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Peptide p277 is a 24-amino acid fragment of the heat shock protein 60 molecule, first discovered to be an antigen for diabetogenic T-cell clones in non-obese diabetic (NOD) mice. Therapeutic vaccination with p277 can arrest the spontaneous diabetogenic process both in NOD mice and in humans associated with a T(h)1 to T(h)2 cytokine shift specific for the autoimmune T cells. We now report that p277 can directly signal human T cells via innate toll-like receptor (TLR)-2, leading to up-regulation of integrin-mediated adhesion to fibronectin, and inhibition of chemotaxis to the chemokine SDF-1alpha in vitro. Resting CD45RA(+) T cells responded to lower concentrations of p277 than resting CD45RO(+) T cells, but activation of CD45RO(+) T cells greatly increased their sensitivity to p277. Mouse T cells, but not macrophages, were also sensitive to the innate effects of peptide p277, and adoptive transfer of diabetes by splenic T cells from NOD mice could be inhibited by p277 treatment before transfer. Thus, T cells do respond innately to p277, and signaling by soluble p277 through TLR2 could contribute to the treatment of type 1 diabetes; p277 may stop the destruction of beta cells by signaling in concert both innate and adaptive receptors on T cells.

Naive non-obese diabetic (NOD/LtJ) mice spontaneously produce natural IgG autoantibodies against self-antigens associated with the experimental autoimmune diseases to which they are susceptible: insulin-dependant diabetes mellitus, systemic lupus erythematosus and experimental autoimmune encephalomyelitis. We discovered recently that NOD/LtJ mice also spontaneously produce IgG antibodies to the acetylcholine receptor (AchR), an antigen that can induce experimental autoimmune myasthenia gravis (EAMG) in susceptible rodents. However, there are no reports indicating that NOD/LtJ mice are susceptible to EAMG. To test whether the presence of spontaneous IgG autoantibodies can predict susceptibility to an autoimmune disease, we challenged NOD/LtJ mice using a standard protocol to induce EAMG. We now report that NOD/LtJ mice developed EAMG, although to a somewhat lesser degree than did C57BL/6 mice, a strain regarded as highly susceptible to the disease. Both strains produced comparable levels of immune antibodies to AchR of the complement-fixing isotypes IgG2a and IgG2b; however, NOD/LtJ mice produced significantly more IgG1. An antigen-specific T cell proliferative response to AchR of the same magnitude was detected in both strains, together with the secretion of similar amounts of IFN-gamma. Thus, NOD/LtJ mice are susceptible to EAMG and disease induction is accompanied by immune responses comparable to those seen in the susceptible strain C57BL/6. These results support the association between specific, natural IgG autoantibodies and susceptibility to the induction of a particular autoimmune disease.

T cells involved in autoimmune diseases have been characterized by the genetic elements used to construct their autoimmune TCR. In the present study, we sequenced the alpha and beta chains of the TCR expressed by a CD4(+) T cell clone, C9, functional in NOD mouse diabetes. Clone C9 can adoptively transfer diabetes or, when attenuated, C9 can be used to vaccinate NOD mice against diabetes. Clone C9 recognizes a peptide epitope (p277) of the 60 kDa heat shock protein (hsp60) molecule. We now report that the C9 TCR beta chain features a CDR3 peptide sequence that is prevalent among NOD mice. This CDR3 element is detectable by 2 weeks of age in the thymus, and later in the spleen and in the autoimmune insulitis. Thus, a TCR CDR3beta sequence appears to be a common idiotope associated with mouse diabetes.

The specificity of peptide binding to MHC molecules is defined by binding motifs composed of several relatively conserved anchor positions. The peptide binding motifs of murine MHC class II I-A molecules are functionally important but poorly characterized. Here we use peptide binding studies and isolation of naturally presented peptides to characterize the peptide binding motif of the MHC class II I-A molecule, RT1.B1, a molecule that is involved in experimental autoimmunity in the Lewis rat. We now report that, similar to other class II motifs, the RT1.B1 motif consists of a nonamer sequence with four major anchor positions (P1, P4, P6 and P9). Residues at P4 and P9, rather than at P1, appeared to be particularly important for binding. Negatively charged residues were favored at P9, consistent with the presence of a serine at position 57 of the RT1.B1 f) chain. This RT1.B1 motif could be observed in the dominant autoantigenic T cell epitopes mapped previously in the Lewis rat. These results highlight a general similarity and some important differences in the organization of MHC class II peptide binding motifs. The reported RT1.B1 motif should facilitate the prediction and design of T cell epitopes for the induction and control of experimental autoimmune diseases in Lewis rat models.

Co-stimulatory signals provided by surface receptors of antigen-presenting cells (APC) are crucial for the activation of CD4+ T cells, classically measured by cell proliferation or IL-2 secretion. The contribution of APC co-stimulatory signals to the acquisition of various effector functions by activated T cells is not fully understood. We have now examined the importance of surface-mediated co-stimulation by APC for activation of the effector potential of T cell clones mediating experimental allergic encephalomyelitis (EAE). We now report that T cell clones can be activated to produce EAE not only with APC but also by antibody-mediated TCR cross-linking in the presence of a mixture of T cell growth factors. Without activation, the T cell clones did not cause EAE. Therefore, at least some types of T cells can be activated to express their effector potential in the absence of any surface co-stimulatory signals requiring intact APC.

NOD mice spontaneously develop type I diabetes resulting from autoimmune destruction of their insulin-producing beta cells. Among the self-antigens targeted by NOD autoimmune T cells is a peptide, p277, from the sequence of the 60 kDa heat shock protein (hsp60). Common to the anti-p277 T cell populations of NOD mice is an idiotope, C9, that spans the CDR3 region of the C9 TCR. We now report: (i) that the C9 idiotope peptide can be presented directly to anti-C9 anti-idiotypic T cells by C9 T cells, (ii) that spontaneous anti-C9 anti-idiotypic T cell activity falls as disease progresses, but immunization can activate the anti-idiotypic T cells to regulate the autoimmune process, (iii) that the anti-idiotypic T cells secrete IFN-gamma, but appear to control the disease by down-regulating the IFN-gamma produced by the pathogenic population of anti-p277 T cells, (iv) that intrathymic administration of the C9 idiotope peptide at 1 week of age can accelerate the disease, and (v) that administering the p277 target peptide can up-regulate the anti-idiotypic T cells and arrest the disease process. Thus, the development of NOD diabetes can be regulated by a balance between anti-idiotypic and anti-target peptide autoimmunity, and anti-idiotypic regulation can lead to changes in the cytokine secretion of the autoimmune T cells involved in the disease process.

To effect the molecular integrity of the individual, the immune system has to gather antigenic information and respond in a meaningful way. Immunologists traditionally have focused their research on analyzing the component parts of the system. The achievements of immunology in its analytical enterprise have now made it possible to begin the task of synthesis. In this paper, we shall consider how the immune system combines germline and somatic information using a chemical language to establish the functional meaning of antigens.

Inflammation is the clinical expression of chemical mediators such as the pro-inflammatory cytokine tumor necrosis factor (TNF-)-alpha produced by macrophages and other cells activated in the immune response. Hence, agents that can inhibit TNF-alpha may be useful in treating arthritis and other diseases resulting from uncontrolled inflammation. We now report that the cleavage of heparin by the enzyme heparinase I generates sulfated disaccharide (DS) molecules that can inhibit the production of TNF-alpha. Administration of nanogram amounts of the sulfated DS molecules to experimental animals inhibited delayed-type hypersensitivity to a skin sensitizer and arrested the joint swelling of immunologically induced adjuvant arthritis. Notably, the sulfated DS molecules showed a bell-shaped dose-response curve in vitro and in vivo: decreased effects were seen using amounts of the DS molecules higher than optimal. Thus, molecular regulators of inflammation can be released from the natural molecule heparin by the action of an enzyme.

The MHC class II molecule of the non-obese diabetic (NOD) mice, I-Ag7, is associated with susceptibility to autoimmune diabetes. To try to understand the molecular basis of this association, we analyzed the peptide binding properties and intracellular behavior of I-Ag7 in comparison with other I-A haplotypes. We found that I-Ag7 molecules manifested normal intracellular trafficking and lifespan, and a small but clearly detectable fraction of I-Ag7 in the cells formed SDS-resistant compact dimers. The binding of an antigenic reference peptide to I-Ag7 was stable and was accompanied by compact dimer formation. Our analysis of the binding specificity of I-Ag7 revealed a peptide binding motif of nine amino acids with a degenerate position at P1 and three conserved anchor positions: P4, P6 and P9. An allele-specific preference for negatively charged residues was found at P9, apparently due to the presence of the rare Ser residue at position 57 of the I-Ag7 beta chain. These findings could have implications for the mechanisms of MHC-mediated susceptibility to autoimmune diabetes in the NOD mice.

The injection of syngeneic activated T cells into rodents can induce a T cell response against activation markers of the T cells, ergotopes. The responding anti-ergotypic T cells have been shown to suppress experimental autoimmune encephalomyelitis (EAE). This paper reports the characteristics of the anti-ergotypic response. It was found that irradiated activated T cells were as good as untreated living activated T cells in inducing anti-ergotypic cells in vivo. Glutardialdehyde-fixed (0.3%) cells were poor stimulators in vivo and non-stimulatory in vitro. Dilution of glutardialdehyde to 0.003% before fixation preserved the stimulatory capacity in vitro. Fixation or irradiation of T cells at different times after activation showed that the stimulatory ergotope appears only after more than 12 h of activation. This ergotope is not secreted by activated T cells, but is a structural component of the activated T cell. Injection of solubilized proteins from activated T cells, but not of supernatants from activated T cells, was able to induce an anti-ergotypic response in vivo. In vitro supernatants from activated T cells also were not stimulatory to anti-ergotypic T cells. The anti-ergotypic response could be measured in draining lymph nodes 3 days after injection, reached a maximum after 7-10 days and subsided thereafter. It was earlier and stronger than the anti-idiotypic response. Induction of the response was dose dependent. As few as 100 cells were able to induce a marked anti-ergotypic response. The ease of the induction and the strength of the anti-ergotypic response suggest a physiological role in immunoregulation.