INFLAMMATORY CHEMOKINE CCL3 IN THE TUMOR MICROENVIRONMENT LEADS TO ENHANCED ANTITUMOR IMMUNE DEVELOPMENT IN THE DRAINING LYMPH NODE

 描述(申请人提供)：炎性趋化因子CCL3(MIP-1α)和CCL4(MIP-1β)在协调免疫淋巴结树突状细胞与非随机初始CD8+T细胞接触中起重要作用，并能促进记忆T细胞的生成。我们推测，在肿瘤微环境中引入CCL3和CCL4可以增强抗肿瘤免疫。使用小鼠结肠肿瘤模型CT26，我们产生了分泌CCL3或CCL4的肿瘤，并将它们接种到幼稚免疫活性受体的足垫中。肿瘤生长动力学测量表明，分泌CCL3的肿瘤要么导致CD8+T细胞依赖的肿瘤进展减缓，要么在免疫活性小鼠中完全消退，并在用作疫苗时保护随后的致命性肿瘤攻击。接下来，我们在肿瘤注射后第3天和第5天用荧光激活细胞分类分析(FACS)分析了淋巴细胞募集对转移瘤引流LN的影响。接种CCL3分泌肿瘤的小鼠在接种第3天时，WT肿瘤的CD4+和CD8+T细胞增加了1.3~1.7倍，CCl4肿瘤增加了1.3倍。到第5天，CCL3肿瘤中的T细胞积聚达到了WT和CCl4肿瘤的相似水平。此外，动态活体双光子显微镜和组织组织学研究进一步表明，肿瘤在早期转移过程中占据B细胞滤泡，外源性CCL3和CCL4梯度可能潜在地改变肿瘤引流LN中的淋巴细胞动态平衡，导致免疫激活而不是对CT26的耐受。我们建议在CCL3或CCL4分泌的CT26肿瘤接种后，探讨CCL3对免疫细胞在引流的LN中的募集、激活、保留和效应功能交付的生物学效应。这些研究包括接触CCL3后树突状细胞和淋巴细胞的功能激活、迁移和趋化因子受体的表达。此外，我们建议在肿瘤接种后的头5天内，研究分泌CCL3、分泌CCL4或野生型CT26的肿瘤细胞与肿瘤浸润性LN中的各种免疫细胞之间的动态细胞-细胞相互作用。这些研究将为探索将炎性趋化因子如CCL3作为抗肿瘤免疫治疗的治疗佐剂提供科学依据。

 DESCRIPTION (provided by applicant): Inflammatory chemokines CCL3 (MIP-1α) and CCL4 (MIP-1β) are important in orchestrating nonrandom naive CD8+ T cell contacts with dendritic cells in vaccinated lymph nodes (LNs) and can enhance memory T cell generation. We hypothesize that incorporating CCL3 and CCL4 into tumor microenvironment can enhance anti-tumor immunity. Using the murine colon tumor model CT26, we generated tumors that secret CCL3 or CCL4 and inoculate them live into the footpad of naïve immunocompetent recipients. Tumor growth kinetic measurements showed that CCL3-secreting tumors resulted in either a CD8+ T cell dependent slowing of tumor progression or complete regression in immunocompetent mice and protected subsequent lethal tumor challenge when used as a vaccine. Next, using fluorescence-activated cell sorting analysis (FACS) we analyzed the effects of lymphocyte recruitment to the LN draining the metastatic tumor by fluorescence- activated cell sorting analysis 3 and 5 days post-tumor injection. Mice inoculated with CCL3-secreting tumors resulted in similar increases in CD4+ and CD8+ T cells ranging from 1.3 to 1.7-fold increase from WT tumors and 1.3-fold increase from CCL4-secreting tumors on day 3. By day 5 the T cell accumulation in CCL3- secreting tumors reached similar levels as that of WT and CCL4-secreting tumors. Furthermore, dynamic intravital 2-photon microscopy and tissue histology studies further revealed that tumors occupy the B-cell follicle during early metastasis, and exogenous CCL3 and CCL4 gradient may potentially alter lymphocyte homeostasis in tumor draining LNs, resulting in immune activation rather than tolerance against CT26. We propose to interrogate biological effect of CCL3 on immune cell recruitment, activation, retention and effector functional delivery in the draining LNs following CCL3- or CCL4-secreting CT26 tumor inoculation. These studies include functional activation, migration and chemokine receptor expression of dendritic cells and lymphocytes upon exposure to CCL3. Furthermore, we propose to study the dynamic cell-cell interaction between CCL3-secreting, CCL4-secreting, or wild type CT26 tumor cells and various immune cells in the tumor-infiltrating LN within the first 5 days following tumor inoculation. These studies will provide scientific rationale for the exploration of incorporating inflammatory chemokines such as CCL3 as a therapeutic adjuvant to anti-tumor immunotherapy.