Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA.
Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital, and Houston Methodist Hospital, Houston, Texas, USA.
J Immunother Cancer. 2023 Apr;11(4). doi: 10.1136/jitc-2022-006267.
The wider application of T cells targeting viral tumor-antigens via their native receptors is hampered by the failure to expand potent tumor-specific T cells from patients. Here, we examine reasons for and solutions to this failure, taking as our model the preparation of Epstein-Barr virus (EBV)-specific T cells (EBVSTs) for the treatment of EBV-positive lymphoma. EBVSTs could not be manufactured from almost one-third of patients, either because they failed to expand, or they expanded, but lacked EBV specificity. We identified an underlying cause of this problem and established a clinically feasible approach to overcome it.
CD45RO+CD45RA- memory compartment residing antigen-specific T cells were enriched by depleting CD45RA positive (+) peripheral blood mononuclear cells (PBMCs) that include naïve T cells, among other subsets, prior to EBV antigen stimulation. We then compared the phenotype, specificity, function and T-cell receptor (TCR) Vβ repertoire of EBVSTs expanded from unfractionated whole (W)-PBMCs and CD45RA-depleted (RAD)-PBMCs on day 16. To identify the CD45RA component that inhibited EBVST outgrowth, isolated CD45RA+ subsets were added back to RAD-PBMCs followed by expansion and characterization. The in vivo potency of W-EBVSTs and RAD-EBVSTs was compared in a murine xenograft model of autologous EBV+ lymphoma.
Depletion of CD45RA+ PBMCs before antigen stimulation increased EBVST expansion, antigen-specificity and potency in vitro and in vivo. TCR sequencing revealed a selective outgrowth in RAD-EBVSTs of clonotypes that expanded poorly in W-EBVSTs. Inhibition of antigen-stimulated T cells by CD45RA+ PBMCs could be reproduced only by the naïve T-cell fraction, while CD45RA+ regulatory T cells, natural killer cells, stem cell memory and effector memory subsets lacked inhibitory activity. Crucially, CD45RA depletion of PBMCs from patients with lymphoma enabled the outgrowth of EBVSTs that failed to expand from W-PBMCs. This enhanced specificity extended to T cells specific for other viruses.
Our findings suggest that naïve T cells inhibit the outgrowth of antigen-stimulated memory T cells, highlighting the profound effects of intra-T-cell subset interactions. Having overcome our inability to generate EBVSTs from many patients with lymphoma, we have introduced CD45RA depletion into three clinical trials: NCT01555892 and NCT04288726 using autologous and allogeneic EBVSTs to treat lymphoma and NCT04013802 using multivirus-specific T cells to treat viral infections after hematopoietic stem cell transplantation.
通过其天然受体靶向病毒肿瘤抗原的 T 细胞的更广泛应用受到未能从患者中扩增出有效肿瘤特异性 T 细胞的阻碍。在这里，我们研究了导致这种失败的原因和解决方案，以 EBV 特异性 T 细胞（EBVST）的制备为例，用于治疗 EBV 阳性淋巴瘤。由于未能扩增，或者它们扩增了，但缺乏 EBV 特异性，几乎三分之一的患者无法制备 EBVST。我们确定了这个问题的根本原因，并建立了一种临床可行的方法来克服它。
通过在 EBV 抗原刺激之前耗尽包括幼稚 T 细胞在内的其他亚群的 CD45RA+外周血单个核细胞（PBMC），从 CD45RO+CD45RA-记忆区室中富集抗原特异性 T 细胞。然后，我们比较了从未分馏的全（W）-PBMC 和 CD45RA 耗尽（RAD）-PBMC 中扩增的 EBVSTs 在第 16 天的表型、特异性、功能和 T 细胞受体（TCR）Vβ库。为了确定抑制 EBVST 生长的 CD45RA 成分，将分离的 CD45RA+亚群添加回 RAD-PBMC 中，然后进行扩增和表征。在自体 EBV+淋巴瘤的小鼠异种移植模型中比较了 W-EBVSTs 和 RAD-EBVSTs 的体内效力。
在抗原刺激前耗尽 CD45RA+PBMC 可增加 EBVST 的体外和体内扩增、抗原特异性和效力。TCR 测序显示，RAD-EBVST 中克隆型的选择性生长较差，而在 W-EBVST 中生长较差。仅幼稚 T 细胞亚群可再现 CD45RA+PBMC 对抗原刺激 T 细胞的抑制作用，而 CD45RA+调节性 T 细胞、自然杀伤细胞、干细胞记忆和效应记忆亚群缺乏抑制活性。至关重要的是，从淋巴瘤患者的 PBMC 中耗尽 CD45RA 使 W-PBMC 未能扩增的 EBVST 得以生长。这种增强的特异性扩展到针对其他病毒的 T 细胞。
我们的研究结果表明，幼稚 T 细胞抑制抗原刺激的记忆 T 细胞的生长，突出了细胞内 T 细胞亚群相互作用的深远影响。通过克服我们无法从许多淋巴瘤患者中生成 EBVST 的问题，我们已经在三项临床试验中引入了 CD45RA 耗尽：NCT01555892 和 NCT04288726 使用自体和同种异体 EBVST 治疗淋巴瘤，以及 NCT04013802 使用多病毒特异性 T 细胞治疗造血干细胞移植后的病毒感染。
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