Search

Background: The diagnosis of drug-induced autoimmune hepatitis (DIAIH) and its differentiation from idiopathic autoimmune hepatitis (AIH) is challenging. This study aimed to differentiate DIAIH from AIH by comparing the biochemical changes, histological features, and frequencies of CD4 + Foxp3 + CD25 +/- regulatory T cells (Tregs) in liver tissues or peripheral blood lymphocytes., Methods: A total of 15 DIAIH patients and 24 AIH patients who underwent liver biopsies at initial presentation were enrolled in this study. The liver histological changes were assessed by HE staining. The phenotypic recognition and distribution of CD4 + Foxp3 + CD25 +/- Tregs in liver tissues were evaluated by single/double immunostains in serial sections. The CD4 + Foxp3 + CD25 +/- Tregs in peripheral blood were analyzed by flow cytometry., Results: The median values of ALT and AST were 404.50 U/L and 454.10 U/L in DIAIH patients and 309.50 U/L and 315.00 U/L in AIH patients, respectively. More importantly, for the first time we found that patients with DIAIH had higher levels of serum ALT and AST, more severe degree of lobular inflammation, higher frequencies of zone 3 necrosis and higher number of lobular CD4 + Foxp3 + CD25 - Tregs compared with AIH (P < 0.05). Furthermore, there were positive correlations in DIAIH between the degree of lobular inflammation and either the AST/ALT level or the number of lobular CD4 + Foxp3 + CD25 - Tregs (P < 0.05). However, the frequency of peripheral blood CD4 + Foxp3 + CD25 +/- Tregs were not significantly different between DIAIH and AIH., Conclusions: The differences of ALT, AST and the number of lobular CD4 + Foxp3 + CD25 - Tregs between patients with DIAIH and those with AIH are clinically helpful in differentiating these two diseases in their early stage., (Copyright © 2018. Published by Elsevier B.V.)

Lymphoid tissues are of intense interest for studies of the pathogenesis of human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques but are relatively difficult to sample non-invasively. Fine needle aspiration (FNA) cytology, conventionally a diagnostic procedure for lymphadenopathy, can be used for longitudinal study of tissue cell subsets during HIV/SIV infection. In this study, we serially sampled lymph node (LN) FNA from pigtail macaques and studied cell subsets in the aspect of absolute count, frequency, and functionality by flow cytometry. The median recovered lymphocyte count from FNA samples was 2.01×10(5) (3.0×10(3) to 2.25×10(6), n=38) and median CD4+ T cell subset recovered was 5.94×10(4) (277 to 6.17×10(5), n=38). Although we observed a relatively large variation in the frequencies of cell subsets of FNA samples taken from different time points, the cell subset composition of FNA samples, in particular T cell and CD4+ T cell frequencies, was broadly comparable to whole excised LNs (n=6) and distinct from peripheral blood. A subset of CD4+ T cells that is located almost exclusively in secondary lymphoid tissues, T follicular helper (TFH) cells, was readily identifiable in LN FNAs and the TFH cell frequencies were strongly correlated with B cell frequencies. In vitro functionality of FNA lymphocytes was demonstrated using polyclonal SEB stimulation, resulting in a median 6% of responding CD4+ T cells, comparable to circulating CD4+ T lymphocytes. We conclude that serial sampling of macaque LNs using FNA is a potentially useful method to study the immunopathogenesis of SIV infection and may be extended to HIV infection., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Introduction: Dental pulp inflammation and repair are closely related. Osteocalcin (OCN), a glycoprotein present in dentin matrix, is expressed by odontoblasts. Although OCN is considered a reparative molecule inside the dental pulp, it is not clear if it is involved in pulpal inflammation. The objective of this study was to localize OCN in reversible and irreversible pulpitis and to describe its possible function in inflammation., Methods: Pulp tissues in the form of reversible and irreversible pulpitis were collected from the endodontic clinic. Those from impacted teeth were used as controls. Immunohistochemistry was used to localize OCN. Samples were analyzed for OCN and inflammatory mediator expression using multiplex assay., Results: OCN in inflamed tissues was localized in cells and matrix around calcification areas and in cells around blood vessels but not in normal tissues. The plex assay (Bio-Plex 200, Bio-Rad Laboratories Ltd, Mississauga, ON, Canada) showed OCN expression in reversible pulpitis significantly higher than in irreversible pulpitis, and both were significantly higher than in the controls. A panel of inflammatory mediators showed an increase in reversible and irreversible pulpitis. Another panel was decreased in both stages compared with the controls. OCN expression in reversible pulpitis was positively correlated to the expression of vascular endothelial growth factor, fibroblast growth factor, macrophage inflammatory protein-1β, monocyte-derived chemokine, monocyte chemoattractant protein-1, interleukin (IL)-17, and soluble IL-2 receptor α and negatively correlated to that of IL-1α, IL-1β, IL-8, granulocyte macrophage colony-stimulating factor, and macrophage inflammatory protein-1α., Conclusions: Profound understanding of the pulp inflammatory process would lead to new molecular treatment strategies. Our data indicate that OCN expression in reversible pulpitis is associated with angiogenic markers, suggesting its potential use in regenerative treatment., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

One of the auto-antigens aberrantly targeted in Multiple sclerosis is myelin basic protein (MBP). In this study, chronic progressive multiple sclerosis (CPMS) patients receiving the experimental drug MBP8298, on a compassionate care trial, were examined before and after high dose peptide treatment for their circulating regulatory T-cell numbers and their responses to the common mitogens, phytohemagglutinin and poke-weed mitogen. Peripheral blood mononuclear cells (PBMCs) isolated from these patients before treatment displayed anergy upon stimulation with phytohemagglutinin; measured through reduced proliferation, IFN-γ and IL-17A secretion in an in vitro cell culture system. 6 Weeks and 6months after treatment their PBMCs displayed a reversal of anergy with phytohemagglutinin stimulation. There was also a marked increase in their CD4(+)CD25(+hi)FoxP3(+) T-cells regulatory T-cells. These results suggest that high dose MBP8298 treatment has a profound effect on the circulating T-cells of CPMS patients, capable of reversing peripheral anergy and establishing T regulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objectives: The purpose of this study was to investigate the association between T cell receptor excision circle levels in peripheral blood mononuclear cells and regulatory T cells that co-express CD25 and Foxp3 in healthy children and adolescents of different ages., Materials and Methods: The quantification of signal-joint T-cell receptor excision circle levels in the genomic DNA of peripheral blood mononuclear cells was performed using real-time quantitative PCR. The analysis of CD4, CD8, CD25, and Foxp3 expression was performed using flow cytometry., Results: Ninety-five healthy controls (46 females and 49 males) ranging in age from 1 to 18 years were analyzed. The mean T-cell receptor excision circle count in all individuals was 89.095 ± 36.790 T-cell receptor excision circles per microgram of DNA. There was an inverse correlation between T-cell receptor excision circles counts and age (r = -0.846; p<0.001) as well as between the proportion of CD4(+)CD25(+)Foxp3(+) T cells and age (r = -0.467; p = 0.04). In addition, we observed a positive correlation between the amount of CD4(+)CD25(+)Foxp3(+) T cells and the amount of T-cell receptor excision circles per microgram of DNA in individuals of all ages (r = -0.529; p = 0.02)., Conclusions: In this study, we observed a decrease in the thymic function with age based on the fact that the level of T-cell receptor excision circles in the peripheral blood positively correlated with the proportion of regulatory T cells in healthy children and adolescents. These findings indicate that although T-cell receptor excision circles and regulatory T cells levels decrease with age, homeostasis of the immune system and relative regulatory T cells population levels are maintained in the peripheral blood.

Background: Autologous haematopoietic stem cells transplantation (HSCT) has been shown to be effective in refractory Crohn's disease., Aim: We analysed the effects of HSCT on the immune response of patients treated for moderate-severe Crohn's disease, refractory or intolerant to multiple drugs., Methods: Unselected peripheral blood stem cells were collected after mobilisation with cyclophosphamide (CTX) and G-CSF. The conditioning regimen included CTX and rabbit antithymocyte globulin. Blood samples for immunological analyses were collected at baseline, after mobilisation, and 3, 6 and 12 months after transplantation. Immunological analyses evaluated: (1) CD4(+)/CD25(high+)/FoxP3(+) regulatory T cells (T-regs); (2) Toll-like receptor 2-(TLR2) and TRL4-expressing monocytes (CD14(+) cells); (3) IL-12, IL-10, TNF-alpha-production by mitogen-stimulated CD14(+) cells and IFN-gamma production by CD4(+) T cells. Immunological results were compared with healthy donors and associated with clinical and endoscopic response during 12 months of follow-up., Results: Overall, T-regs increased, whilst TLR4-expressing cells, as well as TNF-alpha and IL-10, all higher than healthy donors at baseline, significantly decreased after transplantation. Full responders at T(3) had higher T-regs and lower IFN-gamma and IL12. T-regs decreased and IL12 and TLR2 increased in the only relapsed patient., Conclusions: HSCT can induce and maintain clinical and endoscopic remission in refractory Crohn's disease, which is associated with immunomodulation., (Copyright © 2011. Published by Elsevier Ltd.)

Immunomodulatory function of bone marrow derived mesenchymal stem cells in cancer has recently been investigated. But the resident mesenchymal stem cells as whole in cancer and in the breast cancer tissue have not been studied well. In the present work we isolated adipose derived stem cells (ASCs) from breast cancer and normal breast tissues to investigate the expressions of IL-4, IL-10 and transforming growth factor (TGF)-β1 in ASCs and to see if ASCs isolated from patients can modulate the regulatory molecules on peripheral blood lymphocytes. Our results showed that IL-10 and TGF-β1 have significantly higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals (P value <0.05). The culture supernatant of ASCs isolated from breast cancer patients with pathological stage III induced upregulation of the mRNA expression levels of IL-4, TGF-β1, IL-10, CCR4 and CD25 in PBLs. In addition, the percentage of CD4+CD25(high)Foxp3(+) T regulatory cells was increased in vitro. When the same culture supernatant was added to ASCs isolated from normal subjects augmentation of the mRNA expressions of IL-4, IL-10, IL-8, MMP2, VEGF and SDF-1 in normal ASCs was also observed. These data collectively conclude that resident ASCs in breast cancer tissue may have crucial roles in breast tumor growth and progression by inducing regulatory molecules and promoting anti-inflammatory reaction within the tumor microenvironment. Further investigation is required to see if the immune suppression induced by ASCs is an independent property from tumor cells or ASCs gain their immunosuppressive potential from malignant cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Nephrotoxicity limits the use of cisplatin, a widely used chemotherapeutic agent for treatment of various malignancies. Overall, CD4+ T cells mediate cisplatin-induced renal injury; however, the CD4+CD25+ regulatory T-cell subset (CD4+CD25+ Treg) has broad suppressive effects on many different cell types. In this study, we determined whether CD4+CD25+ Treg cells had protective effects against cisplatin-induced acute renal injury in nu/nu mice that lack mature T cells. In these mice, there was marked attenuation of the decreased survival, renal dysfunction and tubular injury, renal tumor necrosis factor-α, and interleukin-1β cytokine levels. Furthermore, renal macrophage accumulation was reduced in CD4+CD25+ Treg cell-adoptive transferred nu/nu mice compared with control mice. Infusion of CD4+CD25+Treg cells into wild-type Balb/c mice reduced serum blood urea nitrogen and creatinine levels equivalent to those in nu/nu mice and extended their survival time after cisplatin injection. In contrast, depletion of CD4+CD25+ Treg cells in wild-type mice exacerbated kidney injury after cisplatin administration. Transcription factor Foxp3-positive cells (Treg cells) were detected in the kidneys of nu/nu mice after cisplatin injection. Our results suggest that CD4+CD25+ Treg cells directly affect cisplatin nephrotoxicity and their modulation represents an additional treatment strategy.

Background & Aims: Immune-enhancing diets (IEDs) contain a mixture of nutrients claimed to have immunological properties. Therefore, it seemed relevant to determine the effect of each of their components. The aim of this study was to examine the role of arginine (Arg) and ω3 polyunsaturated fatty acids (ω3 PUFAs) in the effect of an IED (Crucial(®)) in a validated rat model of inflammation induced by turpentine (TI)., Methods: Forty-two rats were randomized into five groups: AL (ad libitum), TI-EN (TI+standard enteral nutrition (EN): Sondalis(®)HP), TI-EN-Arg (TI+standard EN+Arg in equimolar concentration to Arg in the IED), TI-M-IED (TI+modified IED containing the same ω6/ω3 ratio as in standard EN) and TI-IED (TI+Crucial(®)). Blood was sampled to determine CD25 receptor density on lymphocytes. TNF-α, IL-6 and NO (production and expression) were evaluated on isolated macrophages. Mesenteric lymph nodes, spleen and liver were cultured for analysis of enterobacterial translocation and dissemination., Results: CD25 density was decreased after TI and was corrected in the TI-EN-Arg, TI-M-IED and TI-IED groups (p<0.05). TI induced an alteration of macrophage mRNA expression of IL-6, TNF-α and iNOS, corrected in the TI-EN-Arg and TI-M-IED groups (p<0.05), but not by the IED. Enterobacterial translocation was observed in all treated groups, nevertheless the amount tended (p=0.054) to be lower in the TI-EN-Arg group., Conclusions: Arg and ω3 PUFAs make a major contribution to IED effects, but our study shows interaction between them on macrophage reactivity. This indicates that the individual properties of each pharmaconutrient are not additive in IEDs., (Copyright © 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

CD4+CD25+ regulatory T cells (Tregs) mediate tolerance towards self antigens and prevent the development of autoimmunity. Treg function is typically evaluated by the ability to suppress proliferation and cytokine production of co-cultured CD4+CD25- T cells in Treg suppression assays. Purified Tregs are often obtained using the "Regulatory T Cell isolation kit" from Miltenyi Biotech. Separation can be performed manually using single columns or automated using the AutoMACS Cell Separator. In this Technical Note we present a serious pitfall in Treg suppression assays when evaluating magnetically separated CD4+CD25+ T cells obtained by the "Regulatory T Cell isolation kit" and AutoMACS Cell Separator. The AutoMACS Running Buffer recommended by the manufacturer for separation contains Ethylene-Diamine-Tetra-Acetate (EDTA). Here we show that even minute traces of EDTA in the CD4+CD25+ T cell fraction mediate significant suppression of CD4+CD25- T cell proliferation. The suppressive effect of EDTA is dose-dependent and mimics Treg mediated suppression of CD4+CD25- T cell proliferation. The influence of EDTA can be eliminated by thorough washing of the CD4+CD25+ T cell fraction following the separation. Our observation may have implications for other cell separation methods using EDTA-containing buffers., (2009 Elsevier B.V. All rights reserved.)

Natural CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) control the activation of the immune system and therefore have become a major area of research in immunology. The generation of monoclonal antibodies against human Tregs offers the possibility to discover novel Treg-specific or Treg-associated surface markers and to identify targets for a therapeutic modulation of Tregs. Here we present a methodology optimized to efficiently induce and select mAb against human Tregs by repeated immunization of mice with Tregs from a single donor and a differential two-step flow cytometry-based hybridoma screening procedure., (2010 Elsevier B.V. All rights reserved.)

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.

Cryopreservation of peripheral blood mononuclear cells (PBMC) is essential for many clinical and research assays. Some studies reported consistent changes in PBMC phenotype following cryopreservation. We hypothesized that PBMC freezing may have a negative impact on estimation of the frequency of T regulatory cell (Treg). Treg levels were measured in 6 fresh PBMC samples isolated from 6 healthy donors and these levels were re-measured after freezing for three weeks. Herein, we report a significant reduction in Treg frequency in all samples following cryopreservation.

Human regulatory T cells (Treg) have been variously defined as CD4(+)CD25(+), CD4(+)CD25(high) or CD4(+)CD25(high)FOXP3(+) cells which are responsible for maintaining peripheral tolerance. Their isolation from human peripheral blood or tissues depends on the expression level of CD25(IL-2Ralpha) - a surface marker which is also expressed on activated effector helper T cells. CD39, a cell surface associated ectonucleotidase, can be used to purify Treg with strong suppressor functions. The CD4(+)CD39(+) T cells catalyze cleavage of adenosine triphosphate (ATP) to adenosine monophosphate (AMP), which is then further cleaved to adenosine. CD4(+)CD39(+) T cells largely overlap with CD4(+)CD25(high)FOXP3(+) but not CD4(+)CD25(+) T cell subset, and mediate equally potent immune suppression. Thus, CD39 surface marker can be successfully used for routine isolation of functionally-active human Treg from the peripheral blood of healthy donors or patients with cancer for studies of their role in health and disease.

Recent studies suggest inflammatory mechanisms involved in the pathogenesis of major psychiatric disorders (MPD). T cells play a major role during inflammation, but little is known about T cell subpopulations in the cerebrospinal fluid (CSF). We investigated the frequency of cells positive for the surface markers CD4, CD8, CD25, CD45, CD69, and CD127 in 45 paired cerebrospinal fluid (CSF) and peripheral blood (PB) samples by multiparameter flow cytometry from patients with MPD of the schizophrenic and affective spectrum with normal CSF cell counts and compared them with those from patients with non-inflammatory (NIND), chronic inflammatory (CIND) neurological disorders, and meningitis (MEN). In MEN patients, CD4+ cell frequency in PB, but not in CSF, was significantly increased as compared to CIND and NIND. No difference between patient groups was observed for CD8+. CD4+CD45RO+ double positive cells in PB were significantly lower in CIND than in MEN or NIND. The frequency of CD4+CD25+ cells in PB was significantly higher in MEN than in MPD or CIND. For CSF, the percentage of CD4+CD127(dim) cells was significantly lower in MEN than in MPD. CD4+CD127(dim) in PB and CSF showed overlapping characteristic clusters between MPD and CIND and MEN patients. Overall, the hypothesis of low degree inflammation in a subgroup of MPD is supported. The analysis of lymphocyte subsets in PB and CSF constitutes a novel promising tool to understand underlying pathomechanisms in psychiatric and neurological disorders on an individual case level.

Background: Abnormally high serum uric acid levels have been associated with several disease conditions including gout and kidney stone disease. More recently, it was shown that uric acid crystals stimulate dendritic cell maturation, activate the NALP3 inflammasome, and enhance antigen-specific immune responses. We hypothesize that uric acid can also stimulate T cells directly and in the absence of antigen presentation., Methods: Purified primary human T cells were incubated with and without uric acid at concentrations of 50, 100, 150, and 200 microg/mL. The expression of T-cell activation markers CD25 and CD70 was assessed by flow cytometry. In other experiments, Jurkat T cells were used and the expression of the costimulatory molecule CD70 was determined at the mRNA level., Results: Uric acid directly activates primary human T cells in the absence of antigen presentation. Furthermore, primary human T cells and Jurkat T cells treated with uric acid overexpress the costimulatory molecule CD70, which plays an important role in T cell-B cell interaction and antibody production., Conclusions: The finding that uric acid directly promotes T-cell activation in an antigen-independent system is novel and might play a mechanistic role in the inflammatory response observed in gouty arthritis and other immune-mediated diseases.

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.

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.

Engagement of glucocorticoid-induced TNFR-related protein (GITR) enables the costimulation of both CD25(-)CD4(+) effector (Teff) and CD25(+)CD4(+) regulatory (Treg) cells; however, the effects of GITR-costimulation on Treg function remain controversial. In this study, we examined the effects of GITR ligand (GITRL) binding on the respective functions of CD4(+) T cells. GITRL-P815 transfectants efficiently augmented anti-CD3-induced proliferation and cytokine production by Teff cells. Proliferation and IL-10 production in Treg were also enhanced by GITRL transfectants when exogenous IL-2 and stronger CD3 stimulation was provided. Concomitant GITRL-costimulation of Teff and Treg converted the anergic state of Treg into a proliferating state, maintaining and augmenting their function. Thus, GITRL-costimulation augments both effector and regulatory functions of CD4(+) T cells. Our results suggest that highly activated and increased ratios of Treg reverse the immune-enhancing effects of GITRL-costimulation in Teff, which may be problematic for therapeutic applications using strong GITR agonists.

Recent reports have established an important role of CD4+CD25+ T cells in the immune regulation of infectious diseases, autoimmune disorders and cancer. In the present work, we investigated whether these cells had a regulatory role during Trypanosoma cruzi infection, using the Colombian strain. Inactivation of CD4+CD25+ cells in vivo conferred mice slightly more resistant to infection with the Colombian strain of T. cruzi, as evidenced by lower parasitemia and mortality rates. The augmented resistance to infection with Colombian strain did correlate with increased activation of effector CD4 cells. It was antibody-independent, since no difference in levels of IgM, IgG, IgG1 and IgG2a(b) recognizing T. cruzi antigens was observed throughout the infection of CD25-inactivated and control mice. Regarding pathogenesis, inflammatory infiltrate and frequency of CD4 and CD8 T cells or macrophages in the cardiac tissue was similar in both groups. Together, our data indicate that CD4+CD25+ cells have a limited role on host resistance during early T. cruzi infection. Despite exhaustive investigation, we did not observe any role for these regulatory cells in the pathogenesis of experimental chronic Chagas' disease.

Background: Antibodies that neutralize factor (F) VIII activity, clinically referred to as 'inhibitors', complicate the treatment of hemophilia A patients; current tolerance and bypass strategies are extremely costly and sometimes ineffective. The development of inhibitors requires T-cell help., Objectives: We characterized T-cell responses of a subject with mild hemophilia A with missense genotype A2201P for one year following his initial inhibitor response, with the goals of defining the primary epitope(s) and its (their) MHC Class II restriction. We investigated the possible involvement of regulatory T cells in modulating immune responses., Patients/methods: The subject developed high-titer FVIII-neutralizing antibodies (250 BU mL(-1)) that declined over time to 8 BU ml(-1). His clotting activity was initially impaired (3%) but returned to baseline (8-10%) within four weeks. MHC Class II tetramers were used to analyze his CD4 T cells, which were stimulated with peptides spanning the C2 domain. Responses of total and CD25-depleted CD4 cells to sequences containing A2201 (native), P2201 (hemophilic), and other predicted T-cell epitopes were evaluated., Results and Conclusions: An HLA-DRA-DRB1*0101 restricted T-cell epitope containing the wild-type A2201 sequence was identified. Interestingly, peptides containing A2201 were recognized by CD4 T cells at all time points, whereas a P2201 peptide was recognized only near the initial peak response. The responsiveness of CD25-depleted CD4 cells to an A2201 peptide was enhanced 11 and 19 weeks following inhibitor detection, suggesting the possible involvement of CD4+CD25+ regulatory T cells in modulating immune responses. Patient-derived T-cell clones proliferated in response to C2 protein and to peptides containing A2201 but not P2201.

Background: Plasma plasminogen activator inhibitor-1 (PAI-1) level rises during sepsis and confers a worse prognosis. PAI-1 participation to sepsis has been poorly documented and was mainly associated with fibrin deposits. Beside fibrin deposits, increased tissue PAI-1 expression may contribute to the poor outcome of endotoxemia through other mechanisms., Objective and Methods: During lipopolysaccharide (LPS) challenge, the role of PAI-1 in the early phase of inflammation was examined in the lungs of transgenic mice that either overexpress or lack the PAI-1 gene (PAI-1Tg or PAI-1(-/-))., Results: Analysis of leukocytes revealed that neutrophil and macrophage infiltrations did not differ for PAI-1Tg and wild-type (WT) mice. Remarkably, CD25+ lymphocyte infiltration was totally blunted in PAI-1Tg lungs and inversely correlated with fibrin depositions. In parallel, mRNA levels of the regulatory T cell (Treg) markers FoxP3, CTLA-4, and GITR were significantly lower in PAI-1Tg than in WT lungs after LPS challenge. These data are supported by opposite results in PAI-1(-/-) lungs. The systemic compartments (spleen and peripheral blood) showed no decrease in CD25+, CD4+ CD25+ lymphocytes, and Treg markers in PAI-1Tg mice after LPS injection compared with WT mice. In addition, plasma and lung concentrations of interleukin-6 (IL-6) and macrophage inflammatory protein-1alpha (MIP-1alpha) were significantly higher in PAI-1Tg mice than WT mice., Conclusion: Our results suggest that chronic tissue PAI-1 overexpression influences the early phase of the inflammatory response during endotoxemia through the control of T lymphocyte traffic.

Cumulative evidence suggests the up-regulation of interleukin (IL)-10 and T-regulatory (Treg) cells is implicated in anti-inflammatory effect of heme oxygenase-1 (HO-1). Thus, we postulated that induction of HO-1 could augment IL-10 and transforming growth factor (TGF)-beta production and foxp3+CD4+CD25+ Treg cell function, thereby leading to attenuation of airway inflammation. In this study, CD4+CD25+ Treg cells isolated from mouse spleen were either transfected with a HO-1 expression vector (pcDNA3HO-1) or treated with a HO-1 inducer (hemin). Up-regulation of HO-1 enhanced foxp3 expression and IL-10 secretion in the Treg cells in vitro. Next, BALB/c, C57/B6.129, and IL-10-deficient B6.129P2-Il10tm1Cgn/J mice were challenged by ovalbumin to induce airway inflammation. Consistent with in vitro findings, hemin treatment resulted in induction of HO-1 and foxp3 and production of IL-10 and membrane-bound TGF-beta1 in vivo. This was further correlated with decrease of ovalbumin-specific immunoglobulin E level and eosinophil infiltration in bronchial alveolar lavage fluid from the asthmatic mice. Furthermore, hemin significantly enhanced the biological activity of CD4+CD25+ Treg cells. This protective effect was specifically blocked by Sn-protoporphyrin, a HO-1 enzymatic inhibitor. Finally, hemin failed to up-regulate the function of CD4+CD25+ Treg cells from IL-10-deficient mice. Our study indicates that HO-1 exerts its protective effect on asthma through a mechanism mediated by foxp3+CD4+CD25+ Treg cells, IL-10, and membrane-bound TGF-beta1.

Kidney transplant recipients are monitored for rejection by measurement of serum creatinine and graft biopsies. Biopsy samples are evaluated according to the Banff classification, which states that infiltration of tubules by mononuclear cells is an indicator of acute rejection. However, regulatory T cells play a crucial role in the overall immune response and are also present within transplanted tissue. We hypothesize that infiltration of mononuclear cells within kidney grafts is not always associated with rejection, especially if a high proportion of this infiltrate is regulatory T cells. Using a life-sustaining mouse kidney transplant model, we found that mononuclear cell tubular infiltration can occur in both rejecting and tolerant grafts. However, tolerant kidney grafts demonstrated a higher and sustained level of Foxp3+ regulatory cells. Importantly, a significant proportion of these cells were found within tubules. In cases in which graft function was normal, these cells were not harmful to the kidney and could be said to be mimicking, rather than causing, rejection.

CD4+CD25highFOXP3+ regulatory T (Treg) cells have recently been found at elevated levels in the peripheral blood of tuberculosis patients, compared to Mycobacterium tuberculosis latently infected (LTBI) healthy individuals and non-infected controls. Here, we show that CD4+CD25highFOXP3+ T lymphocytes can be expanded in vitro from peripheral blood mononuclear cells (PBMC) of LTBI individuals, but not of uninfected controls by incubating them with BCG in the presence of TGF-beta. These expanded cells from the PBMC of LTBI subjects expressed CTLA-4, GITR and OX-40, but were CD127low/- and have therefore the phenotype of Treg cells. In addition, they inhibited in a dose-dependant manner the proliferation of freshly isolated mononuclear cells in response to polyclonal stimulation, indicating that they are functional Treg lymphocytes. In contrast, incubation of the PBMC with BCG alone preferentially induced activated CD4+ T cells, expressing CD25 and/or CD69 and secreting IFN-gamma. These results show that CD4+CD25highFOXP3+ Treg cells can be expanded or induced in the peripheral blood of LTBI individuals in conditions known to predispose to progression towards active tuberculosis and may therefore play an important role in the pathogenesis of the disease.

Background: Peroxisome Proliferator-Activated Receptor gamma (PPARgamma) is a nuclear receptor that regulates the transcription of genes associated with lipid and glucose metabolism. Recently, it has been shown that PPARgamma modulates the activity of T cells, resulting in inhibition of T cell proliferation and IL-2 release. In this study we investigated whether the PPARgamma ligand rosiglitazone (R) enhances in vitro the immunosuppressive effects of cyclosporine A (CsA)., Methods: CD4(+) T cells isolated from peripheral blood mononuclear cells of healthy donors were activated either with mitogens or by one-way mixed lymphocyte reaction. The activated T cells were treated with (1) CsA at low and high concentration (50, 150 ng/ml); (2) R (20 muM); (3) R (20 muM) in combination with CsA at low concentration (50 ng/ml). We studied the effects of the various treatments on cell proliferation (incorporation of [(3)H] thymidine), the cell-cycle phases (FACS analysis), IL-2 release (ELISA), and IL-2 receptor (CD25) expression (FACS analysis)., Results: R used alone reduced T cell proliferation and CD25 expression. Low-dose CsA combined with R was significantly more powerful than either high-dose CsA alone or R alone in suppressing IL-2 release, arresting the T cell cycle, and blocking the growth of activated T cells., Conclusion: PPARgamma ligand R potentiates in vitro the inhibitory action of CsA on activated T helper cells. The combined use of PPARgamma ligands and low-dose CsA represents a rationale therapeutic approach aimed to prevent CsA nephrotoxicity while maintaining adequate immunosuppression.

Current therapies in transplantation require continuous immunosuppression and do not result in transplantation tolerance. It is increasingly appreciated that CD4(+)CD25(+) regulatory T-cell (T(REG)) activation is pivotal for the induction and maintenance of peripheral tolerance. To optimally exploit T(REG) in allograft tolerance, we investigated how to further harness their function. In vitro, CD4(+)CD25(+)T cells were expanded by allogeneic bone-marrow derived DC or polyclonal stimulation and were compared in suppressive capacity and phenotype. In vivo, naive allogeneic CD4(+)CD25(+)T cells were analyzed in wild type hosts for proliferative capacity and suppressive capacity upon priming by alloantigen. DC of donor origin were found to potently stimulate alloreactive T(REG)in vitro. This was accompanied by a substantial enhancement of the suppressive capacity of the T(REG) population as a whole, likely due to a proportional rise of alloreactive T(REG) as indicated by CFSE analysis. In vivo analysis of infused naturally occurring allogeneic T(REG) revealed a robust proliferative capacity for T(REG) upon stimulation. Moreover, allogeneic skin transplantation resulted in enhanced capacity of the T(REG) population to suppress the response towards donor antigens. Combining, activation of alloreactive T(REG) is an intrinsic part of the regular alloimmune response and this feature can be exploited for therapeutic purposes. We propose that selectively favoring the effects of alloreactive T(REG) is a pivotal element in inducing graft acceptance.

Aims: Belatacept is a new recombinant molecule (CTLA4-Ig) that interferes with the second activation signal of T lymphocytes. CTLA4-Ig induced T cell allograft tolerance in rodents but not in primates. We examined the changes in peripheral lymphocyte subsets, including regulatory T cells, in renal transplant patients treated with Belatacept., Methods: A cross-sectional immunological study was carried out 6 months after transplantation in 28 patients enrolled in the Belatacept phase II study. Eighteen patients received Belatacept, mycophenolate mofetil and steroids (Belatacept group), while the control group of 10 patients received cyclosporine, mycophenolate mofetil and steroids (CsA group). Lymphocyte subsets were examined by flow cytometry. Foxp3 mRNA expression was measured by quantitative PCR., Results: The number of T lymphocytes and the percentage of CD3+ T cells were similar in both groups. However, the percentage of CD3+ CD4+ T cells was lower in the Belatacept group than in the control CsA group (B=42.5%+/-13.7 vs CsA=52.9%+/-9, p<0.005), and the percentage of CD3+ CD8+ cells was higher in the Belatacept group than in the control (B=32.9%+/-6.7 vs CsA=19.5%+/-8.2, p<0.0002). The percentage of CD19+ cells was similar in both groups. Among CD56+cells, only the percentage of CD16+ cells was significantly higher in the Belatacept group than in the control (B=82%+/-12 vs CsA=59.7%+/-25, p=0.01). Among CD4 and CD8 T cells the percentage of activated lymphocytes expressing CTLA4, HLA-DR or CD40L was similar in both groups. The percentage of CD4+CD25+ T cells was higher in the CsA group. The percentage of regulatory CD4+CD25+ cells with bright CD25 staining was similar in both groups (B=3.6+/-2.3% vs CsA=4.7+/-1.9%, ns) as was the expression of FoxP3., Conclusion: Our results indicated that Belatacept did not induce regulatory T cell expansion in vivo. We suggest that Belatacept treatment should be maintained after transplantation to allow graft acceptance.

We investigate JAK-STAT5 activation and its relationship to full-length Stat5B (FL-Stat5) and constitutive phosphorylated carboxy-truncated Stat5B (ct-pStat5) in four different strains of mouse. Our electrophoresis mobility shift assays data indicate constitutive phosphorylation of full-length-Stat5 (p<0.001) and DNA binding in NOD but not in B6 mice. Our data suggest that the relative ratio of FL-Stat5: ct-Stat5 in NOD is 5- to 8-fold lower (p<0.0001) when compared with normal B6 mice. Additionally, EMSAs data from B6.NOD/c11 suggest contribution of Idd4 susceptibility locus on chromosome 11 in constitutive phosphorylation of Stat5 in NOD mice. The presence of ct-pStat5 in regulatory T cells of NOD mice suggests this form of Stat5 is associated with impaired function of Tregs in NOD mouse. In agreement with our previous report the JAK-Stat5B defective pathway in NOD mice along with other defective factors is associated with the pathogenesis of autoimmune type 1 diabetes in NOD mice.

CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune mediated reactions. The analysis of Treg function in patients with autoimmune, allergic or oncogenic diseases has emerged over the past years. In the present study we describe a CFSE based protocol to measure Treg mediated suppression of CD4(+) T cells. Measuring Treg suppressive capacity towards proliferation of anti-CD3 Ab stimulated CD4(+)CD25(-) T cells in coculture experiments by means of a CFSE based and a classical [(3)H]thymidine incorporation assay gave similar results, provided that CD4(+)CD25(+) T cells were anergic. However, when CD4(+)CD25(+) T cells proliferated upon mitogenic stimulation, data obtained by the CFSE assay allowed the detection of a significant Treg suppression whereas this was clearly underestimated using the [(3)H]thymidine assay. In addition, an indirect CFSE based method was developed to analyze antigen specific responses of total CD4(+) T cells and Treg depleted CD4(+) T cells (i.e. CD4(+)CD25(-) T cells). Our results indicate that, in healthy individuals, CD4(+) T cell responses against the multiple sclerosis (MS) auto-antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), were increased in Treg depleted CD4(+) T cells as compared to total CD4(+) T cells. Our initial data suggest that Tregs in MS patients show an impaired suppression of myelin reactive T cells when compared to healthy controls. Moreover, this experimental setup permits the measurement of cytokine production of the antigen proliferated CFSE(low) T cells by additional flow cytometric analyses. In conclusion, the described CFSE based Treg suppression assay is a valuable tool to study suppressor T cells in (auto)immune disorders.

The characterization of the T-cell receptor (TCR) repertoire of CD4+ regulatory T cells (T(R)) has been limited due to the RNA degradation that results following permeabilization and fixation as routinely used for intracellular staining of Foxp3. In the present study the clonal composition of human umbilical cord blood (UCB) and adult peripheral blood mononuclear cell (PBMC) CD4+ T(R) and non-T(R) was characterized by a DNA-based multiplex PCR which allowed for the consistent clonotypic characterization of cells that have undergone fixation and permeabilization. To validate this method, CD8+ T cells from two HLA A()0201 individuals were sorted and compared clonotypically based upon their ability either to secrete interferon-gamma in response to a CMV pp65 epitope or to bind to the corresponding pMHC I tetramer. Clonotypes shared between the CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- subsets were observed in all 3 UCB and in one adult PBMCs, suggesting that naïve and memory CD4+ T(R) can share the same clonotypes as CD4+ non-T(R) in humans.

Long-term survival is achieved in rat recipients by pre-graft donor-specific blood transfusion. We characterized the immune compartments in long-term survivors and analyzed them for capacity to transfer tolerance and protect against chronic rejection. Splenocytes and spleen T cells from treated recipients transferred long-term graft survival to 100% of secondary recipients. In contrast, blood transferred graft survival to only 50% of recipients whereas blood T cells had no effect. An unaltered TCR repertoire, an increase in suppressive CD4+CD25+ T cells, a decrease in antidonor T-cell proliferative response and normal perforin-granzyme levels were the hallmarks of the spleen T cells. Blood T cells were characterized by a strongly altered CD8+ repertoire, normal CD4+CD25+ T cell number with unchanged antidonor T-cell proliferative response, an activated T-cell phenotype and an increase in perforin-granzyme levels. However, following the transfer of blood or spleen cells into secondary recipients, all grafts displayed chronic rejection. These findings provide evidence that distinct compartments play critical roles in DST recipients. Regulatory cells do not accumulate in blood, which appears to be a reservoir for cytotoxic T cells. Spleen T cells, which display a regulatory-like profile and transfer graft survival, are not able to prevent chronic rejection.

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.

Immunization can sometimes lead to antigen-specific immune suppression. In this study, we investigated this phenomenon by testing several combinations of DNA and protein vaccines directed against various viruses. We find that co-inoculation of mice with combined DNA and protein vaccines induces immune suppression if the two vaccines are "matched" by targeting the same antigen. Conversely, vaccine combinations never lead to immune suppression if they are derived from different viruses and, thus, mismatched antigenically. We have further identified CD4+CD25- T cells as the type of regulatory T cells induced by and are responsible for suppressing T cell activities in an antigen-specific manner in immunized animals. These regulatory T cells are phenotypically unique in their expression of Foxp3, IL-10, and IFN-gamma. Our study thus shows for the first time that co-administration of antigen-matched DNA and protein vaccines can generate this type of adaptive regulatory T cells.

To gain transplantation tolerance between donor organs and hosts is the ultimate goal of all sorts of organ transplantations. Induction of regulatory T cells has been demonstrated to lead to transplantation tolerance. This paper will review subsets of regulatory T cells, the role and mechanisms of CD4(+)CD25(+) regulatory T cells (Tregs) in graft rejection and tolerance, pathway used by Tregs to recognized alloantigens, pathways of Tregs homing into the graft and effects of immunosuppression on Tregs. It was well known that Tregs play a pivotal role in transplantation tolerance. The mechanisms by which Tregs exert their regulatory effect in the induction and maintenance of transplantation tolerance, anthropogenically, consist of physical cell-to-cell contact with potential target cells, autocrine and paracrine properties. ICAM-1, TGF-beta, CTLA-4, GITR and OX40 (CD134), etc. are involved in the regulatory function of Tregs through cell-to-cell contact mechanism. IL-10 and TGF-beta are two important soluble mediators involved in the autocrine mechanism by which Tregs exert their regulatory function. Paracrine properties refer to re-educate potentially destructive alloresponsive T cells to gain regulatory function. All that discussed above could illustrate, at least partially, how naturally occurring Tregs exert their regulatory function in vivo as they constitute only 5-10% of peripheral CD4(+) T cells.

The induction of transplantation tolerance involves a T-cell-mediated process of immune regulation. In clinical transplantation, the use of immunosuppressive drugs that promote or facilitate this process would be highly desirable. Here, we investigated the tolerance-promoting potential of the immunosuppressive drug FK778, currently under development for clinical therapy. Using a human allogeneic in vitro model we showed that, upon T-cell receptor (TCR) triggering, FK778 induced a regulatory phenotype in CD4+ CD25- T cells. Purified CD4+ CD25- T cells primed in the presence of FK778 showed hyporesponsiveness upon restimulation with alloantigen in the absence of the drug. This anergic state was reversible by exogenous interleukin-2 (IL-2) and was induced independent of naturally occurring CD4+ CD25+ regulatory T cells. Pyrimidine restriction was a crucial requirement for the de novo induction of regulatory activity by FK778. The FK778-induced anergic cells showed suppressor activity in a cell-cell contact-dependent manner; were CD25(high), CD45RO+, CD27-, and CD62L-; and expressed cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), and FoxP3. The cells revealed delayed p27(kip1) degradation and enhanced phosphorylation of STAT3. In conclusion, the new drug FK778 shows tolerizing potential through the induction of a regulatory T-cell subset in CD4+ CD25- T cells.

A randomised, double-blind, placebo-controlled study examined the safety, tolerance, immunological effect and anti-human immunodeficiency virus (HIV) activity of sub-cutaneously administered HE2000 (16alpha-bromoepiandrosterone) as monotherapy in treatment-naïve patients with HIV-1. Twenty-four patients received five sequential daily doses of 50 or 100 mg of HE2000 or placebo every 6 weeks for up to three courses, and were followed thereafter for 3 months. HE2000 was safe, with transient injection site reactions being the main side-effect. Peripheral blood samples, collected serially, were analysed for changes in immune cell phenotypes. Significant increases were observed in the numbers of circulating dendritic cells, early activated (CD69+ CD25-) CD8 T-cells and T-NK cells after administration of 50-mg doses of HE2000 (p < 0.05). Gene expression in peripheral blood mononuclear cells was analysed by real-time RT-PCR. Before treatment, HIV-1-infected patients had significantly elevated transcripts for a number of inflammatory mediators (p < 0.012). After 50 mg or 100 mg HE2000, but not after placebo, there were significant sustained decreases in IL-1beta, TNF-alpha, IL-6 and Cox-2 transcripts (p < 0.05). There were no significant differences in CD4 cell numbers, although patients receiving 50-mg doses demonstrated a significant decrease in viral load (- 0.6 log; p < 0.01). Anti-HIV-1 T-cell responses were analysed serially using GAG-peptides to stimulate cytoplasmic IFN-gamma responses. After three courses, the 50-mg dose group demonstrated a significant increase in CD8 T-cell response against two distinct GAG peptide pools (p < 0.03). These findings suggest that immune-based therapies may be able to impact viral load by decreasing inflammation and/or stimulating CD8 T-cells.