149 General discussion and future perspectives is fundamentally altered due to effective B-cell depletion for up to 6 months after a single infusion of rituximab, which is underlined by a decrease in the total number of clones, an increase in number and impact of dominant clones, a decrease in the diversity of the BCR repertoire and a subsequent increase of unmutated BCR clones over time, suggesting re-establishment of the BCR repertoire after B-cell repopulation. This re-establishment of the repertoire is best visible between 6 and 12 months post-rituximab-infusion. These findings are in line with our earlier data in established RA, as described in Chapter 5. In the DOMINO study, a novel cohort of at-risk individuals was established with similar inclusion criteria to the individuals randomized for treatment intervention in the PRAIRI study. This allowed us to investigate the dynamics of the BCR repertoire and dominant clones over time to potentially identify the antigen(s) recognized by the BCRs and involved in the autoimmune repose during the natural course of (potential) arthritis development. This study was also used to determine specificity of BCRs using quantitative BCR repertoire analysis in combination with fluorescence activated cell sorting (FACS) for phenotypical analysis. Unfortunately, no BCR repertoire related parameters that associate with RA development could be identified in the peripheral blood. The BCR repertoire turned out to be very unstable over time, with dominant clones continuously changing from timepoint to timepoint. Intriguingly, in the at-risk individuals participating in the DOMINO study we saw that once dominant clones, at screening or baseline visit, disappeared over time, they seemed to “switch” to a lower frequency with a memory B-cell phenotype. This could be part of the “secondary immune response” by which memory B-cells recognize an antigen and initiate an immune response. During this secondary immune response memory B-cells are activated via antigen rechallenge and have (at least) two distinct fates: they either differentiate into long-lived plasma cells or enter germinal centers to undergo rounds of population expansion, somatic hypermutation and selection, ending up to restock the memory B-cells pool [39]. In the current study, these “switching” clones could potentially be BCR clones involved in autoantibody production. Bioinformatic techniques coupled with high throughput screening are needed to determine the specificity of such clones. It is important to also note here that some of the BCR clones were detected in both the plasmablast/plasma cell subset and the memory B-cell subset. Whether these clones are in transition from one phenotype to another or have a common precursor remains to be determined. It seems plausible that memory B-cells that have recognized a certain antigen, might carry the same BCR as the plasmablasts/plasma cells that have evolved from these cells and produce antibodies against such an antigen. Furthermore, it would be 7
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