Cantoni C, Biassoni R, Pende D, Sivori S, Accame L, Pareti L, Semenzato G, Moretta L, Moretta A, Bottino C
Istituto Scientifico Tumori and Centro Biotecnologie Avanzate, Genova, Italy.
Eur J Immunol. 1998 Jan;28(1):327-38. doi: 10.1002/(SICI)1521-4141(199801)28:01<327::AID-IMMU327>3.0.CO;2-O.
Inhibitory receptor complexes formed by CD94 and NKG2-A (Kp43) molecules have been implicated in HLA class I recognition by human natural killer (NK) cells. Additional forms of CD94 receptors have recently been described in NK cells characterized by the lack of NKG2-A expression. These CD94 receptors were shown to display activating functions. Immunoprecipitation with anti-CD94 monoclonal antibodies (mAb) led to the identification, in these cells, of a 39-kDa (Kp39) molecule that was originally believed to represent an activating isoform of the CD94 molecules. In the present study we show that the Kp39 molecule is covalently associated with CD94 and displays a protein backbone (26 kDa) similar to that of NKG2-A (Kp43) glycoproteins. Peptide mapping analysis indicates that Kp39 and NKG2-A glycoproteins belong to the same molecular family. A novel NKG2-specific mAb (termed P25) has been generated that specifically reacts with both NKG2-A and NKG2-C molecules, but fails to recognize NKG2-E molecules. Analysis of polyclonal and clonal NK cells shows that P25 mAb reacts with all NKG2-A+ cells and with a fraction of CD94+ cells lacking the expression of NKG2-A. These data indicate that NKG2-C molecules are indeed expressed only in a subset of cells lacking the expression of NKG2-A. The CD94-associated Kp39 molecule can be detected only in NKG2-A- P25+ cells, i.e. cells expressing NKG2-C molecules. Indeed, reverse transcription-polymerase chain reaction analysis performed on a large panel of NK clones indicates that NKG2-A- P25+ NK clones express the NKG2-C transcript. Notably, the cytolytic activity of these clones can be triggered by the P25 mAb in redirected killing analysis. Finally, biochemical analysis of COS7 cells cotransfected with CD94 and NKG2-C demonstrates the identity between Kp39 and NKG2-C molecules. Altogether, our data demonstrate that NKG2-C molecules associate with CD94 to form an activating receptor complex in a subset of human NK cells.
由CD94和NKG2-A（Kp43）分子形成的抑制性受体复合物与人类自然杀伤（NK）细胞识别HLA I类分子有关。最近在NK细胞中发现了其他形式的CD94受体，其特征是缺乏NKG2-A表达。这些CD94受体显示出激活功能。用抗CD94单克隆抗体（mAb）进行免疫沉淀，在这些细胞中鉴定出一种39 kDa（Kp39）分子，最初认为它代表CD94分子的一种激活异构体。在本研究中，我们表明Kp39分子与CD94共价结合，并显示出与NKG2-A（Kp43）糖蛋白相似的蛋白质骨架（26 kDa）。肽图谱分析表明，Kp39和NKG2-A糖蛋白属于同一分子家族。已产生一种新型的NKG2特异性mAb（称为P25），它能与NKG2-A和NKG2-C分子特异性反应，但不能识别NKG2-E分子。对多克隆和克隆NK细胞的分析表明，P25 mAb与所有NKG2-A+细胞以及一部分缺乏NKG2-A表达的CD94+细胞反应。这些数据表明，NKG2-C分子确实仅在缺乏NKG2-A表达的细胞亚群中表达。与CD94相关的Kp39分子仅在NKG2-A- P +细胞中被检测到，即表达NKG2-C分子的细胞。实际上，对大量NK克隆进行的逆转录-聚合酶链反应分析表明，NKG2-A- P25+ NK克隆表达NKG2-C转录本。值得注意的是，在重定向杀伤分析中，这些克隆的细胞溶解活性可由P25 mAb触发。最后，对共转染CD94和NKG2-C的COS7细胞进行生化分析，证明了Kp39和NKG2-C分子的同一性。总之，我们的数据表明，NKG2-C分子与CD94结合，在一部分人类NK细胞中形成一种激活受体复合物。
