Centre of Expertise for Lupus-, Vasculitis-, and Complement-Mediated Systemic Autoimmune Diseases (LuVaCs), Department of Internal Medicine, section Nephrology, Leiden University Medical Center, Leiden, Netherlands.
Immunomonitoring group, Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, Netherlands.
Front Immunol. 2020 Dec 15;11:566732. doi: 10.3389/fimmu.2020.566732. eCollection 2020.
B-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.
EuroFlow-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 stimulated with CpG, IL-2, and IL-21 to induce antibody-secreting cells (ASC). (ANCA)-IgG was measured in these supernatants by ELISA.
By 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 CD20CD138 plasmablasts (PBs) were rapidly and strongly reduced, while CD20CD138 PrePC and CD20CD138 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 ANCA production by AAV PBMCs, 24 and 48 weeks after RTX treatment reflecting MRA in the memory compartment of AAV patients.
We 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.
利妥昔单抗（RTX）诱导的 B 细胞耗竭是治疗抗中性粒细胞胞质抗体（ANCA）相关性血管炎（AAV）的有效方法。然而，RTX 后频繁复发，常伴有 B 细胞再增殖，提示尽管进行了治疗，但仍存在残留的自身反应性 B 细胞。因此，本研究旨在鉴定接受 RTX 治疗的 AAV 患者 B 细胞群中的最小残留自身免疫（MRA）。
采用基于 EuroFlow 的高灵敏度流式细胞术（HSFC）在 RTX 治疗前后深入研究 AAV 患者的 B 细胞和浆细胞（PC）亚群。此外，还培养了这些接受 RTX 治疗的 AAV 患者的外周血单核细胞（PBMC），并用 CpG、IL-2 和 IL-21 刺激诱导分泌抗体的细胞（ASC）。通过 ELISA 测量这些上清液中的（ANCA）-IgG。
通过使用基于 EuroFlow 的 HSFC，我们在 RTX 治疗后所有时间点均检测到循环 CD19 B 细胞，而不是传统的低灵敏度流式细胞术。前生发中心（Pre-GC）B 细胞、记忆 B 细胞和 CD20CD138 浆母细胞（PBs）迅速且强烈减少，而 CD20CD138 PrePC 和 CD20CD138 成熟（m）PC 减少较慢且仍可检测到。记忆 B 细胞和 CD20 PC 在 RTX 后仍可检测到。血清 ANCA-IgG 在 RTX 后显著下降。ANCA 水平的变化与幼稚、转换的 CD27 和 CD27（双阴性）记忆 B 细胞的变化强烈相关，但与浆细胞无关。最后，我们证明了 AAV PBMC 在 RTX 治疗后 24 和 48 周产生 ANCA，反映了 AAV 患者记忆区的 MRA。
我们证明 RTX 诱导了循环 B 细胞的强烈减少，但从未导致完全的 B 细胞耗竭。尽管 RTX 后 B 细胞数量明显减少，但在 AAV 患者中仍可检测到 ANCA 特异性记忆 B 细胞。因此，在 AAV 中可以鉴定出 MRA，并为 AAV 患者的 RTX 治疗个体化提供潜在的新方法。
