Use of sequence-specific antisense oligodeoxynucleotides to determine the protozoan parasite antigen recognized by nonspecific cytotoxic cells

The antigen on the protozoan parasite Tetrahymena pyriformis recognized by catfish nonspecific cytotoxic cells (NCC) is a 46- to 48-kDa protein referr ed to as NKTag. The complete cDNA-derived amino acid sequence of NKTag has been obtained. The antigenic determinant of NKTag corresponding to the NCC binding site has been determined with synthetic peptides in target cell com petition experiments. To more directly characterize the mechanism of parasi te:effector cell interaction, we applied NKTag sequence-specific antisense oligodeoxynucleotides to Tetrahymena in vitro. NKTag mRNA translation by Te trahymena was blocked by specific antisense (AS) oligodeoxynucleotides. 5'- 3' sense (S) oligodeoxynucleotide sequences were synthesized corresponding to the first 17 N-terminal amino acids of NKTag tin addition to -2 untransl ated codons plus the start codon). Complimentary AS oligodeoxynucleotides w ere likewise synthesized. To determine the optimum in vitro conditions for AS treatment, we tested parasites at various phases of their growth cycle f or the effects of a single AS treatment. At 9 h post-AS treatment (during t he linear phase of the growth curve), maximum reduction in membrane express ion of NKTag was observed. Eighty-five percent of Tetrahymena were positive for expression of NKTag at 0 time post-AS treatment versus 13% positive at 9 h. Membrane expression of AS-treated parasites returned to normal levels by 24 h post-treatment. In cold target inhibition experiments, the reduced NKTag expression by Tetrahymena at 9 h AS treatment was confirmed by obser ving a complete inability (compared with S-treated parasites) to compete wi th IM-9 cells for binding with NCC. These data demonstrated a unique experi mental in vitro system to define the antigen determinant on target cells re sponsible for recognition by cytotoxic effector cells that participate in i nnate immune responses.