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Background: Adaptive immune cells, including CD4 + CD69 + and CD4 + CD25 + FoxP3 + regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4 + CD25 + FoxP3 + Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4 + CD69 + and CD4 + CD25 + FoxP3 + T cells and interleukin profiles in a pristane-induced SLE experimental model., Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant., Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4 + CD69 + T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4 + CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038)., Conclusion: Increased numbers of CD4 + CD69 + T cells and reduced numbers of CD4 + CD25 + FoxP3 + Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

Objective: To determine whether latency can be established and reversed in both proliferating and nonproliferating CD4+ T cells in the same model in vitro., Methods: Activated CD4+ T cells were infected with either a nonreplication competent, luciferase reporter virus or wild-type full-length enhanced green fluorescent protein (EGFP) reporter virus and cultured for 12 days. The cells were then sorted by flow cytometry to obtain two distinct T-cell populations that did not express the T-cell activation markers, CD69, CD25 and human leukocyte antigen (HLA)-DR: CD69CD25HLA-DR small cells (nonblasts) that had not proliferated in vitro following mitogen stimulation and CD69CD25HLA-DR large cells (which we here call transitional blasts) that had proliferated. The cells were then reactivated with latency-reversing agents and either luciferase or EGFP quantified., Results: Inducible luciferase expression, consistent with latent infection, was observed in nonblasts and transitional blasts following stimulation with either phorbol-myristate-acetate/phytohemagglutinin (3.8 ± 1 and 2.9 ± 0.5 fold above dimethyl sulfoxide, respectively) or romidepsin (2.1 ± 0.6 and 1.8 ± 0.2 fold above dimethyl sulfoxide, respectively). Constitutive expression of luciferase was higher in transitional blasts compared with nonblasts. Using wild-type full-length EGFP reporter virus, inducible virus was observed in nonblasts but not in transitional blasts. No significant difference was observed in the response to latency-reversing agents in either nonblasts or transitional blasts., Conclusion: HIV latency can be established in vitro in resting T cells that have not proliferated (nonblasts) and blasts that have proliferated (transitional blasts). This model could potentially be used to assess new strategies to eliminate latency.

Previous studies established that GM-CSF-deficient (Csf2-deficient) mice exhibit profound resistance to experimental autoimmune encephalomyelitis. This study addressed whether the resistance of Csf2-deficient mice was a result of a requirement for GM-CSF in controlling the functional balance between effector and regulatory T cell subsets during experimental autoimmune encephalomyelitis. The main observation was that treatment with the anti-CD25 mAb PC61 rendered Csf2-deficient mice fully susceptible to severe, chronic experimental autoimmune encephalomyelitis, with disease incidences and severities equivalent to that of C57BL/6 mice. When both donors and recipients were treated with PC61 in a passive model of experimental autoimmune encephalomyelitis, adoptive transfer of myelin-specific Csf2-deficient T cells into Csf2-deficient recipients resulted in a nonresolving chronic course of severe paralytic experimental autoimmune encephalomyelitis. The peripheral Csf2-deficient T cell repertoire was marked by elevated CD3 + T cell frequencies that reflected substantial accumulations of naïve CD44 null-low CD4 + and CD8 + T cells but essentially normal frequencies of CD4 + CD25 + forkhead box P3 + T cells among the CD3 + T cell pool. These findings suggested that Csf2-deficient mice had secondary deficiencies in peripheral T cell sensitization to environmental antigens. In accordance, myelin oligodendrocyte glycoprotein 35-55/CFA-sensitized Csf2-deficient mice exhibited deficient peripheral sensitization to myelin oligodendrocyte glycoprotein, whereas pretreatment of Csf2-deficient mice with PC61 enabled the robust induction of myelin oligodendrocyte glycoprotein-specific T cell responses in the draining lymphatics. In conclusion, the experimental autoimmune encephalomyelitis resistance of Csf2-deficient mice, at least in part, reflects a deficient induction of effector T cell function that cannot surmount normal regulatory T cell barriers. Experimental autoimmune encephalomyelitis effector responses, however, are unleashed upon depletion of regulatory CD25 + T cells., (© Society for Leukocyte Biology.)

Unlabelled: Interleukin 2 (IL-2) signaling through the IL-2 receptor alpha chain (CD25) facilitates HIV replication in vitro and facilitates homeostatic proliferation of CD25(+) FoxP3(+) CD4(+) T cells. CD25(+) FoxP3(+) CD4(+) T cells may therefore constitute a suitable subset for HIV infection and plasma virion production. CD25(+) FoxP3(+) CD4(+) T cell frequencies, absolute numbers, and the expression of CCR5 and cell cycle marker Ki67 were studied in peripheral blood from HIV(+) and HIV(-) study volunteers. Different memory CD4(+) T cell subsets were then sorted for quantification of cell-associated HIV DNA and phylogenetic analyses of the highly variable EnvV1V3 region in comparison to plasma-derived virus sequences. In HIV(+) subjects, 51% (median) of CD25(+) FoxP3(+) CD4(+) T cells expressed the HIV coreceptor CCR5. Very high frequencies of Ki67(+) cells were detected in CD25(+) FoxP3(+) memory CD4(+) T cells (median, 27.6%) in comparison to CD25(-) FoxP3(-) memory CD4(+) T cells (median, 4.1%; P < 0.0001). HIV DNA content was 15-fold higher in CD25(+) FoxP3(+) memory CD4(+) T cells than in CD25(-) FoxP3(-) T cells (P = 0.003). EnvV1V3 sequences derived from CD25(+) FoxP3(+) memory CD4(+) T cells did not preferentially cluster with plasma-derived sequences. Quasi-identical cell-plasma sequence pairs were rare, and their proportion decreased with the estimated HIV infection duration. These data suggest that specific cellular characteristics of CD25(+) FoxP3(+) memory CD4(+) T cells might facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. The contribution of this cell population to plasma virion production remains unclear., Importance: Despite recent advances in the understanding of AIDS virus pathogenesis, which cell subsets support HIV infection and replication in vivo is incompletely understood. In vitro, the IL-2 signaling pathway and IL-2-dependent cell cycle induction are essential for HIV infection of stimulated T cells. CD25(+) FoxP3(+) memory CD4 T cells, often referred to as regulatory CD4 T cells, depend on IL-2 signaling for homeostatic proliferation in vivo Our results show that CD25(+) FoxP3(+) memory CD4(+) T cells often express the HIV coreceptor CCR5, are significantly more proliferative, and contain more HIV DNA than CD25(-) FoxP3(-) memory CD4 T cell subsets. The specific cellular characteristics of CD25(+) FoxP3(+) memory CD4(+) T cells probably facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. However, the contribution of this cell subset to plasma viremia remains unclear., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Signaling lymphocytic activation molecule family 3 (SLAMF3/Ly9) is a coregulatory molecule implicated in T-cell activation and differentiation. Systemic lupus erythematosus (SLE) is characterized by aberrant T-cell activation and compromised IL-2 production, leading to abnormal regulatory T-cell (Treg) development/function. Here we show that SLAMF3 functions as a costimulator on CD4(+) T cells and influences IL-2 response and T helper cell differentiation. SLAMF3 ligation promotes T-cell responses to IL-2 via up-regulation of CD25 in a small mothers against decapentaplegic homolog 3 (Smad3)-dependent mechanism. This augments the activation of the IL-2/IL-2R/STAT5 pathway and enhances cell proliferation in response to exogenous IL-2. SLAMF3 costimulation promotes Treg differentiation from naïve CD4(+) T cells. Ligation of SLAMF3 receptors on SLE CD4(+) T cells restores IL-2 responses to levels comparable to those seen in healthy controls and promotes functional Treg generation. Taken together, our results suggest that SLAMF3 acts as potential therapeutic target in SLE patients by augmenting sensitivity to IL-2., Competing Interests: The authors declare no conflict of interest.

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‑γ 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.

Hematopoietic stem cell transplantation from anti-cytomegalovirus immunoglobulin G (anti-CMV-IgG) positive donors facilitated immunological recovery post-transplant, which may indicate that chronic CMV infection has an effect on the immune system. This can be seen in the recipients after reconstitution with donor lymphocytes. We evaluated the composition of lymphocytes at hematologic recovery in 99 patients with hematologic malignancies post hematopoietic stem cell transplantation (HSCT). Anti-CMV-IgG seropositivity of the donor was associated with higher proportions of CD4+ (227.963 ± 304.858 × 106 vs. 102.050 ± 17.247 × 106 cells/L, p = 0.009) and CD4+CD25high (3.456 ± 0.436 × 106 vs. 1.589 ± 0.218 × 106 cells/L, p = 0.003) lymphocytes in the blood at hematologic recovery. The latter parameter exerted a diverse influence on the risk of acute graft-versus-host disease (GvHD) if low (1.483 ± 0.360 × 106 vs. 3.778 ± 0.484 × 106 cells/L, p < 0.001) and de novo chronic GvHD (cGvHD) if high (3.778 ± 0.780 × 106 vs. 2.042 ± 0.261 × 106 cells/L, p = 0.041). Higher values of CD4+ lymphocytes in patients who received transplants from anti-CMV-IgG-positive donors translated into a reduced demand for IgG support (23/63 vs. 19/33, p = 0.048), and these patients also exhibited reduced susceptibility to cytomegalovirus (CMV), Epstein-Barr virus (EBV) and/or human herpes 6 virus (HHV6) infection/reactivation (12/50 vs. 21/47, p = 0.032). Finally, high levels (³0.4%) of CD4+CD25high lymphocytes were significantly associated with better post-transplant survival (56% vs. 38%, four-year survival, p = 0.040). Donors who experience CMV infection/reactivation provide the recipients with lymphocytes, which readily reinforce the recovery of the transplanted patients' immune system.

In the current study, we investigate changes in CD4+CD25+ cells in chickens during infectious bursal disease virus (IBDV) infection. The percentage of CD4+CD25+ cells in lymph organs, e.g., the thymus, spleen, bursa of Fabricius and peripheral blood, during the first 1-5 days post infection (dpi) was assessed by flow cytometry. The data revealed a remarkable decrease in the percentage of CD4+CD25+ cells in the thymus from 1 to 5 dpi and in the spleen during early infection. An increase of the percentage of CD4+CD25+ cells among peripheral blood lymphocytes was observed during the first two days of IBDV infection. Additionally, CD4+CD25+ cells infiltrated the bursa along with CD4+ cells after IBDV infection. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to measure the mRNA levels of immune-related cytokines in IBDV-infected thymus and bursa of Fabricius tissues. The data revealed that IBDV caused a significant increase in interleukin (IL)-10 mRNA levels, with the Harbin-1 strain (vvIBDV) inducing higher IL-10 expression than the Ts strain. Taken together, our data suggest that chicken CD4+CD25+ cells may participate in IBDV pathogenicity by migrating from their sites of origin and storage, the thymus and spleen, to the virally targeted bursa of Fabricius during IBDV infection.

CD4(+)CD25(+)FOXP3(+) regulatory T cells have long been shown to mediate susceptibility to Leishmania infection, mainly via interleukin 10 production. In this work, we showed that the main sources of interleukin 10 in peripheral blood mononuclear cells (PBMCs) from patients with cutaneous leishmaniasis due to Leishmania braziliensis are CD4(+)CD25(-)CD127(-/low)FOXP3(-) cells. Compared with uninfected controls, patients with CL had increased frequencies of circulating interleukin 10-producing CD4(+)CD25(-)CD127(-/low) cells, which efficiently suppressed tumor necrosis factor α production by the total PBMC population. Also, in CL lesions, interleukin 10 was mainly produced by CD4(+)CD25(-) cells, and interleukin 10 messenger RNA expression was associated with interleukin 27, interleukin 21, and interferon γ expression, rather than with FOXP3 or transforming growth factor β expressions. Active production of both interleukin 27 and interleukin 21, together with production of interferon γ and interleukin 10, was also detected in the lesions. Since these cytokines are associated with the differentiation and activity of Tr-1 cells, our results suggest that this cell population may play an important role in the immunomodulation of CL. Therefore, development of treatments that interfere with this pathway may lead to faster parasite elimination., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Tolerance to the early acquisition of Helicobacter pylori is suggested because of a biased ratio of regulatory to effector T cells in a mice model. This study investigated whether the CD4CD25 regulatory T (Treg) and CD4+CD25- effector T (Teff) cell responses after H. pylori exposure determine H. pylori susceptibility in children., Methods: Treg and Teff cells from peripheral blood mononuclear cells (PBMCs) of H. pylori-infected and non-infected children were incubated with H. pylori protein. The cytokine levels and fraction of FOXP3+ to T cells were measured. FOXP3 expression was assessed by Western blotting and immunohistochemistry of gastric biopsies from dyspeptic children., Results: The fraction of FOXP3+ to CD4+CD25 high cells in PBMCs, FOXP3-positive staining and translation level in gastric tissues were higher in H. pylori-infected children than in controls (P < 0.05). The translation levels of TGF-β1 in gastric tissues were higher in H. pylori-infected children than in controls (P < 0.05). After H. pylori challenge, H. pylori-infected children had a positive net-change in TGF-β1 from Treg cells, and a negative net-change of IFN-γ from Teff cells. Paradoxically, the non-infected controls had a negative net-change in TGF-β1 from Treg cells, and a positive net-change of IFN-γ from Teff cells., Conclusions: The host response of Treg cells with increases in FOXP3 and TGF-β1 combined with a reduction in IFN-γ by Teff cells may contribute to H. pylori susceptibility in children.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complex genetic predisposing factors involved. Ets-1 transcription factor plays an important role in the suppressive activity of CD4(+)CD25(+) Treg cells and stable expression of FOXP3. To find its potential role in the pathogenesis of SLE, we investigate the mRNA expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25(+) Treg cells from patients with SLE. Real-time transcription-polymerase chain reaction analysis was used to determine the expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25(+) Treg cells from 36 patients with SLE and 18 sex-and-age-matched healthy controls. The Ets-1 mRNA expression level was decreased in patients with SLE [0.225 (0.135, 0.337)] than healthy controls [0.528 (0.303, 0.681)] (P < 0.001). The expression levels of FOXP3 mRNA were lower in SLE patients [0.608 (0.272, 1.164)] than healthy controls [0.919 (0.690, 1.223)], but the difference was not significant (P = 0.106). Significant reduction in Ets-1 and FOXP3 expression was also found in new-onset SLE subgroup when compared with healthy controls (P < 0.001). The level of Ets-1 and FOXP3 mRNA was not significantly different in hyperactive and lower active SLE group when compared with inactive SLE group, respectively (P > 0.05). There were no significant differences between SLE with lupus nephritis (LN) and SLE without LN either (P > 0.05). Associations of Ets-1 and FOXP3 mRNA expression levels with major clinical and laboratory parameters of SLE patients were also analyzed. However, no significant association was found. Significant positive correlation was found between Ets-1 and FOXP3 mRNA expression in CD4(+)CD25(+) Treg cells from SLE patients (r = 0.698, P < 0.001). Our results found that the expression levels of Ets-1 mRNA were decreased in SLE patients and Ets-1 expression was positively correlated with the expression of FOXP3. It indicated that Ets-1 may play an important role in the stable expression of FOXP3 in CD4(+)CD25(+) Treg cells.

We aimed to verify whether CD4(+)/CD25(+) T cells suppress CD4(+) T cells and secrete Granzyme B (GZB) during acute and chronic hepatitis C (CHC) infection. We enrolled 50 subjects: 20 patients with CHC (Group A), 15 healthy individuals (Group B), 10 patients with acute hepatitis C later evolved to persistent infection (Group C) and five patients who resolved hepatitis C virus infection during acute phase (Group D). We analysed, on enrolled subjects CD4(+)/CD25(+) T cells and related GZB production as well as Annexin V activity. Patients from Groups A and C had higher frequency and function of peripheral Treg cells than healthy individuals. Groups A and C showed an increase in spot-forming colonies (SFCs) of GZB compared with Group B (P < 0.01, Mann-Whitney U-test). CD4(+)/CD25(+) T cells in Group D had a lower number of GZB SFCs compared with Groups A and C but higher number than Group B (P < 0.01 Mann-Whitney U-test). Annexin V production was higher in Groups A and C than B or D. Patients having acute and chronic hepatitis C have a higher Treg frequency and function in peripheral blood than healthy controls or those resolving the infection in acute phase secreting GZB, probably inducing apoptosis., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Background: Clinical investigation of antigen-specific T cells in potentially immunodeficient patients is an important and often challenging aspect of patient diagnostic work up. Methods for detection of microbial exposure to the T-cell compartment exist but are laborious and time consuming. Recently, a whole blood technique involving flow cytometry and detection of CD25 and OX40 (CD134) expression on the surface of activated CD4+ T cells was shown to be accurate and concordant when compared with more traditional methods of antigen-specific T-cell detection., Methods: Whole heparinized blood was collected from healthy donors and set up using the "OX40" assay to detect antigen-specific CD4+ T-cell responses to Varicella Zoster Virus, Epstein-Barr Virus (EBV), Cytomegalovirus, Candida albicans, and Streptococcus pneumoniae., Results: The "OX40" assay technique was clinically validated for routine use in an NHS clinical immunology laboratory by analysis of incubation length (40-50 h), sample transport time (up to 24 h at room temperature), concordance with serology testing, proliferation and interferon-gamma production. In addition, 63 healthy controls (age range 21-78) were tested for responses to generate a healthy control reference range., Conclusions: The OX40 assay, as presented in this report, represents an economical, rapid, robust whole blood technique to detect antigen-specific T cells, which is suitable for clinical immunology diagnostic laboratory use., (© 2014 Clinical Cytometry Society.)

CD4(+) T cells expressing the immunotolerizing molecule HLA-G have been described as a unique human thymus-derived regulatory T (tTreg) cell subset involved in immunoregulation and parenchymal homeostasis during infectious and autoimmune inflammation. We compared properties and molecular characteristics of human CD4(+)HLA-G(+) with those of CD4(+)CD25(+)FoxP3-expressing tTreg cells using in vitro studies of T-cell receptor (TCR) signaling, single-cell electrophysiology, and functional in vivo studies. Both tTreg populations are characterized by alterations in proximal-signaling pathways on TCR stimulation and a hyperpolarization of the plasma membrane when compared to conventional CD4(+) T cells. However, both clearly differ in phenotype and pattern of secreted cytokines, which results in distinct mechanisms of suppression: While CD4(+)HLA-G(+) cells secrete high levels of inhibitory molecules (IL-10, soluble HLA-G, IL-35), CD4(+)CD25(+)FoxP3(+) cells express these molecules at significantly lower levels and seem to exert their function mainly in a contact-dependent manner via cyclic adenosine-monophosphate. Finally we demonstrate that human CD4(+)HLA-G(+) tTreg cells significantly ameliorated graft-versus-host disease in a humanized mouse model as a first proof of their in vivo relevance. Our data further characterize and establish CD4(+)HLA-G(+) cells as a potent human tTreg population that can modulate polyclonal adaptive immune responses in vivo and thus being a promising candidate for potential clinical applications in the future., (© FASEB.)

An immune role of neural stem/progenitor cells (NSPCs) has been proposed in many recent studies; however much still remains to be elucidated. In the current investigation, we report that NSPCs possess the ability to convert encephalitogenic T cells into CD4(+)-CD25(+)-FOXP3(+) regulatory T cells (T(reg)). Encephalitogenic and nonencephalitogenic T cells isolated from sham and Japanese encephalitis virus (JEV) infected animals were co-cultured with mouse NSPCs. Post co-culture, significant increase in the number of T(regs) was observed from encephalitogenic T cells co-cultured with NSPCs. This increased conversion was found to be dependent on direct contact between T cells and NSPCs. Neutralization of TGF-β and IFN-γ in NSPC cultures abrogated this increased conversion of encephalitogenic T cells into T(regs). Flow cytometric, quantitative RT-PCR, and immunoblot analysis of both T cells and NSPCs revealed surface and intracellular changes post co-culture. Co-stimulatory molecules (B7) and ICAM-1 were increased on NSPCs post co-culture; levels of TGFβ, IFNγ, and TGFβR1 were also increased in NSPCs. This study provides a basic insight into the interaction between CNS-infiltrating encephalitogenic T cells and NSPCs during viral encephalitis. Conversion of encephalitogenic T cells into CD4(+)-CD25(+)-FOXP3(+) T(regs) through interaction with NSPCs indicates an attempt in regulation of excessive inflammation in the CNS.

Background: We and others have previously reported that cell membrane-bound TGFβ (mTGFβ) on activated T regulatory (Treg) cells mediates suppressor function. Current findings suggest that a novel protein known as Glycoprotein A Repetitions Predominant (GARP) anchors mTGFβ to the Treg cell surface and facilitates suppressor activity. Recently, we have described that GARP+TGFβ+ Treg cells expand during the course of FIV infection. Because Treg cells are anergic and generally exhibit poor proliferative ability, we asked how Treg homeostasis is maintained during the course of feline immunodeficiency virus (FIV) infection., Results: Here, we report that Treg cells from FIV+ cats express GARP and mTGFβ and convert T helper (Th) cells into phenotypic and functional Treg cells. Th to Treg conversion was abrogated by anti-TGFβ or anti-GARP treatment of Treg cells or by anti-TGFβRII treatment of Th cells, suggesting that Treg cell recruitment from the Th pool is mediated by TGFβ/TGFβRII signaling and that cell-surface GARP plays a major role in this process., Conclusions: These findings suggest Th to Treg conversion may initiate a cascade of events that contributes to the maintenance of virus reservoirs, progressive Th cell immunosuppression, and the development of immunodeficiency, all of which are central to the pathogenesis of AIDS lentivirus infections.

Background: Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25(+)FOXP3(+) regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples., Methodology/principle Findings: Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3(+), TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4(+)CD25(+)FOXP3(+) T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25(+) cells of the CD4(+) but not that of CD8(+) T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients., Conclusions/significance: Our results indicate that FOXP3(+) iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.

Background/purpose(s): We investigated whether CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are induced in patients suffering from early-stage septic shock and distinguish them from noninfectious patients with systemic inflammatory response., Methods: The study included 37 patients with early-stage septic shock, 15 patients with noninfectious systemic inflammatory response syndrome (SIRS), and 24 heath controls. We prospectively assayed the fraction of Tregs expressing high levels of CD25 and forkhead box P3 (Foxp3) as well as the plasma levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), and soluble CD25 in all the subjects studied., Results: Compared with the control groups, the plasma levels of IFN-γ [66.10 (45.23-85.08) pg/mL vs. 20.97 (17.58-26.21) pg/mL, p < 0.001] and IL-4 [100.69 (77.41-127.68) pg/mL vs. 70.40 (64.14-80.15) pg/mL, p < 0.001] as well as the IFN-γ/IL-4 ratio [0.66 (0.62-0.67) vs. 0.30 (0.27-0.33), p < 0.001] were significantly elevated in the patients with early-stage septic shock, but there was no difference between patients with sepsis and patients with SIRS. We found that the proportion of CD4(+)CD25(+)Foxp3(+) T cells was significantly increased in the patients with early-stage septic shock [(66.82 ± 21.79%) vs. (51.79 ± 21.79%) vs. (56.45 ± 10.68%), p = 0.003] in comparison with the SIRS and control groups, which could be differentiated from the patients with SIRS. The plasma levels of soluble CD25 were also increased, and positively correlated with the proportion of Tregs in patients with early-stage septic shock (Spearman correlation coefficient = 0.390, p = 0.003)., Conclusion: Our findings indicate that the proportion of CD4(+)CD25(+)Foxp3(+) T cells could be an indicator for the early diagnosis of sepsis. This proportion can also facilitate the evaluation of the patient's immune status and guide suitable immunoregulatory therapy., (Copyright © 2012. Published by Elsevier B.V.)

Purpose: Cell therapy may become an option for lung injury treatment. However, no data are available on the alveolar presence and time course of CD4+ CD25 + Foxp3 + T-regulatory lymphocyte cells (Tregs) in acute respiratory distress syndrome (ARDS). Accordingly, we (1) measured the ratio of CD4 + CD25 + Foxp3 + Tregs to all (CD4+) lymphocytes in the bronchoalveolar lavage (BAL) of ARDS patients and of control subjects without lung disease and (2) assessed their impact on 30-day mortality., Methods: In a prospective study, the ratios of CD4 + CD25 + Foxp3 + T-regulatory cells to all CD4+ cells were measured (FACS) within 24 h of the patients' ICU referral in the BAL and in the blood of 47 patients with ARDS (32 males, 15 females; mean age 44 years ±13) as well as in 8 controls undergoing elective abdominal surgery (5 men, 3 women; mean age 49 years ±4). BAL concentrations of several cytokines were also measured in ARDS patients., Results: Tregs were detected in the BAL of control subjects and ARDS patients. However, the mean ratio of Tregs to all CD4+ lymphocytes was threefold greater in ARDS non-survivors (16.5%; p = 0.025) and almost twofold greater in ARDS survivors (9.0%; p = 0.015) compared to controls (5.9%). Multivariate Cox regression analysis revealed the ratio of CD4 + CD25 + Foxp3 + T-regulatory lymphocytes to all CD4+ lymphocytes in the BAL to be an important and independent prognostic factor for 30-day survival (HR 6.5; 95% CI, 1.7-25; p = 0.006)., Conclusion: An increased T-regulatory cell ratio in the admission BAL of patients with ARDS is an important and independent risk factor for 30-day mortality.

Lentivirus infection activates CD4(+) CD25(+) T regulatory (Treg) cells. Activation of Treg cells may be due to direct virus infection or chronic antigenic stimulation. Herein we demonstrate that in vitro feline immunodeficiency virus (FIV) infection, but not UV-inactivated virus, activates Treg cells as measured by immunosuppressive function and upregulation of GARP, FoxP3, and membrane-bound transforming growth factor β (TGF-β). These data demonstrate for the first time that AIDS lentiviruses infect and activate Treg cells, potentially contributing to immune dysfunction.

HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develops HAM/TSP. CD4⁺ T cells are the main target of infection and play a pivotal role in regulating immunity to HTLV and are hypothesized to participate in the pathogenesis of HAM/TSP. The CD39 ectonucleotidase receptor is expressed on CD4⁺ T cells and based on co-expression with CD25, marks T cells with distinct regulatory (CD39⁺CD25⁺) and effector (CD39⁺CD25⁻) function. Here, we investigated the expression of CD39 on CD4⁺ T cells from a cohort of HAM/TSP patients, HTLV-1 asymptomatic carriers (AC), and matched uninfected controls. The frequency of CD39⁺ CD4⁺ T cells was increased in HTLV-1 infected patients, regardless of clinical status. More importantly, the proportion of the immunostimulatory CD39⁺CD25⁻ CD4⁺ T-cell subset was significantly elevated in HAM/TSP patients as compared to AC and phenotypically had lower levels of the immunoinhibitory receptor, PD-1. We saw no difference in the frequency of CD39⁺CD25⁺ regulatory (Treg) cells between AC and HAM/TSP patients. However, these cells transition from being anergic to displaying a polyfunctional cytokine response following HTLV-1 infection. CD39⁻CD25⁺ T cell subsets predominantly secreted the inflammatory cytokine IL-17. We found that HAM/TSP patients had significantly fewer numbers of IL-17 secreting CD4⁺ T cells compared to uninfected controls. Taken together, we show that the expression of CD39 is upregulated on CD4⁺ T cells HAM/TSP patients. This upregulation may play a role in the development of the proinflammatory milieu through pathways both distinct and separate among the different CD39 T cell subsets. CD39 upregulation may therefore serve as a surrogate diagnostic marker of progression and could potentially be a target for interventions to reduce the development of HAM/TSP.

CD4(+) T cells are the main targets of HIV-1 and play a central role during the progression of AIDS, but the mechanism has not been clearly elucidated. In the present study, blood samples were collected from HIV-1-infected Chinese individuals, including typical progressors (TPs) and long-term nonprogressors (LTNPs). More HIV-1 productively infected CD4(+) T cells were obtained through co-cultures and the infected CD4(+) T cells were discriminated from bystander cells by intracellular p24 staining. The activation level and dynamics of cytokine expression of CD4(+) T cells were analyzed. After stimulating the freshly isolated PBMCs with PHA, the frequencies of CD69(+)CD4(+) T cells/CD25(+)CD4(+) T cells were higher in TP than in LTNP group and were positively correlated with viral load and negatively correlated with CD4(+) T cell counts. The activation level of CD4(+) T cells in the co-cultured PBMCs was higher in TP than in LTNP group, and HIV-1 productively infected CD4(+) T cells were more activated than bystander CD4(+) T cells. The expression of Th1 cytokines (IL-2 and IFN-γ) and the frequency of Th1 cells in co-cultured PBMCs were lower in TP than in LTNP group. HIV-1 productively infected CD4(+) T cells expressed higher level of Th1/Th2 cytokines than bystander cells. More productive HIV-1 infection occurred in Th1 than in Th2 cells, followed by Th0 cells. The present results suggest that the excessive activation level of CD4(+) T cells and the preferential replication of HIV-1 in Th1 cells that lead to the shift of Th1 to Th2 are likely crucial to AIDS progression in HIV-1-infected Chinese individuals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Marek's disease virus (MDV) is a cell-associated and highly oncogenic alphaherpesvirus that infects chickens. During lytic and latent MDV infection, a CXC chemokine termed viral interleukin-8 (vIL-8) is expressed. Deletion of the entire vIL-8 open reading frame (ORF) was shown to severely impair disease progression and tumor development; however, it was unclear whether this phenotype was due to loss of secreted vIL-8 or of splice variants that fuse exons II and III of vIL-8 to certain upstream open reading frames, including the viral oncoprotein Meq. To specifically examine the role of secreted vIL-8 in MDV pathogenesis, we constructed a recombinant virus, vΔMetvIL-8, in which we deleted the native start codon from the signal peptide encoding exon I. This mutant lacked secreted vIL-8 but did not affect Meq-vIL-8 splice variants. Loss of secreted vIL-8 resulted in highly reduced disease and tumor incidence in animals infected with vΔMetvIL-8 by the intra-abdominal route. Although vΔMetvIL-8 was still able to spread to naïve animals by the natural route, infection and lymphomagenesis in contact animals were severely impaired. In vitro assays showed that purified recombinant vIL-8 efficiently binds to and induces chemotaxis of B cells, which are the main target for lytic MDV replication, and also interacts with CD4(+) CD25(+) T cells, known targets of MDV transformation. Our data provide evidence that vIL-8 attracts B and CD4(+) CD25(+) T cells to recruit targets for both lytic and latent infection.

Background: Feline immunodeficiency virus (FIV) is a lentivirus of cats that establishes a lifelong persistent infection with immunologic impairment., Results: In an approximately 2 year-long experimental infection study, cats infected with a biological isolate of FIV clade C demonstrated undetectable plasma viral loads from 10 months post-infection onward. Viral DNA was detected in CD4+CD25+ and CD4+CD25- T cells isolated from infected cats whereas viral RNA was not detected at multiple time points during the early chronic phase of infection. Viral transcription could be reactivated in latently infected CD4+ T cells ex vivo as demonstrated by detectable FIV gag RNA and 2-long terminal repeat (LTR) circle junctions. Viral LTR and gag sequences amplified from peripheral blood mononuclear cells during early and chronic stages of infection demonstrated minimal to no viral sequence variation., Conclusions: Collectively, these findings are consistent with FIV latency in peripheral blood CD4+ T cells isolated from chronically infected cats. The ability to isolate latently FIV-infected CD4+ T lymphocytes from FIV-infected cats provides a platform for the study of in vivo mechanisms of lentiviral latency.

Chronic Q fever, which principally manifests as endocarditis, is characterized by Coxiella burnetii persistence and an impaired cell-mediated immune response. The long-term persistence of pathogens has been associated with the expansion of regulatory T cells (Tregs), the CD4(+) T-cell subset that is characterized by the expression of CD25 and Foxp3. We investigated the presence of Tregs in patients with acute Q fever (n = 17), known to exhibit an efficient immune response, patients with Q fever endocarditis (n = 54) and controls (n = 27) by flow cytometry. The proportion of CD3(+) , CD4(+) and CD8(+) T cells was similar in controls and patients with Q fever. The percentage of CD4(+) T cells that expressed CD25 was similar in controls and patients with Q fever. The population of CD4(+) T cells that expressed both CD25 and Foxp3 was significantly (P < 0.001) increased in patients with Q fever endocarditis compared with controls. Our data suggest that the expansion of Tregs may be critical for the chronic evolution of Q fever., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Helicobacter pylori blocks the proliferation of human CD4(+) T cells, facilitated by vacuolating exotoxin (VacA) and γ-glutamyl transpeptidase (GGT). H. pylori-triggered T-cell reactions in mice correlate with bacterial cholesterol and cholesterol α-glucoside content but their role in human cells is unclear. We characterized the effect of VacA, GGT, and cholesterol on T-helper 1, T-helper 2, T-regulatory and T-helper 17 associated cytokines and T-cell proliferation. VacA, GGT, and bacterial cholesterol content exhibited differential and synergistic inhibitory effects on the expression of activation markers CD25 and CD69 and on interleukin 2, interleukin 4, interleukin 10, and interferon γ production. These factors did not affect the H. pylori-mediated abrogation of transforming growth factor β secretion or increased interleukin 6 production. Cholesterol α-glucosyltransferase-deficient bacteria exerted strongly reduced antiproliferative effects on primary human CD4(+) T cells. In conclusion, H. pylori shapes rather than suppresses human CD4(+) T-cell responses, and glucosylated cholesterol is a relevant bacterial component involved in this modulation.

Objective: Natural CD4+CD25+FoxP3+ Treg cells play a crucial role in maintaining immune homeostasis and controlling autoimmunity. In patients with juvenile idiopathic arthritis (JIA), inflammation occurs despite the increased total numbers of Treg cells in the synovial fluid (SF) compared to the peripheral blood (PB). This study was undertaken to investigate the phenotype of CD4+ T cells in PB and SF from JIA patients, the function of synovial Treg cells, and the sensitivity of PB and SF CD4+CD25- effector T cells to the immunoregulatory properties of Treg cells, and to study the suppression of cytokine secretion from SF effector T cells by Treg cells., Methods: The phenotypes of effector T cells and Treg cells of PB and SF from JIA patients and healthy donors were determined by flow cytometry. The functionality of isolated Treg cells and effector T cells was quantified in (3) H-thymidine proliferation assays. Cytokine levels were analyzed using Bio-Plex Pro assay., Results: Compared to PB, SF showed significantly elevated numbers of activated and differentiated CD4+CD45RO+ T cells. Sensitivity of SF effector T cells to the suppressive effects of Treg cells from both PB and SF was impaired, correlating inversely with the expression of CD69 and HLA-DR. However, SF effector T cell cytokine secretion was partly suppressed by SF Treg cells., Conclusion: Our findings indicate that regulation is impaired in the SF of patients with JIA, as shown by the resistance of effector T cells to immunoregulation by functional Treg cells. This resistance of the SF effector T cells might be due to their activated phenotype., (Copyright © 2011 by the American College of Rheumatology.)

In areas where Plasmodium falciparum is endemic, pregnancy is associated with accumulation of infected red blood cells (RBCs) in the placenta, a condition referred to as placental malaria (PM). Infants born to PM-positive mothers are at an increased risk of malaria, which is putatively related to the transplacental passage of parasite-derived antigens, with consequent tolerization of the fetal immune system. Here we addressed the impact of PM on the regulation of neonatal T cell responses. We found that the frequency of regulatory CD25(+) CD127(-/low) Foxp3(+) CD4(+) T cells was significantly decreased in neonates born to mothers with high levels of P. falciparum-induced placental inflammation, consisting mainly of primigravid mothers. However, at the individual level, the ratio between regulatory and effector (CD25(+) CD127(+) Foxp3(-)) CD4(+) T cells was unaffected by PM. In addition, parasite-induced CD4(+) T cell activation and production of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and IL-10 were strongly reduced in neonates born to PM-positive mothers. Thus, our results show that active PM at delivery is associated with a marked suppression of P. falciparum-specific cellular neonatal immune responses, affecting secretion of both pro- and anti-inflammatory cytokines. Additionally, our results suggest that, as in adults, effector and regulatory CD4(+) T cell populations are tightly coregulated in all neonates, irrespective of the maternal infection status.

We observed by flow cytometry that the frequency of both gastric infiltrating Tregs and PD-1-positive CD4 T cells is correlated with the density of Helicobacter pylori, suggesting that cellular immunity against this pathogen is inhibited.

Background: Chandipura virus produces acute infection in mice. During infection drastic reduction of CD4+, CD8+ and CD19 + cell was noticed. Depletion of lymphocytes also noticed in spleen. The reduction may be due to the regulatory mechanism of immune system to prevent the bystander host tissue injury. There are several mechanisms like generation of regulatory cells, activation induced cell death (ACID) etc were indicated to control the activation and maintain cellular homeostasis. Role of regulatory cells in homeostasis has been described in several viral diseases. This study was undertaken to characterize CD4+T regulatory cells from the infected mice., Method: In this study we purified the CD4+ T cells from Chandipura virus infected susceptible Balb/c mice. CD4+ T regulatory cells were identified by expression of cell surface markers CD25, CD127 and CTLA-4 and intracellular markers Foxp3, IL-10 and TGF-beta. Antigen specificity and ability to suppress the proliferation of other lymphocytes were studied in vitro by purified CD4+CD25+T regulatory cells from infected mice. The proliferation was calculated by proliferation module of Flow Jo software. Expression of death receptors on regulatory cells were studied by flowcytometer., Results: The CD4+ T cells isolated from infected mice expressed characteristic markers of regulatory phenotype at all post infective hours tested. The CD4+ T regulatory cells were proliferated when stimulated with Chandipura virus antigen. The regulatory cells did not suppress the proliferation of splenocytes stimulated with anti CD3 antibody when co cultured with them. Interesting observation was, while purification of CD4+ T cells by negative selection, the population of cells negative for CD4 also co purified along with CD4+ T cell. Flow cytometry analysis and light microscopy revealed that CD4 negative cells were of different size and shape (atypical) compared to the normal lymphocytes. Greater percentage of these atypical lymphocytes expressed Fas Ligand and Programmed Death1 (PD-1) receptor., Conclusion: From these results we concluded that virus specific CD4+T regulatory cells are generated during Chandipura virus infection in mice and these cells might control the activated lymphocytes during infection by different mechanism.

CD4+CD25+ regulatory T cells (Tregs) are known to inhibit immune responses to antigens. Since, the process of antigen uptake by dendritic cells (DC) is central to induction of immune responses, we analyzed the effect of Tregs on the expression of endocytic receptors on DC and its repercussion on antigen uptake. Our results demonstrate that Tregs down-regulate the expression and uptake of antigens via C-type lectin-like receptors CD206 and DC-SIGN, restrain the pinocytosis process of DC and augment the expression of FcγRIIB, an inhibitory Fcγ receptor the engagement of which by IgG-bound antigens leads to inhibition of DC activation. Our results thus provide an additional insight on the pertinence of strategies aimed at blocking Treg functions towards improved vaccination protocols., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Heligmosomoides bakeri infection in mice is associated with a dominant CD4(+) T-cell response and with the activity of natural Treg cells with CD4(+) CD25(+) phenotype. The polarization of Th2 T-cell phenotype and the increase in the CD4(+) CD25(+) T cell population are regulated by glucocorticoids that induce apoptosis in CD4(+) CD25(-) T cells and inhibit apoptosis in CD4(+) CD25(+) T cells. However, exposure of mice to H. bakeri antigen induces a high glucocorticoid concentration in serum and a reduction in the number of CD4-positive; CD4(+) CD25(-) and CD4(+) CD25(+) apoptotic T cells in mesenteric lymph node cells. In this study to evaluate the in vitro effect of the anti-apoptotic property of H. bakeri antigen on T cells, apoptosis of these cells was induced by glucocorticoids-dexamethasone (Dex). Excretory-secretory (ES) antigen of the nematode prevented Dex-induced apoptosis in CD4-positive T cells with CD4(+) CD25(-) and CD4(+) CD25(High) phenotype by Bcl-2 protein expression. Contrary to the effect on CD4-positive T cells, survival of CD8(+) T cells was not connected with expression of Bcl-2 protein. This suggest that H. bakeri antigen modulates CD4-positive T cell sensitivity to glucocorticoid-induced apoptosis by induction of Bcl-2 protein., (© 2011 Blackwell Publishing Ltd.)

Rheumatoid arthritis (RA) is a chronic inflammatory disease, with a clinical manifestation both systemic and in joints. It has been suggested that age at disease onset and/or patients' age have influence on disease activity and clinical outcome. The reasons for the different course of RA in older people are not known; however, the activation status of peripheral blood lymphocytes could be responsible. Our aim was to relate expression of activation markers in peripheral blood CD4(+) T cells of RA patients with patients' age and/or onset age and disease activity measured by DAS28. Seventy RA patients were included into the immunological study. Two separation criteria were performed: based on age of RA onset and on the biological age of patients. We examined different activation markers, CD69, CD25, CD95 and human leucocyte antigen D-related (HLA-DR), on the CD4(+) T cell surface. Division of RA patients in 10-year intervals at 40, 50 and 60 years revealed that RA patients with later disease onset were characterized by higher DAS28. This phenomenon was not limited to the division at 60 years of age but, surprisingly, the major differences were found for the 40-year onset division. Analysis of all four components of DAS28 revealed that disease activity in older disease onset was dependent on all components. Older-onset RA patients had a higher percentage of CD4(+) CD25(+) and CD4(+) CD95(+) T cells. Summarizing the major differences in DAS28 and activation status of CD4(+) T cells observed for onset of disease at 40 years seems to be the most informative about the immunological status of RA patients., (© 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.)

Natural regulatory T cells (CD4(+) CD25(+) Foxp3(+)), natural regulatory T cells (nTreg), play an important role in the regulation of inflammatory immune responses. However, the immunosuppressive properties of nTreg may unfavourably affect the host's ability to clear certain infections. In human visceral leishmaniasis (VL), reports on the frequency and function of nTreg are not conclusive. A limitation of our own previous studies that did not indicate a major role for Foxp3(+) nTreg in VL pathogenesis was that Foxp3 was measured by mRNA expression alone, as other tools were not available at the time. We have in this study assessed CD4(+)CD25(+)Foxp3(+) cells in splenic aspirates and peripheral blood mononuclear cells (PBMC) from an extensive series of patients with VL and endemic controls (EC) by flow cytometry (FACS). The results do not show increased frequencies of Foxp3(+) cells in patient with VL pre- and post-treatment, neither were they elevated when compared to PBMC of EC. We conclude that active VL is not associated with increased frequencies of peripheral Foxp3 Treg or accumulation at the site of infection.

Attenuated CD4+ T-cell-mediated immune responses are involved in persistence of HCV infection, but the mechanism remains undefined. In this study, the proportions of CD4+ T cell subsets, naïve, central memory, effector memory and effector cells, along with CD25 (IL-2R alpha) and CD127 (IL-7R alpha) expression on different CD4+ T cell subsets, were measured by polychromatic flow cytometry in 24 chronic HCV-infected individuals and 21 healthy controls. A significant decrease in naïve CD4+ T cells and an increase of central memory and effector memory CD4+ T cells were found in HCV-infected patients compared with healthy controls. HCV-infected patients showed a lower level of CD127 expression in all CD4+ T cells subsets, especially in central memory and effector CD4+ T cells. In terms of total CD4+ T cells, an increase in CD25+ regular T cells (CD4+ CD25+ CD127lo) was found in HCV-infected patients. Interestingly, naïve CD4+ T cells showed increased CD25 expression, while effector memory and effector CD4+ T cells had lower CD25 expression. These data indicated that variations in different fractions of CD4+ T cells, including the phenotypic profile and expression level of CD25 and CD127, may be associated with low efficiency of immune response in chronic HCV infection. These results will strengthen the understanding of pathogenesis and dysfunction of CD4+ T cell immunity during long-term HCV persistence.

The roles of regulatory T cells (Tregs) and PD-1 in hepatitis B have not been clearly described. Also, the role of B and T lymphocyte attenuator (BTLA), which serves as a negative regulator of T-cell activation, is still unknown in hepatitis B. In this study, we analyzed the frequency of circulating CD4(+)CD25(high) Tregs in patients with chronic hepatitis B (CHB), and subsequently investigated expression of PD-1 and BTLA on CD4(+) T cells, as well as their relationships with the clinical index of CHB patients. A total of 39 CHB patients and 19 healthy persons as controls were enrolled in the study. We found that the frequency of CD4(+)CD25(high) Tregs and PD-1 expression on CD4(+) T cells was significantly increased in CHB patients compared with normal controls. However, BTLA expression on CD4(+) T cells showed no significant difference between the two groups. The frequency of Tregs was significantly higher in patients with HBV DNA titers >or=10(8) than in those with HBV DNA titers <10(8). Circulating CD4(+)CD25(high) Treg frequency and PD-1 expression on CD4(+) T cells correlated positively with serum HBV DNA load in CHB patients. Our findings suggest that the increased frequency of CD4(+)CD25(high) Tregs and PD-1 expression on CD4(+) T lymphocytes may inhibit the cellular immune response against HBV and affect viral clearance, leading to the persistence of chronic HBV infection.

Background and Aims: Coeliac disease is a common small intestinal inflammatory disorder that results from a breach of intestinal tolerance to dietary gluten proteins, driven by gluten-reactive effector T cells. We aimed to assess the pathogenic role of gluten-reactive T cells and to generate a model of gluten-induced enteropathy., Methods: CD4+CD25- T cell fractions were adoptively transferred into lymphopenic mice, leading to "baseline" small intestinal inflammation., Results: Rag1-/- recipients of gliadin-presensitised CD4+CD45RBlowCD25- T cells, but not CD4+CD45RBhigh naive T cells, gained less weight and suffered from more severe duodenitis when challenged with oral gluten than recipients on gluten-free diet, or recipients of control (ovalbumin)-presensitised T cells. This was accompanied by deterioration of mucosal histological features characteristic of coeliac disease, and increased Th1/Th17 cell polarisation in the duodenum and the periphery. Interestingly, reintroduction of a gluten-free diet led to weight gain, improvement of histological duodenitis, and a decrease in duodenal interferon gamma and interleukin 17 transcripts. Moreover, B cell-competent nude recipients of gliadin-presensitised CD4+CD45RBlowCD25- T cells produced high levels of serum anti-gliadin immunoglobulin A (IgA) and IgG1/IgG2c only when challenged with oral gluten., Conclusions: CD4+ T cell immunity to gluten leads to a breach of oral gluten tolerance and small intestinal pathology in lymphopenic mice, similar to human coeliac disease. This model will be useful for the study of coeliac disease pathogenesis, and also for testing novel non-dietary therapies for coeliac disease.

The vagus nerve is a conduit for bidirectional signaling between the brain and the viscera. Vagal signaling has been shown to downregulate gastrointestinal inflammation, and the mechanism is thought to involve acetylcholine binding to the alpha-7 subunit of the nicotinic acetylcholine receptor on macrophages. The aims of this study were to quantify the impact of vagotomy in vivo by visualizing nuclear factor (NF)-kappaB activity and to determine if the proinflammatory impact of vagotomy could be transferred by lymphocytes. Real-time biophotonic imaging revealed that subdiaphragmatic vagotomy resulted in increased levels of NF-kappaB in vivo. NF-kappaB activation was further exaggerated in vivo following exposure to 4% DSS for 5 days. Vagotomized animals also exhibited higher disease activity scores and secreted more proinflammatory cytokines. Adoptive transfer of CD4(+) T cells from vagotomized animals (but not CD4(+) T cells from sham-operated controls) to naive dextran sulfate sodium (DSS)-treated recipients resulted in increased inflammatory scores. Further examination of the CD4(+) T cells revealed that adoptive transfer of the CD25(-) population alone from vagotomized donors (but not sham-operated donors) was sufficient to aggravate colitis in DSS-treated recipients. Increased DSS-induced inflammation was associated with reduced CD4(+)CD25(+)Foxp3(+) regulatory T cell numbers in recipients. This study clearly demonstrates the ability of the vagus nerve to modulate activity of the proinflammatory transcription factor NF-kappaB in vivo. The proinflammatory effect of vagotomy is transferable using splenic T cells and highlights a previously unappreciated cellular mechanism for linking central parasympathetic processes with mucosal inflammation and immune homeostasis.

Besides iatrogenic immunosuppression, endogenous suppression by regulatory T cells (Tregs) may also mediate inhibition of effector T cells after transplantation. Here we determined the effect of common immunosuppressive drug regimens on both Treg and effector T cells. Tregs and cytomegalovirus (CMV)-specific T cells were quantified in 88 renal transplant recipients, 58 hemodialysis patients, and 22 controls. T cell dynamics were longitudinally assessed within 20 weeks after transplantation. The number of Tregs was quantified by measurement of CD25 and/or FOXP3-positive cells and by functional assays. CMV-specific T cells were quantified by stimulation-induced intracellular cytokine analysis. Treg frequencies in transplant recipients were significantly lower compared to those in hemodialysis patients and controls. These lower Treg levels were associated with a less pronounced suppression of effector function. Treg levels decreased within the first weeks after transplantation and remained low in the long term. In contrast, although decreased at early post-transplant, long-term levels of CMV-specific T cells normalized to levels found in hemodialysis patients and controls. These studies suggest that there is an initial decrease of Tregs and effector T cells as a consequence of a direct inhibitory effect of immunosuppressive drugs. In the long term, persistently low Treg levels may favor normalization of effector T cells to ensure sufficient pathogen control.

Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.

The mechanisms of action of extracorporeal photochemotherapy (ECP) in cutaneous T-cell lymphoma (CTCL) are poorly understood. Recently, ECP has been shown to induce an increase in regulatory T cell (Treg) expression and functional activities in Graft-versus-host-disease (GvHD), whereas no data are available in CTCL patients. The aim of this study is to evaluate whether ECP is able to modulate the expression levels of the circulating CD4+CD25+bright subset in CTCL patients and whether these modifications are related to the disease course. The patient population included 43 CTCL and 15 chronic GvHD patients treated by ECP at our institutions since 1992. The expression of the circulating CD4+CD25+bright subset was analysed at baseline and sequentially during treatment by flow-cytometry. Fifty healthy donors were used as controls. The baseline circulating CD4+CD25+bright percentage values in CTCL (median: 4.3 percent) were similar to those of healthy donors, whereas GvHD showed significantly lower values (median: 1.5 percent; p<0.001). During treatment, CTCL patients were characterised by an early decrease (from 4.3 percent to 2.4 percent median after 6 months). The CD4+CD25+bright decrease was associated to the disease course, as it occurred in 91.3 percent of responding but in only 25 percent of PD patients (p=0.0001). On the other hand, a significant increase of CD4+CD25+bright cells was observed in GvHD. ECP induces a reciprocal modulation of the circulating CD4+CD25+bright cells in CTCL and GvHD, with a downregulation in CTCL potentially associated with the response mechanisms.

Staphylococcal enterotoxin B (SEB) activates T cells via non-canonical signalling through the T cell receptor and is an established model for T cell unresponsiveness in vivo. In this study, we sought to characterize the suppressive qualities of SEB-exposed CD4(+) T cells and correlate this with genetic signatures of anergy and suppression. SEB-exposed CD25(+) and CD25(-)Vbeta8(+)CD4(+) T cells expressed forkhead box P3 (FoxP3) at levels comparable to naive CD25(+) T regulatory cells and were enriched after exposure in vivo. Gene related to anergy in lymphocytes (GRAIL), an anergy-related E3 ubiquitin ligase, was up-regulated in the SEB-exposed CD25(+) and CD25(-)FoxP3(+)Vbeta8(+)CD4(+) T cells and FoxP3(-)CD25(-)Vbeta8(+)CD4(+) T cells, suggesting that GRAIL may be important for dominant and recessive tolerance. The SEB-exposed FoxP3(+)GRAIL(+) T cells were highly suppressive and non-proliferative independent of CD25 expression level and via a glucocorticoid-induced tumour necrosis factor R-related protein-independent mechanism, whereas naive T regulatory cells were non-suppressive and partially proliferative with SEB activation in vitro. Lastly, adoptive transfer of conventional T cells revealed that induction of FoxP3(+) regulatory cells is not operational in this model system. These data provide a novel paradigm for chronic non-canonical T cell receptor engagement leading to highly suppressive FoxP3(+)GRAIL(+)CD4(+) T cells.

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates diverse cellular functions. It has been found that CD4(+)CD25(+) regulatory T (T(REG)) cells exert their suppressor function by transferring cAMP to responder T cells. Here, we show that miR-142-3p regulates the production of cAMP by targeting adenylyl cyclase (AC) 9 messenger RNA in CD4(+)CD25(-) T cells and CD4(+)CD25(+) T(REG) cells. miR-142-3p limits the level of cAMP in CD4(+)CD25(-) T cells by inhibiting AC9 production, whereas forkhead box P3 (FOXP3) downregulates miR-142-3p to keep the AC9/cAMP pathway active in CD4(+)CD25(+) T(REG) cells. These findings reveal a new molecular mechanism through which CD4(+)CD25(+) T(REG) cells contain a high level of cAMP for their suppressor function, and also suggest that the microRNA controlling AC expression might restrict the final level of cAMP in various types of cells.

In areas where schistosomiasis is endemic, a negative correlation is observed between atopy and helminth infection, associated with a low prevalence of asthma. We investigated whether Schistosoma mansoni infection or injection of parasite eggs can modulate airway allergic inflammation in mice, examining the mechanisms of such regulation. We infected BALB/c mice with 30 S. mansoni cercariae or intraperitoneally injected 2,500 schistosome eggs, and experimental asthma was induced by ovalbumin (OVA). The number of eosinophils in bronchoalveolar lavage fluid was higher in the asthmatic group than in asthmatic mice infected with S. mansoni or treated with parasite eggs. Reduced Th2 cytokine production, characterized by lower levels of interleukin-4 (IL-4), IL-5, and immunoglobulin E, was observed in both S. mansoni-treated groups compared to the asthmatic group. There was a reduction in the number of inflammatory cells in lungs of S. mansoni-infected and egg-treated mice, demonstrating that both S. mansoni infection and the egg treatment modulated the lung inflammatory response to OVA. Only allergic animals that were treated with parasite eggs had increased numbers of CD4(+) CD25(+) Foxp3(+) T cells and increased levels of IL-10 and decreased production of CCL2, CCL3, and CCL5 in the lungs compared to the asthmatic group. Neutralization of IL-10 receptor or depletion of CD25(+) T cells in vivo confirmed the critical role of CD4(+) CD25(+) Foxp3(+) regulatory T cells in experimental asthma modulation independent of IL-10.

Previous studies have demonstrated that distinct immune response profiles can be correlated with the development/maintenance of different clinical forms of human schistosomiasis. We have previously shown that individuals with the more severe clinical forms of the disease such as those presenting different levels of fibrosis or with the hepatosplenic (HS) clinical form of the disease show significantly different immune response when compared with those with the intestinal clinical form (INT). To better understand the immune mechanisms associated with the clinical form of the schistosomiasis, in this study, we present the results of the evaluation, at a single cell level, of the cytokine patterns as well as the chemokine receptors expression by T-cell subsets after in vitro short-term stimulation with soluble egg antigens as well as the ex vivo frequency analysis of putative regulatory CD4+CD25HIGH+ T-cell subset in the peripheral blood mononuclear cells. We observed an increase on IL-4+, IL-5+ and IL-10+ cells both in the CD4+ and CD8+ lymphocytes in INT and a significant decrease on the number of IL-4+, IL-5+ and IL-10+ T-lymphocytes for HS. However, patients with detectable fibrosis presented decrease on IL-10+ (both CD4+ and CD8+ lymphocytes) and basal levels of IL-4 and IL-5. These data suggested that although INT group is under the influence of an effective immunoregulated immune response, mainly due to the high percentage of IL-10+ cells, it presents a mixed type (Type1/Type-2) immune profile. Moreover, the chemokine receptors expression demonstrated that CXCR3 and CXCR4 by CD4+ T-cells in INT may dictate the selective profile of IL-10 associated with the immunomodulatory events in human schistosomiasis. Additionally, the ex vivo analysis also suggests that higher levels of CD4+CD25HIGH+ T-cells may play a role in controlling morbidity in chronic human schistosomiasis. Taken together, these data suggest a major role of IL-10-producing CXCR4+ CD4+ T-cell subset for the asymptomatic outcome of the disease.

Generating and using regulatory T cells (Tregs) to modulate inflammatory disease represents a valuable approach to therapy but has not yet been applied as a means to control virus-induced immunopathological reactions. In this report, we developed a simplified technique that used unfractionated splenocytes as a precursor population and showed that stimulation under optimized conditions for 5 days with solid-phase anti-CD3 monoclonal antibody in the presence of transforming growth factor beta (TGF-beta) and interleukin-2 could induce up to 90% of CD4(+) T cells to become Foxp3(+) and able to mediate suppression in vitro. CD11c(+) dendritic cells were intricately involved in the conversion process and, once modified in the presence of TGF-beta, could convert Foxp3(-) CD4(+) cells into Foxp3(+) CD4(+)cells by producing TGF-beta. The converted cells had undergone cell division, and the majority of them expressed activation markers along with surface molecules that would facilitate their migration into tissue sites. The primary reason for our study was to determine if such in vitro-converted Tregs could be used in vivo to influence the outcome of a virus-induced immunoinflammatory lesion in the eye caused by herpes simplex virus infection. We could show in three separate models of herpetic stromal keratitis that adoptive transfers of in vitro-converted Tregs effectively diminished lesion severity, especially when given in the initial phases of infection. The suppression effect in vivo appeared to be polyspecific. The protocol we have developed could provide a useful additional approach to control virus-induced inflammatory disease.

Costimulatory signals are important for development of effector and regulatory T cells. In this case, CD28 signaling is usually considered inert in the absence of signaling through the TCR. By contrast, mitogenic rat CD28 mAb reportedly expand regulatory T cells without TCR stimulation. We found that a commercially available human CD28 mAb (ANC28) stimulated PBMC without TCR co-ligation or cross-linking; ANC28 selectively expanded CD4(+)CD25(+)FOXP3(-) (Teff) and CD4(+)CD25(+)FOXP3(+) (Treg) cells. ANC28 stimulated the CD45RO(+) CD4(+) (memory) population, whereas CD45RA(+)CD4(+) (naive) cells did not respond. ANC28 also induced inflammatory cytokines. Treg induced by ANC28 retain the Treg phenotype longer than costimulated Treg. Treg induced by ANC28 suppressed CD25(-) T cells through a contact-dependent mechanism. Purity influenced the response of CD4(+)CD25(+ )cells because bead-purified CD4(+)CD25(+ )cells (85-90% pure) responded strongly to ANC28, whereas 98% pure FACS-sorted CD4(+)CD25(bright) (Treg) did not respond. Purified CD4(+)CD25(int) cells responded similarly to the bead-purified CD4(+)CD25(+) cells. Thus, pre-activated CD4(+) T cells (CD25(int)) respond to ANC28 rather than Treg (CD25(bright)). The ability of ANC28 to expand both effectors producing inflammatory cytokines as well as suppressive regulatory T cells might be useful for ex vivo expansion of therapeutic T cells.

The infection with Trypanosoma cruzi leads to a vigorous and apparently uncontrolled inflammatory response in the heart. Although the parasites trigger specific immune response, the infection is not completely cleared out, a phenomenon that in other parasitic infections has been attributed to CD4+CD25+ T cells (Tregs). Then, we examined the role of natural Tregs and its signaling through CD25 and GITR in the resistance against infection with T. cruzi. Mice were treated with mAb against CD25 and GITR and the parasitemia, mortality and heart pathology analyzed. First, we demonstrated that CD4+CD25+GITR+Foxp3+ T cells migrate to the heart of infected mice. The treatment with anti-CD25 or anti-GITR resulted in increased mortality of these infected animals. Moreover, the treatment with anti-GITR enhanced the myocarditis, with increased migration of CD4+, CD8+, and CCR5+ leukocytes, TNF-alpha production, and tissue parasitism, although it did not change the systemic nitric oxide synthesis. These data showed a limited role for CD25 signaling in controlling the inflammatory response during this protozoan infection. Also, the data suggested that signaling through GITR is determinant to control of the heart inflammation, parasite replication, and host resistance against the infection.

The forkhead/winged helix transcription factor (Foxp3) is expressed as two different isoforms in humans: the full-length isoform (Foxp3FL) and an alternative-splicing product lacking the exon 2 (Foxp3DeltaE2). We here studied the cellular distribution of Foxp3 isoforms by quantitative PCR and evaluated the functional outcome of retroviral transduction of Foxp3FL and Foxp3DeltaE2 genes into CD4(+)CD25(-) cells. In PBMC, both isoforms were preferentially expressed in CD4(+)CD25(hi) cells. In single-cell-sorted and expanded Treg, both Foxp3 isoforms were expressed simultaneously but without a fixed ratio. Forced expression of Foxp3FL or Foxp3DeltaE2 genes in CD4(+)CD25(-) T cells induced bona fide Treg that not only displayed Treg phenotype but also were anergic and mediated significant suppressive activity against CD3-activated CD4(+)CD25(-) cells. GFP(-) nontransduced cells or cells transduced with an empty vector showed no Treg phenotype, anergy or suppressive activities. In conclusion, our results reveal that both Foxp3 isoforms possess similar capacities to induce Treg; however, unnaturally high expression levels are required to convey Treg functions to CD4(+)CD25(-) cells. As both Foxp3 isoforms appear to be expressed in an independent fashion, studies aiming at quantification of Treg in peripheral blood or in tissue samples can benefit from determination of total Foxp3 levels rather than one of the isoforms.

Human CD4+CD25+ regulatory T (T(R)) cells express the transcription factor forkhead box p3 (FOXP3) and have potent immunosuppressive properties. While naturally occurring T(R) cells develop in the thymus, adaptive T(R) cells develop in the periphery from naive CD4+ T cells. Adaptive T(R) cells may express cyclooxygenase type 2 (COX-2) and suppress effector T cells by a PGE(2)-dependent mechanism, which is reversible with COX inhibitors. In this study we have characterized the differentiation of naive CD4+ T cells into adaptive T(R) cells in detail during 7 days of continuous antigen stimulation. After 2 days of stimulation of CD4+CD25- T cells, the cells expressed FOXP3 and COX-2 and displayed potent immunosuppressive properties. The suppressive phenotype was present at all observed time-points from Day 2, although suppression was merely present at Day 7. The adaptive T(R) cells expressed cell surface markers consistent with an activated phenotype and secreted high levels of TGF-beta, IL-10, and PGE(2). However, the suppressive phenotype was found exclusively in cells that proliferated upon activation. These data support the notion that activation of naive CD4+ T cells leads to concomitant acquisition of effector and suppressive properties.