Fig 2: The FBP–IL-27–COX-2 regulatory axis in decidua is insufficient in patients with unexplained RSA.(A) The mRNA levels of FBP1 and PFK1 between DSCs from normal pregnant women (n = 10) or patients with unexplained RSA (n = 10) were detected by RT-PCR. (B and C) Expression of FBP1 and PFK1 between decidua tissues from normal pregnant women (n = 12) or patients with unexplained RSA (n = 12) were detected by immunohistochemistry. (D) The FBP levels in the blood plasma from nonpregnant (NP) or pregnant (P) normal women or patients with unexplained RSA (n = 10 per group) were detected. (E) The mRNA level of IL-27 between DSCs from normal pregnant women (n = 10) or patients with unexplained RSA (n = 10) was detected by RT-PCR. (F and G) Expression of IL-27 between decidua tissues from normal pregnant women (n = 20) or patients with unexplained RSA (n = 20) was detected by immunohistochemistry. (H and I) The levels of COX-2 (H), CD80, CD86, INF-γ, CD206, CD209, and IRF4 (I) in dMφs from normal pregnant women (n = 12) or patients with unexplained RSA (n = 12) were detected by FCM. Data were presented as means ± SEM and analyzed by t test or one-way ANOVA test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3: CD45 phosphatase activity defines high-affinity germinal center (GC) PC precursors in a T cell-dependent vaccination system(A) Representative dot plots showing LN GC B cells (B220+GL7+CD38-) and high- and low-affinity HA-reactive cells; HA binding versus IgG expression is shown.(B) Histograms show CD45 activity (left) and IRF4 expression (right) in GC low- (blue-filled histograms) and high-affinity (red) B cells.(C) BM PCs gated as Blimp1+CD138+ cells (green) overlayed on histograms of low- (blue) and high-affinity (red) GC B cells showing CD45 activity (left) and Blimp1 (middle) and CD138 (right) expression levels.(D) Dot plots show BM PC gate from forward and side scatter (F/SSC)-gated cells (left) and from B220+SSClo-gated cells (right).(E) PCs were backgated on dot plots depicting CD45 activity and Blimp1 (left) and CD45 activity and IRF4 (right); PCs are depicted as red dots, BM B220+ B cells are represented in blue.(F) PCs (red) and B220+ B cells (blue) were analyzed for CD45 activity (left) and HA binding (right).(G and H) Graphs show MFI of CD45 activity of low-affinity (LoAff) and high-affinity (HiAff) GC B cells and BM PCs relative to B220+ B cells within the same sample (LN or BM).(I) Graph shows MFI ratio of HA normalized to IgG. Data are representative of two experiments including seven immunized mice in addition to immunization and staining controls.Related to Figure S6. *p < 0.05, **p < 0.01.

Fig 4: Differentiation of antibody-secreting cells by Th signals is CD45-dependent(A) Flow cytometry plots show gated CD38hiCD138hi ASCs (upper panel) and CD45 activity versus CD138 expression (lower panel) in the presence of Th signals and CD45 inhibitor as indicated.(B) Graph shows % CD38hiCD138hi ASC B cells (n= 8).(C) Histograms show the CD45-dependent regulation of ASC-associated markers and transcription factors. CTR+VEH (gray), CD40L+VEH (blue), CD40L+CD45 inhibitor (red).(D) Human peripheral B cells differentiated toward ASCs in the presence of CTR siRNA (upper panels) or CD45 siRNA (lower panels).(E) Graph shows % IRF4+BLIMP1+ ASCs upon delivery of CTR or CD45 siRNA (n = 5).(F) Flow cytometry histograms show expression of CD45 activity (upper left panel), CD45 surface expression (upper right panel), BLIMP1 (lower left panel), and IRF4 (lower right panel) in purified B cells differentiated toward ASCs in the presence of CTR siRNA (gray) or CD45 siRNA (blue).Related to Figure S3. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Galectin-1 enhances CD45 phosphatase activity and PC differentiation(A) Flow cytometry histogram showing surface staining of Galectin-1 in naive (gray), memory (blue), and PCs (red) in human peripheral blood B cells (left panel). Isotype control is shown by gray-dotted histogram. Graph shows fold increase in surface Galectin-1 staining (MFI) relative to naive B cells (n = 4). Contour plot depicts CD45 activity versus Galectin-1 surface staining in B cells (blue) and PCs (CD138hiCD38hi) (red) backgated to CD45 activityhiGalectin-1hi cells (right panel).(B) Histograms show CD45 activity (left panel), Galectin-1 staining (middle panel), and MEM-55 staining (right panel) in CTR- or NA-treated B cells. Graphs show fold increase in MFI of CD45 phosphatase activity (left lower panel) and Galectin-1 staining (right lower panel) relative to CTR-treated B cells (n = 6).(C) Upper panels: Dot plots show expression of IRF4 and BLIMP1 in naive B cells and MBCs differentiated toward ASCs in the presence of 10 and 100 µM OTX008 or VEH. Graph shows % IRF4+BLIMP1+ B cells in MBC cultures. Lower panels: Galectin-1 surface expression (left panel) and CD45 phosphatase activity (right panel) in VEH-treated (blue histogram) and OTX-treated (blue dotted histogram) MBCs. Graphs show MFI values of Galectin-1 staining (left) and CD45 activity (right) (n = 5).(D) Flow cytometry dot plots show CD45 activity versus Galectin-1 surface staining in the presence of medium or rhGAL-1 (left panels). Histogram shows CD45 phosphatase activity of Galectin-1hi B cells with rhGAL1 (blue) and total B cells (gray) (middle panel). Graph shows MFI values of CD45 activity in total B cells (gray histogram) and GAL-1hi (blue open histogram) B cells (right panel) (n = 6).(E) Representative localization of Galectin-1 relative to pSyk, pCAP-SP1, and CD45 in human B cells. The panels (left) present signals from the individual fluorescence detectors, and the center image is a merge of all four channels. The graph (right) shows the average (z axis) fluorescence intensity for each (x axis) pixel number in the indicated area (below graph and dotted area on center figure).(F) CoIP of CD45 of B cell lysates and immunoblotted with anti-CD45 (left) or anti-Galectin-1 (right).(G) Flow cytometry histogram showing CD45 phosphatase activity in gated live Raji B cells with CRISPR-Cas9 knockdown of CD45 (blue), Galectin-1 (green), and wild-type (red).Related to Figures S7 and S8. *p < 0.05, **p < 0.01.