We have previously shown that conformational change in the β 2 -integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8 + T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4 + T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the β 2 -integrin. The kinetics of β 2 -integrin activation was different on CD4 + and CD8 + T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4–6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4 + and CD8 + T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein–Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining β 2 -integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4 + T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4 + and CD8 + T cell reactivities, with versatile applicability in clinical and vaccination studies.
【저자키워드】 SARS-CoV-2, CD4+ T cells, CD8+ T cells, integrin activation, antigen specificity, 【초록키워드】 coronavirus, vaccination, protocol, T cells, monoclonal antibody, CD4, CD8, memory, Antigen, Peripheral blood, HBV, T cell, Whole blood, T cell responses, CMV, EBV, marker, Hepatitis B virus, Ligand, Frequency, PBMCs, conformational change, Epstein–Barr virus, cell type, mononuclear cell, Ex vivo, acute respiratory syndrome, Activation, vaccinated individuals, fluorescent, applicability, clone, MONITOR, CD154, shown, functional, intercellular, 【제목키워드】 detection, CD4, CD8, sensitive, application,