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Abstract The transcription factor FOXP3 is essential for the development and function of CD4+CD25hiFOXP3+ regulatory T (Treg) cells, but also expressed in activated human helper T cells without acquisition of a regulatory phenotype. This comment focuses on glycoprotein-A repetitions predominant (GARP or LRRC32) recently identified as specific marker of activated human Treg cells, which may provide the missing link toward a better molecular definition of the regulatory phenotype. Reviewers: Dr Jim Di Danto, Dr Benedita Rocha and Dr Werner Solbach.

Abstract Background Aplastic anemia (AA) is a bone marrow failure syndrome mostly characterized by an immune-mediated destruction of marrow hematopoietic progenitor/stem cells. The resulting hypocellularity limits a detailed analysis of the cellular immune response. To overcome this technical problem we performed a microarray analysis of CD3+ T-cells derived from bone marrow aspirates and peripheral blood samples of newly diagnosed AA patients and healthy volunteers. Two AA patients were additionally analyzed after achieving a partial remission following immunosuppression. The regulation of selected candidate genes was confirmed by real-time RT-PCR. Results Among more than 22.200 transcripts, 583 genes were differentially expressed in the bone marrow of AA patients compared to healthy controls. Dysregulated genes are involved in T-cell mediated cytotoxicity, immune response of Th1 differentiated T-cells, and major regulators of immune function. In hematological remission the expression levels of several candidate genes tend to normalize, such as immune regulators and genes involved in proinflammatory immune response. Conclusion Our study suggests a pivotal role of Th1/Tc1 differentiated T-cells in immune-mediated marrow destruction of AA patients. Most importantly, immune regulatory genes could be identified, which are likely involved in the recovery of hematopoiesis and may help to design new therapeutic strategies in bone marrow failure syndromes.

The article by Faal et al. [1] describes differences in the level of FOXP3 mRNA expression in the conjunctiva of patients with Chlamydia trachomatis infection. The authors suggest that their [...]

During sepsis, a relative increase of regulatory T (Treg) cells has been reported. Its persistence is associated with lymphocyte anergy, immunoparalysis and a poor prognosis. Currently, an exact quantification of human Treg cells based on protein expression of marker molecules is ambiguous, as these molecules are expressed also by activated non-regulatory T cells. Furthermore, no firm criteria for flow cytometer gate settings exist so far. Recently, a specific DNA methylation pattern within FOXP3-TSDR has been reported that allows distinguishing Treg and non-regulatory T cells, independent of their activation status. Using this epigenetic marker, we established a single-tube real-time PCR based methylation assay (QAMA) for relative quantification of Treg cells. Validation was performed on defined ratios of methylated and unmethylated target sequence and on mixtures of Treg and non-regulatory T cells. DNA-methylation was measured in CD4+ T cells isolated from blood samples of 30 septic patients and 30 healthy subjects and compared with results of Treg cell quantification by flow cytometry based on CD4+ CD25hi CD127low measurement. In septic patients both methods showed an increased ratio of Treg cells to all CD4+ T cells. In healthy individuals, the results obtained by both methods were clearly positively correlated. However, the correlation between both methods in septic patients was only weak. We showed that quantification of Treg cells by QAMA detects CD4+ T cells with unmethylated FOXP3-TSDR, hidden in the CD25med/low fraction of flow cytometry. Given that unmethylated FOXP3-TSDR is the most specific feature of Treg cells to date, our assay precisely quantifies Treg cells, as it additionally detects those committed Treg cells, hidden in the CD25med/low fraction of CD4+ cells. Furthermore, QAMA is a reliable method, which is easier to standardize among laboratories and can thus improve reproducibility of Treg cell quantification. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) are essential for controlling peripheral tolerance by the active suppression of various immune cells including conventional T effector cells (Teffs). Downstream of the T cell receptor (TCR), more than 500 protein kinases encoded by the human genome have to be considered in signaling cascades regulating the activation of Tregs and Teffs, respectively. Following TCR engagement, Tregs posses a number of unique attributes, such as constitutive expression of Foxp3, hyporesponsiveness and poor cytokine production. Furthermore, recent studies showed that altered regulation of protein kinases is important for Treg function. These data indicate that signaling pathways in Tregs are distinctly organized and alterations at the level of protein kinases contribute to the unique Treg phenotype. However, kinase-based signaling networks in Tregs are poorly understood and necessitate further systematic characterization. In this study, we analyzed the differential expression of kinases in Tregs and Teffs by using a kinase-selective proteome strategy. In total, we revealed quantitative information on 185 kinases expressed in the human CD4+ T cell subsets. The majority of kinases was equally abundant in both T cell subsets, but 11 kinases were differentially expressed in Tregs. Most strikingly, Tregs showed an altered expression of cell cycle kinases including CDK6. Quantitative proteomics generates first comparative insight into the kinase complements of the CD4+ Teff and Treg subset. Treg-specific expression pattern of 11 protein kinases substantiate the current opinion that TCR-mediated signaling cascades are altered in Tregs and further suggests that Tregs exhibit significant specificities in cell-cycle control and progression. [ABSTRACT FROM AUTHOR]

Aim: Glycoprotein A repetitions predominant (GARP or LRRC32) represents a human regulatory CD4+ CD25hi FOXP3+ T (Treg) cell-specific receptor that controls FOXP3. Ectopic expression of GARP in helper T (Th) cells has been shown to be sufficient for the induction of FOXP3 and generation of a stable regulatory phenotype. Since expression of FOXP3 in Treg cells is epigenetically controlled by a conserved motif, the so-called Treg-specific demethylated region (TSDR), we asked whether GARP-mediated upregulation of FOXP3 in Th cells is similarly accompanied by demethylation of the TSDR.Methods: DNA methylation of the FOXP3 TSDR was analyzed by direct sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. Results: Although GARP-transduced Th cells exhibit constitutive FOXP3 expression and a regulatory phenotype, the FOXP3 TSDR is completely methylated as in naive Th cells. GARP-mediated FOXP3 upregulation in Th cells is not associated with Treg-specific demethylation of the FOXP3 TSDR.Conclusion: Although GARP-engineered Th cells exhibit stable FOXP3 expression and a phenotypic reprogramming towards Treg cells in vitro, these cells do not completely mimic the epigenotype of natural Treg cells. Thus, concepts based on the genetic modification of Th cells as cellular therapies to treat autoimmune diseases or to control transplantation tolerance should be critically tested before any clinical application. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The transcription factor FOXP3 is essential for the development and function of CD4+CD25hiFOXP3+ regulatory T (Treg) cells, but also expressed in activated human helper T cells without acquisition of a regulatory phenotype. This comment focuses on glycoprotein-A repetitions predominant (GARP or LRRC32) recently identified as specific marker of activated human Treg cells, which may provide the missing link toward a better molecular definition of the regulatory phenotype. [ABSTRACT FROM AUTHOR]

Background: Aplastic anemia (AA) is a bone marrow failure syndrome mostly characterized by an immune-mediated destruction of marrow hematopoietic progenitor/stem cells. The resulting hypocellularity limits a detailed analysis of the cellular immune response. To overcome this technical problem we performed a microarray analysis of CD3+ T-cells derived from bone marrow aspirates and peripheral blood samples of newly diagnosed AA patients and healthy volunteers. Two AA patients were additionally analyzed after achieving a partial remission following immunosuppression. The regulation of selected candidate genes was confirmed by real-time RTPCR. Results: Among more than 22.200 transcripts, 583 genes were differentially expressed in the bone marrow of AA patients compared to healthy controls. Dysregulated genes are involved in T-cell mediated cytotoxicity, immune response of Th1 differentiated T-cells, and major regulators of immune function. In hematological remission the expression levels of several candidate genes tend to normalize, such as immune regulators and genes involved in proinflammatory immune response. Conclusion: Our study suggests a pivotal role of Th1/Tc1 differentiated T-cells in immunemediated marrow destruction of AA patients. Most importantly, immune regulatory genes could be identified, which are likely involved in the recovery of hematopoiesis and may help to design new therapeutic strategies in bone marrow failure syndromes. [ABSTRACT FROM AUTHOR]

CD4CD25 regulatory T cells (T cells) control immune responsiveness to a large variety of antigens. The isolation and therapeutic manipulation of T cells requires the use of reliable surface receptors that are selectively up-regulated in T cells. On the basis of global gene expression studies, we identified neuropilin-1 (Nrp1) as a specific surface marker for CD4CD25 T cells. Nrp1, a receptor involved in axon guidance, angiogenesis, and the activation of T cells, is constitutively expressed on the surface of CD4CD25 T cells independently of their activation status. In contrast, Nrp1 expression is down-regulated in naive CD4CD25 T cells after TCR stimulation. Furthermore, CD4Nrp1 T cells express high levels of Foxp3 and suppress CD4CD25 T cells. Thus, Nrp1 constitutes a useful surface marker to distinguish T cells from both naive and recently activated CD4CD25 non-regulatory T cells. [ABSTRACT FROM AUTHOR]