MHC CLASS I STRUCTURES CONTROLLING NK CELLS

背景 自然杀伤（NK）细胞消除早期癌症， 血行转移 NK介导的杀伤被靶细胞抑制 HLA-I类分子可能通过不同的HLA受体识别 相关的HLA I类等位基因，可能是通过收缩相对保守的 α-螺旋位点 每个NK细胞表达几种交叉反应性HLA 受体并识别多种宿主HLA分子。 承认 靶细胞HLA抑制NK介导的杀伤、增殖和细胞因子 生产 初步研究。 HLA-B*0702保护转染的靶细胞免于 外周血单核细胞）PBMC）NK介导的杀伤。 B*0702中的突变允许显著增加的杀伤 肽结合沟，包括B口袋，和在溶剂中- 可接近的T细胞受体接触位点。 同样的突变也会影响杀戮 由溶细胞性T淋巴细胞介导。 HPLC分离的肽洗脱自 B*0702+细胞抑制NK介导的对B*0702+肽转运的杀伤 缺乏T2细胞。 我们的初步研究表明，NK细胞接触 HLA结合肽和附近的HLA α-螺旋残基。 实验计划。 我们将用PBMC NK检测添加物B*0702突变体 细胞和NK克隆以进一步细化NK MHC接触位点。 我们将 研究携带NKB 1假定HLA-B受体和NK细胞的PBMC NK细胞如何 克隆区分三种模型系统中的HLA等位基因：B*5101-B*0702， B*2705-B*0702和Cw*0301-Cw*0601。 如果不同的HLA分子抑制NK细胞 细胞在数量上相似的方式，然后表达一个，两个，或 三种HLA基因将对NK介导的杀伤具有累加效应。 我们 已经设计了几种实验方法来识别HLA结合 抑制NK细胞的肽。 肽缺乏T2或酸剥离 靶细胞将与结合HLA的合成或细胞 肽并在NK介导的杀伤测定中测试。 或者，T2细胞 将用单独的小基因或小基因文库转染 编码HLA结合肽。 一旦鉴定出保护性肽， 将测试丙氨酸取代的合成肽的HLA结合， 抑制NK细胞，使我们能够推断NK受体接触位点。 此外，我们将测试NK介导的杀伤、增殖和 干扰素γ产生由HLA和肽协同调节 变体。 只要可行，我们将同时使用淋巴和转化 口腔角化细胞靶细胞。 对口腔癌的意义 由于“田野癌变”，口腔癌 患者经常患有第二种恶性肿瘤， 加强对北朝鲜的监视。 控制NK的HLA结构的鉴定 介导的杀伤将有助于表征新描述的NK受体。 产生“分裂反应”的HLA或肽变体的鉴定 分离NK介导的杀伤、增殖和细胞因子产生， 提出了更好的方法来操纵NK细胞攻击口腔癌。 保护 HLA结合肽可以抑制NK对正常细胞的攻击，但不能抑制癌症 细胞

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.