CD70-silenced dendritic cells induce immune tolerance in immune thrombocytopenia patients

The hyper-response of dendritic cell (DC) is believed to participate in the pathogenesis of immune thrombocytopenia (ITP). The CD70 expression on the surface of DCs that takes part in the CD27-CD70 costimulation pathway is an important element of DC ‘licensing’, which may initiate a series of autoreactive immune responses. To elucidate the roles CD70 molecules play in the DCs of ITP patients, we first stimulated the CD70 molecules on the DCs of ITP patients and normal controls, and found that the stimulated DCs from ITP patients exhibited higher ability to induce CD4⁺CD25⁻ T lymphocytes proliferation, while lowering the ability of the induction of regulatory T cells (Tregs) from CD4⁺CD25⁻ T lymphocytes. Meanwhile, higher IFN-γ and lower IL-10 levels were found in the co-culture system of stimulated DCs and CD4⁺CD25⁻ T cells. We then silenced the CD70 genes on the induced DCs of ITP patients and normal controls by siRNA, and confirmed our suggestion that the silence of CD70 expression on the surface of DCs from ITP patients would decrease the CD4⁺CD25⁻ T lymphocytes proliferation and Tregs differentiation, simultaneously inducing higher IL-10 and lower IFN-γ levels. Thus, the interference with the CD27-CD70 costimulatory pathway might lead to the alleviation of the consequent immune reactions, polarisation of Th2, induction of immune tolerance as well as shed new light on treatment of autoimmune diseases.     

Ascaris suum infection modulates inflammation: Implication of CD4+ CD25high Foxp3+ T cells and IL‐10

Helminth infections have the ability to modulate host's immune response through mechanisms that allow the chronic persistence of the worms in the host. Here, we investigated the mechanisms involved on the suppressive effect of Ascaris suum infection using a murine experimental model of LPS‐induced inflammation. We found that infection with A. suum markedly inhibited leucocyte influx induced by LPS into air pouches, suppressed secretion of pro‐inflammatory cytokines (IL‐1β, TNF‐α and IL‐6) and induced high levels of IL‐10 and TGF‐β. Augmented frequency of CD4⁺ CD25^high Foxp3⁺ T cells was observed in the mesenteric lymph nodes of infected mice. Adoptive transfer of purified CD4⁺ CD25⁺ T cells to recipient uninfected mice demonstrated that these cells were able to induce a suppressive effect in the LPS‐induced inflammation in air pouch model. In addition, adoptive transfer of CD4⁺ CD25⁺ T cells derived from IL‐10 knockout mice suggests that this suppressive effect of A. suum infection involves IL‐10 cytokine. In conclusion, our results demonstrated that A. suum experimental infection was capable of suppressing LPS‐induced inflammation by mechanisms, which seem to be dependent on responses of CD4⁺ CD25⁺ T cells and secretion of IL‐10 cytokine.     

Induction of regulatory T cells by Opisthorchis viverrini

Opisthorchis viverrini causes public health problems in South‐East Asia. Recently, TGF‐β and IL‐10 have been reported to increase in O. viverrini‐infected hamsters but the sources of these cytokines are still unknown. In this study, the CD4⁺ T cells in infected hamsters were investigated. It was demonstrated that IL‐4⁺CD4⁺ T cells were significantly increased in hamster spleens and mesenteric lymph nodes (MLNs) during chronic infection. Interestingly, IL‐10⁺CD4⁺ T cells were also discovered at a significant level while Treg (T regulatory)‐like TGF‐ β⁺CD4⁺ T cells were in MLNs of infected hamsters. Moreover, the CD4⁺CD25⁺Foxp3⁺ Treg cell response was significantly found both in spleens and MLNs in infected hamsters. The findings were then confirmed by development of T‐cell clones against crude somatic antigens (CSAg) in immunized BALB/c mice. Five clones named TCC21, TCC23, TCC35, TCC41 and TCC108 were established. The TCC21 was found to be the TGF‐β⁺CD4⁺ while TCC35, TCC41 and TCC108 were IL‐4⁺CD4⁺ and TCC23 was IFN‐γ⁺CD4⁺. This TGF‐β⁺CD4⁺ T clone showed an inhibitory function in vitro in mononuclear cell proliferation via TGF‐β‐mediated mechanisms. This study indicated that O. viverrini‐infected hamsters could induce TGF‐β⁺ CD4⁺ Treg‐like cells. The CSAg‐specific Tregs secreted high TGF‐β, and limited immune cell proliferation.     

Low percentages of regulatory T cells in common variable immunodeficiency (CVID) patients with autoimmune diseases and its association with increased numbers of CD4+CD45RO+ T and CD21low B cells

BackgroundCommon variable immunodeficiency (CVID) is a heterogeneous group of primary antibody deficiencies defined by marked reductions in serum IgG, IgA and/or IgM levels and recurrent bacterial infections. Some patients are associated with defects in T cells and regulatory T cells (Tregs), resulting in recurrent viral infections and early-onset autoimmune disease.MethodsWe analyzed whether there is an association between Tregs cells (CD4+CD25+CD127^low and CD4+CD25+FoxP3+); memory T cells (CD4+CD45RO+); memory B cells (CD19+CD27-IgD-); and CD21^low B cells (CD19+CD38^lowCD21^low); as well as autoimmune manifestations in 36 patients with CVID (25 women and 11 men, mean age 24 years), all by flow cytometry.ResultsFourteen patients presented with autoimmune diseases (AI) (39%), including 11 with autoimmune thrombocytopenia (ITP) (31%); two with vitiligo (6%); one with systemic lupus erythematosus (LES) (3%); and one with multiple sclerosis (MS) (3%). CVID patients with AI had a reduced proportion of Tregs (both CD4+CD25+CD127^low and FoxP3+ cells) compared with healthy controls. CVID patients with AI had expanded CD21^low B cell populations compared with patients who did not have AI. A correlation between increased CD4+CD45RO T cell populations and reduced Tregs was also observed.ConclusionsOur results showed that 39% of patients with CVID had AI and reduced Tregs populations. Research in this area might provide noteworthy data to better understand immune dysfunction and dysregulation related to CVID.     

Heligmosomoides bakeri antigen rescues CD4-positive T cells from glucocorticoid-induced apoptosis by Bcl-2 protein expression

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

Generation and functional characterization of human dendritic cells derived from CD34+ cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34⁺ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34⁺ cells, compared to that of BM CD34⁺ precursors in response to GM-CSF and TNF-α with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34⁺ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34⁺ cells enhanced both the percentage of total CD1a⁺ cells and CD1a⁺CD14^? cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34⁺ DC precursors into PB and circulating CD34⁺ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediate-late-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.     

Upregulation of CD72 expression on CD19+CD27+ memory B cells by CD40L in primary immune thrombocytopenia

CD72 is a co‐receptor of B cells and regulates B cell activation. Although aberrant expression of CD72 has been reported in primary immune thrombocytopenia (ITP), it is uncertain whether this aberrant expression is restricted to specific B cell subsets. Furthermore, the mechanisms that regulate CD72 expression are unknown. In this study, we found higher frequency of CD19⁺ B cells, CD19⁺CD27⁺ memory B cells and lower frequency of CD19⁺CD27⁻ naive B cells in active ITP patients compared with controls and patients in remission. CD72 expression on CD19⁺CD27⁺ cells was upregulated in active ITP patients and correlated with platelet count and anti‐platelet autoantibodies. In vitro, CD40L could specifically induce CD72 upregulation on CD19⁺CD27⁺ B cells. In combination with CD40L, interleukin (IL) 10 and BAFF (also termed TNFSF13B) further enhanced CD72 expression on CD19⁺CD27⁺ B cells, whereas IL21 reduced CD72 upregulation. CD72mRNA expression after CD40L stimulation was increased in ITP patients and controls. Significant increase of CD40L on CD4⁺ T cells was correlated with CD72 expression on CD19⁺CD27⁺ B cells in ITP patients. In conclusion, upregulation of CD72 expression on CD27⁺memory B cells might take part in the pathogenesis of ITP. Elevated CD40L on CD4⁺ cells combined with cytokines might contribute to the upregulation of CD72 expression on CD27⁺memory B cells.     

Increased IL-4- and IL-17-producing CD8+ cells are related to decreased CD39+CD4+Foxp3+ cells in allergic asthma

Objective: In allergic asthma, regulatory T cell (Treg) number and function are decreased. Antigen-primed CD8⁺ T cells play an indispensable role in the full development of airway inflammation and airway hyper-responsiveness (AHR) occurring in asthma. In this study, we investigated the relationship between subpopulations of CD8⁺ T cells and CD39⁺ Tregs. Methods: Female C57BL/6 mice were used to develop the model of allergic asthma. Experimental mice were immunized with ovalbumin (OVA) by intra-peritoneal (i.p) injection and then challenged with OVA by intra-tracheal administration. Control mice were immunized with vehicle by i.p injection and challenged with OVA. Airway inflammation was determined by histology and AHR was measured by an invasive method. Levels of interferon (IFN)-γ, IL-4, and IL-17 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The frequencies of CD8⁺IFN-γ⁺ cells (Tc1), CD8⁺IL-4⁺ cells (Tc2), CD8⁺IL-17⁺cells (Tc17), and CD39⁺Tregs were measured by flow cytometry. The correlation between CD39⁺Tregs and Tc subsets was analyzed by Pearson’s test. Results: Experimental mice displayed phenotypes of allergic asthma, including inflammatory cell infiltration into the lungs, goblet cell hyperplasia, increased airway resistance, and increased IL-4 and IL-17 in BALF. Compared to control mice, experimental mice displayed lower CD39⁺Tregs and Tc1 but higher Tc2 and Tc17. There was a negative correlation between CD39⁺Tregs and Tc2 or Tc17. Conclusion: In allergic asthma, increased Tc2 and Tc17 are possibly related to insufficient CD39⁺Tregs.     

Generation of regulatory dendritic cells and CD4+Foxp3+ T cells by probiotics administration suppresses immune disorders

The beneficial effects of probiotics have been described in many diseases, but the mechanism by which they modulate the immune system is poorly understood. In this study, we identified a mixture of probiotics that up-regulates CD4⁺Foxp3⁺ regulatory T cells (Tregs). Administration of the probiotics mixture induced both T-cell and B-cell hyporesponsiveness and down-regulated T helper (Th) 1, Th2, and Th17 cytokines without apoptosis induction. It also induced generation of CD4⁺Foxp3⁺ Tregs from the CD4⁺CD25⁻ population and increased the suppressor activity of naturally occurring CD4⁺CD25⁺ Tregs. Conversion of T cells into Foxp3⁺ Tregs is directly mediated by regulatory dendritic cells (rDCs) that express high levels of IL-10, TGF-β, COX-2, and indoleamine 2,3-dioxygenase. Administration of probiotics had therapeutical effects in experimental inflammatory bowel disease, atopic dermatitis, and rheumatoid arthritis. The therapeutical effect of the probiotics is associated with enrichment of CD4⁺Foxp3⁺ Tregs in the inflamed regions. Collectively, the administration of probiotics that enhance the generation of rDCs and Tregs represents an applicable treatment of inflammatory immune disorders.     

Idiotype-specific T lymphocytes in monoclonal gammopathies: evidence for the presence of CD4+ and CD8+ subsets

Tumour-specific CD4⁺ T helper (Th) and CD8⁺ T cytotoxic (Tc) cells may participate in the control and eradication of tumour cells. In the present study, idiotype-specific stimulation of CD4⁺ and CD8⁺ blood T cells from patients with monoclonal gammopathy of undetermined significance and patients with untreated multiple myeloma stage I was examined. Activation was measured in the CD4⁺ and CD8⁺ subsets enriched by magnetic microbeads as the incorporation of ³H-thymidine and the secretion of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 by single cells using the enzyme-linked immunospot assay. Idiotype-specific T cells were found in four of seven patients. Stimulation was mainly confined to the CD4⁺ subset in three of the four responding patients. This type of response was major histocompatibility complex (MHC) class II restricted as it could be inhibited by monoclonal antibodies against MHC class II (HLA-DR), but not against class I (HLA-ABC) molecules. Idiotype-specific CD8⁺ T cells were also demonstrated in these patients although at a lower frequency. One patient showed a strong and dominating activation of CD8⁺ T cells which could be blocked by antibodies against HLA-ABC but not against HLA-DR. Idiotype-specific CD4⁺ or CD8⁺ T cells were mainly of the type-1 subsets as judged by their secretion of IFN-γ and IL-2. Thus, this study provides evidence for the presence of idiotype-specific and MHC-restricted CD4⁺ and CD8⁺ T cells of the type-1 subsets in patients with monoclonal gammopathies. Such T cells with the potential to control the growth of tumour B cells may be a suitable target for immunotherapeutic interventions in patients.     

Suppressive activity and altered conventional phenotype markers/mediators of regulatory T cells in patients with self‐limiting hepatitis E

Hepatitis E virus (HEV) is a major cause of self‐limiting acute viral hepatitis in several developing countries. Elevated levels of peripheral CD4⁺CD25⁺Foxp3⁺, CD4⁺CD25⁻Foxp3⁺ and rise in IL‐10 in hepatitis E have been associated with the involvement of regulatory T cells (Treg). The functional role of the same is yet elusive. In the current study, we have assessed (i) Foxp3 expression by real‐time PCR and by flow cytometry, (ii) the levels of antigen‐specific IL‐10 and TGF‐β by ELISA, (iii) functional analysis of Treg cells and (iv) expression of Treg‐associated conventional phenotypes by flow cytometry in 54 acute patients, 44 recovered individuals from hepatitis E and in 33 healthy controls. Foxp3 mRNA elevation in the acute compared with recovered group and elevation in Foxp3⁺ cells in both patient groups were significantly elevated. The levels of IL‐10 and TGF‐β in the acute patients and TGF‐β in the recovered individuals were elevated. Significantly higher expression of CTLA‐4, PD1, GITR, CD95, CD103 and CD73 on Treg and T effector (Teff) cells was detected in the patient groups. Treg cells of acute patients and recovered individuals exhibited suppressive activity indicating that the Treg cells of hepatitis E patients are functional. The suppressive capacity of Treg cells in acute hepatitis E patients was significantly higher compared with the recovered individuals. Based on our findings, the suppressive functionality of these key markers associated with hepatitis E Treg function need further exploration to get a better understanding of the mechanisms of Treg‐mediated suppression.     

Regulatory T cells and CD20+ B cells in pediatric very severe aplastic anemia: possible clinical markers for evaluating the therapeutic efficacy and prognosis

ABSTRACT

Objectives: To investigate the immune status of children with very severe aplastic anemia (VSAA), and evaluate the frequencies of CD20⁺ B cells and Regulatory T cells (Tregs) as potential markers for evaluating the therapeutic efficacy and prognosis.

Methods: We systematically analyzed CD20⁺ B cells and Tregs using Flow Cytometry in 36 children with VSAA (14 newly diagnosed cases and 22 cases in remission after therapy with HDIVIG?+?r-ATG?+?CSA).

Results: In newly diagnosed VSAA patients, the percentage of CD20⁺ B cells was higher than that in healthy children (P?<?.01), whereas the percentage of Tregs was lower than that in healthy children (P?<?.001). After treatment with HDIVIG?+?r-ATG?+?CSA, the percentage of CD20⁺ B cells in peripheral blood was decreased obviously, and the percentage of Tregs was significantly increased.

Conclusion: There is a moderate negative correlation between the percentage of Tregs and CD20⁺ B cells in our study. Our results shed light on the roles of Tregs and CD20⁺ B cells as therapeutic efficacy and prognostic markers of pediatric VSAA. Moreover, the mechanism underlying the decrease of blood Tregs and increase of CD20⁺ B cells in pediatric VSAA patients have been discussed, indicating that Tregs may suppress B cell responses.     

Induction of CD4+CD8+ double positive T cells and increase in CD5+ B cells in efferent lymph in sheep infected with Trypanosoma evansi

The effects of Trypanosoma evansi on efferent lymphocyte phenotypes draining from a lymph node primed with Pasteurella haemolytica vaccine were studied in sheep. The prefemoral efferent lymphatic ducts of the infected sheep along with those of two uninfected sheep were surgically cannulated. Lymph was collected and lymphocytes recovered from it analysed by two-colour indirect immunofluorescence staining and cytofluoremetry in a fluorescence activated cell analyser (FACSCAN). The study showed the appearance and persistence of T. evansi in the efferent lymph for a long period of time and the appearance of CD4⁺CD8⁺ (double positive, DP) T lymphocytes in the efferent lymph of infected animals. The infection also resulted in increases in CD5⁺ B cells in the prefemoral efferent lymph. In addition, there were decreases in the output of conventional B cells, CD5⁺ and CD4⁺ T cell subsets but large increases in CD8⁺ cells followed by terminal depletion of all cell subsets. In contrast, inoculation of sheep with pasteurella vaccine antigen alone produced little alterations in the proportions, but large increases in the numbers of all T cell subsets except that of CD8⁺ cells which also showed little variation; and there was a concurrent increase in the numbers and proportions of efferent B cells. In addition, the abnormal expression of DP and CD5⁺ B cells did not occur in the uninfected vaccinated sheep. It is concluded that these abnormal changes in the kinetics of efferent lymphocyte phenotypes are likely to play a role in the genesis of the generalized immunosuppression seen in trypanosome-infected hosts.     

The role of regulatory T cells during Plasmodium chabaudi chabaudi AS infection in BALB/c mice

An inappropriate immune response to parasite infection is one of the primary drivers of malaria pathogenesis. Regulatory T cells (Tregs), an important subset of CD4⁺ T cells, can maintain self‐tolerance and prevent autoimmune diseases. However, there is little consensus about their role in malaria pathogenesis. In this study, we transiently depleted Tregs (CD25⁺ T cells) using an anti‐CD25 mAb (7D4 clone) at different time points following Plasmodium chabaudi chabaudi AS infection in BALB/c mice and investigated the effect of depletion of Tregs in this model. In control mice, Tregs proliferated significantly and their suppressive function was enhanced after infection. IL‐10 was increased drastically during infection. Depletion of Tregs at various time points can lead to divergent outcomes. When Tregs were depleted prior to or during the early phase of infection, most mice survived and had a robust Th1 immune response. In contrast, when Tregs were depleted close to peak parasitemia, all mice died as a result of inflammation. Taken together, these data suggest that in P. c. chabaudi AS‐infected BALB/c mice, Tregs inhibit the Th1 response and macrophage activation, leading to increased parasite load; however, they also control inflammation‐mediated pathology by secreting high levels of IL‐10.     

A prospective study of T‐ and B‐lymphocyte subpopulations,CD81 expression levels on B cells and regulatory CD4+CD25+CD127low/−FoxP3+ T cells in patients with chronic HCV infection during pegylated interferon‐alpha2a plus ribavirin treatment

Summary. Resolution of hepatitis C virus (HCV) infection requires a complex interplay between innate and adaptative immune responses. The role of lymphocyte subpopulations during combined antiviral treatment remains to be defined. This study was conducted to assess the effect of pegylated interferon‐alpha2a (pegIFN‐α2a) and ribavirin treatment on peripheral blood lymphocytes, mainly on CD81 expression on B cells and CD4⁺CD25⁺CD127^low/?FoxP3⁺ regulatory T cells (Tregs) in patients with chronic HCV infection. Thirty‐five patients with chronic HCV infection who started pegIFN‐α2a and ribavirin treatment were enrolled. Peripheral blood mononuclear cells (PBMC) were obtained at baseline before treatment (BT), mid‐treatment (MT), the end of treatment (ET) and 24 weeks post‐treatment (PT). During combined antiviral treatment, a significant decrease in the percentage of CD3⁺, CD8⁺, CD3⁺gamma/delta (γδ)⁺, CD19⁺ lymphocyte subpopulations and Tregs was observed. There was also a significant increase in the percentage of the CD4⁺ lymphocyte subpopulation and in CD81 expression levels on CD19⁺ B cells when BT was compared with ET (all P < 0.05). Seventeen patients were nonresponders (NR) and 18 had a sustained virological response (SVR). At baseline, NR patients had higher CD81 expression levels on CD19⁺ B cells (P = 0.017) and a higher Tregs percentage (P = 0.025) than SVR patients. Our results suggest that immunomodulation fluctuates during antiviral treatment and that percentage CD81 expression levels on B cells and Tregs might be useful as an immunological prognostic factor for pegIFN‐α2a and ribavirin treatment response in chronic HCV infection.     

CD8+CD122+CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity

The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8⁺CD122⁺ cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8⁺CD122⁻ population, were previously shown to include cells with regulatory activity and could be separated into CD49d^low cells and CD49d^high cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122⁺ cells. Regulatory activity was observed in CD8⁺CD122⁺CD49d^low but not in CD8⁺CD122⁺CD49d^high cells, indicating that the regulatory cells in the CD8⁺CD122⁺ population could be narrowed down to CD49d^low cells. CD8⁺CD122⁻ cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122⁺ Tregs. CD122⁺ Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8⁺CD122⁻ cells, indicating that the regulation by CD122⁺ Tregs is Fas/FasL-dependent. CD122⁺ Tregs taken from IL-10–deficient mice could regulate CD8⁺CD122⁻ cells as equally as wild-type CD122⁺ Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122⁺ Tregs in vitro. CD122⁺ Tregs also regulated CD4⁺ cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122⁺ Tregs that kill activated T cells to maintain immune homeostasis.Fas (CD95) and its ligand FasL (CD178) compose a unique system that is strongly related to programmed cell death (1). FasL has been considered one of the effector molecules involved in the killing of target cells by cytotoxic T lymphocytes (CTLs) (2). When Fas binds to FasL, it induces downstream signal transduction pathways that subsequently activate cell death induction pathways (3, 4). Thus, the Fas/FasL system appears to act as an effector for CTL-mediated killing of virus-infected or cancer cells, similar to the perforin–granzyme system (5, 6). However, because Fas-mutant (lpr) and FasL-deficient (gld) mice show lymphoproliferative changes, it has been suggested that the Fas/FasL system is important for suppression/regulation of activated effector T cells (7, 8).Molecular mechanisms after Fas activation have been thoroughly investigated, and the signal transduction pathway has been largely elucidated (9, 10). However, research on the cellular events that use the Fas/FasL system has progressed comparatively slowly. There are only a few relevant reports in this respect, mostly of human CD4⁺Foxp3⁺ Tregs that use the Fas/FasL system for their regulatory activity. [In this article, we use the term “Treg(s)” for any kind of T cells that show immune regulatory activity.] To complicate matters further, some contradictory reports claim that such Fas/FasL-engaging Tregs do not exist (8, 11, 12). No reliable reports suggesting that CD8⁺ T cells use the Fas/FasL system for their regulatory action are available. Therefore, it is not clear precisely which subset of T cells express FasL or where Fas/FasL-dependent CD8⁺ Tregs, if such cells exist, are located and at which point they function. Thus, the ultimate pathophysiological role of the Fas/FasL system is still unknown.Regulation of the immune reaction is of pivotal importance for maintaining health in multicellular organisms. In highly developed animals, Tregs, a subset of T lymphocytes especially known as CD4⁺CD25⁺Foxp3⁺ cells, are the main regulators of the immune response (13–16). However, it is not quite clear whether CD8⁺ regulatory T cells exist; there are only few and contradictory reports on their existence, in contrast to the reports on CD4⁺ Tregs. A population of predominantly CD8⁺ suppressor T cells has been described and debated in the 1980s (17, 18). In the 2000s, we found and reported that the cells of central memory phenotype (CD8⁺CD122⁺) also possess the regulatory function, and some other reports regarding CD8⁺ Tregs with some contradictory findings have been published. To date, more than 10 CD8⁺ Treg populations with different markers have been reported (19).One of the best characterized CD8⁺ Treg populations is the CD8⁺CD122⁺ Treg (122⁺ Treg) population. To confirm the existence of CD8⁺ Tregs, markers that may be possible to distinguish Tregs from other T cells were examined. We previously found that CD49d can separate CD8⁺CD122⁺ cells into at least two subsets (CD49d^low and CD49d^high) (20). To judge which cells have a stronger regulatory activity, we prepared two experimental systems: an in vitro system, based on cell culture, and an in vivo system, based on adoptive transfer of T cells.Interestingly, CD8⁺ Tregs express CD122 (IL-2/IL-15 receptor β chain) in contrast to CD4⁺ Tregs, which express CD25 (IL-2 receptor α chain), indicating the fundamental importance of IL-2 at the development/maturation of both CD4⁺ and CD8⁺ Tregs. Suzuki and coworkers generated CD122-deficient mice using gene targeting (21) and used them to identify 122⁺ Tregs (22). In this study, which is a follow-up study to our previous report, we performed in vitro and in vivo experiments to gain further understanding of these cells. We found that CD49d might be a good marker for classifying CD8⁺ T cells into naïve, resting T cells (CD62L⁺CD122⁻), effector memory T cells (CD62L⁻), and T cells of central memory phenotype (CD122⁺CD49d^low), by using multicolor staining of CD62L, CD122, and CD49d. To our knowledge, this is the first report on the role of the Fas/FasL system in the action of CD8⁺CD122⁺ Tregs (122⁺ Tregs) (22–25). Additionally, we show that CD8⁺ Tregs are included in the CD49d^low population, which corresponds to the central memory T-cell population, and that their mechanism of suppression depends on the cytotoxicity mediated by the Fas and FasL interaction.     

CD4+ T cells mediate mucosal and systemic immune responses to experimental hookworm infection

Hookworm infection is associated with anaemia and malnutrition in many resource‐limited countries. Ancylostoma hookworms have previously been shown to modulate host cellular immune responses through multiple mechanisms, including reduced mitogen‐mediated lymphocyte proliferation, impaired antigen presentation/processing, and relative reductions in CD4⁺ T cells in the spleen and mesenteric lymph nodes. Syrian hamsters were depleted of CD4⁺ for up to 9 days following intraperitoneal injection (200 μg) of a murine anti‐mouse CD4 monoclonal IgG (clone GK1·5). CD4⁺ T‐cell‐depleted hamsters infected with the hookworm Ancylostoma ceylanicum exhibited a threefold higher mean intestinal worm burden and more severe anaemia than animals that received isotype control IgG. In addition, depletion of CD4⁺ T cells was associated with impaired cellular and humoral (serum and mucosal) immune responses to hookworm antigens. These data demonstrate an effector role for CD4⁺ T cells in hookworm immunity and disease pathogenesis. Ultimately, these studies may yield important insights into the relationship between intestinal nematode infections and diseases that are associated with CD4⁺ T‐cell depletion, including HIV.     

The effect of various disease-modifying anti-rheumatic drugs on the suppressive function of CD4+CD25+ regulatory T cells

Accumulating evidence suggests that defects in the function of CD4⁺CD25⁺ regulatory T cells (Tregs) are important in immune-mediated diseases such as rheumatoid arthritis. Here, we investigated the effects of various disease-modifying anti-rheumatic drugs (DMARDs) on Treg function. Tregs and CD4⁺CD25^? effector T cells (Teffs) were isolated from peripheral blood mononuclear cells obtained from healthy adults. Isolated Tregs were cultured with the DMARDs methotrexate (MTX), sulfasalazine (SSZ), leflunomide (LEF), or infliximab (INF). We found that each DMARD had a different effect on Treg function. SSZ and LEF inhibited the anti-proliferative function of Tregs on cocultured Teffs and reduced Treg expression of Foxp3 mRNA, whereas MTX and INF did not.     

Control of type 1 diabetes by CD4+Foxp3+ regulatory T cells: lessons from mouse models and implications for human disease

In recent years, there has been a revival of the concept of CD4⁺ regulatory T (T_reg) cells as being a central control point in various immune responses, including autoimmune responses and immunity to transplants, allergens, tumours and infectious microbes. The current literature suggests that T_reg cells are diverse in their phenotype and mechanism(s) of action, and as such, may constitute a myriad of naturally occurring and induced T cell precursors with variable degrees of regulatory potential. In this review, we summarize research from various laboratories, including our own, showing that CD4⁺Foxp3⁺ T_reg cells are critical in the control of type 1 diabetes (T1D) in mouse models and humans. In this review, we also discuss cellular and molecular determinants that impact CD4⁺Foxp3⁺ T_reg cell development and function and consequential resistance to organ‐specific autoimmune disease. Recent advances in the use of CD4⁺Foxp3⁺ T_reg cellular therapy to promote immunological tolerance in the absence of long‐term generalized immunosuppression are also presented. Copyright © 2009 John Wiley & Sons, Ltd.