Journal: Inflammation and Regeneration

Article Title: Evaluation of immunosuppression protocols for MHC-matched allogeneic iPS cell-based transplantation using a mouse skin transplantation model

doi: 10.1186/s41232-021-00190-7

Figure Lengend Snippet: Blockade of leukocyte costimulatory molecules permits long-term engraftment of mouse skin grafts from MHC-matched allogeneic donors. ( A ) CB-based immunoregulation protocol. Immunosuppression was performed by using a protocol of administrating a combination of anti-CD40L mAb and CTLA4-Ig daily (CB) with or without rapamycin on every 3 days from POD 1 (CB + rapa). ( B ) Skin graft survival under immunosuppression with CB ( n = 9, C3129F1; n = 9, BALB/c; n = 9, C57BL/6; n = 9, CBA/N). ( C ) Skin graft survival under immunosuppression with CB + rapa ( n = 9, C3129F1; n = 8, BALB/c; n = 9, C57BL/6; n = 9, CBA/N). ( D ) Macroscopic observation (upper panels) and hematoxylin and eosin stained section (lower panels) of autologous (C3129F1), CBA/N, and C57BL/6 skin grafts on day 100. Scale bars: 100 μm. ( E ) Immunohistochemical staining for CD3 of C57BL/6 skin grafts harvested from CB or CB + rapa treated groups on 100 days post-transplantation. Upper and lower panels represent CD3 specific staining and isotype control stained section, respectively. Scale bars: 100 μm. ( F ) Recipient T cell response in MHC-matched but minor antigen-mismatched skin transplantation. The T cell proliferation rates in each treatment group were normalized to that of C3129F1 mice stimulated with autologous irradiated splenocytes. Error bars indicate standard error of technical triplicates. Similar results were obtained in two independent experiments. * p < 0.05, ** p < 0.01 (Tukey’s HSD test). ( G ) De novo anti-donor antibody production in the recipients. Error bars indicate standard error of biological replicates ( n = 8, naive; n = 4, non-treatment; n = 9, CB; n = 9, CB + rapa). tx, transplantation; mAb, monoclonal antibody; CB, co-stimulatory molecule blocking; rapa, rapamycin; POD, post-operative day

Article Snippet: For co-stimulatory molecule blockade (CB) therapy, anti-CD40 ligand (CD40L) (MR-1 for mouse and #BE0292 for human, BioXcell) and CTLA4Ig (Orencia®, Bristol Myers Squibb for mouse and #BE0099, BioXcell for human) were administered at a dose of 500 μg for anti-CD40L, 400 μg for CTLA4Ig on days 0, 2, 4, and 6 after transplantation.

Techniques: Staining, Immunohistochemical staining, Transplantation Assay, Control, Irradiation, Blocking Assay

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: 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: PLoS ONE

Article Title: Bifidobacterium longum Alleviates Dextran Sulfate Sodium-Induced Colitis by Suppressing IL-17A Response: Involvement of Intestinal Epithelial Costimulatory Molecules

doi: 10.1371/journal.pone.0079735

Figure Lengend Snippet: (a) mRNA expression of costimulatory molecules in IECs was analyzed by quantitative PCR. Levels of mRNA were normalized to β-actin mRNA, and expressed relative to control mice. The data are representative of three experiments. Results are expressed as means ± standard error (n = 6 (Control) or 7 (DSS, and DSS+ B . l )). (b) IECs were stained for cytokeratin and CD80 or CD40 and analyzed by flow cytometry. Debris was gated out by forward and side scatter. Representative plots (upper panel) and the mean and standard error values of the percentage of cytokeratin/CD80 or CD40 positive cells (lower panel) are shown (n = 4). The data are representative of two experiments. (c) Cryosections of colonic tissue were labeled for CD40 (green) and E-cadherin (red). The data are representative of two experiments. Scale bars represent 50 μm. ** p <0.01 versus control mice (Control); # p <0.05 and ## p <0.01 versus DSS-treated mice (DSS).

Article Snippet: In some experiments, IECs and CD4 + T cells were pretreated with anti-CD40L blocking antibody (clone MR1; Abcam, Cambridge, UK), anti-CD80 blocking antibody (clone 1G10; Abcam) and/or anti-CD86 blocking antibody (clone GL1; Abcam) at 37°C for 30 min before co-culture, respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Labeling

Journal: PLoS ONE

Article Title: Bifidobacterium longum Alleviates Dextran Sulfate Sodium-Induced Colitis by Suppressing IL-17A Response: Involvement of Intestinal Epithelial Costimulatory Molecules

doi: 10.1371/journal.pone.0079735

Figure Lengend Snippet: (a) IECs from DSS mice were co-cultured with CD4 + T cells from control mice in the presence of anti-CD3 antibody. In the indicated groups, IECs were pretreated with CD80 or CD86 blocking antibodies, and CD4 + T cells were pretreated with CD40L blocking antibody before co-culturing. The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). ** p <0.01 versus CD4 + T cells alone (CD4 + T); ## p <0.01 versus co-culture of CD4 + T cells and IECs from DSS-treated mice (DSS-IEC). (b) IECs were cultured in the absence (gray bars) and presence (black bars) of CD40 agonist antibody for 24 h. IL-6 concentration in the supernatant was measured by ELISA. The data are representative of three experiments. (c) CD4 + T cells were cultured for 3 days in fresh medium or supernatant from IECs (IECs sup) cultured as described in (b) in the presence of anti-CD3/CD28 antibodies. The IL-17A concentration in the supernatant was measured by ELISA. The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). ** p <0.01 versus the supernatant from IECs stimulated with CD40 agonist antibody (CD40 agonist Ab). (d) CD4 + T cells were cultured with fresh medium or supernatant from CD40-stumulated IEC of DSS mice (DSS-IEC sup) as described in (c) in the presence of IL-6-neutralizing antibody (anti-IL-6) or IgG control antibody (isotype). The IL-17A concentration in the supernatant was measured by ELISA. Results are expressed as means ± standard error (n = 6). ** p <0.01 versus fresh medium; # p <0.05 versus the supernatant from CD40-stumulated IEC of DSS mice (DSS-IEC sup).

Article Snippet: In some experiments, IECs and CD4 + T cells were pretreated with anti-CD40L blocking antibody (clone MR1; Abcam, Cambridge, UK), anti-CD80 blocking antibody (clone 1G10; Abcam) and/or anti-CD86 blocking antibody (clone GL1; Abcam) at 37°C for 30 min before co-culture, respectively.

Techniques: Cell Culture, Blocking Assay, Co-Culture Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Bifidobacterium longum Alleviates Dextran Sulfate Sodium-Induced Colitis by Suppressing IL-17A Response: Involvement of Intestinal Epithelial Costimulatory Molecules

doi: 10.1371/journal.pone.0079735

Figure Lengend Snippet: (a) mRNA expression of CD80 and CD40 was analyzed by quantitative real-time PCR in Colon-26 cells after stimulation with IFN-γ. Levels of mRNA were normalized to β-actin mRNA, and expressed relative to before stimulation (0 h). Results are expressed as means ± standard error (n = 4). * p <0.05 and ** p <0.01 versus before stimulation (0 h). (b) Colon-26 cells were pre-incubated with B. longum JCM 1222 T , and then treated with penicillin and streptomycin and incubated with IFN-γ. Messenger RNA expression of CD80 and CD40 was analyzed after stimulation with IFN-γ for 6 and 72 h, respectively. The data are representative of three experiments. (c) Colon-26 cells were incubated with B. longum JCM 1222 T and IFN-γ. Cell surface protein expression of CD80 and CD40 was analyzed by flow cytometry after stimulation with IFN-γ. Debris was gated out by forward and side scatter. Histograms show a representative experiment (specific antibody stains are shown in filled histograms, and isotype control antibody stains are shown in open histograms) and bar figures are representative of two experiments. ** p <0.01 versus non-treated cells (Control); # p <0.05 and ## p <0.01 versus IFN-γ-stimulated cells.

Article Snippet: In some experiments, IECs and CD4 + T cells were pretreated with anti-CD40L blocking antibody (clone MR1; Abcam, Cambridge, UK), anti-CD80 blocking antibody (clone 1G10; Abcam) and/or anti-CD86 blocking antibody (clone GL1; Abcam) at 37°C for 30 min before co-culture, respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, RNA Expression, Flow Cytometry