Identification of a new method to detect t cell response in type 1 diabetes

Type 1 diabetes (T1D) is the result of a T cell-mediated destruction of pancreatic beta cells in the islets of Langerhans. In diabetic animal models and humans, a correlation between disease onset and cytokines, in particular interferon (INF)-gamma has been observed and these studies support the promotion of inflammatory cellular immune responses by T lymphocytes. The development of T1D is also associated with the major histocompatibility complex (MHC) class II genes. An important role of the highly polymorphic HLA class II immune recognition molecules, DR and DQ, located on chromosome 6 has been consistently confirmed. In T1D, the specific epitopes recognized by pathogenic T cells remain poorly defined and the specific autoreactive T cells are difficult to detect due to the low number in the peripheral blood, the inhibition by populations of regulatory T cells and the inability to assay cells from the inflammatory lesion. Moreover, autoantigen reactive T cells are also detected in healthy individuals; therefore the development of new techniques to identify with high specificity beta cells specific T cells in T1D subjects remains a major goal in the study of the disease. The normal process of MHC class II antigen charging and presentation is highly controlled, in order to assure the presentation of selected peptides. MHC class II epitopes are normally bound to MHC class II molecules in a post-Golgi, antigenic peptide-binding compartment of antigen presenting T cells (dendritic cells, macrophages and B cells). The Ii protein, which is coded by one gene in all humans, forms a trimer at synthesis in the endoplasmatic reticulum. The CLIP (cleaved leupetin-induced peptide) peptide of the Ii protein remains on some MHC class II molecules and might facilitate charging of MHC class II molecules with antigenic peptides. A second immunoregulatory peptide, called the Ii-key peptide, also regulates antigenic peptide binding and release of antigenic peptides, even on cell surface-expressed MHC class II molecules. A peptide of the Ii protein, Ii (77-92; LRMKLPKPKPPKPVSQMR), enhanced in vitro presentation of antigenic peptides. The shortest sequence with half-maximal activity of the most potent peptide LRMKLPK contains only four amino acids (LRMK). These Ii-key peptides appear to act at an allosteric site on MHC class II molecules to facilitate charging and presentation of vaccine peptides into the antigenic binding site. The covalent linkage of the Ii-Key core sequence with the antigenic peptide appears to lead to an increased level of binding and recognition of antigenic determinants. This could occur due to increased representation of the antigenic epitope on the surface of the APC, or to some favorable structure change in the MHC antigenic peptide T cell receptor complex. Hybrids consisting of antigenic peptide-flexible-linker-Ii-Key peptide have up to 250 times the potency of the respective antigenic peptide in a T hybridoma response assay. In type 1 diabetes is widely assumed that the pathogenetic process is orchestrated by autoreactive T cells, but to date the studies can usually make use of peripheral blood, in which islet autoreactive T cells are likely to be rare and the epitopes remain poorly characterized. For these reasons, assays must be highly sensitive. The cytokine enzyme-linked immunosorbent spot (ELISPOT) assay has many of the requisite qualities. Detection sensitives as low as one responder cell per million have been claimed. ELISPOT assay can be used to detect early antigen directed activation of lymphocytes subpopulation at the single cell level. The main objective of this study was to investigate the cellular immune response in T1D by characterising the cytokines secreted by peripheral blood mononuclear cells (PBMCs) in response to the exposure to Ii-key/MHC class II insulin/GAD epitope hybrids. To date, this is the first study using the recently introduced novel technique of Ii-key/MHC class II epitope hybrids in diabetes. The first point of this study was to establish the validity of using Ii-Key/MHC class II insulin epitope hybrids to detect anti-insulin responses in patients with T1D. We focused first on MHC class-II presented epitopes in insulin because computer based analysis revealed a plethora of theoretical epitopes in the sequence of the insulin, which have been reported to contain active epitopes that stimulate responses by CD4+ T cells of type 1 diabetics. We identified five epitopes series. Within a homologous series, the longest and shortest hybrids were taken for initial synthesis plus the shortest peptide of 9 amino acids being considered to be a control. Peptides of insulin shown to be recognized by CD4+ T cells of T1D patients are selected from the literature. ELISPOT assay were performed with BD Pharmingen set for IFN-gamma according to the manufacturer's instructions. The study has been carried out in 23 patients affected by type 1 diabetes and in 2 controls. When sufficient cells were available, all epitopes series were analyzed. Initial data showed that some hybrids, and not the epitope-only peptide, stimulate some reactions in some patients. It has been noted that the responder rate was lower in type 1 diabetes of long-standing than in newly diagnosed patients. There was a strong positive response to specific T cell antigen (PHA or PMA). To confirm these preliminary data, we successively performed the study in 15 patients affected by T1D and in 10 healthy Caucasian nondiabetic control subjects. In our tests we added IL-12, because it’s a uniquely different cytokine that only acts on activated T cells. All cases and control subjects showed a detectable and significant INF-γ response to stimulation with the polyclonal T cell stimulus PMA/Ionomycin. Spontaneous production of INF-γ was present at similar, very low levels in both patients and healthy controls. Control subjects showed no detectable response to stimulation with Ii-key hybrids or peptides. Three out 15 patients (20%) demonstrated a positive response to one or more peptides compared with none of 10 nondiabetic control subjects tested. Tested peptides have been shown to be recognized in DR3/DR4 0302+ve diabetic patients, but not in other patients (DR3 or DR4+ve) or normal subjects. In the third part of the study we investigated the cellular immune response in T1D by characterising the cytokines secreted by peripheral blood mononuclear cells in response to exposure to Ii-key/MHC class II GAD epitope hybrids. We tested 8 patients affected by T1D and 7 control subjects. Two out 8 patients (25%) demonstrated a positive response to one or more Ii-Key/GAD65 hybrids compared to none of 7 nondiabetic control subjects. The Ii-key hybrid technique is a new strategy for augmenting the potency of MHC class-II restricted epitopes resulting in a profound increase in T cell stimulation. This study demonstrated in a first step that some Ii-key insulin hybrids and not the epitope-only peptide, stimulate strong reactivity in T1D subjects. As second step, we demonstrated a specific response to Ii-key/GAD65 hybrids only in T1D. The combined use of insulin and GAD key-hybrids might therefore assure a more selective and sensitive cytokine detection in response to antigen stimulation in T1D offering a new highly specific marker of cell mediated immunity in T1D.