D-2-hydroxyglutarate supports a tolerogenic phenotype with lowered major histocompatibility class II expression in non-malignant dendritic cells and acute myeloid leukemia cells.

D-2-hydroxyglutarate (D-2-HG) accumulates in primary acute myeloid leukemia (AML) patients with mutated isocitrate dehydrogenase (IDH) and other malignancies. D-2-HG suppresses antitumor T cell immunity but little is known about potential effects on non-malignant myeloid cells. Here we show that D-2-HG impairs human but not murine dendritic cell (DC) differentiation, resulting in a tolerogenic phenotype with low major histocompatibility (MHC) class II expression. In line, IDH-mutated AML blasts exhibited lower expression of HLA-DP and were less susceptible to lysis by HLA-DP-specific T cells. Interestingly, D-2-HG reprogrammed metabolism towards increased lactate production in DCs and AML besides its expected impact on DNA demethylation. Vitamin C accelerated DNA demethylation, but only the combination of vitamin C and glycolytic inhibition lowered lactate levels and supported MHC class II expression. Our results indicate an unexpected link between the immunosuppressive metabolites 2-HG and lactic acid and suggest a potentially novel therapeutic strategy with combinations of anti-glycolytic drugs and epigenetic modulators (hypomethylating agents) or other therapeutics for the treatment of AML.

A chemokine network of T cell exhaustion and metabolic reprogramming in renal cell carcinoma.

Renal cell carcinoma (RCC) is frequently infiltrated by immune cells, a process which is governed by chemokines. CD8+ T cells in the RCC tumor microenvironment (TME) may be exhausted which most likely influence therapy response and survival. The aim of this study was to evaluate chemokine-driven T cell recruitment, T cell exhaustion in the RCC TME, as well as metabolic processes leading to their functional anergy in RCC. Eight publicly available bulk RCC transcriptome collectives (n=1819) and a single cell RNAseq dataset (n=12) were analyzed. Immunodeconvolution, semi-supervised clustering, gene set variation analysis and Monte Carlo-based modeling of metabolic reaction activity were employed. Among 28 chemokine genes available, CXCL9/10/11/CXCR3, CXCL13/CXCR5 and XCL1/XCR1 mRNA expression were significantly increased in RCC compared to normal kidney tissue and also strongly associated with tumor-infiltrating effector memory and central memory CD8+ T cells in all investigated collectives. M1 TAMs, T cells, NK cells as well as tumor cells were identified as the major sources of these chemokines, whereas T cells, B cells and dendritic cells were found to predominantly express the cognate receptors. The cluster of RCCs characterized by high chemokine expression and high CD8+ T cell infiltration displayed a strong activation of IFN/JAK/STAT signaling with elevated expression of multiple T cell exhaustion-associated transcripts. Chemokinehigh RCCs were characterized by metabolic reprogramming, in particular by downregulated OXPHOS and increased IDO1-mediated tryptophan degradation. None of the investigated chemokine genes was significantly associated with survival or response to immunotherapy. We propose a chemokine network that mediates CD8+ T cell recruitment and identify T cell exhaustion, altered energy metabolism and high IDO1 activity as key mechanisms of their suppression. Concomitant targeting of exhaustion pathways and metabolism may pose an effective approach to RCC therapy.

Low-density lipoprotein balances T cell metabolism and enhances response to anti-PD-1 blockade in a HCT116 spheroid model.

Introduction: The discovery of immune checkpoints and the development of their specific inhibitors was acclaimed as a major breakthrough in cancer therapy. However, only a limited patient cohort shows sufficient response to therapy. Hence, there is a need for identifying new checkpoints and predictive biomarkers with the objective of overcoming immune escape and resistance to treatment. Having been associated with both, treatment response and failure, LDL seems to be a double-edged sword in anti-PD1 immunotherapy. Being embedded into complex metabolic conditions, the impact of LDL on distinct immune cells has not been sufficiently addressed. Revealing the effects of LDL on T cell performance in tumor immunity may enable individual treatment adjustments in order to enhance the response to routinely administered immunotherapies in different patient populations. The object of this work was to investigate the effect of LDL on T cell activation and tumor immunity in-vitro. Methods: Experiments were performed with different LDL dosages (LDLlow = 50 µg/ml and LDLhigh = 200 µg/ml) referring to medium control. T cell phenotype, cytokines and metabolism were analyzed. The functional relevance of our findings was studied in a HCT116 spheroid model in the context of anti-PD-1 blockade. Results: The key points of our findings showed that LDLhigh skewed the CD4+ T cell subset into a central memory-like phenotype, enhanced the expression of the co-stimulatory marker CD154 (CD40L) and significantly reduced secretion of IL-10. The exhaustion markers PD-1 and LAG-3 were downregulated on both T cell subsets and phenotypical changes were associated with a balanced T cell metabolism, in particular with a significant decrease of reactive oxygen species (ROS). T cell transfer into a HCT116 spheroid model resulted in a significant reduction of the spheroid viability in presence of an anti-PD-1 antibody combined with LDLhigh. Discussion: Further research needs to be conducted to fully understand the impact of LDL on T cells in tumor immunity and moreover, to also unravel LDL effects on other lymphocytes and myeloid cells for improving anti-PD-1 immunotherapy. The reason for improved response might be a resilient, less exhausted phenotype with balanced ROS levels.

Integrated single-cell profiling dissects cell-state-specific enhancer landscapes of human tumor-infiltrating CD8+ T cells.

Despite extensive studies on the chromatin landscape of exhausted T cells, the transcriptional wiring underlying the heterogeneous functional and dysfunctional states of human tumor-infiltrating lymphocytes (TILs) is incompletely understood. Here, we identify gene-regulatory landscapes in a wide breadth of functional and dysfunctional CD8+ TIL states covering four cancer entities using single-cell chromatin profiling. We map enhancer-promoter interactions in human TILs by integrating single-cell chromatin accessibility with single-cell RNA-seq data from tumor-entity-matching samples and prioritize cell-state-specific genes by super-enhancer analysis. Besides revealing entity-specific chromatin remodeling in exhausted TILs, our analyses identify a common chromatin trajectory to TIL dysfunction and determine key enhancers, transcriptional regulators, and deregulated genes involved in this process. Finally, we validate enhancer regulation at immunotherapeutically relevant loci by targeting non-coding regulatory elements with potent CRISPR activators and repressors. In summary, our study provides a framework for understanding and manipulating cell-state-specific gene-regulatory cues from human tumor-infiltrating lymphocytes.

Targeting strategies in the treatment of fumarate hydratase deficient renal cell carcinoma.

Fumarate hydratase (FH) - deficient renal cell carcinoma (FHdRCC) is a rare aggressive subtype of RCC caused by a germline or sporadic loss-of-function mutation in the FH gene. Here, we summarize how FH deficiency results in the accumulation of fumarate, which in turn leads to activation of hypoxia-inducible factor (HIF) through inhibition of prolyl hydroxylases. HIF promotes tumorigenesis by orchestrating a metabolic switch to glycolysis even under normoxia, a phenomenon well-known as the Warburg effect. HIF activates the transcription of many genes, including vascular endothelial growth factor (VEGF). Crosstalk between HIF and epidermal growth factor receptor (EGFR) has also been described as a tumor-promoting mechanism. In this review we discuss therapeutic options for FHdRCC with a focus on anti-angiogenesis and EGFR-blockade. We also address potential targets that arise within the metabolic escape routes taken by FH-deficient cells for cell growth and survival.

Immunometabolic Markers in a Small Patient Cohort Undergoing Immunotherapy.

Although the discovery of immune checkpoints was hailed as a major breakthrough in cancer therapy, generating a sufficient response to immunotherapy is still limited. Thus, the objective of this exploratory, hypothesis-generating study was to identify potentially novel peripheral biomarkers and discuss the possible predictive relevance of combining scarcely investigated metabolic and hormonal markers with immune subsets. Sixteen markers that differed significantly between responders and non-responders were identified. In a further step, the correlation with progression-free survival (PFS) and false discovery correction (Benjamini and Hochberg) revealed potential predictive roles for the immune subset absolute lymphocyte count (rs = 0.51; p = 0.0224 *), absolute basophil count (rs = 0.43; p = 0.04 *), PD-1+ monocytes (rs = -0.49; p = 0.04 *), hemoglobin (rs = 0.44; p = 0.04 *), metabolic markers LDL (rs = 0.53; p = 0.0224 *), free androgen index (rs = 0.57; p = 0.0224 *) and CRP (rs = -0.46; p = 0.0352 *). The absolute lymphocyte count, LDL and free androgen index were the most significant individual markers, and combining the immune subsets with the metabolic markers into a biomarker ratio enhanced correlation with PFS (rs = -0.74; p ≤ 0.0001 ****). In summary, in addition to well-established markers, we identified PD-1+ monocytes and the free androgen index as potentially novel peripheral markers in the context of immunotherapy. Furthermore, the combination of immune subsets with metabolic and hormonal markers may have the potential to enhance the power of future predictive scores and should, therefore, be investigated further in larger trials.

Kynurenine induces T cell fat catabolism and has limited suppressive effects in vivo.

BACKGROUND: L-kynurenine is a tryptophan-derived immunosuppressive metabolite and precursor to neurotoxic anthranilate and quinolinate. We evaluated the stereoisomer D-kynurenine as an immunosuppressive therapeutic which is hypothesized to produce less neurotoxic metabolites than L-kynurenine. METHODS: L-/D-kynurenine effects on human and murine T cell function were examined in vitro and in vivo (homeostatic proliferation, colitis, cardiac transplant). Kynurenine effects on T cell metabolism were interrogated using [13C] glucose, glutamine and palmitate tracing. Kynurenine was measured in tissues from human and murine tumours and kynurenine-fed mice. FINDINGS: We observed that 1 mM D-kynurenine inhibits T cell proliferation through apoptosis similar to L-kynurenine. Mechanistically, [13C]-tracing revealed that co-stimulated CD4+ T cells exposed to L-/D-kynurenine undergo increased ß-oxidation depleting fatty acids. Replenishing oleate/palmitate restored effector T cell viability. We administered dietary D-kynurenine reaching tissue kynurenine concentrations of 19 µM, which is close to human kidney (6 µM) and head and neck cancer (14 µM) but well below the 1 mM required for apoptosis. D-kynurenine protected Rag1-/- mice from autoimmune colitis in an aryl-hydrocarbon receptor dependent manner but did not attenuate more stringent immunological challenges such as antigen mismatched cardiac allograft rejection. INTERPRETATION: Our dietary kynurenine model achieved tissue concentrations at or above human cancer kynurenine and exhibited only limited immunosuppression. Sub-suppressive kynurenine concentrations in human cancers may limit the responsiveness to indoleamine 2,3-dioxygenase inhibition evaluated in clinical trials. FUNDING: The study was supported by the NIH, the Else Kröner-Fresenius-Foundation, Laffey McHugh foundation, and American Society of Nephrology.

Metabolic imbalance of T cells in COVID-19 is hallmarked by basigin and mitigated by dexamethasone.

Metabolic pathways regulate immune responses and disrupted metabolism leads to immune dysfunction and disease. Coronavirus disease 2019 (COVID-19) is driven by imbalanced immune responses, yet the role of immunometabolism in COVID-19 pathogenesis remains unclear. By investigating 87 patients with confirmed SARS-CoV-2 infection, 6 critically ill non-COVID-19 patients, and 47 uninfected controls, we found an immunometabolic dysregulation in patients with progressed COVID-19. Specifically, T cells, monocytes, and granulocytes exhibited increased mitochondrial mass, yet only T cells accumulated intracellular reactive oxygen species (ROS), were metabolically quiescent, and showed a disrupted mitochondrial architecture. During recovery, T cell ROS decreased to match the uninfected controls. Transcriptionally, T cells from severe/critical COVID-19 patients showed an induction of ROS-responsive genes as well as genes related to mitochondrial function and the basigin network. Basigin (CD147) ligands cyclophilin A and the SARS-CoV-2 spike protein triggered ROS production in T cells in vitro. In line with this, only PCR-positive patients showed increased ROS levels. Dexamethasone treatment resulted in a downregulation of ROS in vitro and T cells from dexamethasone-treated patients exhibited low ROS and basigin levels. This was reflected by changes in the transcriptional landscape. Our findings provide evidence of an immunometabolic dysregulation in COVID-19 that can be mitigated by dexamethasone treatment.

Heat-Inactivation of Human Serum Destroys C1 Inhibitor, Pro-motes Immune Complex Formation, and Improves Human T Cell Function.

Heat-inactivation of sera is used to reduce possible disturbing effects of complement factors in cell-culture experiments, but it is controversially discussed whether this procedure is appropriate or could be neglected. Here, we report a strong impact of heat-inactivation of human sera on the activation and effector functions of human CD4+ T cells. While T cells cultured with native sera were characterized by a higher proliferation rate and higher expression of CD28, heat-inactivated sera shaped T cells towards on-blast formation, higher cytokine secretion (interferon γ, tumor necrosis factor, and interleukin-17), stronger CD69 and PD-1 expression, and increased metabolic activity. Heat-inactivated sera contained reduced amounts of complement factors and regulators like C1 inhibitor, but increased concentrations of circulating immune complexes. Substitution of C1 inhibitor reduced the beneficial effect of heat-inactivation in terms of cytokine release, whereas surface-molecule expression was affected by the addition of complex forming anti-C1q antibody. Our data clearly demonstrate a beneficial effect of heat-inactivation of human sera for T cell experiments but indicate that beside complement regulators and immune complexes other components might be relevant. Beyond that, this study further underpins the strong impact of the complement system on T cell function.

Targeting In Vivo Metabolic Vulnerabilities of Th2 and Th17 Cells Reduces Airway Inflammation.

T effector cells promote inflammation in asthmatic patients, and both Th2 and Th17 CD4 T cells have been implicated in severe forms of the disease. The metabolic phenotypes and dependencies of these cells, however, remain poorly understood in the regulation of airway inflammation. In this study, we show the bronchoalveolar lavage fluid of asthmatic patients had markers of elevated glucose and glutamine metabolism. Further, peripheral blood T cells of asthmatics had broadly elevated expression of metabolic proteins when analyzed by mass cytometry compared with healthy controls. Therefore, we hypothesized that glucose and glutamine metabolism promote allergic airway inflammation. We tested this hypothesis in two murine models of airway inflammation. T cells from lungs of mice sensitized with Alternaria alternata extract displayed genetic signatures for elevated oxidative and glucose metabolism by single-cell RNA sequencing. This result was most pronounced when protein levels were measured in IL-17-producing cells and was recapitulated when airway inflammation was induced with house dust mite plus LPS, a model that led to abundant IL-4- and IL-17-producing T cells. Importantly, inhibitors of the glucose transporter 1 or glutaminase in vivo attenuated house dust mite + LPS eosinophilia, T cell cytokine production, and airway hyperresponsiveness as well as augmented the immunosuppressive properties of dexamethasone. These data show that T cells induce markers to support metabolism in vivo in airway inflammation and that this correlates with inflammatory cytokine production. Targeting metabolic pathways may provide a new direction to protect from disease and enhance the effectiveness of steroid therapy.

1,25-dihydroxyvitamin-D3 but not the clinically applied marker 25-hydroxyvitamin-D3 predicts survival after stem cell transplantation.

The serum level of 25-hydroxyvitamin-D3 is accepted as marker for a person's vitamin D status but its role for the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) is controversially discussed. The impact of 1,25-dihydroxyvitamin-D3 on HSCT outcome, however, has never been studied. In a discovery cohort of 143 HSCT patients we repeatedly (day -16 to 100) measured 1,25-dihydroxyvitamin-D3 and in comparison the well-established marker for serum vitamin D status 25-hydroxyvitamin-D3. Only lower 1,25-dihydroxyvitamin-D3 levels around HSCT (day -2 to 7, peritransplant) were significantly associated with higher 1-year treatment-related mortality (TRM) risk (Mann-Whitney U test, P = 0.001). This was confirmed by Cox-model regression without and with adjustment for baseline risk factors and severe acute Graft-versus-Host disease (aGvHD; unadjusted P = 0.001, adjusted P = 0.005). The optimal threshold for 1,25-dihydroxyvitamin-D3 to identify patients at high risk was 139.5 pM. Also in three replication cohorts consisting of altogether 365 patients 1,25-dihydroxyvitamin-D3 levels below 139.5 pM had a 3.3-fold increased risk of TRM independent of severe aGvHD compared to patients above 139.5 pM (Cox-model unadjusted P < 0.0005, adjusted P = 0.001). Our data highlight peritransplant 1,25-dihydroxyvitamin-D3 levels but not the commonly monitored 25-hydroxyvitamin-D3 levels as potent predictor of 1-year TRM and suggest to monitor both vitamin D metabolites in HSCT patients.

CD28 costimulation drives tumor-infiltrating T cell glycolysis to promote inflammation.

Metabolic reprogramming dictates the fate and function of stimulated T cells, yet these pathways can be suppressed in T cells in tumor microenvironments. We previously showed that glycolytic and mitochondrial adaptations directly contribute to reducing the effector function of renal cell carcinoma (RCC) CD8+ tumor-infiltrating lymphocytes (TILs). Here we define the role of these metabolic pathways in the activation and effector functions of CD8+ RCC TILs. CD28 costimulation plays a key role in augmenting T cell activation and metabolism, and is antagonized by the inhibitory and checkpoint immunotherapy receptors CTLA4 and PD-1. While RCC CD8+ TILs were activated at a low level when stimulated through the T cell receptor alone, addition of CD28 costimulation greatly enhanced activation, function, and proliferation. CD28 costimulation reprogrammed RCC CD8+ TIL metabolism with increased glycolysis and mitochondrial oxidative metabolism, possibly through upregulation of GLUT3. Mitochondria also fused to a greater degree, with higher membrane potential and overall mass. These phenotypes were dependent on glucose metabolism, as the glycolytic inhibitor 2-deoxyglucose both prevented changes to mitochondria and suppressed RCC CD8+ TIL activation and function. These data show that CD28 costimulation can restore RCC CD8+ TIL metabolism and function through rescue of T cell glycolysis that supports mitochondrial mass and activity.

The immunological Warburg effect: Can a metabolic-tumor-stroma score (MeTS) guide cancer immunotherapy?

The "glycolytic switch" also known as the "Warburg effect" is a key feature of tumor cells and leads to the accumulation of lactate and protons in the tumor environment. Intriguingly, non-malignant lymphocytes or stromal cells such as tumor-associated macrophages and cancer-associated fibroblasts contribute to the lactate accumulation in the tumor environment, a phenomenon described as the "Reverse Warburg effect." Localized lactic acidosis has a strong immunosuppressive effect and mediates an immune escape of tumors. However, some tumors do not display the Warburg phenotype and either rely on respiration or appear as a mosaic of cells with different metabolic properties. Based on these findings and on the knowledge that T cell infiltration is predictive for patient outcome, we suggest a metabolic-tumor-stroma score to determine the likelihood of a successful anti-tumor immune response: (a) a respiring tumor with high T cell infiltration ("hot"); (b) a reverse Warburg type with respiring tumor cells but glycolytic stromal cells; (c) a mixed type with glycolytic and respiring compartments; and (d) a glycolytic (Warburg) tumor with low T cell infiltration ("cold"). Here, we provide evidence that these types can be independent of the organ of origin, prognostically relevant and might help select the appropriate immunotherapy approach.

Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy.

Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials.

The Transcription Factor Bhlhe40 Programs Mitochondrial Regulation of Resident CD8+ T Cell Fitness and Functionality.

Tissue-resident memory CD8+ T (Trm) cells share core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic, and epigenetic regulation of Trm cell and TIL development and function is largely undefined. Here, we found that the transcription factor Bhlhe40 was specifically required for Trm cell and TIL development and polyfunctionality. Local PD-1 signaling inhibited TIL Bhlhe40 expression, and Bhlhe40 was critical for TIL reinvigoration following anti-PD-L1 blockade. Mechanistically, Bhlhe40 sustained Trm cell and TIL mitochondrial fitness and a functional epigenetic state. Building on these findings, we identified an epigenetic and metabolic regimen that promoted Trm cell and TIL gene signatures associated with tissue residency and polyfunctionality. This regimen empowered the anti-tumor activity of CD8+ T cells and possessed therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights into the local regulation of Trm cell and TIL function.

Topical Diclofenac Reprograms Metabolism and Immune Cell Infiltration in Actinic Keratosis.

Background: Melanoma and squamous cell carcinoma of the skin are characterized by an altered glucose metabolism, but little is known about metabolic changes in precancerous skin lesions such as actinic keratosis (AK). Here, we studied the central carbon metabolism and immune cell infiltrate of actinic keratosis lesions before, under, and 4 weeks after treatment with topical diclofenac (Solaraze®). Methods: This study was designed as a prospective, randomized, controlled, monocentric investigation (ClinicalTrials.gov Identifier: NCT01935531). Myeloid and T cell infiltration was analyzed in skin biopsies from 28 patients by immunohistochemistry. Furthermore, immune cell activation was determined via quantitative real-time PCR (IFN-γ, IL-10, CSF1, TGF-ß, IL-6). Glucose, amino acid and Krebs' cycle metabolism was studied by mass spectrometry prior, during and after treatment with topical diclofenac. Biopsies from sun-exposed, untreated, healthy skin served as controls. Results: Increased lactate and decreased glucose levels suggested accelerated glycolysis in pre-treatment AK. Further, levels of Krebs' cycle intermediates other than citrate and amino acids were elevated. Analysis of the immune infiltrate revealed less epidermal CD1a+ cells but increased frequencies of dermal CD8+ T cells in AK. Treatment with diclofenac reduced lactate and amino acid levels in AK, especially in responding lesions, and induced an infiltration of dermal CD8+ T cells accompanied by high IFN-γ mRNA expression, suggesting improved T cell function. Discussion: Our study clearly demonstrated that not only cancers but also pre-malignant skin lesions, like AK, exhibit profound changes in metabolism, correlating with an altered immune infiltrate. Diclofenac normalizes metabolism, immune cell infiltration and function in AK lesions, suggesting a novel mechanism of action.

Chronic lymphocytic leukemia cells impair mitochondrial fitness in CD8+ T cells and impede CAR T-cell efficacy.

In chronic lymphocytic leukemia (CLL), acquired T-cell dysfunction impedes development of effective immunotherapeutic strategies, through as-yet unresolved mechanisms. We have previously shown that CD8+ T cells in CLL exhibit impaired activation and reduced glucose uptake after stimulation. CD8+ T cells in CLL patients are chronically exposed to leukemic B cells, which potentially impacts metabolic homeostasis resulting in aberrant metabolic reprogramming upon stimulation. Here, we report that resting CD8+ T cells in CLL have reduced intracellular glucose transporter 1 (GLUT1) reserves, and have an altered mitochondrial metabolic profile as displayed by increased mitochondrial respiration, membrane potential, and levels of reactive oxygen species. This coincided with decreased levels of peroxisome proliferator-activated receptor γ coactivator 1-α, and in line with that, CLL-derived CD8+ T cells showed impaired mitochondrial biogenesis upon stimulation. In search of a therapeutic correlate of these findings, we analyzed mitochondrial biogenesis in CD19-directed chimeric antigen receptor (CAR) CD8+ T cells prior to infusion in CLL patients (who were enrolled in NCT01747486 and NCT01029366 [https://clinicaltrials.gov]). Interestingly, in cases with a subsequent complete response, the infused CD8+ CAR T cells had increased mitochondrial mass compared with nonresponders, which positively correlated with the expansion and persistence of CAR T cells. Our findings demonstrate that GLUT1 reserves and mitochondrial fitness of CD8+ T cells are impaired in CLL. Therefore, boosting mitochondrial biogenesis in CAR T cells might improve the efficacy of CAR T-cell therapy and other emerging cellular immunotherapies.

Impaired enolase 1 glycolytic activity restrains effector functions of tumor-infiltrating CD8+ T cells.

In the context of solid tumors, there is a positive correlation between the accumulation of cytotoxic CD8+ tumor-infiltrating lymphocytes (TILs) and favorable clinical outcomes. However, CD8+ TILs often exhibit a state of functional exhaustion, limiting their activity, and the underlying molecular basis of this dysfunction is not fully understood. Here, we show that TILs found in human and murine CD8+ melanomas are metabolically compromised with deficits in both glycolytic and oxidative metabolism. Although several studies have shown that tumors can outcompete T cells for glucose, thus limiting T cell metabolic activity, we report that a down-regulation in the activity of ENOLASE 1, a critical enzyme in the glycolytic pathway, represses glycolytic activity in CD8+ TILs. Provision of pyruvate, a downstream product of ENOLASE 1, bypasses this inactivity and promotes both glycolysis and oxidative phosphorylation, resulting in improved effector function of CD8+ TILs. We found high expression of both enolase 1 mRNA and protein in CD8+ TILs, indicating that the enzymatic activity of ENOLASE 1 is regulated posttranslationally. These studies provide a critical insight into the biochemical basis of CD8+ TIL dysfunction.

Computational Immune Monitoring Reveals Abnormal Double-Negative T Cells Present across Human Tumor Types.

Advances in single-cell biology have enabled measurements of >40 protein features on millions of immune cells within clinical samples. However, the data analysis steps following cell population identification are susceptible to bias, time-consuming, and challenging to compare across studies. Here, an ensemble of unsupervised tools was developed to evaluate four essential types of immune cell information, incorporate changes over time, and address diverse immune monitoring challenges. The four complementary properties characterized were (i) systemic plasticity, (ii) change in population abundance, (iii) change in signature population features, and (iv) novelty of cellular phenotype. Three systems immune monitoring studies were selected to challenge this ensemble approach. In serial biopsies of melanoma tumors undergoing targeted therapy, the ensemble approach revealed enrichment of double-negative (DN) T cells. Melanoma tumor-resident DN T cells were abnormal and phenotypically distinct from those found in nonmalignant lymphoid tissues, but similar to those found in glioblastoma and renal cell carcinoma. Overall, ensemble systems immune monitoring provided a robust, quantitative view of changes in both the system and cell subsets, allowed for transparent review by human experts, and revealed abnormal immune cells present across multiple human tumor types.