T lymphocytes recognize and respond to foreign antigens which have been processed and presented on cell surfaces in association with proteins of the major histocompatibility complex (MHC). As a consequence of this MHC- restricted specificity, most mature T cells are alloreactive, that is, able to react on non-self MHC molecules on tissue allografts and in mixed lymphocyte reactions. The molecular basis of allorecognition is a crucial concept in cancer biology, transplantation biology, and in immunologic understanding of the T cell receptor (TCR), a clonotypic immunoglobulin- like cell surface glycoprotein heterodimer which subserves T cell recognition of antigen/self-MHC complexes and foreign MHC allotypes. The goals of the proposed project are to determine by molecular cloning the structures of T cell receptors from a series of alloreactive human cytotoxic T cell clones specific for the MHC Class I protein HLA-A2. Clones which recognize the male-specific antigen H-Y and are HLA-A2 restricted will also be analyzed. Some clones are available already and their analysis is underway; additional clones will be generated. An innovative rapid cDNA cloning method employing the polymerase chain reaction (PCR), described in the proposal, should greatly facilitate molecular cloning and sequence analysis of a large panel of HLA-A2 specific TCRs. Sequence comparisons of this type have not yet been achieved in humans and should offer unique insights into the structural determinates of HLA-A2 allorecognition, and its related phenomenon HLA-A2 restriction. Structure-function models inspired by the structural data will be tested by stably transfecting receptor-negative T cell mutants with isolated native and specifically in vitro mutagenized TCR-alpha and -beta cDNAs. A system will also be established for transient cell-surface expression of the TCR- alpha/beta heterodimer on COS monkey kidney cells which stably express the invariant CD3 complex. This work will enable molecular cloning of any additional structural components of the TCR/CD3 complex which may be required for surface expression, and it may facilitate development of an elegant rapid assay system for testing specificity of cloned TCR-alpha/beta pairs. High level in vitro TCR-alpha/beta chain expression will be undertaken in CHO cell and insect cell expression systems to facilitate production of monoclonal antibodies as analytical reagents and with the hope of determining the X-ray crystal structure of an HLA-A2 specific TCR heterodimer. The latter goal take s impetus from the recently published crystal structure of the HLA-A2 molecule.
| Hall, C G; Sancho, J; Terhorst, C (1993) Reconstitution of T cell receptor zeta-mediated calcium mobilization in nonlymphoid cells. Science 261:915-8 |
