Selective reactivity of CD8-independent T lymphocytes to a cytotoxic T lymphocyte-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-2Kb structural mutants which are resistant to lysis by a Kb-specific alloreactive CTL clone. In this study we describe the Kb 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 Kb expression, R8.60.14 is not recognized by CD8-dependent, Kb-specific CTL. DNA sequence analysis revealed a single Glu----Lys amino acid substitution at position 222 in the Kb alpha 3-domain of this variant. To determine if a direct correlation exists between CD8 dependence of a Kb specific CTL and its failure to respond to R8.60.14, we examined the lytic response against R8.60.14 by CD8-independent, Kb-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 Kb-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 Kb alpha 3 domain. The functional importance of this Glu222 residue for the interaction between the CD8 molecule on CD8-dependent CTL and the Kb 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.
Shepherd
S E
SE
Department of Microbiology, West Virginia University Health Sciences Center, Morgantown 26506.
Sun
R
R
Nathenson
S G
SG
Sheil
J M
JM
D000818:Animals; D016827:CD8 Antigens / Q000502:physiology*; D003602:Cytotoxicity, Immunologic; D006183:H-2 Antigens / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D013329:Structure-Activity Relationship; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D016827:CD8 Antigens; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse