Integrin Activation Enables Sensitive Detection Of Functional Cd4(+) And Cd8(+) T Cells: Application To Characterize Sars-Cov-2 Immunity

We have previously shown that conformational change in the beta(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 beta(2)-integrin. The kinetics of beta(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 beta(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.