CNRS UPR3572, Immunopathologie et chimie thérapeutique, Laboratory of Excellence Medalis, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
CNRS UMR7242, Biotechnology and Cell Signaling, Laboratory of Excellence Medalis, University of Strasbourg, Strasbourg, France.
Arthritis Rheumatol. 2018 Jul;70(7):1077-1088. doi: 10.1002/art.40470. Epub 2018 May 15.
Phosphopeptide P140 (Lupuzor) is an inhibitor of autophagy currently being evaluated in late-stage clinical trials for the treatment of lupus. This study was undertaken to investigate the effect of P140 ex vivo on human T and B cells.
Human B cells, T cells, and dendritic cells were analyzed by flow cytometry and cellular assays. The expression of autophagy markers was evaluated by immunoblotting and flow cytometry. The levels of B cell receptor (BCR) signaling markers and HLA molecules were assessed by flow cytometry. Toll-like receptor ligands were screened using an assay with transfected HEK 293 cells. P140 cell entry and trafficking were measured by immunofluorescence in the presence of various inhibitors of endosomal pathways.
As was previously observed after intravenous injection of the peptide in a mouse model of lupus, P140 entered human B cells by a clathrin coat-dependent endocytosis process and homed into lysosomes. The peptide displayed no direct effect on BCR signaling of memory, naive mature, transitional, and B1 cells. However, it strongly reduced the overexpression of HLA class II molecules on lupus B cells that were acting as antigen-presenting cells, down-regulated the maturation and differentiation of B cells into plasma cells, and decreased IgG secretion.
These findings show that P140 down-regulates HLA class II overexpression in human lupus B cells, and also that P140 hampers the differentiation of B cells into autoantibody-secreting plasma cells, likely due to the resulting lack of T cell signaling and activation. This mechanism appears to switch off the downstream events leading to secretion of pathogenic autoantibodies, thus explaining the highly promising results obtained in clinical trials of P140 (Lupuzor) for the treatment of lupus.
磷酸肽 P140（Lupuzor）是一种自噬抑制剂，目前正在进行晚期临床试验，用于治疗狼疮。本研究旨在研究 P140 对人 T 和 B 细胞的体外作用。
通过流式细胞术和细胞测定分析人 B 细胞、T 细胞和树突状细胞。通过免疫印迹和流式细胞术评估自噬标志物的表达。通过流式细胞术评估 B 细胞受体 (BCR) 信号标志物和 HLA 分子的水平。使用转染的 HEK 293 细胞的测定筛选 Toll 样受体配体。通过在存在各种内体途径抑制剂的情况下进行免疫荧光测量 P140 细胞进入和转运。
正如先前在狼疮小鼠模型中静脉注射该肽后观察到的那样，P140 通过网格蛋白包被依赖性内吞作用进入人 B 细胞，并归巢到溶酶体中。该肽对记忆 B 细胞、幼稚成熟 B 细胞、过渡 B 细胞和 B1 细胞的 BCR 信号没有直接影响。然而，它强烈降低了作为抗原呈递细胞的狼疮 B 细胞中 HLA Ⅱ类分子的过度表达，下调了 B 细胞向浆细胞的成熟和分化，并减少了 IgG 的分泌。
这些发现表明，P140 下调了人狼疮 B 细胞中 HLA Ⅱ类分子的过度表达，并且 P140 阻碍了 B 细胞分化为自身抗体分泌浆细胞，可能是由于缺乏 T 细胞信号和激活所致。这种机制似乎关闭了导致致病性自身抗体分泌的下游事件，从而解释了 P140（Lupuzor）在狼疮治疗的临床试验中获得的非常有前景的结果。
