Mapping of the epitope recognized by non-specific cytotoxic cells: determination of the fine specificity using synthetic peptides.
NCC recognize a conserved target cell antigen (NKTag) expressed on protozoan parasites and on transformed tumour cells. In the present study, synthetic peptides corresponding to N-terminal, C-terminal and internal NKTag(deduced) amino acid sequences were tested for binding and inhibition of NCC lysis of sensitive target cells. A 20-mer peptide equivalent to amino acids (aa) nos. 55-74 specifically inhibited NCC lysis of human EBV transformed target cells (IM-9). Inhibitory effects were nonreversible and concentration dependent, and 30 min pre-incubation produced optimum inhibition. The inhibitory 20-mer peptide was truncated into 17, 14, 10, 9 and 6-mer peptides and tested for inhibition of cytotoxicity. All produced almost complete inhibition except the 6-mer which had no activity. The NKTag sequence required for NCC binding (minimally) consisted of seven amino acids [aa nos 68-74 (ARG-ASN-LEU-THR-PHE-ILE-LEU-)]. The specificity of inhibition and the distribution of target cells expressing NKTag was determined. A 14-mer peptide composed of aa nos 61-74 inhibited lysis of HL-60, IM-9, DAUDI, YAC-1, U937 and NC-37 target cells. Flanking peptides (aa nos 35-54 and 75-94) were negative. Biotinylated aa nos 61-74 bound to NCC effector cells. The recognition requirements for aa sequence versus aa content were determined. Randomization of the aa in the cognate 9-mer obliterated the inhibitory effects. The 17-mer (cognate) synthetic peptide inhibited conjugate formation between NCC and IM-9 targets. These data demonstrate that NCC recognize a conserved antigen determinant on susceptible target cells consisting of a minimum of 7-9 amino acids in the N-terminal region of NKTag.
Evans
D L
DL
Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens 30602, USA.
Leary
J H
JH
Weisman
Z
Z
Warren
J
J
Jaso-Friedmann
L
L
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D018604:Epitope Mapping; D000939:Epitopes; D005260:Female; D005434:Flow Cytometry; D007059:Ictaluridae; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000596:Amino Acids; D000939:Epitopes; D010455:Peptides