Deep immunophenotyping whole blood and synovial fluid immune cell populations in Rheumatoid Arthritis by mass cytometry

Diagnosis and clinical management of Rheumatoid Arthritis (RA) have improved significantly over the last three decades however, not all patients respond successfully to treatment and currently a cure for RA remains elusive. Chronic inflammation is a key feature of RA and if left untreated or improperly managed, can lead to irreversible joint damage. Understanding the immune dysregulation which occurs in RA is central to improving existing approaches for managing RA. Extensive research directed towards immunophenotyping and functional analysis of immune cell subsets in RA have contributed to our current understanding of RA but the impact of these findings have been restricted by existing technology. In this study, deep immunophenotyping was performed using mass cytometry which is a novel multiparameter, high-dimensional single cell technology, to comprehensively interrogate immune cell subsets present in peripheral blood and synovial fluid from patients with RA using a 37 protein marker panel. This study set out to investigate two main aims, the first of which was to evaluate whether mass cytometry was a suitable technology that could be adopted for large patient cohort immunophenotyping studies which prior to the commencement of this study, had not been reported. Significant effort was invested for protocol validation and optimisation and in addition, with support from UCB Pharma, an automated bioinformatics pipeline was developed to analyse the data without the limitations of traditional gating approaches. The second aim was to assess whether mass cytometry could detect immune cell populations which associated with disease stage or immune signatures which are specific to the local joint microenvironment in RA. 10 palladium isotope tagged barcoded batches were prepared and an internal batch control was included to compare consistency of staining. Data pre-processing steps were applied to obtain a single cell population and discovery hypothesis driven analysis was performed through R Studio using a published pipeline called Diffcyt which assessed immune cell populations across different conditions and between samples. Diffcyt analysis revealed that decreased percentage changes in innate cell populations are evident early on in RA compared to healthy donors. Differential expression analysis revealed that both innate and T cell subsets in RA peripheral blood have an activated phenotype characterised by CD27, CD38, CD28 and HLA-DR suggesting the beginning of a hyper chronic inflammatory environment. In addition, analysis of immune cell populations in synovial fluid further corroborated reports of pathologically expanded memory CD4 T cell populations present in RA synovial fluid compared to peripheral blood. Furthermore, a specific CD8 NK cell immunophenotype was detected in RA synovial fluid suggesting a potential role in crosstalk between innate and adaptive immunity. This research has demonstrated that mass cytometry can be used to comprehensively interrogate the immune landscape in large patient cohorts and using the methodology described here, successfully identifies cell populations that support findings previously reported by other researchers giving confidence in the data obtained by mass cytometry. It is hoped that the methodology for analysing high-dimensional cytometry data will provide a template for future analysis of either this dataset or new datasets and that the cell populations identified here will inform further investigation in RA.