Immunophenotyping of canine T cell activation and proliferation by combined protein and RNA flow cytometry

IF 1.4 3区农林科学 Q4 IMMUNOLOGY

Veterinary immunology and immunopathology Pub Date : 2024-02-28 DOI:10.1016/j.vetimm.2024.110739

Xu Zhu , Kara Rogers , Christine Bono , Zhenyu Wang , Carol Donovan , Changhua Ji

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引用次数: 0

Abstract

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.

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通过蛋白质和 RNA 联合流式细胞术对犬 T 细胞活化和增殖进行免疫分型

犬反应性单克隆抗体的有限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 分析法可能是弥补这一不足的潜在解决方案。在此，我们报告了一种血液免疫分型方法，该方法结合了基于蛋白质和 RNA 的流式细胞术，用于描述犬 T 细胞活化和单个细胞增殖的特征。在这种检测方法中，CD69 的表达由 RNA 探针检测，CD25 和 Ki67 则由抗体检测。犬外周血单核细胞（PBMC）受到三种作用方式不同的制剂刺激：抗 CD3/CD28 抗体、植物血凝素或乙酸磷脂酰肉豆蔻酸酯/洋霉素。检测到了强大的 T 细胞活化（CD25+ 和/或 CD69+）和增殖（Ki67+）。CD69 和 CD25 似乎都是强大而敏感的 T 细胞活化标志物，具有早期诱导和低背景表达的特点。刺激后，T 细胞的增殖晚于 T 细胞的活化，并与 CD25 的表达有关。这种犬 T 细胞活化和增殖免疫分型方法使用 10 只不同小猎犬的 PBMC 进行了 5 次独立实验评估，结果令人满意。该方法可极大地促进犬类非临床药物开发中的免疫疾病发病机制评估和免疫毒性风险评估。

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来源期刊

Veterinary immunology and immunopathology 农林科学-免疫学

CiteScore

3.40

自引率

5.60%

发文量

审稿时长

70 days

期刊介绍： The journal reports basic, comparative and clinical immunology as they pertain to the animal species designated here: livestock, poultry, and fish species that are major food animals and companion animals such as cats, dogs, horses and camels, and wildlife species that act as reservoirs for food, companion or human infectious diseases, or as models for human disease. Rodent models of infectious diseases that are of importance in the animal species indicated above,when the disease requires a level of containment that is not readily available for larger animal experimentation (ABSL3), will be considered. Papers on rabbits, lizards, guinea pigs, badgers, armadillos, elephants, antelope, and buffalo will be reviewed if the research advances our fundamental understanding of immunology, or if they act as a reservoir of infectious disease for the primary animal species designated above, or for humans. Manuscripts employing other species will be reviewed if justified as fitting into the categories above. The following topics are appropriate: biology of cells and mechanisms of the immune system, immunochemistry, immunodeficiencies, immunodiagnosis, immunogenetics, immunopathology, immunology of infectious disease and tumors, immunoprophylaxis including vaccine development and delivery, immunological aspects of pregnancy including passive immunity, autoimmuity, neuroimmunology, and transplanatation immunology. Manuscripts that describe new genes and development of tools such as monoclonal antibodies are also of interest when part of a larger biological study. Studies employing extracts or constituents (plant extracts, feed additives or microbiome) must be sufficiently defined to be reproduced in other laboratories and also provide evidence for possible mechanisms and not simply show an effect on the immune system.