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Background: Oral immunotherapy (OIT) is an emerging treatment for cow's milk protein (CMP) allergy in children. The mechanisms driving tolerance following OIT are not well understood. Regulatory T cells (T REG ) cells are key inhibitors of allergic responses and promoters of allergen-specific tolerance. In an exploratory study, we sought to detect induction of allergen-specific T REG in a cohort of subjects undergoing OIT., Methods: Pediatric patients with a history of allergic reaction to cow's milk and a positive Skin Pick Test (SPT) and/or CMP-specific IgE >0.35 kU, as well as a positive oral challenge to CMP underwent OIT with escalating doses of milk and were followed for up to 6 months. At specific milestones during the dose escalation and maintenance phases, casein-specific CD4 + T cells were expanded from patient blood by culturing unfractionated PBMCs with casein in vitro. The CD4 + T cell phenotypes were quantified by flow cytometry., Results: Our culture system induced activated casein-specific FOXP3 + Helios + T REG cells and FOXP3 - T EFF cells, discriminated by expression of CD137 (4-1BB) and CD154 (CD40L) respectively. The frequency of casein-specific T REG cells increased significantly with escalating doses of milk during OIT while casein-specific T EFF cell frequencies remained constant. Moreover, expanded casein-specific T REG cells expressed higher levels of FOXP3 compared to polyclonal T REG cells, suggesting a more robust T REG phenotype. The induction of casein-specific T REG cells increased with successful CMP desensitization and correlated with increased frequencies of casein-specific Th1 cells among OIT subjects. The level of casein-specific T REG cells negatively correlated with the time required to reach the maintenance phase of desensitization., Conclusions: Overall, effective CMP-OIT successfully promoted the expansion of casein-specific, functionally-stable FOXP3 + T REG cells while mitigating Th2 responses in children receiving OIT. Our exploratory study proposes that an in vitro T REG response to casein may correlate with the time to reach maintenance in CMP-OIT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer BL declared a shared affiliation with one of the authors, LL, to the handling editor at time of review., (Copyright © 2021 Zhang, Li, Genest, Zhao, Ke, Bartolucci, Pavey, Al-Aubodah, Lejtenyi, Torabi, Ben-Shoshan, Mazer and Piccirillo.)

Type 1 regulatory T (Tr1) cells play a fundamental role in maintaining and inducing immune tolerance. Our preliminary study demonstrated that an interleukin- (IL-) 10-mediated pathway is a possible regulatory mechanism underlying the xenoantigen-specific human Treg enhanced suppressive capacity. Here, we developed a feasible protocol for expanding IL-10-induced xenoantigen-specific human Tr1 cells in vitro which would be more efficient in transplantation immunotherapy efficiency. In this study, xenoantigen-specific Tr1 cells are generated from human naive CD4 + T cells expanded for two subsequent xenoantigen-stimulation cycles with recombinant human IL-10. The phenotype and suppressive capacity of xenoantigen-stimulated Tr1 cells are assessed, and the mechanism of their suppression is studied. Tr1 cells can be induced by porcine xenoantigen stimulation combined with IL-10, IL-2, and IL-15, displaying an increased expression of CD49b, CTLA-4, and LAG-3 without expressing Foxp3 which also showed an effector memory Treg phenotype and expressed high levels of CD39. After xenoantigen stimulation, the IL-10 and IL-5 gene expression in Tr1 cells increased, secreting more IL-10, and xenoantigen-stimulated Tr1 cells changed their T cell receptor (TCR) V β repertoire, increasing the expression of TCR V β 2, TCR V β 9, and TCR V β 13. In a pig to human mixed lymphocyte reaction (MLR), xenoantigen-stimulated Tr1 cells displayed enhanced suppressive capacity via CD39 in a dose-dependent manner. Moreover, IL-5 could affect the proliferation of xenoantigen-specific Tr1 cells, but not their phenotypes' expression. This study provides a theory and feasible method for immune tolerance induction in clinical xenotransplantation., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2021 Xiaoting Chen et al.)

The thymus produces self-limiting and self-tolerant T cells through the interaction between thymocytes and thymus epithelial cells (TECs), thereby generating central immune tolerance. The TECs are composed of cortical and medullary thymic epithelial cells, which regulate the positive and negative selection of T cells, respectively. During the process of negative selection, thymocytes with self-reactive ability are deleted or differentiated into regulatory T cells (Tregs). Tregs are a subset of suppressor T cells that are important for maintaining immune homeostasis. The differentiation and development of Tregs depend on the development of TECs and other underlying molecular mechanisms. Tregs regulated by thymic epithelial cells are closely related to human health and are significant in autoimmune diseases, thymoma and pregnancy. In this review, we summarize the current molecular and transcriptional regulatory mechanisms by which TECs affect the development and function of thymic Tregs. We also review the pathophysiological models of thymic epithelial cells regulating thymic Tregs in human diseases and specific physiological conditions., (© 2021 The Scandinavian Foundation for Immunology.)

Regulatory T cells (Treg) are essential to maintain immune homeostasis and prevent autoimmune disorders. While the function and molecular regulation of Foxp3 + CD4 + Tregs are well established, much of CD8 + Treg biology remains to be revealed. Here, we will review the heterogenous subsets of CD8 + T cells have been named "CD8 + Treg" and mainly focus on CD122 hi Ly49 + CD8 + Tregs present in naïve mice. CD122 hi Ly49 + CD8 + Tregs, which depends on transcription factor Helios and homeostatic cytokine IL-15, have been established as a non-redundant regulator of germinal center (GC) reaction. Recently, we have demonstrated that TGF-β (Transforming growth factor-β) and transcription factor Eomes (Eomesodermin) are essential for the function and homeostasis of CD8 + Tregs. In addition, we will discuss several open questions regarding the differentiation, function and true identity of CD8 + Tregs as well as a brief comparison between two regulatory T cell subsets critical to control GC reaction, namely CD4 + T FR (follicular regulatory T cells) and CD8 + Tregs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mishra, Srinivasan, Ma and Zhang.)

Systemic lupus erythematosus (SLE) is a chronic multi-organ autoimmune disease involving the production of a wide range of autoantibodies and complement activation. The production of these high-affinity autoantibodies requires T cell/B cell collaboration as well as germinal center (GC) formation. T follicular regulatory cells (TFRs) are functional specialized T regulatory cells (Tregs) that safeguard against both self-reactive T and B cells. However, recent evidence suggests that TFRs are not always stable and can lose Foxp3 expression to become pathogenic "ex-TFRs" that gain potent effector functions. In this review, we summarize the literature on intrinsic and extrinsic mechanisms of regulation of TFR stability and discuss the potential role of TFR reprogramming in autoantibody production and SLE pathogenesis., Competing Interests: XW consulted for Zhongke Qiyuan (Shenzhen) Biotechnology Co., Ltd. in T cell immunity. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wei, Zhang and Zhou.)

CD4 + CD25 high CD127 low/- FOXP3 + T regulatory cells are responsible for maintaining immune tolerance and controlling excessive immune responses. Treg cell use in pre-clinical animal models showed the huge therapeutic potential of these cells in immune-mediated diseases and laid the foundations for their applications in therapy in humans. Currently, there are several clinical trials utilizing the adoptive transfer of Treg cells to reduce the morbidity in autoimmune disorders, allogeneic HSC transplantation, and solid organ transplantation. However, a large part of them utilizes total Treg cells without distinction of their biological variability. Many studies on the heterogeneity of Treg cell population revealed distinct subsets with different functions in the control of the immune response and induction of peripheral tolerance. Some of these subsets also showed a role in controlling the general homeostasis of non-lymphoid tissues. All these Treg cell subsets and their peculiar properties can be therefore exploited to develop novel therapeutic approaches. This review describes these functionally distinct subsets, their phenotype, homing properties and functions in lymphoid and non-lymphoid tissues. In addition, we also discuss the limitations in using Treg cells as a cellular therapy and the strategies to enhance their efficacy., (© 2020 Wiley-VCH GmbH.)

Introduction: Regulatory T cells (Tregs) are a unique CD4+ T cell subset involved in the regulation of immune responses. The traditional immunophenotype used to define Tregs includes CD4+CD25high and the expression of the transcription factor Forkhead box protein 3 (FoxP3). A complex technique of intracellular staining, transient upregulation of FoxP3 in activated conventional T lymphocytes (Tcons), and the omission of naïve CD45RA+ Tregs with downregulated FoxP3 activity but a demethylated FOXP3 promoter region may lead to inaccurate quantification. In an attempt to meet the need for a reliable and simplified enumeration strategy, we investigated different membrane markers to capture the entire Treg compartment and to identify subpopulations of Tregs., Material and Methods: Analyses were performed on whole blood. Tested gating strategies were based on the expression of the following membrane antigens: CD45, CD3, CD4, CD25, CD127, CD26, CD6, CD39, CD71, HLA-DR, CD45RA and CD31. Double controls with FoxP3 were performed., Results: The final enumeration panel consisted of the membrane markers CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA and CD31. A deep analysis of T cells with the CD4+CD25+CD127low/-CD26low/-CD45RAimmunophenotype revealed high expression of FoxP3 and/or CD39, while cells with the naïve immunophenotype, CD4+CD25+CD127low/-CD26low/-CD45RA+, presented lower expression of suppressor markers. Antigen CD31 is considered to be a valuable membrane marker of thymus-derived Tregs., Conclusions: The presented 9-color panel that can be easily applied in laboratories enables reliable enumeration of Tregs with additional information about the functionality, maturity and origin of T regulatory cells.

Regulatory T (T reg ) cells represent an essential component of peripheral tolerance. Given their potently immunosuppressive functions that is orchestrated by the lineage-defining transcription factor forkhead box protein 3 (FoxP3), clinical modulation of these cells in autoimmunity and cancer is a promising therapeutic target. However, recent evidence in mice and humans indicates that T reg cells represent a phenotypically and functionally heterogeneic population. Indeed, both suppressive and non-suppressive T reg cells exist in human blood that are otherwise indistinguishable from one another using classical T reg cell markers such as CD25 and FoxP3. Moreover, murine T reg cells display a degree of plasticity through which they acquire the trafficking pathways needed to home to tissues containing target effector T (T eff ) cells. However, this plasticity can also result in T reg cell lineage instability and acquisition of proinflammatory T eff cell functions. Consequently, these dysfunctional CD4 + FoxP3 + T cells in human and mouse may fail to maintain peripheral tolerance and instead support immunopathology. The mechanisms driving human T reg cell dysfunction are largely undefined, and obscured by the scarcity of reliable immunophenotypical markers and the disregard paid to T reg cell antigen-specificity in functional assays. Here, we review the mechanisms controlling the stability of the FoxP3 + T reg cell lineage phenotype. Particular attention will be paid to the developmental and functional heterogeneity of human T reg cells, and how abrogating these mechanisms can lead to lineage instability and T reg cell dysfunction in diseases like immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, type 1 diabetes, rheumatoid arthritis and cancer., (© 2019 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

CD4 + regulatory T cells (T reg ) expressing the forkhead box protein 3 (FOXP3) transcription factor (T regs ) are instrumental for the prevention of autoimmune diseases. There is increasing evidence that the human T regulatory population is highly heterogeneous in phenotype and function. Numerous studies conducted in human autoimmune diseases have shown that T reg cells are impaired either in their suppressive function, in number, or both. However, the contribution of the FOXP3 + T reg subpopulations to the development of autoimmunity has not been delineated in detail. Rare genetic disorders that involve deficits in T reg function can be studied to develop a global idea of the impact of partial or complete deficiency in a specific molecular mechanism involved in T reg function. In patients with reduced T reg numbers (but no functional deficiency), the expansion of autologous T reg cells could be a suitable therapeutic approach: either infusion of in-vitro autologous expanded cells, infusion of interleukin (IL)-2/anti-IL-2 complex, or both. T reg biology-based therapies may not be suitable in patients with deficits of T reg function, unless their deficit can be corrected in vivo/in vitro. Finally, it is critical to consider the appropriate stage of autoimmune diseases at which administration of T reg cellular therapy can be most effective. We discuss conflicting data regarding whether T reg cells are more effectual at preventing the initiation of autoimmunity, ameliorating disease progression or curing autoimmunity itself., (© 2019 British Society for Immunology.)

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract 1-4 . The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (T reg ) cells 4-8 , and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease 9 . However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain T reg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce T reg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1β. Macrophages in the small intestine produce IL-1β, and activation of this pathway involves MYD88- and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining T reg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of T reg cells. Our results reveal a previously unappreciated pathway in which a microbiota- and IL-1β-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine.

Regulatory T-cells (Tregs) are a very important subtype of lymphocytes when it comes to self-control in the human immunological system. Tregs are decisive not only in the protection against destruction of own tissues by autoimmune immunocompetent cells but also in the immunological answer to developing cancers. On the other hand, Tregs could be responsible for the progression of acute and chronic leukemias. In our study, we review publications available in the PUMED database concerning acute leukemia, with a particular emphasis on child's leukemias. The percentage of regulatory T-lymphocytes in peripheral blood and bone marrow was elevated compared to those in healthy individuals and correlated with progressive disease. Regulatory T-cells taken from children diagnosed with leukemia showed a higher suppressive capability, which was confirmed by detecting elevated levels of secreted IL-10 and TGF-beta. The possibility of pharmacological intervention in the self-control of the immunological system is now under extensive investigation in many human cancers. Presumably, Treg cells could be a vital part of targeted therapies. Routine Treg determination could be used to assess the severity of disease and prognosis in children with acute lymphoblastic leukemia. This proposition results from the fact that in some studies, higher percentage of Treg cells in peripheral blood was demonstrated. However, observations confirming these facts are scarce; thus, extrapolating them to the population of children with hematological malignancies needs to be verified in additional studies.

Our study aimed to study regulatory T cells (Tregs) and their expression of CD45RA, HLA-DR, and CD39 in preterm and full-term infants. In an observational study, we used a three-color flow cytometry for determination of Tregs and their expression of CD45RA, HLA-DR, and CD39 in preterm and full-term infants. The percentages of CD4 + CD25 +high Foxp3 + , CD39 + Tregs, HLA-DR + Tregs and the expression of Foxp3 + in CD4 + CD25 +high Foxp3 Tregs cells were significantly lower in neonates when compared to healthy adult controls. The levels of naïve resting Tregs (CD45RA + Tregs) were significantly higher in neonates than controls. The percentages of CD4 + CD25 +high Foxp3 + Tregs, total CD4 + CD25 + and CD4 + CD25 +high were significantly higher in preterm infants when compared to the full-term group. Moreover, CD45RA + Tregs were significantly higher in preterm than in term infants. We found significant inverse correlations between the gestational age and the levels of both Tregs (r = - 0.395, p = 0.017) and CD45RA + Tregs (r = - 0.422, p = 0.010). Relative to full-term, the frequencies, and phenotypes of Tregs were affected by prematurity. A larger longitudinal study with a sufficient number of newborns is needed to investigate the Treg pool of term and preterm infants thoroughly and to explore the association between the Treg pool and clinical variables.

Intestinal integrity is maintained by balanced numbers of CD103+ Dendritic cells (DCs), which generate peripherally induced regulatory T cells (iTregs). We have developed a mouse model where DC-specific constitutive CD40 signals caused a strong reduction of CD103+ DCs in the lamina propria (LP) and intestinal lymph nodes (LN). As a consequence, also iTregs were strongly reduced and transgenic mice on the C57Bl/6-background (B6) developed fatal colitis. Here we describe that transgenic mice on a pure Balb/c-background (B/c) do not show any pathologies, while transgenic C57Bl/6 x Balb/c (F1) mice develop weak colon inflammation, without fatal colitis. This graded pathology correlated with the effects of CD40-signalling on DCs in each background, with striking loss of CD103+ DCs in B6, but reduced in F1 and diminished in B/c background. We further show direct correlation of CD103+ DC-numbers with numbers of iTregs, the frequencies of which behave correspondingly. Striking effects on B6-DCs reflected robust loss of surface MHCII, known to be crucial for iTreg induction. Furthermore, elevated levels of IL-23 together with IL-1, found only in B6 mice, support generation of intestinal IFN-γ+IL-17+ Th17 cells and IFN-γ+ Th1 cells, responsible for onset of disease. Together, this demonstrates a novel aspect of colitis-control, depending on genetic background. Moreover, strain-specific environmental sensing might alter the CD103+ DC/iTreg-axis to tip intestinal homeostatic balance to pathology., Competing Interests: The authors have declared that no competing interests exist.

Type 1 diabetes is an autoimmune disease where autoreactive T lymphocytes destroy pancreatic beta cells. We previously reported a defect in CD4+ Tregs cell proliferation and reduced CD4+ Tregs PD-1 expression in patients. Another 'memory-like' regulatory subset, CD8+ Tregs, evaluated as CD8+CD25+FOXP3+, has recently raised interest for their effective suppressive activity. Different CD8+ T cell populations, their proliferation capacity and expression of PD-1 molecule were evaluated by flow-cytometer analysis in newly diagnosed, long-term Type 1 diabetes patients compared to healthy normal donors. Under basal conditions, CD8+ Tregs and CD8+ Teffs were seemingly represented among study groups while there was evidence of diminished expression of PD-1 in Teff subsets of long-term patients. After 3 days of PMA/ionomycin stimulation, patients CD8+ Tregs showed decreased percentage in respect to control group. CD8+ Teffs were instead increased in long-term diabetics versus controls. PD-1+CD8+ Tregs were represented at a much lower percentage in long-term diabetic patients, in respect to controls. Importantly, patients CD8+ Tregs and CD8+ Teffs presented a significant proliferation defect in respect to the control group. In conclusion, our study indicates that a defect of CD8+ Tregs is observed in diabetics. This subset could thus represent a novel target of immunotherapy in patients., Competing Interests: The authors have declared that no competing interests exist.

Naïve CD4 T cells can be converted to double-negative regulatory T cells (DNT) by mature dendritic cells (mDCs) and IL-2 stimulation, with IL-2 enhancing the proliferation and Perforin expression of DNT. However, the molecules that affect the conversion of DNT are still not clear. Here, we investigated the effects of Ox40 on the conversion and function of DNT in vitro and in vivo without IL-2. We found that OX86 (an Ox40 agonist) increased the conversion rate of DNT but failed to enhance the suppressive function of DNT. Ox40 deficiency profoundly decreased the conversion rate and suppressive function of DNT. This suppression decline was caused by effects of Ox40 on proliferation and apoptosis independent of Perforin, Granzyme B and Fas ligand. Ox40 deficiency influenced the regulatory function of DNT through multiple signals, such as Cxcr3, Cd160 and Cd30, independently of Prf, Gzmb and Fasl. In conclusion, we elucidated that Ox40 promotes the conversion and maintenance of DNT. Ox40 deficiency reduced the regulatory function of DNT both in vitro and in vivo by regulating proliferation, apoptosis, and suppression-related genes., (Copyright © 2018. Published by Elsevier B.V.)

Background: Sarcoidosis is characterized by exaggerated immune response to unknown agent and can affect different organs. One of the main players in the pathology of the disease are regulatory T cells (Tregs), however, up to date the mechanisms of the possible molecular alterations of this particular cell subset are not known., Methods: In the current study we looked for the global transcriptomic changes of miRNAs, using predefined array, and mRNAs (RNA seq analysis) of Tregs of patients with the most predominant form of the disease - acute pulmonary sarcoidosis (PS). For this purpose sorted CD4+/CD25+/CD127- Tregs from peripheral blood (PB) and CD4+/CD25 + Tregs from bronchoalveolar lavage (BAL) were used., Results: MiRNA analysis revealed that Tregs isolated from PB and BAL display significantly different miRNA profile, suggesting an important role of the pulmonary microenvironment in creating these changes. Among disease-related miRNAs of PB Tregs we identified miR-155 and miR-223. Moreover, looking at the global transcriptome of PB Tregs, we recognized alterations in TLR-2 signaling pathway and in the downstream of NF-κB apoptosis and proliferation signals. However, induction of TLR-2 expression was found not only in Tregs, but also in the heterogeneous population of peripheral blood mononuclear cells (PBMC) as well as two PBMC subpopulations (CD4+/CD25-and CD4-/CD25-) of patients with PS. This indicates that activation of TLR signaling pathway in sarcoidosis does not occur only in Tregs., Conclusion: Our findings offer a deeper insight into the molecular mechanisms of Tregs reduced suppression and increased apoptosis in patients with PS. Based on the current results, future studies should focus on possible therapeutic effect of TLR-2 signaling inhibition., (Copyright © 2018. Published by Elsevier Ltd.)

Asthma is a chronic disease of airway inflammation due to excessive T helper cell type 2 (Th2) response. Present treatment based on inhalation of synthetic glucocorticoids can only control Th2-driven chronic eosinophilic inflammation, but cannot change the immune tolerance of the body to external allergens. Regulatory T cells (Tregs) are the main negative regulatory cells of the immune response. Tregs play a great role in regulating allergic, autoimmune, graft-versus-host responses, and other immune responses. In this review, we will discuss the classification and biological characteristics, the established immunomodulatory mechanisms, and the characteristics of induced differentiation of Tregs. We will also discuss the progress of Tregs in the field of asthma. We believe that further studies on the regulatory mechanisms of Tregs will provide better treatments and control strategies for asthma.

The immune system regulates itself to establish an appropriate immune response to potentially harmful pathogens while tolerating harmless environmental antigens and self-antigens. A central role in this balance is played by regulatory T cells (Tregs) through various ways of actions. By means of molecule secretion and cell-cell contact mechanisms, Tregs may have the capacity to modulate effector T cells and suppress the action of proinflammatory cytokines across a broad range of cell types. As a result, abnormal regulatory T cell function has been pointed as a main cause in the development of allergic diseases, a major public health problem in industrialized countries, with a high socioeconomic impact. This prevalence and impact have created an international interest in improving the allergy diagnosis and therapy. Additionally, research has sought to gain a better understanding of the molecular mechanisms underlining this kind of disease, in order to a better management. At this respect, the role of Treg cells is one of the most promising areas of research, mainly because of their potential use as new immunotherapeutical approaches. Therefore, the aim of this review is to update the existing knowledge of the role of Tregs in this pathology deepening in their implication in allergen-specific therapy (AIT).

Regulatory T cell (Treg cell) is an important immunosuppressive T cell subset and plays a dominant role in maintaining the immune balance in vivo. The function defects in Treg cells have been involved in the pathogenesis of many autoimmune diseases. The detection of Treg cell suppressive function is important for early diagnosis and prediction of response to treatment for autoimmune diseases. The traditional detection of Treg cell suppressive function needs at least 20 mL peripheral blood sample of patients and the results would be got in sixth day, therefore, it could not be widely applied in clinical. However, to find fast and simple detection method is very important. CD147 is a transmembrane protein and its expression is related to Treg cell suppressive function. Recent research has shown that the Treg cells with high CD147 expression have stronger suppressive function than which with low CD147 expression. In this work, we detected the ratio of CD147 high /CD147 low in CD4 + CD25 + T cells in patients with active AS using fluorescence-activated cell sorter (FACS). The results show the ratio of CD147 high /CD147 low decreased obviously in patients with active AS compared with healthy controls, which reflects the suppressive function deficit of Treg cell. In the same time, the detection of the ratio of CD147 high /CD147 low needs only 150 μL peripheral blood sample and the result would be got in 4 h. We therefore hypothesize that the ratio of CD147 high /CD147 low is a good indicator for the Treg cell function, and it is especially suitable for early diagnosis and prediction of response to therapy targeted recovering Treg cell function in autoimmune diseases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a pathological condition characterized by incapacitating fatigue and a combination of neurologic, immunologic, and endocrine symptoms. At present its diagnosis is based exclusively on clinical criteria. Several studies have described altered immunologic profiles; therefore, we proposed to further examine the more significant differences, particularly T and NK cell subpopulations that could be conditioned by viral infections, to discern their utility in improving the diagnosis and characterization of the patients. The study included 76 patients that fulfilled the revised Canadian Consensus Criteria (CCC 2010) for ME/CFS and 73 healthy controls, matched for age and gender. Immunophenotyping of different T cell and natural killer cell subpopulations in peripheral blood was determined by flow cytometry. ME/CFS patients showed significantly lower values of T regulatory cells (CD4 + CD25 ++(high) FOXP3 + ) and higher NKT-like cells (CD3 + CD16 +/- CD56 + ) than the healthy individuals. Regarding NK phenotypes, NKG2C was significantly lower and NKCD69 and NKCD56 bright were significantly higher in the patients group. A classification model was generated using the more relevant cell phenotype differences (NKG2C and T regulatory cells) that was able to classify the individuals as ME/CFS patients or healthy in a 70% of cases. The observed differences in some of the subpopulations of T and NK cells between patients and healthy controls could define a distinct immunological profile that can help in the diagnostic process of ME/CFS patients, contribute to the recognition of the disease and to the search of more specific treatments. However, more studies are needed to corroborate these findings and to contribute to establish a consensus in diagnosis.

Cancer immunotherapy involving blockade of immune checkpoint molecules, such as CTLA-4 and PD-1, has shown remarkable clinical success across several types of malignancies. However, a fraction of patients experience disease progression after treatment; thus, exploring resistant mechanisms for immune checkpoint inhibitors and improving their treatment outcome with additional modalities are of great importance. CD4 + regulatory T (T reg ) cells characterized by expression of the master regulatory transcription factor FOXP3 are a highly immune-suppressive subset of CD4 + T cells that maintain immune homeostasis. Several preclinical and clinical studies suggest that T reg cells hamper immune surveillance against cancer in healthy individuals, prevent the development of effective antitumor immunity in tumor-bearing patients, and promote tumor progression. Therefore, targeting T reg cells should be crucial to improving the treatment outcomes of cancer immunotherapy. Several clinical studies directly or indirectly targeting T reg cells in combination with immune checkpoint inhibitors are ongoing or being planned. Understanding the characteristics and roles of T reg cells in cancer settings could make disease-specific T reg -targeted therapy more efficacious and reduce the incidence of immune-related adverse effects mediated by T reg cell inhibition., (© 2018 New York Academy of Sciences.)

Objective: To establish a quick analytical method using quantitative PCR for marker gene analysis to identify the functions of iTreg cells and subsequently curtail the harvest time for iTreg cells., Results: The data from the marker gene analysis indicated that varying proportions of iTreg cells could reveal the various expression levels of these genes. FoxP3 expression increased to a considerable degree. By using the same iTreg population, the mixed lymphocyte reaction assay was conducted for 5 days. The suppression percentage of T-cells was dependent on the proportion of iTreg cells, indicating that gene expression levels can represent the biological functions of iTreg cells. By using human peripheral blood mononuclear cells for Treg cell induction, the marker gene expression analysis showed a difference between iTreg cells and uninduced T cells., Conclusion: Marker gene analysis requires only 1 day to identify the functions of human iTreg cells can save time in clinical application and might prevent graft-versus-host disease occurrence effectively.

Aims: Tumour-associated macrophages (TAMs) and regulatory T cells (Tregs) form a special niche supporting tumour progression, and both correlate with worse survival in head and neck cancers. However, the prognostic role of TAM and Tregs in nasopharyngeal carcinoma (NPC) is still unknown. Therefore, we determined differences in TAMs and Tregs in different NPC subtypes, and their prognostic significance., Methods: Tissue of 91 NPCs was assessed for TAMs and Tregs by determination of CD68, CD163, CD206 and FOXP3 expression in the tumour microenvironment. Clinicopathological correlations were assessed using Pearson X 2 test, Fisher's exact test, analysis of variance and Mann-Whitney U test. Survival was analysed using Kaplan-Meier curves and Cox regression., Results: CD68 and FOXP3 counts were higher in Epstein-Barr virus (EBV)-positive NPC, while CD68-/FOXP3-, CD163+/FOXP3- and CD206+/FOXP3- infiltrates were more common in EBV-negative NPC. In the whole NPC group, CD68-/FOXP3- correlated with worse overall survival (OS), and after multivariate analysis high FOXP3 count showed better OS (HR 0.352, 95% CI 0.128 to 0.968). No difference in M2 counts existed between EBV-positive and negative NPC., Conclusions: FOXP3, a Treg marker, seems to be an independent prognostic factor for better OS in the whole NPC group. Therefore, immune-based therapies targeting Tregs should be carefully evaluated. M2 spectrum macrophages are probably more prominent in EBV-negative NPC with also functional differences compared with EBV-positive NPC., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Thymus-derived regulatory T cells of CD4+CD25+Foxp3+ phenotype develop as a functional, mature population playing an essential role in self-tolerance and immune homeostasis, and exhibiting therapeutic potential to inhibit adverse immune response. Despite intensive research on thymus-derived Tregs, the knowledge about agents involved in their generation, survival, proliferation, and biological functions is still insufficient. In this research we have focused on the role of selected cytokines in previously developed in vitro model based on the application of anti-CD3 monoclonal antibodies. We have demonstrated an essential role of IL-7 and TGF-β in the generation of thymus-derived Tregs in the co-culture of thymocytes and JAWS II cells. In addition, in vitro generated Tregs exhibited their suppressive function similarly to Tregs sorted from freshly isolated thymus., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The generation and function of immuno-suppressive regulatory T lymphocytes (Treg), which can differentiate in the thymus (tTreg) or in the periphery (pTreg), are regulated in an age-dependent manner. tTreg are produced at high levels in the first weeks of age, when they expand and colonize secondary lymphoid organs and peripheral tissues to protect the organism from autoimmune diseases and to promote tissue repair. Once this population of Treg is operational in the periphery, at puberty, thymic output of Treg declines, but self-reactive tTreg generated early on in life are maintained over time and play a major role in preserving homeostasis of the immune system. Extra-thymic pTreg differentiation declines later on in life. pTreg generated throughout life mainly protect the organism from chronic inflammation and the semi-allogeneic fetus from rejection. In this review, age-dependent modulation of the production and function of these two populations of Treg is described., (© 2017 S. Karger AG, Basel.)

Follicular regulatory T (T FR ) cells are a newly defined regulatory T cell (Treg) subset that suppresses follicular helper T cell-mediated B cell responses in the germinal center reaction. The precise costimulatory signal requirements for proper T FR cell differentiation and function are still not known. Using conditional knockout strategies of CD28, we previously demonstrated that loss of CD28 signaling in Tregs caused autoimmunity in mice (termed CD28-ΔTreg mice), characterized by lymphadenopathy, accumulation of activated T cells, and cell-mediated inflammation of the skin and lung. In this study, we show that CD28 signaling is required for T FR cell differentiation. Treg-specific deletion of CD28 caused a reduction in T FR cell numbers and function, which resulted in increased germinal center B cells and Ab production. Moreover, residual CD28-deficient T FR cells showed a diminished suppressive capacity as assessed by their ability to inhibit Ab responses in vitro. Surprisingly, genetic deletion of B cells in CD28-ΔTreg mice prevented the development of lymphadenopathy and CD4 + T cell activation, and autoimmunity that mainly targeted skin and lung tissues. Thus, autoimmunity occurring in mice with CD28-deficient Tregs appears to be driven primarily by loss of T FR cell differentiation and function with resulting B cell-driven inflammation., (Copyright © 2017 by The American Association of Immunologists, Inc.)

TGF-β type III receptor (TβRIII) is a co-receptor for TGFβ family members required for high-affinity binding of these ligands to their receptors, potentiating their cellular functions. TGF-βs, Bone Morphogenetic Proteins (BMP2/4) and Inhibins/Activins regulate different checkpoints during T cell differentiation. We have previously reported that TβRIII modulates T cell development by protecting developing thymocytes from apoptosis, however the role of this co-receptor in peripheral lymphocytes still remains elusive. Here we describe a detailed characterization of TβRIII expression in murine and human lymphocyte subpopulations demonstrating that this co-receptor is significantly expressed in T but not B lymphocytes and among them, preferentially expressed on naïve and central memory T cells. TβRIII was upregulated after TCR stimulation, in parallel to other early activation markers. In contrast, natural and induced Tregs downregulated TβRIII in association with FoxP3 upregulation. Finally, anti-TβRIII blocking experiments demonstrated that TβRIII promotes TGFβ-dependent iTreg conversion in vitro, and suggest that this co-receptor may be involved in modulating peripheral T cell tolerance and could be considered as a potential target to boost T cell immune responses., (Copyright © 2017. Published by Elsevier Inc.)

Forkhead box P3 (FoxP3) + regulatory T cells (T regs ) are functionally deficient in systemic lupus erythematosus (SLE), characterized by reduced surface CD25 [the interleukin (IL)-2 receptor alpha chain]. Low-dose IL-2 therapy is a promising current approach to correct this defect. To elucidate the origins of the SLE T reg phenotype, we studied its role through developmentally defined regulatory T cell (T reg ) subsets in 45 SLE patients, 103 SLE-unaffected first-degree relatives and 61 unrelated healthy control subjects, and genetic association with the CD25-encoding IL2RA locus. We identified two separate, uncorrelated effects contributing to T reg CD25. (1) SLE patients and unaffected relatives remarkably shared CD25 reduction versus controls, particularly in the developmentally earliest CD4 + FoxP3 + CD45RO - CD31 + recent thymic emigrant T regs . This first component effect influenced the proportions of circulating CD4 + FoxP3 high CD45RO + activated T regs . (2) In contrast, patients and unaffected relatives differed sharply in their activated T reg CD25 state: while relatives as control subjects up-regulated CD25 strongly in these cells during differentiation from naive T regs , SLE patients specifically failed to do so. This CD25 up-regulation depended upon IL2RA genetic variation and was related functionally to the proliferation of activated T regs , but not to their circulating numbers. Both effects were found related to T cell IL-2 production. Our results point to (1) a heritable, intrathymic mechanism responsible for reduced CD25 on early T regs and decreased activation capacity in an extended risk population, which can be compensated by (2) functionally independent CD25 up-regulation upon peripheral T reg activation that is selectively deficient in patients. We expect that T reg -directed therapies can be monitored more effectively when taking this distinction into account., (© 2017 The Authors. Clinical and Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

As age increases, immune responses and consequently protection following vaccination to seasonal influenza is commonly believed to decrease. Possible drivers of this immune dysfunction include immunosenescence, repeated exposure to the same seasonal influenza antigens, and prior infection with cytomegalovirus (CMV). Here, to determine immune parameters distinguishing vaccine humoral responders (R) from non-responders (NR) following vaccination, we surveyed broad peripheral blood "cellular immune correlates" of older adults vaccinated with Fluad® (an adjuvanted subunit influenza vaccine containing strains H1N1, H3N2 and B). Phenotyping included αβ-T-cells, γδ-T-cells, B-cells and myeloid cells. The frequencies of most of these lymphocyte phenotypes were found to be similar in R and NR, although perhaps counterintuitively, one of the few differences seen between the two groups was higher frequencies of regulatory T-cells in R. These differences were more prominent for responses to the vaccine strains H1N1 and H3N2 than to the B strain, and in CMV-seropositive than CMV-seronegative elderly. Further, frequencies of early-differentiated CD4+ T-cells tended to be higher and frequencies of memory CD4+ T-cells tended to be lower in R than NR. There were also differences in B-cells, with higher frequencies in R compared to NR. To the best of our knowledge, these results are the first to report such differences in elderly people responding or failing to respond to adjuvanted seasonal influenza vaccination., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Repair of the lung epithelium after injury is a critical component for resolution; however, the processes necessary to drive epithelial resolution are not clearly defined. Published data demonstrate that Foxp3 + regulatory T cells (Tregs) enhance alveolar epithelial proliferation after injury, and Tregs in vitro directly promote type II alveolar epithelial cell (AT2) proliferation, in part by a contact-independent mechanism. Therefore, we sought to determine the contribution of Treg-specific expression of a growth factor that is known to be important in lung repair, keratinocyte growth factor (kgf). The data demonstrate that Tregs express kgf and that Treg-specific expression of kgf regulates alveolar epithelial proliferation during the resolution phase of acute lung injury and in a model of regenerative alveologenesis in vivo. In vitro experiments demonstrate that AT2 cells cocultured with Tregs lacking kgf have decreased rates of proliferation compared with AT2 cells cocultured with wild-type Tregs. Moreover, Tregs isolated from lung tissue and grown in culture express higher levels of two growth factors that are important for lung repair (kgf and amphiregulin) compared with Tregs isolated from splenic tissue. Lastly, Tregs isolated from human lung tissue can be stimulated ex vivo to induce kgf expression. This study reveals mechanisms by which Tregs direct tissue-reparative effects during resolution after acute lung injury, further supporting the emerging role of Tregs in tissue repair.

Common variable immunodeficiency (CVID) is a heterogeneous group of primary antibody deficiencies (PAD) which is defined by recurrent infections, hypogammaglobulinemia and defects in B-cell differentiation into plasma cells and memory B cells. T cell abnormalities have also been described in CVID patients. Several studies reported that Treg frequencies and their functional characteristics are disturbed and might account for the aberrant immune responses observed in CVID patients. The aim of this review is to describe phenotypic and functional characteristics of Treg cells, and to review the literature with respect to the reported Treg defects and its association with the clinical manifestation in CVID., (Copyright © 2016. Published by Elsevier Inc.)

FOXP3 + Regulatory T (Treg) cells play a key role in the maintenance of immune homeostasis and tolerance. Disruption of Foxp3 expression results in the generation of instable Treg cells and acquisition of effector T-cell-like function. Here we report that the E3 deubiquitinase USP21 prevents the depletion of FOXP3 at the protein level and restricts the generation of T-helper-1-like Treg cells. Mice depleted of Usp21 specifically in Treg cells display immune disorders characterized by spontaneous T-cell activation and excessive T-helper type 1 (Th1) skewing of Treg cells into Th1-like Treg cells. USP21 stabilizes FOXP3 protein by mediating its deubiquitination and maintains the expression of Treg signature genes. Our results demonstrate how USP21 prevents FOXP3 protein depletion and controls Treg lineage stability in vivo.

Background: Regulatory T cells (Tregs) have important functions in peripheral immune tolerance. Dysfunction of Tregs is considered to be a pivotal cause of autoimmune diseases, including rheumatoid arthritis (RA). However, previous reports describing the proportion of Tregs among CD4+ T cells in RA patients were controversial because a range of markers are used to identify Tregs with little consensus. To clarify the status of Tregs in RA, we investigated the proportion of Tregs with focusing on the definitions of them., Methods: We identified the studies reporting the proportion of Tregs in RA patients using PubMed and Google Scholar. We performed a systematic review of them and a meta-analysis to evaluate the proportion of Tregs (FOXP3-positive and/or CD25-positive) among CD4+ T cells in peripheral blood (PB) and synovial fluid (SF) of RA patients and control subjects., Results: A total 31 studies were selected. The proportion of Tregs defined by all definitions among CD4+ T cells in PB was not significantly different between RA patients and control subjects (-0.65, [-1.30, 0.01]). Then we performed sub-analyses based on individual definitions. The proportion of Tregs defined by either CD25 or FOXP3 alone did not differ between RA patients and control subjects. The proportion of Tregs defined by both FOXP3 and CD25 was lower in RA patients than that in control subjects (-2.42 [-3.49, -1.34]). The proportion of Tregs defined by both FOXP3 and CD25 was higher in SF than that in PB among RA patients (3.27 [0.40, 6.14])., Conclusion: The status of Tregs varied according to the definition system. The proportion of Tregs defined by stricter and functionally validated methods decreased in PB and increased in SF among RA patients. If the proportion of Tregs differs in RA, accurate and functionally relevant definitions of Tregs are necessary to elucidate their status in RA., Competing Interests: The authors have declared that no competing interests exist.

Background: Delayed graft function (DGF) and slow graft function (SGF) are ischemia-reperfusion-associated acute kidney injuries (AKI) that decrease long-term graft survival after kidney transplantation. Regulatory T (Treg) cells are protective in murine AKI, and their suppressive function predictive of AKI in kidney transplantation. The conventional Treg cell function coculture assay is however time-consuming and labor intensive. We sought a simpler alternative to measure Treg cell function and predict AKI., Methods: In this prospective observational cohort study, pretransplant recipient circulating CD4+CD25+CD127lo/- and CD4+CD127lo/- tumor necrosis factor receptor 2 (TNFR2)+ Treg cells were measured by flow cytometry in 76 deceased donor kidney transplant recipients (DGF, n = 18; SGF, n = 34; immediate graft function [IGF], n = 24). In a subset of 37 recipients, pretransplant circulating Treg cell-suppressive function was also quantified by measuring the suppression of autologous effector T-cell proliferation by Treg cell in coculture., Results: The TNFR2+ expression on CD4+CD127lo/- T cells correlated with Treg cell-suppressive function (r = 0.63, P < 0.01). In receiver operating characteristic curves, percentage and absolute number of CD4+CD127lo/-TNFR2+ Treg cell predicted DGF from non-DGF (IGF + SGF) with area under the curves of 0.75 and 0.77, respectively, and also AKI (DGF + SGF) from IGF with area under the curves of 0.76 and 0.72, respectively (P < 0.01). Prediction of AKI (DGF + SGF) from IGF remained significant in multivariate logistic regression accounting for cold ischemic time, donor age, previous transplant, and pretransplant dialysis modality., Conclusions: Pretransplant recipient circulating CD4+CD127lo/-TNFR2+ Treg cell is potentially a simpler alternative to Treg cell function as a pretransplant recipient immune marker for AKI (DGF + SGF), independent from donor and organ procurement characteristics.

Human regulatory T cells (T(reg) cells) that develop from conventional T cells (T(conv) cells) following suboptimal stimulation via the T cell antigen receptor (TCR) (induced T(reg) cells (iT(reg) cells)) express the transcription factor Foxp3, are suppressive, and display an active proliferative and metabolic state. Here we found that the induction and suppressive function of iT(reg) cells tightly depended on glycolysis, which controlled Foxp3 splicing variants containing exon 2 (Foxp3-E2) through the glycolytic enzyme enolase-1. The Foxp3-E2-related suppressive activity of iT(reg) cells was altered in human autoimmune diseases, including multiple sclerosis and type 1 diabetes, and was associated with impaired glycolysis and signaling via interleukin 2. This link between glycolysis and Foxp3-E2 variants via enolase-1 shows a previously unknown mechanism for controlling the induction and function of T(reg) cells in health and in autoimmunity.

Foxp3(+) regulatory T cells (Tregs) are essential for preventing autoimmunity and uncontrolled inflammation, and they modulate immune responses during infection and the development of cancer. Accomplishing these tasks requires the widespread distribution of Tregs in both lymphoid and nonlymphoid tissues, and the selective recruitment of Tregs to different tissue sites has emerged as a key checkpoint that controls tissue inflammation in autoimmunity, infection, and cancer development, as well as in the context of allograft acceptance or rejection. Additionally, Tregs are functionally diverse, and it has become clear that some of this diversity segregates with Treg localization to particular tissue sites. In this article, I review the progress in understanding the mechanisms of Treg trafficking and discuss factors controlling their homeostatic maintenance and function in distinct tissue sites., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Regulatory T cells (Tregs) are an essential component of the cellular immune response, occupying a key role in maintaining immunological tolerance and present an attractive therapeutic target in a range of immunopathologies. Comprehensive analysis of the human Treg compartment has been restricted due to technical limitations. The advent of mass cytometry enables simultaneous assessment of vastly increased phenotypic parameters at single-cell resolution. In this study, we used mass cytometry to examine the complexity of human Tregs using an extensive panel of surface markers associated with Treg function and phenotype. We applied unsupervised clustering analysis, revealing 22 distinct subpopulations of Tregs, representing previously identified and novel subpopulations. Our data represent the most in-depth phenotypic description of the human Treg compartment at single-cell resolution and show a hitherto unrecognized degree of phenotypic complexity among cells of the regulatory lineage., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Chronic schistosomiasis is associated with T cell hypo-responsiveness and immunoregulatory mechanisms, including induction of regulatory T cells (Tregs). However, little is known about Treg functional capacity during human Schistosoma haematobium infection., Methodology: CD4+CD25hiFOXP3+ cells were characterized by flow cytometry and their function assessed by analysing total and Treg-depleted PBMC responses to schistosomal adult worm antigen (AWA), soluable egg antigen (SEA) and Bacillus Calmette-Guérin (BCG) in S. haematobium-infected Gabonese children before and 6 weeks after anthelmintic treatment. Cytokines responses (IFN-γ, IL-5, IL-10, IL-13, IL-17 and TNF) were integrated using Principal Component Analysis (PCA). Proliferation was measured by CFSE., Principal Findings: S. haematobium infection was associated with increased Treg frequencies, which decreased post-treatment. Cytokine responses clustered into two principal components reflecting regulatory and Th2-polarized (PC1) and pro-inflammatory and Th1-polarized (PC2) cytokine responses; both components increased post-treatment. Treg depletion resulted in increased PC1 and PC2 at both time-points. Proliferation on the other hand, showed no significant difference from pre- to post-treatment. Treg depletion resulted mostly in increased proliferative responses at the pre-treatment time-point only., Conclusions: Schistosoma-associated CD4+CD25hiFOXP3+Tregs exert a suppressive effect on both proliferation and cytokine production. Although Treg frequency decreases after praziquantel treatment, their suppressive capacity remains unaltered when considering cytokine production whereas their influence on proliferation weakens with treatment.

Treatment of patients with chronic hepatitis C with the current standard pegylated interferon (PEG-IFN) and ribavirin achieves overall response (SVR) rates of ~55%. A role of CD4+ CD25+ regulatory T cells (Treg cells) has been proposed as they can suppress HCV-specific T cells in HCV-infected patients. Patients with chronic HCV legible for PEG-IFN plus ribavirin treatment, were classified according to their response to treatment into two groups (responders and non-responders, 32 and 27 patients respectively). Blood and plasma samples were collected at the start of treatment and at 12 and 24 weeks during treatment. Immunophenotyping by flow cytometry for Treg cells, the FOXP-3 expression using real-time PCR and measurement of IL-10, TGF-β CXCL-9 and CXCL-10 were performed. Increased expression of Treg cells was detected in patients who didn't respond to treatment before and during treatment. Also, the levels of IL-10, TGF-β, CXCL-9 and CXCL-10 revealed significant increase.in non-responders all through compared to responders group. Evaluation of Treg cells, cytokines (IL-10 & TGF-β) and chemokines (CXCL-9 & CXCL-10) before starting the treatment could be a predictor of response to treatment with PEG-IFN plus ribavirin. The optimum levels which would differentiate between responders and non-responders are needed to be defined before-hand.

Analyses of the regulatory T (Treg) cell TCR repertoire should help elucidate the nature and diversity of their cognate antigens and thus how Treg cells protect us from autoimmune diseases. We earlier identified CD44(hi) CD62L(low) activated/memory (am) Treg cells as a Treg-cell subset with a high turnover and possible self-specificity. We now report that amTreg cells are predominantly distributed in lymph nodes (LNs) draining deep tissues. Multivariate analyses of CDR3 spectratyping first revealed that amTreg TCR repertoire is different from that of naïve Treg cells (nTreg cells) and effector T (Teff) cells. Furthermore, in deep- versus superficial LNs, TCR-β deep sequencing further revealed diversified nTreg-cell and amTreg-cell repertoires, although twofold less diverse than that of Teff cells, and with repertoire richness significantly lower in deep-LN versus superficial-LN Treg cells. Importantly, expanded clonotypes were mostly detected in deep-LN amTreg cells, some accounting for 20% of the repertoire. Strikingly, these clonotypes were absent from nTreg cells, but found at low frequency in Teff cells. Our results, obtained in nonmanipulated mice, indicate different antigenic targets for naïve and amTreg cells and that amTreg cells are self-specific. The data we present are consistent with an instructive component in Treg-cell differentiation., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The use of ultraviolet (UV)-B irradiation after alloantigen immunization is unknown because previous studies focused on UV-B irradiation before immunization. Here, we investigated immunosuppressive effects induced by UV-B irradiation after immunization, and examined the phenotype of induced regulatory T cells and the possible mechanism of induction., Material and Methods: B6 mice (H-2(b)) were intravenously immunized by splenocytes from CBF1 mice (H-2(b/d)). One week after alloantigen immunization, B6 mice received high-dose UV-B irradiation (40 kJ/m(2)). Four weeks after UV-B irradiation, proliferation assays (n=4, in each), transplantations with skin or cardiac allografts (n=5, in each), cytokines in mixed lymphocyte culture (n=6, in each), and adoptive transfer of CD4(+) T cells to naïve B6 mice (n=5, in each) were performed. Mice were divided into 4 groups: untreated control, immunized control, UV-irradiated control, and an immunized and UV-irradiated group. B6C3F1 mice (H-2(b/k)) were used as irrelevant alloantigen with immunization controls. Anti-IL-10 monoclonal antibody was used to block IL-10 before and after UV-B irradiation., Results: Immune responses against the immunizing antigen were markedly suppressed in immunized and UV-irradiated mice in an alloantigen-specific manner. Surprisingly, CD4(+) T cells from immunized and UV-irradiated mice produced significantly larger amounts of IL-10, in an alloantigen-specific manner. Moreover, alloantigen-specific immunosuppression via CD4(+) regulatory T cells was transferable to naïve B6 mice. IL-10 blocking clearly abrogated alloantigen-specific immunosuppression, indicating that UV-B irradiation evoked T regulatory type 1 cells., Conclusions: This study demonstrates for the first time that immunization and UV irradiation induces alloantigen-specific CD4(+) T regulatory type 1 cells, and that IL-10 plays an important role for this induction.

Not only macrophages, T-helper (Th)1 and Th2, but also CD4(+) CD25(high) FoxP3(+) regulatory T cells (T-regs) are involved in immune response to Mycobacterium leprae. We aimed to evaluate serum interleukin (IL)-1β and IL-12p70 (macrophage cytokines), interferon-γ (IFN-γ) (Th1 cytokine), IL-4 (Th2 cytokine) and circulating CD4(+) CD25(high) FoxP3(+) T-regs, in untreated leprosy patients. Forty three patients and 40 controls were assessed for the mentioned cytokines using ELISA. Patients were assessed for circulating T-regs using flow cytometry. Patients were subgrouped into tuberculoid (TT), pure neural leprosy (PNL), borderline cases, lepromatous (LL), type 1 reactional leprosy (RL1) and erythema nodosum leprosum (ENL). Serum IL-12p70, IFN-γ and IL-4 were significantly higher in patients versus controls (P < 0.05). Serum IL-4 was highest in LL and lowest in RL1 (P = 0.003). Serum IL-1β levels was significantly higher in multibacillary versus paucibacillary patients (P = 0.006). Significantly higher T-regs levels was detected in TT, RL1 and PNL, while the lowest levels in ENL(P < 0.001), with significant differences versus controls (P < 0.05). FoxP3 expression% was significantly lower in PNL than other patients and controls (P < 0.05). T-regs/T-effs was lowest in ENL(P < 0.05). IFN-γ correlated positively with T-regs but negatively with IL-1β (P = 0.041&0.046 respectively), which correlated positively with T-effs%( P = 0.05). IL-4 correlated positively with T-regs FoxP3 expression% (P = 0.009). We concluded that: Circulating T-regs were increased in TT, RL1 and PNL patients, known of relatively high cell-mediated immunity. This finding was supported by low FoxP3 expression (in PNL) and correlation between T-regs count and IFN-γ level. Overproduction of IL-4 in LL may infer liability to develop ENL, with disease progression and immune hyperactivation, marked by deficient T-regs and increased T-regs FoxP3 expression%. IL-1β probably has a pro-inflammatory role in multibacillary patients as correlated with T-effs%., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Clinical tolerance induction to permit minimization or cessation of immunosuppressive drugs is one of the key research goals in solid organ transplantation. The use of ex vivo expanded or manipulated immunologic cells, including CD4CD25FOXP3 regulatory T cells (Tregs), to achieve this aim is already a reality, with several trials currently recruiting patients. Tregs are a highly suppressive, nonredundant, population of regulatory cells that prevent the development of autoimmune diseases in mammals. Data from transplanted humans and animal models support the notion that Tregs can mediate both induction and adoptive transfer of transplantation tolerance. However, human Tregs are highly heterogeneous and include subpopulations with the potential to produce the proinflammatory cytokine interleukin-17, which has been linked to transplant rejection. Tregs are also small in number in the peripheral circulation, thus they require ex vivo expansion before infusion into man. Selection of the most appropriate Treg population for cell therapy is, therefore, a critical step in ensuring successful clinical outcomes. In this review, we discuss Treg subpopulations, their subdivision based on nonmutually exclusive criteria of origin, expression of immunologic markers and function, availability in the peripheral blood of patients awaiting transplantation, and their suitability for programs of cell-based therapy.

Strategies to boost the numbers and functions of regulatory T cells (Tregs) are currently being tested as means to treat autoimmunity. While Tregs have been shown to be effective in this role, strategies to manipulate Tregs to effectively suppress later stages of ongoing diseases need to be established. In this study, we evaluated the ability of TGF-β-induced Tregs (iTregs) specific for the major self-antigen in autoimmune gastritis to suppress established autoimmune gastritis in mice. When transferred into mice during later stages of disease, iTregs demethylated the Foxp3 promoter, maintained Foxp3 expression, and suppressed effector T cell proliferation. More importantly, these iTregs were effective at stopping disease progression. Untreated mice had high numbers of endogenous Tregs (enTregs) but these were unable to stop disease progression. In contrast, iTregs, were found in relatively low numbers in treated mice, yet were effective at stopping disease progression, suggesting qualitative differences in suppressor functions. We identified several inhibitory receptors (LAG-3, PD-1, GARP, and TNFR2), cytokines (TGF-β1 and IL12p35), and transcription factors (IRF4 and Tbet) expressed at higher levels by iTregs compared to enTregs isolated form mice with ongoing disease, which likely accounts for superior suppressor ability in this disease model. These data support efforts to use iTregs in therapies to treat establish autoimmunity, and show that iTregs are more effective than enTregs at suppressing inflammation in this disease model.

Pregnancy still represents one of the most fascinating paradoxical phenomena in science. Immediately after conception, the maternal immune system is challenged by the presence of foreign paternal antigens in the semen. This triggers mechanisms of recognition and tolerance that all together allow the embryo to implant and later the fetus to develop. Tolerance mechanisms to maintain pregnancy are of special interest as they defy the classical immunology rules. Several cell types, soluble factors, and immune regulatory molecules have been proposed to contribute to fetal tolerance. Within these, regulatory T cells (Treg) are one of the most studied immune cell populations lately. They are reportedly involved in fetal acceptance. Here, we summarize several aspects of Treg biology in normal and pathologic pregnancies focusing on Treg frequencies, subtypes, antigen specificity, and activity as well as on factors influencing Treg generation, recruitment, and function. This review also highlights the contribution of fetal Treg in tolerance induction and addresses the role of Treg in autoimmune diseases and infections during gestation. Finally, the potential of Treg as a predictive marker for the success of assisted reproductive techniques and for therapeutic interventions is discussed., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Increased numbers of regulatory T (Treg) cells are found in synovial fluid from patients with rheumatoid arthritis (RASF) compared with peripheral blood. However, Treg cells in RASF have been shown to have a decreased capacity to suppress T cells. Here we phenotypically classified CD4+ T cells in RASF into six subsets based on the expression of CD45RA, CCR7, CD27 and CD28, and demonstrated that the CCR7-CD45RA-CD27+CD28+ TEM subset was significantly increased in synovial fluid compared with peripheral blood. In addition, the proportion of Foxp3+ Treg cells in the CCR7-CD45RA-CD27+CD28+ TEM subset was significantly increased in RASF. Furthermore, most of the Foxp3+ Treg cells in RASF were non-suppressive CD45RA-Foxp3(low) non-Treg cells, and the frequency of the non-Treg cells in the CCR7-CD45RA-CD27+CD28+ TEM subset was significantly increased in RASF. Our findings suggest that the pro-inflammatory environment in RA joints may induce the increase of CD45RA-Foxp3(low) non-Treg cells in synovial fluid., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival, which is mediated by the induction of CD8+CD45RClo regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II-derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.Aa/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.Aa/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vβ11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function.

Regulatory T (Treg) cells are potent suppressors that maintain immune homeostasis. Accumulation of Treg can inhibit effective immune responses in cancer patients, leading to tumor development and progression. Despite direct cytotoxicity, several chemotherapeutic drugs have been reported to deplete Treg cells for better prognosis for cancer patients. Treg cells are a heterogenous population with at least three different subsets, nonsuppressive, resting, and activated Treg cells. However, the characteristics of Treg cell subsets in lung cancer patients and how chemotherapy affects Treg cells remain elusive. In this study, we first analyzed Treg cell subsets in peripheral blood samples from 40 nonsmall cell lung cancer (NSCLC) patients and 20 healthy donors. Treg cells, specifically activated Treg cell subset, significantly increased in patients with NSCLC. Compared to nonsuppressive Treg cells, activated Treg cells expressed higher level of CD39 and predominantly produced inhibitory cytokines. In vitro assay showed that docetaxel reduced all three subsets of Treg cells. More importantly, we found docetaxel-based chemotherapy significantly decreased all three Treg subsets after 4 cycles of treatment in 17 NSCLC patients. Taken together, this study revealed dynamic changes of various Treg cell subsets in NSCLC patients before and after chemotherapy, providing activated Treg cells as a potential target for chemotherapy.