ALLOREACTIVE CYTOTOXIC LYMPHOCYTES-T RECOGNIZE EPITOPES DETERMINED BY BOTH THE ALPHA-HELICES AND BETA-SHEETS OF THE CLASS-I PEPTIDE BINDING-SITE

A chimeric class I glycoprotein was created to investigate the functional contribution of the alpha-helices and the beta-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 alpha-helices of the L(d) gene with the corresponding sequences from the K(b) gene. Serologic analysis of transfected L cells that expressed the chimeric molecule (K(alpha)(b)L(beta)d) revealed that the engineered class I glycoprotein retains two conformational epitopes associated with the alpha-helices of K(b), as defined by monoclonal antibodies K10.56 and 28-13-3. These results demonstrate that the alpha-helices of K(b) can associate with the beta-pleated sheets of Ld to form a stable structure, which is expressed on the cell surface. To address the role of the alpha-helices of the ARS in determining T cell crossreactivity, alloreactive cytotoxic T lymphocytes (CTL) were used to analyze L cells expressing K(alpha)(b)L(beta)d. CTL raised against K(b) or L(d) as alloantigens showed little, if any, ability to lyse L cells expressing K(alpha)(b)L(beta)d). Thus, alloreactive CTL did not recognize structures determined by the alpha-helices alone or by the beta-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(alpha)(b)L(beta)d. In addition to the K(b) alpha-helices, the K(bm8) ARS shares a single polymorphic amino acid at position 24 with K(alpha)(b)L(beta)d. Amino acid 24 is located on the beta-2 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(alpha)(b)L(beta)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 a helices and beta-sheets of the ARS. These interactions are best explained by the influence of the alpha-helices and beta-sheets on the peptide-binding properties of these antigen-presenting molecules.