Fig. 6: Digital spatial profiling (DSP) confirms the positional identity of AXL and MERTK.

A Schematic representation of the Digital Spatial Profiling (DSP) approach, including the selection of three regions of interest (ROI): aggregate (characterised by the presence of CD3⁺ and CD20⁺ cells), deep sublining (characterised by the absence of CD3⁺ and CD20⁺ cells) and lining with superficial sublining (characterised by the presence of CD68⁺ cells). Scale bar = 100 μm. DSP was performed on 14 aggregates, 25 lining, and 33 sublining regions. B Three-way radial plot showing differential and overlapping genes across aggregate (green), lining (blue) and sublining (red) regions. AXL and MERTK genes have been labelled showing a significantly higher presence of AXL in both lining and sublining regions and a significant presence of MERTK in the lining region. Significance was internally estimated by the volcano3D package combining significance (q < 0.05) from both one-way ANOVA and pairwise T test. C Double immunostaining of Axl (red) and MerTK (yellow) in the aggregate, sublining and lining synovial areas of RA patients. Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. Representative images of n = 32 samples stained. D Expression of selected individual genes included in the AXL and/or MERTK networks in the aggregate (n = 14, including 4 non-responder and 10 responder patients), sublining (n = 33, including 12 non-responder and 21 responder patients) and lining (n = 25, including 8 non-responder and 17 responder patients) synovial areas of responders (light blue) and non-responders (orange) RA patients to either tocilizumab or rituximab. Boxplots represent the median and first and third quartiles, and whiskers span to the minimum and maximum. A paired Wilcoxon test was undertaken to compare responders and non-responders.