CD4+ regulatory T cells in gastric cancer mucosa are proliferating and express high levels of IL-10 but little TGF-ß.

BACKGROUND: An increase of regulatory T cells, defined as CD25high- and/or FOXP3+-expressing CD4+ T cells, within tumors has been reported in several studies. Tregs promote tumor growth by modulating the antitumor immune response, mainly through inhibition of T-cell-mediated tumor cell killing: this has been suggested to be dependent on IL-10 and/or TGF-ß. In stomach cancer, the mechanisms behind the accumulation of Tregs in tumor tissue has not been fully elucidated, and neither has Treg gene expression in situ. MATERIALS AND METHODS: Stomach tissue from gastric cancer patients undergoing gastric resection was analyzed using flow cytometry and cell sorting, followed by RT-PCR. RESULTS: We observed that stomach CD4+ FOXP3+ T cells proliferated to a higher degree than CD4+ FOXP3- T cells, which may contribute to Treg accumulation in the mucosa. By analyzing DNA methylation, we demonstrated that both proliferating and nonproliferating FOXP3+ T cells exhibited complete demethylation of the FOXP3 gene, indicating a stable FOXP3 expression in both cell populations. Furthermore, analysis of T-cell populations isolated directly from the tumor and tumor-free mucosa demonstrated that CD4+ CD25high T cells have a higher IL-10/IFN-γ gene expression ratio but express lower levels of TGF-ß than CD4+ CD25low/- T cells. CONCLUSION: We demonstrate strong proliferation among regulatory CD4+ FOXP3+ CD25high T cells in the gastric cancer mucosa. These local Treg express a suppressive cytokine profile characterized by high IL-10 and low TGF-ß and IFN-γ production.

Profiling of CD4+ T cells with epigenetic immune lineage analysis.

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.

The inflammatory milieu in the rheumatic joint reduces regulatory T-cell function.

Regulatory T cells (Tregs) are important for maintaining immune homeostasis, but many studies suggest that Tregs are functionally impaired in autoimmune and chronic inflammatory disorders. In addition, effector T cells may vary in sensitivity toward Treg suppression. Herein, we have studied the interplay between T effectors and Tregs in the rheumatic joint. Synovial Tregs demonstrated a high degree of FOXP3 demethylation and displayed only marginal IL-17 and virtually no IFN-γ production following in vitro stimulation, altogether indicating suppressive capacity. Still, the frequency of FOXP3 expression could not predict the degree of suppression. Instead, the inflammatory milieu in the joint, i.e. proliferative capacity of effector T cells and in situ levels of pro-inflammatory cytokines influenced Treg function. Indeed, blocking IL-6 or TNF increased the suppression by Tregs in co-cultures. Additionally, approximately 30% of the synovial FOXP3(+) T cells were Ki67(+) and hence actively dividing, but proliferation did not overlap with cytokine production, suggesting that these cells represent functional Tregs having met their cognate antigen and expanded in an attempt to alleviate joint inflammation. Overall, our data argue against a general functional deficit in joint-derived Tregs and instead emphasize the importance of the inflammatory milieu to set the threshold for immune regulation.

MiR-21 is up-regulated in psoriasis and suppresses T cell apoptosis.

MicroRNAs are short non-coding RNAs that regulate gene expression. Previously, in a genome-wide screen, we found deregulation of microRNA expression in psoriasis skin. MicroRNA-21 (miR-21) is one of the microRNAs significantly up-regulated in psoriasis skin lesions. To identify the cell type responsible for the increased miR-21 level, we compared expression of miR-21 in epidermal cells and dermal T cells between psoriasis and healthy skin and found elevated levels of miR-21 in psoriasis in both cell types. In cultured T cells, expression of miR-21 increased markedly upon activation. To explore the function of miR-21 in primary human T helper cells, we inhibited miR-21 using a tiny seed-targeting LNA-anti-miR. Specific inhibition of miR-21 increased the apoptosis rate of activated T cells. Our results suggest that miR-21 suppresses apoptosis in activated T cells, and thus, overexpression of miR-21 may contribute to T cell-derived psoriatic skin inflammation.

Maternal MHC regulates generation of pathogenic antibodies and fetal MHC-encoded genes determine susceptibility in congenital heart block.

Congenital heart block develops in fetuses of anti-Ro52 Ab-positive women. A recurrence rate of 20%, despite the persistence of maternal autoantibodies, indicates that there are additional, yet unidentified, factors critical for development of congenital heart block. In this study, we demonstrate that besides the maternal MHC controlling Ab specificity, fetal MHC-encoded genes influence fetal susceptibility to congenital heart block. Using MHC congenic rat strains, we show that heart block develops in rat pups of three strains carrying MHC haplotype RT1(av1) (DA, PVG.AV1, and LEW.AV1) after maternal Ro52 immunization, but not in LEW rats (RT1(l)). Different anti-Ro52 Ab fine specificities were generated in RT1(av1) versus RT1(l) animals. Maternal and fetal influence was determined in an F(2) cross between LEW.AV1 and LEW strains, which revealed higher susceptibility in RT1(l) than RT1(av1) pups once pathogenic Ro52 Abs were present. This was further confirmed in that RT1(l) pups more frequently developed heart block than RT1(av1) pups after passive transfer of RT1(av1) anti-Ro52 sera. Our findings show that generation of pathogenic Ro52 Abs is restricted by maternal MHC, whereas the fetal MHC locus regulates susceptibility and determines the fetal disease outcome in anti-Ro52-positive pregnancies.

MiR-155 is overexpressed in patients with atopic dermatitis and modulates T-cell proliferative responses by targeting cytotoxic T lymphocyte-associated antigen 4.

BACKGROUND: MicroRNAs (miRNAs) are short noncoding RNAs that suppress gene expression at the posttranscriptional level. Atopic dermatitis is a common chronic inflammatory skin disease characterized by the presence of activated T cells within the skin. OBJECTIVE: We sought to explore the role of miRNAs in the pathogenesis of atopic dermatitis. METHODS: Global miRNA expression in healthy and lesional skin of patients with atopic dermatitis was compared by using TaqMan MicroRNA Low Density Arrays. miR-155 expression in tissues and cells was quantified by means of quantitative real-time PCR. The cellular localization of miR-155 was analyzed by means of in situ hybridization. The regulation of cytotoxic T lymphocyte-associated antigen (CTLA-4) by miR-155 was investigated by using luciferase reporter assays and flow cytometry. CTLA-4 expression and functional assays were performed on T(H) cells overexpressing miR-155. RESULTS: miR-155 was one of the highest-ranked upregulated miRNAs in patients with atopic dermatitis. In the skin miR-155 was predominantly expressed in infiltrating immune cells. miR-155 was upregulated during T-cell differentiation/activation and was markedly induced by T-cell activators in PBMCs in vitro and by superantigens and allergens in the skin in vivo. CTLA-4, an important negative regulator of T-cell activation, was identified as a direct target of miR-155. Overexpression of miR-155 in T(H) cells resulted in decreased CTLA-4 levels accompanied by an increased proliferative response. CONCLUSION: miR-155 is significantly overexpressed in patients with atopic dermatitis and might contribute to chronic skin inflammation by increasing the proliferative response of T(H) cells through the downregulation of CTLA-4.

At the crossroads of T helper lineage commitment-Epigenetics points the way.

The immune system has the capacity to respond to various types of pathogens including bacteria, viruses, tumors and parasites. This requires a flexible immune system, which in part depends on the development of alternative effector T helper cells, with different cytokine repertoires that direct the overall immune response. The reciprocal effects of the T helper subtypes Th1 and Th2 are well documented, but the mechanisms involved in alternative cytokine expression and silencing are less well defined. Introduction of advances within the field of chromatin folding and epigenetic regulation of transcription has begun to explain some of the fundamental principles of T helper cell development. In addition, epigenetic regulation has proven essential also for the more recently discovered T helper cell subtypes; regulatory T cells and the Th17 lineage. As the importance of proper epigenetic regulation becomes evident, attention is also focused on the potential harmfulness of epigenetic dysregulation. Autoimmunity and allergy are two clinical situations that have been implicated as results of imperfect cytokine silencing. This review will address recent advances in the field of epigenetic regulation of T lymphocytes and their maturation from naive cells into different effector T cell lineages. In particular, epigenetic involvement in regulation of key effector cytokines and specific transcription factors determining the CD4(+) T lymphocyte lineage commitment will be discussed.

The expression of microRNA-203 during human skin morphogenesis.

MicroRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally and play important roles in various biological processes. We previously identified miR-203 as a skin- and keratinocyte-specific microRNA. Moreover, miR-203 has been implicated in repressing 'stemness' in epidermal progenitors. Here, we investigate the expression of miR-203 and two of its targets, p63 and suppressor of cytokine signalling-3, during human skin morphogenesis. MiR-203 in situ hybridization was performed on sections of human foetal skin ranging from 14 to 22 weeks' gestation and adult skin. MiR-203 was barely detectable at 14 weeks. Its expression became prominent from week 17 and was most pronounced in the suprabasal layers of the epidermis, while p63 and SOCS-3 were preferentially expressed in the basal layer. Differentiation markers such as involucrin and filaggrin were expressed mainly in the suprabasal layers of epidermis, similar to miR-203. Our results support the involvement of miR-203 in skin morphogenesis.

CpG methylation of the IFNG gene as a mechanism to induce immunosuppression [correction of immunosupression] in tumor-infiltrating lymphocytes.

The execution of appropriate gene expression patterns during immune responses is of eminent importance where CpG methylation has emerged as an essential mechanism for gene silencing. We have charted the methylation status of regulatory elements in the human IFNG gene encoding the signature cytokine of the Th1 response. Surprisingly, human naive CD4(+) T lymphocytes displayed hypermethylation at the IFNG promoter region, which is in sharp contrast to the completely demethylated status of this region in mice. Th1 differentiation induced demethylation of the IFNG promoter and the upstream conserved nucleotide sequence 1 enhancer region, whereas Th2-differentiated lymphocytes remained hypermethylated. Furthermore, CD19(+) B lymphocytes displayed hypomethylation at the IFNG promoter region with a similar pattern to Th1 effector cells. When investigating the methylation status among tumor-infiltrating CD4(+) T lymphocytes from patients with colon cancer, we found that tumor-infiltrating lymphocytes cells are inappropriately hypermethylated, and thus not confined to the Th1 lineage. In contrast, CD4(+) T cells from the tumor draining lymph node were significantly more demethylated than tumor-infiltrating lymphocytes. We conclude that there are obvious interspecies differences in the methylation status of the IFNG gene in naive CD4(+) T lymphocytes, where Th1 commitment in human lymphocytes involves demethylation before IFNG expression. Finally, investigations of tumor-infiltrating lymphocytes and CD4(+) cells from tumor draining lymph node demonstrate methylation of regulatory regions within key effector genes as an epigenetic mechanism of tumor-induced immunosuppression.

Antithrombotic treatment and risk of complications after head and neck full thickness skin graft surgery.

Head and neck skin cancer surgery using a full thickness skin graft is a common procedure. Evidence concerning the effects of perioperative antithrombotic treatment on complications is limited. The aim of this study was to evaluate whether perioperative antithrombotic treatment is associated with risk of necrosis, bleeding or infection after full thickness skin graft surgery. Retrospective single-center cohort study with medical records review. Patients operated with a head and neck full thickness skin graft in 2010 and 2013-2015 had available data and were included. Any antithrombotic treatment was continued and all patients were routinely followed-up on days 7-10 after surgery. Data on demographics, concurrent disease, clinical characteristics, antithrombotic medications and postoperative necrosis, bleeding and infection were collected from electronic medical records. Associations with complications were examined using multivariate logistic regression adjusted for age, sex, reoperation, size of excision, site of surgery and concurrent disease. In total, 302 patients (53% women) were included. Antithrombotic treatment (n = 125 patients) was not associated with higher adjusted risk of total complications in multivariate analysis (OR 0.70; 95% CI: 0.34-1.46). In subgroup analyses, the total risk was not increased in patients on aspirin (OR 0.76; 95% CI: 0.39-1.48) or warfarin (OR 1.20; 95% CI: 0.47-3.10). In the warfarin subgroup (N = 26), there was a statistically non-significant trend towards increased risk of graft necrosis. This study supports that aspirin and warfarin should not be discontinued prior to head and neck full thickness skin graft surgery.

Epigenetics--the key to understand immune responses in health and disease.

PROBLEM: Development of alternative CD4(+) T cells provides flexibility to the immune system. This is crucial for the initiation of appropriate effector mechanisms to protect against various pathogens such as bacteria, viruses, tumors, and parasites. METHOD OF STUDY: Review of research on the epigenetic regulation of T-cell subsets. RESULTS: Studies of the epigenetic modulation of T-cell subset function/dysfunction during the past years have increased our understanding of how the alternative effector populations arise and how their identity is maintained during clonal expansion. CONCLUSIONS: The recent advances in epigenetic research within the field of immunology have also raised questions on how immunology is regulated during pregnancy and early life and how epigenetic regulation of the immune system during prenatal development is related to diseases later in life such as autoimmunity and allergy.

FOXP3 promoter demethylation reveals the committed Treg population in humans.

BACKGROUND: Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs. METHODOLOGY/PRINCIPAL FINDINGS: Human CD4(+)CD25(hi) Tregs displayed a demethylated FOXP3 promoter (1.4%+/-0.95% SEM methylated) in contrast to CD4(+)CD25(lo) T cells which were partially methylated (27.9%+/-7.1%). Furthermore, stimulated CD4(+)CD25(lo) T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level. CONCLUSIONS/SIGNIFICANCE: The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

MicroRNAs: novel regulators involved in the pathogenesis of psoriasis?

MicroRNAs are a recently discovered class of posttranscriptional regulators of gene expression with critical functions in health and disease. Psoriasis is the most prevalent chronic inflammatory skin disease in adults, with a substantial negative impact on the patients' quality of life. Here we show for the first time that psoriasis-affected skin has a specific microRNA expression profile when compared with healthy human skin or with another chronic inflammatory skin disease, atopic eczema. Among the psoriasis-specific microRNAs, we identified leukocyte-derived microRNAs and one keratinocyte-derived microRNA, miR-203. In a panel of 21 different human organs and tissues, miR-203 showed a highly skin-specific expression profile. Among the cellular constituents of the skin, it was exclusively expressed by keratinocytes. The up-regulation of miR-203 in psoriatic plaques was concurrent with the down-regulation of an evolutionary conserved target of miR-203, suppressor of cytokine signaling 3 (SOCS-3), which is involved in inflammatory responses and keratinocyte functions. Our results suggest that microRNA deregulation is involved in the pathogenesis of psoriasis and contributes to the dysfunction of the cross talk between resident and infiltrating cells. Taken together, a new layer of regulatory mechanisms is involved in the pathogenesis of chronic inflammatory skin diseases.