The immune system is equipped with mechanisms to prevent the activation of lymphocytes with specificity for self antigens. The potential consequence of a breakdown in these mechanisms is the initiation of autoimmune disease. Results from a compelling number of studies have suggested that tolerance to many self, as well as to many exogenous, antigens is regulated by CD8+ T cells and the antigen-specific soluble factors they produce. The nature of CD8+ T cells which function solely to suppress immune responses and the soluble factors they produce has not, however, been clearly defined. This laboratory has demonstrated the activity of CD8+ T cells in down-regulating T cell-mediated responses to dinitrophenylated (DNP-) self determinants. These regulatory T cells (Ts) produce DNP-specific/class 1 MHC-restricted soluble molecules which mediate the immunoregulatory effect. Serological and molecular results in this model have suggested that the regulatory molecules produced by these Ts are related to the alpha/Beta T cell receptor (TcR). The long term objective of this project is to clearly define the relationship between these soluble molecules and the alpha/Beta TcR on the surface of the Ts. The first goal of this proposal is to determine the role of the TcR alpha chain gene in encoding these soluble immunoregulatory molecules. The alpha chain gene will be cloned using a cDNA library from a Ts hybridoma that constitutively produces a DNP/H- 2Kd-specific soluble immunoregulatory molecule (SSF) and uses the Valpha4 and VBeta8.2 gene segments to encode its surface receptor. Full-length Valpha4/Calpha cDNA clones will first be subcloned into an expression vector and then transfected into TcR alpha chain- deletion mutants of the parental Ts hybridoma. The ability of the TcR alpha chain cDNAs to reconstitute the expression of the surface TcR and production of SSF will be assessed. The results will indicate if the TcR alpha chain gene encodes one of the chains of these soluble molecules. In a case where this is found to occur, the results may also give some indication of how the alpha chain is produced as a soluble peptide. The second goal of this proposal is to compare the molecular nature of the surface receptor and the soluble immunoregulatory molecule. The molecular characteristics of each isolated molecule will be determined including: 1) molecular weight; 2) subunit structure; 3) antigenic determinants in immunoblotting studies; and 4) extent and types of glycosylation. Furthermore, peptide mapping studies will be performed in order to define structural differences in the two proteins. The results from this study will not only clearly establish the relationship between the surface TcR and the soluble molecule but will also define the molecular nature of an antigen/MHC-specific immunoregulatory molecule. In the event that a relationship between the surface TcR and SSF is established, the results will be important to further understanding of the synthesis and biology of the alpha/Beta TcR. The results generated from these studies should also be useful in engineering the manipulation of immune response to antigenic determinants other than DNP.

National Institute of Health (NIH)
National Cancer Institute (NCI)
First Independent Research Support & Transition (FIRST) Awards (R29)
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Immunological Sciences Study Section (IMS)
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Cleveland Clinic Lerner
United States
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