Reverse immunology has open the door to innovative cancer immunotherapy strategies such as immunogenic antigen-based vaccination and transgenic T cell receptor (TCR)-based adoptive cell transfer. This approach enables the identification of immunogenic tumor specific antigen derived peptides. One of the major challenges is the rapid selection of antigen-specific CD8+ T cell clones. Thus, IFNγ-producing CD8+ T cells magnetic sorting combined with limiting dilution cloning approach represents the most common method of specific T cell cloning. However, during plate setup several wells will not contain T cells whereas others will contain mixed population of T cells. In this case, a re-cloning step is required which make limiting dilution based cloning a laborious, inefficient, expensive and a time-consuming method. To address these obstacles, here we present a novel 2-step workflow combining simple, affordable and gentle magnetic cell separation followed by single cell isolation using a device called DispenCell-S1. We aimed to compare this new workflow with the traditional limiting dilution method using in vitro generated antigen-specific CD8+ T cells. Herein, we reported the reliability of DispenCell-S1 method and its efficiency in T cell clones isolation.

中文翻译：

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一种结合磁性细胞分离和基于阻抗的细胞分配的新工作流程，用于温和、简单和可靠地克隆特定的 CD8+ T 细胞。

反向免疫学为创新的癌症免疫治疗策略打开了大门，例如基于免疫原性抗原的疫苗接种和基于转基因 T 细胞受体 (TCR) 的过继细胞转移。这种方法能够识别免疫原性肿瘤特异性抗原衍生肽。主要挑战之一是快速选择抗原特异性 CD8+ T 细胞克隆。因此，产生 IFNγ 的 CD8+ T 细胞磁分选结合有限稀释克隆方法代表了特异性 T 细胞克隆的最常用方法。然而，在板设置过程中，几个孔将不包含 T 细胞，而其他孔将包含混合的 T 细胞群。在这种情况下，需要重新克隆步骤，这使得基于有限稀释的克隆成为一种费力、低效、昂贵且耗时的方法。为了解决这些障碍，在这里，我们提出了一种新颖的两步工作流程，结合了简单、经济和温和的磁性细胞分离，然后使用名为 DispenCell-S1 的设备进行单细胞分离。我们旨在将这种新的工作流程与使用体外生成的抗原特异性 CD8+ T 细胞的传统有限稀释方法进行比较。在此，我们报告了 DispenCell-S1 方法的可靠性及其在 T 细胞克隆分离中的效率。