Hackett J, Tutt M, Lipscomb M, Bennett M, Koo G, Kumar V
J Immunol. 1986 Apr 15;136(8):3124-31.
Cells bearing the NK-specific marker NK-1.1 were purified from mouse spleens by utilizing a monoclonal anti-NK-1.1 antibody and cell sorting. In normal adult mice, all of the splenic NK activity against YAC-1 cells was found in the NK-1.1+ fraction, whereas NK-1.1- cells were depleted of NK activity. The NK activity of sorted NK-1.1+ cells was enriched 15- to 30-fold over unfractionated spleen cells. Light and electron microscopic studies of purified NK-1.1+ cells showed a homogeneous population of cells, each containing one to four cytoplasmic granules. Mice whose bone marrow has been destroyed by chronic exposure to 17-beta-estradiol have very low NK activity. However, spleen cells of estradiol-treated mice contained a normal frequency of NK-1.1+ cells which bound to YAC-1 cells, but failed to lyse them even after purification and subsequent exposure to interferon-alpha/beta in vitro. It appears, therefore, that in the absence of intact bone marrow, NK-1.1+ cells may be arrested in a nonlytic and interferon-unresponsive state. Spleens of neonatal mice which have low NK activity were analyzed to ascertain whether immature NK-1.1+ cells, similar to those found in estradiol-treated mice, could be demonstrated. Spleens of 8- to 9-day-old mice also contained NK-1.1+ cells which had very low NK activity even after purification. Sorted NK-1.1+ cells were examined for cytotoxicity in mice whose NK activity was suppressed by pretreatment with Corynebacterium parvum (-15 days). In contrast to cells from estradiol-treated and neonatal mice, NK-1.1+ from mice treated with C. parvum had normal functional activity. Similarly, although NK activity of unfractionated bone marrow cells is low, sorted NK-1.1+ cells were greatly enriched for lytic activity. Thus, we conclude that cell sorting with monoclonal anti-NK-1.1 antibody provides a powerful tool for examining the mechanisms underlying various states of low NK activity, and there exist NK-1.1+, nonlytic, interferon-unresponsive cells which apparently require an intact marrow microenvironment for differentiation into mature, lytic NK cells.
利用抗NK-1.1单克隆抗体和细胞分选技术，从小鼠脾脏中纯化出带有NK特异性标志物NK-1.1的细胞。在正常成年小鼠中，所有针对YAC-1细胞的脾脏NK活性都存在于NK-1.1+组分中，而NK-1.1-细胞则缺乏NK活性。分选得到的NK-1.1+细胞的NK活性比未分级的脾细胞富集了15至30倍。对纯化的NK-1.1+细胞进行光镜和电镜研究，结果显示细胞群体均一，每个细胞含有1至4个细胞质颗粒。长期暴露于17-β-雌二醇而导致骨髓被破坏的小鼠，其NK活性非常低。然而，经雌二醇处理的小鼠的脾细胞中，与YAC-1细胞结合的NK-1.1+细胞频率正常，但即使在纯化并随后体外暴露于α/β干扰素后，这些细胞仍无法裂解YAC-1细胞。因此，似乎在缺乏完整骨髓的情况下，NK-1.1+细胞可能停滞在非裂解和对干扰素无反应的状态。对NK活性较低的新生小鼠的脾脏进行分析，以确定是否能证明存在与经雌二醇处理的小鼠中发现的类似的未成熟NK-1.1+细胞。8至9日龄小鼠的脾脏中也含有NK-1.1+细胞，即使在纯化后其NK活性也非常低。对用微小棒状杆菌预处理（-15天）以抑制其NK活性的小鼠的分选NK-1.1+细胞进行细胞毒性检测。与经雌二醇处理的小鼠和新生小鼠的细胞不同，用微小棒状杆菌处理的小鼠的NK-1. +细胞具有正常的功能活性。同样，尽管未分级的骨髓细胞的NK活性较低，但分选得到的NK-1.1+细胞的裂解活性大大富集。因此，我们得出结论，用抗NK-1.1单克隆抗体进行细胞分选为研究各种低NK活性状态的潜在机制提供了一个强大的工具，并且存在NK-1.1+、非裂解、对干扰素无反应的细胞，这些细胞显然需要完整的骨髓微环境才能分化为成熟的、具有裂解活性的NK细胞。
