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

项目总结 CD8+T细胞是抗肿瘤反应的基本成分；然而，肿瘤浸润性CD8+T细胞 肿瘤微环境使肿瘤细胞(TIL)功能紊乱。CD8+TIL呈耗竭表型 细胞因子表达减少，共抑制受体(IR)表达增加，如PD-1和 蒂姆-3。IRS的获得标志着功能障碍的TIL从祖细胞(PD1lo)向终末期进展 力竭(PD1hiTim3+)。我们已经描述了在终末耗尽的CD8+中二价基因的明显增加 TIL，其特征是存在活性标记H3K4me3和抑制标记H3K27me3， 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，并被描述为 作为一种“稳定的”状态，新的研究表明，它并不是干细胞特有的。二价体的增加 终末耗尽细胞染色质提示对肿瘤的反应中甲基化增加 微环境。我们怀疑低氧对组蛋白修饰物的调节与此有关。 二价基因的增加，并在调节功能障碍中发挥关键作用。理解二价键的作用 基因及其调控将提供有关肿瘤中耗竭调节的关键信息。 微环境。这项建议寻求使用传统免疫学技术和 以测序为基础的实验，以更好地了解这些染色质功能的调节。我们 假设H3K27me3的染色质修饰物失调导致二价体和耗竭 终末耗尽的T细胞，可靶向提高抗肿瘤免疫力。我们将对此进行测试 假设有两个目的；我们将1)确定在低氧条件下抑制H3K27去甲基化是否充分 使用去甲基酶抑制剂和创新的T细胞体外检测来驱动二价性和终末耗竭 疲惫不堪。我们将确定Ezh2是否可以作为目标，以使用两者来防止耗尽中的二价 分别在体外和体内系统中抑制Ezh2的活性和诱导Ezh2的缺失。这个 终末耗尽的TIL的二价基因的存在支持这样一种观点，即虽然耗尽的细胞 对免疫治疗有表观遗传抗性，如抗PD1治疗、染色质和转录 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.