Effect of wash media type during PBMC isolation on downstream characterization of SARS-CoV-2-specific T cells.
Effect of wash media type during PBMC isolation on downstream characterization of SARS-CoV-2-specific T cells.
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DOI：
10.1016/j.jim.2023.113520
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发表时间：
2023-08
影响因子：
2.2
通讯作者：
Pannaraj, Pia S.
中科院分区：
文献类型：
--
作者：
Congrave-Wilson, Zion;Kim, Minjun;Sutherland, Aaron;Jumarang, Jaycee;Lee, Yesun;Del Valle, Jennifer;Cheng, Wesley A.;Antunes, Ricardo da Silva;Pannaraj, Pia S.
各个实验室从全血中分离外周血单个核细胞（PBMCs）的方法差异很大，特别是在已发表的关于感染和接种后sars - cov -2特异性T细胞反应的研究中。关于PBMC分离过程中不同洗涤介质类型或离心速度和制动器使用对下游T细胞活化和功能的影响的研究有限。采用不同的PBMC分离方法处理26例covid -19疫苗接种者的血液样本，分别使用PBS或RPMI作为高离心速度和制动器的洗涤介质或RPMI作为低速和制动器的洗涤介质（RPMI+法）。通过基于流式细胞术的激活诱导标记（AIM）试验和干扰素-γ (IFNγ)荧光斑点试验对SARS-CoV-2刺突特异性T细胞进行定量和表征，并比较不同处理方法的反应。用RPMI洗涤的样品比用PBS洗涤的样品显示出更高的AIM+ CD4 T细胞反应，并显示出从naïve向效应记忆表型的转变。激活标记物OX40在rpmi洗涤的CD4 T细胞上显示出更高的SARS-CoV-2尖峰诱导的上调，而CD137的上调在处理方法之间的差异很小。不同处理方法对AIM+ CD8 T细胞的反应程度相似，但表现出更高的刺激指数。在pbs洗涤的样品中，CD69+ CD8 T细胞的背景频率增加，并且在FluoroSpot试验中与更高的ifn γ-生成细胞的基线数量相关。RPMI+方法中较慢的制动并没有提高对sars - cov -2特异性T细胞的检测，而且导致处理时间延长。因此，在PBMC分离的洗涤步骤中，使用具有完全离心制动器的RPMI介质被发现是最有效和高效的。需要进一步的研究来阐明rpmi介导的下游T细胞活性保存的途径。
Protocols for the isolation of peripheral blood mononuclear cells (PBMCs) from whole blood vary greatly between laboratories, especially in published studies of SARS-CoV-2-specific T cell responses following infection and vaccination. Research on the effects of different wash media types or centrifugation speeds and brake usage during the PBMC isolation process on downstream T cell activation and functionality is limited. Blood samples from 26 COVID-19-vaccinated participants were processed with different PBMC isolation methods using either PBS or RPMI as the wash media with high centrifugation speed and brakes or RPMI as the wash media with low speed and brakes (RPMI+ method). SARS-CoV-2 spike-specific T cells were quantified and characterized via a flow cytometry-based activation induced markers (AIM) assay and an interferon-γ (IFNγ) FluoroSpot assay and responses were compared between processing methods. Samples washed with RPMI showed higher AIM+ CD4 T cell responses than those washed with PBS and showed a shift away from naïve and towards an effector memory phenotype. The activation marker OX40 showed higher SARS-CoV-2 spike-induced upregulation on RPMI-washed CD4 T cells, while differences in CD137 upregulation were minimal between processing methods. The magnitude of the AIM+ CD8 T cell response was similar between processing methods but showed higher stimulation indices. Background frequencies of CD69+ CD8 T cells were increased in PBS-washed samples and were associated with higher baseline numbers of IFNγ-producing cells in the FluoroSpot assay. Slower braking in the RPMI+ method did not improve detection of SARS-CoV-2-specific T cells and caused longer processing times. Thus, the use of RPMI media with full centrifugation brakes during the wash steps of PBMC isolation was found to be most effective and efficient. Further studies are needed to elucidate the pathways involved in RPMI-mediated preservation of downstream T cell activity.
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影响因子：
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作者：
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通讯作者：
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DOI：
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发表时间：
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期刊：
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