Tohoku University School of Medicine Sendai, Japan
Ken Yasaka1, Tomohide Yamazaki2, Hiroko Sato1, Tsuyoshi Shirai1, Hiroshi Fujii1, Tomonori Ishii3 and Hideo Harigae4, 1Department of Rheumatology, Tohoku University Hospital, Sendai, Japan, 2SBI Biotech, Tokyo, Japan, 3Clinical Research, Innovation and Education Center, Tohoku University Hospital, Sendai, Japan, 4Department of Hematology, Tohoku University Hospital, Sendai, Japan
Background/Purpose: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by various autoantibodies. In particular, targeting autoreactive B cells could be a promising therapy with minimal adverse effects and dependence on corticosteroids. Some phenotypically distinct B cell populations including IgD-, CD27-, CD11c+, and CXCR5lo double negative 2 (DN2) B cells, are expanded in lupus peripheral blood. DN2 B cells, which includes autoreactive B cells capable of differentiating into antibody secreting cells (ASCs), exhibit high intracellular expression of T-bet. Aiming to identify therapeutic targets in SLE, we found that phospholipase D4 (PLD4) is expressed on the surface of most plasmacytoid dendritic cells (pDCs) and less on the surface of B cells in healthy donors (HDs). PLD4 is believed to be an endolysosomal nuclease that regulates signals of toll-like receptors(TLRs) 7,8, and 9. We aimed to investigate the surface expression of PLD4 on B cells in SLE and to test PLD4+ B cells as a possible target for SLE treatment.
Methods: We developed monoclonal antibodies against PLD4. Flow cytometry was used to analyze PLD4 expression on several populations among peripheral blood mononuclear cells (PBMCs) from individuals with SLE (n = 40) and HDs (n = 11) classified by CD19, CD3, CD14, CD16, CD303, IgD, CD27, CD38, CD43, CD11c, and CXCR5. All SLE patients met ACR 1997 classification criteria. The prevalence of PLD4+ CD19+ B cells was compared between individuals with SLE and HDs, and their correlation with disease status was assessed. Furthermore, we investigated the intracellular expression of T-bet in SLE PLD4+ B cells. Certain compartments of B cells were isolated for culture to test their abilities to differentiate into ASCs by ELISpot.
Results: Among PBMCs, PLD4 was expressed only on pDCs and B cells. The ratio of PLD4+ CD19+ B cells to the whole CD19+ B cells was significantly higher in individuals with SLE than in HDs (mean ± SEM%, 11 ± 1.2% in SLE vs 2.1 ± 0.37% in HD, P < 1.0×10-6). In terms of cell size represented on forward scatter area density plot (FSC-A,) a subset of PLD4+ B cells was comparable to CD38+ CD43+ plasmablasts. We considered PLD4+ large B cells as distinct blastic B cell populations and named them "PLD4+ blasts." Up to 90% of PLD4+ blasts were CD11c+ and CXCR5lo DN2, but few were plasmablasts. The frequencies of PLD4+ blasts significantly correlated with those of DN2 (spearman = +0.79, P < 1.0 × 105). When divided into four compartments by PLD4 and FSC-A (PLD4+/- and blast/small), PLD4+ blasts were exclusively and highly positive for intracellular T-bet expression ( >75% in PLD4+ blast, < 50% in the other compartments, n = 2). In some cases of new-onset SLE, PLD4+ blasts significantly decreased after immunosuppressive therapy (5.0% vs 2.0%, P < 1.0×104, paired t-test). Finally, when sorted and cultured in vitro with TLR 7 or 9 ligands, PLD4+ blasts differentiated into ASCs secreting comparable or higher levels of IgG than whole CD19+ B cells.
Conclusion: PLD4+ B cells are expanded in SLE and those blastic highly overlap with DN2. Targeting cell surface PLD4 could be a novel therapeutic strategy for SLE. The prevalence of PLD4+ B cells in CD19+ B cells was compared between individuals with SLE and healthy donors. SLE showed significant expansion of PLD4+ blasts.
Representative figures of flowcytometry on SLE B cells. A) “PLD4+ blasts” expanded in SLE are as large as plasmablasts, but include few plasmablasts. B) PLD4+ blasts are mostly composed of DN2 B cells. C) The frequencies of DN2 and PLD4+blasts are significantly correlated.
The prevalence of PLD4+ blasts was measured before and after immunosuppressive treatment in 11 new-onset SLE Patients. PLD4+ blasts significantly decreased by treatment. Disclosures: K. Yasaka, None; T. Yamazaki, SBI Biotech Co., Ltd.; H. Sato, None; T. Shirai, None; H. Fujii, None; T. Ishii, None; H. Harigae, None.