Evaluation of T follicular regulatory cells as novel cellular targets of cancer immunotherapy

项目摘要 靶向CTLA-4或PD-1的免疫检查点阻断（ICB）迅速发挥了标准治疗的作用 对于实体瘤，可以导致戏剧性的，持久的反应;尽管如此，只有不到30%的患者 对任何一种药物的单药治疗有反应。联合治疗可获得更好的长期生存结果， 而且还导致更频繁和严重的免疫相关不良事件（irAE）。几种新型 目前正在探索免疫疗法以评估其抗肿瘤能力。但关键是，大多数 这些靶点会受到靶点上、细胞外效应的影响，因为其他免疫细胞类型可以表达高水平的这些靶点。 分子。因此，由于总体应答率低、细胞外效应和广泛的免疫相关毒性， 限制治疗效果和单药治疗和联合治疗选择，迫切需要开发 新的免疫治疗靶点，表现出更受限制的表达谱。我们最近展示了 PD-1+滤泡调节性T（TFR）细胞在人类几种癌症类型的肿瘤组织中普遍存在， 和小鼠，并且它们是抗PD-1治疗功效的关键细胞决定因素。TFR细胞主要是 位于三级淋巴结构（TLS）内，并表现出优越的上级抑制能力， 与调节性T（Treg）细胞相比，TFR细胞的持续性，表明TFR细胞在损害患者的免疫功能中起关键作用。 存活和阻碍免疫疗法治疗功效。虽然我们已经证明肿瘤内TFR细胞来源于 从TREG前体细胞，驱动这一分化步骤的机制和转录因子（TF） 大部分都是未知的。在Aim 1中，我们建议采用单细胞RNA-seq、单细胞ATAC-seq和微- 规模化的ChIP测定，以充分表征肿瘤浸润性TREG细胞的转录组学特征， （4-1BB+）Treg细胞和TFR细胞。阐明TREG不同发育阶段的增强子谱， TFR分化可能为指导这一分化步骤的TF提供重要的见解。这些 分析将确定基因和转录因子，这些基因和转录因子对提高TFR细胞的抑制能力至关重要， 至于它们的维持或分化。耗尽TREG细胞或抑制其功能的策略， 增强抗肿瘤免疫的目的正在被深入研究。然而，关键是，其中大多数 方法基于抗体（即，针对TREG细胞耗尽进行ADCC优化;即抗CTLA-4），其具有 体内半衰期固有较长，因此增加了引起irAE的可能性。相反，我们的数据表明， 磷酸肌醇3-激酶δ（PI 3 K δ）抑制剂的替代给药方案可能提供靶向TFR的途径 细胞更具体。我们认为PI 3 K δ是一个新的、有吸引力的免疫治疗靶点， 肿瘤的在目标2和目标3中，我们将评估PI 3 Kd抑制剂的替代给药方案是否可以被应用于临床。 用于有效和安全地利用PI 3 K δ抑制剂在实体癌中的免疫调节作用， TFR细胞的瞬时消耗或抑制是否足以限制免疫抑制环境， 从而驱动抗肿瘤免疫而不引起毒性。

Project Summary Immune checkpoint blockade (ICB) targeting CTLA-4 or PD-1 rapidly assumed its role as a standard treatment for solid tumors and can lead to dramatic, long-lasting responses; nonetheless, fewer than 30% of patients respond to monotherapy with either agent. Combination therapy results in better long-term survival outcomes, but also causes more frequent and severe immune-related adverse events (irAEs). Several novel immunotherapies are currently being explored to evaluate their anti-tumor capacity. Crucially however, most of these targets suffer from on-target, off-cell effects, as other immune cell types can express high levels of these molecules. Hence, as low overall response rates, off-cell effects and widespread immune related toxicity severely limit both treatment efficacy and monotherapy and combination therapy options, there is urgent need to develop novel immunotherapy targets that exhibit a more restricted expression profile. We have recently demonstrated that PD-1+ follicular regulatory T (TFR) cells were prevalent in tumor tissues of several cancer types in humans and mice, and that they were critical cellular determinants of anti-PD-1 treatment efficacy. TFR cells were primarily located within tertiary lymphoid structures (TLS) and exhibited superior suppressive capacity and in vivo persistence when compared to regulatory T (TREG) cells, suggesting a key role for TFR cells in impairing patient survival and impeding immunotherapy treatment efficacy. While we have shown that intratumoral TFR cells derive from TREG precursor cells, the mechanisms and transcription factors (TFs) that are driving this differentiation step are largely unknown. In Aim1, we propose to employ single-cell RNA-seq, single-cell ATAC-seq and micro- scaled ChIP assays to fully characterize the transcriptomic signatures of tumor-infiltrating TREG cells, transitioning (4-1BB+) TREG cells and TFR cells. Elucidating the enhancer profiles in different developmental stages of TREG to TFR differentiation is likely to provide crucial insights into the TFs that instruct this differentiation step. These analyses will define genes and TFs that are pivotal for the heightened suppressive capacity of TFR cells, as well as for their maintenance or differentiation. Strategies to deplete TREG cells or to curb their functionality with the aim of enhancing anti-tumor immunity are being intensively investigated. Crucially however, most of these approaches are based on antibodies (i.e., ADCC-optimized for TREG cell depletion; i.e. anti-CTLA-4), which have an inherently long half-life in vivo, thus increasing the likelihood of causing irAEs. Conversely, our data imply that alternative dosing regimens of Phosphoinositide 3-kinase δ (PI3Kδ) inhibitors might offer a pathway to target TFR cells more specifically. We propose that PI3Kδ represents a novel and appealing immunotherapy target in solid tumors. In Aim 2 and Aim 3, we will assess whether alternative dosing regimens of PI3Kd inhibitors can be utilized to effectively and safely exploit the immunomodulatory impact of PI3Kδ inhibitors in solid cancers and whether a transient depletion or inhibition of TFR cells might suffice to restrict the immunosuppressive milieu in the tumor and thus drive anti-tumor immunity without causing toxicity.