Proper transcriptional control of pro- and anti-inflammatory responses of the immune system is important for a fine-tuned balance between protection and tolerance. Emerging evidence suggests a key role for epigenetic regulation in governing the Th cell differentiation, where effector cytokines direct the overall immune response. In this study, we describe a method to pinpoint the location of isolated human CD4+ T cells on any T cell effector axis based on specific CpG methylation of cytokine and transcription factor loci. We apply the method on CD4+ cells obtained from rheumatoid arthritis and multiple sclerosis patients and show that synovial fluid infiltrating CD4+ T cells are committed toward both Th1 and regulatory T cell phenotype, whereas the Th2 response is suppressed. Furthermore, we show that the IL-17A gene is regulated by promoter methylation and that Th17 commitment is not a common feature in the inflamed joints of rheumatoid arthritis patients. We conclude that the method described in this paper allows for accurate profiling of Th lineage commitment in ex vivo-isolated CD4+ T cells.

Regulatory T cells (Treg) have long been considered one-sided suppressors of antitumor immune responses and hence associated with poor patient outcome in cancer. However, evidence is mounting of a paradoxical positive prognostic effect of Tregs on certain malignancies, including urinary bladder cancer (UBC). This discrepancy has partly been attributed to the shear misidentification of Tregs, but also to the inflammatory profile of the tumor. Our aim was to determine whether tumor-infiltrating Forkhead box P3 (FOXP3) cells confer a stable Treg phenotype and to investigate putative beneficial Treg functions, focusing on tumor-promoting inflammatory pathways in UBC. Patients ( = 52) with suspected UBC were prospectively included. We show, by using a broad range of analytical approaches, that tumor-infiltrating CD4FOXP3 T cells in UBC phenotypically, functionally, and epigenetically represent a true Treg population. At the invasive front of UBC tumors, we found an inverse relationship between Treg frequency and expression of matrix metalloproteinase 2 (MMP2), a key proinvasive factor induced by tumor-promoting inflammation. Correspondingly, a significant, dose-dependent Treg-mediated downregulation of MMP2 protein and mRNA expression was observed in both macrophages and UBC cells. Also, we found that Treg frequency specifically at the invasive front positively correlated with survival. Thus, we identify Treg-mediated suppression of MMP2 in the tumor microenvironment as a mechanism explaining the paradoxical positive prognostic impact of tumor-infiltrating Tregs in UBC. .

Proinflammatory CD4+CD28null T cells are frequently found in the circulation of RA patients, but are less common in the rheumatic joint. In the present study we sought to identify functional differences between CD4+CD28null T cells from blood and synovial fluid in comparison to conventional CD28 expressing CD4+ T cells. 44 RA patients, displaying a distinct CD4+CD28null T cell population in blood, were recruited for this study and the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN-γ, TNF, IL-17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4+CD28null T cells were significantly more hypomethylated in the CNS-1 region of the IFNG locus than conventional CD4+CD28+ T cells and produced higher levels of both IFN-γ and TNF after TCR crosslinking. CD4+CD28null T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL-17A production could hardly be detected in CD4+CD28null cells from the blood, a significant production was observed in CD4+CD28null T cells from synovial fluid. CD4+CD28null T cells were not only found to differ from conventional CD4+CD28+ T cells in the circulation, but we could also demonstrate that synovial CD4+CD28null T cells showed additional effector functions (IL-17 co-production) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28null population.

OBJECTIVES:It has been demonstrated that circulating monocytes relocate to the intestinal mucosa during intestinal inflammation, but the phenotype and inflammatory mechanisms of these monocytes remain poorly understood. Here, we have investigated blood monocytes expressing high levels of HLA-DR and CCR9 in patients with inflammatory bowel disease (IBD).METHODS:Fifty-one patients with mild to severe ulcerative colitis (UC; n=31; UC-DAI 3–12) or Crohn's disease (CD; n=20; Harvey–Bradshaw indices (HBI) 2–16) were included together with 14 controls, during IBD therapy for four consecutive weeks. The frequency of CD14+HLA-DRhi monocytes was monitored weekly in peripheral blood, using flow cytometry. The surface phenotype and cytokine profile of these monocytes were established using flow cytometry and real-time PCR. Clinical parameters were assessed weekly in all patients.RESULTS:The frequency of circulating CD14+HLA-DRhi monocytes was significantly higher in IBD patients with moderate to severe disease compared with healthy controls (P<0.001). During treatment with corticosteroids and granulocyte/monocyte apheresis, the proportion of circulating CD14+HLA-DRhi monocytes was significantly reduced. CD14+HLA-DRhi monocytes produced high levels of inflammatory mediators, such as tumor necrosis factor (TNF)-α, and expressed the gut-homing receptor CCR9. Furthermore, we found that the CCR9 ligand, CCL25/TECK, was expressed at high levels in the colonic mucosa in IBD patients with active disease.CONCLUSIONS:CD14+HLA-DRhi blood monocytes were increased in patients with active IBD. These monocytes exhibit a pro-inflammatory, gut-homing phenotype with regard to their TNF-α production and expression of CCR9. Our results suggest that these monocytes are important in mediating intestinal inflammation, and provide potential therapeutic targets in IBD.