Integrin Activation Enables Sensitive Detection of Functional CD4+ and CD8+ T Cells: Application to Characterize SARS-CoV-2 Immunity

Front Immunol. 2021 Mar 29;12:626308. doi: 10.3389/fimmu.2021.626308. eCollection 2021.


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.

Keywords: CD4+ T cells; CD8+ T cells; SARS-CoV-2; antigen specificity; integrin activation.

MeSH terms

  • Adult
  • Aged
  • Amino Acid Sequence
  • Binding Sites
  • CD4-Positive T-Lymphocytes / immunology*
  • CD4-Positive T-Lymphocytes / metabolism*
  • CD8-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / metabolism*
  • COVID-19 / genetics
  • COVID-19 / immunology
  • COVID-19 / metabolism
  • COVID-19 / virology
  • Carrier Proteins / chemistry
  • Cytokines / metabolism
  • Cytomegalovirus / immunology
  • Epitopes, T-Lymphocyte / chemistry
  • Epitopes, T-Lymphocyte / immunology
  • Female
  • HLA Antigens / chemistry
  • HLA Antigens / immunology
  • Host-Pathogen Interactions / genetics
  • Host-Pathogen Interactions / immunology*
  • Humans
  • Immunohistochemistry
  • Immunophenotyping
  • Integrins / genetics
  • Integrins / metabolism*
  • Intercellular Adhesion Molecule-1 / chemistry
  • Intercellular Adhesion Molecule-1 / metabolism
  • Lymphocyte Activation / immunology
  • Male
  • Middle Aged
  • Protein Binding
  • Protein Multimerization
  • SARS-CoV-2 / immunology
  • T-Cell Antigen Receptor Specificity
  • T-Lymphocyte Subsets / immunology
  • T-Lymphocyte Subsets / metabolism


  • Carrier Proteins
  • Cytokines
  • Epitopes, T-Lymphocyte
  • HLA Antigens
  • ICAM1 protein, human
  • Integrins
  • Intercellular Adhesion Molecule-1