Human T cell receptors have now been defined on inducer, suppressor and class I and class II specific cytotoxic T lymphocytes as 90KD T3-associated clonotypic molecules. These are comprised of one 49-51KD Alpha and 43KD Beta subunit which are disulfide linked. Peptide map analysis studies suggested that each subunit is comprised of constant and variable domains. Moreover, the precise molecular basis for this variability in the Beta subunit has been defined by DNA cloning studies, showing that similar to immunoglobulin, specific V, D, J and C segments fuse to form an active Ti Beta gene. In the thymus, such disulfide linked heterodimers are restricted in expression to a minor population of cells which are surface T3+. However, the precise molecular details regarding synthesis of Alpha and Beta subunits and the existence of cytoplasmic and surface forms has not been defined due to lack of appropriate reagents capable of reacting with the individual isolated subunits. To understand in greater detail the nature of human T cell receptor expression during intrathymic ontogeny, we will address three areas: 1) production and characterization of anti-peptide antisera to constant regions of the Ti Alpha and Beta subunit employing both heterologous immunization and monoclonal antibody techniques; 2) analysis of intrathymic ontogeny of Ti Alpha and Beta subunits with specific probes; and 3) utilization of anti-peptide antisera to further delineate the structure of the T3-Ti molecular complex. Firstly, peptides corresponding to regions of individual subunits likely to be immunogenic based on Kyte-Doolittle and Hopps and Wood predictions will be synthesized and utilized for production of heteroantisera and monoclonal antibodies. Reagents will then be screened against the immunizing peptide as well as denatured isolated subunits and subsequently assessed for reactivity with the native molecules on the surface of thymocytes and T lymphocytes. Secondly, once monoclonal and heteroantisera reagents are defined, the intrathymic expression of the subunits will be examined. Specifically, it will be determined whether surface and intracytoplasmic forms of the Ti Alpha and Beta subunits exist and where they appear in intrathymic T cell differentiation relative to one another and known T cell surface molecules including T3, T4, T8, T11 and T6. In addition, the localization of cells expressing one or both subunits will be defined. Finally, these anti-peptide reagents will be utilized to investigate some of the structural features of the T3-Ti molecular complex.
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