cd40l blocking  (R&D Systems)

Journal: Nature

Article Title: Transient silencing of hypermutation preserves B cell affinity during clonal bursting

doi: 10.1038/s41586-025-08687-8

Figure Lengend Snippet: a , Proposed model for AID and/or SHM activity. b , Detecting CDK2 activity through subcellular translocation of a DHB reporter. c , Rosa26 DHB-tdTomato knock-in (KI) allele design. Asterisk denotes a silent mutation introduced to prevent sgRNA binding in the KI template. d , DHB–tdTomato (red) and H2B–GFP (green) in cultured GC B cells, with corresponding C/N ratios. Scale bars, 5 μm. e , Snapshots from time-lapse imaging of GC B cells in Nojima cultures expressing H2B–GFP (green) and DHB–tdTomato (red at the top, greyscale at the bottom; Supplementary Video ). Scale bar, 10 µm. f , CDK2 activity traces (grey) with mean ± s.d. (red). Cells treated with anti-CD40L blocking antibody (blue) provide a CDK2 low reference. g , Duration of the C/N ratio below 1.0 (S-phase entry) after anaphase. Each symbol represents one daughter cell. h , DHB–tdTomato (red) and H2B–GFP (green) in GC B cells. CD35 expression (yellow dotted line) delineates the LZ (Supplementary Video ). Arrowheads in insets highlight nuclear DHB–tdTomato in LZ cells and cytoplasmic expression in DZ cells. Scale bars, 50 µm (top); 20 µm (bottom). i , DHB–tdTomato (grey) after treatment with anti-DEC-OVA. Arrowheads highlight nuclear DHB–tdTomato at 72 h. Scale bars, 20 µm. j , Violin plot of CDK2 activity with box plot overlay (median, interquartile range, range). The grey box indicates the CDK2 low state (median of untreated LZ cells). Individual GC data are in Extended Data Fig. . k , Fraction of CDK2 low cells in paired LZ and DZ cells in untreated GCs and DZ B cells after treatment with anti-DEC-OVA. P values by Student’s t -test. l , Snapshots of DHB–tdTomato (red) and H2B–GFP (green) from intravital time-lapse imaging at 0 h or 36 h after treatment with anti-DEC-OVA (Supplementary Videos and ). Images are aligned to the time of anaphase, determined by sister chromatid separation (arrowheads). CDK2 activity is indicated for each daughter cell. Scale bars, 10 µm. m , In vivo CDK2 activity traces in DZ cells with or without treatment with anti-DEC-OVA, summarized with mean and s.d. Non-dividing LZ cells (blue) serve as a CDK2 low reference. P value by Student’s t-test.

Article Snippet: Anti-CD40L blocking antibody (25 μg ml −1 , Bio X Cell) was added as indicated.

Techniques: Activity Assay, Translocation Assay, Knock-In, Mutagenesis, Binding Assay, Cell Culture, Imaging, Expressing, Blocking Assay, In Vivo

Journal: bioRxiv

Article Title: Transfusion of allogenic murine HOD red blood cells preferentially induces low-affinity, short-lived IgG antibodies that are germinal center independent

doi: 10.1101/2025.01.16.633377

Figure Lengend Snippet: (A) Experimental approach for CD40L blocking experiments. WT mice were either transfused with HOD RBCs or vaccinated with HEL-OVA/Alum. They received CD40L blocking antibody 4 days after immunization and subsequently on days 7 and 10 pi. Sera were collected 2 weeks after immunization for assessment of IgG levels by limiting dilution ELISA. (B) IgG titers of transfused mice. (C) IgG titers of vaccinated mice. (D) Experimental approach for experiments that utilized BCL6-BKO mice. WT or BCL6-BKO mice were either transfused with HOD RBCs or vaccinated with HEL-OVA/Alum. Sera were collected 2 weeks after immunization for assessment of IgG levels by limiting dilution ELISA. (E) IgG titers of transfused mice. (F) IgG titers of vaccinated mice. Each data point represents on mouse. Bars on scatter plots are median values. Figure shows a representative experiment out of 3. Groups of interest were compared using Mann-Whitney U tests preceded by Kruskal-Wallis tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns P>0.5.

Article Snippet: For CD40L blocking experiments, mice were given 250 μg of the anti-mouse CD40L blocking antibody or isotype control via IP injection (BioXCell, BE0017-1 or BE0091) on days 4, 7, 10, and 14 post-immunization.

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY