1 6522 114 TRANSCRIPTIONAL AND EPIGENETIC REGULATION OF T CELL HYPORESPONSIVENESS. NAIVE CD8(+) T CELLS DIFFERENTIATE INTO EFFECTOR AND MEMORY CYTOLYTIC T CELLS (CTLS) DURING AN ACUTE INFECTION. IN CONTRAST, IN SCENARIOS OF PERSISTENT ANTIGEN STIMULATION, SUCH AS CHRONIC INFECTIONS AND CANCER, ANTIGEN-SPECIFIC CTLS SHOW A GRADUAL DECREASE IN EFFECTOR FUNCTION, A PHENOMENON THAT HAS BEEN TERMED CD8(+) T CELL "EXHAUSTION" OR "DYSFUNCTION." ANOTHER HYPORESPONSIVE STATE, TERMED "ANERGY", IS OBSERVED WHEN T CELLS ARE ACTIVATED IN THE ABSENCE OF POSITIVE COSTIMULATORY SIGNALS. AMONG THE MANY NEGATIVE REGULATORS INDUCED IN HYPORESPONSIVE T CELLS ARE INHIBITORY CELL-SURFACE RECEPTORS, SUCH AS PD-1, LAG-3, CTLA-4, AND TIM-3; "CHECKPOINT BLOCKADE" THERAPIES THAT INVOLVE TREATMENT OF PATIENTS WITH CANCER WITH BLOCKING ANTIBODIES TO THOSE RECEPTORS SHOW CONSIDERABLE PROMISE IN THE CLINIC BECAUSE THE BLOCKING ANTIBODIES CAN MITIGATE HYPORESPONSIVENESS AND PROMOTE TUMOR REJECTION. IN THIS REVIEW, WE DESCRIBE RECENT ADVANCES IN OUR MOLECULAR UNDERSTANDING OF THESE HYPORESPONSIVE STATES. WE REVIEW EVIDENCE FOR THE INVOLVEMENT OF DIVERSE TRANSCRIPTION FACTORS, METABOLIC PROGRAMS, AND CHROMATIN ACCESSIBILITY CHANGES IN HYPORESPONSIVE T CELLS, AND WE DISCUSS HOW CHECKPOINT BLOCKADE THERAPIES AFFECT THE MOLECULAR PROGRAM OF CD8(+) T CELL EXHAUSTION. 2017 2 5477 34 RESTORING THE FUNCTIONAL IMMUNOGENICITY OF CHRONIC LYMPHOCYTIC LEUKEMIA USING EPIGENETIC MODIFIERS. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS A MALIGNANCY ARISING FROM IMMUNE CELLS (B-LYMPHOCYTES) ENDOWED WITH INTRINSIC ANTIGEN-PRESENTING CAPABILITIES. SUCH A FUNCTION HOWEVER IS LOST DURING MALIGNANT TRANSFORMATION AND CLL CELLS ARE WELL KNOWN FOR THEIR INABILITY TO PROCESS AND PRESENT ANTIGENS TO THE T-CELL ARM OF THE IMMUNE SYSTEM. INSTEAD, MALIGNANT CLL CELLS ELICIT A VAST ARRAY OF IMMUNE REGULATORY MECHANISMS CONDUCIVE TO T-CELL DYSFUNCTION AND IMMUNOSUPPRESSION. PREVIOUSLY, WE HAVE SHOWN THAT TREATMENT OF CLL CELLS WITH THE DEMETHYLATING AGENT 5-AZA-2'-DEOXYCYTIDINE UNLEASHED TARGET ANTIGEN EXPRESSION. HERE WE SHOW FOR THE FIRST TIME THAT COMBINING TWO EPIGENETIC MODIFIERS, 5-AZA-2'-DEOXYCYTIDINE AND THE HISTONE DEACETYLASE INHIBITOR LAQ824 EFFECTIVELY RESTORES THE IMMUNOGENICITY OF CLL CELL LINES AS WELL AS PRIMARY CELLS OBTAINED FROM CLL PATIENTS. INDEED, SUCH A COMBINATION INDUCES THE EXPRESSION OF NOVEL AND HIGHLY ANTIGENIC CANCER-TESTIS ANTIGENS (CTAS) AND COSTIMULATORY MOLECULES. THESE CHANGES FACILITATE THE FORMATION OF ROBUST SUPRAMOLECULAR ACTIVATION COMPLEXES (SMAC) BETWEEN CLL CELLS AND RESPONDER T-CELLS LEADING TO INTRACELLULAR SIGNALING, LYTIC GRANULE MOBILIZATION, AND POLARIZATION OF FUNCTIONAL AND RELEVANT T-CELL RESPONSES. THIS CASCADE OF T-CELL ACTIVATING EVENTS TRIGGERED BY CLL CELLS WITH RESTORED APC FUNCTION, POINTS TO COMBINED EPIGENETIC MODIFIER TREATMENT AS A POTENTIAL IMMUNOTHERAPEUTIC STRATEGY FOR CLL PATIENTS. 2011 3 744 41 CANNABINOID WIN55,212-2 REPROGRAMS MONOCYTES AND MACROPHAGES TO INHIBIT LPS-INDUCED INFLAMMATION. INTRODUCTION: CHRONIC OR UNCONTROLLED ACTIVATION OF MYELOID CELLS INCLUDING MONOCYTES, MACROPHAGES AND DENDRITIC CELLS (DCS) IS A HALLMARK OF IMMUNE-MEDIATED INFLAMMATORY DISORDERS. THERE IS AN URGENT NEED FOR THE DEVELOPMENT OF NOVEL DRUGS WITH THE CAPACITY TO IMPAIR INNATE IMMUNE CELL OVERACTIVATION UNDER INFLAMMATORY CONDITIONS. COMPELLING EVIDENCE POINTED OUT CANNABINOIDS AS POTENTIAL THERAPEUTIC TOOLS WITH ANTI-INFLAMMATORY AND IMMUNOMODULATORY CAPACITY. WIN55,212-2, A NON-SELECTIVE SYNTHETIC CANNABINOID AGONIST, DISPLAYS PROTECTIVE EFFECTS IN SEVERAL INFLAMMATORY CONDITIONS BY MECHANISMS PARTIALLY DEPENDING ON THE GENERATION OF TOLEROGENIC DCS ABLE TO INDUCE FUNCTIONAL REGULATORY T CELLS (TREGS). HOWEVER, ITS IMMUNOMODULATORY CAPACITY ON OTHER MYELOID CELLS SUCH AS MONOCYTES AND MACROPHAGES REMAINS INCOMPLETELY UNDERSTOOD. METHODS: HUMAN MONOCYTE-DERIVED DCS (HMODCS) WERE DIFFERENTIATED IN THE ABSENCE (CONVENTIONAL HMODCS) OR PRESENCE OF WIN55,212-2 (WIN-HMODCS). CELLS WERE STIMULATED WITH LPS, COCULTURED WITH NAIVE T LYMPHOCYTES AND THEIR CYTOKINE PRODUCTION AND ABILITY TO INDUCE T CELL RESPONSES WERE ANALYSED BY ELISA OR FLOW CYTOMETRY. TO EVALUATE THE EFFECT OF WIN55,212-2 IN MACROPHAGE POLARIZATION, HUMAN AND MURINE MACROPHAGES WERE ACTIVATED WITH LPS OR LPS/IFNGAMMA, IN THE PRESENCE OR ABSENCE OF THE CANNABINOID. CYTOKINE, COSTIMULATORY MOLECULES AND INFLAMMASOME MARKERS WERE ASSAYED. METABOLIC AND CHROMATIN IMMUNOPRECIPITATION ASSAYS WERE ALSO PERFORMED. FINALLY, THE PROTECTIVE CAPACITY OF WIN55,212-2 WAS STUDIED IN VIVO IN BALB/C MICE AFTER INTRAPERITONEAL INJECTION WITH LPS. RESULTS: WE SHOW FOR THE FIRST TIME THAT THE DIFFERENTIATION OF HMODCS IN THE PRESENCE OF WIN55,212-2 GENERATES TOLEROGENIC WIN-HMODCS THAT ARE LESS RESPONSIVE TO LPS STIMULATION AND ABLE TO PRIME TREGS. WIN55,212-2 ALSO IMPAIRS THE PRO-INFLAMMATORY POLARIZATION OF HUMAN MACROPHAGES BY INHIBITING CYTOKINE PRODUCTION, INFLAMMASOME ACTIVATION AND RESCUING MACROPHAGES FROM PYROPTOTIC CELL DEATH. MECHANISTICALLY, WIN55,212-2 INDUCED A METABOLIC AND EPIGENETIC SHIFT IN MACROPHAGES BY DECREASING LPS-INDUCED MTORC1 SIGNALING, COMMITMENT TO GLYCOLYSIS AND ACTIVE HISTONE MARKS IN PRO-INFLAMMATORY CYTOKINE PROMOTERS. WE CONFIRMED THESE DATA IN EX VIVO LPS-STIMULATED PERITONEAL MACROPHAGES (PMPHIS), WHICH WERE ALSO SUPPORTED BY THE IN VIVO ANTI-INFLAMMATORY CAPACITY OF WIN55,212-2 IN A LPS-INDUCED SEPSIS MOUSE MODEL. CONCLUSION: OVERALL, WE SHED LIGHT INTO THE MOLECULAR MECHANISMS BY WHICH CANNABINOIDS EXERT ANTI-INFLAMMATORY PROPERTIES IN MYELOID CELLS, WHICH MIGHT WELL CONTRIBUTE TO THE FUTURE RATIONAL DESIGN OF NOVEL THERAPEUTIC STRATEGIES FOR INFLAMMATORY DISORDERS. 2023 4 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 5 771 47 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 6 6481 38 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 7 5902 28 T-HELPER 17 CELL POLARIZATION IN PULMONARY ARTERIAL HYPERTENSION. BACKGROUND: INFLAMMATION MAY CONTRIBUTE TO THE PATHOBIOLOGY OF PULMONARY ARTERIAL HYPERTENSION (PAH). DECIPHERING THE PAH FINGERPRINT ON THE INFLAMMATION ORCHESTRATED BY DENDRITIC CELLS (DCS) AND T CELLS, KEY DRIVER AND EFFECTOR CELLS, RESPECTIVELY, OF THE IMMUNE SYSTEM, MAY ALLOW THE IDENTIFICATION OF IMMUNOPATHOLOGIC APPROACHES TO PAH MANAGEMENT. METHODS: USING FLOW CYTOMETRY, WE PERFORMED IMMUNOPHENOTYPING OF MONOCYTE-DERIVED DCS (MODCS) AND CIRCULATING LYMPHOCYTES FROM PATIENTS WITH IDIOPATHIC PAH AND CONTROL SUBJECTS. WITH THE SAME TECHNIQUE, WE PERFORMED CYTOKINE PROFILING OF BOTH POPULATIONS FOLLOWING STIMULATION, COCULTURE, OR BOTH. WE TESTED THE IMMUNOMODULATORY EFFECTS OF A GLUCOCORTICOID (DEXAMETHASONE [DEX]) ON THIS IMMUNOPHENOTYPE AND CYTOKINE PROFILE. USING AN EPIGENETIC APPROACH, WE CONFIRMED THE IMMUNE POLARIZATION IN BLOOD DNA OF PATIENTS WITH PAH. RESULTS: THE PROFILE OF MEMBRANE COSTIMULATORY MOLECULES OF PAH MODCS WAS SIMILAR TO THAT OF CONTROL SUBJECTS. HOWEVER, PAH MODCS RETAINED HIGHER LEVELS OF THE T-CELL ACTIVATING MOLECULES CD86 AND CD40 AFTER DEX PRETREATMENT THAN DID CONTROL MODCS. THIS WAS ASSOCIATED WITH AN INCREASED EXPRESSION OF IL-12P40 AND A REDUCED MIGRATION TOWARD CHEMOKINE (C-C MOTIF) LIGAND 21. MOREOVER, BOTH WITH AND WITHOUT DEX, PAH MODCS INDUCED A HIGHER ACTIVATION AND PROLIFERATION OF CD4+ T CELLS, ASSOCIATED WITH A REDUCED EXPRESSION OF IL-4 (T HELPER 2 RESPONSE) AND A HIGHER EXPRESSION OF IL-17 (T HELPER 17 RESPONSE). PURIFIED PAH CD4+ T CELLS EXPRESSED A HIGHER LEVEL OF IL-17 AFTER ACTIVATION THAN DID THOSE OF CONTROL SUBJECTS. LASTLY, THERE WAS SIGNIFICANT HYPOMETHYLATION OF THE IL-17 PROMOTER IN THE PAH BLOOD DNA AS COMPARED WITH THE CONTROL BLOOD. CONCLUSIONS: WE HAVE HIGHLIGHTED T HELPER 17 CELL IMMUNE POLARIZATION IN PATIENTS WITH PAH, AS HAS BEEN PREVIOUSLY DEMONSTRATED IN OTHER CHRONIC INFLAMMATORY AND AUTOIMMUNE CONDITIONS. 2015 8 991 42 CHRONIC STIMULATION DRIVES HUMAN NK CELL DYSFUNCTION AND EPIGENETIC REPROGRAMING. A POPULATION OF NATURAL KILLER (NK) CELLS EXPRESSING THE ACTIVATING RECEPTOR NKG2C AND THE MATURATION MARKER CD57 EXPANDS IN RESPONSE TO HUMAN CYTOMEGALOVIRUS (HCMV) INFECTION. CD3-CD56DIMCD57+NKG2C+ NK CELLS ARE SIMILAR TO CD8+ MEMORY T CELLS WITH RAPID AND ROBUST EFFECTOR FUNCTION UPON RE-STIMULATION, PERSISTENCE, AND EPIGENETIC REMODELING OF THE IFNG LOCUS. CHRONIC ANTIGEN STIMULATION DRIVES CD8+ MEMORY T CELL PROLIFERATION WHILE ALSO INDUCING GENOME-WIDE EPIGENETIC REPROGRAMING AND DYSFUNCTION. WE HYPOTHESIZED THAT CHRONIC STIMULATION COULD SIMILARLY INDUCE EPIGENETIC REPROGRAMING AND DYSFUNCTION IN NK CELLS. HERE WE SHOW THAT CHRONIC STIMULATION OF ADAPTIVE NK CELLS THROUGH NKG2C USING PLATE-BOUND AGONISTIC ANTIBODIES IN COMBINATION WITH IL-15 DROVE ROBUST PROLIFERATION AND ACTIVATION OF CD3-CD56DIMCD57+NKG2C+ NK CELLS WHILE SIMULTANEOUSLY INDUCING HIGH EXPRESSION OF THE CHECKPOINT INHIBITORY RECEPTORS LAG-3 AND PD-1. MARKED INDUCTION OF CHECKPOINT INHIBITORY RECEPTORS WAS ALSO OBSERVED ON THE SURFACE OF ADAPTIVE NK CELLS CO-CULTURED WITH HCMV-INFECTED ENDOTHELIAL CELLS. CHRONICALLY STIMULATED ADAPTIVE NK CELLS WERE DYSFUNCTIONAL WHEN CHALLENGED WITH TUMOR TARGETS. THESE CELLS EXHIBITED A PATTERN OF EPIGENETIC REPROGRAMING, WITH GENOME-WIDE ALTERATIONS IN DNA METHYLATION. OUR STUDY HAS IMPORTANT IMPLICATIONS FOR CANCER IMMUNOTHERAPY AND SUGGEST THAT EXHAUSTED NK CELLS COULD BE TARGETED WITH INHIBITORY CHECKPOINT RECEPTOR BLOCKADE. 2019 9 3288 42 HIERARCHICAL TRANSCRIPTIONAL NETWORK GOVERNING HETEROGENEOUS T CELL EXHAUSTION AND ITS IMPLICATIONS FOR IMMUNE CHECKPOINT BLOCKADE. THE FUNDAMENTAL PRINCIPLE OF IMMUNE CHECKPOINT BLOCKADE (ICB) IS TO PROTECT TUMOR-INFILTRATING T CELLS FROM BEING EXHAUSTED. DESPITE THE REMARKABLE SUCCESS ACHIEVED BY ICB TREATMENT, ONLY A SMALL GROUP OF PATIENTS BENEFIT FROM IT. CHARACTERIZED BY A HYPOFUNCTIONAL STATE WITH THE EXPRESSION OF MULTIPLE INHIBITORY RECEPTORS, EXHAUSTED T (TEX) CELLS ARE A MAJOR OBSTACLE IN IMPROVING ICB. T CELL EXHAUSTION IS A PROGRESSIVE PROCESS WHICH ADAPTS TO PERSISTENT ANTIGEN STIMULATION IN CHRONIC INFECTIONS AND CANCERS. IN THIS REVIEW, WE ELUCIDATE THE HETEROGENEITY OF TEX CELLS AND OFFER NEW INSIGHTS INTO THE HIERARCHICAL TRANSCRIPTIONAL REGULATION OF T CELL EXHAUSTION. FACTORS AND SIGNALING PATHWAYS THAT INDUCE AND PROMOTE EXHAUSTION ARE ALSO SUMMARIZED. MOREOVER, WE REVIEW THE EPIGENETIC AND METABOLIC ALTERATIONS OF TEX CELLS AND DISCUSS HOW PD-1 SIGNALING AFFECTS THE BALANCE BETWEEN T CELL ACTIVATION AND EXHAUSTION, AIMING TO PROVIDE MORE THERAPEUTIC TARGETS FOR APPLICATIONS OF COMBINATIONAL IMMUNOTHERAPIES. 2023 10 559 28 BACH2 ENFORCES THE TRANSCRIPTIONAL AND EPIGENETIC PROGRAMS OF STEM-LIKE CD8(+) T CELLS. DURING CHRONIC INFECTION AND CANCER, A SELF-RENEWING CD8(+) T CELL SUBSET MAINTAINS LONG-TERM IMMUNITY AND IS CRITICAL TO THE EFFECTIVENESS OF IMMUNOTHERAPY. THESE STEM-LIKE CD8(+) T CELLS DIVERGE FROM OTHER CD8(+) SUBSETS EARLY AFTER CHRONIC VIRAL INFECTION. HOWEVER, PATHWAYS GUARDING STEM-LIKE CD8(+) T CELLS AGAINST TERMINAL EXHAUSTION REMAIN UNCLEAR. HERE, WE SHOW THAT THE GENE ENCODING TRANSCRIPTIONAL REPRESSOR BACH2 IS TRANSCRIPTIONALLY AND EPIGENETICALLY ACTIVE IN STEM-LIKE CD8(+) T CELLS BUT NOT TERMINALLY EXHAUSTED CELLS EARLY AFTER INFECTION. BACH2 OVEREXPRESSION ENFORCED STEM-LIKE CELL FATE, WHEREAS BACH2 DEFICIENCY IMPAIRED STEM-LIKE CD8(+) T CELL DIFFERENTIATION. SINGLE-CELL TRANSCRIPTOMIC AND EPIGENOMIC APPROACHES REVEALED THAT BACH2 ESTABLISHED THE TRANSCRIPTIONAL AND EPIGENETIC PROGRAMS OF STEM-LIKE CD8(+) T CELLS. IN ADDITION, BACH2 SUPPRESSED THE MOLECULAR PROGRAM DRIVING TERMINAL EXHAUSTION THROUGH TRANSCRIPTIONAL REPRESSION AND EPIGENETIC SILENCING. THUS, OUR STUDY REVEALS A NEW PATHWAY THAT ENFORCES COMMITMENT TO STEM-LIKE CD8(+) LINEAGE AND PREVENTS AN ALTERNATIVE TERMINALLY EXHAUSTED CELL FATE. 2021 11 6851 42 [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 12 5704 29 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 13 6121 28 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 14 1759 33 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 15 2879 49 FUNDAMENTALS TO THERAPEUTICS: EPIGENETIC MODULATION OF CD8(+) T CELL EXHAUSTION IN THE TUMOR MICROENVIRONMENT. IN THE SETTING OF CHRONIC ANTIGEN EXPOSURE IN THE TUMOR MICROENVIRONMENT (TME), CYTOTOXIC CD8(+) T CELLS (CTLS) LOSE THEIR IMMUNE SURVEILLANCE CAPABILITIES AND ABILITY TO CLEAR TUMOR CELLS AS A RESULT OF THEIR DIFFERENTIATION INTO TERMINALLY EXHAUSTED CD8(+) T CELLS. IMMUNE CHECKPOINT BLOCKADE (ICB) THERAPIES REINVIGORATE EXHAUSTED CD8(+) T CELLS BY TARGETING SPECIFIC INHIBITORY RECEPTORS, THUS PROMOTING THEIR CYTOLYTIC ACTIVITY TOWARDS TUMOR CELLS. DESPITE EXCITING RESULTS WITH ICB THERAPIES, MANY PATIENTS WITH SOLID TUMORS STILL FAIL TO RESPOND TO SUCH THERAPIES AND PATIENTS WHO INITIALLY RESPOND CAN DEVELOP RESISTANCE. RECENTLY, THROUGH NEW SEQUENCING TECHNOLOGIES SUCH AS THE ASSAY FOR TRANSPOSASE-ACCESSIBLE CHROMATIN WITH SEQUENCING (ATAC-SEQ), EPIGENETICS HAS BEEN APPRECIATED AS A CONTRIBUTING FACTOR THAT ENFORCES T CELL DIFFERENTIATION TOWARD EXHAUSTION IN THE TME. IMPORTANTLY, SPECIFIC EPIGENETIC ALTERATIONS AND EPIGENETIC FACTORS HAVE BEEN FOUND TO CONTROL CD8(+) T CELL EXHAUSTION PHENOTYPES. IN THIS REVIEW, WE WILL EXPLAIN THE BACKGROUND OF T CELL DIFFERENTIATION AND VARIOUS EXHAUSTION STATES AND DISCUSS HOW EPIGENETICS PLAY AN IMPORTANT ROLE IN THESE PROCESSES. THEN WE WILL OUTLINE SPECIFIC EPIGENETIC CHANGES AND CERTAIN EPIGENETIC AND TRANSCRIPTION FACTORS THAT ARE KNOWN TO CONTRIBUTE TO CD8(+) T CELL EXHAUSTION. WE WILL ALSO DISCUSS THE MOST RECENT METHODOLOGIES THAT ARE USED TO STUDY AND DISCOVER SUCH EPIGENETIC MODULATIONS. FINALLY, WE WILL EXPLAIN HOW EPIGENETIC REPROGRAMMING IS A PROMISING APPROACH THAT MIGHT FACILITATE THE DEVELOPMENT OF NOVEL EXHAUSTED T CELL-TARGETING IMMUNOTHERAPIES. 2022 16 1007 41 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 5223 44 PRIMARY MURINE CD4+ T CELLS FAIL TO ACQUIRE THE ABILITY TO PRODUCE EFFECTOR CYTOKINES WHEN ACTIVE RAS IS PRESENT DURING TH1/TH2 DIFFERENTIATION. CONSTITUTIVE RAS SIGNALING HAS BEEN SHOWN TO AUGMENT IL-2 PRODUCTION, REVERSE ANERGY, AND FUNCTIONALLY REPLACE MANY ASPECTS OF CD28 CO-STIMULATION IN CD4+ T CELLS. THESE DATA RAISE THE POSSIBILITY THAT INTRODUCTION OF ACTIVE RAS INTO PRIMARY T CELLS MIGHT RESULT IN IMPROVED FUNCTIONALITY IN PATHOLOGIC SITUATIONS OF T CELL DYSFUNCTION, SUCH AS CANCER OR CHRONIC VIRAL INFECTION. TO TEST THE BIOLOGIC EFFECTS OF ACTIVE RAS IN PRIMARY T CELLS, CD4+ T CELLS FROM COXSACKIE-ADENOVIRUS RECEPTOR TRANSGENIC MICE WERE TRANSDUCED WITH AN ADENOVIRUS ENCODING ACTIVE RAS. AS EXPECTED, ACTIVE RAS AUGMENTED IL-2 PRODUCTION IN NAIVE CD4+ T CELLS. HOWEVER, WHEN CELLS WERE CULTURED FOR 4 DAYS UNDER CONDITIONS TO PROMOTE EFFECTOR CELL DIFFERENTIATION, ACTIVE RAS INHIBITED THE ABILITY OF CD4+ T CELLS TO ACQUIRE A TH1 OR TH2 EFFECTOR CYTOKINE PROFILE. THIS DIFFERENTIATION DEFECT WAS NOT DUE TO DEFICIENT STAT4 OR STAT6 ACTIVATION BY IL-12 OR IL-4, RESPECTIVELY, NOR WAS IT ASSOCIATED WITH DEFICIENT INDUCTION OF T-BET AND GATA-3 EXPRESSION. IMPAIRED EFFECTOR CYTOKINE PRODUCTION IN ACTIVE RAS-TRANSDUCED CELLS WAS ASSOCIATED WITH DEFICIENT DEMETHYLATION OF THE IL-4 GENE LOCUS. OUR RESULTS INDICATE THAT, DESPITE AUGMENTING ACUTE ACTIVATION OF NAIVE T CELLS, CONSTITUTIVE RAS SIGNALING INHIBITS THE ABILITY OF CD4+ T CELLS TO PROPERLY DIFFERENTIATE INTO TH1/TH2 EFFECTOR CYTOKINE-PRODUCING CELLS, IN PART BY INTERFERING WITH EPIGENETIC MODIFICATION OF EFFECTOR GENE LOCI. ALTERNATIVE STRATEGIES TO POTENTIATE RAS PATHWAY SIGNALING IN T CELLS IN A MORE REGULATED FASHION SHOULD BE CONSIDERED AS A THERAPEUTIC APPROACH TO IMPROVE IMMUNE RESPONSES IN VIVO. 2014 18 590 39 BET BROMODOMAIN PROTEIN INHIBITION REVERSES CHIMERIC ANTIGEN RECEPTOR EXTINCTION AND REINVIGORATES EXHAUSTED T CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA. CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS HAVE INDUCED REMARKABLE ANTITUMOR RESPONSES IN B CELL MALIGNANCIES. SOME PATIENTS DO NOT RESPOND BECAUSE OF T CELL DEFICIENCIES THAT HAMPER THE EXPANSION, PERSISTENCE, AND EFFECTOR FUNCTION OF THESE CELLS. WE USED LONGITUDINAL IMMUNE PROFILING TO IDENTIFY PHENOTYPIC AND PHARMACODYNAMIC CHANGES IN CD19-DIRECTED CAR T CELLS IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). CAR EXPRESSION MAINTENANCE WAS ALSO INVESTIGATED BECAUSE THIS CAN AFFECT RESPONSE DURABILITY. CAR T CELL FAILURE WAS ACCOMPANIED BY PREEXISTING T CELL-INTRINSIC DEFECTS OR DYSFUNCTION ACQUIRED AFTER INFUSION. IN A SMALL SUBSET OF PATIENTS, CAR SILENCING WAS OBSERVED COINCIDENT WITH LEUKEMIA RELAPSE. USING A SMALL MOLECULE INHIBITOR, WE DEMONSTRATED THAT THE BROMODOMAIN AND EXTRA-TERMINAL (BET) FAMILY OF CHROMATIN ADAPTERS PLAYS A ROLE IN DOWNREGULATING CAR EXPRESSION. BET PROTEIN BLOCKADE ALSO AMELIORATED CAR T CELL EXHAUSTION AS MANIFESTED BY INHIBITORY RECEPTOR REDUCTION, ENHANCED METABOLIC FITNESS, INCREASED PROLIFERATIVE CAPACITY, AND ENRICHED TRANSCRIPTOMIC SIGNATURES OF T CELL REINVIGORATION. BET INHIBITION DECREASED LEVELS OF THE TET2 METHYLCYTOSINE DIOXYGENASE, AND FORCED EXPRESSION OF THE TET2 CATALYTIC DOMAIN ELIMINATED THE POTENCY-ENHANCING EFFECTS OF BET PROTEIN TARGETING IN CAR T CELLS, PROVIDING A MECHANISM LINKING BET PROTEINS AND T CELL DYSFUNCTION. THUS, MODULATING BET EPIGENETIC READERS MAY IMPROVE THE EFFICACY OF CELL-BASED IMMUNOTHERAPIES. 2021 19 1313 40 DELETING DNMT3A IN CAR T CELLS PREVENTS EXHAUSTION AND ENHANCES ANTITUMOR ACTIVITY. CHIMERIC ANTIGEN RECEPTOR (CAR) T CELL THERAPY IS REVOLUTIONIZING CANCER IMMUNOTHERAPY FOR PATIENTS WITH B CELL MALIGNANCIES AND IS NOW BEING DEVELOPED FOR SOLID TUMORS AND CHRONIC VIRAL INFECTIONS. ALTHOUGH CLINICAL TRIALS HAVE DEMONSTRATED THE CURATIVE POTENTIAL OF CAR T CELL THERAPY, A SUBSTANTIAL AND WELL-ESTABLISHED LIMITATION IS THE HEIGHTENED CONTRACTION AND TRANSIENT PERSISTENCE OF CAR T CELLS DURING PROLONGED ANTIGEN EXPOSURE. THE UNDERLYING MECHANISM(S) FOR THIS DYSFUNCTIONAL STATE, OFTEN TERMED CAR T CELL EXHAUSTION, REMAINS POORLY DEFINED. HERE, WE REPORT THAT EXHAUSTION OF HUMAN CAR T CELLS OCCURS THROUGH AN EPIGENETIC REPRESSION OF THE T CELL'S MULTIPOTENT DEVELOPMENTAL POTENTIAL. DELETION OF THE DE NOVO DNA METHYLTRANSFERASE 3 ALPHA (DNMT3A) IN T CELLS EXPRESSING FIRST- OR SECOND-GENERATION CARS UNIVERSALLY PRESERVED THE CELLS' ABILITY TO PROLIFERATE AND MOUNT AN ANTITUMOR RESPONSE DURING PROLONGED TUMOR EXPOSURE. THE INCREASED FUNCTIONALITY OF THE EXHAUSTION-RESISTANT DNMT3A KNOCKOUT CAR T CELLS WAS COUPLED TO AN UP-REGULATION OF INTERLEUKIN-10, AND GENOME-WIDE DNA METHYLATION PROFILING DEFINED AN ATLAS OF GENES TARGETED FOR EPIGENETIC SILENCING. THIS ATLAS PROVIDES A MOLECULAR DEFINITION OF CAR T CELL EXHAUSTION, WHICH INCLUDES MANY TRANSCRIPTIONAL REGULATORS THAT LIMIT THE "STEMNESS" OF IMMUNE CELLS, INCLUDING CD28, CCR7, TCF7, AND LEF1. LAST, WE DEMONSTRATE THAT THIS EPIGENETICALLY REGULATED MULTIPOTENCY PROGRAM IS FIRMLY COUPLED TO THE CLINICAL OUTCOME OF PRIOR CAR T CELL THERAPIES. THESE DATA DOCUMENT THE CRITICAL ROLE EPIGENETIC MECHANISMS PLAY IN LIMITING THE FATE POTENTIAL OF HUMAN T CELLS AND PROVIDE A ROAD MAP FOR LEVERAGING THIS INFORMATION FOR IMPROVING CAR T CELL EFFICACY. 2021 20 570 35 BCR-ABL INDEPENDENT MECHANISMS OF RESISTANCE IN CHRONIC MYELOID LEUKEMIA. NOT ALL CHRONIC MYELOID LEUKEMIA (CML) PATIENTS ARE CURED WITH TYROSINE KINASE INHIBITORS (TKIS), AND A PROPORTION OF THEM DEVELOP RESISTANCE. RECENTLY, CONTINUOUS BCR-ABL GENE EXPRESSION HAS BEEN FOUND IN RESISTANT CELLS WITH UNDETECTABLE BCR-ABL PROTEIN EXPRESSION, INDICATING THAT RESISTANCE MAY OCCUR THROUGH KINASE INDEPENDENT MECHANISMS, MAINLY DUE TO THE PERSISTENCE OF LEUKEMIA STEM CELLS (LSCS). LSCS RESIDE IN THE BONE MARROW NICHE IN A QUIESCENT STATE, AND ARE CHARACTERIZED BY A HIGH HETEROGENEITY IN GENETIC, EPIGENETIC, AND TRANSCRIPTIONAL MECHANISMS. NEW APPROACHES BASED ON SINGLE CELL GENOMICS HAVE OFFERED THE OPPORTUNITY TO IDENTIFY DISTINCT SUBPOPULATIONS OF LSCS AT DIAGNOSIS AND DURING TREATMENT. IN THE ONE HAND, TKIS ARE NOT ABLE TO EFFICIENTLY KILL CML-LSCS, BUT THEY MAY BE RESPONSIBLE FOR THE MODIFICATION OF SOME LSCS CHARACTERISTICS, THUS CONTRIBUTING TO HETEROGENEITY WITHIN THE TUMOR. IN THE OTHER HAND, THE BONE MARROW NICHE IS RESPONSIBLE FOR THE INTERACTIONS BETWEEN SURROUNDING STROMAL CELLS AND LSCS, RESULTING IN THE GENERATION OF SPECIFIC SIGNALS WHICH COULD FAVOR LSCS CELL CYCLE ARREST AND ALLOW THEM TO PERSIST DURING TREATMENT WITH TKIS. ADDITIONALLY, LSCS MAY THEMSELVES ALTER THE NICHE BY EXPRESSING VARIOUS COSTIMULATORY MOLECULES AND SECRETING SUPPRESSIVE CYTOKINES, ABLE TO TARGET METABOLIC PATHWAYS, CREATE AN ANTI-APOPTOTIC ENVIRONMENT, AND ALTER IMMUNE SYSTEM FUNCTIONS. ACCORDINGLY, THE PRODUCTION OF AN IMMUNOSUPPRESSANT MILIEU MAY FACILITATE TUMOR ESCAPE FROM IMMUNE SURVEILLANCE AND INDUCE CHEMO-RESISTANCE. IN THIS REVIEW WE WILL FOCUS ON BCR-ABL-INDEPENDENT MECHANISMS, ANALYZING ESPECIALLY THOSE WITH A POTENTIAL CLINICAL IMPACT IN THE MANAGEMENT OF CML PATIENTS. 2019