CDK4/6 inhibition during CD8 T cell priming potentiates memory formation in mice and humans

项目摘要 癌症免疫治疗的标志性成就是延长了总体生存期， 在少数患者中缓解。然而，癌症患者记忆性CD 8 T细胞如何形成的问题， 是很难理解的。我们的长期目标是了解细胞周期调控 和CD 8 T细胞记忆命运。我们检测了在T细胞免疫过程中小鼠和人CD 8 T细胞中的CDK 4/6抑制。 启动并发现CD 8 T细胞向记忆命运的戏剧性倾斜。处理的抗原特异性CD 8 T细胞 在过继转移到小鼠中后，离体CDK 4/6 i显示出增加的长寿命记忆应答。我们 进一步使用单细胞转录谱分析来评估来自以下患者血液的最近活化的人CD 8 T细胞： CDK 4/6 i palbociclib或abemaciclib治疗前和治疗中的乳腺癌患者。TCR克隆型 追踪和RNA速度分析显示，CDK 4/6抑制增加了中枢记忆与 效应CD 8 T细胞前体。配对样本的分析表明，MYC靶点被下调， CDK 4/6抑制剂治疗在人类中的作用，与转录抑制因子MXD 4的增加一致。我们 中心假设是小鼠和人的CD 8 T细胞中的CDK 4/6抑制增加了长期的 保护性免疫为了验证这一点，我们将使用最先进的技术，包括：1）TRP 1跨核小鼠 由我们实验室开发，用于研究抗原特异性TCR，其对内源性 抗原; 2）scRNAseq和TCR克隆型追踪以分析大型纵向血液库和匹配的 来自II期试验患者的组织样品; 3）ChIPseq和免疫共沉淀，以确定 将CDK 4/6抑制与CD 8 T细胞中的记忆细胞命运联系起来的分子基础。在目标1中，我们将确定 将CDK 4和/或CDK 6与诱导记忆细胞命运联系起来的分子机制。减缓细胞周期 其他细胞周期抑制剂或利用细胞分裂速率的天然异质性不会增强 CD 8 T细胞中的记忆形成，暗示CDK 4和/或CDK 6在赋予记忆细胞命运中的独特作用。 我们将确定受CDK 4/6抑制影响的转录调节因子，重点是MYC和RUNX 3。 CDK 4/6抑制剂与PD-1阻断剂在临床中联合使用导致意外的治疗限制 毒性在目的2中，我们提出短期的CDK 4/6抑制将成功地增强CD 8 T细胞 因此，允许CDK 4/6抑制剂的停止以最小化或消除与PD-1阻断的重叠。我们 将使用我们的TRP 1 CD 8 T细胞过继转移模型在小鼠中测试这一假设，并使用29 来自CDK 4/6抑制与PD-1阻断的I期试验的配对血液和组织样品。我们将使用高- 最近激活的CD 8 T细胞的分辨率单细胞转录谱分析，以跟踪单个TCR克隆型 以及它们在CDK 4/6抑制剂治疗前和治疗中的血液和组织样品中的转录谱。影响：A 对CD 4/6在CD 8 T细胞命运决定中的机制作用的基本理解将允许更好地设计 使用CDK 4/6 i作为佐剂促进CD 8 T细胞记忆的癌症或传染病临床试验。

Project Summary Cancer immunotherapy’s signature achievement is extension of overall survival with long-term durable remissions in a minority of patients. However, the question of how memory CD8 T cells form in cancer patients is poorly understood. Our long-term goal is to understand the relationship between cell cycle regulation and CD8 T cell memory fate. We examined CDK4/6 inhibition in mouse and human CD8 T cells during T cell priming and found a dramatic skewing of CD8 T cells toward a memory fate. Antigen-specific CD8 T cells treated ex vivo with CDK4/6i displayed increased long-lived memory responses upon adoptive transfer into mice. We further used single cell transcriptional profiling to evaluate recently activated human CD8 T cells from blood of breast cancer patients pre- and on-treatment with the CDK4/6i palbociclib or abemaciclib. TCR clonotype tracking and RNA velocity analysis revealed that CDK4/6 inhibition increased the ratio of central memory to effector CD8 T cell precursors. Analysis of paired samples showed that MYC targets were downregulated by CDK4/6 inhibitor therapy in humans, consistent with an increase in the transcriptional repressor MXD4. Our central hypothesis is that CDK4/6 inhibition in CD8 T cells in both mice and humans augments long-term protective immunity. To test this, we will use state-of-the-art technologies including: 1) TRP1 transnuclear mice developed by our lab to study antigen-specific TCRs with a physiologic range of affinities for an endogenous antigen; 2) scRNAseq and TCR clonotype tracking to analyze a large bank of longitudinal blood and matched tissue samples from patients on a Phase II trial; 3) ChIPseq, and co-immunoprecipitations to determine the molecular basis linking CDK4/6 inhibition to memory cell fate in CD8 T cells. In Aim 1 we will identify the molecular mechanism linking CDK4 and/or CDK6 to induction of memory cell fate. Slowing the cell cycle using other cell cycle inhibitors or by taking advantage of natural heterogeneity in cell division rates does not potentiate memory formation in CD8 T cells, implying a unique role for CDK4 and/or CDK6 in conferring a memory cell fate. We will identify the transcriptional regulators affected by CDK4/6 inhibition, with a focus on MYC and RUNX3. Combination of CDK4/6 inhibitors with PD-1 blockade in the clinic results in unexpected treatment-limiting toxicity. In Aim 2 we propose that a short course of CDK4/6 inhibition would successfully potentiate CD8 T cell memory, thus allowing cessation of the CDK4/6 inhibitor to minimize or obviate overlap with PD-1 blockade. We will test this hypothesis in mice using our TRP1 CD8 T cell adoptive transfer model, and in humans using 29 paired blood and tissue samples from a Phase I trial of CDK4/6 inhibition with PD-1 blockade. We will use high- resolution single-cell transcriptional profiling of recently activated CD8 T cells to track individual TCR clonotypes and their transcriptional profiles in CDK4/6 inhibitor pre- and on-treatment blood and tissue samples. Impact: A basic understanding of the mechanistic role of CD4/6 in CD8 T cell fate decisions will allow for better design of clinical trials in cancer or infectious disease using CDK4/6i as adjuvants to promote CD8 T cell memory.