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

1.用MiniMACS分离试剂盒从纯合子供体（A2402/、B 0702/、Cw 0702/）中纯化NK细胞，每两周免疫BALB/c小鼠三次。使用人NK细胞系NK 92（A0301/A1101、B 0702/B44031、Cw 0702/Cw 1602）通过流式细胞术筛选2000个克隆。本研究建立了17株抗人NK细胞和NY细胞共同抗原的单克隆抗体。3株mAbS（4-4-24-5.4-2-44-18.4-2-44-28）均为IgG 1型，与NK细胞和小部分T细胞反应。mAb 4-4-24-5部分抑制NK 92细胞对K562细胞的杀伤作用. Ly-49 A是小鼠NK细胞受体Ly-49家族的成员，其在识别其配体（靶细胞表面上的主要组织相容性复合物（MHC）I类分子）时抑制细胞毒性。其抑制功能的机制知之甚少。我们在此证明，抗体介导的B细胞受体（BCR）与转染的Lv-49 A分子的共连接导致BCR诱导的白细胞介素-2（IL-2）分泌的消除以及B细胞系A20中Erk 1/2和p38 MAP激酶活化的显著降低。令人惊讶。BCR与Ly-49 A交联不影响BCR诱导的钙动员。用苯丙氨酸取代ITIM中的单个酪氨酸残基并不导致抑制功能的完全丧失，如通过BCR诱导的IL-2分泌所测量的。N-末端37个氨基酸肽（包括ITIM）的缺失确实消除了抑制活性。免疫共沉淀实验表明，在诱导酪氨酸磷酸化。Ly-49 A表达酪氨酸磷酸酶SHP-1，但不表达肌醇磷酸酶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-49 A利用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.