Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance.

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-ß hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3+ CD4+ CD25high CD127low Foxp3+ ) in blood that resulted from peripheral proliferation (Ki67+ ), possibly new thymic emigration (CD31+ ), and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4+ and CD8+ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.

Foxp3(+) regulatory T cells are expanded in severe atopic dermatitis patients.

Regulatory T cells (Tregs) are known to play critical roles in homeostasis and immune responses in the skin. Whether Treg frequencies are altered in atopic dermatitis (AD) patients has been addressed by several studies, leading to conflicting results. The detection of Tregs by FOXP3 expression may lead to false-positive results as activated T cells without regulatory function may transiently upregulate this transcription factor. In contrast, measurement of the DNA methylation status of a region within the FOXP3 locus that is selectively demethylated only in bona fide Tregs (Treg-specific demethylated region, TSDR) represents a reliable method to quantify Tregs. Here, we measured circulating Treg frequencies of adult patients and detected a positive correlation with disease severity. Subsequent surface marker analysis revealed higher frequencies of CD45RA(+) CCR7(-) tissue-homing Tregs in the patient group with a tendency of reduced expression of CD39 compared with healthy donors, a marker for the highly suppressive TREM subtype.

Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors.

Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients' bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II-IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus-mycophenolate-ATG-F-rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.

Polymorphism of the IL-8 gene and the risk of ovarian cancer.

Ovarian cancer still represents a challenge in gynecological oncology. Most patients are diagnosed in an advanced tumor stage. No specific screening or prevention strategies for ovarian cancer exist as of yet. Interleukin 8 (IL-8) is a pro-inflammatory chemokine known for its angiogenetic activity, and is supposedly responsible for tumor-associated angiogenesis in several malignant tumors. The aim of the study was to investigate the susceptibility of patients with an IL-8 gene polymorphism to developing ovarian cancer. Four single nucleotide polymorphisms (SNPs) (IL-8 -251, IL-8 +781, IL-8 +1633 and IL-8 +2767) of the IL-8 gene were screened, using the PCR method in 268 patients with ovarian cancer and 426 healthy women as a control group. Significant associations were noted in patients with the IL-8 +781 (T/T) genotype (p=0.0048) with increased frequencies of ovarian cancer, while women with the IL-8 +781 (C/C) allele suffer from ovarian cancer significantly less frequently (p=0.0003). Furthermore, the IL-8 +2767 (T/T) genotype is also associated with a higher risk of ovarian cancer (p=0.0177). Our results indicate, for the first time, that IL-8 polymorphism is associated with ovarian cancer.

BRCA1 gene promoter methylation status in high-grade serous ovarian cancer patients--a study of the tumour Bank ovarian cancer (TOC) and ovarian cancer diagnosis consortium (OVCAD).

BACKGROUND: Mutations in BRCA1/2 genes are involved in the pathogenesis of breast and ovarian cancer. Inactivation of these genes can also be mediated by hypermethylation of CpGs in the promoter regions. Aim of this study was to analyse the clinical impact of BRCA1 promoter gene methylation status in a homogenous cohort of high-grade serous ovarian cancer (HGSOC) patients. METHODS: The cohort included 257 primary HGSOC patients treated by cytoreduction and platinum-based chemotherapy. DNA was extracted from fresh frozen tissue samples. BRCA1 gene promoter methylation rate was assessed using polymerase chain reaction (PCR). RESULTS: 14.8% of patients presented hypermethylation within a selected region of the BRCA1 promoter. The rate of hypermethylation was significantly higher in younger patients (20.8% hypermethylation in the age group ⩽ 58 years versus 8.7% hypermethylation in the age group >58 years; p = 0.008). Optimal tumour debulking could be reached in 63% of patients, without significant differences in the extent of residual disease with respect to the methylation status. No impact of BRCA1 gene promoter methylation status on progression free- and overall-survival rates was found. No significant differences within BRCA1 promoter methylation status between primary and metastatic tissue could be observed. These results on BRCA1 promoter methylation status were also confirmed in a subgroup of 107 patients found negative for BRCA1 exon 11 mutations. CONCLUSIONS: Our data suggest that BRCA1 methylation determines the earlier onset of HGSOC. Furthermore our study supports the idea that BRCAness is not only due to mutations but also to epigenetic changes in BRCA1 promoter gene.

An optimized multi-parameter flow cytometry protocol for human T regulatory cell analysis on fresh and viably frozen cells, correlation with epigenetic analysis, and comparison of cord and adult blood.

Multi-parameter flow cytometry analysis of T regulatory (Treg) cells is a widely used approach in basic and translational research studies. This approach has been complicated by a lack of specific markers for Treg cells and lack of uniformity in the quantification of Treg cells. Given the central role of Treg cells in the inception and perpetuation of diverse immune responses as well as its target as a therapeutic, it is imperative to have established methodologies for Treg cell analysis that are robust and usable for studies with multiple subjects as well as multicenter studies. In this study, we describe an optimized multi-parameter flow cytometry protocol for the quantification of human Treg cells from freshly obtained and viably frozen samples and correlations with epigenetic Treg cell analysis (TSDR demethylation). We apply these two methodologies to characterize Treg cell differences between cord blood and adult peripheral blood. In summary, the optimized protocol appears to be robust for Treg cell quantification from freshly isolated or viably frozen cells and the multi-parameter flow cytometry findings are strongly positively correlated with TSDR demethylation thus providing several options for the characterization of Treg cell frequency and function in large translational or clinical studies.

Maternal and newborn vitamin D status and its impact on food allergy development in the German LINA cohort study.

BACKGROUND: Vitamin D levels are known to be associated with atopic disease development; however, existing data are controversial. The aim of this study was to investigate whether corresponding maternal and cord blood vitamin D levels are associated with atopic outcomes in early infancy. METHODS: Within the LINA cohort study (Lifestyle and environmental factors and their Influence on Newborns Allergy risk), 25(OH)D was measured in blood samples of 378 mother-child pairs during pregnancy and at birth. Information about children's atopic manifestations during the first 2 years of life was obtained from questionnaires filled out by the parents during pregnancy and annually thereafter. Cord blood regulatory T cells (Treg) were detected by methylation-specific PCR using a Treg-specific demethylated region in the FOXP3 gene. RESULTS: The median maternal 25(OH)D(3) level was 22.19 ng/ml (IQR 14.40-31.19 ng/ml); the median cord blood 25(OH)D(3) 10.95 ng/ml (6.99-17.39 ng/ml). A high correlation was seen between maternal and cord blood 25(OH)D(3) levels, both showing a seasonal distribution. Maternal and cord blood 25(OH)D(3) was positively associated with children's risk for food allergy within the first 2 years. Further, higher maternal 25(OH)D(3) resulted in a higher risk for sensitization against food allergens at the age of two. Cord blood 25(OH)D(3) levels were negatively correlated with regulatory T cell numbers. CONCLUSION: Our study demonstrates that high vitamin D levels in pregnancy and at birth may contribute to a higher risk for food allergy and therefore argues against vitamin D supplement to protect against allergy.

Enrichment of regulatory T cells in acutely rejected human liver allografts.

Acute cellular rejection (ACR) occurs frequently after liver transplantation and can usually be controlled. Triggering of allospecific immune responses and lack of immunoregulation are currently suggested as a cause of ACR, but there are no investigations of intrahepatic immune responses during ACR. Therefore we prospectively analyzed the intrahepatic T cell infiltration pattern in correlation to the severity of ACR in a cohort of patients with graft hepatitis (n = 151). While CD4(+) cells dominated the portal infiltrates in mild-moderate ACR, CD8(+) cells prevailed in severe ACR. Furthermore portal CD8(+) and not CD4(+) infiltration correlated with serum transaminases and with the likelihood of subsequent ACRs. Surprisingly, the rise of portal effector T cells density during ACR was surpassed by the increase in portal infiltration of regulatory T cells by a factor of two. Thus ACRs rather showed an increase and not a lack of regulation, as was suggested by analysis of peripheral blood mononuclear cells. Despite the pattern of enhanced immunoregulation, patients with severe ACR had a higher risk for subsequent rejections and showed a trend to a reduced survival. Thus, patients with severe rejections might need a modification of their immunosuppression to improve prognosis.

Demethylation analysis of the FOXP3 locus shows quantitative defects of regulatory T cells in IPEX-like syndrome.

Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome is a unique example of primary immunodeficiency characterized by autoimmune manifestations due to defective regulatory T (Treg) cells, in the presence of FOXP3 mutations. However, autoimmune symptoms phenotypically resembling IPEX often occur in the absence of detectable FOXP3 mutations. The cause of this "IPEX-like" syndrome presently remains unclear. To investigate whether a defect in Treg cells sustains the immunological dysregulation in IPEX-like patients, we measured the amount of peripheral Treg cells within the CD3(+) T cells by analysing demethylation of the Treg cell-Specific-Demethylated-Region (TSDR) in the FOXP3 locus and demethylation of the T cell-Specific-Demethylated-Region (TLSDR) in the CD3 locus, highly specific markers for stable Treg cells and overall T cells, respectively. TSDR demethylation analysis, alone or normalized for the total T cells, showed that the amount of peripheral Treg cells in a cohort of IPEX-like patients was significantly reduced, as compared to both healthy subjects and unrelated disease controls. This reduction could not be displayed by flow cytometric analysis, showing highly variable percentages of FOXP3(+) and CD25(+)FOXP3(+) T cells. These data provide evidence that a quantitative defect of Treg cells could be considered a common biological hallmark of IPEX-like syndrome. Since Treg cell suppressive function was not impaired, we propose that this reduction per se could sustain autoimmunity.

Cord blood Tregs with stable FOXP3 expression are influenced by prenatal environment and associated with atopic dermatitis at the age of one year.

BACKGROUND: Regulatory T cells (Tregs) with stable FOXP3 expression are characterized by a specific demethylated region in the FOXP3 gene (Treg-specific demethylated region, TSDR). The aim of this study was to analyse the influence of prenatal factors on cord blood Treg numbers, as detected by changes in the TSDR demethylation, and the subsequent risk for allergic diseases. METHODS: Analyses were performed within the LINA study in blood samples from pregnant women (34th gestational week) and in cord blood (n = 346 mother-child pairs). Treg numbers were detected via DNA demethylation in the FOXP3 TSDR. At age 1, total and specific IgE was measured in children's blood. In addition, maternal cytokine production (Th1/Th2/Th17) was analysed. Exposure and disease outcomes were assessed by questionnaires. RESULTS: Boys had lower Treg numbers compared with girls (P < 0.001). Parental atopy history, particularly maternal hay fever and paternal asthma were related to lower Treg numbers in cord blood (adj. MR = 0.81, 95% CI = 0.68-0.97; adj. MR = 0.60, 95% CI = 0.45-0.81). Maternal cytokines (IL-13, IL-17E and IFN-γ) and maternal smoking/exposure to tobacco smoke during pregnancy were also associated with decreased cord blood Treg numbers (adj. MR = 0.89, 95% CI = 0.97-1.00). Children with lower Treg numbers at birth had a higher risk to develop atopic dermatitis (adj. OR = 1.55, 95% CI = 1.00-2.41) and sensitization to food allergens (adj. OR = 1.55, 95% CI = 1.06-2.25) during the first year of life. CONCLUSIONS: These results indicate that both genetic and environmental factors presumably influence the development of foetal Tregs. Low cord blood Treg numbers may predict early atopic dermatitis.

Intragraft regulatory T cells in protocol biopsies retain foxp3 demethylation and are protective biomarkers for kidney graft outcome.

Presence of subclinical rejection (SCR) with IF/TA in protocol biopsies of renal allografts has been shown to be an independent predictor factor of graft loss. Also, intragraft Foxp3+ T(reg) cells in patients with SCR has been suggested to differentiate harmful from potentially protective infiltrates. Nonetheless, whether presence of Foxp3 T(reg) cells in patients with SCR and IF/TA may potentially protect from a deleterious graft outcome has not yet been evaluated. This is a case-control study in which 37 patients with the diagnosis of SCR and 68 control patients with no cellular infiltrates at 6-month protocol biopsies matched for age and time of transplantation were evaluated. We first confirmed that numbers of intragraft Foxp3-expressing T cells in patients with SCR positively correlates with Foxp3 demethylation at the T(reg) -specific demethylation region. Patients with SCR without Foxp3+ T(reg) cells within graft infiltrates showed significantly worse 5-year graft function evolution than patients with SCR and Foxp3+ T(reg) cells and those without SCR. When presence of SCR and IF/TA were assessed together, presence of Foxp3+ T(reg) could discriminate a subgroup of patients showing the same graft outcome as patients with a normal biopsy. Thus, presence of Foxp3+ T(reg) cells in patients with SCR even with IF/TA is associated with a favorable long-term allograft outcome.

The adenosine deaminase-binding region is distinct from major anti-CD26 mAb epitopes on the human dipeptidyl peptidase IV(CD26) molecule.

CD26 or dipeptidyl peptidase IV (DPP-IV) is a cell surface protease involved in T cell activation. Monoclonal antibodies (mAbs) directed against the CD26 molecule are able to stimulate CD26-expressing T cells. Although many different CD26-specific mAbs exist which are able to provide a triggering signal in T cells, little is known about their specific epitopes on the CD26 molecule. Whereas some mAbs were shown to compete with each other and to inhibit the association of adenosine deaminase (ADA) and human immunodeficiency virus 1 (HIV-1)-derived Tat protein with CD26, other CD26-specific mAbs obviously bind to distinct regions on DPP-IV. In the present study we have generated truncated versions of the human CD26 molecule and expressed them in COS-1 cells to study the binding pattern of a panel of 14 CD26-specific mAbs in confocal microscopy and, thus, correlated the CD26-specific mAbs epitopes with the binding region of ADA. We show that the majority of anti-CD26 mAbs is directed against the glycosylation-rich region of the molecule whereas the ADA-binding site could be located in the cysteine-rich region of DPP-IV. In contrast to binding experiments with purified ADA, which revealed a specific association with CD26 on CD26-positive Jurkat cells, HIV-derived Tat protein did not interact specifically with CD26 on transfected Jurkat cells, nor could Tat binding be competed by anti-CD26-specific mAbs.

The N-terminal structure of HIV-1 Tat is required for suppression of CD26-dependent T cell growth.

Evidence exists that the human immunodeficiency virus-1 (HIV-1) transactivator Tat occurs extracellularly and is involved in the immunosuppression of non-HIV-1-infected T cells of acquired immunodeficiency syndrome (AIDS) patients. The mechanism of this immunosuppressive activity of Tat has been controversially discussed. Interestingly, Tat binds to the T cell activation marker CD26, which has been shown to play a key role in the regulation of growth of lymphocytes and to inhibit its dipeptidyl peptidase IV (DP IV) activity. Here we show that the N-terminal nonapeptide MDPVDPNIE of Tat is a competitive inhibitor of DP IV and suppresses DNA synthesis of tetanus toxoid-stimulated peripheral blood mononuclear cells. Amino acid exchanges at positions 5 and 6 strongly weaken these effects. 1H nuclear magnetic resonance and molecular dynamics simulations of Tat(1-9), I5-Tat(1-9), and L6-Tat(1-9) suggest a similar backbone conformation for Tat(1-9) and L6-Tat(1-9). The solution conformation of I5-Tat(1-9) considerably differs from the other two. However, Tat(1-9) fits into our previously proposed active site model of DP IV in contrast to I5-Tat(1-9) and L6-Tat(1-9). Conformational alterations with regard to the parent peptide and spatial hindrances between these both compounds and DP IV can explain the loss of inhibitory activity. Our data suggest that the N-terminal residues of HIV-1 Tat do interact directly with the active site of DP IV and that DP IV does mediate Tat's immunosuppressive effects.