Deciphering the molecular control of intratumoral dendritic cells

项目摘要 树突状细胞（DC）对肿瘤免疫有重要影响。DC耗竭消除肿瘤免疫， 在许多临床前肿瘤模型中对PD 1（α PD 1）免疫治疗的反应和人类肿瘤中的DC浸润 与临床结果呈正相关。不幸的是，我们仍然缺乏有效的策略来利用DC， 刺激肿瘤免疫，这在很大程度上是因为我们不完全了解肿瘤DC的控制。 为了解决这一缺点，我们对人类和小鼠肺肿瘤病变进行了scRNA-seq。值得注意的是， 我们在人和小鼠肺肿瘤中鉴定了一种独特的且几乎相同的DC群， 与DC成熟相关的上调基因，如CD 40和IL 12，以及免疫调节，包括 PD-L1和CD 200。这使我们将簇注释为“富含免疫调节分子的成熟DC” （mregDC）（Maier等人Nature 2020）。引人注目的是，mregDC是携带肿瘤抗原（Ag）的DC;这意味着这些 DC负责肿瘤Ag呈递。我们假设肿瘤DC的免疫刺激潜力是 在mreg模块中被上调的基因抑制，这阻碍了肿瘤免疫和应答的诱导 αPD1。我们建议通过靶向特定的mreg基因，我们可以解耦调节和刺激程序 增强DC对肿瘤反应性T细胞的活化，促进肿瘤免疫和α PD 1反应。支持 根据我们的假设，阻断mreg模块中上调的基因之一IL 4 R的信号传导， 活化、扩增肿瘤浸润T细胞和降低NSCLC小鼠模型中的肿瘤负荷。 此外，Birc 2/3的抑制（也在mregDC中上调）导致显著增强的DC活化。 为了验证我们的假设并达到我们的目的，我们将：（1）确定IL 4 R在诱导 mregDC状态和肿瘤免疫。我们将敲除小鼠和人DC中的IL 4 R，并确定这是如何发生的。 影响mreg诱导、Ag呈递和肿瘤免疫。我们还将结合联合收割机抗IL 4 R和抗PD 1来评估 在临床前模型中控制肿瘤生长的协同作用。(2)在生理学上评价Birc 2/3抑制， 肿瘤内DC的分子状态和免疫刺激活性。我们将检验这个假设， Birc 2/3的药理学抑制将增强DC产生IL-12以及共刺激分子， 促进癌细胞死亡和肿瘤Ag摄取，并导致强大的肿瘤免疫。(3)解卷积 DC表型的内在调节因子和mreg基因模块。我们将利用第一种CRISPR 我们开发了一个基因组学平台，用于KO 37个转录相关因子（TrF）基因中的每一个，这些基因被 mregDC，并确定每个如何影响肿瘤DC激活和分子状态。该项目的成果 将通过确定特异性免疫球蛋白的作用，为我们理解肿瘤内DC生物学提供重大进展。 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， 靶向mregDC中两种不同途径的化合物，并确定其他分子， 以增强DC活性。

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.