Autoimmunity to HSP60 during diet induced obesity in mice.

Adaptive immunity has been implicated in adipose tissue inflammation, obesity and its adverse metabolic consequences. No obesity-related autoantigen has yet been identified, although heat shock protein 60 (HSP60) has been implicated in other autoimmune diseases. We investigated whether feeding a high-fat diet to C57BL/6J mice would cause autoimmunity to HSP60 and whether immunomodulation with peptides from HSP60 would reverse the resulting obesity or metabolic dysfunction. Obese mice had higher circulating levels of HSP60 associated with increased T-lymphocyte proliferation responses and the emergence of circulating IgG1 and IgG2c antibody levels against HSP60. Treatment with escalating doses of a mixture of three proven immunomodulatory HSP60 peptides did not reduce weight but completely reversed the increase in VLDL/LDL levels and partially reversed the glucose intolerance in obese mice. Obese mice mount an autoimmune response to HSP60, which partly underlies the resulting metabolic disturbances.

Fusion between human mesenchymal stem cells and rodent cerebellar Purkinje cells.

AIMS: we explored whether cellular fusion and heterokaryon formation between human and rodent cells in the cerebellum of mice occurs after intravenous injection of human bone marrow-derived mesenchymal stem cells (MSCs). The influence of central nervous system inflammation on this process was also assessed. In addition, we examined whether tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma, factors associated with inflammation, increase cellular fusion between human MSCs and rodent cerebellar neurons in vitro. METHODS AND RESULTS: human MSCs were intravenously injected into mice with experimental autoimmune encephalomyelitis (EAE) and control mice. After 22 days, mouse Purkinje cells expressing human Golgi Zone were found within the Purkinje cell layer of the cerebellum, indicating that fusion and heterokaryon formation had occurred. The numbers of heterokaryons in the cerebellum were markedly increased in mice with EAE compared with control mice. Rodent cerebellar neuronal cells labelled with enhanced green fluorescent proteinin vitro were co-cultured with human bone marrow-derived MSCs in the presence of TNF-alpha and/or IFN-gamma to determine their influence on fusion events. We found that fusion between MSCs and cerebellar neurons did occur in vitro and that the frequency of cellular fusion increased in the presence of TNF-alpha and/or IFN-gamma. CONCLUSIONS: we believe that this is the first paper to define fusion and heterokaryon formation between human MSCs and rodent cerebellar neurons in vivo. We have also demonstrated that fusion between these cell populations occurs in vitro. These findings indicate that MSCs may be potential therapeutic agents for cerebellar diseases, and other neuroinflammatory and neurodegenerative disorders.

Negative selection during the peripheral immune response to antigen.

Thymic selection depends on positive and negative selective mechanisms based on the avidity of T cell interaction with antigen-major histocompatibility complex complexes. However, peripheral mechanisms for the recruitment and clonal expansion of the responding T cell repertoire remain obscure. Here we provide evidence for an avidity-based model of peripheral T cell clonal expansion in response to antigenic challenge. We have used the encephalitogenic, H-2 A(u)-restricted, acetylated NH(2)-terminal nonameric peptide (Ac1-9) epitope from myelin basic protein as our model antigen. Peptide analogues were generated that varied in antigenic strength (as assessed by in vitro assay) based on differences in their binding affinity for A(u). In vivo, these analogues elicited distinct repertoires of T cells that displayed marked differences in antigen sensitivity. Immunization with the weakest (wild-type) antigen expanded the high affinity T cells required to induce encephalomyelitis. In contrast, immunization with strongly antigenic analogues led to the elimination of T cells bearing high affinity T cell receptors by apoptosis, thereby preventing disease development. Moreover, the T cell repertoire was consistently tuned to respond to the immunizing antigen with the same activation threshold. This tuning mechanism provides a peripheral control against the expansion of autoreactive T cells and has implications for immunotherapy and vaccine design.

The role of polymorphic I-Ak beta chain residues in presentation of a peptide from myelin basic protein.

Proteins encoded by genes in the MHC are highly polymorphic. For class II proteins the highest level of polymorphism is found in distinct regions of variability, notably in the membrane-distal domains. To investigate the role of such residues in antigen presentation, we have tested cells transfected with wild-type or mutant I-Ak beta chains for their ability to present the NH2-terminal peptide of myelin basic protein to a panel of T cell clones. We were unable to detect a gross effect on peptide binding, in that all of the mutant cell lines presented antigen to at least one of the cloned T cells. However, the results imply that the more NH2-terminal residues, particularly 12 and 14, are involved in peptide interactions. Mutations at these residues presented antigen only at high antigen concentrations. Furthermore, residues of the more COOH-terminal regions appear to determine TCR interactions. Mutations in the predicted alpha-helical regions of the beta chain affected antigen presentation without abolishing peptide binding.

Inhibition of T cell and antibody responses to house dust mite allergen by inhalation of the dominant T cell epitope in naive and sensitized mice.

Antigen-specific CD4+ T cells play an important role in the allergic immune response to house dust mite (HDM) allergens in humans. The group 1 allergen of Dermatophagoides spp. is a major target antigen in both B and T cell recognition of HDM. In vitro studies have shown that the presentation of peptides to human T cells under appropriate conditions may lead to a state of specific nonresponsiveness. Therefore, to determine if peptides are able to modulate the function of allergen-reactive T cells in vivo, we have used a murine model of T cell recognition of the HDM allergen Der p 1. The results demonstrate that inhalation of low concentrations of peptide containing the major T cell epitope of Der p 1 (residues 111-139), induces tolerance in naive C57BL/6J mice such that they become profoundly unresponsive to an immunogenic challenge with the intact allergen. When restimulated in vitro with antigen, lymph node T cells isolated from tolerant mice secrete very low levels of interleukin 2, proliferative poorly, and are unable to provide cognate help to stimulate specific antibody production. Furthermore, intranasal peptide therapy was able to inhibit an ongoing immune response to the allergen in mice and this has potential implications in the development of allergen-based immunotherapy.

Preparing for first-in-man studies: the challenges for translational immunology post-TGN1412.

Current immunology research is generating many new approaches to immunotherapy. However, the recent disaster surrounding the testing of TGN1412, has unsettled regulators and the pharmaceutical industry regarding new immunotherapies and highlighted the complexities of conducting clinical trials with agents that target the immune system. Here we discuss the critical role for immunologists in ensuring that the development of new immunotherapies continues.

Immunotherapy of autoimmune disease.

T-cell recognition of autoantigens stands as the primary target for immune intervention in autoimmune disease. Experiments in animal models, in combination with a number of clinical trials completed in the last year, have helped to clarify the pathogenesis of various autoimmune diseases and indicate future strategies for immunotherapy.

Peptide-MHC interaction in autoimmunity.

Our increased understanding of the molecular basis of autoimmunity owes much to an appreciation of general principles governing peptide-MHC interactions. Such understanding may help resolve long-standing questions concerning autoimmune diseases and aid development of improved therapeutic strategies for their treatment.

Identification of an indirectly presented epitope in a mouse model of skin allograft rejection.

BACKGROUND: The indirect pathway of allorecognition involves the processing and presentation of donor molecules by recipient antigen-presenting cells to alloreactive CD4+ T cells. Our objective was to assess the occurrence and significance of the indirect presentation of allogeneic major histocompatibility complex molecules in the rejection of major histocompatibility complex class I-disparate skin. METHODS: A mouse model of allograft rejection was developed in which tail skin from C57.BL/10 (H2b) donors was transplanted onto B10.A(5R) recipients resulting in an allogeneic mismatch at the D locus. T-cell depletion studies were used to characterize T-cell subset involvement in rejection. B10.A(5R) mice were immunized with pools of overlapping peptides spanning the polymorphic region of Db in order to identify Db-derived epitopes involved in rejection. The relevance of these epitopes was assessed through immunization of recipient mice with peptides before skin grafting to observe the effect of presensitization on the kinetics of rejection. RESULTS: Rejection of Db-disparate skin by B10.A(5R) was delayed by CD4 and CD8+ T-cell depletion, indicating the significance of both cell types in rejection. At least six immunogenic peptides were identified, all of which contained a cryptic T-cell epitope. One peptide, however, was able to accelerate the rejection of Db-disparate skin. Presensitization of B10.A(5R) mice with this peptide also resulted in an increase in alloantibody, indicating the presence of a physiological as well as a cryptic epitope. Presensitization of mice with a peptide containing a distinct cryptic epitope, however, failed to influence rejection. CONCLUSIONS: These findings demonstrate a significant role for the indirect pathway of antigen presentation in allograft rejection.

Prolongation of murine vascularized heart allograft survival by recipient-specific anti-major histocompatibility complex class II antibody.

BACKGROUND: Antibodies targeting recipient major histocompatibility complex (MHC) class II molecules have been demonstrated to be effective at prolonging allograft survival. However, antigen-presenting cell depletion would explain this effect and has not been definitively excluded as the mechanism of action of such antibodies. We have studied an anti-MHC class II antibody (OX6) proven to be noncytotoxic in the recipient strain used. METHODS: Antibody was administered the day before, 2 hr before, and the day after grafting. RESULTS: Antibody administration on the day before, 2 hr before, and the day after grafting significantly prolonged vascularized cardiac allograft survival. Importantly, treatment recognizing recipient MHC was effective, whereas a similar regimen recognizing donor MHC was not. CONCLUSIONS: Noncytotoxic recipient MHC class II-specific antibodies modify allograft rejection. Possible mechanisms for this therapeutic effect are discussed.

T cell recognition in experimental autoimmune encephalomyelitis: prospects for immune intervention with synthetic peptides.

Peptide binding and lymph node T cell activation studies have been used to characterize T cell recognition of an encephalitogenic T cell autoantigen from myelin basic protein in mice of the H-2u haplotype. An important role for MHC class II molecules in "determinant selection" is revealed. Amino acids which determine interactions with either the restriction element of the major histocompatibility complex (MHC) or the encephalitogenic T cell receptor are defined. This information enables the design of peptides which bind MHC yet do not crossreact with the autoantigen. Two such peptides compete with the autoantigen for binding to the disease associated class II molecule and inhibit induction of experimental autoimmune encephalomyelitis in H-2u mice. Prospects for peptide mediated therapy are discussed.

Antigen-specific tolerance induction and the immunotherapy of experimental autoimmune disease.

Antigen-specific tolerance induction is the ultimate goal for specific immunotherapy of autoimmune diseases. Here we will discuss recent experiments designed to induce tolerance following mucosal administration of antigens in a mouse model of experimental autoimmune encephalomyelitis (EAE). We were unable to induce oral tolerance either with whole myelin, myelin basic protein (MBP) or the immunodominant peptide antigen. Oral tolerance was possible, however, with an analogue of the immunodominant peptide modified to increase its affinity for the restricting major histocompatibility complex (MHC) antigen. By contrast, intranasal deposition of peptide antigen proved highly effective for both prevention and treatment of EAE. Prevention of disease was directly related to the antigenic property of the peptide which, in itself, was related to affinity for MHC. Notably, administration of a single peptide was shown to inhibit disease involving multiple epitopes. We investigated the resulting bystander regulation by studying the cellular basis of peripheral tolerance in a transgenic model. These studies indicate that bystander regulation may be the consequence of selective cytokine secretion.

Mucosal tolerance in a murine model of experimental autoimmune encephalomyelitis.

Attempts to induce oral tolerance in a murine model of EAE with either the dominant T-cell epitope or whole protein have failed. These results may, in part, be due to the extraordinarily low affinity for class II MHC displayed by the dominant T-cell epitope. This belief is supported by experiments using a high-affinity analogue of the peptide that was capable of inducing tolerance at a high dose. By contrast, peptide inhalation has proven an effective route for induction of mucosal tolerance in this model. Most importantly, the inhalation of a single peptide could inhibit disease induced by the complex mixture of antigens found in whole myelin. Peptide inhalation was effective both before and after disease induction, and there was a positive correlation between affinity of class II binding and tolerogenicity of a panel of analogues of the N-terminal peptide of myelin basic protein.

The recognition of influenza A virus- infected cells by cytotoxic T lymphocytes.

Cytotoxic T cells (Tc) are important in the immune destruction of virus-infected cells. Recent studies, reviewed here by David Wraith, have disclosed much about the proteins of inlfuenza virus that are recognized by Tc and the nature of the recognition process. These findings have implications for an understanding of antigen presentation and the design of influenza vaccines.

Induction of antigen-specific unresponsiveness with synthetic peptides: specific immunotherapy for treatment of allergic and autoimmune conditions.

Current drug-based therapies for autoimmune and allergic conditions are non-specific and often associated with severe side effects. Recent advances in our knowledge of how T cells see antigens points to an improved strategy. T lymphocytes recognise processed forms of antigen which can be mimicked by synthetic peptides designed and built in the laboratory. It is clear from recent work that these synthetic peptides, when given systemically in solution, induce a state of hyporesponsiveness in naive T cells thereby specifically preventing a subsequent immune response. Moreover systemic administration of soluble peptides can inhibit ongoing immune responses. Taken together this new information offers great promise for future development of antigen-based drugs for the treatment of autoimmune and allergic diseases.