Identifying Immune Mechanisms for Microbiota-Inhibition of Anti-PD-L1 Tumor Response

项目总结 免疫检查点阻断是一种最近的癌症治疗策略，已在15- 40%的患者通过许可CD8+肿瘤浸润性淋巴细胞杀死肿瘤细胞来治疗几种癌症。尽管 这些药物戏剧性地改变了部分患者的临床病程，但未能在 大多数癌症患者。人类微生物区系被认为调节免疫基调或反应性，并且 因此，提高检查站封锁响应率是一个有希望的、可修改的目标。事实上，响应率 对于表皮恶性肿瘤来说，抗PD-1与粪便微生物多样性有关，而患者的 抗生素的使用。此外，已经在小鼠身上发现了特定的细菌分离株，这些细菌可以 不成比例地促进抗CTLA-4和抗PD-L1治疗后的肿瘤免疫反应。 虽然微生物区系操纵对检查站封锁反应的潜在重要性是显而易见的，但 要确定这些依赖微生物区系的癌症免疫反应是如何进行的，还有工作要做。 生成并维护。为了开始了解肠道中的微生物群免疫界面如何 为了促进非粘膜恶性肿瘤的免疫活性和抗PD-L1反应，我建立了一种 抗PD-L1治疗黑色素瘤的人源化灵芝模型。我已经证明了一种被定义的微生物 社区可抑制B16黑色素瘤对抗PD-L1抗体的反应。这笔赠款的目的是剖析 介导这种微生物区系依赖的肿瘤反应抑制的未知机制 通过评估几种免疫细胞群的功能和表型来抗PD-L1 肿瘤和肿瘤引流淋巴结。目标1-根据我的初步数据，我们选择了两个定义 当定植到无菌B16中时，导致不同肿瘤生长速度的微生物群落 携带黑色素瘤的小鼠。目的：评价肠道微生物区系对抗肿瘤T细胞的影响。 通过探讨肿瘤免疫的可能差异来探讨检查点阻断治疗后的反应 结构，T细胞增殖和肿瘤细胞杀伤的修饰，关键T细胞亚群的耗尽和 体内调节因子，以及抗PD-L1治疗前后的元基因组图谱。目标2--适应性 免疫反应依赖于髓系细胞的启动和激活，而髓系细胞反过来又被 微生物传感机械。我们假设髓系细胞功能的改变和 表型调节对抗PD-L1的适应性反应。为了评估这些细胞，我将进行预成型 肿瘤和引流淋巴中巨噬细胞和树突状细胞的转录和蛋白质组学分析 结节，通过髓系亚集耗竭评估反应因果依赖，并评估启动能力 有反应和无反应的灵知生菌小鼠的树突状细胞。通过研究这些灵知生物体动物 通过对检查点封锁的不同临床反应，我们希望揭示导致 检查点阻断反应，并可能识别新的免疫治疗靶点或生物标志物。

PROJECT SUMMARY Immune checkpoint blockade is a recent cancer therapeutic strategy that has enabled durable responses in 15- 40% of patients for several cancers by licensing CD8+ tumor infiltrating lymphocytes to kill tumor cells. Despite dramatically altering the clinical course in a subset of patients, these drugs fail to elicit durable response in the majority of cancer patients. The human microbiota is thought to regulate immune tone or responsiveness, and thus is a promising, modifiable target to improve checkpoint blockade response rates. Indeed, response rates for epidermal malignancies to anti-PD-1 have been associated with fecal microbial diversity, and patients’ prior use of antibiotics. Moreover, particular bacterial isolates have been identified in mice that can disproportionally contribute to tumor immune responses following anti-CTLA-4 and anti-PD-L1 therapy. While the potential importance of microbiota manipulation for checkpoint blockade response is clear, much work remains to be done to determine how these microbiota-dependent immune responses to cancer are generated and maintained. To begin understanding how the microbiota-immune interface in the gut can contribute to the immune activity and anti-PD-L1 response of non-mucosal malignancy, I have established a humanized gnotobiotic model of anti-PD-L1 treated melanoma. I have demonstrated that a defined microbial community can inhibit B16 melanoma response to anti-PD-L1. This grant aims to dissect the yet unknown mechanisms that mediate this microbiota-dependent inhibition of tumor response to anti-PD-L1 by evaluating the functions and phenotypes of several immune cell populations in the tumor and tumor draining lymph node. Aim 1—Based on my preliminary data we have selected two defined microbial communities that result in contrasting tumor growth rates when colonized into germfree, B16 melanoma-bearing mice. I aim to evaluate the influence of intestinal microbiota on anti-tumor T cell responses following checkpoint blockade therapy by exploring possible differences in tumor-immune architecture, modifications to T cell proliferation and killing of tumor cells, depletion of key T cell subsets and regulators in vivo, and metagenomic profiling before and after anti-PD-L1 treatment. Aim 2—The adaptive immune response is dependent upon priming and activation by myeloid cells, which in turn are modified by microbial sensing machinery. We hypothesize that alterations in myeloid cell functions and phenotypes regulate the adaptive response to anti-PD-L1. To evaluate these cells I will preform transcriptomic and proteomic analysis of macrophages and dendritic cells in the tumor and draining lymph node, assess response causal dependency through myeloid subset depletion, and evaluate the priming ability of dendritic cells from responding and nonresponding gnotobiotic mice. By studying these gnotobiotic animals with different clinical responses to checkpoint blockade, we hope to uncover the mechanisms that contribute to checkpoint blockade response, and possibly identify new immunotherapy targets or biomarkers.