ANTIGEN RECEPTOR GENES FROM GAT-SPECIFIC TH AND TS CELLS

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The long-term goal of this project is the characterization of the antigen-receptors used by the family of murine B lymphocytes and T lymphocytes involved in the immune response to a single antigen. We will isolate genes encoding antigen-binding polypeptides from both helper (TH) and suppressor (TS) T cells specific for the polypeptide antigen, GAT. Several classes of GAT-specific lymphocytes have been cloned, including B cells whose primary function is the synthesis of GAT-binding immunoglobulin (Ig), helper T cells (TH) that function to augment GAT-Ig production, and two subsets of suppressor T cells (TS1 and TS2) that diminish the immune response to GAT. The genes encoding B-cell GAT-Ig have been isolated previously. To clone the receptor genes from GAT-TS cells, TS1 and TS2 RNA will be used in the construction of cDNA libraries. The cDNA will be cloned into Lambdagtll, an expression vector, and screened with 1) serological reagents to the I-J, idiotypic, and Tsu/Tind determinants on TS cells, 2) oligonucleotide probes complementary to the mRNA predicted from the sequences of secreted antigen-binding suppressive factors, and 3) subtractive probes enriched for TS-specific transcripts. Isolated cDNA clones that are transcribed only in T cells will then be confirmed using functional criteria. The receptor genes from GAT-TH cells will be isolated from a genomic library by hybridization to a TH-probe isolated from non-GAT-TH cells. The T cell receptor genes will be used to characterize possible DNA rearrangements, the genomic organization, and the mechanisms for generating diversity. The chromosomal locations of the genes encoding TS antigen receptors and the I-J, idiotypic, and Tsu/Tind determinants will be ascertained using mouse-hamster hybrid lines. Also, the receptor genes used by several different TH cells that bind GAT, but differ in their MHC restriction, will be compared. Initial studies of the regulation of transcription of the receptor genes in TH cells using various antigenic and cellular stimuli are also described. By comparing the genetic sequences used by the various lymphocyte classes to enclode GAT-binding polypeptides, we will be in a unique position to study the molecular bases of antigen recognition, MHC restriction, immune cellular interactions, and T and B lymphocyte activation. A better understanding of the molecular controls operating in the immune system is of primary importance in determining basic mechanisms and defects in immune disease states.