Mechanism of Action of CpG-ODN in Generating Tumor Immunity in the Aged

描述(申请人提供)：我们先前已经鉴定HER-2/neu转基因小鼠与人类白细胞抗原A2.1/KB转基因小鼠(A2xneu)杂交，对HER-2/neu/A2.1免疫优势表位具有免疫耐受。我们还证明，多肽或HER-2/neu蛋白免疫并不能有效地对抗A2xneu小鼠的肿瘤。HER-2/neu小鼠与HLA-A2.1/KB小鼠杂交的结果之一是，在这些动物中，当动物20-22个月大时，就会出现自发肿瘤。因此，A2xneu小鼠模型提供了一个独特的机会来评估针对同时存在衰老和耐受的自身抗原的抗肿瘤免疫反应。我们已经证明，衰老极大地改变了老龄小鼠的免疫系统。因此，识别和优化疫苗接种策略，有效地刺激免疫反应，从而在幼年和老年耐受动物中产生肿瘤排斥反应，是至关重要的。有大量证据表明，以不同类型的佐剂靶向APC可以诱导抗肿瘤免疫反应。我们比较了不同的TLR配体(Poly I：C、脂多糖、鞭毛蛋白、咪喹莫特、CpG-ODN)对年轻和老年A2xneu小鼠的抗肿瘤作用。结果表明，只有瘤内(I.T.)注射CpG-ODN可诱导A2xneu幼鼠和老年小鼠肿瘤排斥反应。尽管年轻的和老年的A2xneu小鼠在接受CpG-ODN注射治疗后可以排斥原发肿瘤，但这些动物不能产生保护性记忆反应。对注射CpG-ODN后肿瘤微环境的分析表明：1)APC的激活；2)促炎症的Th1型反应；3)CD4、CDS T细胞和NK细胞的激活；4)T-regs的数量急剧减少。综上所述，这些结果表明，IT。注射CpG-ODN影响有利于抗肿瘤反应的肿瘤微环境。然而，我们还不完全了解注射CpG-ODN是如何诱导肿瘤排斥反应的。该方案的目的是确定CpG-ODN在诱导抗肿瘤反应中的作用机制，并开发一种在老年耐受宿主中产生记忆反应的策略。在这些研究中，我们将：1)评估和鉴定CpG-ODN在老龄小鼠诱导免疫反应中的作用；2)评价CpG-ODN和T-regs之间的关系，以及T-regs在调节老龄小鼠免疫反应和激活免疫反应中的关系；3)优化使用抗neu-CpG-ODN来靶向肿瘤部位的CpG-ODN治疗老龄小鼠肿瘤。相关性：我们的结果首次表明，在肿瘤部位靶向CpG-ODN可以恢复老年小鼠的免疫反应。确定CpG-ODN对免疫系统的影响将最终导致该疗法的优化。此外，随着抗neu-CpG-ODN分子的产生，我们可以针对体内任何地方的CpG-ODN，从而有可能开发出治疗癌症的通用疫苗策略。总体而言，从这些研究中获得的信息将揭示控制和操纵免疫系统的策略，以在年轻和老年耐受宿主中开发更有效的免疫疗法。

DESCRIPTION (provided by applicant): We have previously identified that Her-2/neu transgenic mice crossed with the HLA-A2.1/Kb transgenic mice (A2xneu) are immunotolerant against Her-2/neu/A2.1 immunodominant epitopes. We have also demonstrated that immunization with peptides or Her-2/neu proteins does not provide an effective antitumor response against tumors in A2xneu mice. One of the consequences of crossing the Her-2/neu mice with the HLA-A2.1/Kb mice is that in these animals spontaneous tumors appear when animals are 20-22 months old. Therefore, the A2xneu mouse model provides a unique opportunity to evaluate antitumor immune responses against a self antigen where aging and tolerance are present at the same time. We have demonstrated that aging drastically altered the immune system of old mice. Therefore, it is critical to identify and optimize a vaccination strategy that effectively stimulates an immune response resulting in tumor rejection in young and old tolerant animals. There is plenty of evidence indicating that targeting APCs with different types of adjuvants results in the induction of an antitumor immune response. We compared the antitumor effect of different TLR-ligands (Poly I:C, LPS, flagellin, imiquimod, CpG-ODN) in young and old A2xneu mice. The result indicated that only intratumoral (i.t.) injections of CpG-ODN induced the rejection of tumors in young and old A2xneu mice. Although young and old A2xneu mice could reject the primary tumor after treatment with i.t injections of CpG-ODN, these animals could not develop a protective memory response. Analysis of the tumor microenvironment following i.t injections of CpG-ODN indicated: 1) activation of APCs; 2) a pro-inflammatory Th1 type response; 3) activation of CD4, CDS T cells and NK cells; and 4) the numbers of T-regs were drastically reduced. Taken together, these results indicate that i.t. injections of CpG-ODN influence the tumor microenvironment that favors the antitumor response. However, we do not yet completely understand how CpG-ODN injections induce the rejection of tumors. The goal of this proposal is to determine the mechanism of action of CpG-ODN in the induction of antitumor responses and, to develop a strategy to generate memory responses in old tolerant hosts. In these studies we will: 1) evaluate and characterize the effect of CpG-ODN on the induction of immune responses in old mice; 2) evaluate the relationship between CpG-ODN and T-regs, and the relationship of T-regs in regulating the repertoire and activation of immune responses in old mice; and 3) optimize the use of anti-neu-CpG-ODN to target the CpG-ODN at the tumor site for the treatment of tumors in old mice. Relevance: For the first time our results show that targeting CpG-ODN at the tumor site restores the immune response in old mice. Characterizing the effect of CpG-ODN on the immune system will ultimately lead to the optimization of this therapy. Furthermore, with the generation of the anti-neu-CpG-ODN molecule we can target the CpG-ODN anywhere in the body making it possible to develop a universal vaccine strategy for the treatment of cancer. Overall, the information gained from these studies will reveal strategies for controlling and manipulating the immune system to develop more effective immunotherapies in young and old tolerant hosts.