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 ot helices of the Ld gene with the corresponding sequences from the Kb gene. Serologic analysis of transfected L cells that expressed the chimeric molecule (KbαLdβ) revealed that the engineered class I glycoprotein retains two conformational epitopes associated with the α helices of Kb, as defined by monoclonal antibodies K10.56 and 28-13-3. These results demonstrate that the α helices of Kb can associate with the β-pleated sheets of Ld to form a stable structure, which is expressed on the cell surface. To address the role of the ot helices of the ARS in determining T cell crossreactivity, alloreactive cytotoxic T lymphocytes (CTL) were used to analyze L cells expressing KbαLdβ. CTL raised against Kb or Ld as alloantigens showed little, if any, ability to lyse L cells expressing KbαLdβ. Thus, alloreactive CTL did not recognize structures determined by the ot helices alone or by the β sheets of the ARS alone. However, bulk and cloned alloreactive CTL that were generated against the mutant Kb glycoprotein Kbm8 reacted strongly with KbαLdβ. In addition to the Kb α helices, the Kbm8 ARS shares a single polymorphic amino acid at position 24 with KbαLdβ. 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 Kbm8 mutant alloantigenicity. The functional significance of this position in determining crossreactivity between bm8 and KbαLdβ identifies pocket B as a strong anchor for allogenic selfpeptides. These findings demonstrate that determinants recognized by CTL on class I aUoantigens 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.
ASJC Scopus subject areas
- Immunology and Allergy