Abstract
We have previously shown that conformational change in the β2-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 β2-integrin. The kinetics of β2-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 β2-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.
Highlights
One of the first events that can be detected upon T cell receptor (TCR)-mediated stimulation of T cells is the activation of membrane-bound β2-integrins
Following donors were selected for the study: one person with detectable CD4+ T cells binding to the HLA-DRB1*11 multimer refolded with the CMV peptide HPTFTSQYRIQGKLE; two HLA-A*02+ people with detectable CD8+ T cells binding to the HLA-A*02:01 multimer refolded with the CMV NLVPMVATV; one HLA-A*02+ person with detectable CD8+ T cells binding to the HLA-A*02:01 multimer refolded with the Epstein–Barr virus (EBV) BRLF1 peptide YVLDHLIVV and a CD4+ T cell reactivity to an in house-made HLA-class II peptidepool containing nine CMV, EBV and Flu-derived epitopes
We have previously shown that antigen-specific CD8+ T cells are successfully detected by staining of activated β2integrins with fluorescent intercellular adhesion molecule (ICAM)-1 multimers [5]
Summary
One of the first events that can be detected upon T cell receptor (TCR)-mediated stimulation of T cells is the activation of membrane-bound β2-integrins. This activation involves a conformational change (from the bent, inactive form to the open, high-affinity conformation) and a clustering of integrin molecules on the cell membrane. Using fluorescent multimers of ICAM-1, we have previously shown that effector CD8+ T lymphocytes can be and very rapidly detected upon antigen-specific T cell stimulation. We observed that the binding of ICAM-1 multimers to β2-integrins on CD8+ T cells identifies functional effectors that will later degranulate and produce effector cytokines.
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