Mutant study of the structure-function relationship of murine class I H-2K(b) molecule and the bound VSV peptide on the V-alpha and V-beta usage of the specific cytotoxic T cell repertoire

Abstract

Cytotoxic T lymphocytes (CTLs) recognize oligopeptides bound in the antigen binding grooves of the class I MHC molecules before they are activated to eliminate foreign cells or pathogen infected cells. To examine the structural requirements for proper MHC-peptide recognition by T cells, an MHC mutant selection strategy using CTL clones as selecting agents was developed to isolate murine H-2K{dollar}\sp{lcub}\rm b{rcub}{dollar} structural variants with single point mutations. Using PCR amplification of the cDNAs transcribed from the mutant K{dollar}\sp{lcub}\rm b{rcub}{dollar} RNAs and direct nucleotide sequencing of the PCR products, we have identified the mutations of 6 H-2K{dollar}\sp{lcub}\rm b{rcub}{dollar} mutants thus selected and mapped the mutations to the crystal structure of the K{dollar}\sp{lcub}\rm b{rcub}{dollar} molecule. By correlating these mutations with the change of phenotypes of the MHC structural variants, we have established that the TCR must interact with both the {dollar}\alpha{dollar} helices of the class I MHC molecules in order to recognize them; that mutations in the MHC antigen binding groove may disrupt CTL recognition, possibly through changing the peptides bound to the MHC molecule; and that a ridge on the MHC {dollar}\alpha{dollar}3 domain is responsible for the binding of CD8 molecule which is crucial for the recognition of class I MHC by most CTL clones. In testing 54 different CTL clones against a panel of 15 K{dollar}\sp{lcub}\rm b{rcub}{dollar} mutants, including the above selected mutants plus previous antibody selected variants, we have further established that TCRs contact the K{dollar}\sp{lcub}\rm b{rcub}{dollar} molecule within a narrow and diagonally stretching area on the top surface.;In the second project directed at understanding how TCRs recognize MHC-peptide antigens and the structural basis for the TCR V{dollar}\alpha{dollar} and V{dollar}\beta{dollar} usage, synthetic peptides were used to immunize C57BL/6 mice to initiate CTL responses. By analyzing the V{dollar}\alpha{dollar} and V{dollar}\beta{dollar} distributions within the various bulk CTL populations, we have found that a single residue change at position 4 of the VSV peptide is capable of drastically reducing the V{dollar}\alpha{dollar}2 and V{dollar}\beta{dollar}13 dominance found in the VSV specific CTL bulk. Thus as little as a single amino acid change in the peptide antigen may change the entire T cell repertoire. Based on this finding, we propose that the T cell repertoire can be strongly influenced by how the two subunits of TCRs would accommodate the peptide residues they bind; that TCR may bind the MHC-peptide antigen symmetrically; and the V regions of a TCR may define a framework within which the specificity of a TCR may be fine tuned by the two joining regions of the TCR subunits.