Autoantibodies and associated T-cell responses to determinants within the 831-860 region of the autoantigen IA-2 in Type 1 diabetes.

B-cells influence T-cell reactivity by facilitating antigen presentation, but the role of autoantibody-secreting B-cells in regulating T-cell responses in Type 1 diabetes is poorly defined. The aims of this study were to characterise epitopes on the IA-2 autoantigen for three monoclonal antibodies from diabetic patients by amino acid substitutions of selected residues of IA-2, establish contributions of these epitopes to binding of serum antibodies in Type 1 diabetes and relate B- and T-cell responses to overlapping determinants on IA-2. The monoclonal antibodies recognised overlapping epitopes, with residues within the 831-860 region of IA-2 contributing to binding; substitution of Glu836 inhibited binding of all three antibodies. Monoclonal antibody Fab fragments and substitution of residues within the 831-836 region blocked serum antibody binding to an IA-2 643-937 construct. IL-10-secreting T-cells responding to peptides within the 831-860 region were detected by cytokine-specific ELISPOT in diabetic patients and responses to 841-860 peptide were associated with antibodies to the region of IA-2 recognised by the monoclonal antibodies. The study identifies a region of IA-2 frequently recognised by antibodies in Type 1 diabetes and demonstrates that these responses are associated with T-cells secreting IL-10 in response to a neighbouring determinant.

Autoantibodies to GAD65 and IA-2 in canine diabetes mellitus.

Diabetes mellitus in dogs shares many characteristics with the human type 1 disease and virtually all diabetic dogs require insulin therapy to control hyperglycaemia. Insulin deficiency is suspected to result from immune-mediated destruction of pancreatic beta cells in some cases. Human patients suffering from Type 1A (immune-mediated) diabetes or latent autoimmune diabetes of the adult (LADA) demonstrate circulating autoantibodies against the 65kDa isoform of glutamic acid decarboxylase (GAD65) and/or insulinoma antigen-2 (IA-2). The aims of the current study were to develop radio-immunoassays to detect serum antibodies against recombinant canine GAD65 and IA-2 and to identify diabetic dogs showing serological evidence of autoreactivity to these pancreatic beta cell antigens. Canine GAD65 and the 3' end of IA-2 (coding for amino acids 771-979 of the intracellular domain) were amplified by PCR from cDNA prepared from canine insulinoma tissue and cloned into the pCRII vector. The canine sequences were later confirmed by identifying GAD2 and PTPRN genes from the dog genome assembly. Recombinant (35)S-methionine-radiolabelled canine GAD65 and IA-2 (771-979) proteins were used in radio-immunoprecipitation assays to screen sera from 30 newly diagnosed diabetic dogs and 30 control dogs. Four of 30 canine diabetic patients had significant GAD65 autoreactivity (p<0.01) compared to controls and 3 dogs were positive for autoantibodies to IA-2 (771-979). Two diabetic dogs showed dual autoantigen reactivity. These preliminary data indicate that serological reactivity to GAD65 and IA-2 is present in a proportion of diabetic dogs and suggests that, in some cases, canine diabetes is associated with an autoimmune response to these antigens.

Antigen presentation by MHC class II molecules.

The treamendous explosion in the field of MHC research in the last 5 years has significantly advanced our understanding of antigen processing pathways, particularly with regard to details of MHC class II-mediated antigen presentation. MHC class II molecules at the surface of antigen presenting cells present antigenic peptides to CD4+ T helper cells. However for effective cell surface antigen presentation, a number of highly synchronized events must first take place intracellulary. The monomorphic protein, invariant chain (Ii), is a crucial participant in MHC class II antigen presentation. Acting as a molecular chaperone, this molecule escorts the newly synthesized class II heterodimers from the endoplasmic reticulum into the endosomal system. During this manoeuvre, the interaction of li with class II serves to prevent premature association of antigenic peptide. Once the complex reaches the acidic environment of the endosomes, li is proteolytically degraded and dissociates, leaving the class II binding site available for binding antigenic peptide derived from exogenous proteins. The final Ii fragment to be displaced. CLIP (class II-associated invariant chain peptides), must be physically removed from the class II binding groove with assistance from another MHC-encoded molecule, DM. The interaction of DM with class II also aids in the subsequent rapid loading of high-affinity antigen-derived peptides into the MHC class II groove. The stable peptide-loaded complexes are now ready to exit the endocytic compartments to present their peptide antigen to specific T helper cells at the cell surface.

A continuous central motif of invariant chain peptides, CLIP, is essential for binding to various I-A MHC class II molecules.

Invariant chain (li) associates with MHC class II molecules and performs a number of crucial functions in antigen presentation. A nested set of class II-associated li peptides (CLIP) has been isolated, comprising the li sequence between residues 82 and 107. Recently, X-ray crystallographic analysis has revealed that residues 87-101 occupy the HLA-DR3 peptide-binding groove. Based on our previous results, Lee and McConnell have also proposed a model for the binding of CLIP to various mouse I-A molecules in the binding groove. CLIP sequences are able to bind many MHC class II molecules but the molecular basis of this promiscuity has not yet been resolved. We have shown recently that CLIP binding to I-A class II molecules is generally tolerant to side chain substitutions, suggesting that the backbone structure of CLIP may provide the features critical for its interaction with class II. In pursuit of this, backbone stereochemical disruptions by serial D-alanine substitutions in CLIP86-104 have been used in competitive binding assays to I-A class II molecules. These studies have revealed that the phylogenetically conserved central continuous region, CLIP91-99, is intolerant to such configurational substitutions. Experiments with truncated and frame-shift analogues of CLIP showed that for effective binding to class II, the sequence element CLIP90-100 must be incorporated into a peptide of 13 or more residues including at least three residues N-terminal to this motif. Additionally, it appears that different I-A molecules accommodate CLIP in different binding frames. These investigations of the relationship between the structure and binding of CLIP analogues lead us to propose that there is a general backbone motif of a periodic nature within the CLIP sequence that minimizes deleterious contacts and allows promiscuous binding to class II molecules.