1 6481 123 TOX IS EXPRESSED BY EXHAUSTED AND POLYFUNCTIONAL HUMAN EFFECTOR MEMORY CD8(+) T CELLS. CD8(+) T CELL EXHAUSTION IS A HALLMARK OF MANY CANCERS AND CHRONIC INFECTIONS. IN MICE, T CELL FACTOR 1 (TCF-1) MAINTAINS EXHAUSTED CD8(+) T CELL RESPONSES, WHEREAS THYMOCYTE SELECTION-ASSOCIATED HMG BOX (TOX) IS REQUIRED FOR THE EPIGENETIC REMODELING AND SURVIVAL OF EXHAUSTED CD8(+) T CELLS. HOWEVER, IT HAS REMAINED UNCLEAR TO WHAT EXTENT THESE TRANSCRIPTION FACTORS PLAY ANALOGOUS ROLES IN HUMANS. IN THIS STUDY, WE MAPPED THE EXPRESSION OF TOX AND TCF-1 AS A FUNCTION OF DIFFERENTIATION AND SPECIFICITY IN THE HUMAN CD8(+) T CELL LANDSCAPE. HERE, WE DEMONSTRATE THAT CIRCULATING TOX(+) CD8(+) T CELLS EXIST IN MOST HUMANS, BUT THAT TOX IS NOT EXCLUSIVELY ASSOCIATED WITH EXHAUSTION. EFFECTOR MEMORY CD8(+) T CELLS GENERALLY EXPRESSED TOX, WHEREAS NAIVE AND EARLY-DIFFERENTIATED MEMORY CD8(+) T CELLS GENERALLY EXPRESSED TCF-1. CYTOLYTIC GENE AND PROTEIN EXPRESSION SIGNATURES WERE ALSO DEFINED BY THE EXPRESSION OF TOX. IN THE CONTEXT OF A RELENTLESS IMMUNE CHALLENGE, EXHAUSTED HIV-SPECIFIC CD8(+) T CELLS COMMONLY EXPRESSED TOX, OFTEN IN CLUSTERS WITH VARIOUS ACTIVATION MARKERS AND INHIBITORY RECEPTORS, AND EXPRESSED LESS TCF-1. HOWEVER, POLYFUNCTIONAL MEMORY CD8(+) T CELLS SPECIFIC FOR CYTOMEGALOVIRUS (CMV) OR EPSTEIN-BARR VIRUS (EBV) ALSO EXPRESSED TOX, EITHER WITH OR WITHOUT TCF-1. A SIMILAR PHENOTYPE WAS OBSERVED AMONG HIV-SPECIFIC CD8(+) T CELLS FROM INDIVIDUALS WHO MAINTAINED EXCEPTIONAL IMMUNE CONTROL OF VIRAL REPLICATION. COLLECTIVELY, THESE DATA DEMONSTRATE THAT TOX IS EXPRESSED BY MOST CIRCULATING EFFECTOR MEMORY CD8(+) T CELL SUBSETS AND NOT EXCLUSIVELY LINKED TO EXHAUSTION. 2020 2 5704 41 SINGLE-CELL RNA-SEQ REVEALS TOX AS A KEY REGULATOR OF CD8(+) T CELL PERSISTENCE IN CHRONIC INFECTION. PROGENITOR-LIKE CD8(+) T CELLS MEDIATE LONG-TERM IMMUNITY TO CHRONIC INFECTION AND CANCER AND RESPOND POTENTLY TO IMMUNE CHECKPOINT BLOCKADE. THESE CELLS SHARE TRANSCRIPTIONAL REGULATORS WITH MEMORY PRECURSOR CELLS, INCLUDING T CELL-SPECIFIC TRANSCRIPTION FACTOR 1 (TCF1), BUT IT IS UNCLEAR WHETHER THEY ADOPT DISTINCT PROGRAMS TO ADAPT TO THE IMMUNOSUPPRESSIVE ENVIRONMENT. BY COMPARING THE SINGLE-CELL TRANSCRIPTOMES AND EPIGENETIC PROFILES OF CD8(+) T CELLS RESPONDING TO ACUTE AND CHRONIC VIRAL INFECTIONS, WE FOUND THAT PROGENITOR-LIKE CD8(+) T CELLS BECAME DISTINCT FROM MEMORY PRECURSOR CELLS BEFORE THE PEAK OF THE T CELL RESPONSE. WE DISCOVERED A COEXPRESSION GENE MODULE CONTAINING TOX THAT EXHIBITED HIGHER TRANSCRIPTIONAL ACTIVITY ASSOCIATED WITH MORE ABUNDANT ACTIVE HISTONE MARKS IN PROGENITOR-LIKE CELLS THAN MEMORY PRECURSOR CELLS. MOREOVER, THYMOCYTE SELECTION-ASSOCIATED HIGH MOBILITY GROUP BOX PROTEIN TOX (TOX) PROMOTED THE PERSISTENCE OF ANTIVIRAL CD8(+) T CELLS AND WAS REQUIRED FOR THE PROGRAMMING OF PROGENITOR-LIKE CD8(+) T CELLS. THUS, LONG-TERM CD8(+) T CELL IMMUNITY TO CHRONIC VIRAL INFECTION REQUIRES UNIQUE TRANSCRIPTIONAL AND EPIGENETIC PROGRAMS ASSOCIATED WITH THE TRANSCRIPTION FACTOR TOX. 2019 3 2421 42 EPIGENETIC SIGNATURE OF PD-1+ TCF1+ CD8 T CELLS THAT ACT AS RESOURCE CELLS DURING CHRONIC VIRAL INFECTION AND RESPOND TO PD-1 BLOCKADE. WE HAVE RECENTLY DEFINED A NOVEL POPULATION OF PD-1 (PROGRAMMED CELL DEATH 1)+ TCF1 (T CELL FACTOR 1)+ VIRUS-SPECIFIC CD8 T CELLS THAT FUNCTION AS RESOURCE CELLS DURING CHRONIC LCMV INFECTION AND PROVIDE THE PROLIFERATIVE BURST SEEN AFTER PD-1 BLOCKADE. SUCH CD8 T CELLS HAVE BEEN FOUND IN OTHER CHRONIC INFECTIONS AND ALSO IN CANCER IN MICE AND HUMANS. THESE CD8 T CELLS EXHIBIT STEM-LIKE PROPERTIES UNDERGOING SELF-RENEWAL AND ALSO DIFFERENTIATING INTO THE TERMINALLY EXHAUSTED CD8 T CELLS. HERE WE COMPARED THE EPIGENETIC SIGNATURE OF STEM-LIKE CD8 T CELLS WITH EXHAUSTED CD8 T CELLS. ATAC-SEQ ANALYSIS SHOWED THAT STEM-LIKE CD8 T CELLS HAD A UNIQUE SIGNATURE IMPLICATING ACTIVITY OF HMG (TCF) AND RHD (NF-KAPPAB) TRANSCRIPTION FACTOR FAMILY MEMBERS IN CONTRAST TO HIGHER ACCESSIBILITY TO ETS AND RUNX MOTIFS IN EXHAUSTED CD8 T CELLS. IN ADDITION, REGULATORY REGIONS OF THE TRANSCRIPTION FACTORS TCF7 AND ID3 WERE MORE ACCESSIBLE IN STEM-LIKE CELLS WHEREAS PRDM1 AND ID2 WERE MORE ACCESSIBLE IN EXHAUSTED CD8 T CELLS. WE ALSO COMPARED THE EPIGENETIC SIGNATURES OF THE 2 CD8 T CELL SUBSETS FROM CHRONICALLY INFECTED MICE WITH EFFECTOR AND MEMORY CD8 T CELLS GENERATED AFTER AN ACUTE LCMV INFECTION. BOTH CD8 T CELL SUBSETS GENERATED DURING CHRONIC INFECTION WERE STRIKINGLY DIFFERENT FROM CD8 T CELL SUBSETS FROM ACUTE INFECTION. INTERESTINGLY, THE STEM-LIKE CD8 T CELL SUBSET FROM CHRONIC INFECTION, DESPITE SHARING KEY FUNCTIONAL PROPERTIES WITH MEMORY CD8 T CELLS, HAD A VERY DISTINCT EPIGENETIC PROGRAM. THESE RESULTS SHOW THAT THE CHRONIC STEM-LIKE CD8 T CELL PROGRAM REPRESENTS A SPECIFIC ADAPTATION OF THE T CELL RESPONSE TO PERSISTENT ANTIGENIC STIMULATION. 2019 4 6121 31 THE EPIGENETIC LANDSCAPE OF T CELL EXHAUSTION. EXHAUSTED T CELLS IN CANCER AND CHRONIC VIRAL INFECTION EXPRESS DISTINCTIVE PATTERNS OF GENES, INCLUDING SUSTAINED EXPRESSION OF PROGRAMMED CELL DEATH PROTEIN 1 (PD-1). HOWEVER, THE REGULATION OF GENE EXPRESSION IN EXHAUSTED T CELLS IS POORLY UNDERSTOOD. HERE, WE DEFINE THE ACCESSIBLE CHROMATIN LANDSCAPE IN EXHAUSTED CD8(+) T CELLS AND SHOW THAT IT IS DISTINCT FROM FUNCTIONAL MEMORY CD8(+) T CELLS. EXHAUSTED CD8(+) T CELLS IN HUMANS AND A MOUSE MODEL OF CHRONIC VIRAL INFECTION ACQUIRE A STATE-SPECIFIC EPIGENETIC LANDSCAPE ORGANIZED INTO FUNCTIONAL MODULES OF ENHANCERS. GENOME EDITING SHOWS THAT PD-1 EXPRESSION IS REGULATED IN PART BY AN EXHAUSTION-SPECIFIC ENHANCER THAT CONTAINS ESSENTIAL RAR, T-BET, AND SOX3 MOTIFS. FUNCTIONAL ENHANCER MAPS MAY OFFER TARGETS FOR GENOME EDITING THAT ALTER GENE EXPRESSION PREFERENTIALLY IN EXHAUSTED CD8(+) T CELLS. 2016 5 5593 37 ROLE, FUNCTION AND REGULATION OF THE THYMOCYTE SELECTION-ASSOCIATED HIGH MOBILITY GROUP BOX PROTEIN IN CD8(+) T CELL EXHAUSTION. THYMOCYTE SELECTION-ASSOCIATED HIGH MOBILITY GROUP BOX PROTEIN (TOX), A MEMBER OF THE HIGH-MOTILITY GROUP BOX (HMG) PROTEIN SUPERFAMILY, IS AN EVOLUTIONARILY CONSERVED DNA-BINDING PROTEIN. IT FUNCTIONS AS A TRANSCRIPTION FACTOR THAT MODULATES TRANSCRIPTIONAL PROGRAMS BY BINDING TO DNA IN A STRUCTURE-DEPENDENT MANNER. IT HAS BEEN WELL ESTABLISHED THAT TOX IS REQUIRED FOR THE DEVELOPMENT OF CD4(+) T CELLS, NATURAL KILLER (NK) CELLS AND INNATE LYMPHOID CELLS (ILCS), AS WELL AS THE AUTOIMMUNITY MEDIATED BY CD8(+) T CELLS. RECENTLY, EMERGING EVIDENCE SUPPORTS AN ESSENTIAL ROLE FOR TOX IN THE INDUCTION OF T CELL EXHAUSTION IN THE SETTING OF TUMOR OR CHRONIC VIRAL INFECTION BY MEDIATING TRANSCRIPTIONAL AND EPIGENETIC CHANGES, WHICH ARE CARDINAL HALLMARKS OF EXHAUSTED T CELLS. MOREOVER, TOX PLAYS A KEY ROLE IN THE PERSISTENCE OF ANTIGEN-SPECIFIC T CELLS AND IN THE MITIGATION OF TISSUE DAMAGE CAUSED BY IMMUNOPATHOLOGY OVER THE COURSE OF TUMORIGENESIS AND CHRONIC INFECTION. ADDITIONALLY, TOX CONTRIBUTES TO THE HIGH LEVEL OF PROGRAMMED CELL DEATH PROTEIN 1 (PD-1) ON THE CELL SURFACE BY PARTICIPATING IN THE PROCESS OF ENDOCYTIC RECYCLING OF PD-1. IN THIS REVIEW, WE SUMMARIZE THE MOST RECENT INFORMATION ABOUT THE ROLE OF TOX IN THE PROCESS OF T CELL EXHAUSTION, WHICH ENRICHES OUR UNDERSTANDING OF THE MOLECULAR MECHANISMS OF CD8(+) T CELL EXHAUSTION UPON CHRONIC ANTIGEN STIMULATION AND REVEALS PROMISING THERAPEUTIC TARGETS FOR PERSISTING INFECTION AND CANCER. 2021 6 1759 37 EARLY PRECURSOR T CELLS ESTABLISH AND PROPAGATE T CELL EXHAUSTION IN CHRONIC INFECTION. CD8(+) T CELLS RESPONDING TO CHRONIC INFECTIONS OR TUMORS ACQUIRE AN 'EXHAUSTED' STATE ASSOCIATED WITH ELEVATED EXPRESSION OF INHIBITORY RECEPTORS, INCLUDING PD-1, AND IMPAIRED CYTOKINE PRODUCTION. EXHAUSTED T CELLS ARE CONTINUOUSLY REPLENISHED BY T CELLS WITH PRECURSOR CHARACTERISTICS THAT SELF-RENEW AND DEPEND ON THE TRANSCRIPTION FACTOR TCF1; HOWEVER, THEIR DEVELOPMENTAL REQUIREMENTS ARE POORLY UNDERSTOOD. IN THE PRESENT STUDY, WE DEMONSTRATE THAT HIGH ANTIGEN LOAD PROMOTED THE DIFFERENTIATION OF PRECURSOR T CELLS, WHICH ACQUIRED HALLMARKS OF EXHAUSTION WITHIN DAYS OF INFECTION, WHEREAS EARLY EFFECTOR CELLS RETAINED POLYFUNCTIONAL FEATURES. EARLY PRECURSOR T CELLS SHOWED EPIGENETIC IMPRINTING CHARACTERISTIC OF T CELL RECEPTOR-DEPENDENT TRANSCRIPTION FACTOR BINDING AND WERE RESTRICTED TO THE GENERATION OF CELLS DISPLAYING EXHAUSTION CHARACTERISTICS. TRANSCRIPTION FACTORS BACH2 AND BATF WERE KEY REGULATORS WITH OPPOSING FUNCTIONS IN THE GENERATION OF EARLY PRECURSOR T CELLS. OVERALL, WE DEMONSTRATE THAT EXHAUSTION MANIFESTS FIRST IN TCF1(+) PRECURSOR T CELLS AND IS PROPAGATED SUBSEQUENTLY TO THE POOL OF ANTIGEN-SPECIFIC T CELLS. 2020 7 2410 28 EPIGENETIC SCARS OF CD8(+) T CELL EXHAUSTION PERSIST AFTER CURE OF CHRONIC INFECTION IN HUMANS. T CELL EXHAUSTION IS AN INDUCED STATE OF DYSFUNCTION THAT ARISES IN RESPONSE TO CHRONIC INFECTION AND CANCER. EXHAUSTED CD8(+) T CELLS ACQUIRE A DISTINCT EPIGENETIC STATE, BUT IT IS NOT KNOWN WHETHER THAT CHROMATIN LANDSCAPE IS FIXED OR PLASTIC FOLLOWING THE RESOLUTION OF A CHRONIC INFECTION. HERE WE SHOW THAT THE EPIGENETIC STATE OF EXHAUSTION IS LARGELY IRREVERSIBLE, EVEN AFTER CURATIVE THERAPY. ANALYSIS OF CHROMATIN ACCESSIBILITY IN HCV- AND HIV-SPECIFIC RESPONSES IDENTIFIES A CORE EPIGENETIC PROGRAM OF EXHAUSTION IN CD8(+) T CELLS, WHICH UNDERGOES ONLY LIMITED REMODELING BEFORE AND AFTER RESOLUTION OF INFECTION. MOREOVER, CANONICAL FEATURES OF EXHAUSTION, INCLUDING SUPER-ENHANCERS NEAR THE GENES TOX AND HIF1A, REMAIN 'EPIGENETICALLY SCARRED.' T CELL EXHAUSTION IS THEREFORE A CONSERVED EPIGENETIC STATE THAT BECOMES FIXED AND PERSISTS INDEPENDENT OF CHRONIC ANTIGEN STIMULATION AND INFLAMMATION. THERAPEUTIC EFFORTS TO REVERSE T CELL EXHAUSTION MAY REQUIRE NEW APPROACHES THAT INCREASE THE EPIGENETIC PLASTICITY OF EXHAUSTED T CELLS. 2021 8 5015 38 PERSISTENCE OF SELF-REACTIVE CD8+ T CELLS IN THE CNS REQUIRES TOX-DEPENDENT CHROMATIN REMODELING. SELF-REACTIVE CD8(+) T CELLS ARE IMPORTANT MEDIATORS OF PROGRESSIVE TISSUE DAMAGE IN AUTOIMMUNE DISEASES, BUT THE MOLECULAR PROGRAM UNDERLYING THESE CELLS' FUNCTIONAL ADAPTATION IS UNCLEAR. HERE WE CHARACTERIZE THE TRANSCRIPTIONAL AND EPIGENETIC LANDSCAPE OF SELF-REACTIVE CD8(+) T CELLS IN A MOUSE MODEL OF PROTRACTED CENTRAL NERVOUS SYSTEM (CNS) AUTOIMMUNITY AND COMPARE IT TO POPULATIONS OF CNS-RESIDENT MEMORY CD8(+) T CELLS EMERGING FROM ACUTE VIRAL INFECTION. WE FIND THAT AUTOIMMUNE CD8(+) T CELLS PERSISTING AT SITES OF SELF-ANTIGEN EXHIBIT CHARACTERISTIC TRANSCRIPTIONAL REGULATION TOGETHER WITH DISTINCT EPIGENETIC REMODELING. THIS SELF-REACTIVE CD8(+) T CELL FATE DEPENDS ON THE TRANSCRIPTIONAL REGULATION BY THE DNA-BINDING HMG-BOX PROTEIN TOX WHICH REMODELS MORE THAN 400 GENOMIC REGIONS INCLUDING LOCI SUCH AS TCF7, WHICH IS CENTRAL TO STEMNESS OF CD8(+) T CELLS. CONTINUOUS EXPOSURE TO CNS SELF-ANTIGEN SUSTAINS TOX LEVELS IN SELF-REACTIVE CD8(+) T CELLS, WHEREAS GENETIC ABLATION OF TOX IN CD8(+) T CELLS RESULTS IN SHORTENED PERSISTENCE OF SELF-REACTIVE CD8(+) T CELLS IN THE INFLAMED CNS. OUR STUDY ESTABLISHES AND CHARACTERIZES THE GENETIC DIFFERENTIATION PROGRAM ENABLING CHRONIC T CELL-DRIVEN IMMUNOPATHOLOGY IN CNS AUTOIMMUNITY. 2021 9 6319 40 THE ROAD LESS TAKEN: LESS APPRECIATED PATHWAYS FOR MANIPULATING CD8(+) T CELL EXHAUSTION. EXHAUSTED CD8(+) T (TEX) CELLS ARE A DISTINCT CELL POPULATION THAT ARISE DURING PERSISTENT ANTIGEN EXPOSURE IN THE CONTEXT OF CHRONIC INFECTIONS AND CANCERS. ALTHOUGH CHARACTERIZED BY PROGRESSIVE LOSS OF EFFECTOR FUNCTIONS, HIGH AND SUSTAINED INHIBITORY RECEPTOR EXPRESSION AND DISTINCT TRANSCRIPTIONAL AND EPIGENETIC PROGRAMS, TEX CELLS ARE HETEROGENEOUS. AMONG THESE, A SELF-RENEWING TCF-1(+) TEX POPULATION, HAVING UNIQUE CHARACTERISTICS AND THE ABILITY TO RESPOND TO IMMUNE-CHECKPOINT BLOCKADE, GIVES RISE TO TCF-1(-) TERMINALLY TEX CELLS. THESE TCF-1(+) CELLS HAVE STEM CELL-LIKE PROPERTIES SIMILAR TO MEMORY T CELL POPULATIONS, BUT THE SIGNALS THAT REGULATE THE DEVELOPMENTAL PATHWAYS AND RELATIONSHIPS AMONG EXHAUSTED CELL POPULATIONS ARE STILL UNCLEAR. HERE, WE REVIEW OUR CURRENT UNDERSTANDING OF TEX CELL BIOLOGY, AND DISCUSS SOME LESS APPRECIATED MOLECULES AND PATHWAYS AFFECTING T CELL EXHAUSTION. WE HIGHLIGHT TWO CO-STIMULATORY RECEPTORS, CD226 AND CD137, AND THEIR ROLE IN INDUCING OR RESTRAINING T CELL EXHAUSTION, AS WELL AS SIGNALING PATHWAYS THAT MAY BE AMENABLE TO PHARMACOLOGICAL INHIBITION WITH A FOCUS ON PHOSPHOINOSITIDE-3 KINASE AND IL-2 PARTIAL AGONISTS. FINALLY, WE DISCUSS NOVEL METHODS THAT MAY INCREASE TCF-1(+) POPULATIONS AND THEREFORE IMPROVE IMMUNOTHERAPY RESPONSIVENESS. UNDERSTANDING FEATURES OF AND PATHWAYS TO EXHAUSTION HAS IMPORTANT IMPLICATIONS FOR THE SUCCESS OF IMMUNOTHERAPY, INCLUDING CHECKPOINT BLOCKADE AND ADOPTIVE T-CELL TRANSFER THERAPIES. 2022 10 771 39 CD8(+) T CELL EXHAUSTION. CD8(+) T CELLS ARE IMPORTANT FOR THE PROTECTIVE IMMUNITY AGAINST INTRACELLULAR PATHOGENS AND TUMOR. IN THE CASE OF CHRONIC INFECTION OR CANCER, CD8(+) T CELLS ARE EXPOSED TO PERSISTENT ANTIGEN AND/OR INFLAMMATORY SIGNALS. THIS EXCESSIVE AMOUNT OF SIGNALS OFTEN LEADS CD8(+) T CELLS TO GRADUAL DETERIORATION OF T CELL FUNCTION, A STATE CALLED "EXHAUSTION." EXHAUSTED T CELLS ARE CHARACTERIZED BY PROGRESSIVE LOSS OF EFFECTOR FUNCTIONS (CYTOKINE PRODUCTION AND KILLING FUNCTION), EXPRESSION OF MULTIPLE INHIBITORY RECEPTORS (SUCH AS PD-1 AND LAG3), DYSREGULATED METABOLISM, POOR MEMORY RECALL RESPONSE, AND HOMEOSTATIC PROLIFERATION. THESE ALTERED FUNCTIONS ARE CLOSELY RELATED WITH ALTERED TRANSCRIPTIONAL PROGRAM AND EPIGENETIC LANDSCAPE THAT CLEARLY DISTINGUISH EXHAUSTED T CELLS FROM NORMAL EFFECTOR AND MEMORY T CELLS. T CELL EXHAUSTION IS OFTEN ASSOCIATED WITH INEFFICIENT CONTROL OF PERSISTING INFECTIONS AND CANCERS, BUT RE-INVIGORATION OF EXHAUSTED T CELLS WITH INHIBITORY RECEPTOR BLOCKADE CAN PROMOTE IMPROVED IMMUNITY AND DISEASE OUTCOME. ACCUMULATING EVIDENCES SUPPORT THE THERAPEUTIC POTENTIAL OF TARGETING EXHAUSTED T CELLS. HOWEVER, EXHAUSTED T CELLS COMPRISE HETEROGENOUS CELL POPULATION WITH DISTINCT RESPONSIVENESS TO INTERVENTION. UNDERSTANDING MOLECULAR MECHANISM OF T CELL EXHAUSTION IS ESSENTIAL TO ESTABLISH RATIONAL IMMUNOTHERAPEUTIC INTERVENTIONS. 2019 11 1379 32 DEVELOPMENTAL RELATIONSHIPS OF FOUR EXHAUSTED CD8(+) T CELL SUBSETS REVEALS UNDERLYING TRANSCRIPTIONAL AND EPIGENETIC LANDSCAPE CONTROL MECHANISMS. CD8(+) T CELL EXHAUSTION IS A MAJOR BARRIER TO CURRENT ANTI-CANCER IMMUNOTHERAPIES. DESPITE THIS, THE DEVELOPMENTAL BIOLOGY OF EXHAUSTED CD8(+) T CELLS (TEX) REMAINS POORLY DEFINED, RESTRAINING IMPROVEMENT OF STRATEGIES AIMED AT "RE-INVIGORATING" TEX CELLS. HERE, WE DEFINED A FOUR-CELL-STAGE DEVELOPMENTAL FRAMEWORK FOR TEX CELLS. TWO TCF1(+) PROGENITOR SUBSETS WERE IDENTIFIED, ONE TISSUE RESTRICTED AND QUIESCENT AND ONE MORE BLOOD ACCESSIBLE, THAT GRADUALLY LOST TCF1 AS IT DIVIDED AND CONVERTED TO A THIRD INTERMEDIATE TEX SUBSET. THIS INTERMEDIATE SUBSET RE-ENGAGED SOME EFFECTOR BIOLOGY AND INCREASED UPON PD-L1 BLOCKADE BUT ULTIMATELY CONVERTED INTO A FOURTH, TERMINALLY EXHAUSTED SUBSET. BY USING TRANSCRIPTIONAL AND EPIGENETIC ANALYSES, WE IDENTIFIED THE CONTROL MECHANISMS UNDERLYING SUBSET TRANSITIONS AND DEFINED A KEY INTERPLAY BETWEEN TCF1, T-BET, AND TOX IN THE PROCESS. THESE DATA REVEAL A FOUR-STAGE DEVELOPMENTAL HIERARCHY FOR TEX CELLS AND DEFINE THE MOLECULAR, TRANSCRIPTIONAL, AND EPIGENETIC MECHANISMS THAT COULD PROVIDE OPPORTUNITIES TO IMPROVE CANCER IMMUNOTHERAPY. 2020 12 2146 36 EPIGENETIC MANIPULATION RESTORES FUNCTIONS OF DEFECTIVE CD8(+) T CELLS FROM CHRONIC VIRAL INFECTION. FUNCTIONAL EXHAUSTION OF ANTIGEN-SPECIFIC T CELLS IS A DEFINING CHARACTERISTIC OF MANY CHRONIC INFECTIONS, BUT THE UNDERLYING MECHANISMS OF T CELL DYSFUNCTION ARE NOT WELL UNDERSTOOD. EPIGENETICS PLAYS AN IMPORTANT ROLE IN THE CONTROL OF T CELL DEVELOPMENT, DIFFERENTIATION, AND FUNCTION. TO EXAMINE IF EPIGENETICS ALSO PLAYS A ROLE IN T CELL EXHAUSTION, WE ANALYZED CHROMATIN REMODELING IN CD8(+) T CELLS FROM MICE WITH CHRONIC LYMPHOCYTIC CHORIOMENINGITIS VIRUS INFECTION. WE OBSERVED DOWNREGULATION OF DIACETYLATED HISTONE H3 IN BOTH VIRUS-SPECIFIC AND TOTAL CD8(+) T CELLS, AND FUNCTIONAL DEFECTS NOT ONLY IN VIRUS-SPECIFIC CD8(+) T CELLS BUT ALSO WITHIN THE TOTAL CD8(+) T CELL POPULATION. IN VITRO TREATMENT OF THESE EXHAUSTED CD8(+) T CELLS WITH HISTONE DEACETYLASE INHIBITORS RESTORED DIACETYLATED HISTONE H3 LEVELS, AND IMPROVED THEIR IMMUNE FUNCTIONS. UPON ADOPTIVE TRANSFER, THESE TREATED CD8(+) T CELLS DEVELOPED INTO FUNCTIONAL MEMORY T CELLS IN VIVO THAT ENHANCED PROTECTIVE IMMUNITY. THESE RESULTS DEFINE A ROLE OF EPIGENETICS IN T CELL EXHAUSTION AND SUGGEST EPIGENETIC MANIPULATION AS A NOVEL MOLECULAR THERAPY TO RESTORE IMMUNE FUNCTIONS. 2014 13 2718 35 EXHAUSTED T CELLS AND EPIGENETIC STATUS. EXHAUSTED T CELLS ARE A GROUP OF DYSFUNCTIONAL T CELLS, WHICH ARE PRESENT IN CHRONIC INFECTIONS OR TUMORS. THE MOST SIGNIFICANT CHARACTERISTICS OF EXHAUSTED T CELLS ARE ATTENUATED EFFECTOR CYTOTOXICITY, REDUCED CYTOKINE PRODUCTION, AND UPREGULATION OF MULTIPLE INHIBITORY MOLECULAR RECEPTORS (E.G., PD-1, TIM-3, AND LAG-3). THE INTRACELLULAR METABOLIC CHANGES, ALTERED EXPRESSION OF TRANSCRIPTION FACTORS, AND A UNIQUE EPIGENETIC LANDSCAPE CONSTITUTE THE EXHAUSTION PROGRAM. RECENTLY, RESEARCHERS HAVE MADE PROGRESS IN UNDERSTANDING EXHAUSTED T CELLS, WITH THE DEFINITION AND IDENTIFICATION OF EXHAUSTED T CELLS CHANGING FROM PHENOTYPE-BASED TO BEING CLASSIFIED AT THE TRANSCRIPTIONAL AND EPIGENETIC LEVELS. RECENT STUDIES HAVE REVEALED THAT EXHAUSTED T CELLS CAN BE SEPARATED INTO TWO SUBGROUPS, NAMELY TCF1(+)PD-1(+) PROGENITOR-LIKE PRECURSOR EXHAUSTED CELLS AND TCF1(-)PD-1(+) TERMINALLY DIFFERENTIATED EXHAUSTED T CELLS. MOREOVER, THE PROGENITOR-LIKE PRECURSOR CELL POPULATION MAY BE A SUBSET OF T CELLS THAT CAN RESPOND TO IMMUNOTHERAPY. STUDIES HAVE ALSO FOUND THAT TOX INITIATES AND DOMINATES THE DEVELOPMENT OF EXHAUSTED T CELLS AT THE TRANSCRIPTIONAL AND EPIGENETIC LEVELS. TOX ALSO MAINTAINS T CELL SURVIVAL AND MAY AFFECT DECISIONS REGARDING TREATMENT STRATEGIES. IN THIS REVIEW, WE DISCUSS THE LATEST DEVELOPMENTS IN T CELL EXHAUSTION IN REGARDS TO DEFINITIONS, SUBPOPULATIONS, DEVELOPMENT MECHANISMS, DIFFERENCES IN DIVERSE DISEASES, AND TREATMENT PROSPECTS FOR EXHAUSTED T CELLS. FURTHERMORE, WE HYPOTHESIZE THAT THE EPIGENETIC STATE REGULATED BY TOX MIGHT BE THE KEY POINT, WHICH CAN DETERMINE THE REVERSIBILITY OF EXHAUSTION AND THE EFFICACY OF IMMUNOTHERAPY. 2020 14 770 35 CD8(+) T CELL DYSFUNCTION BY TOX INTOXICATION: A PROTUMORIGENIC EVENT IN THE TUMOR MICROENVIRONMENT. ACCUMULATING EVIDENCE SUGGESTS THE ROLE OF CELLULAR COMPONENTS IN ACHIEVING ANTITUMOR TO PROTUMOR MICROENVIRONMENTS. AMONG THE VARIOUS TYPES OF CELLS WITHIN THE TUMOR NICHE, THE STATE OF CD8(+) T CELLS APPARENTLY CHANGES FROM CYTOTOXIC T EFFECTOR CELLS AND MEMORY T CELLS TO EXHAUSTED CD8(+) T CELLS. THESE CHANGES IN THE PHENOTYPE OF CD8(+) T CELLS PROMOTE THE PROTUMOR MICROENVIRONMENT. RECENTLY, COMPREHENSIVE EXPERIMENTAL DATA DELINEATED THE ROLE OF THYMOCYTE SELECTION-ASSOCIATED HIGH-MOBILITY GROUP-BOX PROTEIN (TOX), WHICH REGULATES THE TRANSCRIPTIONAL PROCESS AND EPIGENETIC REMODELING, WITH IMPLICATIONS IN TUMOR AND CHRONIC VIRAL INFECTIONS. THIS PERSPECTIVE SUMMARIZES THE MOLECULAR MECHANISMS THAT LINK CD8(+) T CELLS, TOX, AND TRANSCRIPTIONAL AND EPIGENETIC REPROGRAMMING AS WELL AS FUTURE DIRECTIONS FOR DETERMINING NEW AVENUES OF CANCER THERAPEUTICS. 2021 15 6851 36 [MOLECULAR PROFILES OF EXHAUSTED T CELLS AND THEIR IMPACT ON RESPONSE TO IMMUNE CHECKPOINT BLOCKADE]. T CELL EXHAUSTION IS INDUCED IN THE CONTEXT OF CHRONIC VIRUS INFECTION AND TUMOR MICROENVIRONMENT, IN WHICH CYTOTOXIC T CELLS ARE REPEATEDLY EXPOSED TO THE TARGET ANTIGEN AND DEPRIVED OF THEIR EFFECTOR FUNCTIONS. MULTIPLE STUDIES HAVE ALREADY SHOWN THE SIGNIFICANT IMPACT OF IMMUNE CHECKPOINT MOLECULES SUCH AS PD1 ON FUNCTIONAL PROPERTIES OF EXHAUSTED T CELLS. IN ADDITION TO THESE SIGNALS, EXHAUSTED T CELLS POSSESS DISTINCT TRANSCRIPTIONAL AND EPIGENETIC PROFILES COMPARED WITH CONVENTIONAL EFFECTOR AND MEMORY T CELLS. IMPORTANTLY, MOST OF THESE FEATURES ARE NOT AFFECTED BY IMMUNE CHECKPOINT BLOCKADE, SUGGESTING THAT GENETIC AND EPIGENETIC REMODELING OF T CELLS IS AN UNDERLYING MOLECULAR MECHANISM ESSENTIAL FOR T CELL EXHAUSTION. MOREOVER, IT HAS NOW BEEN EVIDENT THAT EXHAUSTED T CELLS ARE A HETEROGENEOUS CELL POPULATION COMPOSED OF DISTINCT T CELL SUBSETS, AND THESE FUNCTIONAL DIFFERENCES PROFOUNDLY AFFECT THERAPEUTIC EFFICACY OF CANCER IMMUNOTHERAPY. IN THIS REVIEW, I WILL DISCUSS RECENT STUDIES INVESTIGATING MOLECULAR MECHANISMS OF T CELL EXHAUSTION, INCLUDING NOVEL KEY MOLECULES ESSENTIALLY ASSOCIATED WITH T CELL EXHAUSTION. THESE FINDINGS ARE POTENTIALLY APPLICABLE TO REINVIGORATE EFFECTOR FUNCTIONS OF EXHAUSTED T CELLS. 2022 16 1007 33 CHRONIC VIRUS INFECTION ENFORCES DEMETHYLATION OF THE LOCUS THAT ENCODES PD-1 IN ANTIGEN-SPECIFIC CD8(+) T CELLS. FUNCTIONALLY EXHAUSTED T CELLS HAVE HIGH EXPRESSION OF THE PD-1 INHIBITORY RECEPTOR, AND THERAPIES THAT BLOCK PD-1 SIGNALING SHOW PROMISE FOR RESOLVING CHRONIC VIRAL INFECTIONS AND CANCER. BY USING HUMAN AND MURINE SYSTEMS OF ACUTE AND CHRONIC VIRAL INFECTIONS, WE ANALYZED EPIGENETIC REGULATION OF PD-1 EXPRESSION DURING CD8(+) T CELL DIFFERENTIATION. DURING ACUTE INFECTION, NAIVE TO EFFECTOR CD8(+) T CELL DIFFERENTIATION WAS ACCOMPANIED BY A TRANSIENT LOSS OF DNA METHYLATION OF THE PDCD1 LOCUS THAT WAS DIRECTLY COUPLED TO THE DURATION AND STRENGTH OF T CELL RECEPTOR SIGNALING. FURTHER DIFFERENTIATION INTO FUNCTIONAL MEMORY CELLS COINCIDED WITH PDCD1 REMETHYLATION, PROVIDING AN ADAPTED PROGRAM FOR REGULATION OF PD-1 EXPRESSION. IN CONTRAST, THE PDCD1 REGULATORY REGION WAS COMPLETELY DEMETHYLATED IN EXHAUSTED CD8(+) T CELLS AND REMAINED UNMETHYLATED EVEN WHEN VIRUS TITERS DECREASED. THIS LACK OF DNA REMETHYLATION LEAVES THE PDCD1 LOCUS POISED FOR RAPID EXPRESSION, POTENTIALLY PROVIDING A SIGNAL FOR PREMATURE TERMINATION OF ANTIVIRAL FUNCTIONS. 2011 17 1319 45 DEMETHYLATION OF THE PD-1 PROMOTER IS IMPRINTED DURING THE EFFECTOR PHASE OF CD8 T CELL EXHAUSTION. PD-1 IS AN INHIBITORY RECEPTOR THAT HAS A MAJOR ROLE IN T CELL DYSFUNCTION DURING CHRONIC INFECTIONS AND CANCER. WHILE DEMETHYLATION OF THE PD-1 PROMOTER DNA IS OBSERVED IN EXHAUSTED T CELLS ISOLATED FROM CHRONICALLY INFECTED INDIVIDUALS, LITTLE IS KNOWN ABOUT WHEN THIS STABLE DEMETHYLATION OF PD-1 PROMOTER DNA IS PROGRAMMED DURING THE COURSE OF A CHRONIC INFECTION. TO ASSESS IF PD-1 PROMOTER DNA DEMETHYLATION IS IMPACTED BY PROLONGED STIMULATION DURING EFFECTOR PHASE OF CHRONIC INFECTION, WE ADOPTIVELY TRANSFERRED VIRUS-SPECIFIC DAY 8 EFFECTOR CD8 T CELLS FROM MICE INFECTED WITH LYMPHOCYTIC CHORIOMENINGITIS VIRUS (LCMV) CLONE 13 INTO RECIPIENT MICE THAT HAD CLEARED AN ACUTE INFECTION. WE OBSERVED THAT LCMV-SPECIFIC CD8 T CELLS FROM CHRONICALLY INFECTED MICE MAINTAINED THEIR SURFACE EXPRESSION OF PD-1 EVEN AFTER TRANSFER INTO ACUTE IMMUNE MICE UNTIL DAY 45 POSTTRANSFER. INTERESTINGLY, THE PD-1 TRANSCRIPTIONAL REGULATORY REGION CONTINUED TO REMAIN UNMETHYLATED IN THESE DONOR CD8 T CELLS GENERATED FROM A CHRONIC INFECTION. THE OBSERVED MAINTENANCE OF PD-1 SURFACE EXPRESSION AND THE DEMETHYLATED PD-1 PROMOTER WERE NOT A RESULT OF RESIDUAL ANTIGEN IN THE RECIPIENT MICE, BECAUSE SIMILAR RESULTS WERE SEEN WHEN CHRONIC INFECTION-INDUCED EFFECTOR CELLS WERE TRANSFERRED INTO MICE INFECTED WITH A VARIANT STRAIN OF LCMV (LCMV V35A) BEARING A MUTATION IN THE COGNATE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I (MHC-I) EPITOPE THAT IS RECOGNIZED BY THE DONOR CD8 T CELLS. IMPORTANTLY, THE MAINTENANCE OF PD-1 PROMOTER DEMETHYLATION IN MEMORY CD8 T CELLS WAS COUPLED WITH IMPAIRED CLONAL EXPANSION AND HIGHER PD-1 RE-EXPRESSION UPON SECONDARY CHALLENGE. THESE DATA SHOW THAT THE IMPRINTING OF THE EPIGENETIC PROGRAM OF THE INHIBITORY RECEPTOR PD-1 OCCURS DURING THE EFFECTOR PHASE OF CHRONIC VIRAL INFECTION. IMPORTANCE: SINCE PD-1 IS A MAJOR INHIBITORY RECEPTOR REGULATING T CELL DYSFUNCTION DURING CHRONIC VIRAL INFECTION AND CANCERS, A BETTER UNDERSTANDING OF THE MECHANISMS THAT REGULATE PD-1 EXPRESSION IS IMPORTANT. IN THIS WORK, WE DEMONSTRATE THAT THE PD-1 EPIGENETIC PROGRAM IN ANTIGEN-SPECIFIC CD8 T CELLS IS FIXED DURING THE PRIMING PHASE OF CHRONIC INFECTION. 2016 18 1262 38 CUTTING EDGE: PROLONGED EXPOSURE TO HIV REINFORCES A POISED EPIGENETIC PROGRAM FOR PD-1 EXPRESSION IN VIRUS-SPECIFIC CD8 T CELLS. AG-SPECIFIC CD8 T CELLS PLAY A CRITICAL ROLE IN CONTROLLING HIV INFECTION BUT EVENTUALLY LOSE ANTIVIRAL FUNCTIONS IN PART BECAUSE OF EXPRESSION AND SIGNALING THROUGH THE INHIBITORY PROGRAMMED DEATH-1 (PD-1) RECEPTOR. TO BETTER UNDERSTAND THE IMPACT OF PROLONGED TCR LIGATION ON REGULATION OF PD-1 EXPRESSION IN HIV-SPECIFIC CD8 T CELLS, WE INVESTIGATED THE CAPACITY OF VIRUS-SPECIFIC CD8 T CELLS TO MODIFY THE PD-1 EPIGENETIC PROGRAM AFTER REDUCTION IN VIRAL LOAD. WE OBSERVED THAT THE TRANSCRIPTIONAL REGULATORY REGION WAS UNMETHYLATED IN THE PD-1(HI) HIV-SPECIFIC CD8 T CELLS, WHEREAS IT REMAINED METHYLATED IN DONOR-MATCHED NAIVE CELLS AT ACUTE AND CHRONIC STAGES OF INFECTION. SURPRISINGLY, THE PD-1 PROMOTER REMAINED UNMETHYLATED IN HIV-SPECIFIC CD8 T CELLS FROM SUBJECTS WITH A VIRAL LOAD CONTROLLED BY ANTIVIRAL THERAPY FOR >2 Y OR FROM ELITE CONTROLLERS. TOGETHER, THESE DATA DEMONSTRATE THAT THE EPIGENETIC PROGRAM AT THE PD-1 LOCUS BECOMES FIXED AFTER PROLONGED EXPOSURE TO HIV VIRUS. 2013 19 438 35 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. 2021 20 6530 39 TRANSCRIPTIONAL REGULATION AND T CELL EXHAUSTION. PURPOSE OF REVIEW: THIS REVIEW HIGHLIGHTS THE CONTROL OF TRANSCRIPTIONAL NETWORKS, INCLUDING INDUCTION OF INHIBITORY RECEPTORS, BY T CELL-SPECIFIC TRANSCRIPTION FACTORS IN EXHAUSTED T CELLS THAT ACCUMULATE IN CHRONIC VIRAL INFECTIONS INCLUDING HIV. RECENT FINDINGS: TRANSCRIPTIONAL PROFILING HAS ESTABLISHED DISTINCT MOLECULAR PHENOTYPES FOR EXHAUSTED CD4 AND CD8 T CELLS IN CHRONIC VIRAL INFECTION MODELS. THERE EXISTS A SUBSET OF TRANSCRIPTION FACTORS ASSOCIATED WITH EXHAUSTION, NOTABLY BLIMP-1, BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR, ATF-LIKE AND HELIOS. EPIGENETIC PHENOMENA ARE LIKELY IMPORTANT IN REGULATING GENE EXPRESSION NETWORKS DURING EXHAUSTION AS ILLUSTRATED BY PROGRAMMED DEATH 1 PROMOTER METHYLATION PATTERNS. SUMMARY: FOLLOWING CHRONIC VIRAL INFECTIONS, CD4 AND CD8 T CELLS DEFINED FUNCTIONALLY AND PHENOTYPICALLY AS EXHAUSTED HAVE DISTINCT TRANSCRIPTIONAL PROFILES. THESE STUDIES HAVE IDENTIFIED A CORE SET OF TRANSCRIPTION FACTORS THAT HAVE BEEN IMPLICATED IN PROMOTING EXHAUSTION. HOWEVER, NO SINGLE FACTOR APPEARS TO BE AN EXHAUSTION DETERMINING FACTOR, SUGGESTING THAT T CELL EXHAUSTION REFLECTS A COMBINATORIAL MECHANISM WITH MULTIPLE TRANSCRIPTION FACTORS INTERACTING TO INFLUENCE THE DEVELOPMENT OF FUNCTIONALLY EXHAUSTED T CELLS AS WELL AS DIFFERENT T EFFECTOR POPULATIONS. 2014