Enhancing Immunotherapeutic Value of Natural Killer (NK) Cells

CD16，也被称为IgG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。CD16由大多数NK细胞表达，能够通过其与CD3和FcRI链的关联转导激活信号。先前对原代NK细胞的研究表明，当CD16与特异性单克隆抗体（mAb）交联时，受体通过atp独立的机制迅速内化，这需要一个完整的肌动蛋白细胞骨架。其他研究表明，特异性单克隆抗体介导的CD16下调被Zn2+依赖性金属蛋白酶抑制剂1,10-菲罗啉阻断，这表明CD16从细胞表面的下调更多是金属蛋白酶诱导的受体切割的结果，而不是内化。PKC激活剂phorbol 12-肉豆蔻酸13-醋酸酯（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，结合CD16的不同单克隆抗体在调节CD16表达方面的作用不同。一种单抗B73.1似乎在没有额外刺激的情况下增强了细胞表面CD16的下调，而另外两种单抗则没有诱导任何显著的下调。我们目前正在研究B73.1中受体表达的缺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，用这种单抗培养细胞，在不同的时间点上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下调需要完整的肌动蛋白细胞骨架，受体不会再循环回到细胞表面，Arf6似乎在这种下调中起着至关重要的作用。我们惊讶地发现，在原代NK细胞用氟化铝（AlF4-）（一种Arf-6功能抑制剂）处理后，几乎96%的CD16细胞表面表达丢失。奇怪的是，最近的数据显示，用MMPs抑制剂治疗能够恢复多达55%的AlF4-治疗时丢失的CD16表面表达，这表明在某种程度上，Arf-6介导的CD16下调是由MMPs控制的。我们正在深入研究Arf-6调控CD16表达的机制。我们还在研究在肌动蛋白破坏药物治疗中观察到的CD16下调是否可以在MMPs抑制剂的存在下恢复。

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.