Rheumatoid factor-producing CD21low anergic clonal B-cells in essential mixed cryoglobulinaemia: a model for autoantigen-driven pathogenesis of infectious and non-infectious cryoglobulinaemias

Objective. Essential mixed cryoglo- bulinaemia (EMC) is a disorder of B-cells producing rheumatoid factor (RF), and is clinically and immuno- logically similar to mixed cryoglobuli- naemia (MC) related to hepatitis C vi- rus (HCV-MC). We report here the first comprehensive analysis of B-cell clon- ality, phenotype and function in EMC. Methods. The study population in- cluded 16 patients with EMC and 24 patients with HCV-MC. Molecular analysis was done for the detection of circulating clonal B cells and for B cell receptor sequencing. B-cell phenotype, proliferative response, apoptosis and ERK signaling were analysed by flow cytometry. Results. Molecular analysis of im- munoglobulin genes rearrangements revealed circulating B-cell clones in about half of patients, on average of smaller size than those found in HCV-MC patients. Sequence analy- sis showed usage of the same stereo- typed RF-encoding B-cell receptors frequently expressed in HCV-MC and in primary Sjögren’s syndrome. B-cells with low expression of CD21 (CD21low) and unusual homing and inhibitory re- ceptors were increased in EMC and in HCV-MC, but at a significantly lower extent in the former. The CD21low B- cells of EMC and HCV-MC patients shared functional features of exhaus- tion and anergy, namely reduced pro- liferation upon ligation of Toll-like re- ceptor 9, high constitutive expression of phosphorylated ERK, and proneness to spontaneous apoptosis. Conclusion. Our findings suggest a common pathogenetic mechanism in EMC, HCV-MC and primary Sjögren’ssyndrome, consisting of autoantigen- driven clonal expansion and exhaus- tion of selected RF-producing B-cells. The more massive clonal expansion in HCV-MC may be due to co-stimulatory signals provided by the virus.