Highly Sensitive Flow Cytometric Detection of Residual B-Cells After Rituximab in Anti-Neutrophil Cytoplasmic Antibodies-Associated Vasculitis Patients

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.

CD19-targeting CAR T cells protect from ANCA-induced acute kidney injury

“…First, CD19 CAR T cells were highly efficient and depleted B cells (nearly) completely. In contrast, high-sensitive flow cytometry shows residual B cells, including ANCA-specific memory B cells at all time-points after rituximab administration in AAV patients 31. Second, we found that CD19 CAR T cells engrafted in lymphoid organs and inflamed target organs, such as kidneys.…”

“…In contrast, high-sensitive flow cytometry shows residual B cells, including ANCA-specific memory B cells at all time-points after rituximab administration in AAV patients. 31 Second, we found that CD19 CAR T cells engrafted in lymphoid organs and inflamed target organs, such as kidneys. This finding provides an advantage over B cell-targeting antibodies that result in incomplete tissue B cell depletion.…”

CAR-T-Zell-Therapie bei autoimmunen Erkrankungen

“…Allerdings profitieren nicht alle Patienten gleichermaßen, wie nicht zuletzt die hinter den Erwartungen zurückbleibenden Phase-II/III-Studien zu Rituximab und SLE demonstrierten [18,19]. Als Gründe werden die Persistenz autoreaktiver B-Zellen sowohl im peripheren Blut als auch in Organstrukturen diskutiert [20][21][22]. Abzuwarten bleibt, ob die neuere Generation an anti-CD20-Antikörpern dies ändern wird.…”

One year in review 2021: systemic vasculitis

“…As a result of this, the last recommendations do not support the use of tailored-administration strategy as a first-line maintenance treatment, suggesting that more data on this field are needed. On this respect, van Dam et al employed highly-sensitive flow cytometry (HSFC), traditionally used to detect minimal residual disease in haematologic neoplasia, to perform a phenotypic analysis of the B-cell compartment in patients undergoing RTX treatment for either induction or remission maintenance (92). They showed that RTX induced strong reductions in circulating Bcells, but never resulted in complete Bcell depletion when the serum samples were analysed by HSFC, in contrast to conventional low-sensitive flow cytometry.…”