AUTHOR=Jansen Diahann T. S. L. , Ramnoruth Nishta , Loh Khai L. , Rossjohn Jamie , Reid Hugh H. , Nel Hendrik J. , Thomas Ranjeny 

TITLE=Flow Cytometric Clinical Immunomonitoring Using Peptide–MHC Class II Tetramers: Optimization of Methods and Protocol Development

JOURNAL=Frontiers in Immunology

VOLUME=Volume 9 - 2018

YEAR=2018

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2018.00008

DOI=10.3389/fimmu.2018.00008

ISSN=1664-3224

ABSTRACT=With the advent of novel strategies to induce tolerance in autoimmune and autoimmune-like conditions, clinical trials of antigen-specific tolerising immunotherapy have become a reality. Besides safety, it will be essential to gather mechanistic data on responding CD4+ T cells in order to assess the effects of various immunomodulatory approaches in early-phase trials. Peptide-MHCII 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 PBMC samples, which we suggest should be tested across various laboratories to standardise immune-monitoring results.