Abstract
Antigen-specific T cells can serve as a response biomarker in non-clinical or clinical immunotherapy studies in autoimmune disease. There are protocols with optimized multimer staining methods to detect peptide (p)MHCII+ CD4+ T cells, and some qualified and validated protocols for pMHCI+ CD8+ T cells. However, no protocol is fully or partially qualified to enumerate and characterise antigen-specific pMHCII+ CD4+ T cells from patient samples. Implementing such an assay requires a desired level of specificity and precision, in terms of assay repeatability and reproducibility. In transgenic type II collagen (CII)-immunised HLA-DR1/DR4 humanised mouse models of collagen-induced arthritis (CIA), CII259-273-specific T cells dominantly expand. Therefore antigen-specific T cells recognising this epitope presented by rheumatoid arthritis (RA)-associated risk HLA-DR allomorphs are of interest to understand disease progression and responses to immunotherapy in RA patients. Using HLA-DRB1*04:01 or *01:01-collagen type II (CII)259–273 tetramers, we evaluated parameters influencing precision and reproducibility of an optimized flow cytometry-based method for antigen-specific CD4+ T cells and eight specific subpopulations with and without tetramer positivity. We evaluated specificity, precision, and reproducibility for research environments and non-regulated laboratories. The assay has excellent overall precision with %CV<25% for intra-assay repeatability, inter-analyst precision, and inter-assay reproducibility. The precision of the assay correlated negatively with the cell viability after thawing, indicating that post-thaw viability is a critical parameter for reproducibility. This assay is suitable for longitudinal analysis of treatment response and disease activity outcome in RA patients, and adaptable for translational or immunotherapy clinical trial settings.
Type II collagen is a target antigen of clonally expanded T cells in the synovium of patients with rheumatoid arthritis