Co-ordinator report, Juvenile Diabets Research Foundation (JDRF), Innovative USA Grant - "Role of T1DM-associated CTLA-4 Polymorphisms on protein Trafficking and Compartmentalization"

Final Report - Increasing evidence both in humans and in animal models, has shown that defect(s) in immunoregulatory mechanisms contribute to the pathogenesis of type 1 diabetes (T1DM); however the molecular basis and defects of this impaired immunoregulation have not been elucidated. CTLA-4 has an important role in the regulation of T lymphocyte activation. Interestingly, some polymorphisms of CTLA-4 are more frequent in diabetic patients as demonstrated by genetic studies. The main goal of this project is to study the role of these gene variations on the function of the CTLA-4 product. In other words we are interested in evaluating whether modifications in the leader peptide or in the 3' untranslated region (UTR) of the CTLA-4 gene may have an effect on the immune response of T lymphocytes. Using several molecular and cellular approaches we are studying the functional role of polymorphisms of the CTLA-4 gene that are associated with diabetes. CTLA-4 mutants were produced to study the effect of these variations on the stability of the transcribed messenger RNA and the localization in the cell of both the RNA and the CTLA-4 gene product. CTLA-4 expression on the cell surface of T cells should be tightly regulated because, together with other molecules, CTLA-4 modulates the immune response. Thus modifications of the levels of CTLA-4 expression or sequestration into the cell may have important effects on T cell activation and function. Preliminary results suggest that the polymorphic allele of CTLA-4 in the leader sequence that is more frequent in the diabetic patients interferes with the localization of CTLA-4 on the surface of T cells. Decreased levels of CTLA-4 on the cell surface may thus dysregulate the function of the molecule in T cell activation. Similar results have recently been reported by Anjos et al., 2002. Moreover, in vitro studies indicate that also the polymorphism in the 3'UTR of the CTLA-4 messenger RNA may have an important effect either on the stability or on the translation of the CTLA-4 mRNA. Both alterations could lead to a decrease in the level of CTLA-4 protein expression and to alterations of the immune response. Our future plans are to use several biochemical and molecular biology approaches to evaluate the effect of the A/T repeat alleles in the 3'UTR of CTLA-4 associated to diabetes, on CTLA-4 mRNA and protein expression. Genetic studies strongly point to an association of the CTLA-4 gene with T1DM and other autoimmune diseases. Defects in immunoregulatory mechanisms play an important role in the development of autoimmune diseases. A crucial goal for future efforts in the genetics of T1DM will be the transition from association studies to the identification of disease genes and functional characterization of disease pathways. For these reasons we strongly believe that studies of the effect of CTLA-4 gene variations on its biological function are going in the right direction and should contribute to the understanding of immunoregulation and disease development.

LAY ABSTRACT - Objective Alterations in the immune response contribute to the pathogenesis of type 1 diabetes (T1DM). CTLA-4 has an important role in the activation of T lymphocytes: Interestingly some variations of this gene are more frequent in diabetic patients as demonstrated by several genetic studies. The main goal of this project is to study the role of these variations on the funcion of the CTLA-4 product. In other words we are interested in evaluating whether modification in the gene may have an effect on the immune response of T lymphocytes. Background/Rationale T1DM is a polygenic disease caused by the immune-mediated destruction of islet insulin-secreting-cells. Genetic predisposition, enviromental factors and immune (dys) regulation participate in the development of autoimmune diseases. These complex diseases are not caused by a defect in a single gene, as in cystic fibrosis, rather they arise from the interaction of multiple genes and enviromental factors. Apart from HLA that regulates the specificity of the immune response, genetic studies indicate the presence of a disease region on chromosome 2q33 associated with T1DM and other autoimmune diseases in several populations. CTLA-4 gene located in this region has been intensively studied and evidence of its association with T1DM as well with other autoimmune diseases is sufficiently established. It is a challenge to study the effect of T1DM-associated CTLA-4 variations on its biological function and on the pathogenesis of diabetes. T cell activation results from the integration of signals generated through the receptor of T lymphocytes with those from additional positive and negative regulatory pathways such as CTLA4. Disruption of this balance leads to a defective immune response or alternative over-activation of the immune system as observed in several human diseases. Increasing evidence both in humans and in animal models have shown that defect(s) in immunoregulatory mechanisms underlie autoimmune diabetes, however the molecular basis and defects of this impaired immunoregulation have not been elucidated. Description of Project By using several molecular and cellular approaches we will study the variations of the CTLA-4 gene that are associated with diabetes. Several mutants will be produced to study the effect of these variations on the stability of the transcribed messenger RNA, the localization in the cell of both the RNA and the CTLA-4 gene product. CTLA-4 expression on the cell surface of T cells should be tightly regulated because, together with other molecules, CTLA-4 modulates the immune response. Thus modifications of CTLA-4 expression or sequestration into the cell may have important effects on T cell activation and function. Anticipated Outcome We should be able to evaluate the effect of disease associated CTLA-4 gene variations on the function of this important immunoregulatory molecule. These studies should contribute to the understanding of diabetes pathogenesis. Relevance to Type I Diabetes Genetic studies strongly point to an association of the CTLA-4 gene with T1DM and other autoimmune diseases. Defects in immunoregulatory mechanisms play an important role in the development of autoimmune diseases. A crucial goal for future efforts in the genetics of T1DM will be the transition from association studies to the identification of disease genes and functional characterization of disease pathways. For these reasons we strongly believe that studies of the effect of CTLA-4 gene variations on its biological function are going in the right direction and should contribute to the understanding of immunoregulation and disease development.