Bruno Gran

Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: Results of a phase II clinical trial with an altered peptide ligand

Myelin-specific T lymphocytes are considered essential in the pathogenesis of multiple sclerosis. The myelin basic protein peptide (a.a. 83–99) represents one candidate antigen; therefore, it was chosen to design an altered peptide ligand, CGP77116, for specific immunotherapy of multiple sclerosis. A magnetic resonance imaging-controlled phase II clinical trial with this altered peptide ligand documented that it was poorly tolerated at the dose tested, and the trial had therefore to be halted. Improvement or worsening of clinical or magnetic resonance imaging parameters could not be demonstrated in this small group of individuals because of the short treatment duration. Three patients developed exacerbations of multiple sclerosis, and in two this could be linked to altered peptide ligand treatment by immunological studies demonstrating the encephalitogenic potential of the myelin basic protein peptide (a.a. 83–99) in a subgroup of patients. These data raise important considerations for the use of specific immunotherapies in general.

Suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated T cells.

Excessive inflammation occurs during infection and autoimmunity in mice lacking the α-subunit of the interleukin 27 (IL-27) receptor. The molecular mechanisms underlying this increased inflammation are incompletely understood. Here we report that IL-27 upregulated IL-10 in effector T cells that produced interferon-γ and expressed the transcription factor T-bet but did not express the transcription factor Foxp3. These IFN-γ+T-bet+Foxp3− cells resembled effector T cells that have been identified as the main source of host-protective IL-10 during inflammation. IL-27-induced production of IL-10 was associated with less secretion of IL-17, and exogenous IL-27 reduced the severity of adoptively transferred experimental autoimmune encephalomyelitis by a mechanism dependent on IL-10. Our data show that IL-27-induced production of IL-10 by effector T cells contributes to the immunomodulatory function of IL-27.

Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS)

Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model for the human inflammatory demyelinating disease, multiple sclerosis (MS). EAE is a complex condition in which the interaction between a variety of immunopathological and neuropathological mechanisms leads to an approximation of the key pathological features of MS: inflammation, demyelination, axonal loss and gliosis. The counter‐regulatory mechanisms of resolution of inflammation and remyelination also occur in EAE, which, therefore can also serve as a model for these processes. Moreover, EAE is often used as a model of cell‐mediated organ‐specific autoimmune conditions in general. EAE has a complex neuropharmacology, and many of the drugs that are in current or imminent use in MS have been developed, tested or validated on the basis of EAE studies. There is great heterogeneity in the susceptibility to the induction, the method of induction and the response to various immunological or neuropharmacological interventions, many of which are reviewed here. This makes EAE a very versatile system to use in translational neuro‐ and immunopharmacology, but the model needs to be tailored to the scientific question being asked. While creating difficulties and underscoring the inherent weaknesses of this model of MS in straightforward translation from EAE to the human disease, this variability also creates an opportunity to explore multiple facets of the immune and neural mechanisms of immune‐mediated neuroinflammation and demyelination as well as intrinsic protective mechanisms. This allows the eventual development and preclinical testing of a wide range of potential therapeutic interventions.

IL-12p35-deficient mice are susceptible to experimental autoimmune encephalomyelitis : evidence for redundancy in the IL-12 system in the induction of central nervous system autoimmune demyelination

Experimental autoimmune encephalomyelitis (EAE) serves as a model for multiple sclerosis and is considered a CD4+, Th1 cell-mediated autoimmune disease. IL-12 is a heterodimeric cytokine, composed of a p40 and a p35 subunit, which is thought to play an important role in the development of Th1 cells and can exacerbate EAE. We induced EAE with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 (MOG35–55) in C57BL/6 mice and found that while IL-12p40-deficient (−/−) mice are resistant to EAE, IL-12p35−/− mice are susceptible. Typical spinal cord mononuclear cell infiltration and demyelination were observed in wild-type and IL-12p35−/− mice, whereas IL-12p40−/− mice had normal spinal cords. A Th1-type response to MOG35–55 was observed in the draining lymph node and the spleen of wild-type mice. A weaker MOG35–55-specific Th1 response was observed in IL-12p35−/− mice, with lower production of IFN-γ. By contrast, a Th2-type response to MOG35–55 correlated with disease resistance in IL-12p40−/− mice. Production of TNF-α by microglia, CNS-infiltrating macrophages, and CD4+ T cells was detected in wild-type and IL-12p35−/−, but not in IL-12p40−/−, mice. In addition, NO production was higher in IL-12p35−/− and wild-type mice than in IL-12p40−/− mice. These data demonstrate a redundancy of the IL-12 system in the induction of EAE and suggest that p40-related heterodimers, such as the recently cloned IL-23 (p40p19), may play an important role in disease pathogenesis.

Induction of Experimental Autoimmune Encephalomyelitis in IL-12 Receptor-β2-Deficient Mice: IL-12 Responsiveness Is Not Required in the Pathogenesis of Inflammatory Demyelination in the Central Nervous System

IL-12 is thought to be involved in the susceptibility to experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disorder of the CNS. IL-12 signals through a heterodimeric receptor (IL-12Rβ1/IL-12Rβ2), whose β2-chain is up-regulated on activated, autoreactive Th1 cells. Contrary to the expectation that the absence of IL-12Rβ2 would protect from EAE, we found that IL-12Rβ2-deficient mice developed earlier and more severe disease, with extensive demyelination and CNS inflammation. The inflammatory cells were mainly comprised of CD4+ T cells, monocyte/macrophages, and dendritic cells. Compared to wild-type mice, IL-12Rβ2-deficient mice exhibited significantly increased autoantigen-induced proliferative response and increased production of TNF-α, GM-CSF, IL-17, IL-18/IL-18Rα, and NO. In addition, we found significantly increased levels of IL-23p19 mRNA expression in spleen cells from immunized IL-12Rβ2−/− mice compared with wild-type mice. These findings indicate that IL-12 responsiveness is not required in the pathogenesis of inflammatory demyelination in the CNS, and that, in the absence of IL-12Rβ2, increased IL-23 and other inflammatory molecules may be responsible for increased severity of EAE.

Suppressive effect of IL-27 on encephalitogenic Th17 cells and the effector phase of experimental autoimmune encephalomyelitis

IL-27 has been shown to play a suppressive role in experimental autoimmune encephalomyelitis (EAE) as demonstrated by more severe disease in IL-27R-deficient (WSX-1−/−) mice. However, whether IL-27 influences the induction or effector phase of EAE is unknown. This is an important question as therapies for autoimmune diseases are generally started after autoreactive T cells have been primed. In this study, we demonstrate maximal gene expression of IL-27 subunits and its receptor in the CNS at the effector phases of relapsing-remitting EAE including disease peak and onset of relapse. We also show that activated astrocyte cultures secrete IL-27p28 protein which is augmented by the endogenous factor, IFN-γ. To investigate functional significance of a correlation between gene expression and disease activity, we examined the effect of IL-27 at the effector phase of disease using adoptive transfer EAE. Exogenous IL-27 potently suppressed the ability of encephalitogenic lymph node and spleen cells to transfer EAE. IL-27 significantly inhibited both nonpolarized and IL-23-driven IL-17 production by myelin-reactive T cells thereby suppressing their encephalitogenicity in adoptive transfer EAE. Furthermore, we demonstrate a strong suppressive effect of IL-27 on active EAE in vivo when delivered by s.c. osmotic pump. IL-27-treated mice had reduced CNS inflammatory infiltration and, notably, a lower proportion of Th17 cells. Together, these data demonstrate the suppressive effect of IL-27 on primed, autoreactive T cells, particularly, cells of the Th17 lineage. IL-27 can potently suppress the effector phase of EAE in vivo and, thus, may have therapeutic potential in autoimmune diseases such as multiple sclerosis.

Identification of candidate T-cell epitopes and molecular mimics in chronic Lyme disease.

Elucidating the cellular immune response to infectious agents is a prerequisite for understanding disease pathogenesis and designing effective vaccines. In the identification of microbial T-cell epitopes, the availability of purified or recombinant bacterial proteins has been a chief limiting factor. In chronic infectious diseases such as Lyme disease, immune-mediated damage may add to the effects of direct infection by means of molecular mimicry to tissue autoantigens. Here, we describe a new method to effectively identify both microbial epitopes and candidate autoantigens. The approach combines data acquisition by positional scanning peptide combinatorial libraries and biometric data analysis by generation of scoring matrices. In a patient with chronic neuroborreliosis, we show that this strategy leads to the identification of potentially relevant T-cell targets derived from both Borrelia burgdorferi and the host. We also found that the antigen specificity of a single T-cell clone can be degenerate and yet the clone can preferentially recognize different peptides derived from the same organism, thus demonstrating that flexibility in T-cell recognition does not preclude specificity. This approach has potential applications in the identification of ligands in infectious diseases, tumors and autoimmune diseases.

Exploring immunological specificity using synthetic peptide combinatorial libraries

The definition of epitopes for human B and T cells is fundamental for the understanding of the immune response mechanism and its role in the prevention and cause of human disease. This understanding can be applied to the design of diagnostics and synthetic vaccines. In recent years, the understanding of the specificity of B and T cells has been advanced significantly by the development and use of combinatorial libraries made up of thousands to millions of synthetic peptides. The use of this approach has had four major effects: first, the definition of high affinity ligands both for T cells and antibodies; second, the application of alternative means for identifying immunologically relevant peptides for use as potential preventive and therapeutic vaccines; third, a new appreciation of the requirements for TCR interactions with peptide-MHC complexes in immunogenicity; fourth, the establishment of new principles regarding the level of cross-reactivity in immunological recognition.

Mechanisms of immunomodulation by glatiramer acetate.

Objective To define the mechanism of action of glatiramer acetate (GA; formerly known as copolymer-1) as an immunomodulatory treatment for MS. Background The proposed mechanisms of action of GA include 1) functional inhibition of myelin-reactive T cells by human leukocyte antigen (HLA) blocking, 2) T-cell receptor (TCR) antagonism, and 3) induction of T helper 2 (Th2) immunomodulatory cells. In this report, the authors examined the effects of GA on the functional activation of human T-cell clones (TCC) specific for myelin basic protein (MBP) and for foreign antigens. Several questions were addressed: Is the inhibitory effect of GA specific for autoantigens? Is it mediated by blocking the interaction between peptide and HLA molecule? Is GA a partial agonist or TCR antagonist, or does it induce anergy? Does it induce Th2 modulatory T cells? Methods The effects of GA on antigen-induced activation of human TCC specific for MBP, influenza virus hemagglutinin, and Borrelia burgdorferi were studied by proliferation and cytokine measurements, TCR downmodulation, and anergy assays. GA-specific TCC were generated in vitro from the peripheral blood of patients and healthy controls by limiting dilution. Results GA more strongly inhibited the proliferation of MBP, as compared with foreign antigen-specific TCC; in some MBP-specific TCC, the production of Th1-type cytokines was preferentially inhibited. In addition to HLA competition, the induction of anergy, but not direct TCR antagonism, was observed. Numerous GA-specific TCC were generated from the peripheral blood of both MS patients and normal controls, and a fraction of these showed a Th2 phenotype. Conclusions This study confirms a preferential inhibitory effect of GA on autoreactive TCC. With respect to cellular mechanisms, although HLA competition appears to play the most important role in functional inhibition in vitro, a direct effect on the TCR may be involved at least in some autoreactive T cells as shown by anergy induction. Although not confirmed at the clonal level, it is demonstrated further that GA induces T cells that crossreact with myelin proteins. GA-specific, Th2-modulatory cells may play an important role in mediating the effect of the drug in vivo.

IDO Upregulates Regulatory T Cells via Tryptophan Catabolite and Suppresses Encephalitogenic T Cell Responses in Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a Th1 and Th17 cell-mediated autoimmune disease of the CNS. IDO and tryptophan metabolites have inhibitory effects on Th1 cells in EAE. For Th17 cells, IDO-mediated tryptophan deprivation and small molecule halofuginone-induced amino acid starvation response were shown to activate general control nonrepressed 2 (GCN2) kinase that directly or indirectly inhibits Th17 cell differentiation. However, it remains unclear whether IDO and tryptophan metabolites impact the Th17 cell response by mechanisms other than the GCN2 kinase pathway. In this article, we show that IDO-deficient mice develop exacerbated EAE with enhanced encephalitogenic Th1 and Th17 cell responses and reduced regulatory T cell (Treg) responses. Administration of the downstream tryptophan metabolite 3-hydroxyanthranillic acid (3-HAA) enhanced the percentage of Tregs, inhibited Th1 and Th17 cells, and ameliorated EAE. We further demonstrate that Th17 cells are less sensitive to direct suppression by 3-HAA than are Th1 cells. 3-HAA treatment in vitro reduced IL-6 production by activated spleen cells and increased expression of TGF-β in dendritic cells (DCs), which correlated with enhanced levels of Tregs, suggesting that 3-HAA–induced Tregs contribute to inhibition of Th17 cells. By using a DC–T cell coculture, we found that 3-HAA–treated DCs expressed higher levels of TGF-β and had properties to induce generation of Tregs from anti-CD3/anti-CD28–stimulated naive CD4+ T cells. Thus, our data support the hypothesis that IDO induces the generation of Tregs via tryptophan metabolites, such as 3-HAA, which enhances TGF-β expression from DCs and promotes Treg differentiation.