Functional Phenotyping of Lung Mouse CD4⁺ T Cells Using Multiparametric Flow Cytometry Analysis

Area of Science:

• Immunology
• Virology
• Cellular Biology

Background:

• Gammaherpesviruses, including Epstein-Barr virus (EBV), significantly influence host immune responses.
• Ly6Chi monocytes play a role in modulating effector CD4+ T cell function during viral infections.
• Murid gammaherpesvirus 4 infection in mice serves as a model for human EBV infection.

Purpose of the Study:

• To develop and validate an intracellular staining workflow for multiparametric flow cytometry.
• To analyze the polyfunctional properties of CD4+ T cell subsets, including cytotoxic and regulatory functions.
• To investigate the functional heterogeneity of lung CD4+ T cells following viral infection.

Main Methods:

• Preparation of single-cell suspensions from mouse lungs post-infection.
• Ex vivo culture of lung cells with protein transport inhibitors and specific stimulation media.
• Multicolor surface and intracellular staining for cytokines and cytotoxic mediators, followed by flow cytometry analysis.

Main Results:

• A high-resolution method was established to profile functional properties of lung-infiltrating CD4+ T cells.
• The workflow enables simultaneous quantification of cytotoxic mediators and pro- or anti-inflammatory cytokines.
• Deciphered functional heterogeneity of CD4+ T cells recruited to the lungs after viral challenge.

Conclusions:

• The developed flow cytometry protocol provides a detailed understanding of CD4+ T cell responses during viral infections.
• This analysis enhances comprehension of CD4+ T cell regulation's importance in preventing virus-induced lung immunopathology.
• The study offers a comprehensive approach for analyzing immune cell function at the single-cell level.