Dato M E, Kim Y B
Department of Microbiology and Immunology, University of Health Sciences, Chicago Medical School, IL 60064.
J Immunol. 1990 Jun 1;144(11):4452-62.
A mAb, porcine NK-inhibitory mAb (PNK-I) that inhibits porcine NK activity without affecting antibody-dependent cellular cytotoxicity (ADCC) has been developed. PNK-I acts at the level of the effector cell and inhibition of NK activity is independent of complement. Inhibitory effects are seen against various human and murine NK-susceptible targets. Addition of PNK-I antibody up to 60 min after assay initiation was effective at inhibiting NK activity. Furthermore PNK-I does not inhibit E:T conjugation and inhibits during the Ca2(+)-dependent phase of NK cytolysis. PNK-I Ag is present on virtually all PBL showing a bimodal distribution with 74% "dim" and 15% "bright" by flow cytometry. Monocytes and granulocytes stain with an intermediate intensity with greater than 90% and 95% staining positively, respectively. F(ab')2 fragments of PNK-I antibody show identical staining and functional activity as the whole molecule indicating that PNK-I acts independently of FcR. PNK-I immunoprecipitates molecules of molecular mass of 166, 155, 95 kDa under reducing and nonreducing conditions. PNK-I appears to be recognizing an epitope on a CD18 molecule. The CD18 molecule (beta-chain of CD11a,b,c) is ubiquitous on the surface of leukocytes and is implicated in a variety of cellular functions. Dim and bright populations were sorted and assessed functionally for NK and ADCC activity. It is demonstrated that PNK-I+ bright lymphocytes contain all detectable NK and ADCC activity in porcine PBL. Furthermore PNK-I+ bright lymphocytes contain the cytokine responsive NK cells capable of stimulation by IL-2, porcine NK-activating factor, and porcine natural killer-enhancing mAb. PNK-I+ dim cells were devoid of all baseline as well as inducible NK and ADCC activity. Giemsa stain of sorted populations show PNK-I+ bright cells containing the large granular lymphocytes whereas dim are devoid of these. Two color analysis show that PT4+ cells are PNK-I+ dim whereas PT8+ lymphocytes are divided between PNK-I+ bright and dim populations. Our results indicate that we are able to isolate all active as well as inducible NK and ADCC effector cells from porcine PBL based on relative Ag expression of CD18. Therefore quantitative as well as qualitative antigen expression is important in NK/ADCC-mediated cytotoxicity.
已研发出一种单克隆抗体，即猪自然杀伤细胞抑制性单克隆抗体（PNK-I），它能抑制猪自然杀伤细胞活性，而不影响抗体依赖性细胞毒性（ADCC）。PNK-I在效应细胞水平发挥作用，对自然杀伤细胞活性的抑制不依赖补体。对各种人类和小鼠自然杀伤细胞敏感靶标均可见抑制作用。在检测开始后长达60分钟添加PNK-I抗体可有效抑制自然杀伤细胞活性。此外，PNK-I不抑制效应细胞与靶细胞结合，并在自然杀伤细胞溶解的钙离子依赖性阶段发挥抑制作用。PNK-I抗原几乎存在于所有外周血淋巴细胞上，通过流式细胞术显示出双峰分布，其中74%为“暗淡型”，15%为“明亮型”。单核细胞和粒细胞染色强度中等，阳性染色率分别大于90%和95%。PNK-I抗体的F(ab')2片段显示出与整个分子相同的染色和功能活性，表明PNK-I的作用独立于FcR。在还原和非还原条件下，PNK-I免疫沉淀出分子量为166、155、95 kDa的分子。PNK-I似乎识别CD18分子上的一个表位。CD18分子（CD11a、b、c的β链）普遍存在于白细胞表面，并参与多种细胞功能。对暗淡型和明亮型群体进行分选，并对其自然杀伤细胞和ADCC活性进行功能评估。结果表明，PNK-I + 明亮淋巴细胞包含猪外周血淋巴细胞中所有可检测到的自然杀伤细胞和ADCC活性。此外，PNK-I + 明亮淋巴细胞包含能够被白细胞介素-2、猪自然杀伤细胞激活因子和猪自然杀伤增强单克隆抗体刺激的细胞因子反应性自然杀伤细胞。PNK-I + 暗淡细胞缺乏所有基线以及诱导性自然杀伤细胞和ADCC活性。分选群体的吉姆萨染色显示，PNK-I + 明亮细胞含有大颗粒淋巴细胞，而暗淡细胞则没有。双色分析表明，PT4 + 细胞为PNK-I + 暗淡型，而PT8 + 淋巴细胞则分布在PNK-I + 明亮型和暗淡型群体之间。我们的结果表明，基于CD18的相对抗原表达，我们能够从猪外周血淋巴细胞中分离出所有活性以及诱导性自然杀伤细胞和ADCC效应细胞。因此，在自然杀伤细胞/ADCC介导的细胞毒性中，抗原表达的定量和定性都很重要。
