Circulating immunoglobulin-bound transforming growth factor beta at a late tumour-bearing stage impairs antigen-specific responses of CD4+ T cells

In order to elucidate the mechanisms by which tumour‐specific CD4⁺ T‐cell responses are impaired during tumour development, an attempt was made to identify factors which impair CD4⁺ T‐cell responses at a late tumour‐bearing stage. Plasma from mice bearing B16 melanoma for 30 days (plasma d30) showed a more profound immunosuppressive effect on the in vitro proliferation of unrelated antigen‐specific CD4⁺ T cells in the presence of both antigen and antigen‐presenting cells (APC) than plasma from naïve mice. The level of plasma transforming growth factor (TGF)‐β was elevated in mice bearing B16 melanoma for 30 days compared with naïve mice, and the suppressive effect of plasma d30 was partially diminished by the neutralization of TGF‐β. Interestingly, immunoglobulin (IgG)‐bound TGF‐β, but not IgG‐unbound TGF‐β, in plasma d30 was suggested to be responsible for the immunosuppressive activity. In addition, no suppressive effect of plasma d30 was observed when antigen was added as a class II peptide, thus suggesting that the impaired proliferation of CD4⁺ T cells in the presence of plasma d30 was due to a dysfunction of antigen uptake/processing by APC. Furthermore, dissociation between IgG and TGF‐β resulted in a loss of the suppressive activity of plasma d30. Taken together, these results suggest that circulating IgG‐bound TGF‐β is, at least in part, responsible for the impaired responses of CD4⁺ T cells at the late tumour‐bearing stage by suppressing antigen uptake/ processing by APC.     

Inhibition of phosphoantigen-mediated gammadelta T-cell proliferation by CD4+ CD25+ FoxP3+ regulatory T cells

Tumour growth promotes the expansion of CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) which suppress various arms of immune responses and might therefore contribute to tumour immunosurveillance. In this study, we found an inverse correlation between circulating Treg frequencies and phosphoantigen-induced gammadelta T-cell proliferation in cancer patients, which prompted us to address the role of Tregs in controlling the gammadelta T-cell arm of innate immune responses. In vitro, human Treg-peripheral blood mononuclear cell (PBMC) co-cultures strongly inhibited phosphoantigen-induced proliferation of gammadelta T cells and depletion of Tregs restored the impaired phosphoantigen-induced gammadelta T-cell proliferation of cancer patients. Tregs did not suppress other effector functions of gammadelta T cells such as cytokine production or cytotoxicity. Our experiments indicate that Tregs do not mediate their suppressive activity via a cell-cell contact-dependent mechanism, but rather secrete a soluble non-proteinaceous factor, which is independent of known soluble factors interacting with amino acid depletion (e.g. arginase-diminished arginine and indolamine 2,3-dioxygenase-diminished tryptophan) or nitric oxide (NO) production. However, the proliferative activity of alphabeta T cells was not affected by this cell-cell contact-independent suppressive activity induced by Tregs. In conclusion, these findings indicate a potential new mechanism by which Tregs can specifically suppress gammadelta T cells and highlight the strategy of combining Treg inhibition with subsequent gammadelta T-cell activation to enhance gammadelta T cell-mediated immunotherapy.     

Depletion of CD4+CD25+ regulatory T cells enhances natural killer T cell‐mediated anti‐tumour immunity in a murine mammary breast cancer model

Both invariant natural killer T (NK T) cells and CD4⁺CD25⁺ T regulatory cells (T_regs) regulate the immune system to maintain homeostasis. In a tumour setting, NK T cells activated by α‐galactosylceramide (α‐GalCer) execute anti‐tumour activity by secreting cytokines. By contrast, T_regs intrinsically suppress antigen‐specific immune responses and are often found to be elevated in tumour patients. In this study, we have shown that T_regs regulate NK T cell function negatively in vitro, suggesting a direct interaction between these cell types. In a murine mammary tumour model, we demonstrated that administration of either α‐GalCer or anti‐CD25 antibody alone markedly suppressed tumour formation and pulmonary metastasis, and resulted in an increase in the survival rate up to 44% (from a baseline of 0%). When treatments were combined, depletion of T_regs boosted the anti‐tumour effect of α‐GalCer, and the survival rate jumped to 85%. Our results imply a potential application of combining T_reg cell depletion with α‐GalCer to stimulate NK T cells for cancer therapy.     

A role for CD4+CD25+ T cells in regulation of the immune response during human tuberculosis

Active tuberculosis (TB) is associated with prolonged suppression of Mycobacterium tuberculosis (MTB)-specific immune responses, but mechanisms involved are understood incompletely. We investigated a potential role for CD4+CD25+ regulatory T cells in depressed anti-MTB immunity by evaluating serially CD4 cell phenotype and interferon (IFN)-gamma production by mononuclear cells from patients with TB. At diagnosis, frequencies of CD4+CD25+ T cells were increased in blood from TB patients compared to healthy purified protein derivative (PPD)-positive controls (with a history of prior TB exposure), and remained elevated at completion of therapy (6 months). By contrast, expression of another activation marker, CD69, by CD4 T cells was increased at diagnosis, but declined rapidly to control levels with treatment. Among CD4+CD25+ T cells from TB patients at diagnosis those expressing high levels of CD25, probably representing regulatory T cells, were increased 2.9-fold when compared to control subjects, while MTB-stimulated IFN-gamma levels in whole blood supernatants were depressed. A role for CD4+CD25+ T cells in depressed IFN-gamma production during TB was substantiated in depletion experiments, where CD25+-depleted CD4 T cells produced increased amounts of IFN-gamma upon MTB stimulation compared to unseparated T cells. At follow-up, IFN-gamma production improved most significantly in blood from TB patients with high baseline frequencies of CD4+CD25+ T cells (more than threefold higher than controls for both total and CD25hi+ CD4 T cells), who also had a significant drop in frequencies of both total and 'regulatory' CD4+CD25+ T cells in response to treatment. Expansion of CD4+CD25+ regulatory T cells during active TB may play a role in depressed T cell IFN-gamma production.     

Augmented induction of CD8+ cytotoxic T-cell response and antitumour resistance by T helper type 1-inducing peptide

The effector CD8(+) T cells recognize major histocompatibility complex (MHC) class I binding altered self-peptides expressed in tumour cells. Although the requirement for CD4(+) T helper type 1 (Th1) cells in regulating CD8(+) T cells has been documented, their target epitopes and functional impact in antitumour responses remain unclear. We examined whether a potent immunogenic peptide of Mycobacterium tuberculosis eliciting Th1 immunity contributes to the generation of CD8(+) T cells and to protective antitumour immune responses to unrelated tumour-specific antigens. Peptide-25, a major Th epitope of Ag85B from M. tuberculosis preferentially induced CD4(+) Th1 cells in C57BL/6 mice and had an augmenting effect on Th1 generation for coimmunized unrelated antigenic peptides. Coimmunization of mice with Peptide-25 and ovalbumin (OVA) or Peptide-25 and B16 melanoma peptide [tyrosinase-related protein-2 (TRP-2)] for MHC class I led to a profound increase in CD8(+) T cells specific for OVA and TRP-2 peptides, respectively. This heightened response depended on Peptide-25-specific CD4(+) T cells and interferon-gamma-producing T cells. In tumour protection assays, immunization with Peptide-25 and OVA resulted in the enhancement of CD8(+) cytotoxic cell generation specific for OVA and the growth inhibition of EL-4 thymoma expressing OVA peptide leading to the tumour rejection. These phenomena were not achieved by immunization with OVA alone. Peptide-25-reactive Th1 cells counteractivated dendritic cells in the presence of Peptide-25 leading them to activate and present OVA peptide to CD8(+) cytotoxic T cells.     

CD11c+ CD103+ cells of Mycobacterium tuberculosis‐infected C57BL/6 but not of BALB/c mice induce a high frequency of interferon‐γ‐ or interleukin‐17‐producing CD4+ cells

The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4⁺ populations generated after M. tuberculosis infection. Thirty days post-infection, C57BL/6 mice, which generate a stronger interferon-γ (IFN-γ)-mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c⁺ CD11b⁻ CD103⁺ cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c⁺ CD11b⁺ CD103⁻ cells. CD11c⁺ CD11b⁻ CD103⁺ cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN-γ-producing or interleukin-17 (IL-17)-producing CD4⁺ cells. Moreover, CD4⁺ cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN-γ-producing or IL-17-producing CD4⁺ cells is dependent on CD11c⁺ CD11b⁻ CD103⁺ cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.     

CD4+ CD25+ regulatory T cells in human pregnancy: development of a Treg-MLC-ELISPOT suppression assay and indications of paternal specific Tregs

The current study was aimed at developing a one-way mixed leucocyte culture-enzyme-linked immunospot (MLC-ELISPOT) assay for the study of CD4(+) CD25(+) regulatory T (T(reg)) cells and applying this method in the study of antifetal immune reactions during human pregnancy. Twenty-one pregnant women and the corresponding fathers-to-be, and 10 non-pregnant control women and men, participated in the study. CD4(+) CD25(+) cells were isolated from peripheral blood mononuclear cells (PBMC) by immunomagnetic selection. Maternal/control PBMC were stimulated with paternal or unrelated PBMC in MLC. Secretion of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) from responder cells, with or without the presence of autologous T(reg) cells, was analysed by ELISPOT. PBMC from pregnant women showed increased secretion of IL-4 compared to controls. In pregnant and non-pregnant controls, T(reg) cells suppressed IFN-gamma reactivity against paternal and unrelated alloantigens. Interestingly, T(reg) cells suppressed IL-4 secretion against paternal but not unrelated alloantigens during pregnancy. We have successfully developed a model for studying T(reg) cells in antifetal cytokine reactions during pregnancy. Results indicate that T(reg) cells contribute to strict regulation of both T helper type 1-like and type 2-like antifetal immune reactions. Interestingly, T helper type 2-like cells specific to unrelated alloantigens are able to escape the suppression of T(reg) cells, which would allow for IL-4, alongside CD4(+) CD25(+) T(reg) cells, to control potentially detrimental IFN-gamma reactions during pregnancy.     

CD4+ T-cell help amplifies innate signals for primary CD8+ T-cell immunity

CD8⁺ T cells provide an important component of protection against intracellular infections and cancer. Immune responses by these T cells involve a primary phase of effector expansion and differentiation, followed by a contraction phase leading to memory formation and, if antigen is re-encountered, a secondary expansion phase with more rapid differentiation. Both primary and secondary phases of CD8⁺ T-cell immunity have been shown to depend on CD4⁺ T-cell help, although during certain infections the primary phase is variable in this requirement. One explanation for such variability relates to the strength of associated inflammatory signals, with weak signals requiring help. Here, we focus on our studies that have dissected the requirements for help in the primary phase of the CTL response to herpes simplex virus, elucidating intricate interactions and communications between CD4⁺ T cells, various dendritic cell subsets, and CD8⁺ T cells. We place our studies in the context of others and describe a simple model of help where CD40 signaling amplifies innate signals to enable efficient CD8⁺ T-cell expansion and differentiation. This model facilitates CTL induction to various different agents, without altering the qualitative innate signals that direct other important arms of immunity.     

PARP-1 inhibitor-AG14361 suppresses acute allograft rejection via stabilizing CD4+FoxP3+ regulatory T cells

Acute allograft rejection is the most common complication in organ transplantation leading to organ loss. Treg cells play an important role in preventing acute rejection, but they are unstable and easily lose function. Poly(ADP-ribose) polymerase 1(PARP-1) is involved in the differentiation stabilization of Treg cells, it has been suggested that PARP-1 inhibition could prevent acute rejection and prolong allograft survival. This study investigated AG14361 effects on acute allograft rejection. We used a fully MHC-mismatched murine heart transplantation model to compare the effect of PARP-1 inhibitor-AG14361 on alloimmunity to the control. Mice treated with PARP-1 inhibitors showed a longer median survival time of allografts (MS14 compared with the control group, MST was 8 days, and AG14361 was 6 days, P = 0.019). The combination of sirolimus and AG14361 significantly delayed allograft MST (AG14361 + sirolimus for 30 days, sirolimus for 16 days, P = 0.002). AG14361 markedly augmented the number of the CD25+FoxP3+ Treg cells in the graft and periphery. In addition, it could enhance the suppressive function of Treg cells by upregulating the level of CTLA-4, PD-1 and ICOS. In vivo, the Treg/Th17 ratio increased significantly in the AG14351 group compared to the control. In the combination with sirolimus treatment, AG14361 promoted the long-term allograft survival. Our results highlight novel effects of a PARP-1 inhibitor. PARP-1 inhibitor AG14361 may be a promising agent to attenuate acute allograft rejection as it can maintain the number and function of Treg cells in allografts.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

Expansion of CD4+ CD25+ Foxp3+ T cells by bone marrow-derived dendritic cells

Dendritic cells (DCs) are the most important antigen-presenting cells of the immune system and have a crucial role in T-lymphocyte activation and adaptive immunity initiation. However, DCs have also been implicated in maintaining immunological tolerance. In this study, we evaluated changes in the CD4(+) CD25(+) Foxp3(+) T-cell population after co-culture of lymph node cells from BALB/c mice with syngeneic bone marrow-derived DCs. Our results showed an increase in CD4(+) CD25(+) Foxp3(+) T cells after co-culture which occurred regardless of the activation state of DCs and the presence of allogeneic apoptotic cells; however, it was greater when DCs were immature and were pulsed with the alloantigen. Interestingly, syngeneic apoptotic thymocytes were not as efficient as allogeneic apoptotic cells in expanding the CD4(+) CD25(+) Foxp3(+) T-cell population. In all experimental settings, DCs produced high amounts of transforming growth factor (TGF)-beta. The presence of allogeneic apoptotic cells induced interleukin (IL)-2 production in immature and mature DC cultures. This cytokine was also detected in the supernatants under all experimental conditions and enhanced when immature DCs were pulsed with the alloantigen. CD4(+) CD25(+) Foxp3(+) T-cell expansion during co-culture of lymph node cells with DCs strongly suggested that the presence of alloantigen enhanced the number of regulatory T cells (Tregs) in vitro. Our data also suggest a role for both TGF-beta and IL-2 in the augmentation of the CD4(+) CD25(+) Foxp3(+) population.     

Type I interferon supports primary CD8+ T-cell responses to peptide-pulsed dendritic cells in the absence of CD4+ T-cell help

CD8(+) T-cell responses to non-pathogen, cell-associated antigens such as minor alloantigens or peptide-pulsed dendritic cells (DC) are usually strongly dependent on help from CD4(+) T cells. However, some studies have described help-independent primary CD8(+) T-cell responses to cell-associated antigens, using immunization strategies likely to trigger natural killer (NK) cell activation and inflammatory cytokine production. We asked whether NK cell activation by MHC I-deficient cells, or administration of inflammatory cytokines, could support CD4(+) T-cell help-independent primary responses to peptide-pulsed DC. Injection of MHC I-deficient cells cross-primed CD8(+) T-cell responses to the protein antigen ovalbumin (OVA) and the male antigen HY, but did not stimulate CD8(+) T-cell responses in CD4-depleted mice; hence NK cell stimulation by MHC I-deficient cells did not replace CD4(+) T-cell help in our experiments. Dendritic cells cultured with tumour necrosis factor-α (TNF-α) or type I interferon-α (IFN-α) also failed to prime CD8(+) T-cell responses in the absence of help. Injection of TNF-α increased lymph node cellularity, but did not generate help-independent CD8(+) T-cell responses. In contrast, CD4-depleted mice injected with IFN-α made substantial primary CD8(+) T-cell responses to peptide-pulsed DC. Mice deficient for the type I IFN receptor (IFNR1) made CD8(+) T-cell responses to IFNR1-deficient, peptide-pulsed DC; hence IFN-α does not appear to be a downstream mediator of CD4(+) T-cell help. We suggest that primary CD8(+) T-cell responses will become help-independent whenever endogenous IFN-α secretion is stimulated by tissue damage, infection, or autoimmune disease.     

Elevated circulating Th17 and follicular helper CD4+ T cells in patients with rheumatoid arthritis

It remains not fully elucidated the potential functions of Th17 cells and follicular helper T (Tfh) cells and secreting cytokines in the pathogenesis of rheumatoid arthritis (RA) and their association with disease activity. In this study, the frequencies of Th17 and Tfh cells were determined by flow cytometry, and the levels of interleukin (IL)‐17, IL‐21, and IL‐22 were measured by ELISA in RA patients with different disease activities. The dynamic changes of cell subsets were also detected in response to disease‐modify antirheumatic drugs (DMARDs) therapy. The percentages of CD3⁺CD4⁺IL‐17A⁺ (Th17) cells and CD3⁺CD4⁺CXCR5⁺ICOS^high (Tfh) cells, as well as the concentrations of IL‐17, IL‐21, and IL‐22 were significantly elevated in RA patients than those in healthy individuals. Furthermore, Tfh cells, IL‐21, and IL‐22 in the serum was positively correlated with the values of disease activity score. Concentrations of IL‐21 and IL‐22 in the serum were remarkably reduced following the DMARDs therapies. Our data suggested that Th17 cells, Tfh cells as well as the secreting cytokines may be involved in the pathogenesis of RA. The frequency of circulating Tfh cells and the productions of IL‐21 and IL‐22 were associated with the disease activity of RA patients, and might be potential therapeutic targets for treatment of RA.     

Cytokine-dependent anti-viral role of CD4-positive T cells in therapeutic vaccination against chronic hepatitis B viral infection

There are several lines of evidence suggesting that specific vaccine therapy with a standard hepatitis B virus (HBV) vaccination reduces HBV replication. The aim of this study was to investigate the anti-viral mechanism of vaccine therapy in chronic hepatitis B patients. Nineteen patients were assigned to receive either vaccine therapy (n = 13) or no treatment as a control (n = 6). Vaccinated patients were analyzed for T cell proliferative responses specific for envelope antigen and cytokine production by antigen-specific T cells. ELISPOT and cytotoxicity assays also were carried out for limited blood samples. Serum HBV DNA levels decreased significantly at 3 months after completion of therapy and thereafter as compared to the baseline ones, and were significantly lower in vaccinated patients than in controls at 12 and 18 months after completion of therapy. Vaccination induced antigen-specific CD4+ T cell proliferative responses in four patients (30.8%). The production of high levels of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) by antigen-specific T cells was found in six patients (46.0%) who showed significantly lower HBV DNA levels in serum at 6 (P = 0.04) and 18 months (P = 0.005) after completion of therapy than those without high levels of cytokine production. Vaccination did not induce antigen-specific CD8+ T cells or cytotoxic T cells. These results suggest that envelope-specific CD4+ T cells may control directly HBV replication by producing anti-viral cytokines rather than providing help for cytotoxic T cells in therapeutic vaccination against chronic HBV infection.     

CD4+ T helper 2 cells--microbial triggers, differentiation requirements and effector functions

Over the past 10 years we have made great strides in our understanding of T helper cell differentiation, expansion and effector functions. Within the context of T helper type 2 (Th2) cell development, novel innate‐like cells with the capacity to secrete large amounts of interleukin‐5 (IL‐5), IL‐13 and IL‐9 as well as IL‐4‐producing and antigen‐processing basophils have (re)‐emerged onto the type 2 scene. To what extent these new players influence αβ⁺ CD4⁺ Th2 cell differentiation is discussed throughout this appraisal of the current literature. We highlight the unique features of Th2 cell development, highlighting the three necessary signals, T‐cell receptor ligation, co‐stimulation and cytokine receptor ligation. Finally, putting these into context, microbial and allergenic properties that trigger Th2 cell differentiation and how these influence Th2 effector function are discussed and questioned.     

CD4+ T cells clear virus but augment disease in mice infected with respiratory syncytial virus. Comparison with the effects of CD8+ T cells.

Respiratory syncytial (RS) virus-specific T cell lines were derived from the spleens of BALB/c mice primed by intranasal infection with RS virus. The lines were expanded by repeated antigenic stimulation in vitro, and separated into CD4+ and CD8+ T cell-enriched fractions by immunomagnetic adhesion. The effects of passive transfer of these fractions into RS virus infected mice were observed. The most severe immunopathological changes were seen in mice receiving CD4+ cells. Transfer of CD4+, CD8+ or both cell fractions caused RS virus-infected mice to become ill and lose weight. Both cell lines caused an increase in the severity of lung pathology (as monitored by bronchoalveolar lavage) with the appearance of lung haemorrhage and polymorphonuclear cell efflux. In addition, recipients of CD4+ cells developed striking pulmonary eosinophilia. In CD4+ cell recipients, 5 x 10(5) cells were sufficient to decrease lung virus titre, whereas 2 x 10(6) CD8+ cells were needed to produce a similar effect. The unseparated T cell line and the CD4+ cell fraction secreted significant amounts of IL-3, IL-4 and IL-5 (P less than 0.001). High levels of IL-2 were produced only by the unseparated T cell line. The CD8+ cell fraction secreted IL-3 only. The results show that, cell-for-cell, CD4+ cells are more anti-viral and more immunopathogenic than CD8+ cells in RS virus infected mice. Such effects may have contributed to the augmented disease seen in some infants vaccinated against RS virus.     

Selective culling of high avidity antigen-specific CD4+ T cells after virulent Salmonella infection

Typhoid fever is a persistent infection caused by host-adapted Salmonella strains adept at circumventing immune-mediated host defences. Given the importance of T cells in protection, the culling of activated CD4+ T cells after primary infection has been proposed as a potential immune evasion strategy used by this pathogen. We demonstrate that the purging of activated antigen-specific CD4+ T cells after virulent Salmonella infection requires SPI-2 encoded virulence determinants, and is not restricted only to cells with specificity to Salmonella-expressed antigens, but extends to CD4+ T cells primed to expand by co-infection with recombinant Listeria monocytogenes. Unexpectedly, however, the loss of activated CD4+ T cells during Salmonella infection demonstrated using a monoclonal population of adoptively transferred CD4+ T cells was not reproduced among the endogenous repertoire of antigen-specific CD4+ T cells identified with MHC class II tetramer. Analysis of T-cell receptor variable segment usage revealed the selective loss and reciprocal enrichment of defined CD4+ T-cell subsets after Salmonella co-infection that is associated with the purging of antigen-specific cells with the highest intensity of tetramer staining. Hence, virulent Salmonella triggers the selective culling of high avidity activated CD4+ T-cell subsets, which re-shapes the repertoire of antigen-specific T cells that persist later after infection.     

The T-cell pool is anergized in patients with multiple sclerosis in remission

Relapsing-remitting multiple sclerosis (RRMS) is a complex autoimmune disease of the central nervous system with oscillating phases of relapse and remission. RRMS has been considered to be driven by T helper type 1 (Th1) lymphocytes but new data indicate the involvement of Th17 responses. In the present study, blood samples from patients (n=48) and healthy individuals (n=44) were evaluated for their immunological status. T cells from patients with RRMS expressed high levels of the activation marker CD28 (P<0.05) and secreted both interferon-gamma (CD8: P<0.05) and interleukin-17 upon polyclonal mitogen or myelin oligodendrocyte glycoprotein antigen stimulation. However, T cells from patients with RRMS in remission, in contrast to relapse, had poor proliferative capacity (P<0.05) suggesting that they are controlled and kept in anergy. This anergy could be broken with CD28 stimulation that restored the T-cell replication. Furthermore, the patients with RRMS had normal levels of CD4(+) Foxp3(+) T regulatory cells but the frequency of Foxp3(+) cells lacking CD127 (interleukin-7 receptor) was lower in patients with MS (mean 12%) compared to healthy controls (mean 29%). Still, regulatory cells (CD25(+) sorted cells) from patients with RRMS displayed no difference in suppressive capacity. In conclusion, patients in relapse/remission demonstrate in vitro T-cell responses that are both Th1 and Th17 that, while in remission, appear to be controlled by tolerogenic mechanisms yet to be investigated.