Role of NKG2D in immune responses to tumors

描述(申请人提供)：NK细胞和T细胞表达先天的“NK受体”，如NKG2D，使细胞能够识别和清除转化的细胞。NKG2D受体激活靶细胞的细胞溶解和NK细胞产生的细胞因子，与5个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。一旦一个细胞表达了细胞表面的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，已有研究表明，一些人类NKG2D配体被蛋白酶从细胞表面切割并在血清中积聚。人们认为脱落的配体可以干扰NKG2D的识别并阻止肿瘤细胞的消除，但我们认为这一命题的许多证据是有缺陷的和不完整的。由于缺乏动物模型，对它的直接测试受到了阻碍。我们已经填补了这一空白，证明了小鼠的NKG2D配体MULT1可以被蛋白酶有效地从细胞中切割出来，并在患有肿瘤和炎症性疾病的小鼠的血液中高浓度积累。切割的MULT1与NKG2D高亲和力结合(~10 nM)。为了解决脱落形式的MULT1的生物学效应，对不表达内源性NKG2D配体的肿瘤细胞进行了改造，以产生类似于脱落形式的分泌型MULT1。虽然我们预计分泌的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。肿瘤排斥反应是由NK细胞和某些情况下的T细胞介导的，需要宿主表达NKG2D。这些发现促使实验表明，将纯化的重组MULT1与B16黑色素瘤细胞一起提供可以在体外诱导NK功能，并在体内产生肿瘤排斥反应。这些数据作为一个整体支持这样一个模型，在该模型中，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MULT1阻断相互作用，阻止这些脱敏相互作用，从而通过识别NK细胞不同的肿瘤细胞配体，恢复NK细胞的功能和肿瘤排斥反应。值得注意的是，初步数据显示，注射封闭的NKG2D抗体增强了几天后在体外测试的NK细胞的反应性，支持这一模型。因此，虽然人们普遍认为脱落的NKG2D配体抑制抗肿瘤免疫反应，但我们的数据表明，脱落的MULT1动员NK细胞对肿瘤的反应。这些发现具有重要的机制意义，同时也表明可溶性NKG2D配体或阻断这种相互作用的抗体具有相当大的免疫治疗潜力。我们提出以下具体目标：(1)记录NKG2D或其配体的可溶性配体和抗体的免疫增强作用；(2)确定免疫增强和肿瘤排斥的机制；(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.