Department of Small Animal Medicine and Surgery, University of Veterinary Medicine, Hannover, Germany.
Department of Small Animal Medicine and Surgery, University of Veterinary Medicine, Hannover, Germany.
Vet Immunol Immunopathol. 2022 Jan;243:110366. doi: 10.1016/j.vetimm.2021.110366. Epub 2021 Dec 7.
Th17 cells are T helper cells which play an important role during inflammation and autoimmune disease. To investigate the role of these cells in diseases in dogs in a clinical setting, methods for fast identification had to be established. Th17 cells are a rare cell population, for their measurement stimulation is recommended. To examine more samples simultaneously and to receive a relatively high purity of cell population of CD3 + CD4+ cells, different methods on various levels of preselection of cells as well as the possibility of storing blood overnight for measuring Th17 cells by flow cytometry were investigated. Firstly, to receive a high number of mononuclear cells, two different density gradients were compared and analysed. Furthermore, the enrichment of CD3 + CD4+ cells via depletion of CD8alpha+, CD11b + and CD21+ cells by cell sorting (autoMACS Pro Separator) was tested. It was also investigated whether stimulation processes led to better interpretation of results and whether there was a significant difference in measurement of directly processed blood samples and samples that had been stored overnight. In conclusion, the use of the density gradient (Lymph24+ Spin Medium) resulted in a purer cell population through a significant decrease in polymorphonuclear cells (*p = 0.01). After cell sorting, a significant difference in cell population purity was detected. Within the target fraction (containing mainly CD3 + CD4+ cells), CD8alpha+, CD21+, CD11b + cell percentages were significantly lower (***p < 0.001, *p < 0.02, ***p < .0001, respectively), and CD3 + CD4+ cell percentage was significantly higher (***p < .0001). There was a significant difference in Th17 cell percentage between unstimulated and stimulated cell populations (***p < .0001), but no significant difference in the percentage of unstimulated Th17 cells (p = 0.29) or stimulated Th17 cells (p = 0.71) in stored blood in comparison to directly processed EDTA blood samples. Finally, a modified protocol that offers an efficient way to investigate samples that were stored overnight by means of flow cytometry was evolved to research the role of Th17 cells in dogs with different diseases or in healthy populations.
Th17 细胞是辅助性 T 细胞，在炎症和自身免疫性疾病中发挥重要作用。为了在临床环境中研究这些细胞在犬疾病中的作用，必须建立快速鉴定方法。Th17 细胞是一种罕见的细胞群体，建议对其进行刺激测量。为了同时检测更多样本并获得相对高纯度的 CD3+CD4+细胞群体，可以在不同水平上对细胞进行预选，并研究通过流式细胞术测量 Th17 细胞过夜存储血液的可能性。首先，为了获得大量的单核细胞，比较并分析了两种不同的密度梯度。此外，通过细胞分选（autoMACS Pro Separator）耗尽 CD8alpha+、CD11b+和 CD21+细胞，对 CD3+CD4+细胞进行了富集。还研究了刺激过程是否会导致更好的结果解释，以及直接处理的血液样本与过夜存储的样本测量之间是否存在显著差异。结论是，使用密度梯度（Lymph24+Spin Medium）通过显著减少多形核细胞（*p=0.01）导致更纯的细胞群体。细胞分选后，检测到细胞群体纯度的显著差异。在目标分数（主要包含 CD3+CD4+细胞）中，CD8alpha+、CD21+、CD11b+细胞的百分比明显较低（***p<0.001、*p<0.02、***p<0.0001），而 CD3+CD4+细胞的百分比明显较高（***p<0.0001）。未刺激和刺激细胞群体之间的 Th17 细胞百分比存在显著差异（***p<0.0001），但与直接处理的 EDTA 血液样本相比，储存血液中未刺激 Th17 细胞（p=0.29）或刺激 Th17 细胞（p=0.71）的百分比没有显著差异。最后，开发了一种改良的方案，该方案提供了一种通过流式细胞术研究过夜储存样本的有效方法，以研究 Th17 细胞在患有不同疾病或健康犬中的作用。
