Table_1_Investigation of activation-induced markers (AIM) in porcine T cells by flow cytometry.XLSX

Activation-induced markers (AIMs) are frequently analyzed to identify re-activated human memory T cells. However, in pigs the analysis of AIMs is still not very common. Based on available antibodies, we designed a multi-color flow cytometry panel comprising pig-specific or cross-reactive antibodies against CD25, CD69, CD40L (CD154), and ICOS (CD278) combined with lineage/surface markers against CD3, CD4, and CD8α. In addition, we included an antibody against tumor necrosis factor alpha (TNF-α), to study the correlation of AIM expression with the production of this abundant T cell cytokine. The panel was tested on peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, Staphylococcus enterotoxin B (SEB) or PBMCs from African swine fever virus (ASFV) convalescent pigs, restimulated with homologous virus. PMA/ionomycin resulted in a massive increase of CD25/CD69 co-expressing T cells of which only a subset produced TNF-α, whereas CD40L expression was largely associated with TNF-α production. SEB stimulation triggered substantially less AIM expression than PMA/ionomycin but also here CD25/CD69 expressing T cells were identified which did not produce TNF-α. In addition, CD40L-single positive and CD25+CD69+CD40L+TNF-α− T cells were identified. In ASFV restimulated T cells TNF-α production was associated with a substantial proportion of AIM expressing T cells but also here ASFV-reactive CD25+CD69+TNF-α− T cells were identified. Within CD8α+ CD4 T cells, several CD25/CD40L/CD69/ICOS defined phenotypes expanded significantly after ASFV restimulation. Hence, the combination of AIMs tested will allow the identification of primed T cells beyond the commonly used cytokine panels, improving capabilities to identify the full breadth of antigen-specific T cells in pigs.

激活诱导标志物（Activation-induced markers, AIMs）常被用于鉴定活化的人类记忆T细胞。然而在猪体内，针对AIMs的相关分析仍较为少见。基于现有可用抗体，我们设计了一款多色流式细胞术检测组合，包含靶向CD25、CD69、CD40L（CD154）及ICOS（CD278）的猪特异性或交叉反应性抗体，联合针对CD3、CD4与CD8α的谱系/表面标志物抗体。此外，我们纳入了针对肿瘤坏死因子α（tumor necrosis factor alpha, TNF-α）的抗体，以探究AIM表达与该丰度较高的T细胞因子产生之间的相关性。
该检测组合分别在经佛波醇12-肉豆蔻酸酯13-乙酸酯（phorbol 12-myristate 13-acetate, PMA）/离子霉素、葡萄球菌肠毒素B（Staphylococcus enterotoxin B, SEB）刺激的外周血单个核细胞（peripheral blood mononuclear cells, PBMCs），以及经同源病毒再刺激的非洲猪瘟病毒（African swine fever virus, ASFV）康复猪PBMCs上完成了验证。
PMA/离子霉素刺激可使共表达CD25与CD69的T细胞数量显著升高，但其中仅少数亚群可产生TNF-α；而CD40L的表达则与TNF-α的产生高度相关。与PMA/离子霉素刺激相比，SEB诱导的AIM表达水平显著更低，但同样可检测到未产生TNF-α的CD25/CD69阳性T细胞。此外，本研究还鉴定出CD40L单阳性以及CD25+CD69+CD40L+TNF-α−的T细胞亚群。
在经ASFV再刺激的T细胞中，TNF-α的产生与相当比例的AIM阳性T细胞相关，但同样可检测到ASFV反应性的CD25+CD69+TNF-α− T细胞。在CD8α+ CD4 T细胞亚群中，经ASFV再刺激后，若干由CD25/CD40L/CD69/ICOS定义的细胞表型发生了显著扩增。
因此，本研究验证的AIM组合可用于鉴定常规细胞因子检测组无法覆盖的致敏T细胞，提升了猪体内抗原特异性T细胞全谱的鉴定能力。