Loss-of-function mutations in genes encoding TET DNA dioxygenase occur frequently in hematopoietic malignancy, but rarely in solid tumors, which instead commonly have reduced activity. The impact of decreased TET activity in solid tumors is not known. Here we show that TET2 mediates the IFN-γ/JAK/STAT signaling pathway to control chemokine and PD-L1 expression, lymphocyte infiltration, and cancer immunity. IFN-γ stimulated STAT1 to bind TET2 and recruit TET2 to hydroxymethylate chemokine and PD-L1 genes. Reduced TET activity was associated with decreased Th1-type chemokines and tumor-infiltrating lymphocytes and the progression of human colon cancer. Deletion of Tet2 in murine melanoma and colon tumor cells reduced chemokine expression and tumor-infiltrating lymphocytes, enabling tumors to evade antitumor immunity and to resist anti–PD-L1 therapy. Conversely, stimulating TET activity by systematic injection of its cofactor ascorbate/vitamin C increased chemokines and tumor-infiltrating lymphocytes, leading to enhanced antitumor immunity and anti–PD-L1 efficacy and extended lifespan of tumor-bearing mice. These results suggest an IFN-γ/JAK/STAT/TET signaling pathway that mediates tumor response to anti–PD-L1/PD-1 therapy and is frequently disrupted in solid tumors. Our findings also suggest TET activity as a biomarker for predicting the efficacy of and patient response to anti–PD-1/PD-L1 therapy, and stimulation of TET activity as an adjuvant immunotherapy of solid tumors.

Introduction: Multiple sclerosis (MS) disease activity is associated with blood‐brain barrier (BBB) disruption, which is mediated by inflammatory cytokines released by CD4+ lymphocytes. To assess the effects of adenosine A2A receptors on BBB permeability in vitro and in vivo.Methods: A2A receptor expression was detected by immunostaining in experimental autoimmune encephalomyelitis (EAE) C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein (MOG)_35–55, and human MS brain. F‐actin and the tight junction protein Claudin‐5 were assessed in endothelial cells treated with an A2A receptor specific agonist (CGS‐21680) after Th1 cytokine stimulation. EAE mice were divided into control and CGS‐21680 (50 µg/kg, i.p., daily) groups. Disease scores were recorded daily to evaluate neurological impairment. The effects of A2A receptor on inflammation and demyelination were assessed after euthanasia by immunostaining or histology; BBB permeability was measured by sodium fluoride (Na‐F) and FITC‐dextran amounts.Results: Endothelial A2A receptor was detected in demyelination areas of MS brain samples. In EAE lesions, A2A receptor was expressed in the endothelium in association with immune cell infiltration. Treatment with CGS‐21680 counteracted the effects of Th1 cytokines on endothelial cells in vitro, preventing the reduction of tight junction protein expression and stress fiber formation. The effects of A2A receptor activation were correlated with MLCK phosphorylation signaling repression. In EAE, A2A receptor agonist decreased BBB permeability and inhibited EAE neurologic deficiency in mice.Conclusions: A2A receptor activation at EAE onset helps reduce the effects of Th1 stimulation on BBB permeability, indicating that A2A receptor mediates BBB function in CNS demyelinated disease.

Aims: To investigate whether DNA active demethylase TET regulates the expression of tight junction proteins in endothelial cells of the blood–brain barrier (BBB). Methods: Correlations between TET2 activity (indicated by its catalytic product 5hmC) and the expression of BBB tight junction proteins were examined in Tet2 knockout mice and post-mortem human brain tissues. In cultured endothelial cells, the impact of changes of TET activity on the expression of tight junction protein, ZO-1, was studied. BBB permeability assays were performed in Tet2 knockout mice. Results: It was found that the level of 5hmC decreased in brain microvascular endothelial cells of aging mice. In Tet2 knockout mice, the level of 5hmC in endothelial cells was weaker and significantly correlated with the reduced expression of tight junction protein ZO-1. In cultured endothelial cells, H₂O₂ significantly decreased the expression of 5hmC and ZO-1. Tet2 knock-down using siRNA significantly downregulated the expression of ZO-1 in endothelial cells. hMeChip-PCR showed that H₂O₂ decreased the level of 5hmC in the ZO-1 promoter region, which was rescued by N-acetyl cysteine (NAC). Consistently, Tet2 knock-down using siRNA significantly downregulated the level of 5hmC in the ZO-1 promoter region. It was also found that the level of 5hmC decreased in endothelial cells of aging human brains compared with that of adult brains, and the level of ZO-1 was positively correlated with that of 5hmC in microvascular endothelial cells. Conclusions: These findings suggest that TET activity is essential in regulating ZO-1 expression of BBB. It might be a potential target for neuroprotection during aging and in diverse neurological conditions.