Introduction

CD19 directed chimeric antigen receptor T cell (CART19) therapy has resulted in unprecedented clinical outcomes. However, its success in chronic lymphocytic leukemia (CLL) has been modest to date. An increasing body of evidence indicates that impaired CART cell fitness is the predominant mechanism of the relative dysfunction in CLL. The immunosuppressive microenvironment in CLL is well known and may be related to the abundance of circulating extracellular vesicles (EVs) bearing immunomodulatory properties. We hypothesized that CLL derived EVs contribute to CART cell dysfunction.

Objectives

We aimed to investigate the interaction between EVs from CLL patients and CART19 cells.

Methods

We characterized circulating EVs from platelet free plasma in untreated patients with CLL and normal controls using nanoscale flow cytometry. CLL derived EVs, CART19, and CLL B cells were imaged with super resolution microscopy. We stimulated CART19 cells with CD19⁺ JeKo1 cells at a 1:1 ratio in the presence of increasing concentrations of CLL derived EVs and measured inhibitory receptors by flow cytometry. To interrogate the transcriptome, we stimulated CART19 cells with irradiated JeKo1 cells in the presence of CLL derived EVs at ratios of 10:1 and 1:1 EV:CART19. Finally, immunocompromised mice were engrafted with the JeKo1 and randomized to treatment with untreated, CART19 cells, or CART19 cells co-cultured ex vivo with CLL derived EVs for six hours.

Results

Our findings indicate that CLL derived EVs impair normal donor CART19 antigen-specific proliferation (Fig 1A). Although there wasn't a difference in total EV particle count (Fig 1B), CLL derived EVs expressed significantly more PD-L1 than normal controls (Fig 1C). On super resolution microscopy, EVs were localized at the T cell-tumor junction (Fig 1D). Furthermore, CLL derived EVs were captured by T cells (Fig 1E).

Having demonstrated this interaction, we sought to establish their functional impact on CART19 cells. There was a significant upregulation of inhibitory receptors on the T cells (Fig 1F), associated with a reduction in CART effector cytokines at higher concentrations of EVs (Fig 1G), suggesting a state of exhaustion. This was further supported by RNA sequencing, which indicated a significant upregulation of AP-1 and YY1 (Figs 1H), known T cell exhaustion pathways.

Finally, to confirm the impact of CLL derived EVs on CART19 functions in vivo, treatment with CART19 cultured with EVs resulted in reduced anti-tumor activity compared to treatment with CART19 alone (Fig 1I).

Conclusion

Our results indicate that CLL derived EVs induce significant CART19 cell dysfunction in vitro and in vivo, by direct interaction with CART cells resulting in a downstream alteration of their exhaustion pathways. These studies illuminate a novel way that circulating and potentially systemic EVs can lead to CART cell dysfunction in CLL patients.

中文翻译：

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循环细胞外囊泡在慢性淋巴细胞白血病（CLL）中诱导嵌合抗原受体T细胞功能障碍

介绍

CD19定向嵌合抗原受体T细胞（CART19）疗法已导致前所未有的临床结果。然而，迄今为止，它在慢性淋巴细胞性白血病（CLL）中的成功还很有限。越来越多的证据表明，CART细胞适应性受损是CLL相对功能障碍的主要机制。CLL中的免疫抑制微环境是众所周知的，并且可能与具有免疫调节特性的循环的细胞外囊泡（EVs）丰富有关。我们假设CLL衍生的电动汽车有助于CART细胞功能障碍。

目标

我们旨在研究CLL患者的EV与CART19细胞之间的相互作用。

方法

我们使用纳米级流式细胞仪对未经治疗的CLL患者和正常对照的无血小板血浆中的循环电动汽车进行了表征。使用超分辨率显微镜对CLL衍生的EV，CART19和CLL B细胞进行成像。在存在浓度越来越高的CLL衍生的EV和通过流式细胞仪测量抑制受体的情况下，我们以1：1的比例用CD19 ⁺ JeKo1细胞刺激了CART19细胞。为了询问转录组，我们在存在CLL衍生的EV的情况下，以10：1和1：1 EV：CART19的比例用照射的JeKo1细胞刺激了CART19细胞。最后，将免疫受损的小鼠植入JeKo1，并随机分配给未处理的CART19细胞或与CLL衍生的EV体外共培养六小时的CART19细胞进行治疗。

结果

我们的发现表明，CLL衍生的电动汽车损害了正常供体CART19抗原特异性增殖（图1A）。尽管总EV颗粒计数没有差异（图1B），但CLL衍生的EV的PD-L1明显高于正常对照（图1C）。在超高分辨率显微镜下，电动汽车位于T细胞-肿瘤交界处（图1D）。此外，CLL衍生的EV被T细胞捕获（图1E）。

在展示了这种相互作用之后，我们试图确定它们对CART19细胞的功能影响。T细胞上的抑制性受体显着上调（图1F），与较高浓度的EV时CART效应细胞因子的减少有关（图1G），表明处于疲惫状态。RNA测序进一步支持了这一点，RNA测序表明AP-1和YY1（图1H）（已知的T细胞衰竭途径）显着上调。

最后，为证实CLL衍生的EV对体内CART19功能的影响，与仅用CART19进行治疗相比，用EV培养的CART19进行治疗可降低抗肿瘤活性（图1I）。

结论

我们的结果表明，CLL衍生的电动汽车通过与CART细胞直接相互作用，导致其精疲力竭途径的下游改变，在体外和体内均诱导了严重的CART19细胞功能异常。这些研究阐明了循环和潜在全身性电动汽车可导致CLL患者CART细胞功能异常的新方法。