Immune cell profiling in the preclinical phase of rheumatoid arthritis

Early rheumatoid arthritis (RA) represents the end point of a preclinical disease continuum.

This begins with the development of anti-citrullinated protein antibodies (ACPA), followed by musculoskeletal (MSK) symptoms. This is followed by subclinical joint inflammation and then clinical arthritis. The specific immunopathological drivers of progression along the preclinical disease continuum remain unclear.

Specifically, the immune events that underpin the transition from autoimmunity to joint inflammation (subclinical and clinical arthritis) are not well defined. Type 1 interferon (IFN-I) is increased in at-risk individuals and early RA. JAK inhibitors, which inhibit IFN-I, are especially effective for pain and fatigue in RA.

At-risk individuals without synovitis frequently report these symptoms, suggesting IFN-I may be implicated. We will commence a trial to investigate the ability of baricitinib to prevent RA in Leeds in 2021.

We hypothesise ACPA+ individuals have distinct immune profiles and gene expression signatures prior to, and following, the development of subclinical and clinical joint inflammation. Furthermore, these signatures may be associated with progression to RA, and response to JAKi.

We will use single cell RNA sequencing (scRNA-seq) in precisely phenotyped preclinical RA cohorts to probe the immunopathology of critical stages in the preclinical RA continuum, including determinants of disease progression and response to JAKi.

We will use blood samples from two unique cohorts: (1) ACPA+ individuals with MSK symptoms (Leeds CCP cohort), the largest worldwide with longitudinal data on >450 individuals; ACPA+ individuals +/- subclinical joint inflammation on ultrasound will be identified as separate groups; (2) ACPA+ at-risk individuals from (1) who receive baricitinib (48 weeks treatment, 48 weeks observation) as part of the Leeds ExiST trial.

Longitudinal data on progression to RA and response to baricitinib will be available.

We will perform multi-parameter flow cytometry on peripheral blood mononuclear cells (PBMCs) from these populations to phenotype immune cell subsets (T cells, B cells, monocytes, NK cells, pDCs) compared with controls and early RA patients.

We will examine activation/ proliferation states, migratory markers and clinically important subsets, which will inform targets for characterisation by bulk and sc-RNA-seq.

IFN-I stimulated genes will be measured by real-time polymerase chain reaction (RT-PCR) in matched PBMCs; the association between IFN-I response scores and immune cell subsets with progression to RA will be assessed. We will use bulk RNA-seq of sorted cell subsets to dissect perturbed pathways within immune cell subsets.

Together, these data will identify abnormal immune cell subsets associated with progression to RA.

We will then use scRNA-seq to define transcriptomic profiles of altered immune cell subsets, which will be integrated at single cell level to define associations between cell states and patient groups, including progression to RA and response to baricitinib. Bulk RNA-seq will be used to validate findings.

This collaborative work across two biomedical research centres combines unique cohorts of individuals at-risk of RA, with laboratory/bioinformatics expertise.

It will further our understanding of key preclinical intervention points, delivering an important advance in the translational pathway toward RA prevention. This is a key priority identified by public and patient involvement and engagement.