Abstract
BackgroundB-cell depletion with rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients. Nevertheless, relapses are frequent after RTX, often preceded by B-cell repopulation suggesting that residual autoreactive B-cells persist despite therapy. Therefore, this study aimed to identify minimal residual autoimmunity (MRA) in the B-cell compartment of AAV patients treated with RTX.MethodsEuroFlow-based highly-sensitive flow cytometry (HSFC) was employed to study B-cell and plasma cell (PC) subsets in-depth in AAV patients before and after RTX treatment. Additionally, peripheral blood mononuclear cells (PBMCs) of these RTX-treated AAV patients were cultured and in vitro stimulated with CpG, IL-2, and IL-21 to induce antibody-secreting cells (ASC). (ANCA)-IgG was measured in these supernatants by ELISA.ResultsBy employing EuroFlow-based HSFC, we detected circulating CD19+ B-cells at all timepoints after RTX treatment, in contrast to conventional low-sensitive flow cytometry. Pre-germinal center (Pre-GC) B-cells, memory B-cells and CD20+CD138− plasmablasts (PBs) were rapidly and strongly reduced, while CD20−CD138− PrePC and CD20-CD138+ mature (m)PCs were reduced slower and remained detectable. Both memory B-cells and CD20− PCs remained detectable after RTX. Serum ANCA-IgG decreased significantly upon RTX. Changes in ANCA levels strongly correlated with changes in naive, switched CD27+ and CD27− (double-negative) memory B-cells, but not with plasma cells. Lastly, we demonstrated in vitro ANCA production by AAV PBMCs, 24 and 48 weeks after RTX treatment reflecting MRA in the memory compartment of AAV patients.ConclusionWe demonstrated that RTX induced strong reductions in circulating B-cells, but never resulted in complete B-cell depletion. Despite strongly reduced B-cell numbers after RTX, ANCA-specific memory B-cells were still detectable in AAV patients. Thus, MRA is identifiable in AAV and can provide a potential novel approach in personalizing RTX treatment in AAV patients.
Highlights
B-cell depletion with rituximab (RTX) is an effective treatment strategy for patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) [1, 2] and is increasingly prescribed as induction and/or maintenance treatment [3,4,5]
Studies that investigated the B-cell compartment of AAV patients after RTX more in-depth found several phenotypes that were associated with relapses, e.g. incomplete B-cell depletion [17]; or B-cell repopulation with relatively high number of plasmablasts (PBs) [18]; switched memory B-cells [19]; relatively low number of naive B-cells [17] or decreased CD5+ regulatory B-cells [20, 21], whereas the latter inversely correlated with ANCA levels [22]
The number of CD19+ Bcells over time was shown for each individual patient after treatment with RTX as measured with low sensitive” flow cytometry (LSFC) (Figure 1B) and with highly-sensitive flow cytometry (HSFC) (Figure 1C)
Summary
B-cell depletion with rituximab (RTX) is an effective treatment strategy for patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) [1, 2] and is increasingly prescribed as induction and/or maintenance treatment [3,4,5]. Despite the success with RTX as remission-induction therapy in AAV patients [1, 2], in the RAVE-trial, one-third of the patients experienced a relapse within 18 months after RTX [8] In this trial, increases in ANCA levels did not predict relapses in either the RTX or the cyclophosphamide treatment group. We recently demonstrated that PR3positivity predicted future relapses in AAV patients after remission-induction therapy with RTX [16], which was supported by another study [13]. Relapses are frequent after RTX, often preceded by B-cell repopulation suggesting that residual autoreactive B-cells persist despite therapy. This study aimed to identify minimal residual autoimmunity (MRA) in the B-cell compartment of AAV patients treated with RTX
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