Deciphering the molecular control of intratumoral dendritic cells

项目总结 树突状细胞(DC)对肿瘤免疫有重要影响。DC耗竭会破坏肿瘤免疫力， 多种临床前肿瘤模型对pD1(αpd1)免疫治疗的反应及人肿瘤中DC的侵袭 与临床结果呈正相关。不幸的是，我们仍然缺乏有效的战略来利用DC来 刺激肿瘤免疫，这在很大程度上是因为我们没有完全了解肿瘤DC的控制。 为了解决这一缺陷，我们对人和小鼠的肺肿瘤病变进行了scRNA-seq。值得注意的是， 我们在人和小鼠的肺肿瘤中发现了一个截然不同且几乎相同的DC群体 与DC成熟相关的基因上调，如CD40和IL12，以及免疫调节，包括 PD-L1和CD200。这使得我们将该簇注释为“富含免疫调节分子的成熟DC” (MregDC)(Maier等人自然2020)。引人注目的是，mregDC是携带肿瘤抗原(Ag)的DC； 树突状细胞负责肿瘤抗原的提呈。我们假设肿瘤DC的免疫刺激潜力是 被mreg模块中上调的基因抑制，这阻碍了肿瘤免疫和反应的诱导 发送到αpd1。我们建议，通过针对特定的mreg基因，我们可以将调节和刺激程序分离。 增强肿瘤反应性T细胞的DC活性，促进肿瘤免疫和αPD1应答。在支持中 在我们的假设中，阻断IL4R的信号，增强了DC，IL4R是mreg模块中上调的基因之一 在NSCLC小鼠模型中，激活、扩大肿瘤浸润性T细胞和降低肿瘤负担。 此外，抑制BIRC2/3，也在mregDC中上调，导致DC激活显著增强。 为了检验我们的假设和达到我们的目标，我们将：(1)确定IL4R在诱导 MregDC状态与肿瘤免疫。我们将在小鼠和人类DC中敲除IL4R，并确定如何 影响mreg诱导、抗原提呈和肿瘤免疫。我们还将联合抗IL4R和抗PD1来评估 临床前模型中控制肿瘤生长的协同作用。(2)对BIRC2/3的抑制作用进行生理评价。 肿瘤内树突状细胞的分子状态和免疫刺激活性。我们将检验这一假设 药物抑制BIRC2/3将增强DC产生IL-12以及共刺激分子，而 促进癌细胞死亡和肿瘤抗原摄取，并导致强大的肿瘤免疫。(3)去卷积 DC表型和mreg基因模块的内在调节。我们将利用首创的CRISPR 我们开发的基因组学平台可以对37个转录相关因子(TRF)基因进行KO，这些基因由 MregDC，并确定每种基因如何影响肿瘤DC的激活和分子状态。这个项目的结果是 将通过确定特定的DC的作用，为我们对肿瘤内DC生物学的理解提供重大进展 抑制肿瘤DC功能的基因和途径，在临床前模型中建立治疗潜力 针对在mregDC中运行的两条不同路径的化合物，并识别可能 有针对性地加强数据中心活动。

PROJECT SUMMARY Dendritic cells (DC) have a major influence on tumor immunity. DC depletion abrogates tumor immunity and response to PD1 (αPD1) immunotherapy in many preclinical tumor models and DC infiltration in human tumors is a positive correlate of clinical outcome. Unfortunately, we still lack effective strategies for harnessing DC to stimulate tumor immunity and this is in large part because we do not fully understand the control of tumor DCs. To address this shortcoming, we performed scRNA-seq on human and mouse lung tumor lesions. Notably, we identified a distinct and nearly identical population of DC in both human and mouse lung tumors which upregulated genes associated with both DC maturation such as CD40 & IL12, and immunoregulation, including PD-L1 & CD200. This led us to annotate the cluster “mature DC enriched in immuno-regulatory molecules” (mregDC)(Maier et al. Nature 2020). Strikingly, mregDC were the DC carrying tumor antigen (Ag); meaning these DC are responsible for tumor Ag presentation. We hypothesize the immunostimulatory potential of tumor DC is dampened by genes upregulated in the mreg module and this thwarts induction of tumor immunity and response to αPD1. We propose that by targeting specific mreg genes we can decouple regulatory & stimulatory programs and enhance DC activation of tumor-reactive T cells and promote tumor immunity and αPD1 response. In support of our hypothesis, blocking signaling of IL4R, one of the upregulated genes in the mreg module, enhanced DC activation, expanded tumor-infiltrating T cells, and reduced tumor burden in a mouse model of NSCLC. Additionally, inhibition of Birc2/3, also upregulated in mregDC, led to substantially enhanced DC activation. To test our hypotheses and reach our objective, we will: (1) Determine the role of IL4R on induction of the mregDC state and tumor immunity. We will knockout IL4R in mouse & human DC and determine how this impacts mreg induction, Ag presentation, & tumor immunity. We will also combine anti-IL4R & anti-PD1 to assess synergy in controlling tumor growth in a preclinical model. (2) Evaluate Birc2/3 inhibition on the physiology, molecular state and immunostimulatory activity of intratumoral DC. We will test the hypothesis that pharmacological inhibition of Birc2/3 will enhance DC production of IL-12, as well co-stimulatory molecules, while facilitating cancer cell death and tumor Ag uptake, and result in robust tumor immunity. (3) Deconvolute the intrinsic regulators of DC phenotype and the mreg gene module. We will utilize a first-of-its-kind CRISPR genomics platform we developed to KO each of the 37 transcriptional related factor (TrF) genes upregulated by mregDC and determine how each impacts tumor DC activation and molecular state. The outcome of this project will provide a major advance in our understanding of intratumoral DC biology by determining the role of specific genes and pathways in dampening tumor DC functions, establish in preclinical models the therapeutic potential of compounds targeting two different pathways operating in mregDC, and identify additional molecules that could be targeted to enhance DC activity.