1 2185 134 EPIGENETIC MECHANISMS UNDERLYING HIV-INFECTION INDUCED SUSCEPTIBILITY OF CD4+ T CELLS TO ENHANCED ACTIVATION-INDUCED FASL EXPRESSION AND CELL DEATH. BACKGROUND: CHRONIC IMMUNE ACTIVATION AND CD4 T CELL DEPLETION ARE SIGNIFICANT PATHOGENIC FEATURES OF HIV INFECTION. EXPRESSION OF FAS LIGAND (FASL), A KEY MEDIATOR OF ACTIVATION-INDUCED CELL DEATH IN T CELLS, IS ELEVATED IN PEOPLE LIVING WITH HIV-1 INFECTION (PLWH). HOWEVER, THE EPIGENETIC MECHANISMS UNDERLYING THE ENHANCED INDUCTION OF FASL EXPRESSION IN CD4 T LYMPHOCYTES IN PLWH ARE NOT COMPLETELY ELUCIDATED. HENCE, THE CURRENT WORK EXAMINED THE EFFECT OF HIV INFECTION ON FASL PROMOTER-ASSOCIATED HISTONE MODIFICATIONS AND TRANSCRIPTIONAL REGULATION IN CD4 T LYMPHOCYTES IN PLWH. METHOD: FLOW CYTOMETRIC ANALYSIS WAS PERFORMED TO EXAMINE THE FAS-FASL EXPRESSION ON TOTAL CD4 T CELLS AND NAIVE/MEMORY CD4 T CELL SUBSETS. EPIGENETIC FASL PROMOTER HISTONE MODIFICATIONS WERE INVESTIGATED BY CHROMATIN IMMUNOPRECIPITATION-QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION ANALYSIS USING FRESHLY ISOLATED TOTAL CD4 T LYMPHOCYTES FROM HIV-1 INFECTED AND NONINFECTED INDIVIDUALS. RESULTS: ALL NAIVE/MEMORY CD4 T CELL SUBSETS FROM PLWH SHOWED MARKEDLY GREATER FREQUENCY OF FASL EXPRESSION. NOTABLY, EXAMINATION OF FUNCTIONAL OUTCOME OF FASL/FAS CO-EXPRESSION DEMONSTRATED THE PREFERENTIAL SUSCEPTIBILITY OF TCM AND TEM SUBSETS TO ACTIVATION-INDUCED APOPTOSIS. IMPORTANTLY, THESE CD4 T CELLS COLLECTIVELY DEMONSTRATED A DISTINCT FASL PROMOTER HISTONE PROFILE INVOLVING A COORDINATED CROSS-TALK BETWEEN HISTONE H3 MODIFICATIONS LEADING TO ENHANCED FASL GENE EXPRESSION. SPECIFICALLY, LEVELS OF TRANSCRIPTIONALLY PERMISSIVE HISTONE H3K4-TRIMETHYLATION (H3K4ME3) AND HISTONE H3K9-ACETYLATION (H3K9AC) WERE INCREASED, WITH A CONCOMITANT DECREASE IN THE REPRESSIVE H3K9-TRIMETHYLATION (H3K9ME3). CONCLUSION: THE PRESENT WORK DEMONSTRATES THAT EPIGENETIC MECHANISMS INVOLVING PROMOTER-HISTONE MODIFICATIONS REGULATE TRANSCRIPTIONAL COMPETENCE AND FASL EXPRESSION IN CD4 T CELLS FROM PLWH AND RENDER THEM SUSCEPTIBLE TO ACTIVATION-INDUCED CELL DEATH. 2021 2 2076 21 EPIGENETIC DIFFERENCES OF CHRONIC HEPATITIS B IN DIFFERENT TCM SYNDROMES: PROTOCOL FOR A CASE-CONTROL, NON-INTERVENTIONAL, OBSERVATIONAL CLINICAL STUDY. INTRODUCTION: CHRONIC HEPATITIS B IS A SERIOUS DISEASE CAUSING SERIOUS HARM TO THE HUMAN HEALTH. CHINESE MEDICINE HAS ITS UNIQUE ADVANTAGES IN THE CLINICAL PREVENTION AND TREATMENT, WHILE THE SYNDROME OF CHINESE MEDICINE LACKS THE UNDERSTANDING AT THE MICRO LEVEL. THERE ARE SOME THEORETICAL COMMONALITIES BETWEEN THE EPIGENETICS AND TRADITIONAL CHINESE MEDICINE (TCM) SYNDROMES. THE BIOLOGICAL BASIS OF CHRONIC HEPATITIS B (CHB) SYNDROME DIFFERENTIATION FROM THE PERSPECTIVE OF EPIGENETICS IS OF GREAT SIGNIFICANCE TO DIAGNOSE AND PREVENT THE DISEASES. METHODS: THIS PROTOCOL IS A CASE-CONTROL, NONINTERVENTIONAL, OBSERVATIONAL CLINICAL STUDY. PATIENTS WITH CHB FOR SPLEEN-STOMACH DAMP HEAT AND LIVER DEPRESSION AND SPLEEN DEFICIENCY, WITH 12 EACH AND 11 HEALTHY VOLUNTEERS WERE RECRUITED. PERIPHERAL VENOUS BLOOD WAS COLLECTED FROM THE PARTICIPANTS. DNA METHYLATED TRANSFERASE, GENOMIC DNA METHYLATED SPECTRUM, METHYLATED DNA BINDING PROTEIN MECP2, CHRONIC INFECTION OF HEPATITIS B VIRUS WITH METHYLATED RELATED PROTEINS, AND MIRNA TARGET GENES WERE ANALYZED. OBJECTIVES: FROM THE PERSPECTIVE OF DNA METHYLATION EPIGENETICS, "DNA METHYLATION-MIRNA-TARGET GENE" IS THE MAIN LINE, WHICH FURTHER REVEALS THE ESSENCE OF TCM SYNDROME. TO IMPROVE THE LEVEL OF TCM CLINICAL SYNDROME DIFFERENTIATION AND THE CLINICAL EFFICACY OF TCM, ESPECIALLY IN THE STUDY OF TCM SYNDROMES OF CHB, DISCOVERING ITS UNDERLYING BIOLOGICAL SIGNATURE IS NECESSARY. TRIAL REGISTRATION: CLINICAL TRIALS REGISTRATION: CHICTR1800017365, REGISTERED 26 JULY 2018. 2018 3 2831 27 FOCUS ON T CELL EXHAUSTION: NEW ADVANCES IN TRADITIONAL CHINESE MEDICINE IN INFECTION AND CANCER. IN CHRONIC INFECTIONS AND CANCERS, T LYMPHOCYTES (T CELLS) ARE EXPOSED TO PERSISTENT ANTIGEN OR INFLAMMATORY SIGNALS. THE CONDITION IS OFTEN ASSOCIATED WITH A DECLINE IN T-CELL FUNCTION: A STATE CALLED "EXHAUSTION". T CELL EXHAUSTION IS A STATE OF T CELL DYSFUNCTION CHARACTERIZED BY INCREASED EXPRESSION OF A SERIES OF INHIBITORY RECEPTORS (IRS), DECREASED EFFECTOR FUNCTION, AND DECREASED CYTOKINE SECRETION, ACCOMPANIED BY TRANSCRIPTIONAL AND EPIGENETIC CHANGES AND METABOLIC DEFECTS. THE RISE OF IMMUNOTHERAPY, PARTICULARLY THE USE OF IMMUNE CHECKPOINT INHIBITORS (ICIS), HAS DRAMATICALLY CHANGED THE CLINICAL TREATMENT PARADIGM FOR PATIENTS. HOWEVER, ITS LOW RESPONSE RATE, SINGLE TARGET AND HIGH IMMUNOTOXICITY LIMIT ITS CLINICAL APPLICATION. THE MULTIPLE IMMUNOMODULATORY POTENTIAL OF TRADITIONAL CHINESE MEDICINE (TCM) PROVIDES A NEW DIRECTION FOR IMPROVING THE TREATMENT OF T CELL EXHAUSTION. HERE, WE REVIEW RECENT ADVANCES THAT HAVE PROVIDED A CLEARER MOLECULAR UNDERSTANDING OF T CELL EXHAUSTION, REVEALING THE CHARACTERISTICS AND CAUSES OF T CELL EXHAUSTION IN PERSISTENT INFECTIONS AND CANCERS. IN ADDITION, THIS PAPER SUMMARIZES RECENT ADVANCES IN IMPROVING T CELL EXHAUSTION IN INFECTIOUS DISEASES AND CANCER WITH THE AIM OF PROVIDING A COMPREHENSIVE AND VALUABLE SOURCE OF INFORMATION ON TCM AS AN EXPERIMENTAL STUDY AND THEIR ROLE IN COLLABORATION WITH ICIS THERAPY. 2023 4 1937 30 EOMES IS ESSENTIAL FOR ANTITUMOR ACTIVITY OF CD8(+) T CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA. GENOME-WIDE ASSOCIATION STUDIES IDENTIFIED A SINGLE-NUCLEOTIDE POLYMORPHISM (SNP) AFFECTING THE TRANSCRIPTION FACTOR EOMESODERMIN (EOMES) ASSOCIATED WITH A SIGNIFICANTLY INCREASED RISK TO DEVELOP CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). EPIGENETIC ANALYSES, RNA SEQUENCING, AND FLOW CYTOMETRY REVEALED THAT EOMES IS NOT EXPRESSED IN CLL CELLS, BUT IN CD8(+) T CELLS FOR WHICH EOMES IS A KNOWN MASTER REGULATOR. WE THUS HYPOTHESIZED THAT THE INCREASED CLL RISK ASSOCIATED WITH THE EOMES SNP MIGHT BE EXPLAINED BY ITS NEGATIVE IMPACT ON CD8(+) T-CELL-MEDIATED IMMUNE CONTROL OF CLL. FLOW CYTOMETRY ANALYSES REVEALED A HIGHER EOMES EXPRESSION IN CD8(+) T CELLS OF CLL PATIENTS COMPARED TO HEALTHY INDIVIDUALS, AND AN ACCUMULATION OF PD-1(+) EOMES(+) CD8(+) T CELLS IN LYMPH NODES RATHER THAN BLOOD OR BONE MARROW IN CLL. THIS WAS IN LINE WITH AN OBSERVED EXPANSION OF EOMES(+) CD8(+) T CELLS IN THE SPLEEN OF LEUKEMIC EMICRO-TCL1 MICE. AS EOMES EXPRESSION WAS HIGHEST IN CD8(+) T CELLS THAT EXPRESS INHIBITORY RECEPTORS, AN INVOLVEMENT OF EOMES IN T-CELL EXHAUSTION AND DYSFUNCTION SEEMS LIKELY. INTERESTINGLY, EOMES-DEFICIENCY IN CD8(+) T CELLS RESULTED IN THEIR IMPAIRED EXPANSION ASSOCIATED WITH DECREASED CLL CONTROL IN MICE. OVERALL, THESE OBSERVATIONS SUGGEST THAT EOMES IS ESSENTIAL FOR CD8(+) T-CELL EXPANSION AND/OR MAINTENANCE, AND THEREFORE INVOLVED IN ADAPTIVE IMMUNE CONTROL OF CLL. 2021 5 1192 32 CORRELATION OF CPG METHYLATION OF THE PDCD1 GENE WITH PD-1 EXPRESSION ON CD8(+) T CELLS AND MEDICAL LABORATORY INDICATORS IN CHRONIC HEPATITIS B INFECTION. BACKGROUND: THE NEGATIVE SIGNAL PROVIDED BY SOME CO-INHIBITORY FACTORS SUCH AS PROGRAMMED CELL DEATH-1 (PD-1) HAS BEEN ASSOCIATED WITH CHRONIC HEPATITIS B (CHB) INFECTION INDUCED-T CELL EXHAUSTION, ALTHOUGH THE CORRELATION OF CPG METHYLATION OF THE PDCD1 GENE WITH PD-1 EXPRESSION AND MEDICAL LABORATORY INDICATORS IN CHB INFECTION HAS NOT YET BEEN ELUCIDATED. METHODS: BLOOD SAMPLES FROM 20 CHB INFECTION PATIENTS AND 20 SPONTANEOUS CLEARANCE (SC) PATIENTS WERE COLLECTED. PERCENTAGES OF PD-1-POSITIVE CD8(+) T CELLS WERE ANALYZED BY FLOW CYTOMETRY. THE PERCENTAGE OF CPG METHYLATION AT THE PDCD1 LOCUS WAS ANALYZED BY BISULFITE SEQUENCING. STUDENT'S T TEST, PEARSON AND SPEARMAN'S CORRELATION, AND MANN-WHITNEY TESTS WERE USED IN THE STATISTICAL ANALYSIS. RESULTS: PERCENTAGES OF PD-1-POSITIVE CD8(+) T CELLS IN PERIPHERAL BLOOD T CELLS WERE SIGNIFICANTLY HIGHER IN CHB PATIENTS THAN IN THE SC GROUP (P < 0.001). THE METHYLATION LEVEL OF PDCD1 WAS SIGNIFICANTLY LOWER IN CHB PATIENTS (P < 0.001) AND THE METHYLATION LEVEL OF PDCD1 WAS NEGATIVELY CORRELATED WITH PD-1 EXPRESSION LEVEL IN CD8(+) T CELLS (P < 0.001) AND HEPATITIS-B SURFACE ANTIGEN (HBSAG) (P < 0.001). CONCLUSIONS: THE RESULTS OF THE PRESENT STUDY SUGGEST THAT PDCD1 METHYLATION IS CORRELATED WITH PD-1 EXPRESSION ON CD8(+) T CELLS AND CORRELATED WITH HBSAG AND ALANINE AMINOTRANSFERASE. THE RESULTS MAY PROVIDE NEW IDEAS REGARDING ANTI-PD-1 INHIBITORS, AND EPIGENETIC REGULATORS SUCH AS DEMETHYLATION INHIBITORS COULD REPRESENT MORE SUCCESSFUL THERAPEUTIC STRATEGIES IN HEPATITIS B INFECTION PATIENTS. 2020 6 1467 33 DISTINCT CLINICAL AND GENETIC FEATURES OF HEPATITIS B VIRUS-ASSOCIATED FOLLICULAR LYMPHOMA IN CHINESE PATIENTS. HEPATITIS B VIRUS (HBV) INFECTION HAS BEEN ASSOCIATED WITH AN INCREASED RISK FOR B-CELL LYMPHOMAS. WE PREVIOUSLY SHOWED THAT 20% OF DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL) PATIENTS FROM CHINA, AN ENDEMIC AREA OF HBV INFECTION, HAVE CHRONIC HBV INFECTION (SURFACE ANTIGEN-POSITIVE, HBSAG+) AND ARE CHARACTERIZED BY DISTINCT CLINICAL AND GENETIC FEATURES. HERE, WE SHOWED THAT 24% OF FOLLICULAR LYMPHOMA (FL) CHINESE PATIENTS ARE HBSAG+. COMPARED WITH THE HBSAG- FL PATIENTS, HBSAG+ PATIENTS ARE YOUNGER, HAVE A HIGHER HISTOLOGICAL GRADE AT DIAGNOSIS, AND HAVE A HIGHER INCIDENCE OF DISEASE PROGRESSION WITHIN 24 MONTHS. MOREOVER, BY SEQUENCING THE GENOMES OF 109 FL TUMORS, WE OBSERVED ENHANCED MUTAGENESIS AND DISTINCT GENETIC PROFILE IN HBSAG+ FLS, WITH A UNIQUE SET OF PREFERENTIALLY MUTATED GENES (TNFAIP3, FAS, HIST1H1C, KLF2, TP53, PIM1, TMSB4X, DUSP2, TAGAP, LYN, AND SETD2) BUT LACK OF THE HALLMARK OF HBSAG- FLS (IE, IGH/BCL2 TRANSLOCATIONS AND CREBBP MUTATIONS). TRANSCRIPTOMIC ANALYSES FURTHER SHOWED THAT HBSAG+ FLS DISPLAYED GENE-EXPRESSION SIGNATURES RESEMBLING THE ACTIVATED B-CELL-LIKE SUBTYPE OF DIFFUSE LARGE B-CELL LYMPHOMA, INVOLVING IRF4-TARGETED GENES AND NF-KAPPAB/MYD88 SIGNALING PATHWAYS. FINALLY, WE IDENTIFIED AN INCREASED INFILTRATION OF CD8+ MEMORY T CELLS, CD4+ TH1 CELLS, AND M1 MACROPHAGES AND HIGHER T-CELL EXHAUSTION GENE SIGNATURE IN HBSAG+ FL SAMPLES. TAKEN TOGETHER, WE PRESENT NEW GENETIC/EPIGENETIC EVIDENCE THAT LINKS CHRONIC HBV INFECTION TO B-CELL LYMPHOMAGENESIS, AND HBV-ASSOCIATED FL IS LIKELY TO HAVE A DISTINCT CELL-OF-ORIGIN AND REPRESENT AS A SEPARATE SUBTYPE OF FL. TARGETABLE GENETIC/EPIGENETIC ALTERATIONS IDENTIFIED IN TUMORS AND THEIR ASSOCIATED TUMOR MICROENVIRONMENT MAY PROVIDE POTENTIAL NOVEL THERAPEUTIC APPROACHES FOR THIS SUBGROUP OF PATIENTS. 2022 7 66 34 A KEY ROLE FOR EZH2 IN EPIGENETIC SILENCING OF HOX GENES IN MANTLE CELL LYMPHOMA. THE CHROMATIN MODIFIER EZH2 IS OVEREXPRESSED AND ASSOCIATED WITH INFERIOR OUTCOME IN MANTLE CELL LYMPHOMA (MCL). RECENTLY, WE DEMONSTRATED PREFERENTIAL DNA METHYLATION OF HOX GENES IN MCL COMPARED WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), DESPITE THESE GENES NOT BEING EXPRESSED IN EITHER ENTITY. SINCE EZH2 HAS BEEN SHOWN TO REGULATE HOX GENE EXPRESSION, TO GAIN FURTHER INSIGHT INTO ITS POSSIBLE ROLE IN DIFFERENTIAL SILENCING OF HOX GENES IN MCL VS. CLL, WE PERFORMED DETAILED EPIGENETIC CHARACTERIZATION USING REPRESENTATIVE CELL LINES AND PRIMARY SAMPLES. WE OBSERVED SIGNIFICANT OVEREXPRESSION OF EZH2 IN MCL VS. CLL. CHROMATIN IMMUNE PRECIPITATION (CHIP) ASSAYS REVEALED THAT EZH2 CATALYZED REPRESSIVE H3 LYSINE 27 TRIMETHYLATION (H3K27ME3), WHICH WAS SUFFICIENT TO SILENCE HOX GENES IN CLL, WHEREAS IN MCL H3K27ME3 IS ACCOMPANIED BY DNA METHYLATION FOR A MORE STABLE REPRESSION. MORE IMPORTANTLY, HYPERMETHYLATION OF THE HOX GENES IN MCL RESULTED FROM EZH2 OVEREXPRESSION AND SUBSEQUENT RECRUITMENT OF THE DNA METHYLATION MACHINERY ONTO HOX GENE PROMOTERS. THE IMPORTANCE OF EZH2 UPREGULATION IN THIS PROCESS WAS FURTHER UNDERSCORED BY SIRNA TRANSFECTION AND EZH2 INHIBITOR EXPERIMENTS. ALTOGETHER, THESE OBSERVATIONS IMPLICATE EZH2 IN THE LONG-TERM SILENCING OF HOX GENES IN MCL, AND ALLUDE TO ITS POTENTIAL AS A THERAPEUTIC TARGET WITH CLINICAL IMPACT. 2013 8 2143 38 EPIGENETIC LANDSCAPE OF HIV-1 INFECTION IN PRIMARY HUMAN MACROPHAGE. HUMAN IMMUNODEFICIENCY VIRUS (HIV)-INFECTED MACROPHAGES ARE LONG-LIVED CELLS THAT SUSTAIN PERSISTENT VIRUS EXPRESSION, WHICH IS BOTH A BARRIER TO VIRAL ERADICATION AND CONTRIBUTOR TO NEUROLOGICAL COMPLICATIONS IN PATIENTS DESPITE ANTIRETROVIRAL THERAPY (ART). TO BETTER UNDERSTAND THE REGULATION OF HIV-1 IN MACROPHAGES, WE COMPARED HIV-INFECTED PRIMARY HUMAN MONOCYTE-DERIVED MACROPHAGES (MDM) TO ACUTELY INFECTED PRIMARY CD4 T CELLS AND JURKAT CELLS LATENTLY INFECTED WITH HIV (JLAT 8.4). HIV GENOMES IN MDM WERE ACTIVELY TRANSCRIBED DESPITE ENRICHMENT WITH HETEROCHROMATIN-ASSOCIATED H3K9ME3 ACROSS THE COMPLETE HIV GENOME IN COMBINATION WITH ELEVATED ACTIVATION MARKS OF H3K9AC AND H3K27AC AT THE LONG TERMINAL REPEAT (LTR). MACROPHAGE PATTERNS CONTRASTED WITH JLAT CELLS, WHICH SHOWED CONVENTIONAL BIVALENT H3K4ME3/H3K27ME3, AND ACUTELY INFECTED CD4 T CELLS, WHICH SHOWED AN INTERMEDIATE EPIGENOTYPE. 5'-METHYLCYTOSINE (5MC) WAS ENRICHED ACROSS THE HIV GENOME IN LATENTLY INFECTED JLAT CELLS, WHILE 5'-HYDROXYMETHYLCYTOSINE (5HMC) WAS ENRICHED IN CD4 CELLS AND MDMS. HIV INFECTION INDUCED MULTINUCLEATION OF MDMS ALONG WITH DNA DAMAGE-ASSOCIATED P53 PHOSPHORYLATION, AS WELL AS LOSS OF TET2 AND THE NUCLEAR REDISTRIBUTION OF 5-HYDOXYMETHYLATION. TAKEN TOGETHER, OUR FINDINGS SUGGEST THAT HIV INDUCES A UNIQUE MACROPHAGE NUCLEAR AND TRANSCRIPTIONAL PROFILE, AND VIRAL GENOMES ARE MAINTAINED IN A NONCANONICAL BIVALENT EPIGENETIC STATE. IMPORTANCE MACROPHAGES SERVE AS A RESERVOIR FOR LONG-TERM PERSISTENCE AND CHRONIC PRODUCTION OF HIV. WE FOUND AN ATYPICAL EPIGENETIC CONTROL OF HIV IN MACROPHAGES MARKED BY HETEROCHROMATIC H3K9ME3 DESPITE ACTIVE VIRAL TRANSCRIPTION. HIV INFECTION INDUCED CHANGES IN MACROPHAGE NUCLEAR MORPHOLOGY AND EPIGENETIC REGULATORY FACTORS. THESE FINDINGS MAY IDENTIFY NEW MECHANISMS TO CONTROL CHRONIC HIV EXPRESSION IN INFECTED MACROPHAGES. 2022 9 2392 35 EPIGENETIC REPRESSION OF INTERLEUKIN 2 EXPRESSION IN SENESCENT CD4+ T CELLS DURING CHRONIC HIV TYPE 1 INFECTION. THE MOLECULAR MECHANISMS FOR IL2 GENE-SPECIFIC DYSREGULATION DURING CHRONIC HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) INFECTION ARE UNKNOWN. HERE, WE INVESTIGATED THE ROLE OF DNA METHYLATION IN SUPPRESSING INTERLEUKIN 2 (IL-2) EXPRESSION IN MEMORY CD4(+) T CELLS DURING CHRONIC HIV-1 INFECTION. WE OBSERVED THAT CPG SITES IN THE IL2 PROMOTER OF CD4(+) T CELLS WERE FULLY METHYLATED IN NAIVE CD4(+) T CELLS AND SIGNIFICANTLY DEMETHYLATED IN THE MEMORY POPULATIONS. INTERESTINGLY, WE FOUND THAT THE MEMORY CELLS THAT HAD A TERMINALLY DIFFERENTIATED PHENOTYPE AND EXPRESSED CD57 HAD INCREASED IL2 PROMOTER METHYLATION RELATIVE TO LESS DIFFERENTIATED MEMORY CELLS IN HEALTHY INDIVIDUALS. IMPORTANTLY, EARLY EFFECTOR MEMORY SUBSETS FROM HIV-1-INFECTED SUBJECTS EXPRESSED HIGH LEVELS OF CD57 AND WERE HIGHLY METHYLATED AT THE IL2 LOCUS. FURTHERMORE, THE INCREASED CD57 EXPRESSION ON MEMORY CD4(+) T CELLS WAS INVERSELY CORRELATED WITH IL-2 PRODUCTION. THESE DATA SUGGEST THAT DNA METHYLATION AT THE IL2 LOCUS IN CD4(+) T CELLS IS COUPLED TO IMMUNOSENESCENCE AND PLAYS A CRITICAL ROLE IN THE BROAD DYSFUNCTION THAT OCCURS IN POLYCLONAL T CELLS DURING HIV-1 INFECTION. 2015 10 337 29 ALTERATIONS IN DNA METHYLATION/DEMETHYLATION INTERMEDIATES PREDICT CLINICAL OUTCOME IN CHRONIC LYMPHOCYTIC LEUKEMIA. CYTOSINE DERIVATIVE DYSREGULATIONS REPRESENT IMPORTANT EPIGENETIC MODIFICATIONS WHOSE IMPACT ON THE CLINICAL OUTCOME IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS INCOMPLETELY UNDERSTOOD. HENCE, GLOBAL LEVELS OF 5-METHYLCYTOSINE (5-MCYT), 5-HYDROXYMETHYLCYTOSINE (5-HMCYT), 5-CARBOXYLCYTOSINE (5-CACYT) AND 5-HYDROXYMETHYLURACIL WERE TESTED IN PURIFIED B CELLS FROM CLL PATIENTS (N = 55) AND CONTROLS (N = 17). THE DNA METHYLATION 'WRITERS' (DNA METHYLTRANSFERASES [DNMT1/3A/3B]), 'READERS' (METHYL-CPG-BINDING DOMAIN [MBD2/4]), 'EDITORS' (TEN-ELEVEN TRANSLOCATION [TET1/2/3]) AND 'MODULATORS' (SAT1) WERE ALSO EVALUATED. ACCORDINGLY, PATIENTS WERE STRATIFIED INTO THREE SUBGROUPS. FIRST, A SUBGROUP WITH A GLOBAL DEFICIT IN CYTOSINE DERIVATIVES CHARACTERIZED BY HYPERLYMPHOCYTOSIS, REDUCED MEDIAN PROGRESSION FREE SURVIVAL (PFS = 52 MONTHS) AND SHORTER TREATMENT FREE SURVIVAL (TFS = 112 MONTHS) WAS IDENTIFIED. IN THIS SUBGROUP, MAJOR EPIGENETIC MODIFICATIONS WERE HIGHLIGHTED INCLUDING A REDUCTION OF 5-MCYT, 5-HMCYT, 5-CACYT ASSOCIATED WITH DNMT3A, MBD2/4 AND TET1/2 DOWNREGULATION. SECOND, THE CYTOSINE DERIVATIVE ANALYSIS REVEALED A SUBGROUP WITH A PARTIAL DEFICIT (PFS = 84, TFS = 120 MONTHS), MAINLY AFFECTING DNA DEMETHYLATION (5-HMCYT REDUCTION, SAT1 INDUCTION). THIRD, A SUBGROUP EPIGENETICALLY SIMILAR TO CONTROLS WAS IDENTIFIED (PFS AND TFS > 120 MONTHS). THE PROGNOSTIC IMPACT OF STRATIFYING CLL PATIENTS WITHIN THREE EPIGENETIC SUBGROUPS WAS CONFIRMED IN A VALIDATION COHORT. IN CONCLUSION, OUR RESULTS SUGGEST THAT DYSREGULATIONS OF CYTOSINE DERIVATIVE REGULATORS REPRESENT MAJOR EVENTS ACQUIRED DURING CLL PROGRESSION AND ARE INDEPENDENT FROM IGHV MUTATIONAL STATUS. 2017 11 2421 28 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 12 5058 37 PHENOTYPIC ALTERATION OF CD8+ T CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA IS ASSOCIATED WITH EPIGENETIC REPROGRAMMING. IMMUNOSUPPRESSION IS A PREVALENT CLINICAL FEATURE IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) PATIENTS, WITH MANY PATIENTS DEMONSTRATING INCREASED SUSCEPTIBILITY TO INFECTIONS AS WELL AS INCREASED FAILURE OF AN ANTITUMOR IMMUNE RESPONSE. HOWEVER, MUCH IS CURRENTLY NOT UNDERSTOOD REGARDING THE PRECISE MECHANISMS THAT ATTRIBUTE TO THIS IMMUNOSUPPRESSIVE PHENOTYPE IN CLL. TO PROVIDE FURTHER CLARITY TO THIS PARTICULAR PHENOMENON, WE ANALYZED THE T-CELL PROFILE OF CLL PATIENT SAMPLES WITHIN A LARGE COHORT AND OBSERVED THAT PATIENTS WITH AN INVERTED CD4/CD8 RATIO HAD A SHORTER TIME TO FIRST TREATMENT AS WELL AS OVERALL SURVIVAL. THESE OBSERVATIONS COINCIDED WITH HIGHER EXPRESSION OF THE IMMUNE CHECKPOINT RECEPTOR PD-1 IN CLL PATIENT CD8+ T CELLS WHEN COMPARED TO AGE-MATCHED HEALTHY DONORS. INTERESTINGLY, WE DISCOVERED THAT INCREASED PD-1 EXPRESSION IN CD8+ T CELLS CORRESPONDS WITH DECREASED DNA METHYLATION LEVELS IN A DISTAL UPSTREAM LOCUS OF THE PD-1 GENE PDCD1. FURTHER ANALYSIS USING LUCIFERASE REPORTER ASSAYS SUGGESTS THAT THE IDENTIFIED PDCD1 DISTAL UPSTREAM REGION ACTS AS AN ENHANCER FOR PDCD1 TRANSCRIPTION AND THIS REGION BECOMES DEMETHYLATED DURING ACTIVATION OF NAIVE CD8+ T CELLS BY ANTI-CD3/ANTI-CD28 ANTIBODIES AND IL2. FINALLY, WE CONDUCTED A GENOME-WIDE DNA METHYLATION ANALYSIS COMPARING CD8+ T CELLS FROM CLL PATIENTS AGAINST HEALTHY DONORS AND IDENTIFIED ADDITIONAL DIFFERENTIALLY METHYLATED GENES WITH KNOWN IMMUNE REGULATORY FUNCTIONS INCLUDING CCR6 AND KLRG1. TAKEN TOGETHER, OUR FINDINGS REVEAL THE OCCURRENCE OF EPIGENETIC REPROGRAMMING TAKING PLACE WITHIN CLL PATIENT CD8+ T CELLS AND HIGHLIGHT THE POTENTIAL MECHANISM OF HOW IMMUNOSUPPRESSION IS ACCOMPLISHED IN CLL. 2016 13 2650 29 EPIGENOMIC-GUIDED MASS CYTOMETRY PROFILING REVEALS DISEASE-SPECIFIC FEATURES OF EXHAUSTED CD8 T CELLS. EXHAUSTED CD8 T (TEX) CELLS ARE IMMUNOTHERAPY TARGETS IN CHRONIC INFECTION AND CANCER, BUT A COMPREHENSIVE ASSESSMENT OF TEX CELL DIVERSITY IN HUMAN DISEASE IS LACKING. HERE, WE DEVELOPED A TRANSCRIPTOMIC- AND EPIGENETIC-GUIDED MASS CYTOMETRY APPROACH TO DEFINE CORE EXHAUSTION-SPECIFIC GENES AND DISEASE-INDUCED CHANGES IN TEX CELLS IN HIV AND HUMAN CANCER. SINGLE-CELL PROTEOMIC PROFILING IDENTIFIED 9 DISTINCT TEX CELL CLUSTERS USING PHENOTYPIC, FUNCTIONAL, TRANSCRIPTION FACTOR, AND INHIBITORY RECEPTOR CO-EXPRESSION PATTERNS. AN EXHAUSTION SEVERITY METRIC WAS DEVELOPED AND INTEGRATED WITH HIGH-DIMENSIONAL PHENOTYPES TO DEFINE TEX CELL CLUSTERS THAT WERE PRESENT IN HEALTHY SUBJECTS, COMMON ACROSS CHRONIC INFECTION AND CANCER OR ENRICHED IN EITHER DISEASE, LINKED TO DISEASE SEVERITY, AND CHANGED WITH HIV THERAPY. COMBINATORIAL PATTERNS OF IMMUNOTHERAPY TARGETS ON DIFFERENT TEX CELL CLUSTERS WERE ALSO DEFINED. THIS APPROACH AND ASSOCIATED DATASETS PRESENT A RESOURCE FOR INVESTIGATING HUMAN TEX CELL BIOLOGY, WITH IMPLICATIONS FOR IMMUNE MONITORING AND IMMUNOMODULATION IN CHRONIC INFECTIONS, AUTOIMMUNITY, AND CANCER. 2018 14 1319 35 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 15 5897 29 T FOLLICULAR HELPER CELL-DEPENDENT CLEARANCE OF A PERSISTENT VIRUS INFECTION REQUIRES T CELL EXPRESSION OF THE HISTONE DEMETHYLASE UTX. EPIGENETIC CHANGES, INCLUDING HISTONE METHYLATION, CONTROL T CELL DIFFERENTIATION AND MEMORY FORMATION, THOUGH THE ENZYMES THAT MEDIATE THESE PROCESSES ARE NOT CLEAR. WE SHOW THAT UTX, A HISTONE H3 LYSINE 27 (H3K27) DEMETHYLASE, SUPPORTS T FOLLICULAR HELPER (TFH) CELL RESPONSES THAT ARE ESSENTIAL FOR B CELL ANTIBODY GENERATION AND THE RESOLUTION OF CHRONIC VIRAL INFECTIONS. MICE WITH A T CELL-SPECIFIC UTX DELETION HAD FEWER TFH CELLS, REDUCED GERMINAL CENTER RESPONSES, LACKED VIRUS-SPECIFIC IMMUNOGLOBULIN G (IGG), AND WERE UNABLE TO RESOLVE CHRONIC LYMPHOCYTIC CHORIOMENINGITIS VIRUS INFECTIONS. UTX-DEFICIENT T CELLS SHOWED DECREASED EXPRESSION OF INTERLEUKIN-6 RECEPTOR-ALPHA AND OTHER TFH CELL-RELATED GENES THAT WERE ASSOCIATED WITH INCREASED H3K27 METHYLATION. ADDITIONALLY, TURNER SYNDROME SUBJECTS, WHO ARE PREDISPOSED TO CHRONIC EAR INFECTIONS, HAD REDUCED UTX EXPRESSION IN IMMUNE CELLS AND DECREASED CIRCULATING CD4(+) CXCR5(+) T CELL FREQUENCY. THUS, WE IDENTIFY A CRITICAL LINK BETWEEN UTX IN T CELLS AND IMMUNITY TO INFECTION. 2015 16 5094 38 PLASMA EXTRACELLULAR VESICLE SUBTYPES MAY BE USEFUL AS POTENTIAL BIOMARKERS OF IMMUNE ACTIVATION IN PEOPLE WITH HIV. BACKGROUND: EXTRACELLULAR VESICLES (EVS) ARE INTERCELLULAR MESSENGERS WITH EPIGENETIC POTENTIAL SINCE THEY CAN SHUTTLE MICRORNA (MIRNA). EVS AND MIRNA PLAY A ROLE IN HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION IMMUNOPATHOGENESIS. CHRONIC IMMUNE ACTIVATION AND SYSTEMIC INFLAMMATION DURING HIV INFECTION DESPITE EFFECTIVE ANTIRETROVIRAL THERAPY (ART) ARE ASSOCIATED WITH NON-ACQUIRED IMMUNODEFICIENCY SYNDROME (AIDS) COMORBIDITIES IN PEOPLE LIVING WITH HIV (PLWH). ANALYSIS OF PLASMA EVS AND THEIR MIRNA CONTENT MAY BE USEFUL AS IMMUNE ACTIVATION OR INFLAMMATORY BIOMARKERS IN PLWH RECEIVING ART. IN THIS STUDY, WE HYPOTHESIZED THAT THE NUMBER, SIZE, AND MIRNA OF LARGE AND SMALL EVS COULD REFLECT IMMUNE ACTIVATION ASSOCIATED WITH AN ELEVATED CD8 T-CELL COUNT OR A LOW CD4/CD8 RATIO IN PLWH. METHODS: PLASMA EVS SUBTYPE PURIFIED FROM PLWH AND UNINFECTED CONTROLS WERE SIZED USING DYNAMIC LIGHT SCATTERING AND QUANTIFIED USING FLOW CYTOMETRY AND ACETYLCHOLINE ESTERASE (ACHE) ACTIVITY. EXPRESSION OF MATURE MIRNAS MIR-92, MIR-155, MIR-223 WAS MEASURED BY QUANTITATIVE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN REACTION IN EVS AND LEUCOCYTES. RESULTS: HIV INFECTION INDUCES INCREASED PRODUCTION OF SMALL EVS IN PLASMA. EV SUBTYPES WERE DIFFERENTIALLY ENRICHED IN MIR-92, MIR-155, AND MIR-223. POSITIVE CORRELATIONS BETWEEN CD8 T-CELL COUNT AND LARGE EVS ABUNDANCE AND SMALL EVS ACHE ACTIVITY WERE OBSERVED. CD4/CD8 RATIO WAS NEGATIVELY CORRELATED WITH SMALL EV ACHE ACTIVITY, AND MIRNA-155 LEVEL PER SMALL EV WAS NEGATIVELY CORRELATED WITH CD8 T-CELL COUNT. CONCLUSIONS: THESE FINDINGS SUGGEST THAT QUANTIFYING LARGE OR SMALL EVS AND PROFILING MIRNA CONTENT PER EV MIGHT PROVIDE NEW FUNCTIONAL BIOMARKERS OF IMMUNE ACTIVATION AND INFLAMMATION. 2021 17 1433 34 DIFFERENTIAL GENOME-WIDE ARRAY-BASED METHYLATION PROFILES IN PROGNOSTIC SUBSETS OF CHRONIC LYMPHOCYTIC LEUKEMIA. GLOBAL HYPOMETHYLATION AND REGIONAL HYPERMETHYLATION ARE WELL-KNOWN EPIGENETIC FEATURES OF CANCER; HOWEVER, IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), STUDIES ON GENOME-WIDE EPIGENETIC MODIFICATIONS ARE LIMITED. HERE, WE ANALYZED THE GLOBAL METHYLATION PROFILES IN CLL, BY APPLYING HIGH-RESOLUTION METHYLATION MICROARRAYS (27,578 CPG SITES) TO 23 CLL SAMPLES, BELONGING TO THE IMMUNOGLOBULIN HEAVY-CHAIN VARIABLE (IGHV) MUTATED (FAVORABLE) AND IGHV UNMUTATED/IGHV3-21 (POOR-PROGNOSTIC) SUBSETS. OVERALL, RESULTS DEMONSTRATED SIGNIFICANT DIFFERENCES IN METHYLATION PATTERNS BETWEEN THESE SUBGROUPS. SPECIFICALLY, IN IGHV UNMUTATED CLL, WE IDENTIFIED METHYLATION OF 7 KNOWN OR CANDIDATE TUMOR SUPPRESSOR GENES (EG, VHL, ABI3, AND IGSF4) AS WELL AS 8 UNMETHYLATED GENES INVOLVED IN CELL PROLIFERATION AND TUMOR PROGRESSION (EG, ADORA3 AND PRF1 ENHANCING THE NUCLEAR FACTOR-KAPPAB AND MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS, RESPECTIVELY). IN CONTRAST, THESE LATTER GENES WERE SILENCED BY METHYLATION IN IGHV MUTATED PATIENTS. THE ARRAY DATA WERE VALIDATED FOR SELECTED GENES USING METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION, QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION, AND BISULFITE SEQUENCING. FINALLY, THE SIGNIFICANCE OF DNA METHYLATION IN REGULATING GENE PROMOTERS WAS SHOWN BY REINDUCING 4 METHYLATED TUMOR SUPPRESSOR GENES (EG, VHL AND ABI3) IN IGHV UNMUTATED SAMPLES USING THE METHYL-INHIBITOR 5-AZA-2'-DEOXYCYTIDINE. TAKEN TOGETHER, OUR DATA FOR THE FIRST TIME REVEAL DIFFERENCES IN GLOBAL METHYLATION PROFILES BETWEEN PROGNOSTIC SUBSETS OF CLL, WHICH MAY UNFOLD EPIGENETIC SILENCING MECHANISMS INVOLVED IN CLL PATHOGENESIS. 2010 18 6121 25 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 19 4548 33 MUTATION ANALYSIS OF THE FAS AND TNFR APOPTOTIC CASCADE GENES IN HEMATOLOGICAL MALIGNANCIES. OBJECTIVE: THE EXISTENCE OF PROPERLY FUNCTIONING APOPTOTIC PATHWAYS IS OF UTMOST IMPORTANCE IN THE MAINTENANCE OF A NORMAL CELL COUNT. SEVERAL GROUPS HAVE SEARCHED FOR MUTATIONS IN THE FAS RECEPTOR, A WELL-CHARACTERIZED APOPTOTIC PROTEIN CARRYING A DEATH DOMAIN, AND REPORTED THE EXISTENCE OF RARE MUTATIONS IN MULTIPLE MYELOMA, T-ACUTE LYMPHOBLASTIC LEUKEMIA (T-ALL), AND ADULT T-CELL LEUKEMIA. OUR AIM WAS TO EXPAND THESE SEARCHES BY LOOKING FOR MUTATIONS IN THE DEATH DOMAINS OF FAS, FADD, TNFR, TRADD, AND RIP, IN THE PROMOTER REGION OF FAS, AND IN THE PROTEASE DOMAIN OF CASPASE 10, IN A LARGER VARIETY OF HEMATOLOGICAL MALIGNANCIES, SOME OF WHICH EXPRESS AN APOPTOSIS-RESISTANT PHENOTYPE. METHODS: WE EXTRACTED RNA AND DNA SAMPLES FROM 92 HEMATOLOGICAL MALIGNANCIES: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL; 31 CASES), CHRONIC MYELOGENOUS LEUKEMIA (CML; 28 CASES), ESSENTIAL THROMBOCYTHEMIA (ET; 8 CASES), ACUTE LYMPHOCYTIC LEUKEMIA (ALL; 6 CASES), ACUTE MYELOBLASTIC LEUKEMIA (AML; 6 CASES), HAIRY-CELL LEUKEMIA (HCL; 3 CASES), BURKITT'S LYMPHOMA (3 CASES), POLYCYTHEMIA VERA (PV; 3 CASES), MYELOFIBROSIS (2 CASES), AND CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML; 2 CASES) AND PERFORMED PCR-SSCP AND SEQUENCE ANALYSIS ON THESE SAMPLES. RESULTS: FIVE POLYMORPHIC PATTERNS WERE FOUND: THREE IN THE DEATH DOMAIN OF THE FAS GENE IN CML PATIENTS, ONE IN THE PROMOTER OF THIS GENE IN A CLL PATIENT, AND THE FIFTH IN THE DEATH DOMAIN OF THE TRADD GENE IN A CML PATIENT. NO MUTATIONS, ALTERING AMINO ACIDS, WERE FOUND IN THESE GENES IN ANY OF THE AFOREMENTIONED MALIGNANCIES. CONCLUSIONS: THESE OBSERVATIONS IMPLY THAT MUTATIONS IN THE DEATH DOMAINS OF FAS, FADD, TNFR, TRADD, AND RIP AND IN THE PROTEASE DOMAIN OF CASPASE 10 ARE NOT A MAJOR CAUSE FOR FAILURE OF APOPTOSIS IN HEMATOLOGICAL MALIGNANCIES, MAINLY CML AND CLL. REGULATORY AND EPIGENETIC ABNORMALITIES IN THESE APOPTOTIC CASCADE MEMBERS AND ABERRATIONS IN OTHER COMPONENTS OF ALL DEATH MACHINERY SHOULD BE LOOKED FOR. 2001 20 912 26 CHRONIC EXPOSURE TO WATER POLLUTANT TRICHLOROETHYLENE INCREASED EPIGENETIC DRIFT IN CD4(+) T CELLS. AIM: AUTOIMMUNE DISEASE AND CD4(+) T-CELL ALTERATIONS ARE INDUCED IN MICE EXPOSED TO THE WATER POLLUTANT TRICHLOROETHYLENE (TCE). WE EXAMINED HERE WHETHER TCE ALTERED GENE-SPECIFIC DNA METHYLATION IN CD4(+) T CELLS AS A POSSIBLE MECHANISM OF IMMUNOTOXICITY. MATERIALS & METHODS: NAIVE AND EFFECTOR/MEMORY CD4(+) T CELLS FROM MICE EXPOSED TO TCE (0.5 MG/ML IN DRINKING WATER) FOR 40 WEEKS WERE EXAMINED BY BISULFITE NEXT-GENERATION DNA SEQUENCING. RESULTS: A PROBABILISTIC MODEL CALCULATED FROM MULTIPLE GENES SHOWED THAT TCE DECREASED METHYLATION CONTROL IN CD4(+) T CELLS. DATA FROM INDIVIDUAL GENES FITTED TO A QUADRATIC REGRESSION MODEL SHOWED THAT TCE INCREASED GENE-SPECIFIC METHYLATION VARIANCE IN BOTH CD4 SUBSETS. CONCLUSION: TCE INCREASED EPIGENETIC DRIFT OF SPECIFIC CPG SITES IN CD4(+) T CELLS. 2016