Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response [RNA-seq]

Adoptive transfer of immune cells expressing chimeric antigen receptors (CARs) is an effective therapy for B-lineage malignancies. However, most patients will relapse and this therapeutic has yet to show strong efficacy in other hematologic or solid tumors. One opportunity for improvement lies in the ability to select or generate T cells that have the highest potential for potent anti-tumor responses and T cell persistence. Here, we dissect the biology of CD8+ CAR T cells by controlling whether the T cell has encountered cognate TCR antigen prior to CAR generation. We find that prior antigen experience influences multiple aspects of in vitro and in vivo CAR T cell functionality, boosting effector function and leukemia clearance in the setting of limiting target antigen density. However, this comes at the expense of proliferative capacity, resistance to dysfunction, and clearance of wildtype leukemia in the setting of limiting CAR+ cell dose. Epigenetic and transcriptomic comparisons of these cell populations uncover that modulation of the Runx2 transcription factor differentially impacts CAR T cell functionality depending on prior cell state. Collectively, our data demonstrate that prior antigen experience status determines functional attributes of a CAR T cell, as well as amenability to functional enhancement by transcription factor modulation. Overall design: To investigate the role of prior T cell antigen experience in responses of CD8+ chimeric antigen receptor (CAR) T cells to leukemia, we adoptively transferred C57BL/6-background Rag2-/- OT-I TCR transgenic CD8+ T cells into C57BL/6-background CD45.1+ hosts, followed by vaccination with ovalbumin, anti-mouse CD40, and PolyI:C to generate memory CD8+ T cells ("memory-derived"), or used naïve C57BL/6-background Rag2-/- OT-I TCR transgenic CD8+ T cells from naïve hosts ("naïve-derived"). We then transduced both populations with an anti-mouse CD19 CAR containing CD28 costimulatory domain and CD3zeta domain. Memory or naive-derived T cells were compared by RNAseq at three timepoints, Pre-CAR Transduction ("PreCAR", Day -5, ex vivo), Post-CAR Transduction ("PostCAR", Day 0, in vitro), and 4 days after infusion into C57BL/6-background Rag1-/- mice bearing the mouse CD19+ murine B-cell acute lymphoblastic leukemia cell line, E2A-PBX1 ("Tumor", Day +4, Ex Vivo).

表达嵌合抗原受体（chimeric antigen receptor，CAR）的免疫细胞过继疗法，是B系恶性肿瘤的有效治疗方案。然而多数患者会出现复发，且该疗法在其他血液系统或实体肿瘤中尚未展现出显著疗效。优化该疗法的一个可行方向，在于筛选或培育出具备最强抗肿瘤应答潜能与持久存活能力的T细胞。
本研究通过调控T细胞在CAR构建前是否接触过同源T细胞受体（T cell receptor，TCR）抗原，解析了CD8+ CAR T细胞的生物学特性。研究发现，既往抗原暴露经历会从多个维度影响CAR T细胞的体内外功能：在靶抗原密度受限的情况下，可增强其效应功能与白血病清除能力。但这同时会带来若干负面影响：包括增殖能力下降、抗功能失调能力受损，以及在CAR阳性细胞剂量受限的场景中无法有效清除野生型白血病。
对这些细胞群进行表观基因组与转录组学比较分析后发现，Runx2转录因子的调控对CAR T细胞功能的影响，取决于细胞既往的活化状态。综上，本研究数据表明，T细胞的既往抗原暴露状态，不仅决定了CAR T细胞的功能特性，还影响其通过转录因子调控实现功能增强的可行性。
实验整体设计：为探究T细胞既往抗原暴露经历在CD8+嵌合抗原受体（CAR）T细胞抗白血病应答中的作用，我们将C57BL/6背景的Rag2基因敲除型OT-I TCR转基因CD8+ T细胞，输注至C57BL/6背景的CD45.1+受体小鼠体内，随后通过卵清蛋白、抗小鼠CD40抗体与PolyI:C免疫，诱导生成记忆性CD8+ T细胞（记为"记忆来源型"）；或直接取用未免疫小鼠的C57BL/6背景Rag2基因敲除型OT-I TCR转基因CD8+ T细胞（记为"未成熟来源型"）。随后将这两组细胞均转导携带CD28共刺激结构域与CD3ζ结构域的抗小鼠CD19 CAR。
随后在三个时间点对记忆来源型与未成熟来源型T细胞进行RNA测序（RNA-seq）分析：CAR转导前（"PreCAR"，第-5天，离体样本）、CAR转导后（"PostCAR"，第0天，体外样本），以及输注至携带小鼠CD19阳性B细胞急性淋巴细胞白血病细胞系E2A-PBX1的C57BL/6背景Rag1基因敲除小鼠体内4天后（"Tumor"，第+4天，离体样本）。