Gammaherpesviruses such as Epstein-Barr virus (EBV) are major modulators of the immune responses of their hosts. In the related study (PMID: 35857578), we investigated the role for Ly6Chi monocytes in shaping the function of effector CD4(+) T cells in the context of a murine gammaherpesvirus infection (Murid gammaherpesvirus 4) as a model of human EBV. In order to unravel the polyfunctional properties of CD4(+) T-cell subsets, we used multiparametric flow cytometry to perform intracellular staining on lung cells. As such, we have developed herein an intracellular staining workflow to identify on the same samples the cytotoxic and/or regulatory properties of CD4(+) lymphocytes at the single-cell level. Briefly, following perfusion, collection, digestion, and filtration of the lung to obtain a single-cell suspension, lung cells were cultured for 4 h with protein transport inhibitors and specific stimulation media to accumulate cytokines of interest and/or cytotoxic granules. After multicolor surface labeling, fixation, and mild permeabilization, lung cells were stained for intracytoplasmic antigens and analyzed with a Fortessa 4-laser cytometer. This method of quantifying cytotoxic mediators as well as pro- or anti-inflammatory cytokines by flow cytometry has allowed us to decipher at high resolution the functional heterogeneity of lung CD4(+) T cells recruited after a viral infection. Therefore, this analysis provided a better understanding of the importance of CD4(+) T-cell regulation to prevent the development of virus-induced immunopathologies in the lung. Key features center dot High-resolution profiling of the functional properties of lung-infiltrating CD4(+) T cells after viral infection using conventional multiparametric flow cytometry. center dot Detailed protocol for mouse lung dissection, preparation of single-cell suspension, and setup of multicolor surface/intracellular staining. center dot Summary of optimal ex vivo restimulation conditions for investigating the functional polarization and cytokine production of lung-infiltrating CD4(+) T cells. center dot Comprehensive compilation of necessary biological and technical controls to ensure reliable data analysis and interpretation.
