1 3454 129 HYPOMETHYLATION AT THE REGULATORY T CELL-SPECIFIC DEMETHYLATED REGION IN CD25HI T CELLS IS DECOUPLED FROM FOXP3 EXPRESSION AT THE INFLAMED SITE IN CHILDHOOD ARTHRITIS. THE MAINTENANCE OF FOXP3 EXPRESSION IN CD25(HI) REGULATORY T CELLS (TREGS) IS CRUCIAL TO THE CONTROL OF INFLAMMATION AND ESSENTIAL FOR SUCCESSFUL TREG TRANSFER THERAPIES. COEXPRESSION OF CD25 AND FOXP3 IN COMBINATION WITH A HYPOMETHYLATED REGION WITHIN THE FOXP3 GENE, CALLED THE TREG-SPECIFIC DEMETHYLATED REGION (TSDR), IS CONSIDERED THE HALLMARK OF STABLE TREGS. THE TSDR IS AN EPIGENETIC MOTIF THAT IS IMPORTANT FOR STABLE FOXP3 EXPRESSION AND IS USED AS A BIOMARKER TO MEASURE TREG LINEAGE COMMITMENT. IN THIS STUDY, WE REPORT THAT, UNLIKE IN PERIPHERAL BLOOD, CD4(+) T CELL EXPRESSION OF CD25 AND FOXP3 IS FREQUENTLY DISSOCIATED AT THE INFLAMED SITE IN PATIENTS WITH JUVENILE IDIOPATHIC ARTHRITIS, WHICH LED US TO QUESTION THE STABILITY OF HUMAN TREGS IN CHRONIC INFLAMMATORY ENVIRONMENTS. WE DESCRIBE A NOVEL CD4(+)CD127(LO)CD25(HI) HUMAN T CELL POPULATION THAT EXHIBITS EXTENSIVE TSDR AND PROMOTER DEMETHYLATION IN THE ABSENCE OF STABLE FOXP3 EXPRESSION. THIS POPULATION EXPRESSES HIGH LEVELS OF CTLA-4 AND CAN SUPPRESS T CONVENTIONAL CELL PROLIFERATION IN VITRO. THESE DATA COLLECTIVELY SUGGEST THAT THIS POPULATION MAY REPRESENT A CHRONICALLY ACTIVATED FOXP3(LO) TREG POPULATION. WE SHOW THAT THESE CELLS HAVE DEFECTS IN IL-2 SIGNALING AND REDUCED EXPRESSION OF A DEUBIQUITINASE IMPORTANT FOR FOXP3 STABILITY. CLINICALLY, THE PROPORTIONS OF THESE CELLS WITHIN THE CD25(HI) T CELL SUBSET ARE INCREASED IN PATIENTS WITH THE MORE SEVERE COURSES OF DISEASE. OUR STUDY DEMONSTRATES, THEREFORE, THAT HYPOMETHYLATION AT THE TSDR CAN BE DECOUPLED FROM FOXP3 EXPRESSION IN HUMAN T CELLS AND THAT ENVIRONMENT-SPECIFIC BREAKDOWN IN FOXP3 STABILITY MAY COMPROMISE THE RESOLUTION OF INFLAMMATION IN JUVENILE IDIOPATHIC ARTHRITIS. 2014 2 5908 39 TARGETED DE-METHYLATION OF THE FOXP3-TSDR IS SUFFICIENT TO INDUCE PHYSIOLOGICAL FOXP3 EXPRESSION BUT NOT A FUNCTIONAL TREG PHENOTYPE. CD4+ REGULATORY T CELLS (TREGS) ARE KEY MEDIATORS OF IMMUNOLOGICAL TOLERANCE AND PROMISING EFFECTOR CELLS FOR IMMUNO-SUPPRESSIVE ADOPTIVE CELLULAR THERAPY TO FIGHT AUTOIMMUNITY AND CHRONIC INFLAMMATION. THEIR FUNCTIONAL STABILITY IS CRITICAL FOR THEIR CLINICAL UTILITY AND HAS BEEN CORRELATED TO THE DEMETHYLATED STATE OF THE TSDR/CNS2 ENHANCER ELEMENT IN THE TREG LINEAGE TRANSCRIPTION FACTOR FOXP3. HOWEVER, PROOF FOR A CAUSAL CONTRIBUTION OF THE TSDR DE-METHYLATION TO FOXP3 STABILITY AND TREG INDUCTION IS SO FAR LACKING. WE HERE ESTABLISHED A POWERFUL TRANSIENT-TRANSFECTION CRISPR-CAS9-BASED EPIGENETIC EDITING METHOD FOR THE SELECTIVE DE-METHYLATION OF THE TSDR WITHIN THE ENDOGENOUS CHROMATIN ENVIRONMENT OF A LIVING CELL. THE INDUCED DE-METHYLATED STATE WAS STABLE OVER WEEKS IN CLONAL T CELL PROLIFERATION CULTURES EVEN AFTER EXPRESSION OF THE EDITING COMPLEX HAD CEASED. EPIGENETIC EDITING OF THE TSDR RESULTED IN FOXP3 EXPRESSION, EVEN IN ITS PHYSIOLOGICAL ISOFORM DISTRIBUTION, PROVING A CAUSAL ROLE FOR THE DE-METHYLATED TSDR IN FOXP3 REGULATION. HOWEVER, SUCCESSFUL FOXP3 INDUCTION WAS NOT ASSOCIATED WITH A SWITCH TOWARDS A FUNCTIONAL TREG PHENOTYPE, IN CONTRAST TO WHAT HAS BEEN REPORTED FROM FOXP3 OVEREXPRESSION APPROACHES. THUS, TSDR DE-METHYLATION IS REQUIRED, BUT NOT SUFFICIENT FOR A STABLE TREG PHENOTYPE INDUCTION. THEREFORE, TARGETED DEMETHYLATION OF THE TSDR MAY BE A CRITICAL ADDITION TO PUBLISHED IN VITRO TREG INDUCTION PROTOCOLS WHICH SO FAR LACK FOXP3 STABILITY. 2020 3 556 49 AZACYTIDINE TREATMENT INHIBITS THE PROGRESSION OF HERPES STROMAL KERATITIS BY ENHANCING REGULATORY T CELL FUNCTION. OCULAR INFECTION WITH HERPES SIMPLEX VIRUS 1 (HSV-1) SETS OFF AN INFLAMMATORY REACTION IN THE CORNEA WHICH LEADS TO BOTH VIRUS CLEARANCE AND CHRONIC LESIONS THAT ARE ORCHESTRATED BY CD4 T CELLS. APPROACHES THAT ENHANCE THE FUNCTION OF REGULATORY T CELLS (TREG) AND DAMPEN EFFECTOR T CELLS CAN BE EFFECTIVE TO LIMIT STROMAL KERATITIS (SK) LESION SEVERITY. IN THIS REPORT, WE EXPLORE THE NOVEL APPROACH OF INHIBITING DNA METHYLTRANSFERASE ACTIVITY USING 5-AZACYTIDINE (AZA; A CYTOSINE ANALOG) TO LIMIT HSV-1-INDUCED OCULAR LESIONS. WE SHOW THAT THERAPY BEGUN AFTER INFECTION WHEN VIRUS WAS NO LONGER ACTIVELY REPLICATING RESULTED IN A PRONOUNCED REDUCTION IN LESION SEVERITY, WITH MARKEDLY DIMINISHED NUMBERS OF T CELLS AND NONLYMPHOID INFLAMMATORY CELLS, ALONG WITH REDUCED CYTOKINE MEDIATORS. THE REMAINING INFLAMMATORY REACTIONS HAD A CHANGE IN THE RATIO OF CD4 FOXP3(+) TREG TO EFFECTOR TH1 CD4 T CELLS IN OCULAR LESIONS AND LYMPHOID TISSUES, WITH TREG BECOMING PREDOMINANT OVER THE EFFECTORS. IN ADDITION, COMPARED TO THOSE FROM CONTROL MICE, TREG FROM AZA-TREATED MICE SHOWED MORE SUPPRESSOR ACTIVITY IN VITRO AND EXPRESSED HIGHER LEVELS OF ACTIVATION MOLECULES. ADDITIONALLY, CELLS INDUCED IN VITRO IN THE PRESENCE OF AZA SHOWED EPIGENETIC DIFFERENCES IN THE TREG-SPECIFIC DEMETHYLATED REGION (TSDR) OF FOXP3 AND WERE MORE STABLE WHEN EXPOSED TO INFLAMMATORY CYTOKINES. OUR RESULTS SHOW THAT THERAPY WITH AZA IS AN EFFECTIVE MEANS OF CONTROLLING A VIRUS-INDUCED INFLAMMATORY REACTION AND MAY ACT MAINLY BY THE EFFECTS ON TREG.IMPORTANCE HSV-1 INFECTION HAS BEEN SHOWN TO INITIATE AN INFLAMMATORY REACTION IN THE CORNEA THAT LEADS TO TISSUE DAMAGE AND LOSS OF VISION. THE INFLAMMATORY REACTION IS ORCHESTRATED BY GAMMA INTERFERON (IFN-GAMMA)-SECRETING TH1 CELLS, AND REGULATORY T CELLS PLAY A PROTECTIVE ROLE. HENCE, NOVEL THERAPEUTICS THAT CAN REBALANCE THE RATIO OF REGULATORY T CELLS TO EFFECTORS ARE A RELEVANT ISSUE. THIS STUDY OPENS UP A NEW AVENUE IN TREATING HSV-INDUCED SK LESIONS BY INCREASING THE STABILITY AND FUNCTION OF REGULATORY T CELLS USING THE DNA METHYLTRANSFERASE INHIBITOR 5-AZACYTIDINE (AZA). AZA INCREASED THE FUNCTION OF REGULATORY T CELLS, LEADING TO ENHANCED SUPPRESSIVE ACTIVITY AND DIMINISHED LESIONS. HENCE, THERAPY WITH AZA, WHICH ACTS MAINLY BY ITS EFFECTS ON TREG, CAN BE AN EFFECTIVE MEANS TO CONTROL VIRUS-INDUCED INFLAMMATORY LESIONS. 2017 4 407 42 ANALYSIS OF FOXP3+ REGULATORY T CELLS THAT DISPLAY APPARENT VIRAL ANTIGEN SPECIFICITY DURING CHRONIC HEPATITIS C VIRUS INFECTION. WE REPORTED PREVIOUSLY THAT A PROPORTION OF NATURAL CD25(+) CELLS ISOLATED FROM THE PBMC OF HCV PATIENTS CAN FURTHER UPREGULATE CD25 EXPRESSION IN RESPONSE TO HCV PEPTIDE STIMULATION IN VITRO, AND PROPOSED THAT VIRUS-SPECIFIC REGULATORY T CELLS (TREG) WERE PRIMED AND EXPANDED DURING THE DISEASE. HERE WE DESCRIBE EPIGENETIC ANALYSIS OF THE FOXP3 LOCUS IN HCV-RESPONSIVE NATURAL CD25(+) CELLS AND SHOW THAT THESE CELLS ARE NOT ACTIVATED CONVENTIONAL T CELLS EXPRESSING FOXP3, BUT HARD-WIRED TREG WITH A STABLE FOXP3 PHENOTYPE AND FUNCTION. OF APPROXIMATELY 46,000 GENES ANALYZED IN GENOME WIDE TRANSCRIPTION PROFILING, ABOUT 1% WERE DIFFERENTIALLY EXPRESSED BETWEEN HCV-RESPONSIVE TREG, HCV-NON-RESPONSIVE NATURAL CD25(+) CELLS AND CONVENTIONAL T CELLS. EXPRESSION PROFILES, INCLUDING CELL DEATH, ACTIVATION, PROLIFERATION AND TRANSCRIPTIONAL REGULATION, SUGGEST A SURVIVAL ADVANTAGE OF HCV-RESPONSIVE TREG OVER THE OTHER CELL POPULATIONS. SINCE NO TREG-SPECIFIC ACTIVATION MARKER IS KNOWN, WE TESTED 97 NS3-DERIVED PEPTIDES FOR THEIR ABILITY TO ELICIT CD25 RESPONSE (ASSUMING IT IS A SURROGATE MARKER), ACCOMPANIED BY HIGH RESOLUTION HLA TYPING OF THE PATIENTS. SOME REACTIVE PEPTIDES OVERLAPPED WITH PREVIOUSLY DESCRIBED EFFECTOR T CELL EPITOPES. OUR DATA OFFERS NEW INSIGHTS INTO HCV IMMUNE EVASION AND TOLERANCE, AND HIGHLIGHTS THE NON-SELF SPECIFIC NATURE OF TREG DURING INFECTION. 2009 5 5431 34 REGULATORY T CELLS: PATHOPHYSIOLOGICAL ROLES AND CLINICAL APPLICATIONS. INFLAMMATION AND IMMUNE RESPONSES AFTER TISSUE INJURY PLAY PIVOTAL ROLES IN THE RESOLUTION OF INFLAMMATION, TISSUE RECOVERY, FIBROSIS, AND REMODELING. REGULATORY T CELLS (TREGS) ARE RESPONSIBLE FOR IMMUNE TOLERANCE AND ARE USUALLY ACTIVATED IN SECONDARY LYMPHATIC TISSUES. ACTIVATED TREGS SUBSEQUENTLY REGULATE EFFECTOR T CELL AND DENDRITIC CELL ACTIVATION. FOR CLINICAL APPLICATIONS SUCH AS THE SUPPRESSION OF BOTH AUTOIMMUNE DISEASES AND THE REJECTION OF TRANSPLANTED ORGANS, METHODS TO GENERATE STABILIZED ANTIGEN-SPECIFIC TREGS ARE REQUIRED. FOR THIS PURPOSE, TRANSCRIPTIONAL AND EPIGENETIC REGULATION OF FOXP3 EXPRESSION HAS BEEN INVESTIGATED. IN ADDITION TO CONVENTIONAL TREGS, THERE ARE SOME TREGS THAT RESIDE IN TISSUES AND ARE CALLED TISSUE TREGS. TISSUE TREGS EXHIBIT TISSUE-SPECIFIC FUNCTIONS THAT CONTRIBUTE TO THE MAINTENANCE OF TISSUE HOMEOSTASIS AND REPAIR. SUCH TISSUE TREGS COULD ALSO BE USEFUL FOR TREG-BASED CELL THERAPY. WE RECENTLY DISCOVERED BRAIN TREGS THAT ACCUMULATE IN THE BRAIN DURING THE CHRONIC PHASE OF ISCHEMIC BRAIN INJURY. BRAIN TREGS RESEMBLE OTHER TISSUE TREGS, BUT ARE UNIQUE IN EXPRESSING NEURAL CELL-SPECIFIC GENES SUCH AS THE SEROTONIN RECEPTOR (HTR7); CONSEQUENTLY, BRAIN TREGS RESPOND TO SEROTONIN. HERE, WE DESCRIBE OUR EXPERIENCES IN THE USE OF TREGS TO SUPPRESS GRAFT-VERSUS-HOST DISEASE AND TO PROMOTE NEURAL RECOVERY AFTER STROKE. 2020 6 3662 39 INDUCTION OF STABLE HUMAN FOXP3(+) TREGS BY A PARASITE-DERIVED TGF-BETA MIMIC. IMMUNE HOMEOSTASIS IN THE INTESTINE IS TIGHTLY CONTROLLED BY FOXP3(+) REGULATORY T CELLS (TREGS), DEFECTS OF WHICH ARE LINKED TO THE DEVELOPMENT OF CHRONIC CONDITIONS, SUCH AS INFLAMMATORY BOWEL DISEASE (IBD). AS A MECHANISM OF IMMUNE EVASION, SEVERAL SPECIES OF INTESTINAL PARASITES BOOST TREG ACTIVITY. THE PARASITE HELIGMOSOMOIDES POLYGYRUS IS KNOWN TO SECRETE A MOLECULE (HP-TGM) THAT MIMICS THE ABILITY OF TGF-BETA TO INDUCE FOXP3 EXPRESSION IN CD4(+) T CELLS. THE STUDY AIMED TO INVESTIGATE WHETHER HP-TGM COULD INDUCE HUMAN FOXP3(+) TREGS AS A POTENTIAL THERAPEUTIC APPROACH FOR INFLAMMATORY DISEASES. CD4(+) T CELLS FROM HEALTHY VOLUNTEERS WERE EXPANDED IN THE PRESENCE OF HP-TGM OR TGF-BETA. TREG INDUCTION WAS MEASURED BY FLOW CYTOMETRIC DETECTION OF FOXP3 AND OTHER TREG MARKERS, SUCH AS CD25 AND CTLA-4. EPIGENETIC CHANGES WERE DETECTED USING CHIP-SEQ AND PYROSEQUENCING OF FOXP3. TREG PHENOTYPE STABILITY WAS ASSESSED FOLLOWING INFLAMMATORY CYTOKINE CHALLENGE AND TREG FUNCTION WAS EVALUATED BY CELLULAR CO-CULTURE SUPPRESSION ASSAYS AND CYTOMETRIC BEAD ARRAYS FOR SECRETED CYTOKINES. HP-TGM EFFICIENTLY INDUCED FOXP3 EXPRESSION (> 60%), IN ADDITION TO CD25 AND CTLA-4, AND CAUSED EPIGENETIC MODIFICATION OF THE FOXP3 LOCUS TO A GREATER EXTENT THAN TGF-BETA. HP-TGM-INDUCED TREGS HAD SUPERIOR SUPPRESSIVE FUNCTION COMPARED WITH TGF-BETA-INDUCED TREGS, AND RETAINED THEIR PHENOTYPE FOLLOWING EXPOSURE TO INFLAMMATORY CYTOKINES. FURTHERMORE, HP-TGM INDUCED A TREG-LIKE PHENOTYPE IN IN VIVO DIFFERENTIATED TH1 AND TH17 CELLS, INDICATING ITS POTENTIAL TO RE-PROGRAM MEMORY CELLS TO ENHANCE IMMUNE TOLERANCE. THESE DATA INDICATE HP-TGM HAS POTENTIAL TO BE USED TO GENERATE STABLE HUMAN FOXP3(+) TREGS TO TREAT IBD AND OTHER INFLAMMATORY DISEASES. 2021 7 1994 34 EPIGENETIC AND GENE EXPRESSION ALTERATIONS OF FOXP3 IN THE T CELLS OF EAE MOUSE MODEL OF MULTIPLE SCLEROSIS. MULTIPLE SCLEROSIS (MS) IS A CHRONIC AUTOIMMUNE DISEASE WITH DEMYELINATION AND NEURODEGENERATION OF THE CENTRAL NERVOUS SYSTEM. IT HAS BEEN SHOWN THAT THE REGULATORY T (TREG) CELLS ARE RESPONSIBLE FOR MAINTAINING TOLERANCE TO SELF-ANTIGENS AND CAN SUPPRESS THE AUTOIMMUNE PROCESS IN SEVERAL ANIMAL MODELS SUCH AS EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE), A MOUSE MODEL OF MS. RECENT BASIC STUDIES HAVE DEMONSTRATED THAT FORKHEAD BOX P (FOXP3) AND BTB DOMAIN AND CNC HOMOLOG 2 (BACH2) ARE THE MASTER TRANSCRIPTION FACTORS OF THESE CELLS PLAYING A PIVOTAL ROLE IN THE POLARIZATION OF NAIVE T CELLS INTO TREG CELLS. IN THE CURRENT STUDY, THE EXPRESSION OF FOXP3 AND BACH2 GENES AND FOXP3 PROMOTER METHYLATION WERE EVALUATED IN T CELLS OF THE EAE-INDUCED MICE. THE RESULTS OF THIS STUDY SHOWED A PROMINENT AND SIGNIFICANT HYPERMETHYLATION OF THE FOXP3 GENE PROMOTER IN THE EAE-INDUCED MICE COMPARED TO THE SHAM AND CONTROL GROUPS. THE EXPRESSION OF FOXP3 AND BACH2 GENES WAS SIGNIFICANTLY DECREASED IN THE EAE GROUP IN COMPARISON WITH THE SHAM AND CONTROL GROUPS. THIS STUDY SUGGESTS THAT THE EPIGENETIC MODIFICATION OF FOXP3 GENE IS INVOLVED IN THE PATHOGENESIS OF EAE AND THIS COULD BE IMPORTANT IN THERAPY IN AN APPROPRIATE AND LOGICAL STATEMENT. 2017 8 5223 34 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 9 1479 30 DIVERSE TARGETS OF THE TRANSCRIPTION FACTOR STAT3 CONTRIBUTE TO T CELL PATHOGENICITY AND HOMEOSTASIS. STAT3, AN ESSENTIAL TRANSCRIPTION FACTOR WITH PLEIOTROPIC FUNCTIONS, PLAYS CRITICAL ROLES IN THE PATHOGENESIS OF AUTOIMMUNITY. DESPITE RECENT DATA LINKING STAT3 WITH INFLAMMATORY BOWEL DISEASE, EXACTLY HOW IT CONTRIBUTES TO CHRONIC INTESTINAL INFLAMMATION IS NOT KNOWN. USING A T CELL TRANSFER MODEL OF COLITIS, WE FOUND THAT STAT3 EXPRESSION IN T CELLS WAS ESSENTIAL FOR THE INDUCTION OF BOTH COLITIS AND SYSTEMIC INFLAMMATION. STAT3 WAS CRITICAL IN MODULATING THE BALANCE OF T HELPER 17 (TH17) AND REGULATORY T (TREG) CELLS, AS WELL AS IN PROMOTING CD4(+) T CELL PROLIFERATION. WE USED CHROMATIN IMMUNOPRECIPITATION AND MASSIVE PARALLEL SEQUENCING (CHIP-SEQ) TO DEFINE THE GENOME-WIDE TARGETS OF STAT3 IN CD4(+) T CELLS. WE FOUND THAT STAT3 BOUND TO MULTIPLE GENES INVOLVED IN TH17 CELL DIFFERENTIATION, CELL ACTIVATION, PROLIFERATION, AND SURVIVAL, REGULATING BOTH EXPRESSION AND EPIGENETIC MODIFICATIONS. THUS, STAT3 ORCHESTRATES MULTIPLE CRITICAL ASPECTS OF T CELL FUNCTION IN INFLAMMATION AND HOMEOSTASIS. 2010 10 6293 26 THE PRO- AND ANTI-INFLAMMATORY POTENTIAL OF IL-12: THE DUAL ROLE OF TH1 CELLS. THE DIFFERENTIATION OF T-HELPER (TH) LYMPHOCYTES INTO VARIOUS TYPES OF T-HELPER EFFECTOR AND MEMORY CELLS WITH DISTINCT FUNCTIONS DEPENDING ON THE TYPE OF CONCOMITANT SIGNALS THEY RECEIVE UPON ACTIVATION IS A CRITICAL EVENT DETERMINING THE COURSE OF AN IMMUNE REACTION. TH1 CELLS CHARACTERIZED BY THE EXPRESSION OF IFN-GAMMA AND THE RECENTLY DESCRIBED TH17 CELLS PROMOTE INFLAMMATION AND ARE CRITICALLY INVOLVED IN THE INDUCTION AND MAINTENANCE OF AUTOIMMUNITY, WHEREAS THE SECRETION OF IL-4 IS A HALLMARK OF TH2 CELLS MEDIATING PROTECTION FROM PARASITES AND ALLERGY. ORIGINAL STIMULATION IN THE PRESENCE OF IL-12 RESULTS IN THE IMPRINTING OF TH1 MEMORY CELLS FOR THE EXPRESSION OF IFN-GAMMA BY EXPRESSION OF THE TRANSCRIPTION FACTOR T-BET AND EPIGENETIC MODIFICATION OF THE IFNGAMMA GENE. IT HAS BEEN DEMONSTRATED THAT TH1 CELLS ARE POTENT INDUCERS OF INFLAMMATION. HOWEVER, IN THE CHRONIC PHASE OF SUCH INFLAMMATION, THE REGULATORY POTENTIAL OF IL-12 AND TH1 CELLS THEMSELVES MAY PLAY AN IMPORTANT ROLE IN LIMITING IMMUNOPATHOLOGY. 2007 11 6020 36 THE ATTENUATION OF RENAL FIBROSIS BY HISTONE DEACETYLASE INHIBITORS IS ASSOCIATED WITH THE PLASTICITY OF FOXP3(+)IL-17(+) T CELLS. BACKGROUND: THE HISTONE DEACETYLASE (HDAC) INHIBITOR, WHICH HAS POTENTIAL EFFECTS ON EPIGENETIC MODIFICATIONS, HAD BEEN REPORTED TO ATTENUATE RENAL FIBROSIS. CD4(+) FORKHEAD BOX P3 (FOXP3)(+) T REGULATORY (TREG) CELLS MAY BE CONVERTED TO INFLAMMATION-ASSOCIATED T HELPER 17 CELLS (TH17) WITH TISSUE FIBROSIS PROPERTIES. THE ASSOCIATION BETWEEN FOXP3(+)IL-17(+) T CELLS AND THE ATTENUATION OF RENAL FIBROSIS BY THE HDAC INHIBITOR IS NOT CLEAR. METHODS: THIS STUDY EVALUATED THE ROLES OF THE HDAC INHIBITOR, TREG CELLS AND THEIR DIFFERENTIATION INTO TH17 CELLS, WHICH AGGRAVATE CHRONIC INFLAMMATION AND RENAL FIBROSIS IN A UNILATERAL URETERAL OBSTRUCTION (UUO) MOUSE MODEL. THE STUDY GROUPS INCLUDED CONTROL AND UUO MICE THAT WERE MONITORED FOR 7, 14 OR 21 DAYS. RESULTS: JUXTAGLOMERULAR (JG) HYPERPLASIA, ANGIOTENSIN II TYPE 1 RECEPTOR (AT1R) EXPRESSION AND LYMPHOCYTE INFILTRATION WERE OBSERVED IN RENAL TISSUES AFTER UUO BUT WERE DECREASED AFTER TRICHOSTATIN A (TSA) TREATMENT, A HDAC INHIBITOR. THE NUMBER OF CD4(+)FOXP3(+) T CELLS INCREASED PROGRESSIVELY, ALONG WITH THE NUMBER OF FOXP3(+)INTERLEUKIN (IL)-17(+) T CELLS, AFTER 14 DAYS, AND THEIR NUMBERS THEN PROGRESSIVELY DECREASED WITH INCREASING CD4(+)IL-17(+) T CELL NUMBERS, AS DEMONSTRATED BY DOUBLE IMMUNOHISTOCHEMISTRY. PROGRESSIVE RENAL FIBROSIS WAS ASSOCIATED WITH THE LOSS OF CD4(+)FOXP3(+)IL-17(+) T CELLS IN SPLENIC SINGLE-CELL SUSPENSIONS. FOXP3(+)IL-17(+) T CELLS EXPRESSED TGF-BETA1 BOTH IN VITRO AND IN VIVO, AND TGF-BETA1 EXPRESSION WAS SIGNIFICANTLY KNOCKDOWN BY IL-17 SIRNA IN VITRO. THESE CELLS WERE FOUND TO PLAY A ROLE IN CONVERTING TREGS INTO IL-17- AND TGF-BETA1-PRODUCING CELLS. CONCLUSIONS: TSA TREATMENT DECREASED JG HYPERPLASIA, THE PERCENTAGE OF FOXP3(+)IL-17(+) CELLS AND THE DEGREE OF FIBROSIS, SUGGESTING THAT THERAPEUTIC BENEFITS MAY RESULT FROM EPIGENETIC MODIFICATIONS. 2017 12 3893 30 LACTATE INDUCES METABOLIC AND EPIGENETIC REPROGRAMMING OF PRO-INFLAMMATORY TH17 CELLS. INCREASED LACTATE LEVELS IN THE TISSUE MICROENVIRONMENT ARE A WELL-KNOWN FEATURE OF CHRONIC INFLAMMATION. HOWEVER, THE ROLE OF LACTATE IN REGULATING T CELL FUNCTION REMAINS CONTROVERSIAL. HERE, WE DEMONSTRATE THAT EXTRACELLULAR LACTATE PREDOMINANTLY INDUCES DEREGULATION OF THE TH17-SPECIFIC GENE EXPRESSION PROGRAM BY MODULATING THE METABOLIC AND EPIGENETIC STATUS OF TH17 CELLS. FOLLOWING LACTATE TREATMENT, TH17 CELLS SIGNIFICANTLY REDUCED THEIR IL-17A PRODUCTION AND UPREGULATED FOXP3 EXPRESSION THROUGH ROS-DRIVEN IL-2 SECRETION. MOREOVER, WE OBSERVED INCREASED LEVELS OF GENOME-WIDE HISTONE H3K18 LACTYLATION, A RECENTLY DESCRIBED MARKER FOR ACTIVE CHROMATIN IN MACROPHAGES, IN LACTATE-TREATED TH17 CELLS. IN ADDITION, WE SHOW THAT HIGH LACTATE CONCENTRATIONS SUPPRESS TH17 PATHOGENICITY DURING INTESTINAL INFLAMMATION IN MICE. THESE RESULTS INDICATE THAT LACTATE IS CAPABLE OF REPROGRAMMING PRO-INFLAMMATORY T CELL PHENOTYPES INTO REGULATORY T CELLS. 2022 13 2146 30 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 14 6071 38 THE DNA METHYLATION INHIBITOR 5-AZACYTIDINE INCREASES REGULATORY T CELLS AND ALLEVIATES AIRWAY INFLAMMATION IN OVALBUMIN-SENSITIZED MICE. BACKGROUND: ASTHMA IS CHARACTERIZED AS A CHRONIC INFLAMMATORY DISORDER OF THE AIRWAYS ASSOCIATED WITH AN ENHANCED TH2 RESPONSE TO INHALED ALLERGENS. CD4+ T REGULATORY (TREG) CELLS ARE CONTROLLED BY THE MASTER TRANSCRIPTION FACTOR FOXP3 AND STRICTLY MAINTAIN PERIPHERAL IMMUNOTOLERANCE. EPIGENETIC REGULATION OF FOXP3 BY DNA METHYLTRANSFERASE INHIBITORS, SUCH AS 5-AZACYTIDINE (AZA), CAN GENERATE A STEADY SUPPLY OF FUNCTIONAL TREG CELLS. THEREFORE, WE PROPOSE THAT AZA CAN AUGMENT TREG CELLS IN VIVO TO PREVENT THE PATHOGENESIS OF ASTHMA. METHODS: BALB/C MICE WERE SENSITIZED WITH CHICKEN OVALBUMIN (OVA) AND TREATED WITH DIFFERENT DOSES OF AZA. AIRWAY HYPERRESPONSIVENESS TO METHACHOLINE, EOSINOPHILIA IN BRONCHOALVEOLAR LAVAGE FLUID, CIRCULATING TITERS OF OVA-SPECIFIC IGG1 AND IGE, AND STIMULATING LEVELS OF TH2 CYTOKINES FROM SPLENOCYTES WERE THEN DETERMINED. CELLULAR POPULATIONS WERE EXAMINED BY FLOW CYTOMETRY. PC61 ANTIBODY, WHICH DEPLETES CD25+ CELLS, WAS USED TO VERIFY THE ROLE OF CD25+ CELLS IN AZA-INDUCED TOLERANCE. RESULTS: ADMINISTRATION OF AZA TO OVA-SENSITIZED MICE DIMINISHED AIRWAY HYPERREACTIVITY, PULMONARY EOSINOPHILIA, LEVELS OF OVA-SPECIFIC IGG1 AND IGE IN SERUM, AND SECRETION OF TH2 CYTOKINES FROM OVA-STIMULATED SPLENOCYTES IN A DOSE-DEPENDENT MANNER. PERCENTAGES OF CD25+ AND FOXP3+ CELLS IN THE CD4+ CELL POPULATION WERE NOTABLY INCREASED IN AZA-TREATED MICE COMPARED TO SENSITIZED CONTROL MICE. FURTHERMORE, THE MAJOR SYMPTOMS OF ASTHMA WERE EXACERBATED BY DEPLETING CD25+ CELLS IN AZA-TREATED MICE. CONCLUSIONS: AZA MAY HAVE APPLICATIONS AS A NOVEL CLINICAL STRATEGY TO INCREASE THE PRODUCTION OF TREG CELLS IN ORDER TO MODULATE THE AIRWAY INFLAMMATION ASSOCIATED WITH ASTHMA. 2013 15 2389 40 EPIGENETIC REPOLARIZATION OF T LYMPHOCYTES FROM CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS USING 5-AZA-2'-DEOXYCYTIDINE. T CELL IMMUNE DYSFUNCTION HAS AN IMPORTANT ROLE IN THE PROFOUND IMMUNE SUPPRESSION THAT CHARACTERIZES CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). IMPROPER POLARIZATION OF T CELLS HAS BEEN PROPOSED AS ONE OF THE MECHANISM INVOLVED. MOUNTING DATA IMPLICATES CHROMATIN REGULATION, NAMELY PROMOTER METHYLATION, IN THE PLASTICITY OF NAIVE HUMAN T CELLS. RECENT IN VITRO EVIDENCE INDICATES THAT THIS PLASTICITY MAY BE PHENOTYPICALLY ALTERED BY USING METHYLATION INHIBITORS WHICH ARE APPROVED FOR CLINICAL USE IN CERTAIN TYPES OF CANCER. THESE RESULTS BEG THE QUESTION: CAN THE INEFFECTIVE POLARIZATION OF T LYMPHOCYTES IN THE CONTEXT OF CLL BE EFFECTIVELY MODULATED USING METHYLATION INHIBITORS IN A SUSTAINABLE THERAPEUTIC FASHION? TO ANSWER THIS QUESTION OUR LABORATORY HAS STUDIED THE EFFECTS OF 5-AZA-2'-DEOXYCYTIDINE (5A2) IN HELPER AND CYTOTOXIC T LYMPHOCYTES FROM HEALTHY DONORS AND CLL PATIENTS IN WELL CHARACTERIZED MOLECULAR AND EPIGENETIC SIGNALING PATHWAYS INVOLVED IN EFFECTIVE POLARIZATION. MOREOVER, WE SOUGHT TO INVESTIGATE THE CONSEQUENCES OF METHYLATION INHIBITOR TREATMENT ON LYMPHOCYTE SURVIVAL, ACTIVATION INTENSITY, AND NAIVE CELL POLARIZATION. OUR DATA INDICATES THAT 5A2 TREATMENT CAN DEPOLARIZE TH2 CELLS TO EFFECTIVELY SECRETE INTERFERON GAMMA, SIGNAL VIA T-BET, AND ACHIEVE DEMETHYLATION OF CRITICAL TH1 SPECIFIC PROMOTERS. MOREOVER, WE DEMONSTRATE THAT 5A2 CAN FORCE TH1 POLARIZATION OF NAIVE T CELLS DESPITE A STRONG IL-4 STIMULI AND A LACK OF IL-12. IN CONCLUSION OUR DATA SEEKS TO DEFINE A MODALITY IN WHICH IMPROPER OR INEFFECTIVE T CELL POLARIZATION CAN BE ALTERED BY 5AZA AND COULD BE INCORPORATED IN FUTURE THERAPEUTIC INTERVENTIONS. 2011 16 2056 25 EPIGENETIC CONTROL OF CD8(+) T CELL DIFFERENTIATION. UPON STIMULATION, SMALL NUMBERS OF NAIVE CD8(+) T CELLS PROLIFERATE AND DIFFERENTIATE INTO A VARIETY OF MEMORY AND EFFECTOR CELL TYPES. CD8(+) T CELLS CAN PERSIST FOR YEARS AND KILL TUMOUR CELLS AND VIRALLY INFECTED CELLS. THE FUNCTIONAL AND PHENOTYPIC CHANGES THAT OCCUR DURING CD8(+) T CELL DIFFERENTIATION ARE WELL CHARACTERIZED, BUT THE EPIGENETIC STATES THAT UNDERLIE THESE CHANGES ARE INCOMPLETELY UNDERSTOOD. HERE, WE REVIEW THE EPIGENETIC PROCESSES THAT DIRECT CD8(+) T CELL DIFFERENTIATION AND FUNCTION. WE FOCUS ON EPIGENETIC MODIFICATION OF DNA AND ASSOCIATED HISTONES AT GENES AND THEIR REGULATORY ELEMENTS. WE ALSO DESCRIBE STRUCTURAL CHANGES IN CHROMATIN ORGANIZATION THAT AFFECT GENE EXPRESSION. FINALLY, WE EXAMINE THE TRANSLATIONAL POTENTIAL OF EPIGENETIC INTERVENTIONS TO IMPROVE CD8(+) T CELL FUNCTION IN INDIVIDUALS WITH CHRONIC INFECTIONS AND CANCER. 2018 17 1500 37 DNA METHYLATION ANALYSIS OF CD4+ T CELLS IN PATIENTS WITH PSORIASIS. PSORIASIS IS A CHRONIC INFLAMMATORY SKIN DISEASE THAT IS CHARACTERIZED BY ABERRANT CROSS-TALK BETWEEN KERATINOCYTES AND IMMUNE CELLS SUCH AS CD4+ T CELLS, RESULTING IN KERATINOCYTE HYPERPROLIFERATION IN THE EPIDERMIS. DNA METHYLATION, ONE OF SEVERAL EPIGENETIC MECHANISMS, PLAYS AN IMPORTANT ROLE IN GENE EXPRESSION WITHOUT CHANGING THE DNA SEQUENCE. SEVERAL STUDIES HAVE SUGGESTED THE INVOLVEMENT OF EPIGENETIC REGULATION IN SKIN LESIONS FROM PATIENTS WITH PSORIASIS. IN THIS STUDY, WE INVESTIGATED THE GENOME-WIDE DNA METHYLATION STATUS OF CD4+ T CELLS IN PATIENTS WITH PSORIASIS COMPARED WITH HEALTHY SUBJECTS USING METHYLATED DNA IMMUNOPRECIPITATION SEQUENCING (MEDIP-SEQ). THE RESULTS OF MEDIP-SEQ SHOWED THAT THE GLOBAL METHYLATION VALUES OF CD4+ T CELLS ARE HIGHER IN PATIENTS WITH PSORIASIS THAN IN HEALTHY CONTROLS, PARTICULARLY IN THE PROMOTER REGIONS. AMONG THE MOST HYPERMETHYLATED GENES IN THE PROMOTER REGIONS, WE SELECTED THE GENES WHOSE EXPRESSION IS SIGNIFICANTLY REDUCED IN THE CD4+ T CELLS OF PSORIASIS PATIENTS. STUDIES USING THE METHYLATION INHIBITOR 5-AZACYTIDINE IN VITRO METHYLATION ASSAYS HAVE SHOWN THAT THE DIFFERENTIAL EXPRESSION LEVELS WERE ASSOCIATED WITH THE METHYLATION STATUS OF EACH GENE. BISULFITE SEQUENCING OF THE TRANSCRIPTION START REGION OF PHOSPHATIDIC ACID PHOSPHATASE TYPE 2 DOMAIN CONTAINING 3 (PPAPDC3), ONE OF THE SELECTED GENES, SHOWED HYPERMETHYLATION IN THE CD4+ T CELLS OF PSORIASIS PATIENTS. THESE RESULTS SUGGESTED THAT THE METHYLATION STATUS, WHICH IS IDENTIFIED BY MEDIP-SEQ OF THE GENES, WAS CORRELATED WITH THE MRNA EXPRESSION LEVEL OF THE GENES. COLLECTIVELY, THE DNA METHYLATION STATUS IN CD4+ T CELLS MIGHT BE ASSOCIATED WITH THE PATHOGENESIS OF PSORIASIS. 2014 18 855 31 CHROMATIN ACCESSIBILITY LANDSCAPES OF IMMUNE CELLS IN RHEUMATOID ARTHRITIS NOMINATE MONOCYTES IN DISEASE PATHOGENESIS. BACKGROUND: RHEUMATOID ARTHRITIS (RA) IS A CHRONIC, SYSTEMIC AUTOIMMUNE DISEASE THAT INVOLVES A VARIETY OF CELL TYPES. HOWEVER, HOW THE EPIGENETIC DYSREGULATIONS OF PERIPHERAL IMMUNE CELLS CONTRIBUTE TO THE PATHOGENESIS OF RA STILL REMAINS LARGELY UNCLEAR. RESULTS: HERE, WE ANALYSED THE GENOME-WIDE ACTIVE DNA REGULATORY ELEMENTS OF FOUR MAJOR IMMUNE CELLS, NAMELY MONOCYTES, B CELLS, CD4(+) T CELLS AND CD8(+) T CELLS, IN PERIPHERAL BLOOD OF RA PATIENTS, OSTEOARTHRITIS (OA) PATIENTS AND HEALTHY DONORS USING ASSAY OF TRANSPOSASE ACCESSIBLE CHROMATIN WITH SEQUENCING (ATAC-SEQ). WE FOUND A STRONG RA-ASSOCIATED CHROMATIN DYSREGULATION SIGNATURE IN MONOCYTES, BUT NO OTHER EXAMINED CELL TYPES. MOREOVER, WE FOUND THAT SERUM C-REACTIVE PROTEIN (CRP) CAN INDUCE THE RA-ASSOCIATED CHROMATIN DYSREGULATION IN MONOCYTES VIA IN VITRO EXPERIMENTS. AND THE EXTENT OF THIS DYSREGULATION WAS REGULATED THROUGH THE TRANSCRIPTION FACTOR FRA2. CONCLUSIONS: TOGETHER, OUR STUDY REVEALED A CRP-INDUCED PATHOGENIC CHROMATIN DYSREGULATION SIGNATURE IN MONOCYTES FROM RA PATIENTS AND PREDICTED THE RESPONSIBLE SIGNALLING PATHWAY AS POTENTIAL THERAPEUTIC TARGETS FOR THE DISEASE. 2021 19 3791 41 INTERLEUKIN 6 SUPPORTS THE MAINTENANCE OF P53 TUMOR SUPPRESSOR GENE PROMOTER METHYLATION. A STRONG ASSOCIATION EXISTS BETWEEN STATES OF CHRONIC INFLAMMATION AND CANCER, AND IT IS BELIEVED THAT MEDIATORS OF INFLAMMATION MAY BE RESPONSIBLE FOR THIS PHENOMENON. INTERLEUKIN 6 (IL-6) IS AN INFLAMMATORY CYTOKINE KNOWN TO PLAY A ROLE IN THE GROWTH AND SURVIVAL OF MANY TYPES OF TUMORS, YET THE MECHANISMS EMPLOYED BY THIS PLEOMORPHIC CYTOKINE TO ACCOMPLISH THIS FEAT ARE STILL POORLY UNDERSTOOD. ANOTHER IMPORTANT FACTOR IN TUMOR DEVELOPMENT SEEMS TO BE THE HYPERMETHYLATION OF CPG ISLANDS LOCATED WITHIN THE PROMOTER REGIONS OF TUMOR SUPPRESSOR GENES. THIS COMMON EPIGENETIC ALTERATION ENABLES TUMOR CELLS TO REDUCE OR INACTIVATE THE EXPRESSION OF IMPORTANT TUMOR SUPPRESSOR AND CELL CYCLE REGULATORY GENES. HERE WE SHOW THAT IN THE IL-6-RESPONSIVE HUMAN MULTIPLE MYELOMA CELL LINE KAS 6/1, THE PROMOTER REGION OF P53 IS EPIGENETICALLY MODIFIED BY METHYLTRANSFERASES, RESULTING IN DECREASED LEVELS OF EXPRESSION. FURTHERMORE, CELLS TREATED WITH IL-6 EXHIBIT AN INCREASE IN THE EXPRESSION OF THE DNA MAINTENANCE METHYLATION ENZYME, DNMT-1. THE DNA METHYLTRANSFERASE INHIBITOR ZEBULARINE REVERSES THE METHYLATION OF THE P53 PROMOTER, ALLOWING THE RESUMPTION OF ITS EXPRESSION. HOWEVER, WHEN ZEBULARINE IS WITHDRAWN FROM THE CELLS, THE REESTABLISHMENT OF THE ORIGINAL CPG ISLAND METHYLATION WITHIN THE P53 PROMOTER DOES NOT OCCUR IN THE ABSENCE OF IL-6, AND CELLS WHICH DO NOT RECEIVE IL-6 EVENTUALLY DIE, AS P53 EXPRESSION CONTINUES UNCHECKED BY REMETHYLATION. INTERESTINGLY, THIS LOSS OF VIABILITY SEEMS TO INVOLVE NOT THE WITHDRAWAL OF CYTOKINE, BUT THE INABILITY OF THE CELL TO RESILENCE THE PROMOTER. CONSISTENT WITH THIS MODEL, WHEN CELLS THAT EXPRESS IL-6 IN AN AUTOCRINE FASHION ARE SUBJECTED TO IDENTICAL TREATMENT, P53 EXPRESSION IS REDUCED SHORTLY AFTER WITHDRAWAL OF ZEBULARINE. THEREFORE, IT SEEMS IL-6 IS CAPABLE OF MAINTAINING PROMOTER METHYLATION THUS REPRESENTING ONE OF THE POSSIBLE MECHANISMS USED BY INFLAMMATORY MEDIATORS IN THE GROWTH AND SURVIVAL OF TUMORS. 2005 20 1061 54 CLINICAL REMISSION OF SIGHT-THREATENING NON-INFECTIOUS UVEITIS IS CHARACTERIZED BY AN UPREGULATION OF PERIPHERAL T-REGULATORY CELL POLARIZED TOWARDS T-BET AND TIGIT. BACKGROUND: NON-INFECTIOUS UVEITIS CAN CAUSE CHRONIC RELAPSING AND REMITTING OCULAR INFLAMMATION, WHICH MAY REQUIRE HIGH DOSE SYSTEMIC IMMUNOSUPPRESSION TO PREVENT SEVERE SIGHT LOSS. IT HAS BEEN CLASSICALLY DESCRIBED AS AN AUTOIMMUNE DISEASE, MEDIATED BY PRO-INFLAMMATORY TH1 AND TH17 T-CELL SUBSETS. STUDIES SUGGEST THAT NATURAL IMMUNOSUPPRESSIVE CD4(+)CD25(+)FOXP3(+) T-REGULATORY CELLS (TREGS) ARE INVOLVED IN RESOLUTION OF INFLAMMATION AND MAY BE INVOLVED IN THE MAINTENANCE OF CLINICAL REMISSION. OBJECTIVE: TO INVESTIGATE WHETHER THERE IS A PERIPHERAL BLOOD IMMUNOREGULATORY PHENOTYPE ASSOCIATED WITH CLINICAL REMISSION OF SIGHT-THREATENING NON-INFECTIOUS UVEITIS BY COMPARING PERIPHERAL BLOOD LEVELS OF TREG, TH1, AND TH17, AND ASSOCIATED DNA METHYLATION AND CYTOKINE LEVELS IN PATIENTS WITH ACTIVE UVEITIC DISEASE, CONTROL SUBJECTS AND PATIENTS (WITH PREVIOUSLY ACTIVE DISEASE) IN CLINICAL REMISSION INDUCED BY IMMUNOSUPPRESSIVE DRUGS. METHODS: ISOLATED PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) FROM PERIPHERAL BLOOD SAMPLES FROM PROSPECTIVELY RECRUITED SUBJECTS WERE ANALYZED BY FLOW CYTOMETRY FOR CD3, CD4, FOXP3, TIGIT, T-BET, AND RELATED ORPHAN RECEPTOR GAMMAT. EPIGENETIC DNA METHYLATION LEVELS OF FOXP3 TREG-SPECIFIC DEMETHYLATED REGION (TSDR), FOXP3 PROMOTER, TBX21, RORC2, AND TIGIT LOCI WERE DETERMINED IN CRYOPRESERVED PBMC USING A NEXT-GENERATION SEQUENCING APPROACH. RELATED CYTOKINES WERE MEASURED IN BLOOD SERA. FUNCTIONAL SUPPRESSIVE CAPACITY OF TREG WAS ASSESSED USING T-CELL PROLIFERATION ASSAYS. RESULTS: FIFTY PATIENTS WITH UVEITIS (INTERMEDIATE, POSTERIOR, AND PANUVEITIS) AND 10 CONTROL SUBJECTS WERE RECRUITED. THE FREQUENCY OF CD4(+)CD25(+)FOXP3(+) TREG, TIGIT(+) TREG, AND T-BET(+) TREG AND THE RATIO OF TREG TO TH1 WERE SIGNIFICANTLY HIGHER IN REMISSION PATIENTS COMPARED WITH PATIENTS WITH ACTIVE UVEITIC DISEASE; AND TIGIT(+) TREGS WERE A SIGNIFICANT PREDICTOR OF CLINICAL REMISSION. TREG FROM PATIENTS IN CLINICAL REMISSION DEMONSTRATED A HIGH LEVEL OF IN VITRO SUPPRESSIVE FUNCTION COMPARED WITH TREG FROM CONTROL SUBJECTS AND FROM PATIENTS WITH UNTREATED ACTIVE DISEASE. PBMC FROM PATIENTS IN CLINICAL REMISSION HAD SIGNIFICANTLY LOWER METHYLATION LEVELS AT THE FOXP3 TSDR, FOXP3 PROMOTER, AND TIGIT LOCI AND HIGHER LEVELS AT RORC LOCI THAN THOSE WITH ACTIVE DISEASE. CLINICAL REMISSION WAS ALSO ASSOCIATED WITH SIGNIFICANTLY HIGHER SERUM LEVELS OF TRANSFORMING GROWTH FACTOR BETA AND IL-10, WHICH POSITIVELY CORRELATED WITH TREG LEVELS, AND LOWER SERUM LEVELS OF IFNGAMMA, IL-17A, AND IL-22 COMPARED WITH PATIENTS WITH ACTIVE DISEASE. CONCLUSION: CLINICAL REMISSION OF SIGHT-THREATENING NON-INFECTIOUS UVEITIS HAS AN IMMUNOREGULATORY PHENOTYPE CHARACTERIZED BY UPREGULATION OF PERIPHERAL TREG, POLARIZED TOWARD T-BET AND TIGIT. THESE FINDINGS MAY ASSIST WITH INDIVIDUALIZED THERAPY OF UVEITIS, BY INFORMING WHETHER DRUG THERAPY HAS INDUCED PHENOTYPICALLY STABLE TREG ASSOCIATED WITH LONG-TERM CLINICAL REMISSION. 2018