Journal: eLife

Article Title: Tailoring T fh profiles enhances antibody persistence to a clade C HIV-1 vaccine in rhesus macaques

doi: 10.7554/eLife.89395

Figure Lengend Snippet: ( A ) Illustrates gating strategy to identify GC T fh cells. ( B ) Frequencies of T fh cells (CD4 + CD95 + PD-1 + CXCR5 + ) (Top), T fh 1 cells (CD4 + CD95 + PD-1 + CXCR5 + CXCR3 + ) (Middle), and T fh 1/17 cells (CD4 + CD95 + PD-1 + CXCR5 + CXCR3 + CCR6 + ) in lymph nodes. ( C ) Intracellular cytokine staining (ICS) to identify Env-specific T fh cells. ( D ) Co-expression of IL-2 and TNFa in IFNG +versus IFNG- subsets producing IL-21. ( E ) Serum IL-21 post P1. ( F ) Gating of AIM assay.

Article Snippet: Mouse anti-human CD154 (CD40L) (Clone 24–31) , AIM assay , eBioscience , Cat#17154842 RRID: AB_1582215.

Techniques: Staining, Expressing

Journal: eLife

Article Title: Tailoring T fh profiles enhances antibody persistence to a clade C HIV-1 vaccine in rhesus macaques

doi: 10.7554/eLife.89395

Figure Lengend Snippet: Flow cytometry antibodies.

Article Snippet: Mouse anti-human CD154 (CD40L) (Clone 24–31) , AIM assay , eBioscience , Cat#17154842 RRID: AB_1582215.

Techniques: Flow Cytometry

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: 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: FIGURE 3 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 antihuman 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: FIGURE 4 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 antihuman 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: FIGURE 5 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 Figure 4, 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 Figure 2D (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 antihuman 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: FIGURE 6 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 Table 1 and Materials and methods.

Article Snippet: The bound CD40L was detected by adding a non-competitive antihuman 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: FIGURE 7 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 Table 1 and Materials and methods.

Article Snippet: The bound CD40L was detected by adding a non-competitive antihuman 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: FIGURE 8 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 Table 1 and Materials and methods.

Article Snippet: The bound CD40L was detected by adding a non-competitive antihuman 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: BMC Molecular Biology

Article Title: Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

doi: 10.1186/1471-2199-9-90

Figure Lengend Snippet: DCs electroporated with CD40L RNA secrete IL-12 . Panel A: Cytokine secretion profile of DCs transfected with 2 μg/million DC eGFP RNA and matured with the cytokine cocktail. Panel B: time course for the secretion of IL-10 versus IL-12 for DCs matured by transfection with 4 μg/million DC CD40L RNA and cultured in IFN-γ/PGE 2 . Panel C: IL-10 and IL-12 release as well as expression of the DC maturation markers, CD80 and CD83 from immature DC transfected with a range CD40L RNA concentrations, and immediately matured in the presence of IFN-γ.

Article Snippet: 20μL of mouse anti-human CD40L PE and anti-human CD40 APC (BD Biosciences) or mouse IgG1 PE and IgG1 APC (BD Biosciences) was added to each DCs preparation collected and permeabilised at each time point, and incubated at 4°C for 30 minutes in the dark.

Techniques: Transfection, Cell Culture, Expressing

Journal: BMC Molecular Biology

Article Title: Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

doi: 10.1186/1471-2199-9-90

Figure Lengend Snippet: Nucleotide sequence preceding CD40L initiator methionine in various CD40L RNA transcription templates . Upper case: T7 promoter sequence; lower case: the sequence of 5'UTR; Upper case italics: accurate CD40L initiation codon; Bold lower case: upstream initiator codons which are not natural initiator codon for CD40L; Underline: the natural CD40L untranslated sequence (5'UTR).

Article Snippet: 20μL of mouse anti-human CD40L PE and anti-human CD40 APC (BD Biosciences) or mouse IgG1 PE and IgG1 APC (BD Biosciences) was added to each DCs preparation collected and permeabilised at each time point, and incubated at 4°C for 30 minutes in the dark.

Techniques: Sequencing

Journal: BMC Molecular Biology

Article Title: Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

doi: 10.1186/1471-2199-9-90

Figure Lengend Snippet: CD40L protein expression and IL-12 cytokine secretion in DC cultures transfected with various CD40L RNAs . Panel A: intracellular staining of DCs with anti-CD40L antibody at 4 hours post-electroporation with the RNAs. Panel B: cytokine levels measures in DC culture supernatants after overnight incubation. GFP is used as a negative control RNA. Type of cap analogue is indicated by shading back: ARCA and hatched m7G. All RNAs in this experiment were capped co-transcriptionally. All RNAs were polyadenylated in a post-transcriptional reaction and contained >150 nucleotide long poly(A) tail, except for CD40L+5'UTR 64A. For this RNA poly(A) tail was obtained in a transcription reaction by transcribing from templates containing poly(T) stretch after CD40L coding region. 5'UTR 64A+polA tail designates the RNA which in addition to 64 long poly(A) tail were post-transcriptionally polyadenylated. In this case poly(A) tail was >210 nucleotide long.

Article Snippet: 20μL of mouse anti-human CD40L PE and anti-human CD40 APC (BD Biosciences) or mouse IgG1 PE and IgG1 APC (BD Biosciences) was added to each DCs preparation collected and permeabilised at each time point, and incubated at 4°C for 30 minutes in the dark.

Techniques: Expressing, Transfection, Staining, Electroporation, Incubation, Negative Control

Journal: BMC Molecular Biology

Article Title: Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

doi: 10.1186/1471-2199-9-90

Figure Lengend Snippet: Sequence analysis of coding CD40L protein . Panel A: Amino acid sequence of the human CD40L full length polypeptide. In frame internal methionine residues are in bold and underlined. Panel B. SDS-Page Analysis of CD40L polypeptides translated in vitro . Lane 1: CD40L ΔXE; Lane 2: no RNA template control; Lane 3: control of irrelevant positive RNA translation.

Article Snippet: 20μL of mouse anti-human CD40L PE and anti-human CD40 APC (BD Biosciences) or mouse IgG1 PE and IgG1 APC (BD Biosciences) was added to each DCs preparation collected and permeabilised at each time point, and incubated at 4°C for 30 minutes in the dark.

Techniques: Sequencing, SDS Page, In Vitro

Journal: BMC Molecular Biology

Article Title: Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

doi: 10.1186/1471-2199-9-90

Figure Lengend Snippet: Analysis of initiator methionine codon for Kozak composition and its influence on initiation of translation . Panel A. Comparison between the eukaryotic Kozak consensus sequence and the sequence surrounding the CD40L translation initiation site. Nucleotides critical for efficient translation initiation are highlighted. Nucleotide substitutions predicted to enhance CD40L translation initiation without modifying the coding region are shown in red. Panel B. Correlation between CD40L small isoform expression and IL-12 induction. Graph demonstrates quantitative assessment of level of IL-12 cytokine secretion and below are shown SDS PAGE analysis of translation products. WT: wile type CD40L RNA, +Kozak: CD40L sequence with optimized first methionine codon; -Me1: CD40L sequence with knock out of first naturally occurring initiator methionine codon.

Article Snippet: 20μL of mouse anti-human CD40L PE and anti-human CD40 APC (BD Biosciences) or mouse IgG1 PE and IgG1 APC (BD Biosciences) was added to each DCs preparation collected and permeabilised at each time point, and incubated at 4°C for 30 minutes in the dark.

Techniques: Sequencing, Expressing, SDS Page, Knock-Out

Journal: BMC Molecular Biology

Article Title: Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

doi: 10.1186/1471-2199-9-90

Figure Lengend Snippet: CD40L protein lacking first naturally occurring Methionine codon induces highest levels of IL-12 secretion in DCs . Panel A: SDS-Page Analysis of CD40L polypeptides translated in vitro obtained from RNAs with consecutively knocked out 1–4 methionine codons. No RNA: in vitro translation products of reaction containing no RNA template. C: translation of a positive control unrelated RNA Panel D: intracellular staining of DCs with anti-CD40L antibody of DCs harvested at indicated time point post-electroporation with either CD40L WT or CD40L ΔXE Met 1 RNAs as indicated on the bottom of the graph. Panel F: levels of IL-12 cytokine measured by ELISA accumulated in supernatants collected at indicated time point post-electroporation in DC cultures electroporated with either CD40L WT or CD40L ΔXE Met 1 RNAs. Each electroporation was performed in triplicate. All CD40L RNAs were ARCA-capped with a long poly(A) tail. GFP is a negative control RNA.

Article Snippet: 20μL of mouse anti-human CD40L PE and anti-human CD40 APC (BD Biosciences) or mouse IgG1 PE and IgG1 APC (BD Biosciences) was added to each DCs preparation collected and permeabilised at each time point, and incubated at 4°C for 30 minutes in the dark.

Techniques: SDS Page, In Vitro, Positive Control, Staining, Electroporation, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: BMC Molecular Biology

Article Title: Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

doi: 10.1186/1471-2199-9-90

Figure Lengend Snippet: Post-transcriptionally capped CD40L RNAs are 100% capped and induce the highest level of IL-12 secretion . Panel A. Oligonucleotide-directed RNaseH cleavage products from the CD40L MET1 capping efficiency assay analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. Cleavage products from uncapped and capped RNA molecules are denoted by arrows. The control RNA is co-transcriptional ARCA-capped ΔXE-MET1 RNA. Capping efficiency measurements are indicated at the bottom of the gel image. Panel B: IL-12 cytokine secretion induced by ΔXE-MET1 RNA transfection as measured by ELISA. The resulting cap of each RNA is indicated by colour of the bars and as indicated, the type of cap (I or 0) is indicated below the graph. CD40L WT Reference RNA prepared with an ARCA cap by co-transcriptional cap analogue incorporation. GFP is a negative control RNA. All RNAs were polyadenylated post-transcriptionally and have >150 nucleotide tails. non-standard format).

Article Snippet: 20μL of mouse anti-human CD40L PE and anti-human CD40 APC (BD Biosciences) or mouse IgG1 PE and IgG1 APC (BD Biosciences) was added to each DCs preparation collected and permeabilised at each time point, and incubated at 4°C for 30 minutes in the dark.

Techniques: Polyacrylamide Gel Electrophoresis, Autoradiography, Transfection, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: iScience

Article Title: Kinetics of immune responses elicited after three mRNA COVID-19 vaccine doses in predominantly antibody-deficient individuals

doi: 10.1016/j.isci.2022.105455

Figure Lengend Snippet: Frequency of Spike-specific CD4 + T cell using activation-induced markers (A–E) Frequency (left panel) or fold change (right panel) in respect to w0 of Spike-specific CD4+T cells expressing CD69+CD154+ (A), CD69+CD137+ (B) or CD25+OX40+ (C) in CVID (n = 12, black circles), unPAD (n = 9, black triangles), other PADs (CID: n = 1, open triangles and TID: n = 1, open circles), and HC groups (n = 10, black diamonds) after vaccination. Comparison of Spike-specific CD4 + T cell subsets in CVID, unPAD and HC groups at w8 (D), and w28 versus w8 (E). In (D and E), Box-whiskers plots showing Min, Max, median and interquartile range are shown. (A–C) Horizontal dotted line indicates limit of detection and lines indicate median. Data in (A–C) were analyzed using Wilcoxon signed rank test. Data in (D and E) were analyzed using Dunn’s Multiple Comparison Test. ∗p<0.05; ∗∗p<0.01; ∗∗∗p < 0.001.

Article Snippet: BD OptiBuild™ BB700 Mouse Anti-Human CD154 (CD40L) clone trap-1 , BD Biosciences , Cat# 745814; RRID: AB_2743265.

Techniques: Activation Assay, Expressing, Comparison

Journal: iScience

Article Title: Kinetics of immune responses elicited after three mRNA COVID-19 vaccine doses in predominantly antibody-deficient individuals

doi: 10.1016/j.isci.2022.105455

Figure Lengend Snippet:

Article Snippet: BD OptiBuild™ BB700 Mouse Anti-Human CD154 (CD40L) clone trap-1 , BD Biosciences , Cat# 745814; RRID: AB_2743265.

Techniques: Functional Assay, Virus, Enzyme-linked Immunosorbent Assay, Recombinant, Saline, Modification, Expressing, Transfection, Luciferase, Blocking Assay, Enzyme-linked Immunospot, Staining, Software