Reversing immune evasion and enhancing immune detection with topical resiquimod

项目概要/摘要 瑞喹莫特凝胶是一种局部应用的免疫调节剂和TLR 7/8激动剂。它刺激树突状细胞（DC）， 健康和发炎的皮肤，并能有效地增强免疫反应。在12年的一个小型I期试验中， 接受过大量预治疗的难治性皮肤局限性皮肤T细胞淋巴瘤（CTCL）患者，外用瑞喹莫特 逆转免疫逃避，改善免疫检测和诱导效应抗癌反应， 系统地。90%的患者在治疗后的病变中恶性T细胞克隆减少，83%的患者在治疗后的病变中恶性T细胞克隆减少。 治疗和远处未治疗的皮肤病变的消退，表明这种局部治疗增强了 全身免疫反应。这种药物增强局部和全身免疫反应的能力 表明局部瑞喹莫特可能是治疗皮肤感染和癌症的一种有前途的新疗法。我们 建议对时间敏感，首先在新鲜活检标本和血液样本的人类辅助研究中， 行业赞助的，多中心，II期，双盲，安慰剂对照试验的瑞喹莫特凝胶在 IA、IB和IIA期CTCL的治疗。我们假设，局部瑞喹莫特增强先天性和 获得性免疫应答，我们提出了三个目的来研究局部瑞喹莫特对i） 先天免疫系统，ii）适应性免疫系统，以及iii）发现其机制 未治疗的病变消退。我们将使用单细胞RNA测序（scRNA-seq），基于NanoString的基因 表达谱分析、多重免疫染色和TCR测序，以研究治疗前后的皮肤活检 我们将使用飞行时间流式细胞术（CyTOF）、TCR测序和功能性流式细胞术， 分析研究治疗前后的血液样本。我们将评估特定的假设， 瑞喹莫特诱导巨噬细胞极化从M2向M1转变，激活NK细胞，增强 - 将T细胞募集到皮肤中，逆转肿瘤微环境内T细胞的耗竭，和/或 产生新的肿瘤特异性T细胞。我们将确定未治疗病变的消退是否与 可检测水平的I型干扰素或Th 1细胞因子，循环DC或NK细胞的活化，或 肿瘤特异性T细胞通过血流到达未经治疗的病变。这些研究具有时间敏感性，因为 它们需要使用来自正在进行的临床试验的新鲜活检标本。我们的研究将澄清 这种有前途的新药的作用机制，并确定它如何逆转免疫逃避， 增强CTCL中的免疫检测。这些研究可能支持使用瑞喹莫特治疗 其他皮肤癌和感染。

Project Summary/Abstract Resiquimod gel is a topically applied immunomodulator and TLR7/8 agonist. It stimulates dendritic cells (DC) in both healthy and inflamed skin and can potently enhance immune responses. In a small phase I trial of in 12 heavily pretreated patients with refractory, skin limited cutaneous T cell lymphoma (CTCL), topical resiquimod reversed immune evasion, improved immunodetection and induced effector anticancer responses both locally and systemically. 90% of patients had reduction in the malignant T cell clone in treated lesions and 83% had regression of both treated and distant, untreated skin lesions, demonstrating that this topical therapy enhances systemic immune responses. The ability of this drug to enhance both local and systemic immune responses suggests that topical resiquimod could be a promising new treatment for both skin infections and cancer. We propose time sensitive, first in human ancillary studies of fresh biopsy specimens and blood samples from an industry sponsored, multicenter, phase II, double blind, placebo-controlled trial of resiquimod gel in the treatment of stage IA, IB and IIA CTCL. We hypothesize that topical resiquimod enhances both innate and adaptive immune responses and we propose three Aims to study the effects of topical resiquimod on i) the innate immune system, ii) the adaptive immune system, and iii) to discover the mechanisms by which untreated lesions regress. We will use single cell RNA sequencing (scRNA-seq), NanoString based gene expression profiling, multiplex immunostaining and TCR sequencing to study skin biopsies before and after resiquimod therapy and we will use cytometry by time of flight (CyTOF), TCR sequencing and functional assays to study blood samples before and after treatment. We will evaluate specific hypotheses that resiquimod induces a shift in macrophage polarization from M2 to M1, activates NK cells, enhances recruitment of T cells into skin, reverses exhaustion of T cells within the tumor microenvironment and/or generates new tumor-specific T cells. We will determine if regression of untreated lesions is associated with detectable levels of type I interferons or Th1 cytokines, activation of circulating DC or NK cells, or travel of tumor-specific T cells through the bloodstream to untreated lesions. These studies are time sensitive because they require the use of fresh biopsy specimens from an ongoing clinical trial. Our studies will clarify the mechanisms of action of this promising new medication and determine how it reverses immune evasion and enhances immunodetection in CTCL. These studies may support the use of resiquimod in the treatment of other skin cancers and infections.