The aim of the proposed studies is to analyze, at the molecular level, the fine specificity of immune response gene function in mice. Specifically, we will attempt to map the hypervariable regions on the T cell receptor Alpha and Beta genes that determine specificity for antigen and MHC restriction, respectively. Furthermore, we will design a model system to test whether an antigen-MHC molecule interaction takes place during antigen presentation. A more precise understanding of the immune response phenomenon is essential in order to gain direct insight in possible mechanisms of immune disorders causing human diseases, such as some types of cancer and AIDS (acquired immune deficiency syndrome). The proposed studies are based on our detailed analysis of the genetic restriction of T cell recognition of heterologous species of insulin at the cellular level. We have already established libraries of T cell hybridomas with precisely defined antigen epitope specificity and Ia epitope restriction. These T cell hybridomas will be used to clone the cDNAs coding for the T cell receptor Alpha and Beta chain and to study the function of specifically mutagenized Ia molecules. We will prepare cDNA libraries from T cell hybridomas with slightly different Ia epitope and/or antigen epitope recognition. We then will sequence the variable region of the Alpha and Beta gene coding for the T cell receptor in the various T cell lines. With gene transfection experiments we will test the requirement for a functional receptor molecule. The possibility exists that MHC restriction and antigen recognition can be scrambled by combining an Alpha chain of a specific T cell clone with a Beta chain from a T cell clone with diffeent antigen and/or Ia epitope recognition. Finally, we will analyze the mechanism of antigen presentation in conjunction with an Ia molecule; namely we will test Ia molecules that have been altered with site specific mutagenesis at a potential Ia-insulin interaction site. Ideally, only insulin presentation should be affected by such an alteration, while recognition by allo- or autoreactive clones should be left intact.
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