In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

Dongrui Wang; Renate Starr; Darya Alizadeh; Xin Yang; Stephen  J. Forman; Christine  E. Brown

doi:10.3791/59275

JoVE Journal
Cancer Research

This content is Free Access.

JoVE Journal Cancer Research

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

体外肿瘤细胞恢复对 chimeric 抗原受体 t 细胞抗肿瘤功能的预测评价

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February 27, 2019

DOI: 10.3791/59275-v

Dongrui Wang^1,2, Renate Starr¹, Darya Alizadeh¹, Xin Yang¹, Stephen J. Forman*¹, Christine E. Brown*¹

¹Department of Hematology & Hematopoietic Cell Transplantation, T Cell Therapeutics Research Laboratory,City of Hope Beckman Research Institute and Medical Center, ²Irell and Manella Graduate School of Biological Sciences,City of Hope Beckman Research Institute and Medical Center

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Overview

This article presents an in vitro co-culture method for the functional evaluation of CAR T cells through repetitive tumor cell challenges. This approach allows for detailed analysis of T cell activity against glioblastoma cells.

Key Study Components

Area of Science

Immunology
Cell Biology
Cancer Research

Background

Chimeric antigen receptor (CAR) T cells are engineered to target specific tumor antigens.
Evaluating CAR T cell efficacy traditionally involves complex in vivo models.
This study introduces a simpler in vitro method for assessing CAR T cell function.
Understanding T cell responses is crucial for improving cancer immunotherapies.

Purpose of Study

To develop a high-throughput method for evaluating CAR T cell anti-tumor potency.
To analyze the phenotypic and functional characteristics of CAR T cells during co-culture with glioblastoma cells.
To investigate the effects of repetitive tumor challenges on T cell activation and exhaustion.

Methods Used

Co-culture of glioblastoma cells and CAR T cells in a 96-well plate format.
Repetitive challenges with increasing tumor cell numbers over several days.
Flow cytometric analysis to assess T cell activation and exhaustion markers.
Quantification of CAR T cell and tumor cell populations throughout the experiment.

Main Results

Both CD4 and CD8 CAR T cells are activated against glioblastoma cells.
CD4 CAR T cells demonstrate superior expansion and multiple rounds of killing.
CD8 CAR T cells show a higher tendency for exhaustion compared to CD4 CAR T cells.
The presence of CD4 CAR T cells enhances CD8 CAR T cell expansion.

Conclusions

This in vitro method effectively simulates the tumor microenvironment for CAR T cell evaluation.
Repetitive tumor challenges provide insights into T cell dynamics and efficacy.
The findings could inform strategies for optimizing CAR T cell therapies in clinical settings.

Frequently Asked Questions

What is the significance of using an in vitro co-culture method?

It allows for a more controlled environment to evaluate CAR T cell function without the complexities of in vivo models.

How does the repetitive tumor challenge affect T cell behavior?

It provides insights into T cell activation, exhaustion, and memory status over time.

What markers are used to assess T cell activation?

Markers such as CD69, 41BB, PD1, LAG3, and TIM3 are analyzed to evaluate T cell responses.

Can this method be applied to other types of cancer?

Yes, the co-culture method can potentially be adapted for various tumor types to study CAR T cell efficacy.

What are the implications of CD4 and CD8 T cell interactions?

Understanding these interactions can help optimize CAR T cell therapies by enhancing their effectiveness against tumors.

Is this method suitable for high-throughput screening?

Yes, the method is designed for high-throughput applications, allowing for efficient evaluation of multiple CAR T cell products.

在这里, 我们描述了一种体外共培养方法, 递归挑战肿瘤靶向 t 细胞, 允许表型和功能分析抗肿瘤 t 细胞活性。

该协议提供了一个长期，体外，功能方法，通过重复肿瘤细胞挑战评估嵌合抗原受体，或 CAR T细胞。与体内 CAR T 细胞功能评估，该技术更不耗时，且劳动密集型程度更低，同时仍能准确表示真正的抗肿瘤疗效。这种体外技术实现了 CAR T 细胞抗肿瘤效力的高通量筛选和分化。

具有评估 CAR T 细胞产品临床有效性的潜在应用。首先从胶质母细胞瘤肿瘤球培养分离。用一毫升的冷口酶，和30至60秒的移液。

当肿瘤球被破坏时，停止用五毫升温暖的共培养介质的反应。通过离心使肿瘤细胞悬浮液颗粒。将胶质母细胞重新悬浮到每毫升新鲜共培养介质浓度的1.6倍至第五个肿瘤细胞，并稀释从 CAR T 细胞培养中收获的 T 细胞，使同培养中等浓度每毫升的 CAR 阳性 T 细胞的适当百分比。

接下来，在96个井平底组织培养板的每个井中加入100微升稀释肿瘤细胞。和100微升的 CAR T细胞进入肿瘤细胞的每个井与温和的混合。然后将板放在37摄氏度，5%的二氧化碳孵化器中。

在培养的第二天、第四天和第六天，小心地从肿瘤细胞的每个井中去除50微升的培养基，T细胞共培养板根据表进行重新挑战。然后，加入50微升的新鲜胶质母细胞瘤细胞悬浮液，准备刚才证明，但与两倍的初始共培养细胞数到每个井与温和的混合。将盘子返回细胞培养箱。

第一天、第三天、第五天、第七天、第一天、第三天、第五天和七天，将上清液从每一只井中转移到新的96井圆底板中，并按照表将50微升预加热的0.5%Trypsin EDTA加入到每一个介质中耗尽的井中。在37摄氏度下5分钟后，通过光显微镜确认细胞从每一井底部分离出来，并轻轻地将酶溶液移液在每一井底部周围，以重新释放细胞。在将分离电池悬浮液转移到新96孔板的相应相应孔中之前。

通过离心对细胞进行切粒，用200微升荧光细胞的活化细胞分拣站溶液洗涤细胞，或用第二次离心洗涤每井的FFS。将每井100微升SSS的抗体鸡尾酒中的100微升颗粒重新悬浮，在4摄氏度下孵育30分钟。A 孵育结束通过顺序100和200微升FSS洗涤去除任何未绑定的抗体，并在100至200微升DAPI核污渍和FSS中重新悬浮细胞。

然后，根据标准流量细胞计量分析方案分析细胞。用于肿瘤细胞共培养后 CAR T 细胞的功能和表型分析，从流细胞仪中检索数据文件，并门控所有活的 DAPI 阴性细胞。要量化肿瘤细胞，请对CD45负人群进行门控。

要量化 CAR T 细胞门 CD45 阳性 CAR 阳性总体。然后在实验过程中绘制肿瘤和 CAR T 细胞号，通过 41BB 和 CD69 的共表达识别 T 细胞激活。由 PD1、LAG3 和 TIM3 的表达式、T 单元内存状态 LAG3 和 TIM3、T 单元内存状态由 CD45RO 和 CD62 配体表达式表示 T 单元耗尽。

由 CD45RO 和 CD62 配体表达式。CD4阳性和CD8阳性的 CAR T细胞均对表达目标抗原的胶质母细胞瘤细胞进行同样激活，如其CD107a和细胞内细胞因子表达所示。然而，经重复肿瘤挑战SA评估，CD4阳性，但不是CD8阳性，CAR T细胞能够进行多次轮内杀戮。

CD4正 CAR T细胞也实现了更强劲的扩展。当CD4阳性，和CD8阳性的 CAR T细胞以一比一的比例混合时，这个细胞组合出执行CD8阳性，但不是CD4阳性的C-T T细胞在长期细胞毒性方面。此外，CD8阳性 CAR T细胞的膨胀是由CD4阳性T细胞的加入引起的。

而CD4阳性 CAR T细胞在CDa阳性细胞的存在下被抑制。在最初的共培养24小时后，CD4阳性和CD8阳性的C-TT细胞同样被激活。在重复肿瘤挑战中，CD4阳性和CD8阳性的 CAR T细胞都表现出从CD45RO阳性、CD62利根阳性中央记忆表型到CD45RO阳性CD62利根负效应记忆表型的过渡。

CD8阳性 CAR T 细胞也更容易耗尽，相比之下，CD4 阳性 CAR T 细胞在三天共培养后通过 PD1、LAG3 和 TIM3 表达进行评估。此外，在CD4阳性T细胞存在或不存在的情况下，CDa阳性 CAR T细胞衰竭没有差异，表明CD4诱导CD8阳性的C-T T细胞扩张与更好的效应器功能没有关联。本文还可以与T细胞的分类相结合，对转录组、蛋白质组学和感兴趣的特定基因进行进一步分析。

这种重复性肿瘤挑战也可以作为研究汽车T细胞肿瘤识别后生物事件的平台。

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