Exploring the intersection of hypoxia and epigenetic modifiers in tumor-mediated CD8 T cell exhaustion

项目摘要 CD 8 + T细胞是抗肿瘤应答的基本组成部分;然而，肿瘤浸润性CD 8 + T细胞 (TIL)被肿瘤微环境破坏CD 8 + TIL显示衰竭表型 细胞因子表达降低，共抑制受体（IR）表达增加，如PD-1和 蒂姆三号IR的获得标志着功能障碍性TIL从祖细胞（PD 1 lo）到终末细胞（PD 110）的进展。 耗尽（PD 1hiTim 3+）。我们已经描述了在终末耗竭的CD 8+细胞中二价基因的明显增加， TIL的特征在于同时存在活性标记H3 K4 me 3和抑制性标记H3 K27 me 3， 以及低基因表达。虽然这种状态传统上是在多能细胞中发现的， 作为一种“平衡”状态，新的研究表明，它并不是干细胞样细胞所特有的。二价的增加 终末耗竭细胞中的染色质表明对肿瘤的甲基化反应增加 微环境我们怀疑缺氧介导的组蛋白修饰物的调节是造成这一现象的原因 二价基因的增加并在介导功能障碍方面发挥着关键作用。理解二价的作用 基因及其调控将提供有关肿瘤耗竭调控的关键信息， 微环境该提案寻求使用传统免疫学技术的组合， 基于测序的实验，以更好地了解这些染色质特征的调节。我们 假设H3 K27 me 3异常调节染色质修饰物驱动细胞中的二价和耗竭， 终末耗竭的T细胞，并可靶向提高抗肿瘤免疫力。我们将测试这个 假设有两个目的;我们将1）确定在缺氧中抑制H3 K27去甲基化是否足够 使用脱甲基酶抑制剂和创新的T细胞体外测定来驱动二价和末端耗竭， 疲惫不堪我们将2）确定Ezh 2是否可以靶向使用两种方法来防止耗竭中的二价 Ezh 2活性的抑制剂和Ezh 2的可诱导缺失。的 终末耗竭TIL特异性二价基因的存在支持了这样的观点，即当细胞耗竭时， 在表观遗传学上对免疫疗法如抗PD 1治疗、染色质和转录免疫疗法具有抗性。 调节剂可以通过增加关键功能基因的表达来重振疲惫的TIL， 提高抗肿瘤反应。

PROJECT SUMMARY CD8+ T cells are a fundamental component of the anti-tumor response; however, tumor-infiltrating CD8+ T cells (TIL) are rendered dysfunctional by the tumor microenvironment. CD8+ TIL display an exhausted phenotype with decreased cytokine expression and increased expression of co-inhibitory receptors (IRs), such as PD-1 and Tim-3. The acquisition of IRs marks the progression of dysfunctional TIL from progenitors (PD1lo) to terminally exhausted (PD1hiTim3+). We have described a distinct increase in bivalent genes in terminally exhausted CD8+ TIL, which are characterized by presence of both the active mark H3K4me3 and the repressive mark H3K27me3, as well as low gene expression. While this state has traditionally been found in pluripotent cells and described as a “poised” state, new research suggests that it is not specific to stem-like cells. The increase in bivalent chromatin in terminally exhausted cells suggests increased methylation in response to the tumor microenvironment. We suspect that hypoxia-mediated regulation of histone modifiers is responsible for this increase in bivalent genes and plays a critical role in mediating dysfunction. Understanding the role of bivalent genes and their regulation will provide crucial information about the regulation of exhaustion in the tumor microenvironment. This proposal seeks to use a combination of traditional immunology techniques and sequencing-based experiments to better understand the regulation of these chromatin features. We hypothesize that dysregulated chromatin modifiers of H3K27me3 drive bivalency and exhaustion in terminally exhausted T cells and can be targeted to improve anti-tumor immunity. We will test this hypothesis in two aims; we will 1) determine whether inhibition of H3K27 demethylation in hypoxia is sufficient to drive bivalency and terminal exhaustion using demethylase inhibitors and innovative in vitro assays of T cell exhaustion. We will 2) determine whether Ezh2 can be targeted to prevent bivalency in exhaustion using both inhibitors of Ezh2 activity and inducible deletion of Ezh2 in in vitro and in vivo systems, respectively. The presence of bivalent genes specific to terminally exhausted TIL supports the notion that while exhausted cells are epigenetically resistant to immunotherapy, such as anti-PD1 treatment, chromatin and transcriptional regulators can be targeted to reinvigorate exhausted TIL by increasing expression of key functional genes to improve the anti-tumor response.