Epigenetic immune cell quantification for diagnostic evaluation and monitoring of patients with inborn errors of immunity and secondary immune deficiencies.

BACKGROUND: Early detection and monitoring of primary immunodeficiencies (PID) in humans require quantitative determination of immune cells from fresh blood analyzed by flow cytometry. However, epigenetic immune cell quantification allows analysis from fresh, frozen, or dried blood samples. We demonstrate the utility of epigenetic immune cell quantification for patients with PID. METHODS: Epigenetic quantification of basic lymphocyte subpopulations of 259 samples from PID patients were compared to flow cytometric data. Epigenetic analysis was extended to T-cell subsets (Treg, Th17, Tfh, PD-1+, CCR6+) and memory B-cells and compared between venous EDTA and dried blood. RESULTS: A high correlation of >0.9 was observed for basic T- and B-cell subsets. Extended epigenetic analysis showed quantitative trends within PID subgroups, but individually these varied substantially within these groups. Epigenetic analysis of dried blood samples was equivalent to EDTA blood. CONCLUSION: Epigenetic immune cell quantification is suitable for immune cell profiling in PID patients.

Specifications of qPCR based epigenetic immune cell quantification.

OBJECTIVES: Immune monitoring is an important aspect in diagnostics and clinical trials for patients with compromised immune systems. Flow cytometry is the standard method for immune cell counting but faces limitations. Best practice guidelines are available, but lack of standardization complicates compliance with e.g., in vitro diagnostic regulations. Limited sample availability forces immune monitoring to predominantly use population-based reference intervals. Epigenetic qPCR has evolved as alternative with broad applicability and low logistical demands. Analytical performance specifications (APS) have been defined for qPCR in several regulated fields including testing of genetically modified organisms or vector-shedding. METHODS: APS were characterized using five epigenetic qPCR-based assays quantifying CD3+, CD4+, CD8+ T, B and NK cells in light of regulatory requirements. RESULTS: Epigenetic qPCR meets all specifications including bias, variability, linearity, ruggedness and sample stability as suggested by pertinent guidelines and regulations. The assays were subsequently applied to capillary blood from 25 normal donors over a 28-day period. Index of individuality (IoI) and reference change values were determined to evaluate potential diagnostic gains of individual reference intervals. Analysis of the IoI suggests benefits for individual over population-based references. Reference change values (RCVs) show that changes of approx. Fifty percent from prior measurement are suggestive for clinically relevant changes in any of the 5 cell types. CONCLUSIONS: The demonstrated precision, long-term stability and obtained RCVs render epigenetic cell counting a promising tool for immune monitoring in clinical trials and diagnosis.

Epigenetic and immunological indicators of IPEX disease in subjects with FOXP3 gene mutation.

BACKGROUND: Forkhead box protein 3 (FOXP3) is the master transcription factor in CD4+CD25hiCD127lo regulatory T (Treg) cells. Mutations in FOXP3 result in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome. Clinical presentation of IPEX syndrome is broader than initially described, challenging the understanding of the disease, its evolution, and treatment choice. OBJECTIVE: We sought to study the type and extent of immunologic abnormalities that remain ill-defined in IPEX, across genetic and clinical heterogeneity. METHODS: We performed Treg-cell-specific epigenetic quantification and immunologic characterization of severe "typical" (n = 6) and "atypical" or asymptomatic (n = 9) patients with IPEX. RESULTS: Increased number of cells with Treg-cell-Specific Demethylated Region demethylation in FOXP3 is a consistent feature in patients with IPEX, with (1) highest values in those with typical IPEX, (2) increased values in subjects with pathogenic FOXP3 but still no symptoms, and (3) gradual increase over the course of disease progression. Large-scale profiling using Luminex identified plasma inflammatory signature of macrophage activation and TH2 polarization, with cytokines previously not associated with IPEX pathology, including CCL22, CCL17, CCL15, and IL-13, and the inflammatory markers TNF-α, IL-1A, IL-8, sFasL, and CXCL9. Similarly, both Treg-cell and Teff compartments, studied by Mass Cytometry by Time-Of-Flight, were skewed toward the TH2 compartment, especially in typical IPEX. CONCLUSIONS: Elevated TSDR-demethylated cells, combined with elevation of plasmatic and cellular markers of a polarized type 2 inflammatory immune response, extends our understanding of IPEX diagnosis and heterogeneity.

FOXP3 TSDR Measurement Could Assist Variant Classification and Diagnosis of IPEX Syndrome.

Pathogenic FOXP3 variants cause immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, a progressive autoimmune disease resulting from disruption of the regulatory T cell (Treg) compartment. Assigning pathogenicity to novel variants in FOXP3 is challenging due to the heterogeneous phenotype and variable immunological abnormalities. The number of cells with demethylation at the Treg cell-specific demethylated region (TSDR) is an independent biomarker of IPEX. We aimed to investigate if diagnosing IPEX at presentation with isolated diabetes could allow for effective monitoring of disease progression and assess whether TSDR analysis can aid FOXP3 variant classification and predict disease course. We describe a large genetically diagnosed IPEX cohort (n = 65) and 13 individuals with other monogenic autoimmunity subtypes in whom we quantified the proportion of cells with FOXP3 TSDR demethylation, normalized to the number with CD4 demethylation (%TSDR/CD4) and compare them to 29 unaffected controls. IPEX patients presenting with isolated diabetes (50/65, 77%) often later developed enteropathy (20/50, 40%) with a median interval of 23.5 weeks. %TSDR/CD4 was a good discriminator of IPEX vs. unaffected controls (ROC-AUC 0.81, median 13.6% vs. 8.5%, p < 0.0001) with higher levels of demethylation associated with more severe disease. Patients with other monogenic autoimmunity had a similar %TSDR/CD4 to controls (median 8.7%, p = 1.0). Identifying increased %TSDR/CD4 in patients with novel FOXP3 mutations presenting with isolated diabetes facilitates diagnosis and could offer an opportunity to monitor patients and begin immune modulatory treatment before onset of severe enteropathy.

Risk factors for Epstein-Barr virus reactivation after renal transplantation: Results of a large, multi-centre study.

Epstein-Barr virus (EBV) reactivation is a very common and potentially lethal complication of renal transplantation. However, its risk factors and effects on transplant outcome are not well known. Here, we have analysed a large, multi-centre cohort (N = 512) in which 18.4% of the patients experienced EBV reactivation during the first post-transplant year. The patients were characterized pre-transplant and two weeks post-transplant by a multi-level biomarker panel. EBV reactivation was episodic for most patients, only 12 patients showed prolonged viraemia for over four months. Pre-transplant EBV shedding and male sex were associated with significantly increased incidence of post-transplant EBV reactivation. Importantly, we also identified a significant association of post-transplant EBV with acute rejection and with decreased haemoglobin levels. No further severe complications associated with EBV, either episodic or chronic, could be detected. Our data suggest that despite relatively frequent EBV reactivation, it had no association with serious complications during the first post-transplantation year. EBV shedding prior to transplantation could be employed as biomarkers for personalized immunosuppressive therapy. In summary, our results support the employed immunosuppressive regimes as relatively safe with regard to EBV. However, long-term studies are paramount to support these conclusions.

Treatment with rapamycin can restore regulatory T-cell function in IPEX patients.

BACKGROUND: Immune-dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a lethal disease caused by mutations in a transcription factor critical for the function of thymus-derived regulatory T (Treg) cells (ie, FOXP3), resulting in impaired Treg function and autoimmunity. At present, hematopoietic stem cell transplantation is the therapy of choice for patients with IPEX syndrome. If not available, multiple immunosuppressive regimens have been used with poor disease-free survival at long-term follow-up. Rapamycin has been shown to suppress peripheral T cells while sparing Treg cells expressing wild-type FOXP3, thereby proving beneficial in the clinical setting of immune dysregulation. However, the mechanisms of immunosuppression selective to Treg cells in patients with IPEX syndrome are unclear. OBJECTIVE: We sought to determine the cellular and molecular basis of the clinical benefit observed under rapamycin treatment in 6 patients with IPEX syndrome with different FOXP3 mutations. METHODS: Phenotype and function of FOXP3-mutated Treg cells from rapamycin-treated patients with IPEX syndrome were tested by flow cytometry and in vitro suppression assays, and the gene expression profile of rapamycin-conditioned Treg cells by droplet-digital PCR. RESULTS: Clinical and histologic improvements in patients correlated with partially restored Treg function, independent of FOXP3 expression or Treg frequency. Expression of TNF-receptor-superfamily-member 18 (TNFRSF18, glucocorticoid-induced TNF-receptor-related) and EBV-induced-3 (EBI3, an IL-35 subunit) in patients' Treg cells increased during treatment as compared with that of Treg cells from untreated healthy subjects. Furthermore inhibition of glucocorticoid-induced TNF-receptor-related and Ebi3 partially reverted in vitro suppression by in vivo rapamycin-conditioned Treg cells. CONCLUSIONS: Rapamycin is able to affect Treg suppressive function via a FOXP3-independent mechanism, thus sustaining the clinical improvement observed in patients with IPEX syndrome under rapamycin treatment.

Epigenetically quantified immune cells in salivary glands of Sjögren's syndrome patients: a novel tool that detects robust correlations of T follicular helper cells with immunopathology.

OBJECTIVE: To investigate whether epigenetic cell counting represents a novel method to quantify immune cells in salivary glands of patients with different forms of Sjögren's and sicca syndrome and to capture immunopathology and potentially aid in diagnosis. METHODS: DNA from frozen salivary gland tissue sections of sicca patients was used for bisulphite conversion of demethylated DNA cytosine residues, followed by cell-specific quantitative PCR to calculate cell percentages in relation to total tissue cell numbers as quantified by housekeeping gene demethylation. The percentages of epigenetically quantified cells were correlated to RNA expression of matched salivary gland tissue and histological and clinical parameters. RESULTS: The percentages of epigenetically quantified CD3, CD4, CD8, T follicular helper (Tfh) cells, FoxP3+ regulatory T cells and B cells were significantly increased in the salivary glands of patients with SS. Unsupervised clustering using these percentages identified patient subsets with an increased lymphocytic focus score and local B cell hyperactivity and classifies patients different from conventional classification criteria. In particular, Tfh cells were shown to strongly correlate with the expression of CXCL13, lymphocytic focus scores, local B cell hyperactivity and anti-SSA positivity. CONCLUSION: Epigenetic cell counting is a promising novel tool to objectively and easily quantify immune cells in the labial salivary gland of sicca patients, with a relatively small amount of tissue needed. In view of the potential of this technique to include a huge number of (cell-specific) biomarkers, this opens up new standardized ways of salivary gland analysis with high relevance for patient classification, understanding of immunopathology and monitoring of drug responses in clinical trials.

Quantifying circulating Th17 cells by qPCR: potential as diagnostic biomarker for rheumatoid arthritis.

OBJECTIVE: The diagnosis of RA patients remains a challenge, especially in ACPA-negative disease. Novel T-cell subsets, particularly Th17 may be useful, although data on Th17 frequency using flow cytometry in RA are conflicting. We investigated whether a novel epigenetic qPCR assay for the quantification of Th17 could differentiate patients with RA from those with symptoms evolving towards an alternative diagnosis. METHODS: We used a qPCR assay measuring the extent of the methylation at a key position in the IL-17 and CD4 genes. Assays were performed on whole blood from 49 healthy controls (HC) and 165 early arthritis clinic patients. Flow cytometry was further used to detect the expression of CXCR4 on Th17 cells. RESULTS: In 75 inflammatory arthritis patients who progressed to RA, the qPCR assays showed significantly fewer Th17 cells compared with 90 patients who did not (P<0.0001). Regression models demonstrated a high predictive value for RA development (75.8% correct prediction), and particularly for the ACPA-negative group (n = 125) where Th17 and swollen joint count (SJC) were the only predictors (73% correct prediction). The chemokine receptor CXCR4 had significantly higher expression on Th17 from early RA patients (n = 11) compared with HC (n = 15). CONCLUSION: The results of the epigenetic qPCR assay showed that low levels of Th17 cells were predictive of developing RA, particularly in the ACPA-negative patients. This could have value for insights into pathogenesis and management. The results suggest the recruitment of Th17 to the inflammatory disease site, consistent with high CXCR4 expression.

Autologous stem cell transplantation aids autoimmune patients by functional renewal and TCR diversification of regulatory T cells.

Autologous hematopoietic stem cell transplantation (HSCT) is increasingly considered for patients with severe autoimmune diseases whose prognosis is poor with standard treatments. Regulatory T cells (Tregs) are thought to be important for disease remission after HSCT. However, eliciting the role of donor and host Tregs in autologous HSCT is not possible in humans due to the autologous nature of the intervention. Therefore, we investigated their role during immune reconstitution and re-establishment of immune tolerance and their therapeutic potential following congenic bone marrow transplantation (BMT) in a proteoglycan-induced arthritis (PGIA) mouse model. In addition, we determined Treg T-cell receptor (TCR) CDR3 diversity before and after HSCT in patients with juvenile idiopathic arthritis and juvenile dermatomyositis. In the PGIA BMT model, after an initial predominance of host Tregs, graft-derived Tregs started dominating and displayed a more stable phenotype with better suppressive capacity. Patient samples revealed a striking lack of diversity of the Treg repertoire before HSCT. This ameliorated after HSCT, confirming reset of the Treg compartment following HSCT. In the mouse model, a therapeutic approach was initiated by infusing extra Foxp3(GFP+) Tregs during BMT. Infusion of Foxp3(GFP+) Tregs did not elicit additional clinical improvement but conversely delayed reconstitution of the graft-derived T-cell compartment. These data indicate that HSCT-mediated amelioration of autoimmune disease involves renewal of the Treg pool. In addition, infusion of extra Tregs during BMT results in a delayed reconstitution of T-cell compartments. Therefore, Treg therapy may hamper development of long-term tolerance and should be approached with caution in the clinical autologous setting.

Imprinting of Skin/Inflammation Homing in CD4+ T Cells Is Controlled by DNA Methylation within the Fucosyltransferase 7 Gene.

E- and P-selectin ligands (E- and P-ligs) guide effector memory T cells into skin and inflamed regions, mediate the inflammatory recruitment of leukocytes, and contribute to the localization of hematopoietic precursor cells. A better understanding of their molecular regulation is therefore of significant interest with regard to therapeutic approaches targeting these pathways. In this study, we examined the transcriptional regulation of fucosyltransferase 7 (FUT7), an enzyme crucial for generation of the glycosylated E- and P-ligs. We found that high expression of the coding gene fut7 in murine CD4+ T cells correlates with DNA demethylation within a minimal promoter in skin/inflammation-seeking effector memory T cells. Retinoic acid, a known inducer of the gut-homing phenotype, abrogated the activation-induced demethylation of this region, which contains a cAMP responsive element. Methylation of the promoter or mutation of the cAMP responsive element abolished promoter activity and the binding of CREB, confirming the importance of this region and of its demethylation for fut7 transcription in T cells. Furthermore, studies on human CD4+ effector memory T cells confirmed demethylation within FUT7 corresponding to high FUT7 expression. Monocytes showed an even more extensive demethylation of the FUT7 gene whereas hepatocytes, which lack selectin ligand expression, exhibited extensive methylation. In conclusion, we show that DNA demethylation within the fut7 gene controls selectin ligand expression in mice and humans, including the inducible topographic commitment of T cells for skin and inflamed sites.

Genetic Versus Epigenetic BRCA1 Silencing Pathways: Clinical Effects in Primary Ovarian Cancer Patients: A Study of the Tumor Bank Ovarian Cancer Consortium.

OBJECTIVES: The aim of the present study was to assess in a large cohort of primary epithelial ovarian cancer patients the incidence and the clinical effect of BRCA1 genetic and epigenetic silencing mechanisms. METHODS: A total of 188 primary epithelial ovarian cancer patients, treated between 2000 and 2011 at the Charité University Hospital of Berlin, were included. The patients' tumor and blood samples were obtained from the Tumor Bank Ovarian Cancer Network (www.toc-network.de). Direct sequencing of BRCA1 exon 11 was performed to detect germline mutations, whereas tumor samples were assessed for BRCA1 promoter hypermethylation by bisulphite-converted methylation-specific polymerase chain reaction. Basing on their BRCA1 status, patients were compared regarding clinicopathological variables and survival. RESULTS: Twenty-one patients (11.2%) showed hypermethylation in BRCA1 promoter (HMB), and 18 patients (9.6%) presented germline mutations in BRCA1 exon 11 (GMB). Patients with HMB showed a significantly younger age at diagnosis compared with BRCA1 wild type (BWT) patients (54 vs 61 years, P = 0.045), and both GMB and HMB patients were more likely to have high-grade serous ovarian cancer (76.2% and 77.8% vs 52.7%, P = 0.043 and P = 0.043). Positive family history of breast or ovarian cancer (OC) was more frequently reported among GMB patients with respect to BWT patients (44.4% vs 13.5%, P = 0.003); GMB, HMB, and BWT patients did not show significant differences in terms of tumor dissemination pattern, surgical outcomes, platinum response or survival; neither mutational nor hypermethylation BRCA1 status was found to be an independent prognostic factor for OC patients. CONCLUSIONS: Hypermethylation in BRCA1 is associated with earlier occurrence of OC. In addition, the coexistence of both GBM and HMB is an infrequent event, occurring in 0.5% of OC cases. Silencing of BRCA1 through mutation and hypermethylation confers to distinct clinical characteristics of OC patients but similar clinical outcome with respect to BWT patients.

Germline mutations of BRCA1 gene exon 11 are not associated with platinum response neither with survival advantage in patients with primary ovarian cancer: understanding the clinical importance of one of the biggest human exons. A study of the Tumor Bank Ovarian Cancer (TOC) Consortium.

Germline mutations in BRCA1 gene have been reported in up to 20 % of epithelial ovarian cancer (EOC) patients. Distinct clinical characteristics have been attributed to this special EOC population. We hypothesized that mutations in different BRCA1 gene exons may differently affect the clinical course of the disease. The aim of this study was to analyze, in a large cohort of primary EOCs, the clinical impact of mutations in BRCA1 gene exon 11, the largest exon of the gene sequence encoding the 60 % of BRCA1 protein. Two hundred sixty-three primary EOC patients, treated between 2000 and 2008 at Charité University Hospital of Berlin, were included. Patients' blood samples were obtained from the Tumor Ovarian Cancer (TOC) Network ( www.toc-network.de ). Direct sequencing of BRCA1 gene exon 11 was performed for each patient to detect mutations. Based on their BRCA1 exon 11 mutational status, patients were compared regarding clinico-pathological variables and survival. Mutations in BRCA1 exon 11 were found in 18 out of 263 patients (6.8 %). Further 10/263 (3.8 %) cases showed variants of uncertain significance (VUS). All exon 11 BRCA1-positive tumors (100 %) were Type 2 ovarian carcinomas (p = 0.05). Age at diagnosis was significantly younger in Type 2 exon 11 mutated patients (p = 0.01). On multivariate analysis, BRCA1 exon 11 mutational status was not found to be an independent predictive factor for optimal cytoreduction, platinum response, or survival. Mutations in BRCA1 gene exon 11 seem to predispose women to exclusively develop a Type 2 ovarian cancer at younger age. Exon 11 BRCA1-mutated EOC patients showed distinct clinico-pathological features but similar clinical outcome with respect to sporadic EOC patients.

Isolation of human antigen-specific regulatory T cells with high suppressive function.

Adoptive transfer of regulatory T (Treg) cells could be an alternative to chronic immunosuppression for prevention of allogeneic graft rejection. While polyspecific Treg cells can prevent immune responses under lymphopenic conditions, Ag-specific Treg cells are needed to treat autoimmunity and graft rejection. Yet, reliable markers for Ag-specific Treg cells are missing. We report that latency-associated peptide (LAP) and glycoprotein A repetitions predominant (GARP) can identify human Ag-specific Treg cells. In addition, we show that the depletion of CD154(+) cells from LAP(+) or GARP(+) Treg cells increases the Treg-cell purity to over 90%, as assessed by epigenetic analysis. These Ag-specific Treg cells can be isolated magnetically and might contribute to the development of GMP-based protocols. In addition, Ag-specific Treg cells are functionally far superior to CD4(+) CD25(high) or CD4(+) CD25(high) CD127(low) Treg cells in vitro and in preventing strong alloreactions in humanized mice. They could, therefore, have a high therapeutic potential for the control of alloimmune, autoimmune, and allergic immune responses in patients.

Active demethylation of the Foxp3 locus leads to the generation of stable regulatory T cells within the thymus.

Stable expression of Foxp3 in regulatory T cells (Tregs) depends on DNA demethylation at the Treg-specific demethylated region (TSDR), a conserved, CpG-rich region within the Foxp3 locus. The TSDR is selectively demethylated in ex vivo Tregs purified from secondary lymphoid organs, but it is unclear at which stage of Treg development demethylation takes place. In this study, we show that commitment to a stable lineage occurred during early stages of murine thymic Treg development by engraving of lineage-specific epigenetic marks in parallel with establishment of a Treg-specific gene expression profile. TSDR demethylation was achieved through an active mechanism and involved enzymes of the ten-eleven-translocation family and hydroxylation of methylated cytosines, a modification that is implicated as an initiating step of mitosis-independent DNA demethylation pathways and has not yet been observed at specific loci during immune cell differentiation. Together, our results demonstrate that initiating TSDR demethylation during early stages of thymic Treg development commences stabilization of Foxp3 expression and guarantees full functionality and long-term lineage stability of Tregs.

Maternal and cord blood miR-223 expression associates with prenatal tobacco smoke exposure and low regulatory T-cell numbers.

BACKGROUND: There is evidence that microRNAs (miRNAs) are sensitive to environmental stressors, including tobacco smoke. On the other hand, miRNAs are involved in immune regulation, such as regulatory T (Treg) cell differentiation. The aim of the present study was to investigate the association between prenatal tobacco smoke exposure, miRNAs, and Treg cell numbers. METHODS: Within a prospective mother-child study (Lifestyle and Environmental Factors and Their Influence on Newborns Allergy Risk), we analyzed the expression of miR-155 and miR-223 together with Treg cell numbers in maternal blood during pregnancy, as well as in cord blood (n = 441). Tobacco smoke exposure was assessed based on questionnaire answers and maternal urine cotinine levels. Additionally, the concentration of smoking-related volatile organic compounds was measured in dwellings of study participants. RESULTS: Both maternal and cord blood miR-223 expressions were positively correlated with maternal urine cotinine levels. An association was also found between maternal miR-223 expression and indoor concentrations of benzene and toluene. High miR-223 expression was associated with lower Treg cell numbers in maternal and cord blood. Furthermore, children with lower Treg cell numbers at birth had a higher risk of atopic dermatitis during the first 3 years of life. The concentration of the toluene metabolite S-benzylmercapturic acid in maternal urine was associated with decreased cord blood, but not maternal blood, miR-155 expression. A relationship between miR-155 expression and Treg cell numbers was not found. CONCLUSIONS: For the first time, we show that maternal tobacco smoke exposure during pregnancy correlates with the level of miRNA-223 expression in blood, with an effect on children's cord blood Treg cell numbers and subsequent allergy risk.

Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies.

BACKGROUND & AIMS: Autoimmune hepatitis (AIH) is a chronic autoimmune liver disease usually requiring life-long immunosuppression. The mechanisms for disease initiation and chronicity are largely unknown. A contribution of deficient regulatory T cells (Tregs) in the blood was controversially discussed recently. So far investigations in the target organ have been limited to single parameter analysis in untreated AIH. METHODS: We retrospectively analysed the pattern of liver infiltrating T, B and regulatory T cells quantitatively with simultaneous multicolour immunofluorescence before (n=45) and under (n=31) therapy in adult AIH type 1 (AIH-1) patients. RESULTS: Intrahepatic CD4(+) cells dominate over CD8(+) at diagnosis, but with increasing disease activity the CD4(+)/CD8(+) ratio approached one. While there is no change of Tregs in the blood, they are enriched with effector T cells (Teffs) within the liver of patients with untreated AIH-1 with a constant Treg/Teff ratio. Even more importantly, immunosuppression mostly with steroids and azathioprine caused a disproportional loss of intrahepatic Tregs. Patients reaching biochemical remission had higher intrahepatic Treg/Teff and Treg/B cell ratios compared to patients failing to reach remission. In vitro proliferation of Tregs seemed to be more suppressed by prednisolone than expansion of Teffs. Furthermore, intraportal B cells correlated with serum IgG suggesting an autochthonous intrahepatic IgG production. CONCLUSIONS: Intrahepatic Tregs are rather enriched than numerically deficient in untreated AIH-1. The disproportional decrease of intrahepatic Tregs during therapy might explain high relapse rates after discontinuation of immunosuppression. Thus, future therapies increasing intrahepatic immunoregulation might be better suited for long-term control of AIH.

Reduced FOXP3(+) regulatory T cells in patients with primary sclerosing cholangitis are associated with IL2RA gene polymorphisms.

BACKGROUND & AIMS: Recently, genome wide association studies in primary sclerosing cholangitis (PSC) revealed associations with gene polymorphisms that potentially could affect the function of regulatory T cells (Treg). The aim of this study was to investigate Treg in patients with PSC and to associate their numbers with relevant gene polymorphisms. METHODS: Treg frequency in blood was assessed by staining for CD4(+)CD25(high)FOXP3(+)CD127(low) lymphocytes and determination of Treg-specific FOXP3 gene locus demethylation. Single nucleotide polymorphisms (SNP) in the interleukin-2 receptor alpha (IL2RA), the interleukin-2 (IL2) and interleukin-21 (IL21) gene locus were analysed. Liver biopsies taken at the time of diagnosis were stained for FOXP3 and CD3. Treg function was assessed in a CFSE-based suppression assay. RESULTS: The frequency of Treg in peripheral blood of PSC patients was significantly decreased. We confirmed this finding by demonstrating a reduction of non-methylated DNA in the Treg-specific demethylated FOXP3 gene region of peripheral blood cells in PSC patients. Reduced peripheral Treg numbers were significantly associated with homozygosity for the major allele of the SNP "rs10905718" in the IL2RA gene. Intrahepatic FOXP3(+) cell numbers at the time of initial diagnosis were decreased in PSC as compared to PBC. In addition to reduced numbers, the suppressive capacity of Treg isolated from PSC patients seemed to be impaired as compared to healthy controls. CONCLUSIONS: Our findings indicate that Treg impairment may play a role in the immune dysregulation observed in PSC. Reduced Treg numbers in patients with PSC are associated with polymorphisms in the IL2RA gene.

Generation of highly effective and stable murine alloreactive Treg cells by combined anti-CD4 mAb, TGF-ß, and RA treatment.

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long-term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti-CD4 antibody (aCD4). Here, we investigated whether adding TGF-ß and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg-cell generation and function. Murine CD4(+) T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF-ß+RA or aCD4+Rapa. Addition of TGF-ß+RA or Rapa resulted in an increase of CD25(+)Foxp3(+)-expressing T cells. Expression of CD40L and production of IFN-γ and IL-17 was abolished in aCD4+TGF-ß+RA aTreg cells. Additionally, aCD4+TGF-ß+RA aTreg cells showed the highest level of Helios and Neuropilin-1 co-expression. Although CD25(+)Foxp3(+) cells from all culture conditions displayed complete demethylation of the Treg-specific demethylated region, aCD4+TGF-ß+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF-ß+RA aTreg cells suppressed effector T-cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF-ß+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.

Low levels of naturally occurring regulatory T lymphocytes in blood of mares with early pregnancy loss.

Early pregnancy loss is a major reason for low reproductive efficiency in the horse. In humans and mice, low numbers of regulatory T cells (Treg cells) are linked to miscarriage. The percentage of Treg cells in oestrous mares at the start of the breeding season was evaluated in relation to the outcome of subsequent pregnancy. For identification and quantification of Treg cells, a highly sensitive and specific qPCR assay targeting the Treg-specific demethylated region in the equine forkhead box transcription factor (FOXP3) gene was established. In a total of 108 mares, pregnancy was followed until detection of early pregnancy loss (n=17), abortion without identification of an infectious or apparent cause (n=9) or birth of a viable foal (n=82). Measured Treg-cell levels did not significantly differ between mares that conceived (82%; 1.50±0.04%) or did not get pregnant (18%; 1.45±0.10%). The Treg-cell percentage at oestrus before breeding was significantly different (P<0.05) between mares that either underwent early pregnancy loss up to Day 40 of pregnancy (1.29±0.07%) and mares that aborted (1.61±0.15%) or gave birth to a live foal (1.52±0.05%). These results suggest that low levels of Treg cells in mares can contribute to pregnancy loss up to Day 40 after ovulation.

Corrigendum: Epigenetic immune monitoring for COVID-19 disease course prognosis.

[This corrects the article DOI: 10.3389/fimmu.2023.1107900.].