Flow Cytometric Clinical Immunomonitoring Using Peptide-MHC Class II Tetramers: Optimization of Methods and Protocol...

With the advent of novel strategies to induce tolerance in autoimmune and autoimmune-like conditions, clinical trials of antigen-specific tolerizing immunotherapy have become a reality. Besides safety, it will be essential to gather mechanistic data on responding CD4⁺ T cells to assess the effects of various immunomodulatory approaches in early-phase trials. Peptide-MHC class II (pMHCII) multimers are an ideal tool for monitoring antigen-specific CD4⁺ T cell responses in unmanipulated cells directly ex vivo. Various protocols have been published but there are reagent and assay limitations across laboratories that could hinder their global application to immune monitoring. In this methodological analysis, we compare protocols and test available reagents to identify sources of variability and to determine the limitations of the tetramer binding assay. We describe a robust pMHCII flow cytometry-based assay to quantify and phenotype antigen-specific CD4⁺ T cells directly ex vivo from frozen peripheral blood mononuclear cell samples, which we suggest should be tested across various laboratories to standardize immune-monitoring results.