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

1.使用MiniMACS分离试剂盒从纯合子供体（A2402/, B0702/, Cw0702/）中纯化NK细胞，每两周免疫BALB/c小鼠三次。利用人NK细胞系NK92 (A0301/A1101, B0702/B44031, Cw0702/Cw1602)，流式细胞术筛选了2000个克隆。我们建立了17个针对人NK细胞和NY细胞系共同抗原的单抗。3个单克隆抗体（4-4-24-5.4-2-44-18.4-2-44-28）为IgG1，反应NK细胞和小部分T细胞。mAb 4-4-24-5部分抑制NK92细胞对K562细胞的细胞毒性。Ly-49A是小鼠NK细胞受体Ly-49家族的一员，通过识别其配体（靶细胞表面的主要组织相容性复合体（MHC） I类分子）来抑制细胞毒性，尽管Ly-49A在其细胞质尾部具有免疫受体酪氨酸基抑制基序（ITIM）。其抑制功能的机制尚不清楚。我们在这里证明了抗体介导的B细胞受体（BCR）与转染的Lv-49A分子的共连接导致BCR诱导的白介素-2 （IL-2）分泌减少，并显著降低了B细胞系A20中Erk 1/2和p38 MAP激酶的激活。令人惊讶的是。BCR与ly - 49a交联不影响BCR诱导的钙动员。通过bcr诱导的IL-2分泌测量，用苯丙氨酸取代ITIM中的单个酪氨酸残基不会导致抑制功能的完全丧失。n端含有ITIM的37个氨基酸肽的缺失使其抑制活性丧失。共免疫沉淀实验表明，在酪氨酸磷酸化诱导下。Ly-49A抑制酪氨酸磷酸酶SHP-1，但不抑制肌醇磷酸酶SHIP， ITIM中的酪氨酸残基对这种相互作用至关重要。这些结果表明，Ly-49A具有两种不同的抑制机制：itim依赖性和itim非依赖性。

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.