Background: The R4RA trial, the first biopsy-based randomised trial in TNF-i inadequate responder patients with Rheumatoid Arthritis, showed that molecular stratification of RA synovial tissue was associated with clinical response, demonstrating that, in patients with low/absent B-cell lineage signature in synovial-tissue, tocilizumab is superior to rituximab ¹ .

Objectives: Here, we aimed to perform cell-transcript deconvolution of pre-and post-treatment synovial biopsies from the R4RA trial.

Methods: A total of 164 patients underwent pre-treatment synovial biopsy (US-guided or arthroscopic) prior to randomization 1:1 to rituximab (83) or tocilizumab (81). 65 patients had a repeat biopsy at 16 weeks when clinical response was assessed using Clinical Disease Activity Index (CDAI) 50% improvement. RNA extracted from a minimum of 6 synovial samples/patient underwent RNA-sequencing and the abundance of tissue-infiltrating immune and stromal cell populations was estimated using the Microenvironment Cell Populations-counter (MCP-counter) method ( Figure 1a ).

Results: At baseline, while synovial semiquantitative immunohistochemistry scores did not differ between CDAI50% responders and non-responders, both for rituximab and tocilizumab, MCP-counter analysis showed significantly higher CD8 T-cells in responders to rituximab and higher macrophage-monocytes and myeloid dendritic cells (mDC) in responders to tocilizumab ( Figure 1b ). Moreover, when patients were classified according to MCP-counter scores, B-cell poor patients (MCP-counter B cell score <median value) showed significantly higher response rates to tocilizumab, while no difference was found in B-cell rich patients ( Figure 1c ). In contrast, macrophage and myeloid dendritic cell (mDC) rich individuals showed higher responses to tocilizumab ( Figure 1d ). Combined scores for lymphoid and myeloid cells demonstrated that patients poor in B-cells but rich in macrophages/mDC had a significantly higher response to tocilizumab (77% responders to tocilizumab vs 14% responders to rituximab, p=0.017, OR 16.48, 95%CI 1.29-1000.5) ( Figure 1e ). By analysing disease activity over time from baseline to week 16, we found a statistically significant interaction effect between treatments and time in B-cell poor (p=0.003), T-cell poor (p=0.022), mDC rich (p=0.029) and B-cell poor/Macrophages-mDC rich patients (p=0.006) ( Figure 1f -g-h). Finally, by applying MCP-counter on matched pre-and post-treatment biopsies, rituximab-treated patients showed a significant reduction of B-cells, T-cells and monocyte/macrophages, while tocilizumab-treated patients showed a significant reduction of monocyte/macrophages, T-cells, but also neutrophils, myeloid dendritic cells and, interestingly, an increase in fibroblast signature ( Figure 1i ).

Conclusion: In silico deconvolution of the synovial tissue identify pre-treatment lymphoid cell lineages associated with response to rituximab and myeloid cells for tocilizumab. The longitudinal analysis of matched pre- and post-treatment synovial biopsies indicated that both medications have an effect on synovial immune cells, but tocilizumab can also affect stromal cells.

REFERENCES:

[1]Humby et al. Rituximab versus tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis (R4RA): 16-week outcomes of a stratified, biopsy-driven, multicentre, open-label, phase 4 randomised controlled trial Lancet. 2021 Jan 23;397(10271):305-317. doi: 10.1016/S0140-6736(20)32341-2.

Acknowledgements: We would like to thank all patients and the R4RA recruiting centres and principal investigators http://www.r4ra-nihr.whri.qmul.ac.uk/recruiting_centres.php We would also like to acknowledge the UK National Institute of Health Research for funding the R4RA trial (grant reference: 11/100/76) and Versus Arthritis for providing infrastructure support through the Experimental Arthritis Treatment Centre (grant number: 20022).

Disclosure of Interests: None declared.