Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Model of SPD-gp100-CD40L. Amino acids 25 to 596 (sequence KVPRNQD to EAGLGQV) of human gp100 were inserted between amino acids 105 and 106 of murine SPD within the SPD-CD40L fusion construct. (b) Cartoon of expected SPD-gp100-CD40L 4-trimer structure showing trimers of CD40L (gray circles) and trimers of gp100 (black circles) extending from the SPD collagen-like domain. (c) Western blot analysis. 293T cells were transfected with DNA plasmid encoding gp100 or SPD-gp100-CD40L fusion protein. After 48-hour culture, supernatant was collected and run on an SDS-PAGE gel in the presence of reducing agent. Western blot was performed using a polyclonal antibody to gp100.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Sequencing, Construct, Western Blot, Transfection, Plasmid Preparation, SDS Page

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Representative SEC elution profile of SPD-gp100-CD40L as monitored by the scattering intensity, expressed in terms of the excess Rayleigh ratio (R(θ)), at three scattering angles of 42° (tall gray line), 90° (black line) and 138° (short gray line) as a function of elution volume (V). Note that SPD-gp100-CD40L apparently elutes as a major species (megamer) and as a minor species (polymer, evident as a shoulder of the major elution peak). (b) Representative partial Zimm plot obtained from analytical SLS measurements at a specific protein concentration for the SPD-gp100-CD40L complex. The line through the data points—collected at three scattering angles of 42°, 90° and 138°—represents a linear fit. (c) Representative autocorrelation function plot obtained from analytical DLS measurements at a specific protein concentration for the SPD-gp100-CD40L complex. The line through the data points—collected at a scattering angle of 90°—represents non-linear least squares fit.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Polymer, Protein Concentration

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) In vitro activity of SPD-CD40L and SPD-gp100-CD40L was determined using a cell-based CD40 NF-κB enzymatic reporter system. An equivalent amount of 293T supernatant from pcDNA3.1, pSPD-CD40L or pSPD-gp100-CD40L transfected cells was incubated with 293-CD40-SEAP NF-κB reporter cells at various dilutions. (b) In vitro activity of supernatant from SPD-gp100-CD40L transfected 293T cells was evaluated on mouse bone marrow derived mouse DC and compared to supernatant from 293T cells transfected with empty vector pcDNA3.1. Error bars represent mean + SEM (n=3). * p<0.05, ** p<0.01 by Student’s t test compared to pcDNA3.1 supernatant.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: In Vitro, Activity Assay, Transfection, Incubation, Derivative Assay, Plasmid Preparation

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Immunization schedule for B16-F10 tumor challenge and DNA/GVAX therapeutic vaccination, as indicated by arrows. B16-F10 cells (50,000) were injected i.d. into the left flank of mice on day 0. Mice were then immunized by i.m. injection of PBS or pSPD-gp100-CD40L on day 3, 10, and 17. GVAX, B16-F10 tumor cells expressing GM-CSF, were irradiated at 5,000 rad and 1 × 106 cells injected subcutaneously on day 3, 6, and 9. (b) Tumor growth analysis. Each point represents the mean tumor volume per group (n=5). (c) Survival analysis was based on the date of death or when tumor size reached >1500 cm2.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Injection, Expressing, Irradiation

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Immunization schedule for B16-F10 tumor challenge and DNA or GVAX vaccination, as indicated by arrows. B16-F10 cells (50,000) were injected i.d. into the left flank of the mice on day 0. Mice were immunized i.m. with PBS, pSPD-gp100-CD40L + pIL-12, pSPD-gp100-CD40L + pGM-CSF, or pSPD-gp100-CD40L + pIL-12 + pGM-CSF on day 3, 10, and 17. For GVAX therapy, B16-F10 tumor cells expressing GM-CSF (GVAX), were irradiated at 5,000 rad and 1 × 106 cells were injected subcutaneously on day 3, 6, and 9. (b) Tumor growth analysis. Each point represents the mean tumor volume of animals in each group (n=5). (c) Survival analysis of mice. (d) Tumor growth kinetics of individual mice from each treatment arm.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Injection, Expressing, Irradiation

Journal: Vaccine

Article Title: A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

doi: 10.1016/j.vaccine.2015.07.081

Figure Lengend Snippet: (a) Immunization schedule for B16-F10 tumor challenge and DNA vaccination, as indicated by arrows. B16-F10 cells (50,000) were injected into the left flank of the mice on day 0. Mice were then immunized i.m. with PBS, pgp100, pgp100 + pIL-12, pgp100 + pGM-CSF, pgp100 + pIL-12 + pGM-CSF, pgp100-IRES-SPD-CD40L + pIL-12 + pGM-CSF, or pSPD-gp100-CD40L + pIL-12 + pGM-CSF on day 3, 10, and 17. (b) Tumor growth analysis. Each point represents the mean tumor volume of animals in each group (n=5). (c) Survival analysis of mice.

Article Snippet: Following electrophoresis, protein was blotted onto PVDF membrane (Pierce) by electrophoretic transfer, blocked, and probed with goat anti-mouse CD40L antibody (R&D Systems).

Techniques: Injection

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:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: Expression of murine CD40L by recombinant canarypox vector. Recombinant canarypox viruses vCPmCD40L and vCPmSP-D-CD40L were generated as described in Materials and Methods. (A) RT-PCR. CEF cells were infected by vCPmCD40L, vCPmSP-D-CD40L or its parental control ALVAC II at 5 MOI for 3 days. Total RNA was isolated from infected CEF cells using Trizol Reagent and subjected to RT-PCR with specific primers amplifying the coding sequence of murine CD40L or SP-D-CD40L. The identity of the amplified product was further verified by DNA sequencing (not shown). (B) Flow cytometry. Human PBMCs were infected by vCPmCD40L, vCPmSP-D-CD40L or ALVAC II at 5 MOI for 24 hrs and stained with PE-labeled anti-mouse CD40L mAb. (C) Western blot. Hela cells were infected by vCPmCD40L, vCPmSP-D-CD40L, or ALVAC II at 10 MOI for 24–48hrs. Cell lysates or magnetic beads incubated with supernatant of infected cells were subjected to Western blot and detected with a goat anti-mouse CD40L or anti-mouse SP-D antibody. As expected, the membrane CD40L was about 35–40kD and soluble multimeric form of CD40L was about 70kD.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Expressing, Recombinant, Plasmid Preparation, Generated, Reverse Transcription Polymerase Chain Reaction, Infection, Control, Isolation, Sequencing, Amplification, DNA Sequencing, Flow Cytometry, Staining, Labeling, Western Blot, Magnetic Beads, Incubation, Membrane

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus boosts the immunogenicity of an HIV-1 canarypox vaccine in mice. Female Balb/c mice of 6–8 wks age were immunized 3 times with an HIV-1 canarypox vaccine, vCP1452, with either of vCPmCD40L or vCPmSP-D-CD40L or ALVAC II. Six weeks after the last immunization, mice were sacrificed and spleens were taken and used for preparation of splenocytes. (A) IFN-γ ELISpot. Splenocytes were stimulated with P815 cells pulsed with an H-2Kd restricted HIV-1 Gag peptide, AMQMLKETI, for 16 hrs. Cells producing IFN-γ were detected and counted as described in Materials and Methods. (B) and (C) MHC-I tetramer analysis. Splenocytes were stained with anti-mouse CD8 mAb and AMQMLKETI/H-2Kd tetramer. (B) shows representative flow cytometry results for each group of mice. Numbers are percentage of tetramer positive cells in CD8+ cells. (C) shows summary of the tetramer data for each group of mice. (D) to (G) Polyfunctional CD8+ T cells analyzed by flow cytometry. Splenocytes were stained with anti-mouse IFN-γ and anti-mouse TNF-α mAbs and co-expressing cells are measured in (D) and (E) or anti-mouse IFN-γ and anti-mouse CD107a mAbs and co-expressing cells are measured in (F) and (G). (D) and (F) show representative flow cytometry results and (E) and (G) show summary of the polyfunctional CD8+ T cell data for each group of mice. (H) CD8+ T cells producing IFN-γ analyzed by flow cytometry. Splenocytes were stained with anti-mouse CD8 and anti-mouse IFN-γ mAbs. (I) and (J) Memory HIV-1-Gag specific CD8+ cells analyzed by flow cytometry. Splenocytes were stained with anti-mouse CD8, anti-mouse CD127 (IL-7Rα) mAbs, and AMQMLKETI/H-2Kd tetramer. In (I) CD8+/Tetramer+ cells are gated and measured by CD127 (y-axis). CD8+ T cells from naïve mice unstained for CD127 was used as control for CD127 gating. (J) shows summary data of CD127 expression in tetramer+ cells. (K) Lymphocyte proliferation. Splenocytes were stimulated with HIV-1 p24 Gag for 6 days and [3H]-thymidine incorporation was measured. (L) Cytokine production. Splenocytes were stimulated with HIV-1 p24 Gag for 5 days. IFN-γ and IL-4 in the supernatant were measured by ELISA. (M) Serum anti-Gag antibody. HIV-1 p24 Gag was used as antigen, and mice serum anti-Gag antibodies were measured by ELISA. Data shown are mean±SEM. n=4–10. *: P<0.05. **: P<0.01.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Immunopeptidomics, Enzyme-linked Immunospot, Staining, Flow Cytometry, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus enhances TNF-α and IL-12 production of human MDDCs. Human immature MDDCs were infected with recombinant ALVAC virus expressing human CD40L called vA3131-2, an ALVAC-HIV vaccine called vCP205, or a parental control, ALVAC II at an MOI of 10, or incubated with medium alone, or with CD40L trimer (2 μg/ml) as a positive control at 37°C for 24 h. The production of TNF-α and IL-12 was determined by intracellular staining flow cytometry. (A) Data are taken from the HIV-1-infected participant #1 and are representative of experiments with MDDCs derived from two HIV-1-infected and two HIV-1-uninfected individuals. The numbers in each gate represent the percentage of TNF-α or IL-12-positive MDDCs in total MDDCs. (B) Pooled data from all participants are shown. Data shown are mean±SEM. **: P<0.01.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Infection, Expressing, Control, Incubation, Positive Control, Staining, Flow Cytometry, Derivative Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus promotes human MDDCs maturation independent of TNF-α secretion. Immature MDDCs were infected with recombinant ALVAC virus expressing human CD40L, vA3131-2; an ALVAC-HIV vaccine, vCP205; or a parental control, ALVAC II at an MOI of 10, or incubated with medium alone at 37°C for 48 h. Expression of surface molecules was analyzed by flow cytometry. (A) Numbers in CD83 row represent percentage of CD83 positive cells in total MDDCs and numbers in CD86 and CD80 rows represent the mean fluorescence intensity of specific staining (histogram with solid line) subtracted from the value of background staining with matched isotype control mouse mAbs (dotted histogram). Data shown are from HIV-1-infected participant #1 and representative of six experiments performed with MDDCs obtained from three HIV-1-uninfected blood donors and three HIV-1-infected individuals. (B) Pooled data from all six participants studied are shown. Data shown are mean±SEM. **: P<0.01. (C) Immature human MDDCs were infected or not (medium) with vA3131-2 (CD40L expressing), vCP205, or ALVAC II at an MOI of 10 and then incubated in the presence of a blocking anti-human TNF-α Ab or the control isotype immunoglobulin (20 μg/mL). After 24 h incubation, MDDCs were collected and stained for CD83 expression to monitor DC maturation. Numbers in each dot plot represent percentage of CD83 positive cells in total MDDCs. Expression of CD83 on MDDCs treated with isotype immunoglobulin is similar to that on MDDCs treated with medium. Increasing the concentration of blocking anti-human TNF-α Ab up to 100 μg/mL did not further reduce the CD83 expression in all conditions (data not shown). The results represent one of five experiments.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Infection, Expressing, Control, Incubation, Flow Cytometry, Fluorescence, Staining, Blocking Assay, Concentration Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: Effects of ALVAC recombinant vA3131-2 expressing human CD40L on apoptosis of DCs. Immature MDDCs were infected or not (medium control) with either parental ALVAC II, ALVAC recombinant vA3131-2 expressing human CD40L, or an HIV canarypox vaccine vCP205 at an MOI of 10 at 37°C for 48 h. Infected cells were harvested and early apoptotic marker caspase-3 was determined by intracellular staining and flow cytometry using FITC- or PE-conjugated mAb against human caspase-3. (A) MDDCs were intracellularly stained for caspase-3 expression to detect early apoptotic MDDCs. The level of caspase-3 expression (x axis) is shown for each of the conditions. Background staining with FITC-conjugated isotype control Ab is shown by a solid gray histogram; medium-treated MDDCs are shown by a histogram with a dotted line, MDDCs infected with vA3131-2 are represented with a histogram with a dark line, and MDDCs infected with parental ALVAC II are represented with a histogram with a light line. The results represent one of five experiments from two HIV-1-uninfected and three HIV-1-infected donors. (B) Pooled data from all participants are shown. Data shown are mean±SEM. **: P<0.01.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Expressing, Infection, Control, Marker, Staining, Flow Cytometry

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4+ T cell-containing or -depleted PBMCs from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8+ T cells and 51Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow cytometry of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4+ T cell-containing condition and dark bar represent CD4+ T cell -depleted conditions. (C) A representative 51Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4+ T cell -containing condition and dark bars represent CD4+ T cell -depleted conditions. (E) A representative 51Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Activity Assay, Infection, Expressing, Release Assay, Flow Cytometry

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