Corticosteroids do not reverse the inhibitory effect of cyclosporine on regulatory T-cell activity in contrast to mycophenolate mofetil.

BACKGROUND: Inevitable hepatitis C virus recurrence after liver transplantation, a major barrier to survival of the transplanted liver may be promoted by immunosuppression and by CD4(+)CD25(+) regulatory T cells (Treg). Treg cells are essential for the induction and maintenance of immunologic self-tolerance as well as transplant tolerance. Moreover, we have previously described low doses of cyclosporine (CsA) to inhibit Treg activity by inducing interleukin-2 and interfron-γ. We investigated here in, the effect of mycophenolate mofetil (MMF) and corticosteroids, usually used in combination with a calcineurin inhibitor on human CD4(+)CD25(+) Treg cells. METHODS: Human CD4(+)CD25(+) cells isolated from healthy donors were cultured in the presence of CsA +/- corticoids or MMF. Suppressive activity of regulatory T cells was assessed in mixed leukocyte reactions including CD25(+) solvents with autologous activated peripheral blood mononuclear cells (PBMC). RESULTS: MMF and dexamethasone inhibited PBMC and Treg proliferation in dose-dependent fashing, maintaining the suppressive activity of Treg cells. However, the association of corticoids with CsA could not reverse the inhibitory effects of CsA on Treg activity, unlike the MMF and CsA combination. CONCLUSION: We have previously shown CsA to significantly impair the function of CD4(+)CD25(+) Treg cells. Herein we reports that corticoids were not able to reverse this effect, whereas MMF couterbalanced it, suggesting that the combination of MMF with CsA maintains regulatory T cells activity promoting tolerance.

Inhibitory effects of cyclosporine on human regulatory T cells in vitro.

BACKGROUND: Inevitable hepatitis C virus (HCV) recurrence after liver transplantation is a major barrier to the survival of a transplanted liver. It may be promoted by immunosuppression and the emergence of CD4+CD25+ regulatory T cells (Treg). Treg cells can mediate the induction and maintenance of immunological self-tolerance as well as transplant tolerance. We investigated the effects of cyclosporine (CsA), a widely used immunosuppressive agent, on human CD4+CD25+ Treg cells. METHODS: Human CD4+CD25+ cells isolated from healthy donors were cultured in the presence of 40 or 400 ng/mL CsA. The suppressive activity of Treg was assessed in mixed leukocyte reactions (MLR) using CD25+ and autologous activated peripheral blood mononuclear cells (PBMC). Phenotype analysis (flow cytometric, Q-PCR) and cytokine production (ELISA) of Treg cells were then performed on cultures. RESULTS: CsA (40 or 400 ng/mL) inhibited the proliferative capacity of PBMC and CD4+CD25+ Treg in a dose-dependent manner. Interestingly, addition of 40 ng/mL CsA in MLR impaired the suppressive activity of CD4+CD25+ cells, whereas a higher dose of CsA had no effect on Treg function. It appears that a therapeutic dose of CsA (40 ng/mL) did not change the phenotype of CD4+CD25+ T cells, but altered Treg activity by switching the regulatory to an inflammatory cytokine profile. CONCLUSION: CsA significantly impaired the function of CD4+CD25+ Treg cells by inducing interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) secretion. The present studies suggested that CsA may block the induction of immune tolerance and decrease the risk of hepatitis C recurrence.

Increased expression of regulatory Tr1 cells in recurrent hepatitis C after liver transplantation.

Immune response failure during HCV infection has been associated with the activity of regulatory T cells. Hepatitis C-related cirrhosis is the main reason for liver transplantation. However, 80% of transplanted patients present an accelerated recurrence of the disease. This study assessed the involvement of regulatory T-cell subsets (CD4+CD25+ cells: 'Treg' and CD49b+CD18+ cells: 'T regulatory-1' cells), in the recurrence of HCV after liver transplantation, using transcriptomic analysis, ELISA assays on serum samples and immunohistochemistry on liver biopsies from liver recipients 1 and 5 years after transplantation. Three groups of patients were included: stable HCV-negative recipients and those with mild and severe hepatitis C recurrence. At 5 years, Treg markers were overexpressed in all HCV+ recipients. By contrast, Tr1 markers were only overexpressed in patients with severe recurrence. At 1 year, a trend toward the overexpression of Tr1 was noted in patients evolving toward severe recurrence. IL-10 production, a characteristic of the Tr1 subset, was enhanced in severe recurrence at both 1 and 5 years. These results suggest that Tr1 are enhanced during severe HCV recurrence after liver transplantation and could be predictive of HCV recurrence. High levels of IL-10 at 1 year could be predictive of severe recurrence, and high IL-10 producers might warrant more intensive management.

[Regulatory T-cells and hepatocellular carcinoma: implication of the regulatory T lymphocytes in the control of the immune response]. / T régulateurs et carcinome hépatocellulaire : rôle des lymphocytes T régulateurs dans le contrôle de la réponse immune.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and also the third most common cause of cancer-related death. HCC arises most frequently in males with cirrhosis, which is most often a consequence of chronic hepatitis infection (HBV and HCV) or alcohol abuse. To date, the only effective approaches for patients with HCC are resection or liver transplantation. Immunological mechanisms are important in the surveillance of malignancy and control of tumor progression. Tumor-infiltrating lymphocytes (TILs) have been described in HCC, and extensive infiltration has been associated with reduced tumor recurrence following resection. However continued tumor-growth, despite the presence of a lymphocytic infiltration, including tumor-specific T-cells within and surrounding tumors, suggests a failure of immune control. Although, many mechanisms have been proposed for this attenuated immune response, it becomes evident that direct suppression of effector cells, supported by regulatory T-cells could play a pivotal role in the suppression of immune response to tumors. Initially described in context of immune disorders such as inflammatory autoimmune pathologies, regulatory T lymphocytes are characterized by their capacity to inhibit T helper response. To date, several regulatory T-cells are described, however CD4+CD25+ regulatory T-cells and Tr1 subpopulations remain best characterized. Currently, there is no evidence for direct implication of CD4+CD25+ regulatory T-cells in the malignancy and control of HCC progression. However, recent studies showed that both regulatory T-cells subpopulations and particularly Tr1 have been implicated in the modulation of the immune response during HCV chronic infection, supporting HCC progression.

Novel promiscuous HLA-DQ HIV Nef peptide that induces IFN-gamma-producing memory CD4+ T cells.

We describe the highly conserved sequence 56-68 of the HIV Nef protein as the first promiscuous HLA-DQ HIV-derived peptide. The Nef peptide exhibits an albeit rare capacity to bind 6 different HLA-DQ molecules whereas no binding is observed with the 10 HLA-DR molecules tested. In agreement with these data, after immunization with the Nef peptide, HLA-DQ transgenic Abeta degrees mice display a vigorous cellular and humoral response while the specific immune response of HLA-DR expressing mice is minimal. The promiscuous potentiality of the Nef 56-68 peptide in humans has been confirmed by ex vivo immunization experiments with CD4+ T cells from 14 healthy donors expressing different HLA genotypes. Nef 56-68 specific CD4+ T cells rapidly acquire a memory cell phenotype and are characterized by the preferential usage of the TCR Vbeta 6.1 gene segment and predominant production of IFN-gamma. Taken together, these data indicate that the Nef 56-68 peptide constitutes an attractive component of vaccines aiming at inducing or enhancing HIV-specific T cell immunity.

HLA class II polymorphism influences onset and severity of pathology in Schistosoma mansoni-infected transgenic mice.

Genetic factors that might influence susceptibility or resistance in naive individuals and early-stage pathology in schistosomiasis are difficult to study in clinical trials, since in areas where the disease is endemic the first contact with the parasite occurs most often at very early ages. Therefore, four strains (DR1.Abeta degrees, DR2.Abeta degrees, DQ8.Abeta degrees, and DQ6.Abeta degrees ) of major histocompatibility complex class II-deficient mice (Abeta degrees ), transgenic for different HLA alleles, have been used to evaluate the potential role of HLA class II polymorphism in the onset of the infection by Schistosoma mansoni. The survival rates and parasitological and immunological parameters after infection were evaluated and compared against the control values obtained with Abeta degrees mice. All four mouse strains used in this study were able to generate a specific immune response against S. mansoni antigens (cytokine production and antibody production). However, only mice expressing DR alleles survived until the chronic stage of the infection and were able to mount protective granulomatous response avoiding hepatic damage, presenting predominant gamma interferon production. In contrast, strains expressing DQ alleles revealed an impairment in generating effective granulomas, resulting in earlier death, which was associated with an impaired hepatic granulomatous response and liquefactic necrosis, reflecting the influence of HLA polymorphism in the establishment of protective response in the early stage of infection.

Novel hyperbranched glycomimetics recognized by the human mannose receptor: quinic or shikimic acid derivatives as mannose bioisosteres.

The mannose receptor mediates the internalization of a wide range of molecules or microorganisms in a pattern recognition manner. Therefore, it represents an attractive entry for specific drug, gene, or antigen delivery to macrophages and dendritic cells. In an attempt to design novel effective synthetic mannose receptor ligands, quinic and shikimic acid were selected as putative mannose mimics on the basis of X-ray crystallographic data from the related rat mannose-binding lectin. As the mannose receptor preferentially binds to molecules displaying several sugar residues, fluorescein-labeled cluster quinic and shikimic acid derivatives with valencies of two to eight were synthesized. Their mannose receptor mediated uptake was assayed on monocyte-derived human dendritic cells by cytofluorimetric analysis. Mannose-receptor specificity was further assessed by competitive inhibition assays with mannan, by confocal microscopy analysis, and by expression of the mannose receptor in transfected Cos-1 cells. Constructs derived from both quinic and shikimic acid were efficiently recognized by the mannose receptor with an optimum affinity for the molecules with a valency of four. As a result, commercially available quinic and shikimic acids appear as stable mannose bioisosteres, which should prove valuable tools for specific cell delivery.

Schistosomal egg antigen-responsive CD8 T-cell population in Schistosoma mansoni-infected BALB/c mice.

We demonstrated here that schistosomal egg antigen (SEA) is able to stimulate an antigen-specific, cytotoxic CD8+ T-cell response in mice. Indeed, a single i.p. immunization with SEA resulted in the in vivo induction of significant cytotoxic T lymphocyte (CTL) activity in the spleen within 20 days. Effector cells were classic class I major histocompatibility complex (MHC)-restricted CD8+ lymphocytes producing interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), suggesting a type 1 response to SEA. We therefore investigated the relevance of these observations in the context of the Schistosoma mansoni parasite infection. CTL activity against SEA-pulsed target cells was evidenced throughout the infection after in vitro stimulation of recovered splenic cells with SEA demonstrating that SEA-specific CD8+ T cells with cytotoxic potentialities are present during infection. This activity was strongly increased after immunization of mice with SEA like the production of IFN-gamma in the sera. A marked reduction in the number of granulomas and of fibrosis with the presence of cells producing IFN-gamma in the liver was also observed leading to the survival of SEA-immunized mice.

Immunological response of major histocompatibility complex class II-deficient (Abeta(o)) mice infected by the parasite Schistosoma mansoni.

We have characterized the immunological behaviour of major histocompitibility complex (MHC) Class II molecule-deficient (Abeta(o)) mice after infection by Schistosoma mansoni. In Abeta(o) mice, morbidity developed dramatically 7 weeks after infection leading to death, despite the absence of an increase in parasite burden or of eggs trapped in the liver. Histological examination of the liver revealed the absence of a classical granulomatous reaction. Antibodies were produced only against schistosomulum antigens. Specific antibodies against adult worm (SWAP) or egg antigen (SEA) were not detected. Cytokine production (IFN-gamma and IL-4) was absent after in vitro restimulation of splenic cells from infected Abeta(o) mice with parasite antigens. Adoptive transfer of primed splenic cells (total, purified CD4+ or CD8+ T cells) failed to improve survival or to induce a granulomatous reaction in infected Abeta(o) mice. Survival, cellular and humoral responses in CD8+ T-cell-depleted Abeta(o) mice or MHC(o) mice (lacking MHC class I and II molecules) were similar to nondepleted Abeta(o) mice, suggesting that anti-schistosomula antibody production was thymo-independent. Our results demonstrate a high degree of susceptibility of Abeta(o) mice to infection and corroborate the importance of CD4+ T cells in the initiation of the granulomatous response. However, our results do not show evidence for the involvement of CD8+ T cells in response to S. mansoni infection.

Schistosoma mansoni-infected mice show augmented hepatic fibrosis and selective inhibition of liver cytokine production after treatment with anti-NK1.1 antibodies.

Gamma interferon (IFN-gamma) plays an immunoregulatory role at different stages of the experimental Schistosoma mansoni-driven processes in mice through its ability to induce cell cytotoxicity against the parasite larvae and to reduce established hepatic fibrosis. The role of Natural Killer (NK) cells, as possible major source of IFN-gamma, has never been studied during the entire course of murine schistosomiasis. In this paper, we investigated the consequences of in vivo NK cell depletion, maintained during 17 weeks of infection, on both hepatic granuloma development and immunological parameters. We found that NK cell depletion following anti-NK1.1 monoclonal antibody (mAb) injections led to an increase of hepatic collagen content in the late stages of granuloma formation and to the diminution of interleukin 12 (IL-12) p40 and IL-7 mRNA expression in the livers. The hepatic mRNA expression of other cytokines (IFN-gamma, tumor necrosis factor alpha [TNF-alpha] and IL-4), as well as humoral and cytokine responses in sera, were not significantly different between control monoclonal antibody (CmAb) and anti-NK1.1-treated mice. Thus, we demonstrate that the anti-NK1.1 treatment might induce alterations of regulatory mechanisms, detectable at a late stage of a chronic process in immunocompetent mice.

Induction of cytotoxic T-cell activity by the protective antigen of Schistosoma mansoni Sm28GST or its derived C-terminal lipopeptide.

In a previous work the authors demonstrated that immunization with Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28GST) was able to reduce hepatic damage in infected mice and that the adoptive transfer of Sm28GST-specific T cells reproduced the protective effect obtained with the recombinant molecule. In the present paper, the authors show that Sm28GST is also able to stimulate an antigen-specific, cytotoxic T-cell response against Sm28GST-pulsed P815 target cells in normal mice and that effector cells induced in vivo were classical Class I MHC-restricted CD8+ lymphocytes. The authors found no spontaneous CTL activity against Sm28GST-pulsed target cells during the course of the infection by S. mansoni although Sm28GST is expressed at different developmental stages of the parasite. It was observed, however, that immunization with Sm28GST is sufficient to elicit a significant level of CTL response for 6 weeks in infected mice. The role of these Class I MHC-restricted CD8+ lymphocytes in the protection observed precisely at the same period in immunized mice remains to be elucidated. The authors also observe that immunization with the lipopeptide form of the C-terminal peptide of the molecule (190-211 peptide) led to a CTL activation comparable to that observed after immunization with the whole molecule demonstrating the feasibility of using a synthetic lipopeptide as immunogen for a CTL response against Sm28GST epitopes. Moreover, like Sm28GST-specific CTLs, 190-211 lipopeptide-specific cells were also Class I MHC-restricted lymphocytes.

Cellular immune response and pathology in schistosomiasis.

In the present review, some aspects of the cellular response following the murine Schistosoma mansoni infection are described. Due to the peculiar route used by the schistosome to infect its definitive host, the skin appears as a critical site in which the initial events of the host/parasite relationship occur and where the immune response is initiated. Moreover, the induction and the modulation of the granuloma formation, which represent the main aspect of the pathology of this parasitic disease, is under the control of several cellular populations n which CD4 and CD8 T cells play a key role. The cytokines produced in response to the parasite, such as IL7 in the skin and IFN gamma in the liver, seem to influence the further development of immunity against Schistosoma mansoni.

Evaluation of the effect of Sm28GST-derived peptides in murine hepatosplenic schistosomiasis: interest of the lipopeptidic form of the C-terminal peptide.

Among the synthetic peptides derived from the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST), immunization with the C-terminal peptide comprising amino acid residues 190-211 induced a reduction in splenomegaly, in the number of hepatic eggs and in hepatic fibrosis in mice infected by Schistosoma mansoni. The absence of antibodies specific for the Sm28GST or for the 190-211 peptide observed in our conditions of immunization with this peptide argued in favour of the involvement of cellular-dependent mechanisms in the reduction in hepatic pathology. This was confirmed by the passive transfer of 190-211 peptide-specific T-cell enriched spleen cells which reproduced the protective effect conferred by immunization with the 190-211 peptide. These 190-211 peptide-specific cells produced little IL4 and high levels of IFN-gamma, a potent inhibitor of collagen synthesis. Furthermore, the use of a lipopeptidic form of the 190-211 peptide significantly improved the reduction in hepatic pathology obtained with the uncoupled peptide and induced a durable protective response. These results provide encouraging information for the possible use of synthetic peptides in the immunoprophylaxis of Schistosomiasis.

Protective effect of rSm28GST-specific T cells in schistosomiasis: role of gamma interferon.

Immunization with a single dose of 50 micrograms of recombinant Schistosoma mansoni 28-kDa glutathione-S-transferase (rSm28GST) was able to induce a reduction in the worm burden, the number of eggs, and the degree of hepatic fibrosis as quantified by the measurement of collagen content in the liver of S. mansoni-infected mice. No relationship was found between anti-Sm28GST immunoglobulin G and immunoglobulin A titers and the levels of protection obtained. Adoptive transfers of Sm28GST-specific total, CD4+, or CD8+ T cells reproduced the protective effect obtained with the recombinant molecule. Moreover, experiments studying in vivo T-cell depletion demonstrated that anti-CD4- or anti-CD8-treated mice showed a significant decrease in the protective effect conferred, suggesting a role of the two T-cell subpopulations in the expression of Sm28GST-mediated protection against hepatic damage. Sm28GST-specific cells produced little interleukin-4 and high levels of gamma interferon. Treatment of immunized mice with anti-gamma interferon antibody totally suppressed the Sm28GST-induced protective effect and led to the rapid death of infected animals, suggesting a role for this cytokine in the expression of the protective immunity obtained after immunization with rSm28GST.

A radioimmunoassay for the quantification of human ubiquitin in biological fluids: application to parasitic and allergic diseases.

Purified ubiquitin has been shown to share similar biological and physicochemical properties with a previously characterized lymphokine, platelet activity suppressive lymphokine (PASL). This lymphokine, which inhibits the cytotoxic function of activated platelets, is produced during schistosomiasis and Hymenoptera venom hypersensitivity (HVH). We have developed a radioimmunoassay specific for ubiquitin, in order to determine the ubiquitin levels in human sera and plasma from patients with these pathologies. The working range of the assay was between 60 and 500 ng/ml, and the sensitivity was 8-10 ng/ml. The reproducibility, specificity and accuracy were determined under standard condition (PBS-0.3% BSA) and using different biological fluids (human serum, plasma and T lymphocyte supernatant). Using this assay, we found that the ubiquitin concentrations were higher in both schistosomiasis and HVH (up to 150-300 ng/ml) compared with sera and plasma from healthy donors where the ubiquitin levels did not exceed 50 ng/ml.

IFN-gamma treatment of rodents infected with erythrocytic stages of Plasmodium chabaudi: differential effects according to the immunological status.

Recombinant rat interferon-gamma (rrIFN-gamma) was tested for its antimalarial activity in three different models of Plasmodium chabaudi-blood stage malaria. Doses ranging from 1 x 10(4) to 1 x 10(5) U of rrIFN-gamma were used in each model. In BALB/c mice (lethal infection), prophylactic treatment with daily intraperitoneal (i.p.) injections reduced parasitemia and delayed mortality. In contrast, subcutaneous administration of rrIFN-gamma was inefficient, as was curative schedule of i.p. administration. Euthymic Fischer rats, which develop an acute and resolutive infection, were partly protected by i.p. prophylactic administration of rrIFN-gamma. Parasitemia was reduced without being lengthened, resulting in a marked decrease in parasite burden. Subcutaneous administration was less efficient whereas curative schedule was not. Athymic (nude) Fischer rats which present a longlasting and stable infection were treated with prophylactic and curative schedules of i.p. administration of rrIFN-gamma. In each case, rrIFN-gamma-treated nude rats, as control nude rats, were unable to resolve their chronic infection. The conditions required to obtain a beneficial effect are thus restrictive for a therapeutic use in humans. Moreover, these results show that, despite the fact that IFN-gamma is considered as a major component of the immune response, this cytokine alone is not sufficient to induce the totality of the effector mechanisms necessary to cure malarial infections.

Effect of ubiquitin on platelet functions: possible identity with platelet activity suppressive lymphokine (PASL).

In an attempt to clone a suppressive lymphokine of platelet function (PASL), we have obtained a cDNA clone coding for the previously described human ubiquitin-80 amino acid fusion protein. Our clone differs from the described sequence in that it contains the complete amino acid sequence of ubiquitin as well as a short (25 bp) 5' noncoding region. In addition the 3' untranslated region is slightly longer than that previously shown. Like PASL, purified ubiquitin can inhibit the cytotoxic properties of platelets and the production of oxygen metabolites by these cells. Moreover, this molecule is able to act as a proaggregating factor and seems of a great interest in pathologies involving defects in platelet aggregation. Ubiquitin could also have a potential use in the regulation of immunological disorders in which platelets seem to be implicated such as hymenoptera venom hypersensitivity and aspirin-sensitive asthma, since in both situations, ubiquitin is able, as is PASL, to inhibit the cytotoxic function of platelets. Indeed ubiquitin possesses important pharmacological potentialities which have not been previously described. This molecule and PASL share several similarities in their functional and physicochemical properties. PASL could therefore belong to the family of ubiquitins.

Identification and characterization of a functional receptor for interferon-gamma on a megakaryocytic cell line.

We have previously shown that human interferon-gamma (Hu-IFN-gamma) induces platelets to become efficient effector cells, capable of killing young larvae of the parasite Schistosoma mansoni. Recently, binding sites for IFN-gamma on platelets have been characterized. We show here the presence of high-affinity receptors for IFN-gamma on the surface of the human megakaryocytic Dami cell line. Scatchard analysis indicated the presence of about 11,000 binding sites per cell, with a kd of 3 +/- 0.5 x 10(-10) mol/L; the apparent molecular weight of the receptor was 90 Kd. Receptor-bound 125I Hu-recombinant IFN-gamma was rapidly internalized and degraded when the temperature was increased from 4 degrees C to 37 degrees C. The half-life of this receptor was about 7 hours, and pretreatment of cells with IFN-gamma or phorbol myristate acetate had very little effect on the surface receptor number and no detectable effect on IFN-gamma receptor messenger RNA (mRNA) expression. The receptor was functional, because 24 hours of treatment with IFN-gamma led to the increase of HLA class I mRNA expression and to the initiation of HLA class II mRNA expression. These effects were selective because platelet glycoprotein Ib, IIb, or IIIa mRNA expression and cell proliferation were unaffected.

Interleukin-6 is the main mediator of the interaction between monocytes and platelets in the killing of Schistosoma mansoni.

We demonstrate the existence of a cooperation between monocytes and platelets for the killing of Schistosoma mansoni. Indeed, supernatants obtained after a 24 hr adherence of normal human monocytes were able to induce, in a dose dependent manner, the cytotoxicity of normal human platelets towards the young larvae of S.mansoni in vitro. The physicochemical analysis of the supernatants showed that a factor exhibiting a pl of 4.8-4.9 was responsible of this effect, suggesting a role of IL-6, detected in the supernatants, in this induction. This was confirmed by the neutralization of the cytotoxic effect by a polyclonal serum against IL6 whereas polyclonal sera against IL-1 beta or TNF-alpha, the other cytokines present in the supernatants, did not modify the cytotoxicity observed. Finally human recombinant IL-6 induces the platelet cytotoxic function, demonstrating a direct effect of IL6 on blood platelets.