Szopa Iwona Monika, Granica Monika, Bujak Joanna Katarzyna, Łabędź Agata, Błaszczyk Maciej, Paulos Chrystal Mary, Majchrzak-Kuligowska Kinga
Department of Physiological Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland.
Department of Physics and Biophysics, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland.
Front Immunol. 2021 Feb 19;12:604066. doi: 10.3389/fimmu.2021.604066. eCollection 2021.
Expansion protocols for human T lymphocytes using magnetic beads, which serve as artificial antigen presenting cells (aAPCs), is well-studied. Yet, the efficacy of magnetic beads for propagation and functionality of peripheral blood lymphocytes (PBLs) isolated from companion dogs still remains limited. Domestic dog models are important in immuno-oncology field. Thus, we built the platform for induction of canine PBLs function, proliferation and biological activity using nano-sized magnetic beads (termed as MicroBeads) coated with anti-canine CD3 and CD28 antibodies. Herein we reveal that activation of canine PBLs MicroBeads induces a range of genes involved in immediate-early response to T cell activation in dogs. Furthermore, canine T lymphocytes are effectively activated by MicroBeads, as measured by cluster formation and induction of activation marker CD25 on canine T cells as quickly as 24 h post stimulation. Similar to human T cells, canine PBLs require lower activation signal strength for efficient proliferation and expansion, as revealed by titration studies using a range of MicroBeads in the culture. Additionally, the impact of temperature was assessed in multiple stimulation settings, showing that both 37°C and 38.5°C are optimal for the expansion of canine T cells. In contrast to stimulation using plant mitogen Concanavalin A (ConA), MicroBead-based activation did not increase activation-induced cell death. In turn, MicroBeads supported the propagation of T cells with an effector memory phenotype that secreted substantial IL-2 and IFN-γ. Thus, MicroBeads represent an accessible and affordable tool for conducting immunological studies on domestic dog models. Similarities in inducing intracellular signaling pathways further underscore the importance of this model in comparative medicine. Presented herein MicroBead-based expansion platforms for canine PBLs may benefit adoptive immunotherapy in dogs and facilitate the design of next-generation clinical trials in humans.
使用作为人工抗原呈递细胞（aAPCs）的磁珠对人T淋巴细胞进行扩增方案已得到充分研究。然而，磁珠对从伴侣犬分离的外周血淋巴细胞（PBLs）的增殖和功能的功效仍然有限。家犬模型在免疫肿瘤学领域很重要。因此，我们构建了一个平台，用于使用包被抗犬CD3和CD28抗体的纳米级磁珠（称为微珠）诱导犬PBLs的功能、增殖和生物活性。在此我们揭示，犬PBLs微珠的激活诱导了一系列与犬T细胞激活的早期反应相关的基因。此外，通过刺激后24小时内犬T细胞上簇的形成和激活标志物CD25的诱导来测量，微珠可有效激活犬T淋巴细胞。与人类T细胞类似，通过在培养中使用一系列微珠进行滴定研究表明，犬PBLs需要较低的激活信号强度才能有效增殖和扩增。此外，在多种刺激条件下评估了温度的影响，结果表明37°C和38.5°C均最适合犬T细胞的扩增。与使用植物有丝分裂原刀豆蛋白A（ConA）刺激相比，基于微珠的激活不会增加激活诱导的细胞死亡。反过来，微珠支持具有效应记忆表型的T细胞的增殖，这些T细胞分泌大量的IL-2和IFN-γ。因此，微珠是一种用于在家犬模型上进行免疫学研究的便捷且经济实惠的工具。诱导细胞内信号通路的相似性进一步强调了该模型在比较医学中的重要性。本文介绍的基于微珠的犬PBLs扩增平台可能有益于犬的过继免疫疗法，并有助于设计人类的下一代临床试验。
