Production of Interferon Gamma by Regulatory T Cells Mediates Response to Immunotherapy

摘要 最近在癌症免疫学中使用免疫检查点抑制剂（ICI）的成功，例如抗- 程序性细胞死亡蛋白1（PD 1）对癌症患者的总体生存产生了显著影响。 引人注目的是，抗PD 1使黑色素瘤患者的5年生存率增加了一倍，达到34%。的主要机制 ICI归因于对功能失调的CD 8+细胞毒性T淋巴细胞（CTL）的直接作用， 效应子功能的恢复和无反应性细胞状态的消除。激活的CTL产生强效的 细胞因子干扰素γ（IFNγ）通过激活巨噬细胞Th 1诱导抗肿瘤免疫 发育、促进淋巴细胞迁移和增加抗原呈递。然而， IFNγ和ICI对调节性T细胞（Tcells）的作用尚未完全研究。T细胞代表抑制性CD 4 + T细胞 通过预防自身免疫和慢性炎症调节免疫稳态的细胞群 在细胞接触、依赖和非依赖机制中。我们的实验室显示肿瘤完全消退， 小鼠中的T细胞被耗尽，说明T细胞是抗肿瘤免疫的主要屏障。特什哈韦 由叉头盒P3（Foxp 3）转录因子的表达定义，该转录因子已被提出， 对于Treg谱系的抑制功能和定型是必需的。然而，我们最近的发现 他挑战了这一教条，提出了一个中间状态的证据，在这个中间状态下，T细胞失去了抑制功能， 变得更像效应器，但仍保持Foxp 3表达，我们称之为“Treg脆性”。有趣的是， 这些脆弱的T细胞开始产生IFNγ。我们已经在体外证明IFNγ暴露于Tcl 4诱导Treg 脆性，并且Tfngr 1 L/LFoxp 3Cre-YFP上缺乏IFNγ受体的小鼠对抗PD 1具有抗性， 证明了IFNγ诱导的Treg脆性在癌症免疫治疗中的重要性。然而，生产 IFN-γ的IFN-γ活性尚未得到广泛研究。还有待确定（1）。如果IFNγ的产生是由TcB 对免疫疗法的反应是必需的，以及2.）IFNγ+ T细胞在肿瘤中的作用 微环境（TME）。我们假设ICI直接诱导Treg脆弱性，这是失能所必需的。 TME中的免疫抑制并允许CTL用免疫疗法重新激活/活化。 此外，TcR的重编程产生IFNγ，可能增强TcR的抗肿瘤应答。 通过Treg衍生的IFNγ作用于TME中的其他细胞的免疫疗法。然而，仍有待确定， Treg脆性发生在除抗PD 1以外的免疫疗法中。本实验室制作了小鼠模型，通过 CRISPR/Cas9，以使用Cre-Lox重组（IfngL/L）从特定细胞类型中选择性地缺失IFNγ。我们将 使用这种新的模型来确定Treg衍生的IFNγ是否是免疫治疗所需的， 将IFNγ从T细胞中删除（IfngL/LFoxp 3Cre-YFP）。我们还研究了Treg衍生的IFNγ对其他肿瘤细胞的影响。 TME中的细胞。如果成功，这些研究将揭示癌症免疫治疗诱导的效果。 以及Tcirrhosis用于驱动对免疫疗法的抵抗和反应的机制。

ABSTRACT Recent successes in cancer immunology with the use of immune checkpoint inhibitors (ICI), such as anti- programed cell death protein 1 (PD1) have made a significant impact on the overall survival of cancer patients. Strikingly, anti-PD1 doubled the 5-year survival rate for melanoma patients to 34%. The primary mechanism of ICI has been attributed to the direct effects on dysfunctional CD8+ cytotoxic T lymphocytes (CTLs), allowing for reinvigoration of effector function and elimination of the anergic cellular state. Activated CTLs produce the potent cytokine interferon gamma (IFNγ) to induce anti-tumor immunity via activation of macrophages, Th1 development, promotion of lymphocyte migration and increased antigen presentation. However, the effects of IFNγ and ICI on regulatory T cells (Tregs) has not been fully explored. Tregs represent a suppressive CD4+ T cell population that regulates immune homeostasis through prevention of autoimmunity and chronic inflammation in cell contact, -dependent and -independent, mechanisms. Our lab has shown complete tumor regression when Tregs are depleted from mice, illustrating that Tregs represent a major barrier to anti-tumor immunity. Tregs have been defined by the expression of the forkhead box P3 (Foxp3) transcription factor which has been proposed to be necessary for suppressive function and commitment to the Treg lineage. However, our findings recently challenged this dogma with evidence of an intermediate state where Tregs lose suppressive function and become more effector-like, yet maintain Foxp3 expression, a process we termed “Treg fragility”. Interestingly, these fragile Tregs start to produce IFNγ. We have shown in vitro that IFNγ exposure to Tregs induces Treg fragility, and mice that lack the IFNγ receptor on Tregs (Ifngr1L/LFoxp3Cre-YFP) are resistant to anti-PD1, demonstrating the importance of IFNγ-induced Treg fragility in cancer immunotherapy. However, the production of IFNγ by Tregs has not been extensively studied. It remains to be determined, 1.) if IFNγ production by Tregs is required for response to immunotherapy and 2.) what the function of IFNγ+ Tregs is in the tumor microenvironment (TME). We hypothesize that ICI directly induce Treg fragility, which is necessary to disable immune suppression in the TME and allow for CTLs to be reinvigorated/activated with immunotherapy. Additionally, the reprogramming of Tregs to produce IFNγ, may potentiate the anti-tumor response of immunotherapy via Treg-derived IFNγ acting on other cells in the TME. However, it remains to be determined if Treg fragility occurs in immunotherapies other than anti-PD1. Our lab has made a mouse model, via CRISPR/Cas9, to selectively delete IFNγ from specific cell types using Cre-Lox recombination (IfngL/L). We will use this novel model to determine if Treg-derived IFNγ is required for response to immunotherapy by selectively deleting IFNγ from Tregs (IfngL/LFoxp3Cre-YFP). We are also investigating the effect of Treg-derived IFNγ on other cells in the TME. If successful, these studies will uncover the effects that cancer immunotherapeutics induce on Tregs, and the mechanisms used by Tregs to drive resistance and response to immunotherapy.