The Relationship between Mojor Histocompatibility Complex Class I Molccules and Natural Killer Cells

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1.NK cells were purified from a homozygous donor (A2402/, B0702/, Cw0702/) using a MiniMACS separation kit, and were immunized BALB/c mice three time biweekly. 2000 clones were screened using human NK cell line NK92 (A0301/A1101, B0702/B44031, Cw0702/Cw1602) by flow cytometry. We established 17 mAbs against the common antigens between human NK cells and NY cell line. Three mAbS(4-4-24-5.4-2-44-18.4-2-44-28) were IgG1 and reacted NK cells and a small part of T cells. mAb 4-4-24-5 partially inhibited the cytotoxicity of NK92 cells against K562 cells.2. Ly-49A is a member of the Ly-49 family of mouse NK cell receptors that inhibit cytotoxicity upon recognition of their ligands, the major histocompatibility complex (MHC) class I molecules on the target cell surface Although Ly-49A has an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic tail. The mechanisms underlying its inhibitory function are poorly understood. We demonstrate here that antibody-mediated co-ligation of B cell receptor (BCR) with transfected Lv-49A molecule results in abrogation of BCR-induced Interleukin-2 (IL-2) secretion and substantial reduction in activation of Erk 1/2 and p38 MAP kinases in B cell line A20. Surprisingly. BCR-induced calcium mobilization was unaffected by cross-l inking of BCR with Ly-49A.Furthermore. substitution of the single tyrosine residue in the ITIM with phenylatanine did not result in a complete loss of the inhibitory function, as measu red by BCR-induced IL-2 secretion. Deletion of the N-terminal 37 amino acid peptide which includes the ITIM did abrogate the inhibitory activity. Co-immunoprecipitation experiments revealed that, upon induction of tyrosine phosphorylation. Ly-49A rccrnits tyrosine phosphatase SHP-1 but not inositol phosphatase SHIP, and that the tyrosine residue in the ITIM is critical for this interaction. These results suggest that Ly-49A utilizes two different inhibitory mechanisms : ITIM-dependent and ITIM-independent.