SELECTIVE REACTIVITY OF CD8-INDEPENDENT LYMPHOCYTES-T TO A CYTOTOXIC LYMPHOCYTES-T SELECTED H-2KB MUTANT ALTERED AT POSITION-222 IN THE ALPHA-3 DOMAIN

To study the structural basis for specificity and affinity of cytotoxic T lymphocytes for major histocompatibility complex/peptide complexes, we have employed a cytotoxic T lymphocyte (CTL)-mediated immunoselection approach to obtain H-2K(b) structural mutants which are resistant to lysis by a K(b)-specific alloreactive CTL clone. In this study we describe the K(b) structural mutant, designated R8.60.14, recovered following immunoselection using the CD8-dependent CTL clone 60 as a selective agent. Although serologically unaltered with respect to K(b) expression, R8.60.14 is not recognized by CD8-dependent, K(b)-specific CTL. DNA sequence analysis revealed a single Glu --> Lys amino acid substitution at position 222 in the K(b) alpha-3 domain of this variant. To determine if a direct correlation exists between CD8 dependence of a K(b) specific CTL and its failure to respond to R8.60.14, we examined the lytic response against R8.60.14 by CD8-independent, K(b)-specific CTL obtained from long-term culture in the presence of anti-CD8 monoclonal antibody, 3.155. CD8-independent CTL exhibit no difference in their response against the R8 parent and R8.60.14 variant. This study demonstrates unequivocally that K(b)-specific recognition of R8.60.14 by CD8-independent CTL is unaltered, while the response by CD8-dependent CTL is completely abrogated. Thus, the sole basis for emergence of this variant in the CTL-mediated immunoselection approach used in this study resides in the alteration of a single CD8-binding site residue at position 222 in the K(b) alpha-3 domain. The functional importance of this Glu222 residue for the interaction between the CD8 molecule on CD8-dependent CTL and the K(b) alpha-3 domain is further reinforced by virtue of the recovery of the R8.60.14 variant on the basis of its resistance to lysis by a CD8-dependent CTL clone in this CTL-mediated immunoselection approach.