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CD4+ T follicular helper cells (TFH) were assessed in adult patients with common variable immune deficiency (CVID) classified according to the presence of granulomatous disease (GD), autoimmunity (AI), or both GD and AI (Group I) or the absence of AI and GD (Group II). TFH lymphocytes were characterized by expression of CXCR5 and PD-1. TFH were higher (in both absolute number and percentage) in Group I than in Group II CVID patients and normal controls (N). Within CXCR5+CD4+ T cells, the percentage of PD-1 (+) was higher and that of CCR7 (+) was lower in Group I than in Group II and N. The percentages of Treg and TFH reg were similar in both CVID groups and in N. TFH responded to stimulation increasing the expression of the costimulatory molecules CD40L and ICOS as did N. After submitogenic PHA+IL-2 stimulation, intracellular expression of TFH cytokines (IL-10, IL-21) was higher than N in Group I, and IL-4 was higher than N in Group II. These results suggest that TFH are functional in CVID and highlight the association of increased circulating TFH with AI and GD manifestations. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance. Therapeutic manipulation of Tregs is subject to numerous clinical investigations including trials for adoptive Treg transfer. Since the number of naturally occurring Tregs (nTregs) is minute, it is highly desirable to develop a complementary approach of inducing Tregs (iTregs) from naïve T cells. Mouse studies exemplify the importance of peripherally induced Tregs as well as the applicability of iTreg transfer in different disease models. Yet, procedures to generate iTregs are currently controversial, particularly for human cells. Here we therefore comprehensively compare different established and define novel protocols of human iTreg generation using TGF-β in combination with other compounds. We found that human iTregs expressed several Treg signature molecules, such as Foxp3, CTLA-4 and EOS, while exhibiting low expression of the cytokines Interferon-γ, IL-10 and IL-17. Importantly, we identified a novel combination of TGF-β, retinoic acid and rapamycin as a robust protocol to induce human iTregs with superior suppressive activity in vitro compared to currently established induction protocols. However, iTregs generated by these protocols did not stably retain Foxp3 expression and did not suppress in vivo in a humanized graft-versus-host-disease mouse model, highlighting the need for further research to attain stable, suppressive iTregs. These results advance our understanding of the conditions enabling human iTreg generation and may have important implications for the development of adoptive transfer strategies targeting autoimmune and inflammatory diseases. [ABSTRACT FROM AUTHOR]

The suppressive function of regulatory T cells (Tregs) is critical to the maintenance of immune homeostasis in vivo and yet, the specific identification of Tregs by phenotypic markers is not perfect. Tregs were originally identified in the CD4+CD25+ fraction of T cells, but FoxP3 expression was later included as an additional marker of Tregs as FoxP3 expression was identified as being critical to the development and function of these cells. Intracellular expression of FoxP3 makes it difficult in using to isolate live and not permeabilized cells for functional assays. As such CD4+CD25+ fraction is still frequently used for functional assays of Tregs. Although, the CD4+CD25+ fraction substantially overlaps with the FoxP3+ fraction, the minor mismatch between CD4+CD25+ and FoxP3+ fractions may confound the functional characteristics of Tregs. In this study, we isolated CD4+FoxP3+ as well as CD4+CD25+ fractions from Foxp3 knock-in mice, and compared their proliferative and suppressive activity in the presence or absence of various concentrations of IL-2. Our results showed comparable patterns of proliferative and suppressive responses for both fractions, except that contrary to the CD4+CD25+ fraction the FoxP3+ fraction did not proliferate in an autocrine fashion even in response to a strong stimulation. In presence of exogenous IL-2, both CD4+CD25+ and CD4+FoxP3+ fractions were more sensitive than the CD4+CD25- responder cells in proliferative responsiveness. In addition, a low dose IL-2 enhanced whereas a high dose abrogated the suppressive activities of the CD4+CD25+ and CD4+FoxP3+ fractions. These results may provide an additional understanding of the characteristics of the various fractions of isolated Tregs based on phenotype and function and the role of varying levels of exogenous IL-2 on the suppressive activity of these cells. [ABSTRACT FROM AUTHOR]

CD83 is a widely recognized surface marker for mature dendritic cells, which are essential for priming naïve CD4+ T cells into effector cells. However, CD83 is also expressed on activated CD4+ T cells, which remains an enigma in T-cell mediated immunity. Therefore, the identification of the biological features and regulation of the expression of CD83 on activated CD4+ T cells is important in understanding the function of CD83 in the adaptive immune response. The present study revealed a time dependent manner of the expression of CD83 on anti-CD3/CD28-stimulated human CD4+ T cells, which is characterized by the maximum expression at day 2 and a significant decrease at day 3. The reduced expression is not a result of a reduced rate of cell proliferation. The activation of interleukin-2 and secretion of interferon-α accumulated progressively from day 1 to 3. Of note, sustained expression of CD83 was observed when CD4+ T cells were induced by transforming growth factor-β to differentiate into CD4+CD25+ forkhead box P3+ regulatory T (iTreg) cells. Confocal immunofluorescence microscopy analysis demonstrated that CD83 was highly co-localized with CD25 on activated CD4+ T cells. In conclusion, the findings of the present study suggested that the continuous expression of CD83 on activated human CD4+ T cells is correlated with their differentiation into iTreg cells. [ABSTRACT FROM AUTHOR]

MiR-568 targets NFAT5 and inhibits human CD4+ T cells and Treg cellsCD4+ T cells play critical roles in orchestrating adaptive immune responses. Their activation and proliferation are critical steps that occur before they execute their biological functions. Despite the important role of this process, the underlying molecular events are not fully understood. MicroRNAs (miRNAs) have been shown to play important roles in lymphocyte development and function. However, the miRNAs that regulate T-cell differentiation, activation and proliferation are still largely unknown. In our previous study, using a miRNA array, we found that several miRNAs (including miR-202, 33b, 181c, 568 and 576) are differentially expressed between resting and activated CD4+ T cells. In this study, we focused on the function of miR-568 during CD4+ T-cell activation. We showed that the expression level of miR-568 decreased during the activation of T cells, including Jurkat cells and human peripheral blood CD4+ T cells. When Jurkat or human peripheral blood CD4+ T cells were transfected with miR-568 mimics, cell activation was significantly inhibited, as shown by the inhibited expression of activation markers such as CD25, CD69 and CD154; decreased IL-2 production; and inhibited cell proliferation. Using software predictions and confirmatory experiments, we demonstrated that nuclear factor of activated T cells 5 (NFAT5) is a target of miR-568. Treg cells are an important CD4+ T-cell subpopulation, so we also evaluated the function of miR-568 in Treg-cell activation and differentiation. We showed that the miR-568 level decreased, while the NFAT5 protein level increased during CD4+CD25+ Treg-cell activation, and the transfection of miR-568 mimics inhibited the NFAT5 expression, inhibited the production of both TGF-β and IL-10 and also inhibited the proliferation of Treg cells. Our further study showed that over-expression of miR-568 can inhibit Treg-cell differentiation and can inhibit the suppressive effect of these cells on effector cells. In addition, inhibition of NFAT5 by siRNA-mediated knockdown can inhibit the activation and differentiation of Treg cells. These findings reveal that miR-568 can inhibit the activation and function of both CD4+ T cells and Treg cells by targeting NFAT5. Since miR-568 plays an important role in both CD4+ T cells and Treg cells, these findings may provide leads for the development of novel treatments for human inflammatory and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Objective: Herpes simplex virus 1 (HSV-1) is regarded as an important underlying cause of Bell's palsy, but the immunologic mechanism remains unknown. Here, we employed a mouse facial paralysis model to investigate the expressions of CD4 T lymphocytes and interleukin (IL)-2 and -4 in the left draining cervical lymph nodes (LCLN) and spleen, as well as the inhibitory effects of glucocorticoids (GCs). Methods: HSV-1 was inoculated into the surface of the posterior auricle to generate the facial paralysis model. The paralyzed mice were divided into three groups; in one group without any treatment, mice were killed at different time points, and those in the other two groups were injected with methylprednisolone sodium succinate (MPSS) or with a combination of MPSS and GC receptor blocker (RU486). The expression levels of CD4 T lymphocytes and CD4-IL-2 and CD4-IL-4 cells in the LCLN and spleen were detected by fluorescence-activated cell sorting. Results: Expression levels of CD4, IL-2, and IL-4 first increased then decreased in LCLN and spleen and peaked 5 and 7 days, respectively, after the manifestation of facial paralysis. All the data at the peak points were significantly different compared with control ( p < 0.05), and these effects were inhibited by MPSS. Conclusion : Our results suggest that CD4, IL-2, and IL-4 participate in the HSV-1-induced facial paralysis immune response. MPSS can effectively attenuate HSV-1-mediated nervous system damage, which is associated with its inhibitory effect on expression of these inflammatory markers. [ABSTRACT FROM AUTHOR]

Objective: Aging has been reported to be associated with changes in immune function. Although frequent infection and the development of malignancy suggest the decline of immune function with aging, changes toward proinflammatory conditions also develop at the same time. Th17 cells are well known CD4+ T cell subpopulation closely linked to chronic inflammation and autoimmunity. In this study, changes in the Th17 population were investigated to elucidate a possible mechanism for this response with aging. Methods: Splenocytes were isolated from 2-month-old (young) and 20-month-old (aged) mice. CD4+CD44+ memory T cells and CD4+CD62L+ naïve T cells were isolated and sorted using magnetic beads and flow cytometry. The frequency of IL-17-producing cells was measured using flow cytometry. The expression of IL-17 and Th17-related factors at the mRNA level was measured with RT-PCR. IL-17 and Il-1β expression in spleen tissues was additionally assessed using confocal microscopy. Results: The proportion of IL-17-producing CD4+ T cells was higher in the splenocytes among the old mice than those of the young mice. When splenocytes were cultured in Th17 polarizing conditions, the proportion of IL-17 producing CD4+ T cells was higher in aged mice as well. This was consistently observed when naïve and memory cells were isolated and differentiated into Th17 respectively. In addition, the expression of retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt) and other Th17-related factors (AhR, CCR6, and CCL20) increased in the splenocytes of aged mice compared to the young mice. The expression of IL-1β, showing to promote Th17 differentiation, was higher in the aged mice. Likewise, CD4+ T cell expression of IL-1R was higher in the aged mice, suggesting that the CD4+ T cells of the aged mice are readily prepared to differentiate into Th17 cells in response to IL-1β. Confocal microscopy showed that cells positive for IL-1R or IL-1β were more frequent in the spleens of the aged mice. When an anti-IL-2 antibody was applied, the proportion of IL-17-producing cells increased more prominently in the young mice. We observed that IL-2 production and IL-2R expression were reduced in the aged mice, respectively, explaining the blunted response to the anti-IL-2 antibody treatment and the consequent minimal change in the Th17 population. Conclusion: We demonstrated that the proportion of Th17 cells increased in the aged mice both in naïve and memory cell populations. Elevation of IL-1R and IL-1β expression and the reduction in IL-2 and IL-2R expression in aged mice seemed to promote Th17 differentiation. Our results suggest that enhanced Th17 differentiation in aging may have a pathogenic role in the development of Th17-mediated autoimmune diseases. [ABSTRACT FROM AUTHOR]

Highlights: [•] Id1 expression enables naïve T cell proliferation without anti-CD28 co-stimulation. [•] Id1 expression facilitates T cells survival when stimulated with anti-CD3. [•] Elevation of IL-2 production by Id1 contributes increased proliferation and survival. [•] Id1 potentiates NF-κB activation by anti-CD3 stimulation. [ABSTRACT FROM AUTHOR]

Determination of antigen-specific T cell repertoires in human blood has been a challenge. Here, we show a novel integrated approach that permits determination of multiple parameters of antigen-specific T cell repertoires. The approach consists of two assays: the Direct assay and the Cytokine-driven assay. Briefly, human PBMCs are first stimulated with overlapping peptides encoding a given antigen for 48 hours to measure cytokine secretion (Direct assay). Peptide-reactive T cells are further expanded by IL-2 for 5 days; and after overnight starvation, expanded cells are stimulated with the same peptides from the initial culture to analyze cytokine secretion (Cytokine-driven assay). We first applied this integrated approach to determine the type of islet-antigen-specific T cells in healthy adults. Out of ten donors, the Direct assay identified GAD65-specific CD4+ T cells in three adults and zinc transporter 8 (ZnT8)-specific CD4+ T cells in five adults. The intracytoplasmic cytokine staining assay showed that these islet-antigen-specific CD4+ T cells belonged to the CD45RO+ memory compartment. The Cytokine-driven assay further revealed that islet-antigen-specific CD4+ T cells in healthy adults were capable of secreting various types of cytokines including type 1 and type 2 cytokines as well as IL-10. We next applied our integrated assay to determine whether the type of ZnT8-specific CD4+ T cells is different between Type 1 diabetes patients and age/gender/HLA-matched healthy adults. We found that ZnT8-specific CD4+ T cells were skewed towards Th1 cells in T1D patients, while Th2 and IL-10-producing cells were prevalent in healthy adults. In conclusion, the Direct assay and the Cytokine-driven assay complement each other, and the combination of the two assays provides information of antigen-specific T cell repertoires on the breadth, type, and avidity. This strategy is applicable to determine the differences in the quality of antigen-specific T cells between health and disease. [ABSTRACT FROM AUTHOR]

Appropriate expression of IL-2 plays a central role during the priming and differentiation of T cells. A tight balance between IL-2 and the effector cytokine IL-17A is essential for immune homeostasis. Epigenetic mechanisms have been documented as a key component of cytokine regulation during lineage commitment. The molecular mechanisms that induce chromatin remodeling are less well understood. We investigated epigenetic regulators that mediate the diametric expression of IL-2 and IL-17A in naive, central memory, and effector memory CD4+ T cells. We demonstrate that cAMP response modulator (CREM)α contributes to epigenetic remodeling of IL2 in effector memory T cells through the recruitment of DNMT3a. CREMa also reduces CpG-DNA methylation of the IL17A promoter. CREMa expression is regulated at the epigenetic level by CpG-DNA methylation, which allows increased CREMa expression in effector memory CD4+ T cells. T cells from patients with systemic lupus erythematosus (SLE) express increased levels of CREMa and exhibit a phenotype that is similar to effector memory CD4+ T cells with epigenetically predetermined expression patterns of IL-2 and IL-17A. We conclude that CREMa mediates epigenetic remodeling of the 1L2 and IL17A gene during T-cell differentiation in favor of effector memory T cells in health and disease. [ABSTRACT FROM AUTHOR]

The transcription factors T-bet and Bcl-6 are required for the establishment of a T helper type 1 cell (TH1 cell) and follicular helper T cell (TFH cell) gene-expression profile, respectively. Here we found that high concentrations of interleukin 2 (IL-2) inhibited Bcl-6 expression in polarized TH1 cells. Mechanistically, the low concentrations of Bcl-6 normally found in effector TH1 cells did not repress its target genes because a T-bet-Bcl-6 complex masked the Bcl-6 DNA-binding domain. TH1 cells increased their Bcl-6/T-bet ratio in response to limiting IL-2 conditions, which allowed excess Bcl-6 to repress its direct target Prdm1 (which encodes the transcriptional repressor Blimp-1). The Bcl-6-dependent repression of Blimp-1 effectively induced a partial TFH profile because Blimp-1 directly repressed a subset of TFH signature genes, including Cxcr5. Thus, IL-2-signaling regulates the Bcl-6-Blimp-1 axis in TH1 cells to maintain flexibility with a TFH cell-like gene profile. [ABSTRACT FROM AUTHOR]

Nuclear factor of activated T cells (NFAT) is a family of transcription factors composed of five proteins. Among them, NFAT1 is a predominant NFAT protein in CD4+ T cells. NFAT1 positively regulates transcription of a large number of inducible cytokine genes including IL-2, IL-4, IL-5 and other cytokines. However, disruption of NFAT1 results in an unexpected increase of IL-4. In this study, we have investigated the role of NFAT1 in regulation of IL-4 gene expression in T helper 2 cells (Th2) from an epigenetic viewpoint. NFAT1 deficient Th2 cells showed a sustained IL-4 expression while wild type (WT) cells reduced its expression. We tested whether epigenetic maintenance and changes in the chromatin architecture of IL-4 promoter locus play a role in differential IL-4 transcription between in WT and NFAT1 deficient Th2 cells. Compared with WT, NFAT1 deficient CD4+ Th2 cells exhibited enhanced chromatin accessibility with permissive histone modification and DNA demethylation in the IL-4 promoter region. Transcription factors bound to IL-4 promoter region in the absence of NFAT1 were identified by Micro-LC/LC-MS/MS analysis. Among the candidates, preferential recruitment of JUNB to the IL-4 promoter was confirmed by chromatin immunoprecipitation analysis. Overexpression of JUNB together with SATB1 synergistically upregulated IL-4 promoter activity, while knockdown JUNB significantly reduced IL-4 expression. Our results suggest that the prolonged IL-4 expression in NFAT1 deficient Th2 cells is mediated by preferential binding of JUNB/SATB1 to the IL-4 promoter with permissive chromatin architecture. [ABSTRACT FROM AUTHOR]

Relatively little is known about regulatory T (Treg) cells and their functional responses in dogs. We have used the cross-reactive anti-mouse/rat Foxp3 antibody clone FJK-16s to identify a population of canine CD4 FOXP3 T cells in both the peripheral blood (PB) and popliteal lymph node (LN). FOXP3 cells in both PB and LN yielded positive staining with the newly developed anti-murine/human Helios antibody clone 22F6, consistent with the notion that they were naturally occurring Treg cells. Stimulation of mononuclear cells of LN origin with concanavalin A (Con A) in vitro yielded increased proportions and median fluorescence intensity of FOXP3 expression by both CD4 and CD8 T cells. Removal of the Con A and continued culture disclosed a CD4 FOXP3 population, distinct from the CD4 FOXP3 T cells; very few CD8 FOXP3 T cells were observed, though CD8 FOXP3 cells were present in equal abundance to CD4 FOXP3 cells. The CD4 FOXP3 T cells were thought to represent activated Treg cells, in contrast to the FOXP3 cells, which were thought to be a more heterogeneous population comprising predominantly activated conventional T cells. Co-staining with interferon-γ (IFN-γ) supported this notion, because the FOXP3 T cells were almost exclusively IFN-γ, whereas the FOXP3 cells expressed a more heterogeneous IFN-γ phenotype. Following activation of mononuclear cells with Con A and interleukin-2, the 5% of CD4 T cells showing the highest CD25 expression (CD4 CD25) were enriched in cells expressing FOXP3. These cells were anergic in vitro, in contrast to the 20% of CD4 T cells with the lowest CD25 expression (CD4 CD25), which proliferated readily. The CD4 CD25 FOXP3 T cells were able to suppress the proliferation of responder CD4 T cells in vitro, in contrast to the CD4 CD25 cells, which showed no regulatory properties. [ABSTRACT FROM AUTHOR]

OBJECTIVE--In humans, multiple genes in the interleukin (IL)-2/IL-2 receptor (IL-2R) pathway are associated with type 1 diabetes. However, no link between IL-2 responsiveness and CD4[sup +]CD25[sup +]FOXP3[sup +] regulatory T-cells (Tregs) has been demonstrated in type 1 diabetic subjects despite the role of these IL-2-dependent cells in controlling autoimmunity. Here, we address whether altered IL-2 responsiveness impacts persistence of FOXP3 expression in Tregs of type 1 diabetic subjects. RESEARCH DESIGN AND METHODS--Persistence of Tregs was assessed by culturing sorted CD4[sup +]CD25[sup hi] natural Tregs with IL-2 and measuring FOXP3 expression over time by flow cytometry for control and type 1 diabetic populations. The effects of IL-2 on FOXP3 induction were assessed 48 h after activation of CD4[sup +]CD25[sup -] T-cells with anti-CD3 antibody. Cytokine receptor expression and signaling upon exposure to IL-2, IL-7, and IL-15 were determined by flow cytometry and Western blot analysis. RESULTS--Maintenance of FOXP3 expression in CD4[sup +]CD25[sup +] Tregs of type 1 diabetic subjects was diminished in the presence of IL-2, but not IL-7. Impaired responsiveness was not linked to altered expression of the IL-2R complex. Instead, IL-2R signaling was reduced in Tregs and total CD4[sup +] T-cells of type 1 diabetic subjects. In some individuals, decreased signal transducer and activator of transcription 5 phosphorylation correlated with significantly higher expression of protein tyrosine phosphatase N2, a negative regulator of IL-2R signaling. CONCLUSIONS--Aberrant IL-2R signaling in CD4[sup +] T-cells of type 1 diabetic subjects contributes to decreased persistence of FOXP3 expression that may impact establishment of tolerance. These findings suggest novel targets for treatment of type 1 diabetes within the IL-2R pathway and suggest that an altered IL-2R signaling signature may be a biomarker for type 1 diabetes. Diabetes 59:407-415, 2010 [ABSTRACT FROM AUTHOR]

Abstract: Chronic immune activation is the leading event in the pathogenesis of HIV-1 infection and is associated with depletion of CD4+ T cells and Th1/Th2 imbalance. The role of Tregs in HIV infection is still controversial as these cells may control both immune activation and HIV-specific T cell responses. Aim of the present study was to correlate the degree of immune activation with FOXP3 expression and production of IL-2 and IL-10. Peripheral blood was obtained from 10 HIV-1-infected subjects with sustained response to HAART. Four cellular fractions were purified and simultaneously used in further experiments: PBMCs, CD4+CD45RO+, CD4+CD45RO+CD25− and CD4+CD45RO+CD25+. The level of immune activation was measured both by flow cytometry and radiometry as short-term 1-hour spontaneous lymphocyte proliferation; IL-2 and IL-10 production were determined by ELISA in supernatants of cultures non-stimulated or stimulated with PHA and p24 antigen. FOXP3 mRNA expression was detected by RT-PCR. A group of healthy subjects was used as control. Statistical analysis was performed by SPSS. The level of immune activation (total CD25 expression) was found correlated to both CD4+ and Tregs (CD4+CD25high+; FOXP3mRNA) and it was found higher in HIV-1 infected subjects in comparison to the healthy control group. The highest SLP-1 h was measured in the CD4+CD4+CD45RO+ CD25− population (T reg depleted), suggesting a role of Tregs in controlling immune activation during HIV-1 infection. In addition, CD4+ cells from HIV-infected individuals remained responsive to the negative regulation by Tregs (measured as IL-10 production) both spontaneously and upon stimulation. It is worth noting, however, that total PBMCs of these patients also responded well to specific antigen (p24) stimulation even though IL-2 release was impaired. The results obtained suggest that Tregs significantly influence the level of immune activation. At the same time, their function appears to be dependent on IL-2 production by the remaining T cells. These findings suggest that the control of Th1/Th2 balance could be an approach to immune-based therapy for HIV infection. [Copyright &y& Elsevier]