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Vγ9Vδ2 T lymphocytes have been shown to respond to a variety of non-peptide antigens including alkylamines and phosphoantigens. Recently, aminobisphosphonates have also been shown to stimulate this subset of γδ+ T cells. In this study we analyzed the proliferative responses of freshly isolated γδ T lymphocytes obtained from human cord blood when challenged with pyrophosphomonoesters or aminobisphosphonates. Nitrogen-containing aminobisphopsphonates, in contrast to phoshoantigens, readily stimulated expansion of Vδ2Vγ9 cells in human cord blood. Expanded cells displayed an activated mature phenotype, and were capable of producing TNFalpha and IFNgamma but not perforin following secondary stimulation, consistent with the development of a regulatory, as opposed to cytotoxic, phenotype. This approach may provide a useful strategy for a new approach to the treatment of neonatal pathologies.

Renal cell carcinoma (RCC) can inhibit protective immunity by induction of immunosuppressive cells that produce inhibitory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β. If this immunosuppression influences response to kinase inhibitors such as sorafenib is not known. Therefore, we asked for the prognostic influence of cells with immunosuppressive properties in peripheral blood (pB) in a cohort of metastatic clear cell renal cell carcinoma (mRCC) patients uniformly receiving sorafenib treatment. IL-10 and TGF-β mRNA levels, regulatory T-cell (Treg) counts, and frequencies of IL-10/TGF-β producing mononuclear cell subsets were determined in pB from 46 patients with mRCC before receiving sorafenib treatment. Relationship between established clinical and laboratory prognostic factors and outcome were examined by univariate and multivariate Cox regression analysis. IL-10 and TGF-β1 mRNA levels, and frequencies of CD4(+)CD25high/CD3(+) and CD4(+)CD25highFoxP3(+)/CD3(+)Treg cells were significantly higher in mRCC patients compared with healthy individuals. Monocytes were suggested as main producers of IL-10 and TGF-β. In a multivariate analysis low ECOG score and-surprisingly-high TGF-β1 mRNA levels were independently associated with favorable progression-free survival (P=0.005 and P=0.003, respectively) and overall survival (P=0.001 and P=0.039, respectively). In conclusion, mRCC is associated with an immunosuppressive phenotype in peripheral blood. The positive prognostic influence of high TGF-β1 mRNA expression levels may reflect immune promoting functions of TGF-β in combined activity with inflammatory cytokines.

A cross-regulation between two regulatory T cell (T(reg) ) subsets [CD4(+) CD25(+) and invariant natural killer (NK) T - iNK T] has been described to be important for allograft tolerance induction. However, few studies have evaluated these cellular subsets in stable recipients as correlates of favourable clinical outcome after heart transplantation. T(reg) and iNK T cell levels were assayed by flow cytometry in peripheral blood samples from 44 heart transplant recipients at a 2-year interval in 38 patients, and related to clinical outcome. Multi-parameter flow cytometry used CD4/CD25/CD127 labelling to best identify T(reg) , and a standard CD3/CD4/CD8/Vα24/Vβ11 labelling strategy to appreciate the proportions of iNK T cells. Both subtypes of potentially tolerogenic cells were found to be decreased in stable heart transplant recipients, with similar or further decreased levels after 2 years. Interestingly, the patient who presented with several rejection-suggesting incidents over this period displayed a greater than twofold increase of both cell subsets. These results suggest that CD4(+) CD25(+) CD127(low/neg) T(reg) and iNK T cells could be involved in the local control of organ rejection, by modulating immune responses in situ, in clinically stable patients. The measurement of these cell subsets in peripheral blood could be useful for non-invasive monitoring of heart transplant recipients, especially in the growing context of tolerance-induction trials., (© 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.)

Background: Post kala-azar dermal leishmaniasis (PKDL), a sequel to visceral leishamaniasis (VL) in 5-15% cases, constitutes a parasite reservoir important in disease transmission. The precise immunological cause of PKDL outcome remains obscure. However, overlapping counter regulatory responses with elevated IFN-γ and IL-10 are reported., Methodology/principal Findings: Present study deals with ex-vivo mRNA and protein analysis of natural regulatory T cells (nTreg) markers (Foxp3, CD25 and CTLA-4) and IL-10 levels in lesion tissues of PKDL patients at pre and post treatment stages. In addition, correlation of nTreg markers and IL-10 with parasite load in tissue lesions was investigated. mRNA levels of nTreg markers and IL-10 were found significantly elevated in pre-treatment PKDL cases compared to controls (Foxp3, P = 0.0009; CD25 & CTLA-4, P<0.0001; IL-10, P<0.0001), and were restored after treatment. Analysis of nTreg cell markers and IL-10 in different clinical manifestations of disease revealed elevated levels in nodular lesions compared to macules/papules. Further, Foxp3, CD25 and IL-10 mRNA levels directly correlated with parasite load in lesions tissues., Conclusion/significance: Data demonstrated accumulation of nTreg cells in infected tissue and a correlation of both IL-10 and nTreg levels with parasite burden suggesting their role in disease severity in PKDL.

Mastocytosis is a term used to designate a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow (BM), liver, spleen, and lymph nodes, among others. Recent advances in our understanding of mast cell biology and disease resulted in the identification of important differences in the expression of mast cell surface antigens between normal and neoplastic mast cells. Most notably, detection of aberrant expression of CD25 and CD2 on the surface of neoplastic mast cells but not on their normal counterparts lead to the inclusion of this immunophenotypic abnormality in the World Health Organization diagnostic criteria for systemic mastocytosis. Aberrant mast cell surface marker expression can be detected in the bone marrow aspirate by flow cytometry, even in patients lacking histopathologically detectable aggregates of mast cells in bone marrow biopsy sections. These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this chapter, we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, for their phenotypic characterization, and the criteria currently used for correct interpretation of the immunophenotypic results obtained., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Blood transfusion (BT) may elicit both harmful and beneficial immune responses against a subsequent organ graft. Immune parameters of a single, non leukocyte-depleted BT were investigated in two groups: non-human leukocyte antigen (HLA)-sensitized recipients with a one-HLA-DR matched donor (protocolled BT [PBT]) and females with previous exposure to HLA alloantigens through pregnancy (donor-specific transfusion [DST])., Methods: Thirty-five percent of DST recipients and 9.5% of PBT recipients developed HLA antibodies after BT.Phenotypic and functional analyses were performed in pre-BT, 2 weeks post-BT, and more than 10 weeks post-BT samples (PBT: n=10; DST: n=14)., Result: The number of donor-reactive interferon-γ-producing memory T cells increased 2 weeks post-BT, but only in the DST group, increased frequencies persisted beyond 10 weeks (P0.004). In the DST recipients, the proportion of natural killer cells (CD3(-)CD56(+)) significantly increased after BT (P=0.01), whereas in PBT recipients, the proportion of regulatoryT cells (CD4(+)CD25(+)Foxp3(+)CD127 low) significantly increased at 2 weeks post-BT (P=0.039). Microarray analysis confirmed increased activity of genes involved in function of natural killer cells,Tcells, and Bcells in DSTrecipients and increased expression of immune regulatory genes (galectin-1, Foxo3a, and follistatin-like 3) in PBT recipients. Galectin-1 expression by quantitative polymerase chain reaction was significantly enhanced in peripheral blood cells after PBT (P0.05)., Conclusion: Decreased immune effector mechanisms combined with an increased immune regulatory cell signature after HLA-DR-matched BT in nonsensitized patients is in line with clinical observations of improved outcome of a subsequent graft. Previous sensitization, however, may lead to HLA antibody formation and prolonged donor-specific memory T-cell reactivity after BT.

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.

Objective: To investigate the role of CD4(+)CD25(+)forkhead box P 3 (Foxp3)(+) T-regulatory cells (Tregs) in protecting the activation and function of human umbilical vein endothelial cells (HUVECs) induced by proinflammatory stimulus and the mechanisms of it., Methods and Results: ECs play a major role in atherogenic initiation, changing their quiescence into activated phenotypes to support every phase of the inflammatory process. HUVECs were incubated alone, with Tregs or CD4(+)CD25(-) T cells in the presence of anti-CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without oxidized low-density lipoprotein/lipopolysaccharide for an additional 24 hours. Tregs are able to induce alternative expression of immune phenotypic markers of activated HUVECs by down modulating CD86 and to inhibit the adhesion molecule, such as vascular cell adhesion molecule-1 (VCAM-1) and proinflammatory cytokine (eg, monocyte chemoattractant protein-1 and interleukin 6), response of HUVECs to oxidized low-density lipoprotein/lipopolysaccharide. Moreover, Tregs downregulate proinflammatory factor nuclear factor-κB activation and induce resistance to suppression of anti-inflammatory factor Kruppellike factor 2 in HUVECs induced by a proinflammatory stimulus. Mechanism studies reveal that Treg-mediated suppression of HUVEC proinflammatory cytokines and adhesion molecule expression impaired by oxidized low-density lipoprotein/lipopolysaccharide require cell contact by cytotoxic T-lymphocyte antigen-4 and CD86 and by soluble factors (mainly interleukin 10 and transforming growth factor [TGF]-β)., Conclusions: Tregs may exert their protective effects against atherogenesis in part through inducing an immune-inhibitory phenotype of ECs involving cytotoxic T-lymphocyte antigen-4-dependent cell-to-cell contact and also requiring soluble factors (mainly interleukin 10 and TGF-β).

Importance of the Field: Hepatic cirrhosis is a frequent consequence of chronic hepatitis infection (HBV and HCV) or alcohol abuse and the most common cause of hepatocellular carcinoma (HCC). Currently, liver transplantation remains the only effective therapeutic approach for cirrhosis-related HCC patients. The evolution of the pathology strongly depends on immunological mechanisms., Areas Covered in This Review: Despite the presence of specific T cells, viral chronic infection and continuous tumor growth suggest a failure of immune control. It appears that direct suppression of antiviral or antitumor effector cells by regulatory T cells plays a pivotal role in the impairment of immune response. Several types of regulatory T cells have been described, natural regulatory T cells (nTreg) and induced-type 1 regulatory T cells (Tr1) being the best characterized., What the Reader Will Gain: Currently, there is no evidence for a direct implication of regulatory T cells in the evolution of hepatitis, especially concerning chronic infection, cirrhosis late stage and HCC progress. However, recent studies show that regulatory T cells are implicated in the modulation of HBV- and HCV-associated immune response, thus, promoting HCC progress., Take Home Message: Therefore, nTreg and Tr1 cells seem to play an important role in the control of immune response leading to chronic hepatitis infection and progression of the pathology to cirrhosis and HCC.

Introduction: Recently it has been reported that Astragalus membranaceus injection (AMI) inhibits immune responses, but whether it affects alloimmunity is not clear. It has been shown that the CD4(+) CD25(+) regulatory T cells (Treg) down-regulate immune responses. The aim of our study was to investigate the effects of AMI on allograft survival and its relation to Treg., Materials and Methods: Allografted mice were administered AMI for 14 consecutive days with observations of graft survival. The specific recall response, the ratio of Treg, the expression of Foxp3 mRNA, and interleukin (IL)-10 secretion were measured by mixed lymphocyte reactions (MLR), FCM, reverse transcriptase-polymerase chain reaction, and radioimmunoassay, respectively., Results: AMI significantly prolonged allograft survival by up-regulating the Treg ratio and promoting Foxp3 expression (P < .05). The ratio of Tregs, the expression of Foxp3 mRNA, and the IL-10 level in the AMI administration group increased from day 7, to reach a maximum at day 14, recovering to the initial level on day 21. No obvious difference was detected between the AMI and a cyclosporine group., Conclusion: AMI administered in vivo prolonged allograft survival associated with promotion of Treg activities., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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.)

Mother-to-child-transmission (MTCT) of human immunodeficiency virus (HIV) remains a significant cause of new HIV infections in many countries. To examine whether fetal immune activation as a consequence of prenatal exposure to parasitic antigens increases the risk of MTCT, cord blood mononuclear cells (CBMCs) from Kenyan and North American newborns were examined for relative susceptibility to HIV infection in vitro. Kenyan CBMCs were 3-fold more likely to be infected with HIV than were North American CBMCs (P=.03). Kenyan CBMCs with recall responses to malaria antigens demonstrated enhanced susceptibility to HIV when compared with Kenyan CBMCs lacking recall responses to malaria (P=.03). CD4(+) T cells from malaria-sensitized newborns expressed higher levels of CD25 and human leukocyte antigen DR ex vivo, which is consistent with increased immune activation. CD4(+) T cells were the primary reservoir of infection at day 4 after virus exposure. Thus, prenatal exposure and in utero priming to malaria may increase the risk of MTCT.

The increase of CD4+CD25+ regulatory T cells in patients with ovarian carcinoma has been verified. Here we investigated the effects of supernatant derived from ovarian carcinoma cell SKOV3 on peripheral regulatory T cells. Supernatant from SKOV3 was collected and fractionated into three different molecular weight fractions (MWFs). The proliferation of the CD4+CD25+ regulatory T cells cultured in complete RPMI 1640 medium with the different stimulators was detected. The phenotype (GITR and CTLA-4) of natural and expanded CD4+CD25+ T cells was detected by flow cytometry. Foxp3 mRNA expression of low MWF-expanded CD4+CD25+ T cells was detected by RT-PCR. Those expanded CD4+CD25+ regulatory T cells showed enhanced capacity to suppress CD4+CD25- T proliferation and increased expression of GITR and CTLA-4. In brief, low molecular weight fraction of supernatant secreted by SKOV3 could expand peripheral CD4+CD25+ regulatory T cells and enhance their suppressive function.

Regulatory T (Treg) cells are activated and suppress immune responses during infection, and are characterized as CD4(+)CD25(hi)FOXP3(+). Ex vivo studies demonstrate that Treg cells potentially suppress anti-HIV-1 T cell responses. Lentivirus-induced CD4(+)CD25(hi) Treg cells were first described in feline immunodeficiency virus (FIV)-infected cats. In the present study we demonstrate that anti-feline CD25 monoclonal antibody (mAb) therapy depletes Treg cells in FIV-infected cats for 4 weeks and does not exacerbate viral replication or proinflammatory cytokine production. Significant FIV-specific immune responses are revealed in Treg cell-depleted cats. These anti-FIV effector cells exist prior to Treg cell depletion and are not expanded while Treg cells are depleted. Importantly, cats receiving the Treg cell-depleting mAb are able to produce a robust humoral response to new antigen. We propose that short-term in vivo Treg cell depletion during chronic HIV-1 infection could provide a window of opportunity for therapeutic vaccination in individuals with controlled viral replication.

Tubulointerstitial infiltration of immunocompetent cells is often associated with a more rapid progression in IgA nephropathy (IgAN). Using an in vitro Transwell coculture system, we examined the chemotactic response of peripheral blood mononuclear cells to proximal tubular epithelial cells (PTEC) following activation by conditioned medium prepared from mesangial cells cultured with macromolecular IgA1 from 60 patients with multiplex familial IgAN (MpIgAN) and 91 of their asymptomatic relatives; 43 patients with sporadic IgAN (SpIgAN) and 90 of their asymptomatic relatives; and 43 healthy subjects. Compared with SpIgAN patients, PTEC activated by conditioned medium from patients with MpIgAN had elevated gene expression of a spectrum of C-C, C-X-C chemokines and proinflammatory cytokines, with prominent expressions of interleukin-6, interleukin-8, and tumor necrosis factor-alpha. In response to conditioned medium from patients with familial IgAN, there was a significant increase in chemotaxis of CD45+ cells, CD3+, CD4+, CD8+, CD20+ lymphocytes, and monocytes with CD25 expression. Our findings suggest that compared with SpIgAN patients, macromolecular IgA1 taken from MpIgAN patients is more pathogenic to cultured PTEC through a glomerulotubular interaction. A long-term follow-up is needed to better define the prognostic course for familial IgAN and to clarify the risk of developing IgAN in initially asymptomatic relatives from a multiplex IgAN family.

LAKs are used in cancer therapy. A common argument against LAK therapy is the probability of the activation of Tregs by IL-2 alongside with NK cells. In order to evaluate negative impact of Tregs, contents of Foxp3(+)CD4(+)CD25(+) Tregs and their influence on LAKs' cytotoxic activity in the samples of healthy volunteers' PBMCs cultured with or without IL-2 were determined. A marked increase in the proportion of the CD4(+)CD25(+) T cells in the most of the PBMCs cultured with IL-2 was evident. Surprisingly, the contents of Foxp3(+)CD4(+)CD25(+) Tregs showed variable response to IL-2 in blood samples of different people. Their proportions increased, remained on the same level or even decreased compared to PBMCs cultured without exogenous IL-2. Different methods of Treg isolation were compared regarding the purities of the resulting T-cell populations and their enrichment in the Foxp3-expressing Tregs, as well as their viability. Neither of the kits applied provided any enrichment in Foxp3-level in the purified cells compared to the CD4(+)CD25(+) T cells gated on applying flow-cytometric cell analysis in the non-separated LAKs. Moreover, the addition of the purified supposedly Tregs to LAKs never inhibited their cytotoxic reactions, even then they were added in great non-physiological excess. In some of the blood samples, there have been detected another non-identified type of the Foxp3-expressing cells, which lacked CD25, as well as CD4. They may be a new type of regulatory cells, which has not been described before., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

Various immune functions of different types of immune cells are strongly impaired in patients with head and neck squamous cell carcinoma (HNSCC). Regulatory T-lymphocyte cells (Tregs) have been suggested to be involved in the immunomodulation of immune responses and contribute to HNSCC progression and immune escape. 'Naturally' occurring CD4+ CD25+ Tregs represent a small fraction within the different subsets of regulatory T cells, which are known to inhibit numerous immune functions of different types of immune cells. In this study, the cellular ratio of CD4+ CD25(high) Tregs to the entire population of CD4+ T-lymphocytes was analyzed with respect to different stages of tumor progression and disease. Our data indicate a significantly high increased abundance of CD4+ CD25(high) CD127(low) Tregs in the peripheral blood of patients with HNSCC, which in addition show modulated expression levels of various functional proteins. Surprisingly, increased Treg levels were found even in patients with no active disease several years after tumor resection, with no significant correlation to the individual tumor stage. Additionally, increased levels of chemokine CCL22, which mediates migration of Tregs to the tumor, and upregulation of the corresponding receptor protein CCR4 were observed in HNSCC. Our data strongly suggest that HNSCC leads to a permanent shift of Treg levels with hardly recognizable recovery rates.

Objectives: Regulatory T cells (T(REG)) represent a T cell subset able to modulate immune response by suppressing autoreactive T-lymphocytes. The evidence of a reduced number and an impaired function of this cell population in autoimmune/inflammatory chronic diseases led to the hypothesis of its involvement in the pathogenesis of these disorders. Glucocorticoid-induced TNFR-related protein (GITR) is a well known marker of murine T(REG) cells, but little is known in humans. The aim of this study was to investigate the characteristics of T(REG) cells in systemic lupus erythematosus (SLE) and the potential role of GITR as marker of human T(REG)., Methods: Nineteen SLE patients and 15 sex- and age-matched normal controls (NC) were enrolled. CD4(+) T cells were magnetic sorted from peripheral blood by negative selection. Cell phenotype was analyzed through flow-cytometry using primary and secondary antibodies and real time polymerase-chain reaction (PCR) using TaqMan probes., Results: The CD25(high)GITR(high) subset was significantly decreased in SLE patients with respect to NC (0.37±0.21% vs 0.72±0.19%; p<0.05). On the opposite, the CD25⁻GITR(high) cell population was expanded in the peripheral blood of SLE patients (3.5±2.25 vs 0.70±0.32%, p<0.01). Interestingly, FoxP3 at mRNA level was expressed in both CD25⁻GITR(high) and CD25(high)GITR(high) cells, suggesting that both cell subsets have regulatory activity., Conclusions: CD4(+)CD25⁻GITR(high) cells are increased in SLE as compared to NC. The expression of high level of GITR, but not CD25, on FoxP3+ cells appears to point to a regulatory phenotype of this peculiar T cell subset.

Lymphoid nodules are a normal component of the mucosa of the rectum, but little is known about their function and whether they contribute to the host immune response in malignancy. In rectal cancer specimens from patients with local (n=18), regional (n=12) and distant (n=10) disease, we quantified T cell (CD3, CD25) and dendritic cell (CD1a, CD83) levels at the tumour margin as well as within tumour-associated lymphoid nodules. In normal tissue CD3+, but not CD25+, T cells are concentrated at high levels within lymphoid nodules, with significantly fewer cells found in surrounding normal mucosa (P=0.001). Mature (CD83), but not immature (CD1a), dendritic cells in normal tissue are also found clustered almost exclusively within lymphoid nodules (P=<0.0001). In rectal tumours, both CD3+ T cells (P=0.004) and CD83+ dendritic cells (P=0.0001) are also localized preferentially within tumour-associated lymphoid nodules. However, when comparing tumour specimens to normal rectal tissue, the average density of CD3+ T cells (P=0.0005) and CD83+ dendritic cells (P=0.0006) in tumour-associated lymphoid nodules was significantly less than that seen in lymphoid nodules in normal mucosa. Interestingly, regardless of where quantified, T cell and dendritic cell levels did not depend upon the stage of disease. Increased CD3+ T cell infiltration of tumour-associated lymphoid nodules predicted improved survival, independent of stage (P=0.05). Other T cell (CD25) markers and different levels of CD1a+ or CD83+ dendritic cells did not predict survival. Tumour-associated lymphoid nodules, enriched in dendritic cells and T cells, may be an important site for antigen presentation and increased T cell infiltration may be a marker for improved survival.

Antisense RNA technology was employed to specifically inhibit the expression of the protein kinase Cbeta (PKCbeta) isoform in Jurkat cells, to explore its influence on the expression of surface antigens (CD69) and the cytokines interleukin-8 (IL-8), tumour necrosis factor (TNF)-alpha and beta, and to characterise its controversial involvement in the expression of IL-2 and its receptor (IL-2R). Transfection of cells with an antisense PKCbeta construct (as-PKCbeta-pREP3) significantly increased IL-2R/CD25 expression in phorbol 12-myristate 13-acetate (PMA)-stimulated as-PKCbeta-pREP3 transfectants, in contrast to Jurkat cells transfected with a control as-PKCalpha-pREP3 plasmid. IL-2 production, in contrast, was strongly inhibited in both transfectant populations stimulated by PMA plus the calcium ionophore ionomycin. Three clones (asb1/asb2/asb3), selected from as-PKCbeta-pREP3 transfectants, showed decreased PKCbeta protein levels (40 percent, 50 percent and 60 percent, respectively, as determined by western blotting) and mRNA levels. The specific inhibition was confirmed in immunoblots for other PKC (alpha, delta, epsilon, gamma, theta, and lambda lambda/tau) isoforms and in immunoprecipitates from representative (c2/asb2) clones. Stimulation of PKCbeta-depleted clones significantly increased CD25 expression but decreased IL-2 production (similarly to as-PKCbeta-pREP3 transfectants) and IL-2 message levels. CD69 expression and IL-8 secretion were significantly decreased, but TNFbeta message levels were highly increased in asb2/asb3 clones, without affecting TNFalpha secretion. Analysis of the mitogen-activated protein kinase (MAP Kinase) signalling pathway showed unaltered extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation but increased activation of c-Jun N-terminal kinase (JNK1) and its substrate, the transcription factor ATF-2 (activated transcription factor-2), which are involved in IL-2 gene expression. Our results revealed new PKCbeta functions, affecting CD69 expression and IL-8 production, and support the requirement for PKCbeta in IL-2 secretion/transcription and IL-2R regulation.

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.

Mast cell neoplasms are characterized by abnormal growth and focal accumulation of mast cells (MC) in one or more organs. Although several cytokines, including stem cell factor (SCF) and interleukin-9 (IL-9) have been implicated in growth of normal MC, little is known about pro-oncogenic molecules and conditions triggering differentiation and growth of MC far enough to lead to the histopathological picture of overt mastocytosis. The anaplastic lymphoma kinase (ALK) has recently been implicated in growth of neoplastic cells in malignant lymphomas. Here, we describe that transplantation of NPM-ALK-transplanted mouse bone marrow progenitors into lethally irradiated IL-9 transgenic mice not only results in lymphoma-formation, but also in the development of a neoplastic disease exhibiting histopathological features of systemic mastocytosis, including multifocal dense MC-infiltrates, occasionally with devastating growth in visceral organs. Transplantation of NPM-ALK-transduced progenitors into normal mice or maintenance of IL-9-transgenic mice without NPM-ALK each resulted in MC hyperplasia, but not in mastocytosis. Neoplastic MC in mice not only displayed IL-9, but also the IL-9 receptor, and the same was found to hold true for human neoplastic MC. Together, our data show that neoplastic MC express IL-9 receptors, that IL-9 and NPM-ALK upregulate MC-production in vivo, and that both'hits' act in concert to induce a mastocytosis-like disease in mice. These data may have pathogenetic and clinical implications and fit well with the observation that neoplastic MC in advanced SM strongly express NPM and multiple "lymphoid" antigens including CD25 and CD30.

Trichinella spiralis is a helminth that provokes Th2 and anti-inflammatory type responses in an infected host. Our previous studies using Dark Agouti (DA) rats indicated that T. spiralis infection reduced experimental autoimmune encephalomyelitis (EAE) severity in rats. The aim of this study was to analyse the mechanisms underlying EAE suppression driven by T. spiralis infection. Reduced clinical and histological manifestations of the disease were accompanied by increased IL-4 and IL-10 production and decreased IFN-gamma and IL-17 production in draining lymph node cells. This indicates that T. spiralis infection successfully maintains a Th2 cytokine bias regardless of EAE induction. High IL-10 signifies parasite-induced anti-inflammatory and/or regulatory cell responses. Transfer of splenic T cell-enriched population of cells from T. spiralis-infected rats into EAE immunized rats caused amelioration of EAE and in some cases protection from disease development. This population of cells contained higher proportion of CD4(+) CD25(+) Foxp3(+) regulatory cells and produced high level of IL-10 when compared with uninfected rats.

Background: Several lines of research suggest that exposure to cellular material can alter the susceptibility to infection by HIV-1. Because sexual contact often includes exposure to cellular material, we hypothesized that repeated mucosal exposure to heterologous cells would induce an immune response that would alter the susceptibility to mucosal infection. Using the feline immunodeficiency virus (FIV) model of HIV-1 mucosal transmission, the cervicovaginal mucosa was exposed once weekly for 12 weeks to 5,000 heterologous cells or media (control) and then cats were vaginally challenged with cell-associated or cell-free FIV., Results: Exposure to heterologous cells decreased the percentage of lymphocytes in the mucosal and systemic lymph nodes (LN) expressing L-selectin as well as the percentage of CD4+ CD25+ T cells. These shifts were associated with enhanced ex-vivo proliferative responses to heterologous cells. Following mucosal challenge with cell-associated, but not cell-free, FIV, proviral burden was reduced by 64% in cats previously exposed to heterologous cells as compared to media exposed controls., Conclusions: The pathogenesis and/or the threshold for mucosal infection by infected cells (but not cell-free virus) can be modulated by mucosal exposure to uninfected heterologous cells.

Prurigo nodularis is a pruritic dermatosis of unknown origin. Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukaemia/lymphoma. HTLV-1 is not considered to be a cause of prurigo nodularis. A 52-year-old black man, from the French West Indies, who had had prurigo nodularis for 12 years, presented with a distinct micropapular eruption with the typical pathological picture of epidermotropic T-cell lymphoma. Based on HTLV-1-positive serology and monoclonal integration of HTLV-1 we diagnosed smouldering adult T-cell leukaemia/lymphoma. Re-examination of previous skin biopsies revealed that the disease had been evolving for 12 years. Treatment with alpha-interferon, 3 x 106 units three times a week, associated with zidovudine, 1 g daily, resulted in complete remission within 4 months. When investigating a prurigo nodularis, we therefore recommend: (i) performing HTLV-1 serology if the patient comes from an endemic area; (ii) if positive, performing CD25 staining and looking for a HTLV-1 clonal integration; and (iii) if positive, using a treatment targeting HTLV-1.

Background: Systemic sclerosis (SSc) and morphoea are connective tissue diseases characterized by fibrosis of the skin. Although to date their pathogenesis has not been clearly defined, it is thought that autoimmunity may play a role in the development of the skin lesions observed in both these diseases. As regulatory T cells (Tregs) play a key role in the modulation of immune responses, it has recently been suggested that Treg impairment may lead to the development of autoimmune diseases., Objectives: To investigate the presence of Tregs and their immunomodulatory cytokines, transforming growth factor (TGF)-beta and interleukin (IL)-10, in patients with SSc and morphoea., Patients/methods: Fifteen patients with SSc and 15 with morphoea were enrolled. Immunohistochemistry was applied to identify FoxP3+ (forkhead/winged helix transcription factor) Tregs, TGF-beta+ cells and IL-10+ cells in the skin, cytofluorometry to detect CD4+CD25+FoxP3+ Tregs in the blood, and enzyme-linked immunosorbent assays to analyse TGF-beta and IL-10 serum levels., Results: Fewer FoxP3+ Tregs and TGF-beta+ and IL-10+ cells were found in the skin of patients with scleroderma than in controls. Similarly, there were reduced TGF-beta and IL-10 serum levels and fewer circulating CD4+CD25brightFoxP3+ cells in patients with SSc or morphoea, than in controls., Conclusions: The quantitative reduction of Tregs, together with that of TGF-beta and IL-10 serum levels, may be responsible for the loss of tolerance observed in both SSc and morphoea.

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.

Background: Although the therapeutic potential of regulatory T lymphocytes (Tregs) in preventing allograft rejection has been well documented, accumulating evidence indicates that supplemental measures, such as concomitant use of immunosuppressive agents, are essential for effective application of Treg cell therapy in clinical transplantation. Thus, it is important to know the effect of immunosuppressive agents on Treg cell therapy., Methods: We examined the impact of various immunosuppressive agents on the in vivo proliferation and therapeutic efficacy of in vitro-expanded Tregs using the murine graft-versus-host reaction and skin allograft model (BDF1 [H-2] to C57BL/6 [H-2]), respectively., Results: All six immunosuppressive agents tested inhibited the alloantigen-stimulated proliferation of Tregs as efficiently as they inhibited the proliferative response of conventional CD3 T cells. We further show that blockade of the CD40-CD40L interaction by treatment with a MR-1 antibody significantly increased the therapeutic efficacy of Tregs, a synergistic effect that seemed to be related to the strong regulatory activity of adoptively transferred Tregs together with effector T-cell hyporesponsiveness. Although concomitant use of rapamycin marginally augmented the therapeutic effectiveness of Tregs, mycophenolate mofetil and cyclosporine A at their full therapeutic doses exerted an antagonistic effect on Treg cell therapy., Conclusion: These results demonstrate that inhibition of CD40-CD40L interaction or treatment with rapamycin could be successfully combined with in vitro-expanded Treg cell therapy, but the concomitant use of mycophenolate mofetil or cyclosporine A in this type of Treg cell therapy should be carefully considered.

Previously, we found that co-expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3(+) cells present in CD25(high), CD25(low) and even CD25(-) subsets of CD4(+) cells expressed high levels of TNFR2. Consequently, TNFR2-expressing CD4(+)CD25(+) Treg included all of the FOXP3(+) cells present in the CD4(+)CD25(high) subset as well as a substantial proportion of the FOXP3(+) cells present in the CD4(+)CD25(low) subset. Flow cytometric analysis of PB identified five-fold more Treg, determined by FOXP3 expression, in the CD4(+)CD25(+)TNFR2(+) subset than in the CD4(+)CD25(high) subset. In addition, similar levels of FOXP3(+) cells were identified in both the CD4(+)CD25(+)TNFR2(+) and CD4(+)CD25(+)CD127(low/-) subsets. Furthermore, the CD4(+)CD25(+)TNFR2(+) subset expressed high levels of CTLA-4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4(+)CD25(+)TNFR2(+) cells were anergic and markedly inhibited the proliferation and cytokine production of co-cultured T-responder cells. In contrast, CD4(+)CD25(+)TNFR2(-) and CD4(+)CD25(-)TNFR2(+) T cells did not show inhibitory activity. As some non-Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.

Cancer-induced immunotolerance mediated by inducible Treg (iTreg) is a major obstacle to cancer immunotherapy. In a basic study of immunotolerance, injection of an endogenous superantigen, i.e. the minor lymphocyte stimulatory (Mls)-1(a), into specific TCR Vbeta8.1-Tg mice enabled generation of anergic CD25(-) iTreg, the immunosuppressive function of which was maintained by IL-10 production via p38-MAPK activation. Interestingly, although p38-chemical inhibitor (p38-inhibitor) is capable of breaking CD25(-) iTreg-induced immunotolerance, the p38-inhibitor had hardly any immunotolerance breaking effect when CD25(+) Treg were present, suggesting that depletion of CD25(+) Treg is necessary for p38-inhibitor to be effective. Peptide OVA(323-339) iv.-injection into its specific TCR-Tg (OT-II) mice also induced adaptive tolerance by iTreg. Peptide immunotherapy with p38-inhibitor after CD25(+) Treg-depletion was performed in an OVA-expressing lymphoma E.G7-bearing tolerant model established by adoptive transfer of OT-II CD25(-) iTreg, which resulted in suppression of tumor growth. Similarly, the antitumor immunity induced by peptide immunotherapy in colon carcinoma CT26-bearing mice, in which the number of IL-10-secreting iTreg is increased, was augmented by treatment with p38-inhibitor after CD25(+) Treg-depletion and resulted in inhibition of tumor progression. These results suggest that simultaneous inhibition of two distinct Treg-functions may be important to the success of cancer immunotherapy.

Chronic hepatitis B (CHB) virus hepatitis B virus (HBV) infection is the key cause of hepatocellular carcinoma (HCC) in Asians. Recent studies have shown that levels of CD4(+)CD25(+) regulatory T cells (Tregs) were increased and were linked to an impaired immune response in patients with CHB. Evaluating whether Tregs are involved in the progression of CHB to HCC will provide insight into the immunopathogenesis of HCC. In the present study, we showed that circulating and liver-residing Tregs increased in CHB (n = 15) and HCC (n = 49) patients, particularly in the peripheral blood of HCC patients with HBV infection (n = 29). The increased Tregs in CHB patients suppressed the specific immune response induced by not only HBV antigen, but also by HCC tumour antigen. When peripheral blood mononuclear cells (PBMC) were co-cultured with human hepatoma cell lines that are stably transfected with HBV (HepG2.2.15), CD4(+)CD25(+) Treg populations increased and upregulated the expression of forkhead box P3 transcriptional regulator (FoxP3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and glucocorticoid-induced tumour necrosis factor (TNF) receptor family gene (GITR). In contrast, PBMCs co-cultured with HepG2 cells (the parental cell line of HepG2.2.15) did not. CD4(+)CD25(+) Tregs isolated from PBMCs that were co-cultured with HepG2.2.15 cells also had a greater suppressive ability with respect to the tumour antigen-specific immune response induced by NY-ESO-1 or MAGE-A3 compared with CD4(+)CD25(+) Tregs isolated from PBMCs co-cultured with HepG2 cells. The results offer evidence that the expansion of CD4(+)CD25(+) Tregs and the enhancement of the suppressor function of CD4(+)CD25(+) Tregs induced by HBV infection-related factors could suppress the anti-tumour immune response to HCC tumour antigen and inhibit tumour immuno-surveillance against HCC, which may be involved in the immunopathogenesis from CHB to HCC.

Background: The relationship between tonsillar autoimmune response and the pathogenesis of IgA nephropathy (IgAN) has been previously demonstrated. However, the role of CD4+CD25+ cells, which play critical roles in maintaining peripheral tolerance and preventing autoimmunity, has not yet been defined in IgAN., Methods: The lymphocytes from tonsils of all subjects (including 37 IgAN cases and 37 controls without renal diseases) were cultured for 72 hours without stimulation, or with stimulation by alpha-hemolytic streptococcus (HS) isolated from the tonsillar crypts of cases (the HS-IgAN) or controls (HS-controls). The CD4+CD25+ cells were measured by flow cytometry. Expression of J chain mRNA was analyzed by in situ hybridization (ISH) and the dimeric IgA-producing cells were identified by immunofluorescence and fluorescent ISH., Results: The number of CD4+CD25+ cells was significantly lower in cases than in controls (0.98% +/- 0.204% vs. 3.58% +/- 0.554%, 1.37% +/- 0.214% vs. 5.78% +/- 0.562%, and 1.43% +/- 0.202% vs. 6.05% +/- 0.521%, for nonstimulation, HS-controls and HS-cases, respectively). CD4+CD25+ cells from cases showed a significantly lower stimulation index (SI) when stimulated with HS-controls and HS-IgAN than controls (p<0.05), whereas the number of dimeric IgA-producing cells was significantly higher in cases than controls (11.9% +/- 3.1% vs. 6.5% +/- 1.5%, 33.5% +/- 5.7% vs. 13.9% +/- 1.2%, and 35.1% +/- 6.2% vs. 13.9% +/- 1.2%, for nonstimulation, HS-controls and HS-cases, respectively). The dimeric IgA-producing cells from patients with IgAN showed a significantly higher SI when stimulated with HS-controls, or HS-IgAN than those from patients without renal disease (p<0.01). The SI of CD4+CD25+ cells was negatively correlated with that of dimeric IgA-producing cells., Conclusion: The results suggest that CD4+CD25+ cells and dimeric IgA-producing cells in tonsils may be related to the pathogenesis of IgAN.

Background: Epidemiological data show significant associations of vitamin D deficiency and autoimmune diseases. Vitamin D may prevent autoimmunity by stimulating naturally occurring regulatory T cells., Objectives: To elucidate whether vitamin D supplementation increases Tregs frequency (%Tregs) within circulating CD4+ T cells., Methods: We performed an uncontrolled vitamin D supplementation trial among 50 apparently healthy subjects including supplementation of 140,000 IU at baseline and after 4 weeks (visit 1). The final follow-up visit was performed 8 weeks after the baseline examination (visit 2). Blood was drawn at each study visit to determine 25-hydroxyvitamin D levels and %Tregs. Tregs were characterized as CD4+CD25++ T cells with expression of the transcription factor forkhead box P3 and low or absent expression of CD127., Results: Forty-six study participants (65% females, mean age +/- SD 31 +/- 8 years) completed the trial. 25(OH)D levels increased from 23.9 +/- 12.9 ng/ml at baseline to 45.9 +/- 14.0 ng/ml at visit 1 and 58.0 +/- 15.1 ng/ml at visit 2. %Tregs at baseline were 4.8 +/- 1.4. Compared to baseline levels we noticed a significant increase of %Tregs at study visit 1 (5.9 +/- 1.7, P < 0.001) and 2 (5.6 +/- 1.6, P < 0.001)., Conclusions: Vitamin D supplementation was associated with significantly increased %Tregs in apparently healthy individuals. This immunomodulatory effect of vitamin D might underlie the associations of vitamin D deficiency and autoimmune diseases. Hence, our finding provides a rationale for further studies to investigate vitamin D effects on autoimmunological processes.

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.)

The significance of TLR expression and Tregs in HBV infection has not been clearly described. In this report, flow cytometry was performed to assess TLR2/4 expression on monocytes and circulating CD4(+)CD25(+)CD127(low/-) Tregs frequency of 16 acute hepatitis B (AHB), 42 chronic hepatitis B (CHB), 22 asymptomatic HBV carriers (AsC), and 20 normal controls (NC). We found that TLR2 and TLR4 were overexpressed on CD14(+) monocytes in HBV-infected patients as compared with NCs. Upregulation of TLR2 in NCs and TLR4 in CHBs was observed following HBeAg incubation. However, TLR2 and TLR4 expression decreased after HBcAg stimulation. The difference in the proportion of Tregs between NCs and CHBs was significant. Both Pam3Csk4 (TLR2 agonist)- and lipopolysaccharide (TLR4 agonist)-activated CD4(+)CD25(+) Tregs showed enhanced suppression function in CHBs. These results suggest that overexpression of TLR2 and TLR4 may modulate the suppressive function of Tregs, which contribute to the immunotolerance of chronic HBV infection., (Copyright 2009 Elsevier Inc. All rights reserved.)

The protozoan parasite Leishmania mexicana causes chronic cutaneous disease in humans and most mouse strains. We previously showed that STAT4-deficient mice, but not IL-12p40-deficient mice, have more parasites and progressively growing lesions unlike those of wild-type mice, the lesions and parasite burdens of which plateau by 10-12 weeks post-infection. This demonstrates a STAT4-dependent, IL-12/IL-23-independent pathway of parasite control. Type I IFNs are important in viral and other infections and can activate STAT4. We found that IFN-alpha/betaR-deficient mice have a nonpersistent, early IFN-gamma defect, and a persistent, early IL-10 defect, without changes in serum IL-12 or LN-derived nitric oxide. We found less IL-10 per cell in CD25+CD4+ T cells and possibly fewer IL-10-producing cells in the draining LN of IFN-alpha/betaR-deficient vs. wild-type mice. IFN-alpha/betaR-deficient mice have chronic, nonprogressive disease, like wild-type mice, suggesting that IL-10 and IFN-gamma defects may balance each other. Our data indicate that although type I IFNs help promote early Th1 responses, they are not the missing activators of STAT4 responsible for partial control of L. mexicana. Also, the lack of lesion resolution in IFN-alpha/betaR-deficient mice despite lower IL-10 levels indicates that other pathways independent of T cell IL-10 help prevent an IL-12-driven clearance of parasites.

CD8(+) lymphocytes are critical to the control and elimination of viral pathogens. Impaired CD8(+) responses are well recognized in lentiviral infections; however, the mechanisms underlying CD8(+) impairment remain elusive. Using the feline immunodeficiency virus (FIV) model for human AIDS, we reported previously that CD4(+)CD25(+) Treg cells in both the acute and long-term, asymptomatic phase of infection are constitutively activated and suppress CD4(+)CD25(-) T cell responses. In the current study, we have demonstrated that CD4(+)CD25(+) Treg cells suppress CD8(+) responses to immune stimulation during both the acute and chronic, asymptomatic phase of FIV infection and that the mechanism of suppression may be mediated by membrane-associated TGF-beta (mTGF-beta) on CD4(+)CD25(+) lymphocytes. Depletion of CD4(+)CD25(+) lymphocytes from lymph node suspensions significantly enhanced production of IFN-gamma during the acute phase of infection and coculture of CD8(+) lymphocytes with CD4(+)CD25(+) lymphocytes resulted in suppression of CD8(+) IFN-gamma during both the acute and chronic stages of infection. FACS analysis indicated that there was TGF-betaRII upregulation on CD8(+) cells from FIV(+) cats during the acute and chronic stage of infection. In addition, there was upregulation of mTGF-beta on the CD4(+)CD25(+) subset in chronically infected cats. In support of activation of the TGF-beta signaling pathway, Western blotting showed Smad 2 phosphorylation in CD8(+) targets following CD4(+)CD25(+)/CD8(+) coculture. These results demonstrate the suppressive effect CD4(+)CD25(+) Treg cells have on the CD8(+) immune response during the acute and chronic stages of FIV infection and suggest that the mechanism of suppression may be mediated by mTGF-beta.

Groups of 5-month-old lambs which had been trickle infected with Teladorsagia circumcincta for 8 weeks then drenched, and worm-free control lambs were challenged with 50 000 T. circumcincta L3s. From 10 days later fewer parasites were recovered from the previously infected sheep, and secondary cellular and humoral responses were observed in the gastric lymph. Increases in CD4+ and CD25+ T lymphoblast traffic on day 3, followed by CD21+ and IgA+ lymphoblasts on day 5, and an increase in total and parasite specific IgA concentrations peaking on day 6 were observed in previously infected lambs. Similar peaks in lymphoblast output were not observed until days 10-12 in the control lambs. This data was highly comparable with that obtained recently from yearling sheep subjected to an identical infection-challenge regime, and contrasted with that obtained from similar experiments in the 1980s when 4(1/2)-month-old previously infected lambs were more susceptible to and had much weaker immune responses to challenge than 10-month-old sheep. The fact that 40% fewer larvae were given during the trickle infection regime in the four recent trials is offered as an explanation for this difference.

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.

Hepatocellular carcinoma (HCC) represents the third most common cause of cancer-related deaths worldwide and efficient treatment options are urgently needed. Based on its pathogenesis as well as a number of correlative studies, immunotherapy represents a potential therapeutic option for patients with HCC. However, tumors have also evolved numerous immune escape mechanisms, such as the generation of cells with immune suppressor functions, including regulatory T cells and myeloid-derived suppressor cells. It has been shown that these suppressor cells mask tumor-specific immune responses in patients with HCC. We propose that targeting suppressor cells either alone or in combination with conventional immunotherapy should be further evaluated in HCC patients., (Copyright 2010 S. Karger AG, Basel.)

Johne's disease, a chronic enteritis of ruminants, is caused by infection with Mycobacterium avium subsp. paratuberculosis. Three distinct forms have been observed in sheep: paucibacillary disease (PB), multibacillary disease (MB), and asymptomatic infection (AS). In this study, immune parameters for animals naturally infected with M. avium subsp. paratuberculosis and identified postmortem as having PB, MB, or AS were compared to provide a further understanding of the immunological reactivity contributing to or resulting from these different disease states in sheep. PB was associated with strong ex vivo M. avium subsp. paratuberculosis antigen-stimulated gamma interferon responses, pronounced increases in CD25(+) T-cell frequencies in circulation, antibody production, and a B-cell population that expanded significantly upon ex vivo antigenic stimulation. The MB group featured the highest antibody levels and a lack of cellular immune responsiveness to the M. avium subsp. paratuberculosis antigen. The AS group expressed an immunological phenotype intermediate between that for noninfected control animals and that for the PB group. The relationship between immune responses and disease severity within the PB group was investigated more closely; significant positive correlations were observed between disease severity and both the CD8(+) population in the circulating blood and the expression of interleukin-4 mRNA in antigen-stimulated blood samples ex vivo. Together, these data point toward distinct immune profiles in sheep that correspond to different Johne's disease states, which can be determined from circulating blood and/or from localized intestinal tract tissue samples.

Although signaling through certain TLRs is known to modulate the function of T lymphocytes, the effect of TLR7 stimulation on CD4(+)CD25(+) T(reg) cell activity has not yet been elucidated. In this study, we show that mouse CD4(+)CD25(+) T(reg) cells express TLR7 mRNA and protein. We therefore used the TLR7 agonists imiquimod, gardiquimod, and single-stranded poly(U) to show that TLR7 stimulation enhanced the ability of murine T(reg) cells to suppress anti-CD3/anti-CD28 mAb-coated bead-stimulated proliferation of syngeneic CD4(+)CD25(-) T(resp) cells. In contrast, imiquimod failed to enhance the suppressor function of T(reg) cells from mice deficient in the MyD88 adaptor protein involved in TLR7 and other TLR signal transduction. Imiquimod increased murine T(reg) cell-mediated suppression of T(resp) cell proliferation induced by anti-TCRbeta mAb in the presence of syngeneic BMDCs, and T(reg) cells from gardiquimod-treated mice exhibited enhanced in vitro suppressor function. Moreover, levels of T(resp) cell-secreted IL-2 and IFN-gamma were reduced further in the presence of T(reg) cells plus imiquimod in comparison with T(reg) cells alone. In addition, imiquimod treatment increased CD25 expression by T(reg) cells and caused exogenous IL-2 to enhance T(reg) cell suppressor function. Furthermore, combined treatment with imiquimod and IL-2 increased Foxp3 expression by T(reg) cells. Collectively, these findings suggest that TLR7 signaling enhanced the suppressor function of T(reg) cells by sensitizing T(reg) cells to IL-2-induced activation. We speculate that TLR7-stimulated enhancement of T(reg) cell suppressor function may modulate host T cell responses against ssRNA viruses.

A number of age-related changes in the 24-hour hormonal and non-hormonal rhythms have been found in older human beings. Lymphocyte subpopulations present circadian variation of some of their subsets and this variation may influence magnitude and expression of the immune responses. Numerous interactions exist among the nervous, endocrine and immune systems, mediated by neurotransmitters, hormones and cytokines. The aim of this study is to evaluate circadian variations of some endocrine and immune factors in older adults. Cortisol and melatonin serum levels were measured and lymphocyte subpopulation analyses were performed on blood samples collected every four hours for 24 hours from ten healthy young and middle-aged subjects and from ten healthy elderly subjects. There was a statistically significant difference between the groups in the observed values of CD20 (higher in young and middle-aged subjects) and CD25 and DR+ T cells (higher in elderly subjects). In the group of young and middle-aged subjects a clear circadian rhythm was validated for the time-qualified changes of all the factors studied. In the group of elderly subjects a number of rhythms were absent or altered. The results of the current study show that aging is associated with enhanced responsiveness of T cell compartment and alterations of circadian rhythmicity.

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.

Plasmodium chabaudi chabaudi AS blood stage infection results in various degrees of clinical symptoms to malaria depending on the mouse strain. This study aimed to investigate the development of memory responses in susceptible A/J and resistant C57BL/6 mice which differ in the degree of susceptibility to clinical malaria following P. chabaudi AS infection. A rapid increase of activated cells (CD25(+), CD44(high), and CD62L(low)) and production of both interferon-gamma and interleukin-4 was found in spleens of both malaria-infected mouse strains. After the parasitemia had been cleared, these activated cells were converted to their resting phenotypes. Although exhibiting susceptible phenotype and having lower magnitude of cellular changes during primary infection, susceptible A/J mice that had been exposed to malaria parasites and drug-cured were able to generate protective immunity capable of control parasite growth following reinfection to the same level as C57BL/6 mice. This may be due to the capability of susceptible mice to produce parasite-specific antibodies, in particular of the IgG2a and IgG3 isotypes. The results from this study may provide more insights useful for the development of vaccines against malaria.

Human monocytotropic ehrlichiosis (HME), an emerging and often life-threatening tick-transmitted disease, is caused by the obligately intracellular bacterium Ehrlichia chaffeensis. HME is modeled in C57BL/6 mice using Ehrlichia muris, which causes persistent infection, and Ixodes ovatus Ehrlichia (IOE), which is either acutely lethal or sublethal depending on the dose and route of inoculation. A persistent primary E. muris infection, but not a sublethal IOE infection, protects mice against an ordinarily lethal secondary IOE challenge. In the present study, we determined the role of persistent infection in maintenance of protective memory immune responses. E. muris-infected mice were treated with doxycycline or left untreated and then challenged with an ordinarily lethal dose of IOE. Compared to E. muris-primed mice treated with doxycycline, untreated mice persistently infected with E. muris had significantly greater numbers of antigen-specific gamma interferon-producing splenic memory T cells, significant expansion of CD4(+) CD25(+) T regulatory cells, and production of transforming growth factor beta1 in the spleen. Importantly, E. muris-primed mice treated with doxycycline showed significantly greater susceptibility to challenge infection with IOE compared to untreated mice persistently infected with E. muris. The study indicated that persistent ehrlichial infection contributes to heterologous protection by stimulating the maintenance of memory T-cell responses.