Role of NKG2D in immune responses to tumors

描述（由申请人提供）：NK细胞和T细胞表达天然的“NK受体”，如NKG2D，使细胞能够识别和消除转化细胞。NKG2D受体激活NK细胞对靶细胞的细胞溶解和细胞因子的产生，与5种或更多独立的细胞表面配体（NKG2D配体）结合。NKG2D配体在肿瘤细胞中表达上调。一旦细胞表面表达NKG2D配体，NK细胞就可以杀死它。在癌症患者和其他一些综合征中，已经证明一些人类NKG2D配体被蛋白酶从细胞表面切割并积聚在血清中。人们认为，脱落配体可以干扰NKG2D识别并阻止肿瘤细胞消除，但我们认为，这一命题的许多证据是有缺陷和不完整的。由于缺乏动物模型，直接测试一直受到阻碍。我们通过证明小鼠NKG2D配体MULT1可以通过蛋白酶有效地从细胞中切割，并在患有肿瘤和炎症性疾病的小鼠的血液中高浓度积累，填补了这一空白。裂解后的MULT1以高亲和力（~10 nM）与NKG2D结合。为了解决MULT1脱落形式的生物学效应，不表达内源性NKG2D配体的肿瘤细胞被改造成产生与脱落形式相似的分泌形式的MULT1。尽管我们预计分泌MULT1可能会干扰肿瘤排斥反应，但我们发现分泌MULT1的肿瘤细胞被排斥。肿瘤排斥反应由NK细胞介导，在某些情况下也由T细胞介导，并且需要宿主表达NKG2D。这些发现促使实验表明，与B16黑色素瘤细胞一起提供纯化的重组MULT1可诱导NK细胞体外功能和体内肿瘤排斥反应。这些数据总体上支持一个模型，即非肿瘤细胞（如肿瘤相关巨噬细胞）表达NKG2D配体，持续刺激NK细胞，导致NK细胞脱敏；可溶性MULT1阻断相互作用可阻止这些脱敏相互作用，因此通过NK细胞识别不同的肿瘤细胞配体恢复NK细胞功能和肿瘤排斥反应。值得注意的是，初步数据显示，注射阻断性NKG2D抗体增强了NK细胞在体外几天后的反应性，支持了该模型。因此，尽管人们普遍认为shed NKG2D配体抑制抗肿瘤免疫反应，但我们的数据表明，shed MULT1调动NK细胞对肿瘤的反应。这些发现具有重要的机制意义，同时表明可溶性NKG2D配体或阻断相互作用的抗体作为免疫治疗剂具有相当大的潜力。我们提出以下具体目标：（目标1）记录NKG2D或其配体的可溶性配体和抗体的免疫增强作用；（目的2）确定免疫增强和肿瘤排斥反应的机制；（目的3）评估可溶性配体和抗体的免疫治疗潜力。

DESCRIPTION (provided by applicant): NK cells and T cell express innate "NK receptors" such as NKG2D, which enable the cells to recognize and eliminate transformed cells. The NKG2D receptor, which activates target cell cytolysis and cytokine production by NK cells, binds to 5 or more independent cell surface ligands (NKG2D ligands). NKG2D ligands are upregulated in tumor cells. Once a cell expresses cell surface NKG2D ligands, NK cells can kill it. In cancer patients and some other syndromes, it has been shown that some human NKG2D ligands are cleaved from the cell surface by proteases and accumulate in serum. It is believed that the shed ligands can interfere with NKG2D recognition and prevent tumor cell elimination, but we believe that much of the evidence for this proposition is flawed and incomplete. Direct testing of it has been hampered by the lack of an animal model. We have filled this gap by showing that a mouse NKG2D ligand, MULT1, is cleaved efficiently from cells by proteases, and accumulates at high concentrations in the blood of mice with tumors and inflammatory diseases. Cleaved MULT1 binds to NKG2D with high affinity (~10 nM). To address the biological effects of a shed form of MULT1, tumor cells that did not express endogenous NKG2D ligands were engineered to produce a secreted form of MULT1 similar to the shed form. Whereas we expected that secreted MULT1 might interfere with tumor rejection responses, we found instead that tumor cells secreting MULT1 were rejected. Tumor rejection was mediated by NK cells, and in some cases T cells, and required that the host express NKG2D. These findings prompted experiments that showed that provision of purified recombinant MULT1 along with B16 melanoma cells resulted in induction of NK function ex vivo and tumor rejection in vivo. The data as a whole support a model in which expression of NKG2D ligands by non-tumor cells, such as tumor associated macrophages, persistently stimulate NK cells, resulting in desensitization of the NK cells; interruption of the interaction by soluble MULT1 prevents these desensitizing interactions, and therefore restores NK cell functionality and tumor rejection via recognition of distinct tumor cell ligands for NK cells. Remarkably, preliminary data show that injection of a blocking NKG2D antibody enhanced the responsiveness of NK cells tested a few days later, ex vivo, supporting the model. Thus, whereas it is widely assumed that shed NKG2D ligands inhibit anti-tumor immune responses, our data indicate that shed MULT1 mobilizes NK cell responses against tumors. These findings have important mechanistic implications and at the same time suggest that soluble NKG2D ligands, or antibodies that block the interaction, have considerable potential as immunotherapeutic agents. We propose the following specific aims: (Aim 1) To document immune-enhancement by soluble ligands and antibodies to NKG2D or its ligands; (Aim 2) To determine the mechanisms of immune-enhancement and tumor rejection; (Aim 3) To assess the immunotherapeutic potential of soluble ligands and antibodies.