Effect of wash media type during PBMC isolation on downstream characterization of SARS-CoV-2-specific T cells

J Immunol Methods. 2023 Jun 28:113520. doi: 10.1016/j.jim.2023.113520. Online ahead of print.ABSTRACTProtocols 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 ind...
Source: Journal of Immunological Methods - Category: Allergy & Immunology Authors: Source Type: research