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.

[Hodgkin's disease and Epstein-Barr virus]. / Maladie de Hodgkin et virus d'Epstein-Barr: physiopathologie et perspectives thérapeutiques.

Epstein-Barr virus (EBV) seems to use B cell normal differentiation pathways to establish and maintain a persistent infection. This process is effectively controlled by the immune system through the action of EBV-specific T lymphocytes, so that the lifelong chronic infection is free of complications for most individuals. EBV is, however, associated with several malignancies. 30-50% of Hodgkin's lymphomas (HL) are EBV-associated. In EBV-positive HL, the virus is localized to the tumor cells and is clonal. HL is characterized by a type II form of latency with viral antigen expression limited to EBNA1, LMP1 and LMP2. EBV-positive HL is more frequent in childhood, in older patients and in mixed cellularity cases. EBV association may represent a poor prognosis factor in the elderly. The true contribution of EBV to the pathogenesis of HL remains uncertain, but EBV may provide to abnormal B cells survival signals protecting them from apoptosis. Finally, whatever the role that EBV plays in tumor development, the presence of viral antigens in the malignant cells may represent a target for new therapeutic strategies.

Ocular transfer of retinal glial cells transduced ex vivo with adenovirus expressing viral IL-10 or CTLA4-Ig inhibits experimental autoimmune uveoretinitis.

Gene transfer using immunomodulatory molecules is a promising tool for in vivo regulation of immune responses. Experimental autoimmune uveitis (EAU), which serves as a model for human ocular inflammation, is induced by systemic immunization with autoantigens, but its expression is restricted to the eye. Previously, we reported protection of rodents against EAU by intravenous or/and periocular injection of vIL-10-expressing adenovirus. Here, the expression of vIL-10 was targeted into the rat Lewis eye, by intravitreal injection of either the free virus or ex vivo transfected retinal Müller glial cells (RMG-vIL-10). As shown using GFP-expressing adenovirus, a longer expression of transgene was observed in the eye after transfer of transfected syngeneic RMG cells than was seen after injection of free virus. Intravitreal injection of RMG-vIL-10 led to significant decrease in ocular pathological manifestations, compared to control RMG cells. This was observed when cells were injected simultaneously with autoantigen, but also after a delayed administration of transfected cells. Finally, injection of RMG cells transfected with adenovirus expressing CTLA4 had a strongly protective effect. In conclusion, inhibition of antigen presentation at the site of expression of the autoimmune disorders represents an attractive alternative to treat ocular inflammation, and the transfer of ex vivo genetically modified cells provides a promising method to target the factor of interest into the eye.

Inhibition of experimental autoimmune uveoretinitis by systemic and subconjunctival adenovirus-mediated transfer of the viral IL-10 gene.

Pathological ocular manifestations result from a dysregulation in the balance between proinflammatory type 1 cytokines and regulatory type 2 cytokines. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with potent immunosuppressive effects. We have examined the efficiency of viral IL-10 adenovirus (Ad-vIL-10)-mediated gene transfer on experimental autoimmune uveoretinitis (EAU) induced in mice and rats by purified retinal autoantigens, respectively, interphotoreceptor binding protein (IRBP) and S-antigen (S-Ag). B10-A mice that received a single unilateral injection of Ad-vIL-10 in the retro-orbital sinus venosus performed 1 day before immunization with IRBP in the footpads showed high levels of circulating vIL-10 in their sera and a significant reduction in pathological ocular manifestations. Lower levels of IFN-gamma and IL-2 were found in cellular supernatants from IRBP-stimulated splenic cells in these treated mice. The local effect on ocular disease of vIL-10 was neutralized completely by injection of a monoclonal anti-vIL-10 antibody, demonstrating the specificity of the treatment. To determine whether the transfer of the vIL-10 gene within the periocular tissues of the eye could prevent acute EAU, a subconjunctival injection of Ad-vIL-10 was performed in Lewis rats simultaneously with S-antigen in the footpads. This injection determined in situ vIL-10 expression with very low circulating vIL-10 and led to a significant reduction of EAU without affecting the systemic immune response. The present results suggest that Ad-mediated gene transfer resulting in systemic and local expression of vIL-10 provide a promising approach for the treatment of uveitis.

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.

Cutaneous interleukin-7 transgenic mice display a propitious environment to Schistosoma mansoni infection.

Interleukin (IL)-7 is produced early in Schistosoma mansoni-infected human and murine skin and was recently shown to favour parasite development. In the present work, we investigated the participation of keratinocyte-derived IL-7 in this process. Keratinocytes are the predominant cellular constituents of the epidermis and the first tissue encountered by the parasite when it infects the vertebrate host. We therefore infected IL-7 cutaneous transgenic mice and compared several parasitological and immunological parameters to those of infected littermate controls. In transgenic mice, an increased number of total adult worms was observed while egg number and female fecundity remained unchanged. Additionally, transgenic animals displayed a more intensive hepatic fibrosis. In parallel, infected IL-7 transgenic animals showed a dominant Th2-type humoral response towards egg antigens. The results presented here confirm and reinforce the key role play by IL-7 in S. mansoni-vertebrate host interplay, beginning with keratinocyte-derived IL-7.

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.

Fast evolution of interleukin-2 in mammals and positive selection in ruminants.

Interleukin-2 (IL-2) is a cytokine involved in induction and regulation of the immune response in mammals. There have been numerous reports about the search for IL-2 in species other than mammals, and recently an IL-2-like gene has been isolated in chicken. Using PCR, we searched for IL-2 gene sequences in a wide variety of mammals, including marsupials and monotremes, as well as in birds. Although we can readily amplify IL-2 gene fragments in placental mammals, no amplification was obtained in other species. This is best explained by very high substitution rates. This suggest that strategies to isolate IL-2 homologous genes outside mammals should involve functional assays, as for the chicken gene, and not hybridization-based techniques. Nonsynonymous substitution rates are especially high in ruminants, due to positive selection acting on regions important in term of structure-function. We suggest that, although globally similar, the immune response of various mammals is not identical, mainly at the level of cytokine-mediated regulations.

Structural diversity of human class II histocompatibility molecules induced by peptide ligands.

SDS-PAGE analyses of stable HLA-DR1 complexes indicate that the binding of T cell epitopes can lead to multiple conformational variants. Whereas short T epitopes (<14-mer) induce complexes with apparent MW ranging from 47 to 57 kDa, longer peptides form generally high mobility complexes (44-45 kDa). The generation of HLA-DR1 conformational variants appears dependent on core peptide residues fitting inside the groove but can additionally be attributed to the presence of N- and C-terminal flanking residues (PFRs) acting as a complementary mechanism. These PFRs can jointly affect major histocompatibility complex class II conformation and stability, supporting the existence of alternative contacts at a distance from the classical binding site.

Sex-dependent neutralizing humoral response to Schistosoma mansoni 28GST antigen in infected human populations.

The reduction of Schistosoma fecundity observed after experimental vaccination with the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm28GST) antigen has been related to the inhibition of glutathione S-transferase (GST) enzymatic activity by specific antibody. The humoral immune response to the protective antigen Sm28GST and to the epitopes involved in the enzymatic site (amino acid ¿aa sequences 10-43 and 190-211) was evaluated in infected individuals before chemotherapy treatment. The capacity of the serum samples to inhibit GST enzymatic activity was assessed. Specific IgG3 response was predominant in the male population with a low intensity of infection and was associated with maximal GST inhibition. In contrast, the neutralizing activity of serum samples from women with a low intensity of infection was correlated with high specific IgA response specifically directed toward the 190-211 epitope. These results strongly support the hypothesis that GST-neutralizing IgG3 and IgA isotypes are sex dependent. The relationship of this specific acquired immune response with the level of intensity of infection is discussed.

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.

Direct evidence of cytoplasmic delivery of PKC-alpha, -epsilon and -zeta pseudosubstrate lipopeptides: study of their implication in the induction of apoptosis.

Protein kinases C (PKC) are serine/threonine kinase enzymes involved in the mechanism of cell survival. Their pseudosubstrate sequences are autoinhibitory domains, which maintain the enzyme in an inactive state in the absence of allosteric activators, thus representing an attractive tool for the modulation of different PKC isoforms. Here, we report the use of palmitoylated modified PKC-alpha, -epsilon, and -zeta pseudosubstrate peptides, and determine their intracellular distribution together with their respective PKC isoenzymes. Finally, we propose that the differential distribution of the peptides is correlated with a selective induction of apoptosis and therefore argues for different involvement of PKC isoforms in the anti-apoptotic program.

IFN-gamma-derived lipopeptides: influence of lipid modification on the conformation and the ability to induce MHC class II expression on murine and human cells.

Two truncated analogues of a previously identified lipopeptide agonist toward the IFN-gamma receptor were synthesized in an attempt to determine the minimal compound able to induce expression of MHC class II molecules on murine and human cells and to study the role of the lipid tail. Circular dichroism studies were used to probe the induced conformationnal changes. Our results indicate at least a double role for the lipid modification that contributes to the stabilization of helical organization of the associated peptide and to its passive delivery into the cytoplasm. The persistence of biological activity in a truncated peptide of half of the residues present in the lead compound suggests that the lipid tail could also contribute to the stabilization of the peptide-receptor binding through additional hydrophobic interactions. This study allowed to readjust the minimal requirements for intracellular IFN-gamma receptor stimulation. More generally, we suggest that lipidated analogues of functional peptides could be utilized for intracellular target validation in the drug discovery process.

Cellular immune responses of a Senegalese community recently exposed to Schistosoma mansoni: correlations of infection level with age and inflammatory cytokine production by soluble egg antigen-specific cells.

A recently reported epidemic of Schistosoma mansoni infection in Senegal provided an opportunity to study the dynamics of the development of immunity to human schistosomiasis. We report here on the cell-mediated immune response in a population of 99 females and 95 males, with particular emphasis on the relationship between intensity of infection and age. We found that the intensity of infection correlated negatively with age in females but not in males. In men and women, both Th1- and Th2-type cytokines were detected upon in vitro stimulation of PBMCs with soluble egg antigen (SEA) or soluble adult worm antigens (SWAP). In the female group, SEA-induced PBMC proliferation was associated with the production of IFN-gamma, IL-2 and IL-5, all of which correlated negatively with intensity of infection. Most cytokine production correlated positively with age. Spontaneous production of TNF-alpha, IL-6 and IL-10 was higher in the infected population than in an uninfected control group. Our results suggest that immunity to infection could be more pronounced in the female population and associated with a Th0/1 + 2 pattern of cytokine secretion mediated by soluble egg antigen (SEA).

Systemic delivery of an adenovirus expressing EBV-derived vIL-10 in mice infected with Schistosoma mansoni or Leishmania amazonensis: controversial effects on the development of pathological parameters.

Within the context of microorganism/host interactions, those which last over weeks are expected to be sensitive to more or less sustained and targeted immuno-intervention, such as delivery of cytokines known to operate as down-regulators of acute inflammatory processes. IL-10 has received growing attention as a potential tool in immunotherapy, due to its anti-inflammatory and immunosuppressive properties. Therefore, using two experimental models of long-term interactions between parasites and laboratory mice, we monitored some effects of the systemic delivery of an adenovirus (Ad) expressing EBV-derived IL-10 (vIL-10) designated Ad-vIL-10. We first monitored the vIL-10 serum level following intranasal, intraperitoneal, intramuscular and intravenous administration. The i. p. and i.v. delivery of Ad-vIL-10 allowed a high serum level of vIL-10 (= 100 ng/ml), the i.v. route leading to a more sustained expression (up to 3 weeks). As a first model of parasite/mouse interaction, Schistosoma mansoni/C57Bl/6 mouse was selected. Ad-vIL-10 delivery was performed 4 weeks after S. mansoni infection i.e. at the time of egg-laying, and several parameters were monitored: (i) number of adult worms in the mesenteric vein, (ii) number of eggs trapped in the liver and intestine, (iii) liver fibrosis, (iv) serum levels of egg-reactive antibody subclasses, (v) serum content of cytokines, and (vi) cytokine production in the supernatant of antigen-stimulated mesenteric lymph node cells. No apparent effect was observed, either on the different parasitological parameters or on fibrosis development at day 70 of infection. Surprisingly, a marked increase in both Th1 and Th2 type cytokines was observed in the sera of the Ad-vIL-10 injected animals, as well as in the supernatants of their Ag-stimulated mesenteric lymph node cells. Nevertheless, polarization of the humoral response towards a Th2 profile was demonstrated by an increase in the IgE level in the Ad-vIL-10-injected animals. As far as the second model is concerned, namely the Leishmania amazonensis /C57Bl6 mouse interactions, Ad-vIL-10 was delivered intravenously one day before subcutaneous injection of stationary promastigotes and footpad swelling was monitored over 110 days. Under these conditions, vIL-10 exhibited a biphasic effect, decreasing the lesion size at the early stages of infection, but leading to a more pronounced lesion size during the chronic phase. This observation suggests a deactivation of the macrophage host cells under the influence of vIL-10. The results are discussed in the context of immunotherapy and the paradoxical effects observed in immunointervention with vIL-10.