Search

Tolerance to self‐proteins involves multiple mechanisms, including conventional CD4+ T‐cell (Tconv) deletion in the thymus and the recruitment of natural regulatory T cells (nTregs). The significant incidence of autoantibodies specific for the blood coagulation factor VIII (FVIII) in healthy donors illustrates that tolerance to self‐proteins is not always complete. In contrast to FVIII‐specific Tconvs, FVIII‐specific nTregs have never been revealed and characterized. To determine the frequency of FVIII‐specific Tregs in human peripheral blood, we assessed the specificity of in vitro expanded Tregs by the membrane expression of the CD137 activation marker. Amplified Tregs maintain high levels of FOXP3 expression and exhibit almost complete demethylation of the FOXP3 Treg‐specific demethylated region. The cells retained FOXP3 expression after long‐term culture in vitro, strongly suggesting that FVIII‐specific Tregs are derived from the thymus. From eleven healthy donors, we estimated the frequencies of FVIII‐specific Tregs at 0.17 cells per million, which is about 10‐fold lower than the frequency of FVIII‐specific CD4+ T cells we previously published. Our results shed light on the mechanisms of FVIII tolerance by a renewed approach that could be extended to other self‐ or non‐self‐antigens. [ABSTRACT FROM AUTHOR]

Objective: To study the involvement of Treg cells expressing tumor necrosis factor receptor type II (TNFRII) in exerting control of inflammation in experimental models and in the response to anti‐TNF treatments in patients with rheumatoid arthritis (RA) or spondyloarthritis (SpA). Methods: The role of TNFRII in Treg cells was explored using a multilevel translational approach. Treg cell stability was evaluated by analyzing the methylation status of the Foxp3 locus using bisulfite sequencing. Two models of inflammation (imiquimod‐induced skin inflammation and delayed‐type hypersensitivity arthritis [DTHA]) were induced in TNFRII−/− mice, with or without transfer of purified CD4+CD25+ cells from wild‐type (WT) mice. In patients with RA and those with SpA, the evolution of the TNFRII+ Treg cell population before and after targeted treatment was monitored. Results: Foxp3 gene methylation in Treg cells was greater in TNFRII−/− mice than in WT mice (50% versus 36.7%). In cultured Treg cells, TNF enhanced the expression, maintenance, and proliferation of Foxp3 through TNFRII signaling. Imiquimod‐induced skin inflammation and DTHA were aggravated in TNFRII−/− mice (P < 0.05 for mice with skin inflammation and P < 0.0001 for mice with ankle swelling during DTHA compared to WT mice). Adoptive transfer of WT mouse Treg cells into TNFRII−/− mice prevented aggravation of arthritis. In patients with RA receiving anti‐TNF treatments, but not those receiving tocilizumab, the frequency of TNFRII+ Treg cells was increased at 3 months of treatment compared to baseline (mean ± SEM 65.2 ± 3.1% versus 49.1 ± 5.5%; P < 0.01). In contrast, in anti‐TNF–treated patients with SpA, the frequency of TNFRII+ Treg cells was not modified. Conclusion: TNFRII expression identifies a subset of Treg cells that are characterized by stable expression of Foxp3 via gene hypomethylation, and adoptive transfer of TNFRII‐expressing Treg cells ameliorates inflammation in experimental models. Expansion and activation of TNFRII+ Treg cells may be one of the mechanisms by which anti‐TNF agents control inflammation in RA, but not in SpA. [ABSTRACT FROM AUTHOR]

Objective: Systemic lupus erythematosus (SLE) has limited monozygotic twin concordance, implying a role for pathogenic factors other than genetic variation, such as epigenetic changes. Using the disease‐discordant twin model, we investigated genome‐wide DNA methylation changes in sorted CD4+ T cells, monocytes, granulocytes, and B cells in twin pairs with at least 1 SLE‐affected twin. Methods: Peripheral blood obtained from 15 SLE‐affected twin pairs (6 monozygotic and 9 dizygotic) was processed using density‐gradient centrifugation for the granulocyte fraction. CD4+ T cells, monocytes, and B cells were further isolated using magnetic beads. Genome‐wide DNA methylation was analyzed using Infinium HumanMethylation450K BeadChips. When comparing probes from SLE‐affected twins and co‐twins, differential DNA methylation was considered statistically significant when the P value was less than 0.01 and biologically relevant when the median DNA methylation difference was >7%. Findings were validated by pyrosequencing and replicated in an independent case–control sample. Results: In paired analyses of twins discordant for SLE restricted to the gene promoter and start region, we identified 55, 327, 247, and 1,628 genes with differentially methylated CpGs in CD4+ T cells, monocytes, granulocytes, and B cells, respectively. All cell types displayed marked hypomethylation in interferon‐regulated genes, such as IFI44L, PARP9, and IFITM1, which was more pronounced in twins who experienced a disease flare within the past 2 years. In contrast to what was observed in the other cell types, differentially methylated CpGs in B cells were predominantly hypermethylated, and the most important upstream regulators included TNF and EP300. Conclusion: Hypomethylation of interferon‐regulated genes occurs in all major cellular compartments in SLE‐affected twins. The observed B cell promoter hypermethylation is a novel finding with potential significance in SLE pathogenesis. [ABSTRACT FROM AUTHOR]

Background DNA methylation alterations are early events in tumorigenesis and important in the regulation of gene expression in cancer cells. Lung cancer patients have in general a poor prognosis, and a deeper insight into the epigenetic landscape in lung adenocarcinoma tumors and its prognostic implications is needed. Results We determined whole-genome DNA methylation profiles of 164 fresh frozen lung adenocarcinoma samples and 19 samples of matched normal lung tissue using the Illumina Infinium 450K array. A large number of differentially methylated CpGs in lung adenocarcinoma tissue were identified, and specific methylation profiles were observed in tumors with mutations in the EGFR- , KRAS- or TP53 genes and according to the patients' smoking status. The methylation levels were correlated with gene expression and both positive and negative correlations were seen. Methylation profiles of the tumor samples identified subtypes of tumors with distinct prognosis, including one subtype enriched for TP53 mutant tumors. A prognostic index based on the methylation levels of 33 CpGs was established, and was significantly associated with prognosis in the univariate analysis using an independent cohort of lung adenocarcinoma patients from The Cancer Genome Atlas project. CpGs in the HOX B and HOX C gene clusters were represented in the prognostic signature. Conclusions Methylation differences mirror biologically important features in the etiology of lung adenocarcinomas and influence prognosis. [ABSTRACT FROM AUTHOR]

Characterizing genetic contributions to endometriosis might help to shorten the time to diagnosis, especially in the most severe forms, but represents a challenge. Previous genome-wide association studies (GWAS) made no distinction between peritoneal endometriosis (SUP), endometrioma (OMA), and deep infiltrating endometriosis (DIE). We therefore conducted a pooled sample-based GWAS and distinguished histologically confirmed endometriosis subtypes. We performed an initial discovery step on 10-individual pools (two pools per condition). After quality control filtering, a Monte-Carlo simulation was used to rank the significant SNPs according to the ratio of allele frequencies and the coefficient of variation. Then, a replication step of individual genotyping was conducted in an independent cohort of 259 cases and 288 controls. Our approach was very stringent but probably missed a lot of information due to the Monte-Carlo simulation, which likely explained why we did not replicate results from “classic” GWAS. Four variants (rs227849, rs4703908, rs2479037, and rs966674) were significantly associated with an increased risk of OMA. Rs4703908, located close to ZNF366, provided a higher risk of OMA (OR = 2.22; 95% CI: 1.26–3.92) and DIE, especially with bowel involvement (OR = 2.09; 95% CI: 1.12–3.91). ZNF366, involved in estrogen metabolism and progression of breast cancer, is a new biologically plausible candidate for endometriosis. [ABSTRACT FROM AUTHOR]

Radiotherapy (RT) is a central treatment modality for breast cancer patients. The purpose of our study was to investigate the DNA methylation changes in tumors following RT, and to identify epigenetic markers predicting treatment outcome. Paired biopsies from patients with inoperable breast cancer were collected both before irradiation ( n = 20) and after receiving 10-24 Gray (Gy) ( n = 19). DNA methylation analysis was performed by using Illumina Infinium 27K arrays. Fourteen genes were selected for technical validation by pyrosequencing. Eighty-two differentially methylated genes were identified in irradiated ( n = 11) versus nonirradiated ( n = 19) samples (false discovery rate, FDR = 1.1%). Methylation levels in pathways belonging to the immune system were most altered after RT. Based on methylation levels before irradiation, a panel of five genes ( H2AFY, CTSA, LTC4S, IL5RA and RB1) were significantly associated with clinical response ( p = 0.041). Furthermore, the degree of methylation changes for 2,516 probes correlated with the given radiation dose. Within the 2,516 probes, an enrichment for pathways involved in cellular immune response, proliferation and apoptosis was identified (FDR < 5%). Here, we observed clear differences in methylation levels induced by radiation, some associated with response to treatment. Our study adds knowledge on the molecular mechanisms behind radiation response. [ABSTRACT FROM AUTHOR]