Abstract 545: Differential regulation of human T-cells by TGR-1202, a novel PI3Kδ inhibitor
Abstract 545: Differential regulation of human T-cells by TGR-1202, a novel PI3Kδ inhibitor
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摘要 545：新型 PI3Kδ 抑制剂 TGR-1202 对人类 T 细胞的差异调节
DOI：
10.1158/1538-7445.am2016-545
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发表时间：
2016
期刊：
影响因子：
11.2
通讯作者：
J. Pinilla
中科院分区：
文献类型：
--
作者：
Kamira Maharaj;J. Powers;R. Fonseca;H. Miskin;D. Maryanski;E. Sahakian;J. Pinilla
TGR-1202是一种新型的新一代PI3Kδ抑制剂，与先前的小分子PI3Kδ抑制剂相比，具有显著的结构和药理差异。与以往的PI3Kδ抑制剂相比，TGR-1202在治疗不良事件显著不同的B细胞恶性肿瘤方面具有很高的临床疗效，特别是在肝毒性或结肠炎方面，这些不良事件很少或不存在。据推测，这些作用可能是由于T细胞免疫介导的机制。我们假设TGR-1202保留了调节性T细胞（Treg）群的功能，从而减少了治疗后免疫介导的副作用。在这里，我们的目的是比较临床可用的PI3Kδ抑制剂对T细胞的影响，重点是Tregs。方法：我们比较了ideelalisib、duvelisib和TGR-1202作为单一药物在体外对分离的人T细胞的活性。采用CellTiter Blue®、annexin V/PI和CFSE染色检测细胞活力、细胞凋亡和细胞增殖。CBA和qRT PCR检测细胞因子和转录因子。流式细胞术检测亚群比例和表面标记物表达。结果：首先，我们观察到，在用ideelalisib、duvelisib或TGR-1202治疗分离的T细胞群后，细胞毒性从25uM开始呈剂量依赖性增加。在此剂量下，细胞凋亡在48 - 72小时内被诱导。其次，所有抑制剂在刺激后都能降低CD3+ T细胞的细胞因子产生。特别是，TGR-1202治疗后，IFN-γ、IL-10和IL-17a的减少不太明显，表明T细胞反应相对保持。所有抑制剂均降低T-bet （Th1）、gta -3 （Th2）和FoxP3 （Treg）的mRNA表达，但在TGR-1202处理的T细胞中，FoxP3水平始终较高。第三，检测药物治疗后CD4:CD8比值正常，CD4+和CD8+亚群增殖能力未受影响。最后，所有抑制剂都降低了刺激后treg的总百分比（CD4+ CD25HI FoxP3+），同时降低了treg上共抑制分子CTLA-4和PD-1的表达。有趣的是，TGR-1202显著保留了treg接近正常的百分比，以及CTLA-4和PD-1在treg上的表面表达，表明免疫检查点封锁和抑制性表型的保留更大。结论：我们在此报告TGR-1202对人T细胞的影响不同于ideelalisib和duvelisib。TGR-1202维持IL-10的产生、FoxP3 mRNA的表达，维持Treg百分比和免疫检查点分子的表达，提示Treg数量和功能的相对保存。提出的数据开始为免疫介导的细胞机制提供新的见解，这些机制在TGR-1202的临床试验中缺乏副作用。体内模型进一步表征对T细胞室的影响正在进行中。引文格式：Kamira K. Maharaj, John Powers, Renee Fonseca, Hari Miskin, Dave Maryanski, Eva Sahakian, Javier Pinilla-Ibarz。新型PI3Kδ抑制剂TGR-1202对人t细胞的差异调控[摘要]。摘自：第107届美国癌症研究协会年会论文集；2016年4月16-20日；新奥尔良，洛杉矶。费城（PA）： AACR；癌症研究，2016；76（14增刊）：摘要第545期。
INTRODUCTION: TGR-1202 is a novel, next-generation PI3Kδ inhibitor presenting significant structural and pharmacological differences from prior small-molecule PI3Kδ inhibitors. TGR-1202 has high clinical efficacy in treatment of B cell malignancies with a substantially differentiated adverse event profile compared to previous PI3Kδ inhibitors, specifically concerning hepatotoxicity or colitis which have been minimal or non-existent. It has been postulated that these effects may be due to T cell immune-mediated mechanisms. We hypothesized that TGR-1202 preserves the function of the regulatory T cell (Treg) population, translating to decreased immune-mediated side effects after treatment. Here, we aimed to compare effects of clinically available PI3Kδ inhibitors on T cells with an emphasis on Tregs. METHODS: We compared activity of idelalisib, duvelisib, and TGR-1202 as single agents in vitro in isolated human T cells. Viability, apoptosis, and proliferation were determined using CellTiter Blue®, annexin V/PI and CFSE staining. CBA and qRT PCR were used to measure cytokines and transcription factors. Flow cytometry was used to detect subset ratios and surface marker expression. RESULTS: First, we observed comparable dose-dependent increases in cytotoxicity beginning at 25uM following treatment of isolated T cell populations with idelalisib, duvelisib, or TGR-1202. At this dose, apoptosis was induced between 48 and 72h. Second, all inhibitors reduced cytokine production in CD3+ T cells upon stimulation. Particularly, IFN-γ, IL-10 and IL-17a reduction was less pronounced after TGR-1202 treatment, indicating relative conservation of T cell response. All inhibitors lowered mRNA expression of T-bet (Th1), GATA-3 (Th2) and FoxP3 (Treg), however FoxP3 levels were consistently higher in TGR-1202 treated T cells. Third, we detected normal CD4:CD8 ratio and unaffected proliferative capacity of CD4+ and CD8+ subsets after drug treatment. Finally, all inhibitors decreased total percent of Tregs following stimulation (CD4+ CD25HI FoxP3+) accompanied by decreased expression of co-inhibitory molecules CTLA-4 and PD-1 on the Tregs. Interestingly, TGR-1202 significantly preserved the percent of Tregs closer to normal as well as surface expression of CTLA-4 and PD-1 on Tregs, indicating greater retention of immune checkpoint blockade and suppressive phenotype. CONCLUSIONS: We report herein that TGR-1202 affects human T cells differently than idelalisib and duvelisib. TGR-1202 sustains IL-10 production, FoxP3 mRNA expression, and maintains Treg percentage and expression of immune checkpoint molecules, suggesting relative preservation of numbers and function of Tregs. Data presented begin to provide novel insight into immune mediated cellular mechanisms responsible for lack of side effects in clinical trials of TGR-1202. In vivo models to further characterize effects on the T cell compartment are ongoing. Citation Format: Kamira K. Maharaj, John Powers, Renee Fonseca, Hari Miskin, Dave Maryanski, Eva Sahakian, Javier Pinilla-Ibarz. Differential regulation of human T-cells by TGR-1202, a novel PI3Kδ inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 545.