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

描述（由申请人提供）：我们之前已经确定Her-2/neu转基因小鼠与HLA-A2.1/Kb转基因小鼠（A2xneu）杂交对Her-2/neu/A2.1免疫显性表位具有免疫耐受性。我们还证明，在A2xneu小鼠中，用多肽或Her-2/neu蛋白免疫不能提供有效的抗肿瘤反应。Her-2/ new小鼠与HLA-A2.1/Kb小鼠杂交的结果之一是，这些动物在20-22个月大时出现自发性肿瘤。因此，A2xneu小鼠模型提供了一个独特的机会来评估针对自身抗原的抗肿瘤免疫反应，其中衰老和耐受性同时存在。我们已经证明，衰老极大地改变了老年小鼠的免疫系统。因此，确定和优化疫苗接种策略是至关重要的，该策略可以有效地刺激免疫反应，从而在年轻和年老的耐受动物中产生肿瘤排斥反应。有大量证据表明，用不同类型的佐剂靶向apc可诱导抗肿瘤免疫应答。我们比较了不同tlr配体（Poly I:C、LPS、鞭毛蛋白、咪喹莫特、CpG-ODN）对年轻和年老A2xneu小鼠的抗肿瘤作用。结果表明，仅瘤内注射CpG-ODN可诱导年轻和老年A2xneu小鼠的肿瘤排斥反应。尽管年轻和年老的A2xneu小鼠在注射CpG-ODN后可以对原发肿瘤产生排斥反应，但这些动物不能产生保护性记忆反应。注射CpG-ODN后肿瘤微环境分析表明：1)apc活化；2)促炎性Th1型反应；3) CD4、CDS、T细胞和NK细胞的活化；4) T-regs的数量急剧减少。综上所述，这些结果表明，注射CpG-ODN影响肿瘤微环境，有利于抗肿瘤反应。然而，我们还不完全了解CpG-ODN注射是如何诱导肿瘤排斥的。本提案的目的是确定CpG-ODN在诱导抗肿瘤反应中的作用机制，并制定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)评估和表征CpG-ODN在诱导老年小鼠免疫应答中的作用；2)评估CpG-ODN与T-regs的关系，以及T-regs在老龄小鼠免疫反应库调节和激活中的关系；3)优化利用anti- new -CpG-ODN靶向肿瘤部位的CpG-ODN治疗老年小鼠肿瘤。相关性：我们的研究结果首次表明，在肿瘤部位靶向CpG-ODN可恢复老年小鼠的免疫应答。表征CpG-ODN对免疫系统的影响将最终导致该疗法的优化。此外，随着抗新CpG-ODN分子的产生，我们可以靶向体内任何部位的CpG-ODN，从而有可能开发一种治疗癌症的通用疫苗策略。总的来说，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，以开发更有效的免疫疗法，用于年轻和年老的耐受宿主。

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.