Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (A–C) Flow cytometry dot plots showing gating of peripheral blood B cell subsets. (D and E) CD45 phosphatase activity (D) and panCD45 surface expression (E) in human MBC and ASC subpopulations (red) compared to naive B cells (blue). Numbers in the histograms represent MFI ratio relative to naive B cells. (F and G) Graphs show pCAP-SP1 (F) and panCD45 surface expression (G) MFI values relative to naive B cells (n= 12). (H) Human BM CD19 − CD38 hi CD138 + (left panel) and CD19 + CD138 +/− CD38 + (right panel) previously shown to contain LLPCs and SLPCs, respectively. (I) CD45 phosphatase activity (left panel), CD45 surface expression (middle panel), and BLIMP1 expression (right panel) in LLPCs (red) and SLPCs (blue) relative to mature BM B cells (CD19 + SSC lo CD45 hi ) obtained from the same donor (gray). (J) Graphs show fold change in MFI of CD45 phosphatase activity (left panel) and CD45 surface expression (right panel) from human BM PCs relative to mature BM B cells from the same healthy donor (n = 4). Related to . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: FITC Rat monoclonal anti-mouse CD38 (clone 90.4) , Miltenyi Biotec , Cat # 130–102-514; RRID:AB_2657875.

Techniques: Flow Cytometry, Activity Assay, Expressing

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (A) Flow cytometry plots show gated CD38 hi CD138 hi 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 % CD38 hi CD138 hi 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 . *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: FITC Rat monoclonal anti-mouse CD38 (clone 90.4) , Miltenyi Biotec , Cat # 130–102-514; RRID:AB_2657875.

Techniques: Flow Cytometry, Activity Assay, Expressing, Purification

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (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 + SSC lo -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 . *p < 0.05, **p < 0.01.

Article Snippet: FITC Rat monoclonal anti-mouse CD38 (clone 90.4) , Miltenyi Biotec , Cat # 130–102-514; RRID:AB_2657875.

Techniques: Binding Assay, Expressing, Activity Assay, Staining

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (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 (CD138 hi CD38 hi ) (red) backgated to CD45 activity hi Galectin-1 hi 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-1 hi 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-1 hi (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 and . *p < 0.05, **p < 0.01.

Article Snippet: FITC Rat monoclonal anti-mouse CD38 (clone 90.4) , Miltenyi Biotec , Cat # 130–102-514; RRID:AB_2657875.

Techniques: Flow Cytometry, Staining, Control, Activity Assay, Expressing, Fluorescence, CRISPR, Knockdown

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: FITC Rat monoclonal anti-mouse CD38 (clone 90.4) , Miltenyi Biotec , Cat # 130–102-514; RRID:AB_2657875.

Techniques: Negative Control, Recombinant, Purification, Staining, Gene Expression, Lysis, Immunoprecipitation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Cell Isolation, Software, Microscopy