cd40l blocking agent  (Bio X Cell)

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

Journal: Nature Communications

Article Title: A hepatic network of dendritic cells mediates CD4 T cell help outside lymphoid organs

doi: 10.1038/s41467-024-45612-5

Figure Lengend Snippet: a Comparison of the total number of intrahepatic effector gBT-1 T cells at 14 days post-rAAV treatment in recipient mice treated with anti-CD4 depleting, anti-CD40L blocking Ab, or isotype control Ab. b Comparison of the total number of intrahepatic effector gBT-1 T cells at 15 days post-rAAV treatment of recipient mice treated with isotype control, or CD40L blocking Abs beginning day 0 or day 9 post-rAAV. c Comparison of the total number of intrahepatic effector gBT-1 T cells at 15 days post-rAAV treatment of CD4-depleted recipient mice administered agonist anti-CD40 mAb or isotype control 9-days post-rAAV. d Representative IF image of a portal tract cluster containing transferred gBT-1 and P25 T cells interacting with MHCII high cells in the liver 12-days post-rAAV treatment of recipient mice. Data are representative of two independent experiments. n = 4 ( a ) or n = 5 ( b ), n = 4 or 5 ( c ), n = 3 mice per group. Error bars indicate ± SEM. Analysed with one-way ANOVA with Tukey’s multiple comparisons test ( a , b ), or a two-tailed Student’s t test (c). Scale bars, 40 μm/20 μm ( d ). Source data are provided as a Source Data file.

Article Snippet: To block CD40L in vivo, mice were injected i.v. with 500 μg MR1 anti-mouse CD40L blocking mAb (BioXcell, West Lebanon, USA) or polyclonal Armenian hamster IgG isotype control (BioXcell, West Lebanon, USA) in PBS at the indicated time points.

Techniques: Comparison, Blocking Assay, Control, Two Tailed Test

Journal: Nature Communications

Article Title: A hepatic network of dendritic cells mediates CD4 T cell help outside lymphoid organs

doi: 10.1038/s41467-024-45612-5

Figure Lengend Snippet: a – c Representative confocal IF images of the liver of XCR1 Venus/+ rAAV-treated recipient mice at 12-days post rAAV, showing large clusters of XCR1 + cDC1s, gBT-1 and P25 T cells in PT regions Scale bars 50 μm ( a ), 30 μm ( b ), 4 μm/2 μm ( c ). d , e Quantification of the average distance (μm) separating XCR1 + cells with gBT-1 T cells and ( d ), or with P25 T cells ( e ) in portal tracts (PT), peri-central vein (PCV) and sinusoidal (S) liver regions. Data points represent a single region of interest from n = 7 mice from 2 independent experiments. f , g Hepatic XCR1 + cDC1 isolated from recipient mice treated, 12-days prior, with rAAV gB-Ag85b or PBS, were assessed for their ability to activate and induce proliferation (assessed by CD44 upregulation and CTV dilution), of naive gBT-1 ( f ) or P25 ( g ) T cells co-cultured for 72 h. h , i Same as ( f , g ) but recipient mice were also treated with isotype control (black) or anti-CD40L blocking mAb (red) to assess the role of CD40/CD40L interactions. Representative flow plots ( h ), and proportion of proliferating CD44 high CTV low gBT-1 T cells ( i ). j Co-stimulatory molecule expression levels in hepatic cDC1s isolated from rAAV gB-Ag85b –treated mice that also received isotype control (black) or anti-CD4 depleting mAb (red) at 12-days post rAAV. k , l Quantification of Ki67 + gB T-1 T cell numbers forming clusters in PT ( k ) and PCV regions ( l ) at 12-days post-rAAV treatment in XCR1 +/+ (black) and XCR1 DTR/+ (red) recipient mice treated with DTx, 9-days prior. m , n Quantification of total numbers of intrahepatic ( m ) and splenic ( n ) effector gBT-1 cell in recipient mice treated with rAAV, 15-days prior. Comparison between diphtheria toxin treated XCR1 +/+ (black) and XCR1 DTR/+ (red) recipient mice at 9-days post-rAAV.Data representative of two independent experiments. n = 5 ( a – c ), n = 7 ( d , e ), n = 3 or 4 ( f , g ), n = 7 ( i ), n = 4 ( j ) n = 5 ( k , l ) and n = 6 ( m , n ) mice per group. Error bars indicate ± SEM. Analysed with a Kruskal-Wallis test with multiple comparisons ( d , e ), a two-tailed unpaired Student’s t test ( i , j ) or a two tailed unpaired Mann–Whitney U test ( k – n ); ns not statistically significant. Source data are provided as a Source Data file.

Article Snippet: To block CD40L in vivo, mice were injected i.v. with 500 μg MR1 anti-mouse CD40L blocking mAb (BioXcell, West Lebanon, USA) or polyclonal Armenian hamster IgG isotype control (BioXcell, West Lebanon, USA) in PBS at the indicated time points.

Techniques: Isolation, Cell Culture, Control, Blocking Assay, Expressing, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: IL-21 shapes germinal center polarization via light zone B cell selection and cyclin D3 upregulation

doi: 10.1084/jem.20221653

Figure Lengend Snippet: IL-21 regulates light zone GC B cell selection. (A) Wild-type (WT) mice were immunized with SRBC and either treated with blocking anti-CD40L antibody (MR1; 200 μg/i.p. injection on days 5 and 6 after immunization) or received no further treatment (Control). Collated data (left) and representative flow cytometry plots (right) for c-Myc+BATF+IRF4+ splenic light zone (LZ; CXCR4 low CD86 high ) GC B cell (CD19+BCL6+) frequencies 7 d after immunization; color scales define IRF4 expression levels in each cell. (B) WT mice were immunized with SRBC and either treated with agonistic CD40 antibody (FGK4.5; 50 μg i.p on day 5 after immunization) or received no further treatment (Control). Collated data (left) and representative flow cytometry plots (right) for c-Myc+BATF+IRF4+ splenic light zone GC B cell frequencies 6 d after immunization; color scales define IRF4 expression levels in each cell. (C) p-S6 expression was analyzed in splenic GC from SRBC-immunized WT mice 6 d after immunization. Representative histograms showing p-S6 expression mean fluorescence intensity (MFI) in c-Myc+BATF+IRF4+ and c-Myc−BATF−IRF4− light zone GC B cells compared to follicular B cells (CD19+IgD+BCL6−; left). Collated data for p-S6 expression in c-Myc+BATF+IRF4+ and c-Myc−BATF−IRF4− light zone and all dark zone (DZ; CXCR4 high CD86 low ) GC B cells (right). Data are collated from two to four independent experiments; n = 4–14; Mann–Whitney U test or Kruskal–Wallis test. (D) CD19+ cells were isolated from spleens of 10-wk-old WT BALB/c mice using MACS positive selection. Cells were stimulated using 0–60 ng/ml of IL-21 and 20 μg/ml of anti-CD40 mAb for 24 h. Collated data and representative histograms for p-S6 median fluorescence intensity. One-way ANOVA; n = 4. (E) Representative t-SNE plots showing expression of c-Myc, BATF, and IRF4 in concatenated CTLA-4−/− and IL-21R−/−CTLA-4−/− light zone GC B cells; color scales define protein expression levels in each cell. (F) Collated data for c-Myc+BATF+IRF4+ splenic light zone GC B cell frequencies in 17–21-d-old CTLA-4−/− and IL-21R−/−CTLA-4−/− mice. Data are collated from five independent experiments; n = 8–9; Mann–Whitney U test. Mean ± SD are shown; ****, P < 0.0001; **, P < 0.01; *, P < 0.05.

Article Snippet: When indicated, anti-CD40L blocking antibodies (clone MR-1; Bio X Cell) were injected i.p. 5.5 and 6.5 d after SRBC immunization (200 μg per injection), and agonistic anti-CD40 antibodies (clone FGK4.5; Bio X Cell) were injected 5 d after SRBC immunization (50 μg per injection).

Techniques: Selection, Blocking Assay, Injection, Control, Flow Cytometry, Expressing, Fluorescence, MANN-WHITNEY, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Reduced Satb1 expression predisposes CD4 + T conventional cells to Treg suppression and promotes transplant survival

doi: 10.1073/pnas.2205062119

Figure Lengend Snippet: Differential expression of Satb1 in TCR75 T cells from tolerant and rejecting mice. ( A ) Experimental design. Congenic TCR75 CD4 + T cells seeded 1 d before transplantation were sorted on day 35 posttransplantation from spleen and lymph nodes (LNs) or allografts of tolerant or rejecting mice and RNA-seq was performed. ( B ) Principal component analysis. ( C ) Heatmap of differentially expressed transcription factors (fold change ≥ 1.5; adjusted P ≤ 0.05). Color key is normalized log2 transformed counts per million (CPM). n = 3 (memory: TCR75 T cells from 1 mouse/replicate; tolerant: pooled from 5 mice/replicate, except for tolerant allograft n = 1 with TCR75 T cells pooled from 15 allografts). ( D and E ) TCR75 T cells were seeded into host B6 mice 1 d before transplantation with B/c heart ±donor splenocytes and anti-CD154. Mice were euthanized at induction of memory and tolerance (day 3 ± 1 and day 12 ± 2) and Satb1 protein expression (MFI) was measured by flow cytometry of TCR75-gated T cells in spleen ( D ) and allograft ( E ). Naive group in ( D and E ) is the same and shows Satb1 expression of splenic TCR75 T cells before transfer. ( F ) Congenic OTII T cells were seeded 1 d before transplantation of 2W-OVA-expressing skin grafts, and hosts were treated or not with anti-CD154 and 2W-OVA-expressing splenocytes. OTII cells were sorted from spleen on d35 posttransplantation and analyzed by flow cytometry for expression of Satb1. All results are displayed as mean +/− SEM. n = 3 to 9/group ( D ), n = 3 to 5/group ( E ) and n = 3 to 4/group ( F ). * P < 0.05; ** P < 0.01 (ANOVA, two-way for D , one-way for E and F , with pairwise comparisons).

Article Snippet: For induction of tolerance, heart recipients received an intravenous injection of splenocytes from 1/4 of donor spleen on day 0 and were treated with 600 μg of anti-CD154 (MR1) blocking mAb (Bio-X-cell) on days 0 (intravenous), 7 (intraperitoneal), and 14 (intraperitoneal) postheart transplantation.

Techniques: Quantitative Proteomics, Transplantation Assay, RNA Sequencing, Transformation Assay, Expressing, Flow Cytometry