Ortaldo J R, Wiltrout T A, Sayers T J, Yagita H, Winkler-Pickett R T
Laboratory of Experimental Immunology, BRMP, DCT, NCI-FCRDC, Frederick, Maryland 21701-1013, USA.
J Leukoc Biol. 1995 Jun;57(6):897-903. doi: 10.1002/jlb.57.6.897.
Recently, two populations of small lymphocytes (SL) have exhibited non-major histocompatibility complex (MHC) restricted lysis. Recent studies by numerous laboratories have demonstrated that resting T cells triggered through CD3 and CD28 costimulations can result in immediate, non-MHC restricted killing. Our recent studies with CD3-, CD56+ SL demonstrated that although these cells contained no cytoplasmic granules detected with electron microscopy, they mediated potent NK and ADCC activity. In the present study, we have used a Western blotting technique that allows for the detection and quantitation of total cellular levels of pore-forming protein (PFP). We have found that freshly isolated peripheral non-granulated lymphocytes (both CD3+ and CD3-) contain PFP. In addition, CD3-, CD56+ SL contain levels of PFP similar to those of the highly granular CD3- LGL. In search of non-granule PFP, we exploited the rat NK (RNK) cell lines as a source of other potential cytotoxic factors. A membrane associated PFP, based on Western blotting, was isolated from rat RNK cells. Unlike PFP isolated from granules, this PFP was active after culture in Ca(2+)-containing medium. However, the lytic activity isolated from the non-granule PFP of RNK cells was inhibited by monoclonal antibodies to PFP. Collectively, these studies indicate that PFP is present in small agranular lymphocytes (both NK and T cells) and that it is not stored in large cytoplasmic granules. The implication of our results for the acquisition and activation of lytic ability in NK and T cells will be discussed.
最近，两群小淋巴细胞（SL）表现出非主要组织相容性复合体（MHC）限制的细胞溶解作用。众多实验室最近的研究表明，通过CD3和CD28共刺激触发的静息T细胞可导致即刻的、非MHC限制的杀伤作用。我们最近对CD3-、CD56+ SL的研究表明，尽管这些细胞经电子显微镜检测未发现胞质颗粒，但它们介导了强大的自然杀伤（NK）和抗体依赖的细胞介导的细胞毒性（ADCC）活性。在本研究中，我们使用了一种蛋白质印迹技术，该技术可检测和定量穿孔蛋白（PFP）的总细胞水平。我们发现，新鲜分离的外周无颗粒淋巴细胞（CD3+和CD3-均有）含有PFP。此外，CD3-、CD56+ SL所含PFP水平与高度颗粒化的CD3-大颗粒淋巴细胞（LGL）相似。为了寻找非颗粒状PFP，我们利用大鼠NK（RNK）细胞系作为其他潜在细胞毒性因子的来源。基于蛋白质印迹法，从大鼠RNK细胞中分离出一种与膜相关的PFP。与从颗粒中分离出的PFP不同，这种PFP在含Ca（2+）的培养基中培养后仍具有活性。然而，从RNK细胞的非颗粒状PFP中分离出的裂解活性被抗PFP单克隆抗体所抑制。总的来说，这些研究表明PFP存在于小的无颗粒淋巴细胞（NK细胞和T细胞）中，且不储存在大的胞质颗粒中。我们将讨论这些结果对NK细胞和T细胞裂解能力的获得与激活的意义。
