1 3617 91 IN VITRO MODELING OF CD8(+) T CELL EXHAUSTION ENABLES CRISPR SCREENING TO REVEAL A ROLE FOR BHLHE40. IDENTIFYING MOLECULAR MECHANISMS OF EXHAUSTED CD8 T CELLS (T(EX)) IS A KEY GOAL OF IMPROVING IMMUNOTHERAPY OF CANCER AND OTHER DISEASES. HOWEVER, HIGH-THROUGHPUT INTERROGATION OF IN VIVO T(EX) CAN BE COSTLY AND INEFFICIENT. IN VITRO MODELS OF T(EX) ARE EASILY CUSTOMIZABLE AND QUICKLY GENERATE HIGH CELLULAR YIELD, ENABLING CRISPR SCREENING AND OTHER HIGH-THROUGHPUT ASSAYS. WE ESTABLISHED AN IN VITRO MODEL OF CHRONIC STIMULATION AND BENCHMARKED KEY PHENOTYPIC, FUNCTIONAL, TRANSCRIPTIONAL, AND EPIGENETIC FEATURES AGAINST BONA FIDE IN VIVO T(EX). WE LEVERAGED THIS MODEL OF IN VITRO CHRONIC STIMULATION IN COMBINATION WITH CRISPR SCREENING TO IDENTIFY TRANSCRIPTIONAL REGULATORS OF T CELL EXHAUSTION. THIS APPROACH IDENTIFIED SEVERAL TRANSCRIPTION FACTORS, INCLUDING BHLHE40. IN VITRO AND IN VIVO VALIDATION DEFINED A ROLE FOR BHLHE40 IN REGULATING A KEY DIFFERENTIATION CHECKPOINT BETWEEN PROGENITOR AND INTERMEDIATE T(EX) SUBSETS. BY DEVELOPING AND BENCHMARKING AN IN VITRO MODEL OF T(EX), THEN APPLYING HIGH-THROUGHPUT CRISPR SCREENING, WE DEMONSTRATE THE UTILITY OF MECHANISTICALLY ANNOTATED IN VITRO MODELS OF T(EX). 2023 2 3616 87 IN VITRO MODELING OF CD8 T CELL EXHAUSTION ENABLES CRISPR SCREENING TO REVEAL A ROLE FOR BHLHE40. IDENTIFYING NOVEL MOLECULAR MECHANISMS OF EXHAUSTED CD8 T CELLS (T (EX) ) IS A KEY GOAL OF IMPROVING IMMUNOTHERAPY OF CANCER AND OTHER DISEASES. HOWEVER, HIGH-THROUGHPUT INTERROGATION OF IN VIVO T (EX) CAN BE COSTLY AND INEFFICIENT. IN VITRO MODELS OF T (EX) ARE EASILY CUSTOMIZABLE AND QUICKLY GENERATE HIGH CELLULAR YIELD, OFFERING AN OPPORTUNITY TO PERFORM CRISPR SCREENING AND OTHER HIGH-THROUGHPUT ASSAYS. WE ESTABLISHED AN IN VITRO MODEL OF CHRONIC STIMULATION AND BENCHMARKED KEY PHENOTYPIC, FUNCTIONAL, TRANSCRIPTIONAL, AND EPIGENETIC FEATURES AGAINST BONA FIDE IN VIVO T (EX) . WE LEVERAGED THIS MODEL OF IN VITRO CHRONIC STIMULATION IN COMBINATION WITH POOLED CRISPR SCREENING TO UNCOVER TRANSCRIPTIONAL REGULATORS OF T CELL EXHAUSTION. THIS APPROACH IDENTIFIED SEVERAL TRANSCRIPTION FACTORS, INCLUDING BHLHE40. IN VITRO AND IN VIVO VALIDATION DEFINED A ROLE FOR BHLHE40 IN REGULATING A KEY DIFFERENTIATION CHECKPOINT BETWEEN PROGENITOR AND INTERMEDIATE SUBSETS OF T (EX) . BY DEVELOPING AND BENCHMARKING AN IN VITRO MODEL OF T (EX) , WE DEMONSTRATE THE UTILITY OF MECHANISTICALLY ANNOTATED IN VITRO MODELS OF T (EX) , IN COMBINATION WITH HIGH-THROUGHPUT APPROACHES, AS A DISCOVERY PIPELINE TO UNCOVER NOVEL T (EX) BIOLOGY. 2023 3 6482 33 TOX TRANSCRIPTIONALLY AND EPIGENETICALLY PROGRAMS CD8(+) T CELL EXHAUSTION. EXHAUSTED CD8(+) T (T(EX)) CELLS IN CHRONIC INFECTIONS AND CANCER HAVE LIMITED EFFECTOR FUNCTION, HIGH CO-EXPRESSION OF INHIBITORY RECEPTORS AND EXTENSIVE TRANSCRIPTIONAL CHANGES COMPARED WITH EFFECTOR (T(EFF)) OR MEMORY (T(MEM)) CD8(+) T CELLS. T(EX) CELLS ARE IMPORTANT CLINICAL TARGETS OF CHECKPOINT BLOCKADE AND OTHER IMMUNOTHERAPIES. EPIGENETICALLY, T(EX) CELLS ARE A DISTINCT IMMUNE SUBSET, WITH A UNIQUE CHROMATIN LANDSCAPE COMPARED WITH T(EFF) AND T(MEM) CELLS. HOWEVER, THE MECHANISMS THAT GOVERN THE TRANSCRIPTIONAL AND EPIGENETIC DEVELOPMENT OF T(EX) CELLS REMAIN UNKNOWN. HERE WE IDENTIFY THE HMG-BOX TRANSCRIPTION FACTOR TOX AS A CENTRAL REGULATOR OF T(EX) CELLS IN MICE. TOX IS LARGELY DISPENSABLE FOR THE FORMATION OF T(EFF) AND T(MEM) CELLS, BUT IT IS CRITICAL FOR EXHAUSTION: IN THE ABSENCE OF TOX, T(EX) CELLS DO NOT FORM. TOX IS INDUCED BY CALCINEURIN AND NFAT2, AND OPERATES IN A FEED-FORWARD LOOP IN WHICH IT BECOMES CALCINEURIN-INDEPENDENT AND SUSTAINED IN T(EX) CELLS. ROBUST EXPRESSION OF TOX THEREFORE RESULTS IN COMMITMENT TO T(EX) CELLS BY TRANSLATING PERSISTENT STIMULATION INTO A DISTINCT T(EX) CELL TRANSCRIPTIONAL AND EPIGENETIC DEVELOPMENTAL PROGRAM. 2019 4 6417 33 THE SWI/SNF CHROMATIN REMODELING COMPLEXES BAF AND PBAF DIFFERENTIALLY REGULATE EPIGENETIC TRANSITIONS IN EXHAUSTED CD8(+) T CELLS. CD8(+) T CELL EXHAUSTION (TEX) LIMITS DISEASE CONTROL DURING CHRONIC VIRAL INFECTIONS AND CANCER. HERE, WE INVESTIGATED THE EPIGENETIC FACTORS MEDIATING MAJOR CHROMATIN-REMODELING EVENTS IN TEX-CELL DEVELOPMENT. A PROTEIN-DOMAIN-FOCUSED IN VIVO CRISPR SCREEN IDENTIFIED DISTINCT FUNCTIONS FOR TWO VERSIONS OF THE SWI/SNF CHROMATIN-REMODELING COMPLEX IN TEX-CELL DIFFERENTIATION. DEPLETION OF THE CANONICAL SWI/SNF FORM, BAF, IMPAIRED INITIAL CD8(+) T CELL RESPONSES IN ACUTE AND CHRONIC INFECTION. IN CONTRAST, DISRUPTION OF PBAF ENHANCED TEX-CELL PROLIFERATION AND SURVIVAL. MECHANISTICALLY, PBAF REGULATED THE EPIGENETIC AND TRANSCRIPTIONAL TRANSITION FROM TCF-1(+) PROGENITOR TEX CELLS TO MORE DIFFERENTIATED TCF-1(-) TEX SUBSETS. WHEREAS PBAF ACTED TO PRESERVE TEX PROGENITOR BIOLOGY, BAF WAS REQUIRED TO GENERATE EFFECTOR-LIKE TEX CELLS, SUGGESTING THAT THE BALANCE OF THESE FACTORS COORDINATES TEX-CELL SUBSET DIFFERENTIATION. TARGETING PBAF IMPROVED TUMOR CONTROL BOTH ALONE AND IN COMBINATION WITH ANTI-PD-L1 IMMUNOTHERAPY. THUS, PBAF MAY PRESENT A THERAPEUTIC TARGET IN CANCER IMMUNOTHERAPY. 2023 5 75 32 A MURINE CELL LINE BASED MODEL OF CHRONIC CDK9 INHIBITION TO STUDY WIDESPREAD NON-GENETIC TRANSCRIPTIONAL ELONGATION DEFECTS (TEDEFF) IN CANCERS. WE HAVE PREVIOUSLY REPORTED THAT A SUBSET OF CANCERS IS DEFINED BY GLOBAL TRANSCRIPTIONAL DEREGULATIONS WITH WIDESPREAD DEFICIENCIES IN MRNA TRANSCRIPTION ELONGATION (TE)-WE CALL SUCH CANCERS AS TE(DEFF). NOTABLY, TE(DEFF) CANCERS ARE CHARACTERIZED BY SPURIOUS TRANSCRIPTION AND FAULTY MRNA PROCESSING IN A LARGE SET OF GENES, SUCH AS INTERFERON/JAK/STAT AND TNF/NF-KAPPAB PATHWAYS, LEADING TO THEIR SUPPRESSION. THE TE(DEFF) SUBTYPE OF TUMORS IN RENAL CELL CARCINOMA AND METASTATIC MELANOMA PATIENTS SIGNIFICANTLY CORRELATE WITH POOR RESPONSE AND OUTCOME IN IMMUNOTHERAPY. GIVEN THE IMPORTANCE OF INVESTIGATING TE(DEFF) CANCERS-AS IT PORTENDS A SIGNIFICANT ROADBLOCK AGAINST IMMUNOTHERAPY-THE GOAL OF THIS PROTOCOL IS TO ESTABLISH AN IN VITRO TE(DEFF) MOUSE MODEL TO STUDY THESE WIDESPREAD, NON-GENETIC TRANSCRIPTIONAL ABNORMALITIES IN CANCERS AND GAIN NEW INSIGHTS, NOVEL USES FOR EXISTING DRUGS, OR FIND NEW STRATEGIES AGAINST SUCH CANCERS. WE DETAIL THE USE OF CHRONIC FLAVOPIRIDOL MEDIATED CDK9 INHIBITION TO ABROGATE PHOSPHORYLATION OF SERINE 2 RESIDUE ON THE C-TERMINAL REPEAT DOMAIN (CTD) OF RNA POLYMERASE II (RNA POL II), SUPPRESSING THE RELEASE OF RNA POL II INTO PRODUCTIVE TRANSCRIPTION ELONGATION. GIVEN THAT TE(DEFF) CANCERS ARE NOT CLASSIFIED UNDER ANY SPECIFIC SOMATIC MUTATION, A PHARMACOLOGICAL MODEL IS ADVANTAGEOUS, AND BEST MIMICS THE WIDESPREAD TRANSCRIPTIONAL AND EPIGENETIC DEFECTS OBSERVED IN THEM. THE USE OF AN OPTIMIZED SUBLETHAL DOSE OF FLAVOPIRIDOL IS THE ONLY EFFICACIOUS STRATEGY IN CREATING A GENERALIZABLE MODEL OF NON-GENETIC WIDESPREAD DISRUPTION IN TRANSCRIPTION ELONGATION AND MRNA PROCESSING DEFECTS, CLOSELY MIMICKING THE CLINICALLY OBSERVED TE(DEFF) CHARACTERISTICS. THEREFORE, THIS MODEL OF TE(DEFF) CAN BE LEVERAGED TO DISSECT, CELL-AUTONOMOUS FACTORS ENABLING THEM IN RESISTING IMMUNE-MEDIATED CELL ATTACK. 2019 6 3054 30 GENOME-WIDE CRISPR SCREENS OF T CELL EXHAUSTION IDENTIFY CHROMATIN REMODELING FACTORS THAT LIMIT T CELL PERSISTENCE. T CELL EXHAUSTION LIMITS ANTITUMOR IMMUNITY, BUT THE MOLECULAR DETERMINANTS OF THIS PROCESS REMAIN POORLY UNDERSTOOD. USING A CHRONIC STIMULATION ASSAY, WE PERFORMED GENOME-WIDE CRISPR-CAS9 SCREENS TO SYSTEMATICALLY DISCOVER REGULATORS OF T CELL EXHAUSTION, WHICH IDENTIFIED AN ENRICHMENT OF EPIGENETIC FACTORS. IN VIVO CRISPR SCREENS IN MURINE AND HUMAN TUMOR MODELS DEMONSTRATED THAT PERTURBATION OF THE INO80 AND BAF CHROMATIN REMODELING COMPLEXES IMPROVED T CELL PERSISTENCE IN TUMORS. IN VIVO PERTURB-SEQ REVEALED DISTINCT TRANSCRIPTIONAL ROLES OF EACH COMPLEX AND THAT DEPLETION OF CANONICAL BAF COMPLEX MEMBERS, INCLUDING ARID1A, RESULTED IN THE MAINTENANCE OF AN EFFECTOR PROGRAM AND DOWNREGULATION OF EXHAUSTION-RELATED GENES IN TUMOR-INFILTRATING T CELLS. FINALLY, ARID1A DEPLETION LIMITED THE ACQUISITION OF EXHAUSTION-ASSOCIATED CHROMATIN ACCESSIBILITY AND LED TO IMPROVED ANTITUMOR IMMUNITY. IN SUMMARY, WE PROVIDE AN ATLAS OF THE GENETIC REGULATORS OF T CELL EXHAUSTION AND DEMONSTRATE THAT MODULATION OF EPIGENETIC STATE CAN IMPROVE T CELL RESPONSES IN CANCER IMMUNOTHERAPY. 2022 7 5671 37 SHARED AND DISTINCT BIOLOGICAL CIRCUITS IN EFFECTOR, MEMORY AND EXHAUSTED CD8(+) T CELLS REVEALED BY TEMPORAL SINGLE-CELL TRANSCRIPTOMICS AND EPIGENETICS. NAIVE CD8(+) T CELLS CAN DIFFERENTIATE INTO EFFECTOR (T(EFF)), MEMORY (T(MEM)) OR EXHAUSTED (T(EX)) T CELLS. THESE DEVELOPMENTAL PATHWAYS ARE ASSOCIATED WITH DISTINCT TRANSCRIPTIONAL AND EPIGENETIC CHANGES THAT ENDOW CELLS WITH DIFFERENT FUNCTIONAL CAPACITIES AND THEREFORE THERAPEUTIC POTENTIAL. THE MOLECULAR CIRCUITRY UNDERLYING THESE DEVELOPMENTAL TRAJECTORIES AND THE EXTENT OF HETEROGENEITY WITHIN T(EFF), T(MEM) AND T(EX) POPULATIONS REMAIN POORLY UNDERSTOOD. HERE, WE USED THE LYMPHOCYTIC CHORIOMENINGITIS VIRUS MODEL OF ACUTE-RESOLVING AND CHRONIC INFECTION TO ADDRESS THESE GAPS BY APPLYING LONGITUDINAL SINGLE-CELL RNA-SEQUENCING (SCRNA-SEQ) AND SINGLE-CELL ASSAY FOR TRANSPOSASE-ACCESSIBLE CHROMATIN SEQUENCING (SCATAC-SEQ) ANALYSES. THESE ANALYSES UNCOVERED NEW SUBSETS, INCLUDING A SUBPOPULATION OF T(EX) CELLS EXPRESSING NATURAL KILLER CELL-ASSOCIATED GENES THAT IS DEPENDENT ON THE TRANSCRIPTION FACTOR ZEB2, AS WELL AS MULTIPLE DISTINCT TCF-1(+) STEM/PROGENITOR-LIKE SUBSETS IN ACUTE AND CHRONIC INFECTION. THESE DATA ALSO REVEALED INSIGHTS INTO THE RESHAPING OF T(EX) SUBSETS FOLLOWING PROGRAMMED DEATH 1 (PD-1) PATHWAY BLOCKADE AND IDENTIFIED A KEY ROLE FOR THE CELL STRESS REGULATOR, BTG1, IN ESTABLISHING THE T(EX) POPULATION. FINALLY, THESE RESULTS HIGHLIGHTED HOW THE SAME BIOLOGICAL CIRCUITS SUCH AS CYTOTOXICITY OR STEM/PROGENITOR PATHWAYS CAN BE USED BY CD8(+) T CELL SUBSETS WITH HIGHLY DIVERGENT UNDERLYING CHROMATIN LANDSCAPES GENERATED DURING DIFFERENT INFECTIONS. 2022 8 833 14 CHARTING A SHARED EPIGENETIC PATHWAY TO CD8(+) T CELL DYSFUNCTION IN INFECTION AND CANCER. PRITYKIN ET AL. (2021) ESTABLISH A COMPREHENSIVE CHROMATIN ATLAS OF CD8(+) T CELL DYSFUNCTION IN CHRONIC VIRAL INFECTION AND CANCER VIA ANALYSIS OF BULK AND SINGLE-CELL ATAC-SEQ DATASETS ACROSS IMMUNE CHALLENGES. THESE RESULTS UNIFY THE CLASSIFICATION SCHEME AND MOLECULAR PROGRAMS DRIVING CD8(+) T CELL DYSFUNCTION ACROSS DISEASE SETTINGS AND WILL FACILITATE BASIC DISCOVERY AND TRANSLATIONAL EFFORTS IN T CELL IMMUNITY. 2021 9 6121 23 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 10 4190 25 METABOLIC DYSFUNCTION-ASSOCIATED FATTY LIVER DISEASE: ADVANCES IN GENETIC AND EPIGENETIC IMPLICATIONS. PURPOSE OF REVIEW: FATTY LIVER ASSOCIATED WITH METABOLIC DYSFUNCTION, ALSO KNOWN UNDER THE ACRONYM NAFLD (NONALCOHOLIC FATTY LIVER DISEASE) IS THE LEADING GLOBAL CAUSE OF CHRONIC LIVER DISEASE. IN THIS REVIEW, WE ADDRESS THE STATE OF RESEARCH ON GENETICS AND EPIGENETICS OF NAFLD WITH FOCUS ON KEY DISCOVERIES AND CONCEPTUAL ADVANCES OVER THE PAST 2 YEARS. RECENT FINDINGS: THE ANALYSIS OF NAFLD-ASSOCIATED GENETIC VARIANT EFFECTS ON THE WHOLE-TRANSCRIPTOME, INCLUDING QUANTITATIVE TRAIT LOCI (QTL) ASSOCIATED WITH GENE EXPRESSION (EQTL) OR SPLICING (SQTL) MAY EXPLAIN PLEIOTROPIC EFFECTS. FUNCTIONAL EXPERIMENTS ON NAFLD-EPIGENETICS, INCLUDING PROFILING OF LIVER CHROMATIN ACCESSIBILITY QUANTITATIVE TRAIT LOCI (CAQTL) SHOW CO-LOCALIZATION WITH NUMEROUS GENOME-WIDE ASSOCIATION STUDY SIGNALS LINKED TO METABOLIC AND CARDIOVASCULAR TRAITS. NOVEL STUDIES PROVIDE INSIGHTS INTO THE MODULATION OF THE HEPATIC TRANSCRIPTOME AND EPIGENOME BY TISSUE MICROBIOTAS. GENETIC VARIATION OF COMPONENTS OF THE LIVER CELLULAR RESPIRASOME MAY RESULT IN BROAD CELLULAR AND METABOLIC EFFECTS. MITOCHONDRIAL NONCODING RNAS MAY REGULATE LIVER INFLAMMATION AND FIBROGENESIS. RNA MODIFICATIONS AS N6-METHYLADENOSINE MAY EXPLAIN SEX-SPECIFIC DIFFERENCES IN LIVER GENE TRANSCRIPTION LINKED TO LIPID TRAITS. SUMMARY: THE LATEST DEVELOPMENTS IN THE FIELD OF NAFLD-GENOMICS CAN BE LEVERAGED FOR IDENTIFYING NOVEL DISEASE MECHANISMS AND THERAPEUTIC TARGETS THAT MAY PREVENT THE MORBIDITY AND MORTALITY ASSOCIATED WITH DISEASE PROGRESSION. VIDEO ABSTRACT: HTTP://LINKS.LWW.COM/COL/A23. 2022 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 2409 29 EPIGENETIC SCARRING OF EXHAUSTED T CELLS HINDERS MEMORY DIFFERENTIATION UPON ELIMINATING CHRONIC ANTIGENIC STIMULATION. EXHAUSTED CD8 T CELLS (T(EX)) ARE A DISTINCT STATE OF T CELL DIFFERENTIATION ASSOCIATED WITH FAILURE TO CLEAR CHRONIC VIRUSES AND CANCER. IMMUNOTHERAPIES SUCH AS PD-1 BLOCKADE CAN REINVIGORATE T(EX) CELLS, BUT REINVIGORATION IS NOT DURABLE. A MAJOR UNANSWERED QUESTION IS WHETHER T(EX) CELLS DIFFERENTIATE INTO FUNCTIONAL DURABLE MEMORY T CELLS (T(MEM)) UPON ANTIGEN CLEARANCE. HERE, USING A MOUSE MODEL, WE FOUND THAT UPON ELIMINATING CHRONIC ANTIGENIC STIMULATION, T(EX) CELLS PARTIALLY (RE)ACQUIRE PHENOTYPIC AND TRANSCRIPTIONAL FEATURES OF T(MEM) CELLS. THESE 'RECOVERING' T(EX) CELLS ORIGINATED FROM THE T CELL FACTOR (TCF-1(+)) T(EX) PROGENITOR SUBSET. NEVERTHELESS, THE RECALL CAPACITY OF THESE RECOVERING T(EX) CELLS REMAINED COMPROMISED AS COMPARED TO T(MEM) CELLS. CHROMATIN-ACCESSIBILITY PROFILING REVEALED A FAILURE TO RECOVER CORE MEMORY EPIGENETIC CIRCUITS AND MAINTENANCE OF A LARGELY EXHAUSTED OPEN CHROMATIN LANDSCAPE. THUS, DESPITE SOME PHENOTYPIC AND TRANSCRIPTIONAL RECOVERY UPON ANTIGEN CLEARANCE, EXHAUSTION LEAVES DURABLE EPIGENETIC SCARS CONSTRAINING FUTURE IMMUNE RESPONSES. THESE RESULTS SUPPORT EPIGENETIC REMODELING INTERVENTIONS FOR T(EX) CELL-TARGETED IMMUNOTHERAPIES. 2021 13 769 35 CD8 T CELL EXHAUSTION DURING CHRONIC VIRAL INFECTION AND CANCER. EXHAUSTED CD8 T (TEX) CELLS ARE A DISTINCT CELL LINEAGE THAT ARISE DURING CHRONIC INFECTIONS AND CANCERS IN ANIMAL MODELS AND HUMANS. TEX CELLS ARE CHARACTERIZED BY PROGRESSIVE LOSS OF EFFECTOR FUNCTIONS, HIGH AND SUSTAINED INHIBITORY RECEPTOR EXPRESSION, METABOLIC DYSREGULATION, POOR MEMORY RECALL AND HOMEOSTATIC SELF-RENEWAL, AND DISTINCT TRANSCRIPTIONAL AND EPIGENETIC PROGRAMS. THE ABILITY TO REINVIGORATE TEX CELLS THROUGH INHIBITORY RECEPTOR BLOCKADE, SUCH AS ALPHAPD-1, HIGHLIGHTS THE THERAPEUTIC POTENTIAL OF TARGETING THIS POPULATION. EMERGING INSIGHTS INTO THE MECHANISMS OF EXHAUSTION ARE INFORMING IMMUNOTHERAPIES FOR CANCER AND CHRONIC INFECTIONS. HOWEVER, LIKE OTHER IMMUNE CELLS, TEX CELLS ARE HETEROGENEOUS AND INCLUDE PROGENITOR AND TERMINAL SUBSETS WITH UNIQUE CHARACTERISTICS AND RESPONSES TO CHECKPOINT BLOCKADE. HERE, WE REVIEW OUR CURRENT UNDERSTANDING OF TEX CELL BIOLOGY, INCLUDING THE DEVELOPMENTAL PATHS, TRANSCRIPTIONAL AND EPIGENETIC FEATURES, AND CELL INTRINSIC AND EXTRINSIC FACTORS CONTRIBUTING TO EXHAUSTION AND HOW THIS KNOWLEDGE MAY INFORM THERAPEUTIC TARGETING OF TEX CELLS IN CHRONIC INFECTIONS, AUTOIMMUNITY, AND CANCER. 2019 14 568 30 BATF REGULATES PROGENITOR TO CYTOLYTIC EFFECTOR CD8(+) T CELL TRANSITION DURING CHRONIC VIRAL INFECTION. DURING CHRONIC VIRAL INFECTION, CD8(+) T CELLS DEVELOP INTO THREE MAJOR PHENOTYPICALLY AND FUNCTIONALLY DISTINCT SUBSETS: LY108(+)TCF-1(+) PROGENITORS, LY108(-)CX(3)CR1(-) TERMINALLY EXHAUSTED CELLS AND THE RECENTLY IDENTIFIED CX(3)CR1(+) CYTOTOXIC EFFECTOR CELLS. NEVERTHELESS, HOW CX(3)CR1(+) EFFECTOR CELL DIFFERENTIATION IS TRANSCRIPTIONALLY AND EPIGENETICALLY REGULATED REMAINS ELUSIVE. HERE, WE IDENTIFY DISTINCT GENE REGULATORY NETWORKS AND EPIGENETIC LANDSCAPES UNDERPINNING THE FORMATION OF THESE SUBSETS. NOTABLY, OUR DATA DEMONSTRATE THAT CX(3)CR1(+) EFFECTOR CELLS BEAR A STRIKING SIMILARITY TO SHORT-LIVED EFFECTOR CELLS DURING ACUTE INFECTION. GENETIC DELETION OF TBX21 SIGNIFICANTLY DIMINISHED FORMATION OF THE CX(3)CR1(+) SUBSET. IMPORTANTLY, WE FURTHER IDENTIFY A PREVIOUSLY UNAPPRECIATED ROLE FOR THE TRANSCRIPTION FACTOR BATF IN MAINTAINING A PERMISSIVE CHROMATIN STRUCTURE THAT ALLOWS THE TRANSITION FROM TCF-1(+) PROGENITORS TO CX(3)CR1(+) EFFECTOR CELLS. BATF DIRECTLY BOUND TO REGULATORY REGIONS NEAR TBX21 AND KLF2, MODULATING THEIR ENHANCER ACCESSIBILITY TO FACILITATE THE TRANSITION. THESE MECHANISTIC INSIGHTS CAN POTENTIALLY BE HARNESSED TO OVERCOME T CELL EXHAUSTION DURING CHRONIC INFECTION AND CANCER. 2021 15 4178 27 MEMORY T-CELL HETEROGENEITY AND TERMINOLOGY. IMMUNOLOGICAL MEMORY AND EXHAUSTION ARE FUNDAMENTAL FEATURES OF ADAPTIVE IMMUNITY. RECENT ADVANCES REVEAL INCREASING HETEROGENEITY AND DIVERSITY AMONG CD8 T-CELL SUBSETS, RESULTING IN NEW SUBSETS TO ANNOTATE AND UNDERSTAND. HERE, WE REVIEW OUR CURRENT KNOWLEDGE OF DIFFERENTIATION AND MAINTENANCE OF MEMORY AND EXHAUSTED CD8 T CELLS, INCLUDING PHENOTYPIC CLASSIFICATION, DEVELOPMENTAL PATHS, TRANSCRIPTIONAL AND EPIGENETIC FEATURES, AND CELL INTRINSIC AND EXTRINSIC FACTORS. ADDITIONALLY, WE USE THIS OUTLINE TO DISCUSS THE NOMENCLATURE OF EFFECTOR, MEMORY, AND EXHAUSTED CD8 T CELLS. FINALLY, WE DISCUSS HOW NEW FINDINGS ABOUT THESE CELL TYPES MAY IMPACT THE THERAPEUTIC EFFICACY AND DEVELOPMENT OF IMMUNOTHERAPIES TARGETING EFFECTOR, MEMORY, AND/OR EXHAUSTED CD8 T CELLS IN CHRONIC INFECTIONS AND CANCER. 2021 16 790 27 CELLULAR AND MOLECULAR MECHANISMS OF CD8(+) T CELL DIFFERENTIATION, DYSFUNCTION AND EXHAUSTION. T CELLS FOLLOW A TRIPHASIC DISTINCT PATHWAY OF ACTIVATION, PROLIFERATION AND DIFFERENTIATION BEFORE BECOMING FUNCTIONALLY AND PHENOTYPICALLY "EXHAUSTED" IN SETTINGS OF CHRONIC INFECTION, AUTOIMMUNITY AND IN CANCER. EXHAUSTED T CELLS PROGRESSIVELY LOSE CANONICAL EFFECTOR FUNCTIONS, EXHIBIT ALTERED TRANSCRIPTIONAL NETWORKS AND EPIGENETIC SIGNATURES AND GAIN CONSTITUTIVE EXPRESSION OF A BROAD COINHIBITORY RECEPTOR SUITE. THIS REVIEW OUTLINES RECENT ADVANCES IN OUR UNDERSTANDING OF EXHAUSTED T CELL BIOLOGY AND EXAMINES CELLULAR AND MOLECULAR MECHANISMS BY WHICH A STATE OF DYSFUNCTION OR EXHAUSTION IS ESTABLISHED, AND MECHANISMS BY WHICH EXHAUSTED T CELLS MAY STILL CONTRIBUTE TO PATHOGEN OR TUMOUR CONTROL. FURTHER, THIS REVIEW DESCRIBES OUR UNDERSTANDING OF EXHAUSTED T CELL HETEROGENEITY AND OUTLINES THE MECHANISMS BY WHICH CHECKPOINT BLOCKADE DIFFERENTIALLY ENGAGES EXHAUSTED T CELL SUBSETS TO OVERCOME EXHAUSTION AND RECOVER T CELL FUNCTION. 2020 17 2367 27 EPIGENETIC REGULATION OF T CELL EXHAUSTION. CHRONIC ANTIGEN STIMULATION DURING VIRAL INFECTIONS AND CANCER CAN LEAD TO T CELL EXHAUSTION, WHICH IS CHARACTERIZED BY REDUCED EFFECTOR FUNCTION AND PROLIFERATION, AND THE EXPRESSION OF INHIBITORY IMMUNE CHECKPOINT RECEPTORS. RECENT STUDIES HAVE DEMONSTRATED THAT T CELL EXHAUSTION RESULTS IN WHOLESCALE EPIGENETIC REMODELING THAT CONFERS PHENOTYPIC STABILITY TO THESE CELLS AND PREVENTS T CELL REINVIGORATION BY CHECKPOINT BLOCKADE. HERE, WE REVIEW FOUNDATIONAL TECHNOLOGIES TO PROFILE THE EPIGENOME AT MULTIPLE SCALES, INCLUDING MAPPING THE LOCATIONS OF TRANSCRIPTION FACTORS AND HISTONE MODIFICATIONS, DNA METHYLATION AND THREE-DIMENSIONAL GENOME CONFORMATION. WE DISCUSS HOW THESE TECHNOLOGIES HAVE ELUCIDATED THE DEVELOPMENT AND EPIGENETIC REGULATION OF EXHAUSTED T CELLS AND FUNCTIONAL IMPLICATIONS ACROSS VIRAL INFECTION, CANCER, AUTOIMMUNITY AND ENGINEERED T CELL THERAPIES. FINALLY, WE COVER EMERGING MULTI-OMIC AND GENOME ENGINEERING TECHNOLOGIES, CURRENT AND UPCOMING OPPORTUNITIES TO APPLY THESE TO T CELL EXHAUSTION, AND THERAPEUTIC OPPORTUNITIES FOR T CELL ENGINEERING IN THE CLINIC. 2022 18 6530 36 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 19 5620 34 SCHRODINGER'S T CELLS: MOLECULAR INSIGHTS INTO STEMNESS AND EXHAUSTION. T CELL STEMNESS AND EXHAUSTION COEXIST AS TWO KEY CONTRASTING PHENOMENA DURING CHRONIC ANTIGEN STIMULATION, SUCH AS INFECTION, TRANSPLANT, CANCER, AND AUTOIMMUNITY. T CELL EXHAUSTION REFERS TO THE PROGRESSIVE LOSS OF EFFECTOR FUNCTION CAUSED BY CHRONIC ANTIGEN EXPOSURE. EXHAUSTED T (T(EX)) CELLS HIGHLY EXPRESS MULTIPLE INHIBITORY RECEPTORS AND EXHIBIT SEVERE DEFECTS IN CELL PROLIFERATION AND CYTOKINE PRODUCTION. THE TERM T CELL STEMNESS DESCRIBES THE STEM CELL-LIKE BEHAVIORS OF T CELLS, INCLUDING SELF-RENEWAL, MULTIPOTENCY, AND FUNCTIONAL PERSISTENCE. IT IS WELL ACCEPTED THAT NAIVE AND SOME MEMORY T CELL SUBSETS HAVE STEM CELL-LIKE PROPERTIES. WHEN INVESTIGATING THE EXHAUSTIVE DIFFERENTIATION OF T CELLS IN CHRONIC INFECTION AND CANCER, RECENT STUDIES HIGHLIGHTED THE STEMNESS OF "PRECURSORS OF EXHAUSTED" T (T(PEX)) CELLS PRIOR TO THEIR TERMINAL DIFFERENTIATION TO T(EX) CELLS. CLINICALLY SUCCESSFUL CHECKPOINT BLOCKADES FOR CANCER TREATMENT APPEAR TO INVIGORATE ANTITUMOR T(PEX) CELLS BUT NOT T(EX) CELLS. HERE WE DISCUSS THE TRANSCRIPTIONAL AND EPIGENETIC REGULATIONS OF T CELL STEMNESS AND EXHAUSTION, WITH A FOCUS ON HOW SYSTEMS IMMUNOLOGY WAS AND WILL BE UTILIZED TO DEFINE THE MOLECULAR BASIS UNDERLYING THE TRANSITION OF T(PEX) TO T(EX) CELLS. WE SUGGEST A "STEPWISE MODEL" OF T CELL STEMNESS AND EXHAUSTION, IN WHICH LOSS OF STEMNESS AND EXHAUSTION PROGRESSION ARE GRADUAL MULTI-STEP PROCESSES. WE PROVIDE PERSPECTIVES ON THE RESEARCH NEEDED TO DEFINE T CELL STEMNESS AND EXHAUSTION IN THE TRANSPLANTATION SETTING, IN WHICH ALLOGENIC T CELLS ARE ALSO CHRONICALLY EXPOSED TO ALLOANTIGENS. A BETTER UNDERSTANDING OF T CELL STEMNESS AND EXHAUSTION WILL SHED LIGHT ON DEVELOPING NOVEL STRATEGIES FOR IMMUNOTHERAPIES. 2021 20 438 29 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