132 CHAPTER 6 point, and the top-25 clones were variable in every follow-up visit. The majority of these dominant clones originate from cells that exhibit a plasmablast/plasma cell phenotype. This is in line with what is known about mature plasmablasts/plasma cells in peripheral blood: they generally have a short survival time in circulation, yet have a high RNA load, which may result in more dominance in the BCRh repertoire [17–19]. On a more general note: It shows that the BCRh repertoire is constantly changing over time, also in the preclinical phase of RA. This is expected, following B cell depleting therapy, but is also present in placebo treated or untreated RArisk individuals. It suggests a continuous effort to develop new dominant clonal (plasmablast) responses, some of which might be involved in autoantibody production in at-risk patients. Such a continuously renewing response in this phase might explain induction of new (sub)specificities in these autoimmune responses just before the onset of arthritis [20]. Interesting is how clones that initially were dominant at screening visit or at baseline would switch their phenotype in peripheral blood once they became non-dominant over a 6-month period: they could be preferentially retraced in the memory B cell phenotype compartment. This dynamic is probably the net result of differences in production rates in germinal centres for memory B cells and plasma cells in different phases of the response, differences in survival time of both cell types in blood, and differences in the efflux from blood to lymphoid or peripheral tissues. In the current study, these “switching” clones could potentially be BCRh clones involved in autoantibody production. Bioinformatic techniques coupled with flow cytometric methods and high throughput screenings are needed to isolate and test the specificity of such clones. It is important to also note here that some of the BCRh clones were detected in both the plasmablast/plasma cell subset and memory B cell subset. Both cell types might well derive from common clonal precursors during germinal centre reactions. It seems plausible that memory B cells that have recognized a certain antigen, might thus carry the same BCRh as the plasma blasts/plasma cells that produce antibodies against such an antigen. It seems very unlikely that there is contamination originating from the FACS experiments, although this is a possibility. The two sorted phenotypes differ in three surface markers (CD19, CD20 and CD38), as well as in their subsequent gating strategy. Furthermore, setting up of sorting gates as well as other experiments were performed with great care using state-ofthe-art techniques to decrease the risk of contamination. We were not able to determine the phenotype of nine dominant BCRh clones detected in peripheral blood. A possible explanation could be that these clones are regulatory (Bregs) or of another phenotype and our FACs panel did not permit for
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