Journal of IiME Volume 8 Issue 1 predict poor response to anti-CD20 therapy (18, 19). In patients with SLE, we have shown that high serum BAFF levels and possession of autoantibodies with ENA specificity indicated shorter clinical response(<6 months) to RTX (20). Thus, current evidence suggests that tracking of memory B celland plasma cell activity are both important in predicting clinical relapse/response. Whereas serum immunoglobulin levels including autoantibody levels are used to identify plasma cell activity there are no biomarkers that may predict the formation of memory B cells. Our early studies of the kinetics of autoantibody levels following RTX showed a correlation with the ‘delayed’ onset of clinical response (often approximately 1-2 months after B cell depletion induced), characteristic of RTX treatment in patients with RA. Studies by Immunohistochemical studies of synovial biopsies after RTX by Thurlings and colleagues (21) showed that the reduction of plasma cells at 16 weeks post-RTX was the best predictor of clinical improvement at 24 week followup. Although B cell numbers in synovium were also reduced at 16 weeks, they were not correlated with response. The results from studies of biopsied joints following RTX therefore supported our hypothesis that the clinical response to RTX was due at least in part to an indirect effect on plasmablasts/plasma cells associated with autoantibody production (22). Rituximab was therefore possibly working by preventing recruitment of activated autoreactive B cells into secondary lymphoid tissue and to joints by removing circulating B cells. Re-establishment of disease involves the reengagement of pro-inflammatory pathways, which are absent or greatly diminished during the period of B-cell depletion. Relapse after RTX has been found to follow B cell return to the periphery in patients with RA, but this relationship is less clear in patients with SLE. B cell return after RTX mirrors ontogeny with transitional and naïve B cells, many expressing CD5, exiting the bone marrow and expanding in the periphery. Recovery of B cell numbers to within the normal range varies enormously between patients and can be very protracted. Maturation to memory phenotype, and restoration of the normal ratio between naïve and memory B cell compartments is not often achieved for many months or even years in either condition(23, 24). Differentiation towards memory phenotype after B cell return, most commonly associated (but not always) with gaining CD27+ status (25), may however herald relapse (14). Our May 2014 observations also suggest that rises in IgM-RhF are closely associated with impending relapse (26). Therefore, relatively long periods of B cell depletion in the peripheral blood (6-9 months in patients with RA) are associated with reduction in symptoms after RTX. The trigger for relapse can either coincide with, or follow by periods of some months, the exit of new B cells from the bone marrow. The time-course and B cell kinetics during the RTX treatment cycle therefore suggest that pathogenic plasmablasts/plasma cells are mostly short-lived and their removal is necessary for induction of remission. The strong association of rises in autoantibodies, rather that B cell numbers, with clinical relapse, suggests that naïve B cells or resistent memory B cell populations (perhaps expanded by by-stander help as a consequence of T cell dependent or independent pathways), are differentiating into Ig-producing cells. Studies leading to this project: Preliminary studies in our laboratory suggested that the measurement of a serum factor released from B cells as they undergo differentiation to memory phenotype (soluble CD23) , may be a useful surrogate of a) relative rate of differentiation of naïve B cells to a memory phenotype (CD27+) following RTX (Cambridge et al, submitted 2013) and b) a potential biomarker for depletion/response to RTX in RA and SLE patients (preliminary data, Figure 1 attached). Briefly, we found that in 23 RA patients treated with RTX, baseline levels of serum sCD23 were generally within normal limits, decreasing to below the normal range at depletion, demonstrating that most serum sCD23 was derived from B cells. It has previously been shown that sCD23 levels may correlate with disease activity in patients with SLE and SS (27, 28). CD23, the low affinity FcεR, is expressed on mature naïve B cells, lost from germinal centre cells and is expressed only on a low proportion of IgD+ memory B cells and possibly some transitional B cells (29). Expression of CD27 following antigen encounter induces cleavage of CD23 and the soluble receptor, sCD23 is released into the circulation. CD23 and CD27 expression appears to be virtually mutually exclusive; with both antigens possibly only transiently expressed on the same cell. sCD23 is released from the membrane expressed molecule by the action of endogenous αdisintegrin and metalloprotease10. Cleavage from the cell surface can be induced following stimulation in vitro with IL4 and CD40-L. Serum levels above normal limits (>2000ng/ml) in vivo are associated with allergy and atopy. It has recently Invest in ME (Charity Nr. 1114035) www.investinme.org Page 14 of 52

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