Laboratory of Experimental Hematology (LEH), Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.
Antwerp Unit for Data Analysis and Computation in Immunology and Sequencing (AUDACIS), Antwerp, Belgium.
Cytometry A. 2019 Oct;95(10):1096-1107. doi: 10.1002/cyto.a.23867. Epub 2019 Jul 29.
T cell proliferation is routinely identified in vitro using tracking dyes or through detecting intracellular upregulation of the nuclear protein, Ki-67. However, labeling with tracking dyes is cumbersome, associated with cellular toxicity, while Ki-67 cannot be used to identify and isolate viable T cells, and both techniques are incompatible with MACS technology. Here, we introduce a simple tool to identify and isolate in vitro T cell expansion that is tracking dye-independent and allows for sorting of viable T cells. We show that CD71, a transferrin receptor, and CD98, a heterodimer glycoprotein involved in both integrin signaling and amino-acid transport, are both highly upregulated on proliferating T cells upon in vitro stimulation, and that CD71 expression is maximal on the more recent progeny T cells, while CD98 upregulation remains stable across different generations of progeny T cells. Moreover, we demonstrate that the upregulation of CD71 and CD98 identifies CFSE T cells and provides further proof of the antigen-specificity of T cells identified by CD71 and CD98 dual upregulation based on tetramer staining. We further show that CD71 can be used to enrich for in vitro expanding T cells using MACS technology. In conclusion, we show that CD71 and CD98 can be used to identify and isolate expanded T cells following in vitro stimulation and that CD71 is an MACS-compatible alternative to tracking dyes or Ki-67 detection. © 2019 International Society for Advancement of Cytometry.
T 细胞增殖通常通过追踪染料或检测核蛋白 Ki-67 的细胞内上调来体外鉴定。然而，用追踪染料进行标记很繁琐，而且与细胞毒性有关，而 Ki-67 不能用于鉴定和分离活 T 细胞，这两种技术都与 MACS 技术不兼容。在这里，我们引入了一种简单的工具，用于鉴定和分离体外 T 细胞扩增，该工具不依赖于追踪染料，并且允许对活 T 细胞进行分选。我们表明，在体外刺激时，增殖 T 细胞上高度上调的转铁蛋白受体 CD71 和参与整合素信号和氨基酸转运的异二聚体糖蛋白 CD98 均上调，并且 CD71 的表达在最近的 T 细胞后代中最大，而 CD98 的上调在不同代的 T 细胞后代中保持稳定。此外，我们证明 CD71 和 CD98 的上调可识别 CFSE T 细胞，并基于四聚体染色为 CD71 和 CD98 双重上调鉴定的 T 细胞提供抗原特异性的进一步证据。我们进一步表明，CD71 可用于通过 MACS 技术富集体外扩增的 T 细胞。总之，我们表明 CD71 和 CD98 可用于鉴定和分离体外刺激后的扩增 T 细胞，并且 CD71 是追踪染料或 Ki-67 检测的 MACS 兼容替代品。
