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Integrin Activation Enables Sensitive Detection of Functional CD4+ and CD8+ T Cells: Application to Characterize
Anna Schöllhorn1, Juliane Schuhmacher1, Luciana Besedovsky2
1Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen, Germany.
Insights
This study introduces a new assay to detect functional antigen-specific CD4+ and CD8+ T cells. The method uses a modified beta-2 integrin activation marker for sensitive screening in infectious disease and vaccination research.
Area of Science:
- Immunology
- Cellular Biology
- Virology
Background:
- Beta-2 integrin conformational change is an early activation marker for antigen-specific CD8+ T cells.
- Previous methods allowed rapid assessment of CD8+ T cell responses.
Purpose of the Study:
- To develop a modified assay for sensitive detection of functional antigen-specific CD4+ T cells.
- To enable simultaneous screening of both CD4+ and CD8+ T cell reactivities.
Main Methods:
- Utilized a modified protocol employing a monoclonal antibody (m24 Ab) targeting the high-affinity conformation of beta-2 integrin.
- Assessed T cell responses in whole blood or PBMCs from individuals infected/vaccinated with SARS-CoV-2, CMV, EBV, and HBV.
- Employed co-staining with m24 and CD154 antibodies to evaluate polyfunctional CD4+ T cell responses.
Main Results:
- The modified assay effectively detects functional antigen-specific CD4+ T cells.
- Beta-2 integrin activation kinetics differ between CD4+ and CD8+ T cells, but m24 Ab stains both 4-6 hours post-stimulation.
- The assay successfully monitored T cell responses to SARS-CoV-2, CMV, EBV, and HBV.
- Detected extremely low frequencies of polyfunctional CD4+ T cell responses.
Conclusions:
- A novel, sensitive assay allows simultaneous screening of CD4+ and CD8+ T cell reactivities.
- The assay is versatile for clinical and vaccination studies, enabling monitoring of effector and memory T cells.
- This method enhances the assessment of T cell immunity against various viral infections.
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.
