1 1684 149 DRUG-TOLERANT CANCER CELLS SHOW REDUCED TUMOR-INITIATING CAPACITY: DEPLETION OF CD44 CELLS AND EVIDENCE FOR EPIGENETIC MECHANISMS. CANCER STEM CELLS (CSCS) POSSESS HIGH TUMOR-INITIATING CAPACITY AND HAVE BEEN REPORTED TO BE RESISTANT TO THERAPEUTICS. VICE VERSA, THERAPY-RESISTANT CANCER CELLS SEEM TO MANIFEST CSC PHENOTYPES AND PROPERTIES. IT HAS BEEN GENERALLY ASSUMED THAT DRUG-RESISTANT CANCER CELLS MAY ALL BE CSCS ALTHOUGH THE GENERALITY OF THIS ASSUMPTION IS UNKNOWN. HERE, WE CHRONICALLY TREATED DU145 PROSTATE CANCER CELLS WITH ETOPOSIDE, PACLITAXEL AND SOME EXPERIMENTAL DRUGS (I.E., STAUROSPORINE AND 2 PACLITAXEL ANALOGS), WHICH LED TO POPULATIONS OF DRUG-TOLERANT CELLS (DTCS). SURPRISINGLY, THESE DTCS, WHEN IMPLANTED EITHER SUBCUTANEOUSLY OR ORTHOTOPICALLY INTO NOD/SCID MICE, EXHIBITED MUCH REDUCED TUMORIGENICITY OR WERE EVEN NON-TUMORIGENIC. DRUG-TOLERANT DLD1 COLON CANCER CELLS SELECTED BY A SIMILAR CHRONIC SELECTION PROTOCOL ALSO DISPLAYED REDUCED TUMORIGENICITY WHEREAS DRUG-TOLERANT UC14 BLADDER CANCER CELLS DEMONSTRATED EITHER INCREASED OR DECREASED TUMOR-REGENERATING CAPACITY. DRUG-TOLERANT DU145 CELLS DEMONSTRATED LOW PROLIFERATIVE AND CLONOGENIC POTENTIAL AND WERE VIRTUALLY DEVOID OF CD44(+) CELLS. PROSPECTIVE KNOCKDOWN OF CD44 IN DU145 CELLS INHIBITED CELL PROLIFERATION AND TUMOR REGENERATION, WHEREAS RESTORATION OF CD44 EXPRESSION IN DRUG-TOLERANT DU145 CELLS INCREASED CELL PROLIFERATION AND PARTIALLY INCREASED TUMORIGENICITY. INTERESTINGLY, DRUG-TOLERANT DU145 CELLS SHOWED BOTH INCREASES AND DECREASES IN MANY "STEMNESS" GENES. FINALLY, EVIDENCE WAS PROVIDED THAT CHRONIC DRUG EXPOSURE GENERATED DTCS VIA EPIGENETIC MECHANISMS INVOLVING MOLECULES SUCH AS CD44 AND KDM5A. OUR RESULTS THUS REVEAL THAT 1) NOT ALL DTCS ARE NECESSARILY CSCS; 2) CONVENTIONAL CHEMOTHERAPEUTIC DRUGS SUCH AS TAXOL AND ETOPOSIDE MAY DIRECTLY TARGET CD44(+) TUMOR-INITIATING CELLS; AND 3) DTCS GENERATED VIA CHRONIC DRUG SELECTION INVOLVE EPIGENETIC MECHANISMS. 2011 2 1475 21 DISTRIBUTION OF H3K27ME3, H3K9ME3, AND H3K4ME3 ALONG AUTOPHAGY-RELATED GENES HIGHLY EXPRESSED IN STARVED ZEBRAFISH MYOTUBES. THE ZEBRAFISH (DANIO RERIO) REMAINS THE TELEOST FISH OF CHOICE FOR BIOLOGICAL INVESTIGATIONS DUE TO THE VAST ARRAY OF MOLECULAR TOOLS AND RESOURCES AVAILABLE. TO BETTER UNDERSTAND THE EPIGENETIC REGULATION OF AUTOPHAGY, WE UTILIZED A PRIMARY MYOTUBE CULTURE SYSTEM GENERATED FROM ISOLATED MYOGENIC PRECURSOR CELLS (MPCS) FROM ZEBRAFISH GROWN UNDER STARVATION CONDITIONS USING A MEDIA DEVOID OF SERUM AND AMINO ACIDS. HERE, WE REPORT STARVATION-INDUCED REGULATION OF SEVERAL AUTOPHAGY-RELATED GENES (ATG) EXPRESSION AND PROFILE THE DISTRIBUTION OF H3K27ME3, H3K9ME3, AND H3K4ME3 MARKS ALONG LC3B, ATG4B AND P62/SQSTM1 LOCI. THESE DATA SUPPORT EPIGENETIC REGULATION OF AUTOPHAGY IN RESPONSE TO STARVATION THAT SUGGESTS A LEVEL OF REGULATION THAT CAN BE SUSTAINED FOR CHRONIC CONDITIONS VIA CHROMATIN MODIFICATION. 2017 3 4430 36 MOLECULAR BIOLOGY OF ONCOGENIC INFLAMMATORY PROCESSES. I. NON-ONCOGENIC AND ONCOGENIC PATHOGENS, INTRINSIC INFLAMMATORY REACTIONS WITHOUT PATHOGENS, AND MICRORNA/DNA INTERACTIONS (REVIEW). IN SOME INFLAMMASOMES TUMOR CELLS ARE GENERATED. THE INTERNAL ENVIRONMENT OF THE INFLAMMASOME IS CONDUCIVE TO THE INDUCTION OF MALIGNANT TRANSFORMATION. EPIGENETIC CHANGES INITIATE THIS PROCESS. THE SUBVERTED STROMAL CONNECTIVE TISSUE CELLS ACT TO PROMOTE AND SUSTAIN THE PROCESS OF MALIGNANT TRANS-FORMATION. IN ITS EARLY STAGES, THE PREMALIGNANT CELLS DEPEND ON PARACRINE CIRCUITRIES FOR THE RECEPTION OF GROWTH FACTORS. THE LIGANDS ARE DERIVED FROM THE CONNECTIVE TISSUE, AND THE RECEPTORS ARE EXPRESSED ON THE RECIPIENT PREMALIGNANT CELLS. THE INITIAL EVENTS ARE NOT A DIRECT ATTACK ON THE PROTO-ONCOGENES, AND THUS IT MAY BE ENTIRELY REVERSIBLE. EPIGENETIC PROCESSES OF HYPERMETHYLATION OF THE GENES AT THE PROMOTERS OF TUMOR SUPPRESSOR GENES (TO SILENCE THEM), AND DEACETYLATION OF THE HISTONES AIMED AT THE PROMOTERS OF PROTO-ONCOGENES (TO ACTIVATE THEM) ARE ON-GOING. A LARGE NUMBER OF SHORT RNA SEQUENCES (INTERFERING, MICRO-, SHORT HAIRPIN, NON-CODING RNAS) SILENCE TUMOR SUPPRESSOR GENES, BY NEUTRALIZING THEIR MRNAS. IN A SERIAL SEQUENCE ONCOGENES UNDERGO AMPLIFICATIONS, POINT-MUTATIONS, TRANSLOCATIONS AND FUSIONS. IN ITS EARLIEST STAGE, THE PROCESS IS REVERSIBLE BY DEMETHYLATION OF THE SILENCED SUPPRESSOR GENE PROMOTERS (TO REACTIVATE THEM), OR RE-ACETYLATION OF THE HISTONES OF THE ONCOGENE PROMOTERS, THUS DE-ACTIVATING THEM. THE EXTERNAL ADMINISTRATION OF HISTONE DEACETYLASE INHIBITORS USUALLY LEADS TO THE RESTORATION OF HISTONE ACETYLATION. IN TIME, THE UNCORRECTED PROCESSES SOLIDIFY INTO CONSTITUTIVE AND IRREVERSIBLE GENE MUTATIONS. SOME OF THE PATHOGENS INDUCING INFLAMMATIONS WITH CONSQUENTIAL MALIGNANT TRANSFORMATION CONTAIN ONCOGENIC GENE SEQUENCES (PAPILLOMA VIRUSES, EPSTEIN-BARR VIRUS, KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS, HEPATITIS B AND C VIRUSES, MERKEL CELL POLYOMA VIRUS, HELICOBACTER PYLORI, ENTEROTOXIGENIC BACTEROIDES FRAGILIS). THESE INDUCED MALIGNANCIES MAY BE MULTIFOCAL. OTHER PATHOGENS ARE DEVOID OF ANY KNOWN ONCOGENIC GENOMIC SEQUENCES (MYCOPLASMA VAV-CARCINOGENESIS, CHLAMYDIA MALT-LYMPHOMA GENESIS). IN THESE CASES THE HOST'S INFLAMMATORY REACTIONS INDUCE THE MALIGNANT TRANSFORMATION IN SERIAL SEQUENCES OF GENE ALTERATIONS INITIATED BY HYPOXIA AND REACTIVE OXYGEN AND NITROGEN SPECIES GENERATION. CARCINOGENIC INTRINSIC INFLAMMATORY PROCESSES ENDOGENOUSLY INITIATED WITHOUT A PATHOGEN ARE RECOGNIZED. CHRONIC INFLAMMATORY PROCESSES SIGNAL THE RNA/DNA COMPLEX. IN RESPONSE, THE DNA MAY REVERT INTO ITS ANCIENT PRIMORDIAL 'IMMORTAL' FORMAT, WHICH THE CLINICS RECOGNIZE AS 'ONCOGENESIS'. THE DNA REMAINS THE ULTIMATE MASTER OF BIOENGINEERING IN ORDER TO SUSTAIN LIFE. A DISCUSSION ON THE MOST VERSATILE AND RESISTANT PRIMORDIAL RNA/DNA COMPLEX AND THE PRE-, PROTO-, AND UNICELLULAR WORLD IN WHICH THEY CO-EXISTED IS INCLUDED. 2012 4 824 27 CHARACTERIZATION OF COPY NUMBER ALTERATIONS IN A MOUSE MODEL OF FIBROSIS-ASSOCIATED HEPATOCELLULAR CARCINOMA REVEALS CONCORDANCE WITH HUMAN DISEASE. HEPATOCELLULAR CARCINOMA (HCC) IS A PREVALENT HUMAN CANCER WITH RISING INCIDENCE WORLDWIDE. HUMAN HCC IS FREQUENTLY ASSOCIATED WITH CHRONIC LIVER INFLAMMATION AND CIRRHOSIS, PATHOPHYSIOLOGICAL PROCESSES THAT ARE A CONSEQUENCE OF CHRONIC VIRAL INFECTION, DISTURBANCES IN METABOLISM, OR EXPOSURE TO CHEMICAL TOXICANTS. TO BETTER CHARACTERIZE THE PATHOGENESIS OF HCC, WE USED A HUMAN DISEASE-RELEVANT MOUSE MODEL OF FIBROSIS-ASSOCIATED HEPATOCARCINOGENESIS. IN THIS MODEL, MARKED LIVER TUMOR RESPONSE CAUSED BY THE PROMUTAGENIC CHEMICAL N-NITROSODIETHYLAMINE IN THE PRESENCE OF LIVER FIBROSIS WAS ASSOCIATED WITH EPIGENETIC EVENTS INDICATIVE OF GENOMIC INSTABILITY. THEREFORE, WE HYPOTHESIZED THAT DNA COPY NUMBER ALTERATIONS (CNAS), A FEATURE OF GENOMIC INSTABILITY AND A COMMON CHARACTERISTIC OF CANCER, ARE CONCORDANT BETWEEN HUMAN HCC AND MOUSE MODELS OF FIBROSIS-ASSOCIATED HEPATOCARCINOGENESIS. WE EVALUATED DNA CNAS AND CHANGES IN GENE EXPRESSION IN THE MOUSE LIVER (NORMAL, TUMOR, AND NONTUMOR FIBROTIC TISSUES). ADDITIONALLY, WE COMPARED OUR FINDINGS TO DNA CNAS IN HUMAN HCC CASES (TUMOR AND NONTUMOR CIRRHOTIC/FIBROTIC TISSUES) USING PUBLICLY AVAILABLE DATA FROM THE CANCER GENOME ATLAS (TCGA). WE OBSERVED THAT WHILE FIBROTIC LIVER TISSUE IS LARGELY DEVOID OF DNA CNAS, HIGHLY FREQUENTLY OCCURRING DNA CNAS ARE FOUND IN MOUSE TUMORS, WHICH IS INDICATIVE OF A PROFOUND INCREASE IN CHROMOSOMAL INSTABILITY IN HCC. THE CROSS-SPECIES GENE-LEVEL COMPARISON OF CNAS IDENTIFIED SHARED REGIONS OF CNAS BETWEEN HUMAN FIBROSIS- AND CIRRHOSIS-ASSOCIATED LIVER TUMORS AND MOUSE FIBROSIS-ASSOCIATED HCC. OUR RESULTS SUGGEST THAT CNAS MOST COMMONLY ARISE IN NEOPLASTIC TISSUE RATHER THAN IN FIBROTIC OR CIRRHOTIC LIVER, AND DEMONSTRATE THE UTILITY OF THIS MOUSE MODEL IN REPLICATING THE MOLECULAR FEATURES OF HUMAN HCC. 2016 5 150 36 ABERRANT HYPOMETHYLATION OF THE CANCER-TESTIS ANTIGEN PRAME CORRELATES WITH PRAME EXPRESSION IN ACUTE MYELOID LEUKEMIA. PRAME IS A TUMOR-ASSOCIATED ANTIGEN, WHICH BELONGS TO THE FAMILY OF CANCER-TESTIS ANTIGENS (CTA). THE EXPRESSION OF CTA IS MAINLY RESTRICTED TO THE TESTIS AND VARIOUS TUMORS. IN CONTRAST TO OTHER CTA, PRAME EXPRESSION IS ALSO FREQUENTLY DETECTED IN ACUTE AND CHRONIC LEUKEMIAS. DUE TO THIS EXPRESSION PATTERN, PRAME HAS ATTRACTED GREAT INTEREST AS A PROGNOSTIC TUMOR MARKER THAT CAN BE USED FOR THE DETECTION OF MINIMAL RESIDUAL DISEASE AND AS A POTENTIAL TARGET FOR IMMUNOTHERAPY. IN ACUTE MYELOID LEUKEMIA (AML), PRAME EXPRESSION HAS BEEN OBSERVED IN 30-64% OF CASES. TO EVALUATE WHETHER EPIGENETIC MECHANISMS CONTRIBUTE TO PRAME ACTIVATION IN AML, WE STUDIED DNA METHYLATION OF 15 CPG DINUCLEOTIDES WITHIN A CPG-RICH REGION LOCATED IN THE INTRON 1 OF THE PRAME GENE. DNA METHYLATION WAS DETERMINED BY SEQUENCE ANALYSIS OF CLONED PCR PRODUCTS GENERATED FROM BISULFITE-TREATED GENOMIC DNA. METHYLATION PATTERNS WERE CORRELATED WITH PRAME MRNA LEVELS AS DETERMINED BY MICROARRAY ANALYSIS AND REAL-TIME PCR. WE FOUND ALMOST COMPLETE METHYLATION IN MONONUCLEAR BLOOD CELLS FROM TWO HEALTHY DONORS AND IN BONE MARROW CELLS OF FOUR PRAME-NEGATIVE AML PATIENTS. IN CONTRAST, THE DEGREE OF PRAME METHYLATION WAS CLEARLY REDUCED IN FOUR PRAME-POSITIVE AML BONE MARROW SAMPLES. IN PARTICULAR, THESE SAMPLES WERE CHARACTERIZED BY THE PRESENCE OF CLONES, WHICH WERE COMPLETELY DEVOID OF METHYLATION. THE SIGNIFICANT INVERSE CORRELATION BETWEEN THE DEGREE OF METHYLATION AND PRAME EXPRESSION SUGGESTS A CAUSAL ROLE OF DNA METHYLATION IN PRAME REGULATION. SUCH A ROLE IS FURTHER SUPPORTED BY THE OBSERVATION THAT TREATMENT OF PRAME-NEGATIVE CELL LINES U-937 AND THP-1 WITH THE DEMETHYLATING AGENT 5'-AZA-2'DC RESULTED IN A DOSE-RELATED UPREGULATION OF PRAME EXPRESSION. 2008 6 3732 25 INNATE IMMUNE MEMORY AND THE HOST RESPONSE TO INFECTION. UNLIKE THE ADAPTIVE IMMUNE SYSTEM, THE INNATE IMMUNE SYSTEM HAS CLASSICALLY BEEN CHARACTERIZED AS BEING DEVOID OF MEMORY FUNCTIONS. HOWEVER, RECENT RESEARCH SHOWS THAT INNATE MYELOID AND LYMPHOID CELLS HAVE THE ABILITY TO RETAIN MEMORY OF PRIOR PATHOGEN EXPOSURE AND BECOME PRIMED TO ELICIT A ROBUST, BROAD-SPECTRUM RESPONSE TO SUBSEQUENT INFECTION. THIS PHENOMENON HAS BEEN TERMED INNATE IMMUNE MEMORY OR TRAINED IMMUNITY. INNATE IMMUNE MEMORY IS INDUCED VIA ACTIVATION OF PATTERN RECOGNITION RECEPTORS AND THE ACTIONS OF CYTOKINES ON HEMATOPOIETIC PROGENITORS AND STEM CELLS IN BONE MARROW AND INNATE LEUKOCYTES IN THE PERIPHERY. THE TRAINED PHENOTYPE IS INDUCED AND SUSTAINED VIA EPIGENETIC MODIFICATIONS THAT REPROGRAM TRANSCRIPTIONAL PATTERNS AND METABOLISM. THESE MODIFICATIONS AUGMENT ANTIMICROBIAL FUNCTIONS, SUCH AS LEUKOCYTE EXPANSION, CHEMOTAXIS, PHAGOCYTOSIS, AND MICROBIAL KILLING, TO FACILITATE AN AUGMENTED HOST RESPONSE TO INFECTION. ALTERNATIVELY, INNATE IMMUNE MEMORY MAY CONTRIBUTE TO THE PATHOGENESIS OF CHRONIC DISEASES, SUCH AS ATHEROSCLEROSIS AND ALZHEIMER'S DISEASE. 2022 7 3167 30 GROUP 1 METABOTROPIC GLUTAMATE RECEPTOR EXPRESSION DEFINES A T CELL MEMORY POPULATION DURING CHRONIC TOXOPLASMA INFECTION THAT ENHANCES IFN-GAMMA AND PERFORIN PRODUCTION IN THE CNS. WITHIN THE BRAIN, A PRO-INFLAMMATORY RESPONSE IS ESSENTIAL TO PREVENT CLINICAL DISEASE DUE TO TOXOPLASMA GONDII REACTIVATION. INFECTION IN THE IMMUNOCOMPROMISED LEADS TO LETHAL TOXOPLASMIC ENCEPHALITIS WHILE IN THE IMMUNOCOMPETENT, THERE IS PERSISTENT LOW-GRADE INFLAMMATION WHICH IS DEVOID OF CLINICAL SYMPTOMS. THIS SIGNIFIES THAT THERE IS A WELL-BALANCED AND REGULATED INFLAMMATORY RESPONSE TO T. GONDII IN THE BRAIN. T CELLS ARE THE DOMINANT IMMUNE CELLS THAT PREVENT CLINICAL DISEASE, AND THIS IS MEDIATED THROUGH THE SECRETION OF EFFECTOR MOLECULES SUCH AS PERFORINS AND IFN-GAMMA. THE PRESENCE OF COGNATE ANTIGEN, THE EXPRESSION OF SURVIVAL CYTOKINES, AND THE ALTERATION OF THE EPIGENETIC LANDSCAPE DRIVE THE DEVELOPMENT OF MEMORY T CELLS. HOWEVER, SPECIFIC EXTRINSIC SIGNALS THAT PROMOTE THE FORMATION AND MAINTENANCE OF MEMORY T CELLS WITHIN TISSUE ARE POORLY UNDERSTOOD. DURING CHRONIC INFECTION, THERE IS AN INCREASE IN EXTRACELLULAR GLUTAMATE THAT, DUE TO ITS FUNCTION AS AN EXCITATORY NEUROTRANSMITTER, IS NORMALLY TIGHTLY CONTROLLED IN THE CNS. HERE WE DEMONSTRATE THAT CD8(+) T CELLS FROM THE T. GONDII-INFECTED BRAIN PARENCHYMA ARE ENRICHED FOR METABOTROPIC GLUTAMATE RECEPTORS (MGLUR'S). CHARACTERIZATION STUDIES DETERMINED THAT MGLUR(+) EXPRESSION BY CD8(+) T CELLS DEFINES A DISTINCT MEMORY POPULATION AT THE TRANSCRIPTIONAL AND PROTEIN LEVEL. FINALLY, USING RECEPTOR ANTAGONISTS AND AGONISTS WE DEMONSTRATE MGLUR SIGNALING IS REQUIRED FOR OPTIMAL CD8(+) T CELL PRODUCTION OF THE EFFECTOR CYTOKINE IFNGAMMA. THIS WORK SUGGESTS THAT GLUTAMATE IS AN IMPORTANT ENVIRONMENTAL SIGNAL OF INFLAMMATION THAT PROMOTES T CELL FUNCTION. UNDERSTANDING GLUTAMATE'S INFLUENCE ON T CELLS IN THE BRAIN CAN PROVIDE INSIGHTS INTO THE MECHANISMS THAT GOVERN PROTECTIVE IMMUNITY AGAINST CNS-INFILTRATING PATHOGENS AND NEUROINFLAMMATION. 2023 8 2355 31 EPIGENETIC REGULATION OF PRAME GENE IN CHRONIC MYELOID LEUKEMIA. TUMOR ASSOCIATED ANTIGENS (TAA) PROVIDE ATTRACTIVE TARGETS FOR CANCER-SPECIFIC IMMUNOTHERAPY. PRAME IS A TAA GENE UP-REGULATED IN ADVANCED PHASES OF CHRONIC MYELOID LEUKEMIA (CML). TO DATE, MOLECULAR MECHANISMS FOR THE EXPRESSION OF PRAME HAVE NEVER BEEN STUDIED. WE FOUND THAT SOME PH'-POSITIVE CELL LINES DID NOT EXPRESS PRAME. THE EXPRESSION OF PRAME WAS RESTORED IN THESE CELL LINES BY TREATMENT WITH 5'-AZA-2'-DEOXYCYTIDINE, SUGGESTING THAT THE EXPRESSION OF PRAME IS MAINLY SUPPRESSED BY HYPERMETHYLATION. BISULFITE SEQUENCING ANALYSIS OF THE CPG SITES OF THE PRAME EXON 2 IN THESE CANCER CELL LINES REVEALED A CLOSE RELATIONSHIP BETWEEN THE METHYLATION STATUS OF THE PRAME GENE AND ITS EXPRESSION. A METHYLATION-SPECIFIC PCR ANALYSIS DEMONSTRATED THAT HYPOMETHYLATION OF PRAME WAS SIGNIFICANTLY MORE FREQUENT IN CML BLAST CRISIS (70%) THAN IN CHRONIC PHASE (36%) (P=0.01) AND WAS CORRELATED WITH HIGH EXPRESSION LEVELS OF PRAME TRANSCRIPTS (P<0.0001). THESE RESULTS SUGGEST THAT HYPOMETHYLATION OF PRAME UP-REGULATES ITS EXPRESSION IN CML AND MIGHT PLAY A SIGNIFICANT ROLE IN THE PROGRESSION OF THE DISEASE. 2007 9 6376 27 THE ROLE OF NEUTROPHILS IN TRAINED IMMUNITY. THE PRINCIPLE OF TRAINED IMMUNITY REPRESENTS INNATE IMMUNE MEMORY DUE TO SUSTAINED, MAINLY EPIGENETIC, CHANGES TRIGGERED BY ENDOGENOUS OR EXOGENOUS STIMULI IN BONE MARROW (BM) PROGENITORS (CENTRAL TRAINED IMMUNITY) AND THEIR INNATE IMMUNE CELL PROGENY, THEREBY TRIGGERING ELEVATED RESPONSIVENESS AGAINST SECONDARY STIMULI. BM PROGENITORS CAN RESPOND TO MICROBIAL AND STERILE SIGNALS, THEREBY POSSIBLY ACQUIRING TRAINED IMMUNITY-MEDIATED LONG-LASTING ALTERATIONS THAT MAY SHAPE THE FATE AND FUNCTION OF THEIR PROGENY, FOR EXAMPLE, NEUTROPHILS. NEUTROPHILS, THE MOST ABUNDANT INNATE IMMUNE CELL POPULATION, ARE PRODUCED IN THE BM FROM COMMITTED PROGENITOR CELLS IN A PROCESS DESIGNATED GRANULOPOIESIS. NEUTROPHILS ARE THE FIRST RESPONDERS AGAINST INFECTIOUS OR INFLAMMATORY CHALLENGES AND HAVE VERSATILE FUNCTIONS IN IMMUNITY. TOGETHER WITH OTHER INNATE IMMUNE CELLS, NEUTROPHILS ARE EFFECTORS OF PERIPHERAL TRAINED IMMUNITY. HOWEVER, GIVEN THE SHORT LIFETIME OF NEUTROPHILS, THEIR ABILITY TO ACQUIRE IMMUNOLOGICAL MEMORY MAY LIE IN THE CENTRAL TRAINING OF THEIR BM PROGENITORS RESULTING IN GENERATION OF REPROGRAMMED, THAT IS, "TRAINED", NEUTROPHILS. ALTHOUGH TRAINED IMMUNITY MAY HAVE BENEFICIAL EFFECTS IN INFECTION OR CANCER, IT MAY ALSO MEDIATE DETRIMENTAL OUTCOMES IN CHRONIC INFLAMMATION. HERE, WE REVIEW THE EMERGING RESEARCH AREA OF TRAINED IMMUNITY WITH A PARTICULAR EMPHASIS ON THE ROLE OF NEUTROPHILS AND GRANULOPOIESIS. 2023 10 2765 29 EXPRESSION, EPIGENETIC REGULATION, AND HUMORAL IMMUNOGENICITY OF CANCER-TESTIS ANTIGENS IN CHRONIC MYELOID LEUKEMIA. OBJECTIVE: CANCER-TESTIS (CT) ANTIGENS REPRESENT ATTRACTIVE TARGETS FOR TUMOR IMMUNOTHERAPY BASED ON THEIR TUMOR-RESTRICTED EXPRESSION AND IMMUNOGENICITY. HOWEVER, A BROAD PICTURE OF THE EXPRESSION OF CT ANTIGENS AND ASSOCIATED HUMORAL IMMUNE RESPONSES IN CHRONIC MYELOID LEUKEMIA (CML) IS STILL MISSING. METHODS: WE SCREENED CML CELL LINES AND BONE MARROW (BM) SAMPLES FROM HEALTHY DONORS BY RT-PCR FOR THE EXPRESSION OF 31 CT ANTIGENS BEFORE AND AFTER TREATMENT WITH EPIGENETIC AGENTS. EXPRESSION OF TUMOR-RESTRICTED ANTIGENS WAS FURTHER EXAMINED IN 60 CML PATIENTS AND HUMORAL IMMUNE RESPONSES AGAINST 15 CT ANTIGENS WERE SCREENED BY ELISA. RESULTS: IN UNTREATED CELL LINES WE DETECTED THE EXPRESSION OF 17 CT ANTIGENS THAT WERE ABSENT FROM NORMAL BM. EXPRESSION OF MOST ANTIGENS INCREASED FOLLOWING DEMETHYLATING TREATMENT WITH 5'-AZA-2'-DEOXYCYTIDINE. IN THESE SAMPLES, ONLY PRAME WAS REPEATEDLY DETECTED AND EXPRESSION CORRELATED WITH SEVERAL CLINICOPATHOLOGICAL PARAMETERS AND DECREASED OVERALL SURVIVAL. WE FURTHER SHOW THAT A LOWER FREQUENCY OF PRAME-POSITIVE SAMPLES DURING IMATINIB TREATMENT WAS NOT CAUSED BY GENE-SPECIFIC DOWNREGULATION. ANALYZING THE PATIENTS' ANTIBODY RESPONSES WE FOUND THAT THE VAST MAJORITY OF PATIENTS LACKED SPONTANEOUS IMMUNITY AGAINST CT ANTIGENS INCLUDING PRAME. CONCLUSIONS: CT ANTIGEN EXPRESSION CAN BE INCREASED BY THE APPLICATION OF EPIGENETIC AGENTS AND THE EXPRESSION OF PRAME CORRELATES WITH CLINICOPATHOLOGICAL PARAMETERS AND OVERALL SURVIVAL IN PATIENTS WITH CML, BUT DOES NOT LEAD TO HUMORAL IMMUNE RESPONSES. PRAME-SPECIFIC IMMUNOTHERAPY MIGHT REPRESENT A PROMISING APPROACH FOR THE ERADICATION OF RESIDUAL THERAPY-RESISTANT LEUKEMIC CELLS DUE TO ITS FREQUENT EXPRESSION AND STABILITY UNDER IMATINIB TREATMENT. 2010 11 744 44 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 12 5908 30 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 13 1410 34 DIETARY INTERVENTIONS FOR AUTISM SPECTRUM DISORDER: AN UPDATED SYSTEMATIC REVIEW OF HUMAN STUDIES. AUTISM IS A COMPLEX SPECTRUM OF DISORDERS WITH GENETIC, EPIGENETIC, AUTOIMMUNE, OXIDATIVE STRESS, AND ENVIRONMENTAL ETIOLOGIES. TREATMENT OF ASD USING DIETARY APPROACH IS A PROMISING STRATEGY, ESPECIALLY OWING TO ITS SAFETY AND AVAILABILITY. OUR STUDY CRITICALLY ANALYSED THE ROLES AND EFFICACY OF ANTIOXIDANTS, PROBIOTICS, PREBIOTICS, CAMEL MILK AND VITAMIN D. THIS SYSTEMATIC REVIEW PROVIDES AN UPDATED SYNOPSIS OF HUMAN STUDIES THAT INVESTIGATED THERAPEUTIC BENEFITS OF THESE DIETARY INTERVENTIONS IN AUTISM. A TOTAL OF 943 PAPERS WERE IDENTIFIED OUT OF WHICH 21 ARTICLES WERE INCLUDED IN THE SYSTEMATIC REVIEW. THE SELECTED STUDIES INVESTIGATED THE IMPACT OF 5 DIFFERENT DIETARY SUPPLEMENTATIONS IN ASD SYMPTOM AND BEHAVIOURS. THESE AGENTS INCLUDE; ANTIOXIDANTS/POLYPHENOLIC COMPOUNDS, PROBIOTICS, PREBIOTICS, CAMEL MILK AND VITAMIN D. FROM THE RESULTS OF THE PRESENT REVIEW, ANTIOXIDANTS/POLYPHENOLIC COMPOUNDS DECREASED THE LEVELS OF INFLAMMATORY CYTOKINES AND IMPROVED BEHAVIOURAL SYMPTOMS. PROBIOTICS IMPROVED BEHAVIOURAL AND GI SYMPTOMS AS WELL AS RESTORED GUT MICROBIOTA EQUILIBRIUM. PREBIOTICS DECREASED LEVELS OF INFLAMMATORY CYTOKINES, IMPROVED BEHAVIOURAL AND GI SYMPTOMS AND IMPROVED GUT MICROBIOTA. VITAMIN D IMPROVED BEHAVIOURAL SYMPTOMS AND OFFERED PROTECTIVE EFFECTS AGAINST NEUROTOXICITY. CAMEL MILK REDUCED INFLAMMATORY RESPONSES AND OXIDATIVE STRESS. GIVEN THE CHRONIC NATURE AS WELL AS EARLY ONSET OF ASD, DIETARY SUPPLEMENTS BECOME USEFUL TO COMPLEMENT NUTRITIONAL DEFICIENCIES IN CHILDREN WITH ASD. KEY BENEFITS OF THESE AGENTS STEM FROM THEIR ABILITY TO TARGET MULTIPLE PHYSIOLOGICAL AREAS VIA THE GUT BRAIN-AXIS AND ARE DEVOID OF POTENTIAL HARMFUL OR AGGRAVATING EFFECTS ON ASD PATIENTS. THE EVIDENCE COLLATED IN THIS REVIEW PROPOSE THAT DIETARY INTERVENTION MAY PROVIDE A NEW PLATFORM FOR THE MANAGEMENT OF AUTISM. 2022 14 5223 39 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 15 5477 30 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 16 3666 28 INFECTION, STEM CELLS AND CANCER SIGNALS. THE ASSOCIATION OF CANCER WITH PRECEDING PARASITIC INFECTIONS HAS BEEN OBSERVED FOR OVER 200 YEARS. SOME SUCH CANCERS ARISE FROM INFECTION OF TISSUE STEM CELLS BY VIRUSES WITH INSERTION OF VIRAL ONCOGENES INTO THE HOST DNA (MOUSE POLYOMA VIRUS, MOUSE MAMMARY TUMOR VIRUS). IN OTHER CASES THE VIRUS DOES NOT INSERT ITS DNA INTO THE HOST CELLS, BUT RATHER COMMANDEERS THE METABOLISM OF THE INFECTED CELLS, SO THAT THE CELLS CONTINUE TO PROLIFERATE AND DO NOT DIFFERENTIATE (HUMAN PAPILLOMA VIRUS AND CERVICAL CANCER). CYTOPLASMIC EPSTEIN BARR VIRUS INFECTION IS ASSOCIATED WITH A SPECIFIC GENE TRANSLOCATION (IG/C-MYC) THAT ACTIVATES PROLIFERATION OF AFFECTED CELLS (BURKITT LYMPHOMA). IN CHRONIC OSTEOMYELITIS AN INFLAMMATORY REACTION TO THE INFECTION APPEARS TO ACT THROUGH PRODUCTION OF INFLAMMATORY CYTOKINES AND OXYGEN RADICAL FORMATION TO INDUCE EPITHELIAL CANCERS. INFECTION WITH HELICOBACTER PYLORI LEADS TO EPIGENETIC CHANGES IN METHYLATION AND INFECTION BY A PARASITE. CLONORCHIS SINENSIS ALSO ACTS AS A PROMOTER OF CANCER OF THE BILE DUCTS OF THE LIVER (CHOLANIOCARCINOMA). THE COMMON THREAD AMONG THESE DIVERSE PATHWAYS IS THAT THE INFECTIONS ACT TO ALTER TISSUE STEM CELL SIGNALING WITH CONTINUED PROLIFERATION OF TUMOR TRANSIT AMPLIFYING CELLS. 2011 17 1313 39 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 18 1759 26 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 19 6591 27 TUMOR-ASSOCIATED MACROPHAGES AS A PARADIGM OF MACROPHAGE PLASTICITY, DIVERSITY, AND POLARIZATION: LESSONS AND OPEN QUESTIONS. MACROPHAGES ARE PRESENT IN ALL BODY COMPARTMENTS, INCLUDING CANCEROUS TISSUES, AND THEIR FUNCTIONS ARE PROFOUNDLY AFFECTED BY SIGNALS FROM THE MICROENVIRONMENT UNDER HOMEOSTATIC AND PATHOLOGICAL CONDITIONS. TUMOR-ASSOCIATED MACROPHAGES ARE A MAJOR CELLULAR COMPONENT OF CANCER-RELATED INFLAMMATION AND HAVE SERVED AS A PARADIGM FOR THE PLASTICITY AND FUNCTIONAL POLARIZATION OF MONONUCLEAR PHAGOCYTES. TUMOR-ASSOCIATED MACROPHAGES CAN EXERT DUAL INFLUENCE OF CANCER DEPENDING ON THE ACTIVATION STATE, WITH CLASSICALLY ACTIVATED (M1) AND ALTERNATIVELY ACTIVATED (M2) CELLS GENERALLY EXERTING ANTITUMORAL AND PROTUMORAL FUNCTIONS, RESPECTIVELY. THESE ARE EXTREMES IN A CONTINUUM OF POLARIZATION STATES IN A UNIVERSE OF DIVERSITY. TUMOR-ASSOCIATED MACROPHAGES AFFECT VIRTUALLY ALL ASPECTS OF TUMOR TISSUES, INCLUDING STEM CELLS, METABOLISM, ANGIOGENESIS, INVASION, AND METASTASIS. PROGRESS HAS BEEN MADE IN DEFINING SIGNALING MOLECULES, TRANSCRIPTION FACTORS, EPIGENETIC CHANGES, AND REPERTOIRE OF MICRORNAS UNDERLYING MACROPHAGE POLARIZATION. PRECLINICAL AND EARLY CLINICAL DATA SUGGEST THAT MACROPHAGES MAY SERVE AS TOOLS FOR THE DEVELOPMENT OF INNOVATIVE DIAGNOSTIC AND THERAPEUTIC STRATEGIES IN CANCER AND CHRONIC NONRESOLVING INFLAMMATORY DISEASES. 2013 20 3454 37 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