MHC CLASS I STRUCTURES CONTROLLING NK CELLS

MHC I 类结构控制 NK 细胞

• 批准号: 2443683
• 负责人: Charles T. Lutz
• 金额: $ 18.89万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2443683
• 关键词: CD antigens; MHC class I antigen; alleles; binding proteins; cytokine; flow cytometry; gene expression; gene mutation; genetic mapping; high performance liquid chromatography; human subject; interferon gamma; keratinocyte; molecular site; mutant; natural killer cells; oral pharyngeal neoplasm; peptide structure; polymerase chain reaction; synthetic peptide; tissue /cell culture; transfection

Background. Natural killer (NK) cells eliminate incipient cancers and hematogenous metastases. NK mediated killing is inhibited y target cell HLA class i molecules, possibly through diverse HLA receptors recognize related HLA class I alleles, probably by contracting relatively conserved alpha-helical sites. Each NK cell expresses several crossreactive HLA receptors and recognizes multiple host HLA molecules. Recognition of target cell HLA inhibits NK mediated killing, proliferations, and cytokine production. Preliminary Studies. HLA-B*0702 protects transfected target cells from peripheral blood mononuclear cell )PBMC) NK mediated killing. Significantly increased killing is allowed by mutations in the B*0702 peptide binding groove, including the B pocket, and in the solvent- accessible T cell receptor contact site. The same mutations affect killing mediated by cytolytic T lymphocytes. HPLC separated peptides eluted from B*0702+ cells inhibit NK mediated killing of B*0702+ peptide transport deficient T2 cells. Our preliminary studies suggest that NK cells contact HLA-bound peptides and nearby HLA alpha-helical residues. Experimental Plan. We will test additions B*0702 mutants with PBMC NK cells nd NK clones to further refine the NK MHC contact site. We will examine how PBMC NK cells bearing the NKB1 putative HLA-B receptor and NK clones distinguish HLA alleles in three model systems; B*5101-B*0702, B*2705-B*0702, and Cw*0301-Cw*0601. If different HLA molecules inhibit NK cells in a quantitatively similar fashion, then expression of one, two, or three HLA genes will have an additive effect on NK mediated killing. We have devised several experimental approaches to identify HLA-binding peptides that inhibit NK cells. Peptide deficient T2 or acid stripped target cells will be incubated with HLA-binding synthetic or cellular peptides and tested in NK mediated killing assays. Alternatively, T2 cells will be transfected with individual minigenes or minigene libraries encoding HLA-binding peptides. Once protective peptides are identified, alanine substituted synthetic peptides will be tested for HLA binding and inhibition of K cells, allowing us to deduce NK receptor contact sites. Furthermore, we will t est if NK mediated killing, proliferations, and interferon gamma production are coordinately regulated by HLA and peptide variants. Whenever feasible, we will use both lymphoid and transformed oral keratinocyte target cells. Significance to Oral Cancer. Due to "field cancerization", oral cancer patients frequently suffer second malignancies and would benefit from enhanced NK surveillance. Identification of HLA structures that control NK mediated killing will help characterize newly described NK receptors. Identification of HLA or peptide variants that produce a 'split response" separating NK mediated killing, proliferation, and cytokine production, may suggest better ways to manipulate Nk attack on oral cancers. Protective HLA-binding peptides could inhibit NK attack of normal cells but not cancer cells.

背景资料。自然杀伤(NK)细胞消除早期癌症和 血源性转移。NK介导的杀伤被抑制为靶细胞 人类白细胞抗原I类分子可能通过不同的人类白细胞抗原受体识别 相关的人类白细胞抗原I类等位基因，可能通过收缩相对保守的 α-螺旋位置。每个NK细胞表达几种交叉反应的人类白细胞抗原 受体和识别多种宿主的人类白细胞抗原分子。认识到 靶细胞人类白细胞抗原抑制NK介导的杀伤、增殖和细胞因子 制作。 初步研究。人类白细胞抗原B*0702保护转染靶细胞 外周血单个核细胞(PBMC)NK介导的杀伤作用。 B*0702基因突变可显著增加杀伤力 多肽结合槽，包括B袋，并在溶剂中- 可访问的T细胞受体接触部位。同样的突变也会影响杀戮 由细胞溶解T淋巴细胞介导。洗脱物中的高效液相分离多肽 B*0702+细胞抑制NK介导的B*0702+多肽转运杀伤作用 T2细胞缺乏。我们的初步研究表明，NK细胞接触 与人类白细胞抗原结合的多肽和附近的人类白细胞抗原α螺旋残基。 实验计划。我们将用PBMC NK测试添加的B*0702突变体 细胞和NK克隆，以进一步细化NK MHC接触部位。我们会 检测PBMC NK细胞如何携带NKB1可能的人类白细胞抗原B受体和NK细胞 B*5101-B*0702、B*5101-B*0702、 B*2705-B*0702和CW*0301-CW*0601。如果不同的人类白细胞抗原分子抑制NK细胞 数量相似的细胞，然后表达一个、两个或 3个人类白细胞抗原基因对NK介导的杀伤具有相加作用。我们 我设计了几种实验方法来识别人类白细胞抗原结合 抑制NK细胞的多肽。短肽T2或酸剥离 靶细胞将与人类白细胞抗原结合的合成细胞或细胞培养 在NK介导的杀伤实验中进行检测。或者，T2细胞 将被单独的微基因或微基因文库导入 编码人类白细胞抗原结合肽。一旦确定了保护性多肽， 丙氨酸取代的合成肽将测试人类白细胞抗原结合和 抑制K细胞，使我们可以推断NK受体的接触部位。 此外，我们将测试NK是否介导了杀伤、增殖和 干扰素γ的产生受人类白细胞抗原和多肽的协同调节 变种。只要可行，我们将同时使用淋巴和转化 口腔角质形成细胞靶细胞。 对口腔癌的意义。由于“野战癌变”，口腔癌 患者经常患有第二种恶性肿瘤，并将受益于 加强了对朝鲜的监视。控制NK细胞的人类白细胞抗原结构的鉴定 介导性杀伤将有助于新描述的NK受体的特征。 鉴定产生“分裂反应”的人类白细胞抗原或多肽变体 分离NK介导的杀伤、增殖和细胞因子产生可能 建议更好的方法来控制口腔癌的NK攻击。保护性的 HLA结合肽能抑制正常细胞的NK攻击，但不能抑制肿瘤细胞的NK攻击 细胞。