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Mapping of the Epitope Recognized by Non‐Specific Cytotoxic Cells: Determination of the Fine Specificity Using Synthetic Peptides
Author(s) -
EVANS D. L.,
LEARY III J. H.,
WEISMAN Z.,
WARREN J.,
JASOFRIEDMANN L.
Publication year - 1996
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1046/j.1365-3083.1996.d01-81.x
Subject(s) - epitope , cytotoxic t cell , epitope mapping , computational biology , microbiology and biotechnology , biology , chemistry , immunology , antigen , biochemistry , in vitro
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.