate how AML myeloblasts metabolically adapt to BETi by consuming lactate and demonstrate that combining BETi with inhibitors of lactate utilization may be useful in AML treatment., Significance: Lactate utilization allows AML myeloblasts to maintain metabolic integrity and circumvent antileukemic therapy, which supports testing of lactate utilization inhibitors in clinical settings to overcome BET inhibitor resistance in AML. See related commentary by Boët and Sarry, p. 950., (©2024 American Association for Cancer Research.)

Cells in multicellular organisms experience diverse neighbors, signals, and evolving physical environments that drive functional and metabolic demands. To maintain proper development and homeostasis while avoiding inappropriate cell proliferation or death, individual cells interact with their neighbors via "social" cues to share and partition available nutrients. Metabolic signals also contribute to cell fate by providing biochemical links between cell-extrinsic signals and available resources. In addition to metabolic checkpoints that sense nutrients and directly supply molecular intermediates for biosynthetic pathways, many metabolites directly signal or provide the basis for post-translational modifications of target proteins and chromatin. In this review, we survey the landscape of T cell nutrient sensing and metabolic signaling that supports proper immunity while avoiding immunodeficiency or autoimmunity. The integration of cell-extrinsic microenvironmental cues with cell-intrinsic metabolic signaling provides a social metabolic control model to integrate cell signaling, metabolism, and fate., Competing Interests: Declaration of interests J.C.R. consults as a scientific advisory board member of Sitryx Therapeutics., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Serum Ab concentrations, selection for higher affinity BCRs, and generation of higher Ab affinities are important elements of immune response optimization and functions of germinal center (GC) reactions. B cell proliferation requires nutrients to support the anabolism inherent in clonal expansion. Glucose usage by mouse GC B cells has been reported to contribute little to their energy needs, with questions raised as to whether glucose uptake or glycolysis increases in GC B cells compared with their naive precursors. Indeed, metabolism can be highly flexible, such that supply shortage along one pathway may be compensated by increased flux on others. We now show that reduction of the glucose transporter GLUT1 in mice after establishment of a preimmune B cell repertoire, even after initiation of the GC B cell gene expression program, decreased initial GC B cell population numbers, affinity maturation, and plasma cell outputs. Glucose oxidation was heightened in GC B cells, but this hexose flowed more into the pentose phosphate pathway, whose activity was important in controlling reactive oxygen species (ROS) and Ab-secreting cell production. In modeling how glucose usage by B cells promotes the Ab response, the control of ROS appeared insufficient. Surprisingly, the combination of galactose, which mitigated ROS, with provision of mannose, an efficient precursor to glycosylation, supported robust production of and normal Ab secretion by Ab-secreting cells under glucose-free conditions. Collectively, the findings indicate that GCs depend on normal glucose influx, especially in plasma cell production, but reveal an unexpected metabolic flexibility in hexose requirements., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Mononuclear phagocytes (MPs) play a crucial role in tissue homeostasis; however, MPs also contribute to tumor progression and resistance to immune checkpoint blockade (ICB). Targeting MPs could be an effective strategy to enhance ICB efficacy. We report that protein kinase C delta (PKCδ), a serine/threonine kinase, is abundantly expressed by MPs in human and mouse tumors. PKCδ -/- mice displayed reduced tumor progression compared to wild types, with increased response to anti-PD-1. Tumors from PKCδ -/- mice demonstrated T H 1-skewed immune response including increased antigen presentation and T cell activation. Depletion of MPs in vivo altered tumor growth in control but not PKCδ -/- mice. Coinjection of PKCδ -/- M2-like macrophages with cancer cells into wild-type mice markedly delayed tumor growth and significantly increased intratumoral T cell activation compared to PKCδ +/+ controls. PKCδ deficiency reprogrammed MPs by activating type I and type II interferon signaling. Thus, PKCδ might be targeted to reprogram MPs to augment ICB efficacy.

T cell activation is associated with a profound and rapid metabolic response to meet increased energy demands for cell division, differentiation and development of effector function. Glucose uptake and engagement of the glycolytic pathway are major checkpoints for this event. Here we show that the low-affinity, concentration-dependent glucose transporter 2 (Glut2) regulates the development of CD8 + T cell effector responses in mice by promoting glucose uptake, glycolysis and glucose storage. Expression of Glut2 is modulated by environmental factors including glucose and oxygen availability and extracellular acidification. Glut2 is highly expressed by circulating, recently primed T cells, allowing efficient glucose uptake and storage. In glucose-deprived inflammatory environments, Glut2 becomes downregulated, thus preventing passive loss of intracellular glucose. Mechanistically, Glut2 expression is regulated by a combination of molecular interactions involving hypoxia-inducible factor-1 alpha, galectin-9 and stomatin. Finally, we show that human T cells also rely on this glucose transporter, thus providing a potential target for therapeutic immunomodulation., (© 2023. The Author(s).)

Activated T cells undergo metabolic reprogramming to meet anabolic, differentiation, and functional demands. Glutamine supports many processes in activated T cells, and inhibition of glutamine metabolism alters T cell function in autoimmune disease and cancer. Multiple glutamine-targeting molecules are under investigation, yet the precise mechanisms of glutamine-dependent CD8 T cell differentiation remain unclear. We show that distinct strategies of glutamine inhibition by glutaminase-specific inhibition with small molecule CB-839, pan-glutamine inhibition with 6-diazo-5-oxo-l-norleucine (DON), or by glutamine-depleted conditions (No Q) produce distinct metabolic differentiation trajectories in murine CD8 T cells. T cell activation with CB-839 treatment had a milder effect than did DON or No Q treatment. A key difference was that CB-839-treated cells compensated with increased glycolytic metabolism, whereas DON and No Q-treated cells increased oxidative metabolism. However, all glutamine treatment strategies elevated CD8 T cell dependence on glucose metabolism, and No Q treatment caused adaptation toward reduced glutamine dependence. DON treatment reduced histone modifications and numbers of persisting cells in adoptive transfer studies, but those T cells that remained could expand normally upon secondary Ag encounter. In contrast, No Q-treated cells persisted well yet demonstrated decreased secondary expansion. Consistent with reduced persistence, CD8 T cells activated in the presence of DON had reduced ability to control tumor growth and reduced tumor infiltration in adoptive cell therapy. Overall, each approach to inhibit glutamine metabolism confers distinct effects on CD8 T cells and highlights that targeting the same pathway in different ways can elicit opposing metabolic and functional outcomes., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Introduction: We successfully developed a broad spectrum of patient-derived endocrine organoids (PDO) from benign and malignant neoplasms of thyroid, parathyroid, and adrenal glands. In this study, we employed functionally intact parathyroid PDOs from benign parathyroid tissues to study primary hyperparathyroidism (PHPT), a common endocrine metabolic disease. As proof of concept, we examined the utility of parathyroid PDOs for bioenergetic and metabolic screening and assessed whether parathyroid PDO metabolism recapitulated matched PHPT tissues., Methods: Our study methods included a fine-needle aspiration (FNA)-based technique to establish parathyroid PDOs from human PHPT tissues (n=6) in semi-solid culture conditions for organoid formation, growth, and proliferation. Mass spectrometry metabolomic analysis of PHPT tissues and patient-matched PDOs, and live cell bioenergetic profiling of parathyroid PDOs with extracellular flux analyses, were performed. Functional analysis cryopreserved and re-cultured parathyroid PDOs for parathyroid hormone (PTH) secretion was performed using ELISA hormone assays., Results and Discussion: Our findings support both the feasibility of parathyroid PDOs for metabolic and bioenergetic profiling and reinforce metabolic recapitulation of PHPT tissues by patient-matched parathyroid PDOs. Cryopreserved parathyroid PDOs exhibited preserved, rapid, and sustained secretory function after thawing. In conclusion, successful utilization of parathyroid PDOs for metabolic profiling further affirms the feasibility of promising endocrine organoid platforms for future metabolic studies and broader multiplatform and translational applications for therapeutic advancements of parathyroid and other endocrine applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sekhar, Codreanu, Williams, Rathmell, Rathmell, McLean, Sherrod and Baregamian.)

The tumor-associated vasculature imposes major structural and biochemical barriers to the infiltration of effector T cells and effective tumor control. Correlations between stimulator of interferon genes (STING) pathway activation and spontaneous T cell infiltration in human cancers led us to evaluate the effect of STING-activating nanoparticles (STANs), which are a polymersome-based platform for the delivery of a cyclic dinucleotide STING agonist, on the tumor vasculature and attendant effects on T cell infiltration and antitumor function. In multiple mouse tumor models, intravenous administration of STANs promoted vascular normalization, evidenced by improved vascular integrity, reduced tumor hypoxia, and increased endothelial cell expression of T cell adhesion molecules. STAN-mediated vascular reprogramming enhanced the infiltration, proliferation, and function of antitumor T cells and potentiated the response to immune checkpoint inhibitors and adoptive T cell therapy. We present STANs as a multimodal platform that activates and normalizes the tumor microenvironment to enhance T cell infiltration and function and augments responses to immunotherapy.

The tumor immune microenvironment dynamically evolves to support tumor growth and progression. Immunosuppressive regulatory T cells (Treg) promote tumor growth and metastatic seeding in patients with breast cancer. Deregulation of plasticity between Treg and Th17 cells creates an immune regulatory framework that enables tumor progression. Here, we discovered a functional role for Hedgehog (Hh) signaling in promoting Treg differentiation and immunosuppressive activity, and when Hh activity was inhibited, Tregs adopted a Th17-like phenotype complemented by an enhanced inflammatory profile. Mechanistically, Hh signaling promoted O-GlcNAc modifications of critical Treg and Th17 transcription factors, Foxp3 and STAT3, respectively, that orchestrated this transition. Blocking Hh reprogramed Tregs metabolically, dampened their immunosuppressive activity, and supported their transdifferentiation into inflammatory Th17 cells that enhanced the recruitment of cytotoxic CD8+ T cells into tumors. Our results demonstrate a previously unknown role for Hh signaling in the regulation of Treg differentiation and activity and the switch between Tregs and Th17 cells in the tumor microenvironment., (©2023 American Association for Cancer Research.)

Immunometabolism considers the relationship between metabolism and immunity. Typically, researchers focus on either the metabolic pathways within immune cells that affect their function or the impact of immune cells on systemic metabolism. A more holistic approach that considers both these viewpoints is needed. On September 5-8, 2022, experts in the field of immunometabolism met for the Keystone symposium "Immunometabolism at the Crossroads of Obesity and Cancer" to present recent research across the field of immunometabolism, with the setting of obesity and cancer as an ideal example of the complex interplay between metabolism, immunity, and cancer. Speakers highlighted new insights on the metabolic links between tumor cells and immune cells, with a focus on leveraging unique metabolic vulnerabilities of different cell types in the tumor microenvironment as therapeutic targets and demonstrated the effects of diet, the microbiome, and obesity on immune system function and cancer pathogenesis and therapy. Finally, speakers presented new technologies to interrogate the immune system and uncover novel metabolic pathways important for immunity., (© 2023 New York Academy of Sciences.)

The expansion of follicular helper T (Tfh) cells, which is tightly associated with the development of lupus, is reversed by the inhibition of either glycolysis or glutaminolysis in mice. Here we analyzed the gene expression and metabolome of Tfh cells and naive CD4 + T (Tn) cells in the B6. Sle1.Sle2.Sle3 (triple congenic, TC) mouse model of lupus and its congenic B6 control. Lupus genetic susceptibility in TC mice drives a gene expression signature starting in Tn cells and expanding in Tfh cells with enhanced signaling and effector programs. Metabolically, TC Tn and Tfh cells showed multiple defective mitochondrial functions. TC Tfh cells also showed specific anabolic programs including enhanced glutamate metabolism, malate-aspartate shuttle, and ammonia recycling, as well as altered dynamics of amino acid content and their transporters. Thus, our study has revealed specific metabolic programs that can be targeted to specifically limit the expansion of pathogenic Tfh cells in lupus., Competing Interests: Dr. Jeffrey Rathmell is a founder, scientific advisory board member, and stockholder of Sitryx Therapeutics, a scientific advisory board member and stockholder of Caribou Biosciences, a member of the scientific advisory board of Nirogy Therapeutics, has consulted for Merck, Pfizer, and Mitobridge within the past three years, and has received research support from Incyte Corp., Calithera Biosciences, and Tempest Therapeutics. The other authors declare no competing interests., (© 2023 The Authors.)

Neurons require large amounts of energy, but whether they can perform glycolysis or require glycolysis to maintain energy remains unclear. Using metabolomics, we show that human neurons do metabolize glucose through glycolysis and can rely on glycolysis to supply tricarboxylic acid (TCA) cycle metabolites. To investigate the requirement for glycolysis, we generated mice with postnatal deletion of either the dominant neuronal glucose transporter (GLUT3cKO) or the neuronal-enriched pyruvate kinase isoform (PKM1cKO) in CA1 and other hippocampal neurons. GLUT3cKO and PKM1cKO mice show age-dependent learning and memory deficits. Hyperpolarized magnetic resonance spectroscopic (MRS) imaging shows that female PKM1cKO mice have increased pyruvate-to-lactate conversion, whereas female GLUT3cKO mice have decreased conversion, body weight, and brain volume. GLUT3KO neurons also have decreased cytosolic glucose and ATP at nerve terminals, with spatial genomics and metabolomics revealing compensatory changes in mitochondrial bioenergetics and galactose metabolism. Therefore, neurons metabolize glucose through glycolysis in vivo and require glycolysis for normal function., Competing Interests: Declaration of interests M.G.V.H. discloses that he is a scientific advisor for Agios Pharmaceuticals, iTeos Therapeutics, Sage Therapeutics, Faeth Therapeutics, DRIOA Ventures, and Auron Therapeutics. Y.H. discloses that he is a co-founder and scientific advisory board member of GABAeron., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Increased glucose metabolism and uptake are characteristic of many tumors and used clinically to diagnose and monitor cancer progression. In addition to cancer cells, the tumor microenvironment (TME) encompasses a wide range of stromal, innate, and adaptive immune cells. Cooperation and competition between these cell populations supports tumor proliferation, progression, metastasis, and immune evasion. Cellular heterogeneity leads to metabolic heterogeneity because metabolic programs within the tumor are dependent not only on the TME cellular composition but also on cell states, location, and nutrient availability. In addition to driving metabolic plasticity of cancer cells, altered nutrients and signals in the TME can lead to metabolic immune suppression of effector cells and promote regulatory immune cells. Here we discuss how metabolic programming of cells within the TME promotes tumor proliferation, progression, and metastasis. We also discuss how targeting metabolic heterogeneity may offer therapeutic opportunities to overcome immune suppression and augment immunotherapies., Competing Interests: Declaration of interests J.C.R. holds stock equity in Sitryx and Caribou and within the past 2 years has received unrelated research support, travel, or honoraria from Sitryx, Caribou, Nirogy, Kadmon, Calithera, Tempest, Merck, Mitobridge, and Pfizer., (Copyright © 2023 Elsevier Inc. All rights reserved.)

T cells in systemic lupus erythematosus (SLE) exhibit multiple metabolic abnormalities. Excess iron can impair mitochondria and may contribute to SLE. To gain insights into this potential role of iron in SLE, we performed a CRISPR screen of iron handling genes on T cells. Transferrin receptor (CD71) was identified as differentially critical for T H 1 and inhibitory for induced regulatory T cells (iT regs ). Activated T cells induced CD71 and iron uptake, which was exaggerated in SLE-prone T cells. Cell surface CD71 was enhanced in SLE-prone T cells by increased endosomal recycling. Blocking CD71 reduced intracellular iron and mTORC1 signaling, which inhibited T H 1 and T H 17 cells yet enhanced iT regs . In vivo treatment reduced kidney pathology and increased CD4 T cell production of IL-10 in SLE-prone mice. Disease severity correlated with CD71 expression on T H 17 cells from patients with SLE, and blocking CD71 in vitro enhanced IL-10 secretion. T cell iron uptake via CD71 thus contributes to T cell dysfunction and can be targeted to limit SLE-associated pathology.

Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired "browning" of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.

Background: Recent advancements in 3-dimensional patient-derived organoid models have revolutionized the field of cancer biology. There is an urgent need for development of endocrine tumor organoid models for medullary thyroid carcinoma, adrenocortical carcinoma, papillary thyroid carcinoma, and a spectrum of benign hyperfunctioning parathyroid and adrenal neoplasms. We aimed to engineer functionally intact 3-dimensional endocrine patient-derived organoids to expand the in vitro and translational applications for the advancement of endocrine research., Methods: Using our recently developed fine needle aspiration-based methodology, we established patient-derived 3-dimensional endocrine organoid models using prospectively collected human papillary thyroid carcinoma (n = 6), medullary thyroid carcinoma (n = 3), adrenocortical carcinoma (n = 3), and parathyroid (n = 5). and adrenal (n = 5) neoplasms. Multiplatform analyses of endocrine patient-derived organoids and applications in oncoimmunology, near-infrared autofluorescence, and radiosensitization studies under 3-dimensional in vitro conditions were performed., Results: We have successfully modeled and analyzed the complex endocrine microenvironment for a spectrum of endocrine neoplasms in 3-dimensional culture. The endocrine patient-derived organoids recapitulated complex tumor microenvironment of endocrine neoplasms morphologically and functionally and maintained cytokine production and near-infrared autofluorescence properties., Conclusion: Our novel engineered endocrine patient-derived organoid models of thyroid, parathyroid and adrenal neoplasms represent an exciting and elegant alternative to current limited 2-dimensional systems and afford future broad multiplatform in vitro and translational applications, including in endocrine oncoimmunology., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cellular metabolism influences immune cell function, with mitochondrial fatty acid β-oxidation and oxidative phosphorylation required for multiple immune cell phenotypes. Carnitine palmitoyltransferase 1a (Cpt1a) is considered the rate-limiting enzyme for mitochondrial metabolism of long-chain fatty acids, and Cpt1a deficiency is associated with infant mortality and infection risk. This study was undertaken to test the hypothesis that impairment in Cpt1a-dependent fatty acid oxidation results in increased susceptibility to infection. Screening the Cpt1a gene for common variants predicted to affect protein function revealed allele rs2229738_T, which was associated with pneumonia risk in a targeted human phenome association study. Pharmacologic inhibition of Cpt1a increases mortality and impairs control of the infection in a murine model of bacterial pneumonia. Susceptibility to pneumonia is associated with blunted neutrophilic responses in mice and humans that result from impaired neutrophil trafficking to the site of infection. Chemotaxis responsible for neutrophil trafficking requires Cpt1a-dependent mitochondrial fatty acid oxidation for amplification of chemoattractant signals. These findings identify Cpt1a as a potential host determinant of infection susceptibility and demonstrate a requirement for mitochondrial fatty acid oxidation in neutrophil biology., (© 2022. The Author(s).)

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation., (Copyright © 2022 The Authors.)

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy 1 . The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1 -/- Ctla4 +/- mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration 2 . Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1 -/- Ctla4 +/- mice, we identify clonal effector CD8 + T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1 -/- Ctla4 +/- mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8 + T cells. The cardiac-specific protein α-myosin, which is absent from the thymus 3,4 , was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8 + T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)