Alloreactive cytotoxic T lymphocytes recognize epitopes determined by both the α helices and β sheets of the class I peptide binding site

A chimeric class I glycoprotein was created to investigate the functional contribution of the α helices and the β-pleated sheets in forming the antigen recognition site (ARS) of antigen-presenting molecules. This novel molecule was generated by replacing the DNA sequences encoding the α helices of the L^d gene with the corresponding sequences from the K^bk gene. Serologic analysis of transfected L cells that expressed the chimeric molecule (K_α^bL_β^d) revealed that the engineered class I glycoprotein retains two conformational epitopes associated with the α helices of K^b, as defined by monoclonal antibodies K10.56 and 28-13-3. These results demonstrate that the α helices of K^b can associate with the β-pleated sheets of L^d to form a stable structure, which is expressed on the cell surface. To address the role of the a helices of the ARS in determining T cell crossreactivity, alloreactive cytotoxic T lymphocytes (CTL) were used to analyze L cells expressing K_α^bL_β^d. CTL raised against K^b or L^d as alloantigens showed little, if any, ability to lyse L cells expressing K_α^bL_β^d. Thus, alloreactive CTL did not recognize structures determined by the α helices alone or by the β sheets of the ARS alone. However, bulk and cloned alloreactive CTL that were generated against the mutant K^b glycoprotein K^bm8 reacted strongly with K_α^bL_β^d. In addition to the K^b α helices, the K^bm8 ARS shares a single polymorphic amino acid at position 24 with K_α^bL_β^d. Amino acid 24 is located on the β2 strand that forms part of the floor of the ARS and has been identified as a component of pocket B in the HLA class I ARS. The substitution of Glu to Ser at this position was shown previously to be the central determinant of the K^bm8 mutant alloantigenicity. The functional significance of this position in determining crossreactivity between bm8 and K_α^bL_β^d identifies pocket B as a strong anchor for allogenic self-peptides. These findings demonstrate that determinants recognized by CTL on class I alloantigens are formed by interactions involving both the α helices and β sheets of the ARS. These interactions are best explained by the influence of the α helices and β sheets on the peptide-binding properties of these antigen-presenting molecules.