Fig. 4.

Phenotypic characterisation of unstimulated B cells in slow progressors demonstrate increased expression of CD95 among B cells with a switched memory phenotype. Expression of the indicated markers in unstimulated samples was assessed using flow cytometry. (a) SPADE image of pooled B cells from all participants, auto-partitioned into eight annotated areas with node size scaled to the log number of cells in each node, showing median CD95 expression as a heatmap. Based on the expression of CD27 and IgD, the cells in the different areas were designated as follows: areas 1, 2 and 5, switched memory (CD27⁺IgD⁻); area 3, unswitched (CD27^intIgD⁺); areas 4 and 7, naive (CD27⁻IgD⁺); area 6, transitional (CD27^intIgD^int); area 8, naive/switched (CD27⁻IgD^int). (b) SPADE boxplots showing marker distribution in each area or in all areas for the pooled samples depicted in (a). Central red lines indicate median values and the ends of blue boxes indicate interquartile ranges. The dashed horizontal line at the bottom indicates the ‘All’ category. (c) CD95 expression (transformed values) in area 5 for each participant. Horizontal lines indicate mean values and red squares denote slow progressors who tested seropositive for a single islet autoantibody at the time of immune cell analysis. *p < 0.05 for slow progressors vs healthy donors (determined using an unpaired Student’s t test). Results for people with newly diagnosed and long-standing type 1 diabetes are shown in grey to add context but were not included in statistical analysis due to small sample sizes and lack of age matching between newly diagnosed and slow-progressing individuals. AU, arbitrary unit; HD, healthy donors; LS, long-standing type 1 diabetes; MFI, median fluorescence intensity; ND, newly diagnosed type 1 diabetes; SP, slow progressors