Centre de Recherche du CHUM, Montreal, QC H2X 0A9, Canada; Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, QC H2X 0A9, Canada.
Centre de Recherche du CHUM, Montreal, QC H2X 0A9, Canada.
Cell Rep Methods. 2024 Jan 22;4(1):100690. doi: 10.1016/j.crmeth.2023.100690. Epub 2024 Jan 15.
Broadly applicable methods to identify and characterize antigen-specific CD4 and CD8 T cells are key to immunology research, including studies of vaccine responses and immunity to infectious diseases. We developed a multiplexed activation-induced marker (AIM) assay that presents several advantages compared to single pairs of AIMs. The simultaneous measurement of four AIMs (CD69, 4-1BB, OX40, and CD40L) creates six AIM pairs that define CD4 T cell populations with partial and variable overlap. When combined in an AND/OR Boolean gating strategy for analysis, this approach enhances CD4 T cell detection compared to any single AIM pair, while CD8 T cells are dominated by CD69/4-1BB co-expression. Supervised and unsupervised clustering analyses show differential expression of the AIMs in defined T helper lineages and that multiplexing mitigates phenotypic biases. Paired and unpaired comparisons of responses to infections (HIV and cytomegalovirus [CMV]) and vaccination (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) validate the robustness and versatility of the method.
广泛适用于鉴定和表征抗原特异性 CD4 和 CD8 T 细胞的方法是免疫学研究的关键，包括疫苗反应和传染病免疫研究。我们开发了一种多重激活诱导标记物（AIM）检测方法，与单个 AIM 对相比具有多个优势。同时测量四种 AIM（CD69、4-1BB、OX40 和 CD40L）可以创建六个 AIM 对，这些对定义了具有部分和可变重叠的 CD4 T 细胞群体。当将其结合到用于分析的 AND/OR 布尔门控策略中时，与任何单个 AIM 对相比，这种方法可以增强 CD4 T 细胞的检测，而 CD8 T 细胞则主要由 CD69/4-1BB 共表达决定。监督和无监督聚类分析显示，AIM 在定义的辅助性 T 细胞谱系中表达不同，并且多重化减轻了表型偏倚。对感染（HIV 和巨细胞病毒 [CMV]）和疫苗接种（严重急性呼吸综合征冠状病毒 2 [SARS-CoV-2]）的反应的配对和非配对比较验证了该方法的稳健性和多功能性。
