Enhancing Immunotherapeutic Value of Natural Killer (NK) Cells

CD16又称低亲和力免疫球蛋白受体(FcRIIIa)，是诱导NK抗体依赖性细胞毒作用(ADCC)的受体。CD16是由大多数NK细胞表达的，它通过与CD3和FcRI链结合来传递激活信号。先前对原代NK细胞的研究表明，当CD16与特定的单抗(MAb)交联后，受体被一种需要完整的肌动蛋白细胞骨架的ATP非依赖性机制迅速内化。其他研究表明，特异性单抗介导的CD16下调可被锌依赖的金属蛋白酶抑制剂1，10-菲咯啉阻断，这表明CD16从细胞表面的下调更多地是金属蛋白酶诱导的受体切割的结果，而不是内化的结果。在PKC激活剂佛波酯(PMA)处理NK细胞后，也显示CD16的脱落。我们的研究集中在确定内化和脱落对配体介导的CD16下调的有效贡献。我们的初步数据表明，不同的结合CD16的单抗在调节CD16表达方面的作用不同。其中一种单抗B73.1似乎在没有额外刺激的情况下增强了CD16从细胞表面的下调，而另外两种单抗则不能引起任何显著的下调。我们目前正在调查B73.1中受体表达的丧失是由于内化还是由于金属蛋白酶(MMPs)切割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，用这种单抗在不同的时间点孵育细胞，无论是否存在或不存在特定的MMPs抑制剂。同时，我们正在研究控制CD16细胞表面表达和细胞内运输的机制。我们的初步数据表明，CD16的下调需要完整的肌动蛋白细胞骨架，受体没有循环回到细胞表面，Arf6似乎在这种下调中发挥了关键作用。我们惊讶地发现，用Arf-6功能抑制剂氟化铝(AlF4-)处理原代NK细胞后，几乎96%的CD16细胞表面表达消失。奇怪的是，最近的数据显示，用MMPs抑制剂处理能够恢复AlF4处理后丢失的CD16表面表达的55%，这表明在某种程度上，Arf-6介导的CD16下调受MMPs控制。我们正在深入研究Arf-6调节CD16表达的机制。我们还在研究在肌动蛋白干扰药物治疗时观察到的受损的CD16下调是否可以在MMPs抑制剂存在的情况下恢复。 FcmuR是免疫球蛋白基因超家族中的一员，在NK细胞上高度表达，最近发现它是一种IgM特异性的Fc受体。关于这种受体在NK细胞上的功能目前还不清楚。我们的初步结果表明，在原代NK细胞上连接该受体可导致胞浆内酪氨酸残基的磷酸化，并激活细胞内的激酶，表明该受体的连接能够激活NK细胞。此外，在原代NK细胞中加入IgM可增强其对K562肿瘤细胞的杀伤作用。我们还了解到，NK细胞暴露于IL-2可显著下调FcmuR的表达。由于激活的NK细胞分泌IL-2，这种下调可能是为了避免过度炎症，从而可能产生功能性后果。

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.