Investigating CD8 T Cell Exhaustion in Chronic LCMV Infection by Flow Cytometry

Abstract

CD8 T cells and the process of T cell exhaustion are central topics in both basic immunology and translational research, including clinical applications. Here, we provide a comprehensive, step-by-step guide to investigate CD8 T cells in murine in vivo lymphocytic choriomeningitis virus (LCMV) infection models. We outline viral stock preparation, infection, and viral titer evaluation. We put an emphasis on flow cytometry as a method to investigate phenotypical and functional characteristics of CD8 T cell exhaustion, but the protocols provided will also facilitate imaging or transcriptomic analyses. With these resources, we aim to streamline establishment of the LCMV model for new users and to support harmonization and optimization of workflows for laboratories already working with this model. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Virus production

Basic Protocol 2: Virus titer quantification from virus stocks by focus-forming assay

Alternate Protocol 1: Virus titer quantification from infected tissues by focus-forming assay

Basic Protocol 3: Peptide-MHC-I tetramer production

Basic Protocol 4: Naïve CD8 T cell isolation using StemCell^TM kit

Alternate Protocol 2: Naïve CD8 T cell enrichment using MojoSort^TM kit

Basic Protocol 5: Adoptive CD8 T cell transfer and LCMV infection

Basic Protocol 6: Animal husbandry

Basic Protocol 7: Preparation of single-cell suspension from secondary lymphoid organs

Alternate Protocol 3: Preparation of single-cell suspension from peripheral tissues

Basic Protocol 8: Extracellular and intracellular staining

Basic Protocol 9: Restimulation analysis

Basic Protocol 10: Sorting