This is a first submission for an ongoing project submitted by an investigator who is interested in control of expression of the alpha gene of the T-cell antigen receptor (TCRa). The investigator notes that the particular V regions of the alpha genes are preferentially expressed in either CD4 or CD8 T-cell subsets. Interestingly, single amino acid substitutions in the complementarity determining region 1 (CDR1) or CDR2 of a V-alpha (Va) protein can change it from being selected into the CD4 subset to being selected into the CD8 subset. The investigator has shown, additionally, that certain of the alpha proteins are selected better on mutant MHC class I molecules which contain altered residues on the a-1 helix of this molecule. In this application, the investigator describes experiments to investigate the interaction of the Va3 TCRs with mutant forms of class I molecules using genetic techniques and biochemically using soluble protein and surface plasmon resonance. The investigator proposes to prepare cloned CTL expressing Va3.2 which recognizes a particular MHC class I (Kb)-peptide complex. These clones will then be tested for reactivity on Kb molecules which have mutations in residues that have been identified as important in thymic selection. Following this analysis, mutations will be prepared in the Va residues in order to test which parts of the TCR interact with particular regions of class I and how each contributes to CTL activity. Additionally, the investigator proposes to express the mutant Kb molecules and TCR molecules in soluble form to analyze the kinetics of their interaction via BiaCore to determine if there are changes in affinity which correlate with the mutations in both the TCR and MHC molecules.
Specific Aim 2 describes experiments to investigate the role of the TCRa locus in driving differentiation into either CD4 or CD8-positive cells. The investigator notes that while the TCRAb haplotype correlates with a higher level of CD8 cells, TCRAb haplotype correlates with high CD4/CD8 ratio. The investigator speculates that this effect is caused by polymorphisms in the individual Va elements within these loci that results in preferential selection into a class I or class II restricted subsets. Experiments are described which will investigate further this hypothesis.
Specific Aim 3 proposes to investigate the biology of allelic exclusion of the TCR a-chain locus. The investigator notes that although allelic exclusion is noted at the cell surface level, mRNA and intracellular protein often is expressed for both alleles of the a-chain. The investigator proposes to test the hypothesis that allelic exclusion occurs by transcriptional or translational down-regulation of one of the TCR a-chains or conversely is caused by competition between the two alpha-chains and the beta-chain. In addition, the investigator proposes to study whether or not allelic exclusion is maintained in the periphery and whether this correlates with activation state or location of T-cells.
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