Department of Rheumatology, Tohoku University Hospital, 1-1 Seiryo-Machi, Aoba-Ku, Sendai, Miyagi, 980-8574, Japan.
Research and Development Department, Ginkgo Biomedical Research Institute, SBI Biotech Co., Ltd., Tokyo, Japan.
Arthritis Res Ther. 2023 Oct 16;25(1):200. doi: 10.1186/s13075-023-03186-5.
In systemic lupus erythematosus (SLE), autoreactive B cells are thought to develop by-passing immune checkpoints and contribute to its pathogenesis. Toll-like receptor (TLR) 7 and 9 signaling have been implicated in their development and differentiation. Although some B cell subpopulations such as T-bet + double negative 2 (DN2) cells have been identified as autoreactive in the past few years, because the upregulated surface markers of those cells are not exclusive to them, it is still challenging to specifically target autoreactive B cells in SLE patients.
Our preliminary expression analysis revealed that phospholipase D4 (PLD4) is exclusively expressed in plasmacytoid dendritic cells (pDCs) and B cells in peripheral blood mononuclear cells (PBMCs) samples. Monoclonal antibodies against human PLD4 were generated, and flow cytometry analyses were conducted for PBMCs from 23 healthy donors (HDs) and 40 patients with SLE. In vitro cell culture was also performed to study the conditions that induce PLD4 in B cells from HDs. Finally, recombinant antibodies were synthesized from subpopulations of PLD4 + B cells from a patient with SLE, and their antinuclear activity was measured through enzyme-linked immunosorbent assay.
pDCs from both groups showed comparable frequency of surface PLD4 expression. PLD4 + B cells accounted for only a few percent of HD B cells, whereas they were significantly expanded in patients with SLE (2.1% ± 0.4% vs. 10.8% ± 1.2%, P < 0.005). A subpopulation within PLD4 + B cells whose cell size was comparable to CD38 + CD43 + plasmablasts was defined as "PLD4 + blasts," and their frequencies were significantly correlated with those of plasmablasts (P < 0.005). PLD4 + blasts phenotypically overlapped with double negative 2 (DN2) cells, and, in line with this, their frequencies were significantly correlated with several clinical markers of SLE. In vitro assay using healthy PBMCs demonstrated that TLR7 or TLR9 stimulation was sufficient to induce PLD4 on the surface of the B cells. Finally, two out of three recombinant antibodies synthesized from PLD4 + blasts showed antinuclear activity.
PLD4 + B cells, especially "blastic" ones, are likely autoreactive B cells undergoing TLR stimulation. Therefore, PLD4 is a promising target marker in SLE treatment.
在系统性红斑狼疮（SLE）中，自身反应性 B 细胞被认为通过绕过免疫检查点而发展，并有助于其发病机制。Toll 样受体（TLR）7 和 9 的信号转导已被认为与其发育和分化有关。尽管过去几年已经确定了一些 B 细胞亚群，如 T-bet+双阴性 2（DN2）细胞作为自身反应性细胞，但由于这些细胞上调的表面标志物并非它们所特有，因此仍然难以在 SLE 患者中特异性靶向自身反应性 B 细胞。
我们的初步表达分析表明，磷脂酶 D4（PLD4）仅在外周血单个核细胞（PBMC）样本中的浆细胞样树突状细胞（pDC）和 B 细胞中表达。生成了针对人 PLD4 的单克隆抗体，并对来自 23 名健康供体（HD）和 40 名 SLE 患者的 PBMC 进行了流式细胞术分析。还进行了体外细胞培养，以研究诱导 HD 来源的 B 细胞中 PLD4 的条件。最后，从 SLE 患者的 PLD4+B 细胞亚群中合成重组抗体，并通过酶联免疫吸附试验测量其抗核活性。
两组的 pDC 表面 PLD4 表达频率相当。PLD4+B 细胞仅占 HD B 细胞的一小部分，而在 SLE 患者中则显著扩增（2.1%±0.4%对 10.8%±1.2%，P<0.005）。一个与 CD38+CD43+浆母细胞大小相当的 PLD4+B 细胞亚群被定义为“PLD4+blasts”，其频率与浆母细胞的频率显著相关（P<0.005）。PLD4+blasts 表型与 DN2 细胞重叠，与此一致，其频率与 SLE 的几个临床标志物显著相关。使用健康 PBMC 的体外测定表明，TLR7 或 TLR9 刺激足以诱导 B 细胞表面的 PLD4。最后，从 PLD4+blasts 合成的三种重组抗体中的两种显示出抗核活性。
PLD4+B 细胞，特别是“浆母细胞样”B 细胞，可能是经历 TLR 刺激的自身反应性 B 细胞。因此，PLD4 是 SLE 治疗中有希望的靶标标志物。
