INITIATING THE ADAPTIVE IMMUNE RESPONSE TO CANCER

越来越清楚的是，NK细胞可以促进DC的激活和后续免疫反应的极化。本项目将在可移植和自发的小鼠肿瘤模型中评估NK-DC相互作用对抗肿瘤CTL的产生和功能的意义，并将通过控制/操纵这些相互作用来提高对已建立的MHC+/MHC-肿瘤的抗肿瘤活性。我们假设，被激活的NK(A-NK)细胞被输送到肿瘤部位，将杀死肿瘤细胞，从而释放急性炎症介质(如HSP和HMGB1)和DC可以摄取的抗原。此外，DC和NK细胞之间的接触和细胞因子依赖的串扰将进一步激活 这两种类型的细胞，导致产生极化的树突状细胞，其刺激Th1抗肿瘤反应的能力增强。这将在三个动物模型中进行评估：1)一个模型肿瘤抗原表达系统(MO-5，卵清蛋白转基因的B16黑色素瘤)。在这个模型中，通过测量肿瘤、淋巴结、血液和脾中过继转移的卵特异性OT-I(CDS)和OT-II(CD4)细胞的数量来评估有效的NK/DC相互作用；2)野生型B16模型；3)自发黑色素瘤的p16INK4a-/-KO模型。在全身或局部输送A-NK细胞和DC后，我们将改变肿瘤微环境中的细胞因子分布。为了达到这一目的，我们将用含有细胞因子基因的病毒载体转导DC和NK细胞，以产生各种前NK和前DC细胞因子，如IL-2、IL-18、干扰素-α和GM-CSF。此外，将测试多次注射NK/DC以确保持续刺激传入肢体(从而产生CTL)以及局部CTL效应器功能的能力。目的1.明确不同途径注射不同NK/DC比例的荷瘤动物后，NK细胞和DC在体内的存活及其向肿瘤和淋巴结的转运。目的2.在可移植和自发的肿瘤模型中，证实NK细胞和DC的瘤内传递促进CTL的产生和功能。目的3.通过强制NK细胞和DC的细胞因子基因转导来增强肿瘤中的NK/DC功能。

It has become increasingly clear that NK cells can promote DC activation and polarization of the subsequent immune response. This project will evaluate, in transplantable and spontaneous murine tumor models, the significance of NK-DC interactions for the generation and function of anti-tumor CTLs and will improve antitumor activity against established MHC+/MHC- tumors by controlling/manipulating these interactions. We hypothesize that activated NK (A-NK) cells, delivered to tumor sites, will kill tumor cells and thereby liberate both mediators of acute inflammation (such as HSP and HMGB1) and antigens which can be taken up by DCs. In addition, contact- and cytokine-dependent cross-talk between DCs and NK cells will further activate both cell types, leading to the generation of polarized DCs with enhanced capability of stimulating Th1-anti tumor responses. This will be evaluated in three animal models: 1) a model tumor antigen expressing system (the MO-5, ovalbumin transfected B16 melanoma). Effective NK /DC interactions will, in this model be evaluated by measuring the number of adoptively transferred ova-specific OT-I (CDS) and OT-II (CD4) cells in tumors, lymph nodes, blood and spleen; 2) The wild-type B16 model and; 3) in the p16INK4a-/-KO model of spontaneous melanoma. Following systemic or local delivery of A-NK cells and DCs, we will vary the cytokine profile in the tumor microenvironment. To achieve this, DCs and NK cells will be transduced with cytokine gene-containing viral vectors to produce various pro-NK and pro-DC cytokines, e.g., IL2, IL18, IFN-alpha and GM-CSF. In addition, the ability of multiple NK/DC injections to ensure continuous stimulation of the afferent limb (and thereby resultant CTL generation) as well as local CTL effector function, will be tested. Three Specific Aims will be conducted to explore these issues: Aim 1. Define the in vivo survival of NK cells and DCs as well as their traffic to tumors and lymph nodes following injection different NK/DC ratios into tumor-bearing animals via different routes. Aim 2. Demonstrate that intratumoral delivery of NK cells and DCs promotes CTL generation and function in transplantable and spontaneous tumor models.; Aim 3. Enhance NK/DC function in tumors by forced cytokine-gene transduction of NK cells and DCs.