Metabolic reprogramming of tumor-associated macrophages using glutamine antagonist JHU083 drives tumor immunity in myeloid-rich prostate and bladder cancer tumors.

Glutamine metabolism in tumor microenvironments critically regulates anti-tumor immunity. Using glutamine-antagonist prodrug JHU083, we report potent tumor growth inhibition in urologic tumors by JHU083-reprogrammed tumor-associated macrophages (TAMs) and tumor-infiltrating monocytes (TIMs). We show JHU083-mediated glutamine antagonism in tumor microenvironments induces TNF, pro-inflammatory, and mTORC1 signaling in intratumoral TAM clusters. JHU083-reprogrammed TAMs also exhibit increased tumor cell phagocytosis and diminished pro-angiogenic capacities. In vivo inhibition of TAM glutamine consumption resulted in increased glycolysis, a broken TCA cycle, and purine metabolism disruption. Although the anti-tumor effect of glutamine antagonism on tumor-infiltrating T cells was moderate, JHU083 promoted a stem cell-like phenotype in CD8+ T cells and decreased Treg abundance. Finally, JHU083 caused a ubiquitous shutdown in glutamine utilizing metabolic pathways in tumor cells, leading to reduced HIF-1alpha, c-MYC phosphorylation, and induction of tumor cell apoptosis, all key anti-tumor features.

Adoptive transfer of CD49a+ Tissue resident memory cells reverses pulmonary fibrosis in mice.

Pulmonary fibrosis is a devastating disease with no effective treatments to cure, stop or reverse the unremitting, fatal fibrosis. A critical barrier to treating this disease is the lack of understanding of the pathways leading to fibrosis as well as those regulating the resolution of fibrosis. Fibrosis is the pathologic side of normal tissue repair that results when the normal wound healing programs go awry. Successful resolution of tissue injury requires several highly coordinated pathways, and this research focuses on the interplay between these overlapping pathways: immune effectors, inflammatory mediators and fibroproliferation in the resolution of fibrosis. Previously we have successfully prevented, mitigated, and even reversed established fibrosis using vaccinia vaccination immunotherapy in two models of murine lung fibrosis. The mechanism by which vaccinia reverses fibrosis is by vaccine induced lung specific Th1 skewed tissue resident memory (TRMs) in the lung. In this study, we isolated a population of vaccine induced TRMs - CD49a+ CD4+ T cells - that are both necessary and sufficient to reverse established pulmonary fibrosis. Using adoptive cellular therapy, we demonstrate that intratracheal administration of CD49a+ CD4+ TRMs into established fibrosis, reverses the fibrosis histologically, by promoting a decrease in collagen, and functionally, by improving lung function, without the need for vaccination. Furthermore, co-culture of in vitro derived CD49+ CD4+ human TRMs with human fibroblasts from individuals with idiopathic pulmonary fibrosis (IPF) results in the down regulation of IPF fibroblast collagen production. Lastly, we demonstrate in human IPF lung histologic samples that CD49a+ CD4+ TRMs, which can down regulate human IPF fibroblast function, fail to increase in the IPF lungs, thus potentially failing to promote resolution. Thus, we define a novel unappreciated role for tissue resident memory T cells in regulating established lung fibrosis to promote resolution of fibrosis and re-establish lung homeostasis. We demonstrate that immunotherapy, in the form of adoptive transfer of CD49a+ CD4+ TRMs into the lungs of mice with established fibrosis, not only stops progression of the fibrosis but more importantly reverses the fibrosis. These studies provide the insight and preclinical rationale for a novel paradigm shifting approach of using cellular immunotherapy to treat lung fibrosis.

Glutamine metabolism inhibition has dual immunomodulatory and antibacterial activities against Mycobacterium tuberculosis.

As one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomic analysis of lungs from JHU083-treated Mtb-infected mice reveals citrulline accumulation, suggesting elevated nitric oxide (NO) synthesis, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. JHU083-treated macrophages also produce more NO potentiating their antibacterial activity. When tested in an immunocompromised mouse model of Mtb infection, JHU083 loses its therapeutic efficacy suggesting the drug's host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis.

SIKs Regulate HDAC7 Stabilization and Cytokine Recall in Late-Stage T Cell Effector Differentiation.

Understanding the mechanisms underlying the acquisition and maintenance of effector function during T cell differentiation is important to unraveling how these processes can be dysregulated in the context of disease and manipulated for therapeutic intervention. In this study, we report the identification of a previously unappreciated regulator of murine T cell differentiation through the evaluation of a previously unreported activity of the kinase inhibitor, BioE-1197. Specifically, we demonstrate that liver kinase B1 (LKB1)-mediated activation of salt-inducible kinases epigenetically regulates cytokine recall potential in effector CD8+ and Th1 cells. Evaluation of this phenotype revealed that salt-inducible kinase-mediated phosphorylation-dependent stabilization of histone deacetylase 7 (HDAC7) occurred during late-stage effector differentiation. HDAC7 stabilization increased nuclear HDAC7 levels, which correlated with total and cytokine loci-specific reductions in the activating transcription mark histone 3 lysine 27 acetylation (H3K27Ac). Accordingly, HDAC7 stabilization diminished transcriptional induction of cytokine genes upon restimulation. Inhibition of this pathway during differentiation produced effector T cells epigenetically poised for enhanced cytokine recall. This work identifies a previously unrecognized target for enhancing effector T cell functionality.

TSC2 S1365A mutation potently regulates CD8+ T cell function and differentiation and improves adoptive cellular cancer therapy.

MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however, sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show that manipulating TSC2 at Ser1365 potently regulated activated but not basal mTORC1 signaling in CD8+ T cells. Unlike nonstimulated TSC2-KO cells, CD8+ T cells expressing a phosphosilencing mutant TSC2-S1365A (TSC2-SA) retained normal basal mTORC1 activity. PKC and T cell receptor (TCR) stimulation induced TSC2 S1365 phosphorylation, and preventing this with the SA mutation markedly increased mTORC1 activation and T cell effector function. Consequently, SA CD8+ T cells displayed greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also displayed enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, SA CD8+ T cells used as adoptive cell therapy displayed greater antitumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhanced both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.

Serendipitous Discovery of T Cell-Produced KLK1b22 as a Regulator of Systemic Metabolism.

In order to study mechanistic/mammalian target of rapamycin's role in T cell differentiation, we generated mice in which Rheb is selectively deleted in T cells (T-Rheb-/- C57BL/6J background). During these studies, we noted that T-Rheb-/- mice were consistently heavier but had improved glucose tolerance and insulin sensitivity as well as a marked increase in beige fat. Microarray analysis of Rheb-/- T cells revealed a marked increase in expression of kallikrein 1-related peptidase b22 (Klk1b22). Overexpression of KLK1b22 in vitro enhanced insulin receptor signaling, and systemic overexpression of KLK1b22 in C57BL/6J mice also enhances glucose tolerance. Although KLK1B22 expression was markedly elevated in the T-Rheb-/- T cells, we never observed any expression in wild-type T cells. Interestingly, in querying the mouse Immunologic Genome Project, we found that Klk1b22 expression was also increased in wild-type 129S1/SVLMJ and C3HEJ mice. Indeed, both strains of mice demonstrate exceptionally improved glucose tolerance. This prompted us to employ CRISPR-mediated knockout of KLK1b22 in 129S1/SVLMJ mice, which in fact led to reduced glucose tolerance. Overall, our studies reveal (to our knowledge) a novel role for KLK1b22 in regulating systemic metabolism and demonstrate the ability of T cell-derived KLK1b22 to regulate systemic metabolism. Notably, however, further studies have revealed that this is a serendipitous finding unrelated to Rheb.

Sirolimus-eluting airway stent reduces profibrotic Th17 cells and inhibits laryngotracheal stenosis.

Laryngotracheal stenosis (LTS) is pathologic fibrotic narrowing of the larynx and trachea characterized by hypermetabolic fibroblasts and CD4+ T cell-mediated inflammation. However, the role of CD4+ T cells in promoting LTS fibrosis is unknown. The mTOR signaling pathways have been shown to regulate the T cell phenotype. Here we investigated the influence of mTOR signaling in CD4+ T cells on LTS pathogenesis. In this study, human LTS specimens revealed a higher population of CD4+ T cells expressing the activated isoform of mTOR. In a murine LTS model, targeting mTOR with systemic sirolimus and a sirolimus-eluting airway stent reduced fibrosis and Th17 cells. Selective deletion of mTOR in CD4+ cells reduced Th17 cells and attenuated fibrosis, demonstrating CD4+ T cells' pathologic role in LTS. Multispectral immunofluorescence of human LTS revealed increased Th17 cells. In vitro, Th17 cells increased collagen-1 production by LTS fibroblasts, which was prevented with sirolimus pretreatment of Th17 cells. Collectively, mTOR signaling drove pathologic CD4+ T cell phenotypes in LTS, and targeting mTOR with sirolimus was effective at treating LTS through inhibition of profibrotic Th17 cells. Finally, sirolimus may be delivered locally with a drug-eluting stent, transforming clinical therapy for LTS.

Glutamine metabolism inhibition has dual immunomodulatory and antibacterial activities against Mycobacterium tuberculosis.

As one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a novel glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomics analysis of lungs from JHU083-treated Mtb-infected mice revealed reduced glutamine levels, citrulline accumulation suggesting elevated NOS activity, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. When tested in an immunocompromised mouse model of Mtb infection, JHU083 lost its therapeutic efficacy suggesting the drug's host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis.

GOT1 regulates CD8+ effector and memory T cell generation.

T cell activation, proliferation, function, and differentiation are tightly linked to proper metabolic reprogramming and regulation. By using [U-13C]glucose tracing, we reveal a critical role for GOT1 in promoting CD8+ T cell effector differentiation and function. Mechanistically, GOT1 enhances proliferation by maintaining intracellular redox balance and serine-mediated purine nucleotide biosynthesis. Further, GOT1 promotes the glycolytic programming and cytotoxic function of cytotoxic T lymphocytes via posttranslational regulation of HIF protein, potentially by regulating the levels of α-ketoglutarate. Conversely, genetic deletion of GOT1 promotes the generation of memory CD8+ T cells.

Cutting Edge: mTORC2 Regulates CD8+ Effector and Memory T Cell Differentiation through Serum and Glucocorticoid Kinase 1.

The mechanistic target of rapamycin is an essential regulator of T cell metabolism and differentiation. In this study, we demonstrate that serum- and glucocorticoid-regulated kinase 1 (SGK1), a downstream node of mechanistic target of rapamycin complex 2 signaling, represses memory CD8+ T cell differentiation. During acute infections, murine SGK1-deficient CD8+ T cells adopt an early memory precursor phenotype leading to more long-lived memory T cells. Thus, SGK1-deficient CD8+ T cells demonstrate an enhanced recall capacity in response to reinfection and can readily reject tumors. Mechanistically, activation of SGK1-deficient CD8+ T cells results in decreased Foxo1 phosphorylation and increased nuclear translocation of Foxo1 to promote early memory development. Overall, SGK1 might prove to be a powerful target for enhancing the efficacy of vaccines and tumor immunotherapy.

Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug.

6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist that suppresses cancer cell metabolism but concurrently enhances the metabolic fitness of tumor CD8+ T cells. DON showed promising efficacy in clinical trials; however, its development was halted by dose-limiting gastrointestinal (GI) toxicities. Given its clinical potential, we designed DON peptide prodrugs and found DRP-104 [isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate] that was preferentially bioactivated to DON in tumor while bioinactivated to an inert metabolite in GI tissues. In drug distribution studies, DRP-104 delivered a prodigious 11-fold greater exposure of DON to tumor versus GI tissues. DRP-104 affected multiple metabolic pathways in tumor, including decreased glutamine flux into the TCA cycle. In efficacy studies, both DRP-104 and DON caused complete tumor regression; however, DRP-104 had a markedly improved tolerability profile. DRP-104's effect was CD8+ T cell dependent and resulted in robust immunologic memory. DRP-104 represents a first-in-class prodrug with differential metabolism in target versus toxicity tissue. DRP-104 is now in clinical trials under the FDA Fast Track designation.

Interrogation of T Cell-Enriched Tumors Reveals Prognostic and Immunotherapeutic Implications of Polyamine Metabolism.

Metabolic features of the tumor microenvironment (TME) antagonize anti-tumor immunity. We hypothesized that T cell infiltrated tumors with a known antigen should exhibit superior clinical outcomes, though some fare worse given unfavorable metabolic features leveraging T cell-infiltrated (Thi), human papillomavirus-related (HPV+) head and neck squamous cell carcinomas (HNSC) to test this hypothesis. Expression of 2,520 metabolic genes were analyzed among Thi HPV+ HNSCs stratified by high-risk molecular subtype. RNAseq data from The Cancer Genome Atlas (TCGA; 10 cancer types), single cell RNAseq data, and an immunotherapy-treated melanoma cohort were used to test the association between metabolic gene expression and clinical outcomes and contribution of tumor versus stromal cells to metabolic gene expression. Polyamine (PA) metabolism genes were overexpressed in high-risk, Thi HPV+ HNSCs. Genes involved in PA biosynthesis and transport were associated with T cell infiltration, recurrent or persistent cancer, overall survival status, primary site, molecular subtype, and MYC genomic alterations. PA biogenesis gene sets were associated with tumor intrinsic features while myeloid cells in HPV+ HNSCs were enriched in PA catabolism, regulatory, transport, putrescine, and spermidine gene set expression. PA gene set expression also correlated with IFNγ or cytotoxic T cell ssGSEA scores across TCGA tumor types. PA transport ssGSEA scores were associated with poor survival whereas putrescine ssGSEA scores portended better survival for several tumor types. Thi melanomas enriched in PA synthesis or combined gene set expression exhibited worse anti-PD-1 responses. These data address hurdles to anti-tumor immunity warranting further investigation of divergent polyamine metabolism in the TME.

Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge.

Memory CD8+ T cells are characterized by their ability to persist long after the initial antigen encounter and their capacity to generate a rapid recall response. Recent studies have identified a role for metabolic reprogramming and mitochondrial function in promoting the longevity of memory T cells. However, detailed mechanisms involved in promoting their rapid recall response are incompletely understood. Here, we identify a role for the initial and continued activation of the trifunctional rate-limiting enzyme of the de novo pyrimidine synthesis pathway CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) as critical in promoting the rapid recall response of previously activated CD8+ T cells. We found that CAD was rapidly phosphorylated upon naïve T cell activation in an mTORC1-dependent manner, yet remained phosphorylated long after initial activation. Previously activated CD8+ T cells displayed continued de novo pyrimidine synthesis in the absence of mitogenic signals, and interfering with this pathway diminished the speed and magnitude of cytokine production upon rechallenge. Inhibition of CAD did not affect cytokine transcript levels but diminished available pre-rRNA (ribosomal RNA), the polycistronic rRNA precursor whose synthesis is the rate-limiting step in ribosomal biogenesis. CAD inhibition additionally decreased levels of detectable ribosomal proteins in previously activated CD8+ T cells. Conversely, overexpression of CAD improved both the cytokine response and proliferation of memory T cells. Overall, our studies reveal a critical role for CAD-induced pyrimidine synthesis and ribosomal biogenesis in promoting the rapid recall response characteristic of memory T cells.

Sulforaphane exhibits antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses in vitro and in mice.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are limited therapeutic options for the prevention and treatment of SARS-CoV-2 infections. We evaluated the antiviral activity of sulforaphane (SFN), the principal biologically active phytochemical derived from glucoraphanin, the naturally occurring precursor present in high concentrations in cruciferous vegetables. SFN inhibited in vitro replication of six strains of SARS-CoV-2, including Delta and Omicron, as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN should be explored as a potential agent for the prevention or treatment of coronavirus infections.

Re-engineered BCG overexpressing cyclic di-AMP augments trained immunity and exhibits improved efficacy against bladder cancer.

In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed trained immunity. Despite its potential as a broadly protective vaccine, little has been done to determine if BCG-mediated trained immunity levels can be optimized. Here we re-engineer BCG to express high levels of c-di-AMP, a PAMP recognized by stimulator of interferon genes (STING). We find that BCG overexpressing c-di-AMP elicits more potent signatures of trained immunity including higher pro-inflammatory cytokine responses, greater myeloid cell reprogramming toward inflammatory and activated states, and enhances epigenetic and metabolomic changes. In a model of bladder cancer, we also show that re-engineered BCG induces trained immunity and improved functionality. These results indicate that trained immunity levels and antitumor efficacy may be increased by modifying BCG to express higher levels of key PAMP molecules.

Signatures of GVHD and relapse after posttransplant cyclophosphamide revealed by immune profiling and machine learning.

The key immunologic signatures associated with clinical outcomes after posttransplant cyclophosphamide (PTCy)-based HLA-haploidentical (haplo) and HLA-matched bone marrow transplantation (BMT) are largely unknown. To address this gap in knowledge, we used machine learning to decipher clinically relevant signatures from immunophenotypic, proteomic, and clinical data and then examined transcriptome changes in the lymphocyte subsets that predicted major posttransplant outcomes. Kinetics of immune subset reconstitution after day 28 were similar for 70 patients undergoing haplo and 75 patients undergoing HLA-matched BMT. Machine learning based on 35 candidate factors (10 clinical, 18 cellular, and 7 proteomic) revealed that combined elevations in effector CD4+ conventional T cells (Tconv) and CXCL9 at day 28 predicted acute graft-versus-host disease (aGVHD). Furthermore, higher NK cell counts predicted improved overall survival (OS) due to a reduction in both nonrelapse mortality and relapse. Transcriptional and flow-cytometric analyses of recovering lymphocytes in patients with aGVHD identified preserved hallmarks of functional CD4+ regulatory T cells (Tregs) while highlighting a Tconv-driven inflammatory and metabolic axis distinct from that seen with conventional GVHD prophylaxis. Patients developing early relapse displayed a loss of inflammatory gene signatures in NK cells and a transcriptional exhaustion phenotype in CD8+ T cells. Using a multimodality approach, we highlight the utility of systems biology in BMT biomarker discovery and offer a novel understanding of how PTCy influences alloimmune responses. Our work charts future directions for novel therapeutic interventions after these increasingly used GVHD prophylaxis platforms. Specimens collected on NCT0079656226 and NCT0080927627 https://clinicaltrials.gov/.

An engineered IL-2 partial agonist promotes CD8+ T cell stemness.

Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+ T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential.

mTORC1 Signaling Regulates Proinflammatory Macrophage Function and Metabolism.

Metabolic programming is integrally linked to immune cell function. Nowhere is this clearer than in the differentiation of macrophages. Proinflammatory M1 macrophages primarily use glycolysis as a rapid energy source but also to generate antimicrobial compounds, whereas alternatively activated M2 macrophages primarily rely on oxidative phosphorylation for the longevity required for proper wound healing. mTOR signaling has been demonstrated to be a key regulator of immune cell metabolism and function. mTORC2 signaling is required for the generation of M2 macrophages, whereas the role of mTORC1 signaling, a key regulator of glycolysis, has been controversial. By using genetic deletion of mTORC1 signaling in C57BL/6 mouse macrophages, we observed enhanced M1 macrophage function in vitro and in vivo. Surprisingly, this enhancement occurred despite a significant defect in M1 macrophage glycolytic metabolism. Mechanistically, enhanced M1 function occurred because of inhibition of the class III histone deacetylases the sirtuins, resulting in enhanced histone acetylation. Our findings provide a counterpoint to the paradigm that enhanced immune cell function must occur in the presence of increased cellular metabolism and identifies a potential, pharmacologic target for the regulation of inflammatory responses.

Antigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance.

Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.