RECOGNITION OF LIGANDS BY SPECIFIC CYTOTOXIC T LYMPHOCYTES & NATURAL KILLER CELL

由NK细胞和T细胞亚群表达的CD94/NKG2受体被认为是广泛的经典和非经典HLAI类分子的受体。为了检测CD94/NKG2蛋白的配基特异性，制备了一种可溶性的异二聚体形式的CD94/NKG2受体，并将其用于与表达特定的HLAI/肽复合体的细胞的直接结合研究。我们证实CD94/NKG2A与人类白细胞抗原-E具有特异性的相互作用，并证明这种相互作用依赖于人类白细胞抗原-E与多肽的结合。此外，CD94/NKG2A与经典的HLAI类分子之间没有相互作用，通过直接与可溶性受体结合或用CD94/NKG2A+NK细胞进行功能测定。此外，我们还研究了与人类白细胞抗原E相关的多肽在人类白细胞抗原E与CD94/NKG2A相互作用中的作用。所有测试的I类前导序列多肽均与人类白细胞抗原E结合，并被CD94/NKG2A识别。然而，I类前导序列的氨基酸变异影响了人类白细胞抗原-E的稳定性。此外，CD94/NKG2A并不能识别所有检测到的人类白细胞抗原-E/多肽复合体。因此，CD94/NKG2A对人类白细胞抗原-E的识别受两个水平的多肽控制：第一，多肽必须稳定人类白细胞抗原E并促进细胞表面的表达；第二，多肽必须形成CD94/NKG2A的配体。测定了CD94胞内区的晶体结构，发现了一个独特的C型凝集素折叠。该晶体结构可能是其他NK细胞受体的原型，如LY-49、NKR-P1和CD-69。在表达H-2(B)基因和小型猪I类转基因(PD1)的转基因小鼠的原代胚胎成纤维细胞中，用腺病毒12转化导致所有I类复合体的组装和细胞表面表达受到抑制。将多肽转运蛋白基因导入细胞，可恢复PD1在细胞表面的表达。然而，H-2K(B)基因表达的重建还需要将H-2K(B)mRNA的稳态水平提高2倍，这可以通过干扰素处理细胞或通过将H-2K(B)基因导入细胞来实现。对I类分子生物发生的详细分析揭示了I类自由重链的稳定表达，即使在多肽转运蛋白基因过度表达的情况下，这些重链也不会转化为构象复合体。-自然杀伤(NK)细胞，人类白细胞抗原I类受体，人类白细胞抗原E，CD94/NKG2，MHC I类复合体，肽结合，癌症

The CD94/NKG2 receptors expressed by subpopulations of NK cells and T cells have been implicated as receptors for a broad range of both classical and nonclassical HLA class I molecules. To examine the ligand specificity of CD94/NKG2 proteins, a soluble heterodimeric form of the receptor was produced and used in direct binding studies with cells expressing defined HLA class I/peptide complexes. We confirmed that CD94/NKG2A specifically interacts with HLA-E and demonstrated that this interaction is dependent on the association of HLA-E with peptide. Moreover, no interaction between CD94/NKG2A and classical HLA class I molecules were observed, as assayed by direct binding of the soluble receptor or by functionalassays using CD94/NKG2A+ NK cells. The role of the peptide associated with HLA-E in the interaction between HLA-E and CD94/NKG2A was also assessed. All class I leader sequence peptides tested bound to HLA-E and were recognized by CD94/NKG2A. However, amino acid variations in class I leader sequences affected the stability of HLA-E. Additionally, not all HLA-E/peptide complexes examined were recognized by CD94/NKG2A. Thus CD94/NKG2A recognition of HLA-E is controlled by peptide at two levels; first, peptide must stabilize HLA- E and promote cell surface expression and second, the HLA-E/peptide complex must form the ligand for CD94/NKG2A. The crystal structure of the intracellular domain of CD94 was determined revealing a unique C- type lectin fold. The crystal structure is likely a prototype for other NK cell receptors such as Ly-49, NKR-P1 and CD-69.In primary embryonal fibroblasts from transgenic mice expressing H-2(b) genes and a miniature swine class I transgene (PD1), transformation with adenovirus 12 results in suppression of assembly and cell surface expression of all class I complexes. Cell surface expression of PD1 can be recovered by transfecting the cells with peptide transporter genes. However, reconstitution of the H-2K(b) gene expression requires, in addition, a 2-fold increase in the steady state level of the H-2K(b) mRNA that can be attained by treatment of the cells with interferons or by transfecting them with the H-2K(b) gene. A detailed analyses of the biogenesis of class I molecules has revealed the steady state expression of free class I heavy chains that are not converted into conformed complexes even when peptide transporter genes are overexpressed. - Natural Killer (NK) cells, HLA class I receptors, HLA- E, CD94/NKG2, MHC class I complexes, peptide binding, cancer