Journal: Transplantation

Article Title: Differential Regulation of T Cell Immunity and Tolerance by Stromal Laminin Expressed in the Lymph Node

doi: 10.1097/TP.0000000000002774

Figure Lengend Snippet: Laminin α5 and laminin α4 expression differentially regulated in the lymph node following the induction of inflammation, immunity or tolerance. A, Skin LNs from naive, CFA, DST, DST+anti-CD40L and anti-CD40L treated C57BL/6 mice isolated 5 hours after treatment. B, Skin draining and non-draining LNs distal to the s.c. injection site of MC38 tumor cells isolated 48 hours after injection. C, Mesenteric LNs from untreated or DSS treated colitic mice isolated 24 hours after treatment. All LNs sectioned and stained for laminin α4, laminin α5, PNAd and ER-TR7. Representative images, 100X. Percentages of laminin α4 and laminin α5 positive areas, and the ratios of laminin α4: laminin α5 in the cortical ridge (CR) and high endothelial venules (HEV). Data shown as mean ± SEM. n = at least six mice per condition from three independents experiments; 1–4 LNs per mouse, 30–100 HEVs per LN.

Article Snippet: 10 On the day of transplantation, mice were treated with anti-integrin α6 mAb (clone GoH3, 10μg i.v, Biolegend, San Diego, CA) or isotype control (rat IgG2a), anti- α dystroglycan mAb (clone IIH6C4, 10 μg i.v, Merck Millipore, Darstadt, Germany) or isotype control IgM associated or not with anti-CD40L mAb (clone MR1, 0.25 mg i.v., Bio X Cell, West Lebanon, NH).

Techniques: Expressing, Isolation, Injection, Staining

Journal: Transplantation

Article Title: Differential Regulation of T Cell Immunity and Tolerance by Stromal Laminin Expressed in the Lymph Node

doi: 10.1097/TP.0000000000002774

Figure Lengend Snippet: Blocking laminin 511 receptor α6 integrin and αDG prolongs allogenic cardiac graft survival. C57BL/6 mice received BALB/c heterotopic cardiac allografts on day 0 along with anti-α6 integrin or isotype control mAb alone (10 μg i.v, day 0 and every 3 days) (A), anti- αDG or isotype control mAb alone (B), anti-α6 integrin or anti- αDG (10 μg i.v, day 0 and once a week) with anti-CD40L (250 μg i.v. day 0 only) (C). Data presented as percent survival. n = at least five mice per group.

Article Snippet: 10 On the day of transplantation, mice were treated with anti-integrin α6 mAb (clone GoH3, 10μg i.v, Biolegend, San Diego, CA) or isotype control (rat IgG2a), anti- α dystroglycan mAb (clone IIH6C4, 10 μg i.v, Merck Millipore, Darstadt, Germany) or isotype control IgM associated or not with anti-CD40L mAb (clone MR1, 0.25 mg i.v., Bio X Cell, West Lebanon, NH).

Techniques: Blocking Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A) Bright field images (400×) of mature DC1 or DC2 co-cultured in the presence of SEB alone (left panels) or with CD4+ T cells (middle panels), or rhCD40L (right panels) for 24 h. The arrows highlight TNT-like extensions. (B) 24 h co-cultures of SEB Ag-presenting DC1 and CD4+ T cells in the presence of control mAb (top) or CD40L blocking mAb (bottom). (C) Representative Z-series projection image revealing a network of TNT-like membrane connections in fluorescently labeled DC1. (A-C) Images are representative of 6 independent experiments conducted on DC from 3 healthy donors. (D) Representative Z-series projection images of a CD40L-activated DC1 analyzed using the IMARIS ‘Surfaces’ program to determine total cell surface area (left panel), and IMARIS ‘FilamentTracer’ to trace the pathway of each ‘filament’ branch extending from a single origin (blue sphere) at the edge of the cell body (middle panel). Individual segments begin at an origin or branch point (orange spheres) and end at the next branch or terminal point (green spheres). Spatial ‘reach’ was defined as the shortest distance from a filament origin to the distal terminal point of that filament (right panel; white lines connecting 2 points). (E) Total cell surface area comparisons of resting and CD40L-activated DC1 and DC2. (F) Comparison of percentage ‘TNT positive’ resting DC1 and DC2 (media only), defined as those expressing ≥5 individual TNTs per cell that were each >5.0 μm in length. (G) Comparison of percentage ‘reticulation positive’ CD40L-activated DC1 and DC2, delineated as those displaying ≥5 filaments per cell that were each >10.0 μm in sum segment length. (H) ‘Maximum reach’ of filaments, defined as the shortest distance (μm) from the origin to the farthest terminal point of a filament. (E-H) Data were generated from randomly chosen image fields (20–30 per donor) and represented as mean ± SD of 3 healthy donors independently tested. P-values <0.0001, <0.001, <0.01 and <0.05 are represented by ****, ***, **, and *, respectively. See also Supplemental Fig. 1; Video 1; Video 2.

Article Snippet: When used, CD40L blocking mAb (Enzo Life Sciences) or control mAb (BD Biosciences, San Jose, CA) were added to the cultures.

Techniques: Cell Culture, Blocking Assay, Labeling, Expressing, Generated

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A–C) 3D reconstruction images (600×) of differentially matured DC stimulated for 20 h with rhCD40L prior to labeling the cell surface with MHC class I mAb (green) and labeling nuclei (blue), followed by live-cell confocal imaging. Data are representative of 6 independent experiments conducted using DC from 3 healthy donors. (A) Membrane morphologies of mature CD40L-activated DC0, DC1, and DC2 propagated using LPS alone, LPS + IFN-γ, or LPS + PGE2, respectively. (B) Mature CD40L-treated DC0, DC1, and DC2 generated by the respective use of R848 alone, R848 + IFN-γ, or R848 + PGE2. (C) Cell morphologies of DC1 treated with media alone or CD40L. DC1 were generated using the αDC1 cytokine-based cocktail (first two panels on left), or induced by 48 h co-culture of iDC with either 2-signal activated NKh cells (middle) or SEB-activated CD8+ T cells (right).

Article Snippet: When used, CD40L blocking mAb (Enzo Life Sciences) or control mAb (BD Biosciences, San Jose, CA) were added to the cultures.

Techniques: Labeling, Imaging, Generated, Co-Culture Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A) Sequential still frames of DC1 actively reticulating from 4 to 7.6 h post-addition of rhCD40L, captured by live-cell, time-lapse DIC imaging (600×). See also Video 3. (B) Sequential frames of high resolution, time-lapse DIC imaging (600×) of live 8 h rhCD40L-stimulated DC1 showing endogenous cell structures resembling vesicles (arrows) trafficking between neighboring cells through CD40L-induced TNTs. See also Video 4. (C) Confocal reconstruction images (1000×) revealing early endosome- (green, arrows) and F-actin-containing TNTs (red) and nuclei (blue) in fixed CD40L-activated DC1. Data are representative of 3 donors independently tested.

Article Snippet: When used, CD40L blocking mAb (Enzo Life Sciences) or control mAb (BD Biosciences, San Jose, CA) were added to the cultures.

Techniques: Imaging

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A–C) Cy5-labeled (blue), YG latex bead (40 nm)-containing ‘donor’ DC1 or DC2 were co-cultured with the respective non-bead-containing, Cy3-labeled (red) ‘recipient’ DC type for 20 h in the presence or absence of rhCD40L. Data are representative of 2 donors independently tested. (A) Montage of time-lapse, confocal imaging (600×) showing beads (green; arrows) moving rapidly through donor DC1 TNTs (blue) over a time span of 28 min. See also Video 5. (B) Montage tracing the pathway of beads (green; arrows) traveling from the cell body of donor DC1 (blue), through donor cell TNTs and into the connected recipient DC (red), where they finally collect in the recipient cell body (yellow, lower left arrows). (C) Flow cytometric analysis and quantification of intercellular exogenous bead transfer from donor to recipient DC types after treatment with media only or CD40L. (D) IFNγ-ELISPOT assays measuring Ag-specific recall responses of cultured T cells following their in vitro sensitization with FACS sorted, YG bead/Ag positive and negative recipient DC1. Data are represented as mean ± SE of 2 independent experiments, and * represents the p-value <0.05. See also Supplemental Fig. 3.

Article Snippet: When used, CD40L blocking mAb (Enzo Life Sciences) or control mAb (BD Biosciences, San Jose, CA) were added to the cultures.

Techniques: Labeling, Cell Culture, Imaging, Enzyme-linked Immunospot, In Vitro

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

doi: 10.4049/jimmunol.1401832

Figure Lengend Snippet: (A) DC1 monolayers that had been stimulated with rhCD40L for 10 h were exposed to EGFP-expressing Escherichia coli for 2 h. Subsequent time-lapse confocal resonant scanning microscopy shows individual bacterium (green) trafficking bi-directionally along CD40L-induced TNTs (arrows) between mature DC1. See also Video 6. (B) Donor DC1 (blue) pulsed with EGFP-expressing HIV-1-like particles (green) were co-cultured with recipient DC1 (red) in the presence of CD40L for 10 h prior to live-cell imaging. HIV-1-like particles localizing to a donor cell nanotube (blue; arrows) that has formed a connection with a recipient cell body. (C) At 20 h post-CD40L stimulation, HIV-1-like particles can be detected at the interface between blue donor cell TNTs and red recipient cells (arrows), prior to their subsequent transfer to recipient cell bodies. (A-C) Imaging data are representative of 3 donors independently tested.

Article Snippet: When used, CD40L blocking mAb (Enzo Life Sciences) or control mAb (BD Biosciences, San Jose, CA) were added to the cultures.

Techniques: Expressing, Microscopy, Cell Culture, Live Cell Imaging, Imaging

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: List of SPD-CD40L fusions generated for this publication.

Article Snippet: The bound CD40L was detected by adding a non-competitive anti-human CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Generated

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: GS542–CD40L fusions are expressed and bind both CD40 and PDL1. Culture medium of HeLa cells infected/transfected by plasmids encoding the indicated GS542–CD40L fusions (also named sdAb–CD40L) were analyzed for transgene expression by immunoblot using an HRP-conjugated anti-FLAG tag monoclonal antibody (A) ; the ability of the expressed fusion to bind simultaneously CD40 and PDL1 proteins by ELISA (B) . SdAb–CD40L and sdAb–link–CD40L refer to the fusion of GS542 sdAb directly to the CD40L ectodomain or through a (GGGS) 3 linker, respectively.

Article Snippet: The bound CD40L was detected by adding a non-competitive anti-human CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Infection, Transfection, Expressing, Western Blot, FLAG-tag, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: GS542–linker–TNFSF fusions are potent TNFRSF agonists in the presence of PDL1 + cells only. Culture medium of HeLa cells either infected by the indicated recombinant virus (B) or infected/transfected by indicated plasmids (A, C) encoding different GS542–TNFSF fusions were analyzed for their CD40 agonist activity using InvivoGen HEK-blue CD40L cells (A, B) or for their 4-1BB agonist activity using Promega 4-1BB effector cells (C) . SdAb–CD40L and sdAb–link–CD40L refer to the fusion of GS542 sdAb directly to the CD40L ectodomain or through a (GGGS) 3 linker, respectively. Accordingly, VV–sdAb–link–CD40L is a vaccinia virus encoding the sdAb–link–CD40L construct. SdAb–link–4-1BBL refers to the fusion of GS542 sdAb to the 4-1BBL ectodomain through a (GGGS) 3 linker.

Article Snippet: The bound CD40L was detected by adding a non-competitive anti-human CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Infection, Recombinant, Virus, Transfection, Activity Assay, Construct

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: The CD40 agonist activity of sdAb–linker–CD40L is inhibited by coincubation with a PDL1 blocking antibody, and sdAb–linker–CD40L has a PD1/PDL1 blocking activity. CD40 agonist activity of culture supernatants of VV–sdAb–linker–CD40L-infected cells was analyzed, as shown in , in the presence or absence of 100 μg/mL of PDL1 blocking antibody (avelumab). In the case of the addition of avelumab, or of its isotype control, only undiluted supernatants were tested (A) . The PD1/PDL1 blocking activity of the culture medium of A549 cells infected by the mentioned virus was measured, as shown in (B) . VV–sdAb–link–CD40L is a vaccinia virus encoding the sdAb–link–CD40L construct. VV–scDimeric: VV encoding single-chain dimeric sdAb GS542; VV–monomeric: VV encoding monomeric sdAb GS542; and VV–avelumab: VV encoding avelumab.

Article Snippet: The bound CD40L was detected by adding a non-competitive anti-human CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Activity Assay, Blocking Assay, Infection, Control, Virus, Construct

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: SP-D–CD40L fusions are all expressed at approximately the same level and bind CD40 in vitro . The culture medium of HeLa cells infected/transfected by plasmids encoding the indicated SP-D–CD40L fusions were analyzed for transgene expression by immunoblot, in both non-reducing and reducing conditions, and using an HRP-conjugated anti-FLAG tag monoclonal antibody (A) ; the ability of the expressed fusions to bind immobilized CD40 was assessed by ELISA (B) . Blue arrows, closed, and open red arrowheads indicate monomers, trimers, and multimers, respectively (A) . Full length is the fusion of the entire human SP-D deleted of the CRD and fused to the human CD40L ectodomain. 12R COL is the fusion of oligomeric domains of human SPD containing only 12 collagen repeats and fused to the CD40L ectodomain. The 12R COL link is the same construct as previously described but with a (GGGS) 3 linker between the C-terminus of the collagen domain and the N-terminus of the CD40L ectodomain. OR COL and OR COL link are the same constructions described previously except that they are lacking any collagen domain (zero collagen repeat). CD40L is a construction encoding the human CD40L ectodomain. For details on constructions, refer to and Materials and methods.

Article Snippet: The bound CD40L was detected by adding a non-competitive anti-human CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: In Vitro, Infection, Transfection, Expressing, Western Blot, FLAG-tag, Enzyme-linked Immunosorbent Assay, Construct

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: SP-D–CD40L fusions with shortened collagen domains are potent CD40 agonists. Culture medium of HeLa cells either infected by the indicated recombinant virus (B) or infected/transfected by indicated plasmids (A) encoding the different SP-D–CD40L fusions was analyzed for their CD40 agonist activity using InvivoGen HEK-blue CD40L cells (A, B) . Full length is the fusion of the entire human SP-D deleted of the CRD and fused to the human CD40L ectodomain. 12R COL is the fusion of oligomeric domains of human SPD containing only 12 collagen repeats and fused to the CD40L ectodomain. 12R COL link is the same construct as described previously but with a (GGGS) 3 linker between the C-terminus of the collagen domain and the N-terminus of the CD40L ectodomain. OR COL and OR COL link are the same constructions described previously except that they are lacking any collagen domain (zero collagen Repeat). CD40L is a construction encoding the human CD40L ectodomain. For details on constructions, refer to and Materials and methods.

Article Snippet: The bound CD40L was detected by adding a non-competitive anti-human CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Infection, Recombinant, Virus, Transfection, Activity Assay, Construct

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus

doi: 10.3389/fbioe.2023.1247802

Figure Lengend Snippet: SP-D–4-1BBL fusion is expressed at approximately the same level as 4-1BBL but is a much better 4-1BB agonist. Culture medium of HeLa cells infected/transfected (no recombinant virus was generated) by plasmids encoding SP-D–4-1BBL or –4-1BBL was analyzed for transgene expression by immunoblot using an HRP-conjugated anti-FLAG tag monoclonal antibody (A) and their 4-1BB agonist activity using Promega 4-1BB reporter cells (B) . 12R COL–4-1BBL is the fusion of oligomeric domains of human SPD containing only 12 collagen repeats and fused to the 4-1BBL ectodomain. 4-1BBL is a construction encoding human CD40L ectodomain. Irr. refers to a plasmid coding an irrelevant protein. For details on constructions, refer to and Materials and methods.

Article Snippet: The bound CD40L was detected by adding a non-competitive anti-human CD40L monoclonal antibody (Bio-Rad, MCA1561) at 1 μg/mL in saturation buffer.

Techniques: Infection, Transfection, Recombinant, Virus, Generated, Expressing, Western Blot, FLAG-tag, Activity Assay, Plasmid Preparation

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