Enhancing Immunotherapeutic Value of Natural Killer (NK) Cells

CD 16也称为IgG的低亲和力受体（FcRIIIa），是负责诱导NK抗体依赖性细胞毒性（ADCC）的受体。由大多数NK细胞表达的CD 16能够通过其与CD 3和FcRI链的结合来传递活化信号。先前对原代NK细胞的研究表明，在CD 16与特异性单克隆抗体（mAb）交联后，受体通过ATP非依赖性机制快速内化，该机制需要完整的肌动蛋白细胞骨架。其他人已经表明，特异性mAb介导的CD 16下调被Zn 2+依赖性金属蛋白酶抑制剂1，10-菲咯啉阻断，表明细胞表面的CD 16下调更多是金属蛋白酶诱导的受体裂解的结果，而不是内化。在用PKC激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）处理NK细胞后也显示出CD 16的脱落。我们的研究集中于确定内化和脱落对配体介导的CD 16下调的有效贡献。我们的初步数据表明，结合CD 16的不同mAb在调节CD 16表达方面的作用不同。一种mAb B73.1似乎在没有额外刺激的情况下增强了细胞表面的CD 16的下调，而另外两种mAb不诱导任何显著的下调。我们目前正在研究B73.1受体表达的丧失是由于内化还是由于金属蛋白酶（MMPs）的裂解导致受体从细胞表面脱落。为了解决这个问题，我们正在进行实验，在存在或不存在特定MMP抑制剂的情况下，将细胞与该mAb孵育不同的时间点。与此同时，我们正在研究控制细胞表面表达和细胞内运输的CD 16的机制。我们的初步数据表明，CD 16下调需要完整的肌动蛋白细胞骨架，受体不能再循环回细胞表面，Arf 6似乎在这种下调中起着至关重要的作用。我们惊奇地发现，在用氟化铝（AlF 4-）（Arf-6功能的抑制剂）处理原代NK细胞后，几乎96%的CD 16细胞表面表达丧失。令人好奇的是，最近的数据显示，用MMPs抑制剂处理能够恢复高达55%的在AlF 4处理后失去的CD 16的表面表达，这表明在某种程度上，Arf-6介导的CD 16的下调由MMPs控制。我们正在深入研究Arf-6调节CD 16表达的机制。我们也正在研究，如果受损的CD 16下调，我们观察到后，肌动蛋白破坏药物治疗可以恢复在MMPs抑制剂的存在。 FcmuR是免疫球蛋白基因超家族的成员，其在NK细胞上高度表达，并且最近已显示其是IgM特异性Fc受体。目前还不清楚这种受体在NK细胞上的功能。我们的初步结果表明，交联这种受体的原代NK细胞导致细胞质酪氨酸残基的磷酸化，以及细胞内激酶的激活，表明这种受体的连接是能够激活NK细胞。此外，向原代NK细胞添加IgM增强了它们对K562肿瘤细胞的杀伤。我们还了解到NK细胞暴露于IL-2显著下调FcmuR的表达。由于活化的NK细胞分泌IL-2，这种下调可能发生，以避免过度炎症，因此可能具有功能性后果。

CD16, also known as the low affinity receptor for IgG (FcRIIIa), is the receptor responsible for the induction of NK antibody-dependent cellular cytotoxicity (ADCC). CD16, expressed by most NK cells, is able to transduce an activating signal through its association with CD3 and FcRI chains. Previous studies with primary NK cells have shown that upon CD16 cross-linking with specific monoclonal antibodies (mAb), the receptor is rapidly internalized by an ATP-independent mechanism that requires an intact actin cytoskeleton. Others have shown that specific mAb-mediated down-regulation of CD16 is blocked by the Zn2+-dependent metalloproteases inhibitor, 1,10-phenanthroline, suggesting that the down-regulation of CD16 from the cell surface is more a consequence of metalloprotease-induced cleavage of receptor rather than internalization. Shedding of CD16 has also been shown after treatment of NK cells with the PKC activator phorbol 12-myristate 13-acetate (PMA). Our studies are focused on determining the effective contribution of internalization and shedding to the ligand-mediated down-regulation of CD16. Our preliminary data suggest that different mAbs that bind CD16 act differently in regard to regulating CD16 expression. One mAb, B73.1, seems to enhance the down modulation of the CD16 from the cell surface without additional stimuli, whereas two other mAbs do not induce any significant down modulation. We are currently investigating whether this loss of receptor expression in case of B73.1 is due to internalization or to the shedding of the receptor from the cell surface due to cleavge by metallo-proteases (MMPs). To address this, we are performing experiments incubating the cells with this mAb for different time points in presence or not or specific MMPs inhibitors. In parallel, we are investigating the mechanisms that control the cell surface expression and intracellular trafficking of CD16. Our preliminary data indicate that CD16 down modulation requires and intact actin cytoskeleton, that the receptor is not recycling back to the cell surface and that Arf6 seems to play a crucial in this down modulation. We surprisingly found that almost 96% of the CD16 cell surface expression is lost upon treatment of primary NK cells with aluminium fluoride(AlF4-), an inhibitor of Arf-6 function. Curiously, very recent data show that treatment with MMPs inhibitors is able to restore up to 55% of the surface expression of CD16 lost upon AlF4- treatment, suggesting that somehow, the Arf-6 mediated down-modulation of CD16 is controlled by MMPs. we are intensely investigating the mechanism by which Arf-6 is regulating CD16 expression. We are also investigating if the impaired CD16 down modulation that we observe upon actin disrupting drug treatment can be restored in the presence of MMPs inhibitors. FcmuR is a member of immunoglobulin gene superfamily that is highly expressed on NK cells and recently it has been shown to be an IgM-specific Fc receptor. Nothing is known about the function of this receptor on NK cells. Our preliminary results show that crosslinking this receptor on primary NK cells leads to phosphorylation of cytoplasmic tyrosine residues, as well as to the activation of intracellular kinases, indicating that ligation of this receptor is capable of activating NK cells. Moreover, the addition of IgM to primary NK cells enhances their killing of K562 tumor cells. We have also learned that exposure of NK cells to IL-2 markedly down-regulates the expression of FcmuR. Since activated NK cells secrete IL-2, this down-modulation may occur in order to avoid excess inflammation and thus may have functional consequences.