The limited availability of canine-reactive monoclonal antibodies restricts the analyses of immune cell subsets and their functions by flow cytometry. The PrimeFlow™ RNA Assay may serve as a potential solution to close this gap. Here we report a blood immunophenotyping method utilizing combined protein- and RNA-based flow cytometry to characterize canine T cell activation and proliferation within individual cells. In this assay, CD69 expression was detected by an RNA probe and CD25 and Ki67 were detected by antibodies. Canine peripheral blood mononuclear cells (PBMCs) were stimulated with three agents with different modes of action, anti-CD3/CD28 antibodies, phytohemagglutinin, or phorbol myristate acetate /ionomycin. Robust T cell activation (CD25+ and/or CD69+) and proliferation (Ki67+) were detected. Both CD69 and CD25 appear to be robust and sensitive T cell activation markers with early induction and low background expression. Upon stimulation, T cell proliferation occurred later than T cell activation and was associated with CD25 expression. This canine T cell activation and proliferation immunophenotyping method was evaluated in 5 independent experiments using PBMCs from 10 different beagle dogs with satisfactory assay performance. This method can greatly facilitate the evaluation of immune disease pathogenesis and immunotoxicity risk assessment in nonclinical drug development in canine.

中文翻译：

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通过联合蛋白质和 RNA 流式细胞术对犬 T 细胞活化和增殖进行免疫表型分析

犬反应性单克隆抗体的有限可用性限制了流式细胞术对免疫细胞亚群及其功能的分析。 PrimeFlow™ RNA 检测可作为弥补这一差距的潜在解决方案。在这里，我们报告了一种血液免疫表型分析方法，利用基于蛋白质和 RNA 的组合流式细胞术来表征犬 T 细胞在单个细胞内的活化和增殖。在此测定中，通过 RNA 探针检测 CD69 表达，通过抗体检测 CD25 和 Ki67。用三种具有不同作用模式的药物刺激犬外周血单核细胞（PBMC）：抗CD3/CD28抗体、植物血凝素或佛波醇肉豆蔻酸酯/离子霉素。检测到强劲的 T 细胞激活（CD25+ 和/或 CD69+）和增殖（Ki67+）。 CD69 和 CD25 似乎都是稳健且敏感的 T 细胞激活标记物，具有早期诱导和低背景表达的特点。刺激后，T 细胞增殖发生晚于 T 细胞激活，并且与 CD25 表达相关。这种犬 T 细胞活化和增殖免疫表型方法在 5 个独立实验中使用来自 10 只不同比格犬的 PBMC 进行了评估，具有令人满意的测定性能。该方法可以极大地促进犬类非临床药物开发中免疫疾病发病机制的评价和免疫毒性风险评估。