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

摘要 免疫检查点抑制剂(ICI)的使用在癌症免疫学中的最新成功，如抗- 程序性细胞死亡蛋白1(PD1)对癌症患者的总体生存有重要影响。 引人注目的是，抗PD1使黑色素瘤患者的5年生存率翻了一番，达到34%。其主要作用机制是 ICI被归因于对功能失调的CD8+细胞毒性T淋巴细胞(CTL)的直接作用，允许 重振效应器功能，消除细胞无能状态。激活的CTL产生有效的 细胞因子干扰素γ通过激活巨噬细胞Th1诱导抗肿瘤免疫 发展，促进淋巴细胞迁移和增加抗原呈递。然而，它的影响 干扰素、γ和ICI对调节性T细胞(Treg)的影响尚未得到充分研究。Treg代表抑制性的CD4+T细胞 通过预防自身免疫和慢性炎症调节免疫动态平衡的细胞群 在细胞接触中，依赖和独立的机制。我们的实验室显示当肿瘤完全消退时 小鼠体内的Tregs被耗尽，这表明Tregs是抗肿瘤免疫的主要障碍。Tregs有 由叉头盒P3(Foxp3)转录因子的表达定义，该转录因子已被提出 对于抑制功能和对Treg血统的承诺是必要的。然而，我们最近的发现 用中间状态的证据挑战了这一教条，在中间状态中，特雷格失去了压制功能 变得更像效应器，同时保持Foxp3的表达，这一过程我们称之为“Treg脆弱性”。有趣的是， 这些脆弱的树开始产生干扰素γ。我们在体外已经证明，干扰素γ暴露于Treg可以诱导Treg 易感性，缺乏树上干扰素γ受体的小鼠(Ifngr1L/LFoxp3Cre-YFP)对抗PD1具有抵抗力， 证明干扰素γ诱导的Treg脆性在癌症免疫治疗中的重要性。然而，生产 Tregs对干扰素γ的作用尚未得到广泛研究。还有待确定，1.)如果Tregs产生干扰素γ 对免疫治疗的反应是必需的和2。)干扰素γ+Treg s在肿瘤中的作用 微环境(TME)。我们假设ICI直接导致Treg脆弱性，这是禁用该漏洞所必需的 TME中的免疫抑制，并允许CTL通过免疫疗法重新激活/激活。 此外，重新编程Treg以产生干扰素γ，可能会增强Tregs的抗肿瘤反应 通过作用于TME中其他细胞的Treg来源的干扰素γ进行免疫治疗。然而，还有待确定的是， Treg脆性出现在抗PD1以外的免疫疗法中。我们的实验室已经制作了一个小鼠模型，通过 CRISPR/Cas9，利用Cre-Lox重组技术选择性删除特定细胞类型的干扰素γ(IfngL/L)。我们会 使用这一新的模型通过选择性地确定免疫治疗是否需要Treg来源的干扰素γ 从树上删除干扰素γ(IfngL/LFoxp3Cre-Yfp)。我们还在研究Treg衍生的干扰素γ对其他 TME中的细胞。如果成功，这些研究将揭示癌症免疫疗法所产生的影响。 关于Tregs，以及Tregs用来驱动抵抗和对免疫治疗的反应的机制。

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.