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

主要组织相容性复合物I类分子与自然杀伤细胞的关系

• 批准号: 08670519
• 负责人: KIURA Katsuyuki
• 金额: $ 1.22万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670519/
• 关键词: Natural Killer Cells; HLA Class Molecules; NK Cell Receptors; Killer Inhibitory Receptors; Negative Signal; HLA ClassI分子; NK細胞リセプター; ClassI; Negative signal; NK細胞Receptor; GL183; EB6; NKB1; Class I; NK細胞receptor; NKBI

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.

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。

项目成果

Ueoka H et al.: "A randomized trial of hybrid administration of cyclophosphamide. doxorubicin. and vincristine (CAV) /cisplatin and etoposide (PVP) versus sequential administration of CAV-PVP for the treatment of patients with small cell lung carcinoma :

Ueoka H 等人：“环磷酰胺、阿霉素和长春新碱 (CAV)/顺铂和依托泊苷 (PVP) 混合给药与 CAV-PVP 序贯给药治疗小细胞肺癌患者的随机试验：

K.Kiura,S.Watarai H.Ueoka et al: "Analteration of ganglioside composition in cisplatin-resistant Lung cancer cell line" Anticancer Research. 18. 2957-2960 (1998)

K.Kiura，S.Watarai H.Ueoka 等：“顺铂耐药肺癌细胞系中神经节苷脂成分的改变”抗癌研究。

W.Zheng, K.Kiura, I.Nakamura et al.: "Murine NK cell allospecificity-1 is defined by inhibitory ligands" The Journal of Immunology. 156. 4651-4655 (1996)

W.Zheng、K.Kiura、I.Nakamura 等人：“小鼠 NK 细胞同种异体特异性 1 由抑制性配体定义”《免疫学杂志》。

W-P Zheng,K Kiura I NAKAMURA et.al.: "Murine NK cell allospecificity-1 is defined by inhibitory ligands" The Journal of Immunology. 156. 4651-4665 (1996)

W-P Cheng、K Kiura I NAKAMURA 等人：“小鼠 NK 细胞同种异体特异性 1 由抑制性配体定义”《免疫学杂志》。

W.Zeng, K.Kiura, I.NAKAMURA et al: "Murine NK cell allospecificity-1 is defined by inhibitory ligand" The Journal of Immunology. 156. 4651-4655 (1996)

W.Zeng、K.Kiura、I.NAKAMURA 等人：“小鼠 NK 细胞同种异体特异性 1 由抑制性配体定义”《免疫学杂志》。