经常分析活化诱导的标志物(AIM)以鉴定再活化的人记忆T细胞。然而,在猪中,对AIM的分析仍然不是很常见。基于现有的抗体,我们设计了一个包含猪特异性或交叉反应性抗CD25,CD69,CD40L(CD154)的多色流式细胞术小组,和ICOS(CD278)结合针对CD3、CD4和CD8α的谱系/表面标志物。此外,我们包括针对肿瘤坏死因子α(TNF-α)的抗体,研究AIM表达与这种丰富的T细胞细胞因子产生的相关性。在用佛波醇12-肉豆蔻酸酯13-乙酸酯(PMA)/离子霉素刺激的外周血单核细胞(PBMC)上测试了面板,来自非洲猪瘟病毒(ASFV)恢复期猪的葡萄球菌肠毒素B(SEB)或PBMC,用同源病毒再刺激。PMA/离子霉素导致CD25/CD69共表达T细胞的大量增加,其中只有一个亚群产生TNF-α,而CD40L的表达在很大程度上与TNF-α的产生有关。SEB刺激引发的AIM表达明显低于PMA/离子霉素,但这里也鉴定了不产生TNF-α的表达CD25/CD69的T细胞。此外,鉴定CD40L-单阳性和CD25+CD69+CD40L+TNF-α-T细胞。在ASFV再刺激的T细胞中,TNF-α的产生与相当大比例的表达AIM的T细胞相关,但在此也鉴定了ASFV反应性CD25+CD69+TNF-α-T细胞。在CD8α+CD4T细胞内,几个CD25/CD40L/CD69/ICOS定义的表型在ASFV再刺激后显著扩增。因此,所测试的AIM组合将允许鉴定超出常用细胞因子组的引发T细胞,提高识别猪抗原特异性T细胞的能力。
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.