Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: The expression of 6Ckine, IFNγ and 6Ckine/IFNγ fusion protein in HepG2 cells mediated by adenovirus vector (L, Ad-LacZ; C, Ad-6Ckine; I, Ad-IFNγ; F, Ad-6Ckine/IFNγ; M, marker; +, positive control; N, mock-transfection). The HepG2 cells were transfected with recombinant adenoviruses at 10 MOI or mock-transfection (N) for 48 h, and then the expression and biological activity of 6Ckine, IFNγ and 6Ckine/IFNγ were assessed. a RT-PCR analysis for 6Ckine, IFNγ and 6Ckine/IFNγ mRNA. β-actin was used as an internal control (6Ckine 405 bp, IFNγ 501 bp, 6Ckine/IFNγ 849 bp, T-bet 450 bp, and β-actin 245 bp). b Immunofluorescent staining (×200). Red fluorescence indicated positive cells. c Western blot analysis. d Interferon activity was analyzed following the protocol from NICPBP. e 6Ckine function was detected by microchemotaxis assay

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Expressing, Plasmid Preparation, Marker, Positive Control, Transfection, Recombinant, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Fluorescence, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: The influences of recombinant adenoviruses infection on the endocytosis of DCs (C, Ad-6Ckine-DCs; I, Ad-IFNγ-DCs; F, Ad-6Ckine/IFNγ-DCs; L, Ad-LacZ-DCs; N, Non-transfected DCs. The percentage of FITC-Dextran uptake by the DCs was assessed with FACS analysis after 24 h of adenoviruses infection. The data represent three separate experiments. P > 0.05 when compared among all groups

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Recombinant, Infection, Transfection

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Chemokines secretion of DCs and the recruiting function to T cells. a and b DCs were pulsed with HepG2 cell lysates, and incubated at 37°C for 24 h. Supernatants from each DC group were harvested, and then 6Ckine (a) and RANTES (b) concentration was measured by the specific ELISA assay. The amounts of chemokines from five separate experiments were shown in nanograms per million DCs 48 h after transfection. c Microchemotaxis assay. The above-mentioned culture supernatant was collected, and the chemotaxis activity to naïve T cells was analyzed by a Transwells® apparatus. RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μl of recombinant human 6Ckine protein was used as positive or negative control individually. Supernatant pre-incubated with recombinant human anti-6Ckine monoclonal antibody was used for chemotaxis blocking assay (results represent three independent experiments). *P < 0.05 when compared with other groups; **P < 0.05 compared with medium control; ***P < 0.05 compared with Ad-6Ckine/IFNγ-DCs and Ad-6Ckine-DCs, P > 0.05 compared with Ad-IFNγ-DCs Ad-LacZ-DCs and NTDCs

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Chemotaxis Assay, Activity Assay, Recombinant, Negative Control, Blocking Assay, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Cytokine secretion of gene modified-DCs (C, Ad-6Ckine-DCs; I, Ad-IFNγ-DCs; F, Ad-6Ckine/IFNγ-DCs; L, Ad-LacZ-DCs; N, non-transfected-DCs). DCs were pulsed with 100 μg/ml of HepG2 cell lysates, and incubated for 24 h. The supernatant from each DC group was harvested, and then the level of IFNγ (a), IL-12p70 (b) and IL-10 (c) was detected by LiquidChip assay, while TNFα (d) were measured by ELISA. The values referring to the cytokine production were obtained from the average of five separate experiments. *P < 0.01 when compared with Ad-LacZ-DCs and non-transfected-DCs

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Modification, Transfection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Ad-6Ckine/IFNγ-DCs promote T cell activation. a Autologous T cell reaction. Following inactivation with mitomycin, antigen-loaded DCs were co-cultured with autologous T cells at a DC:T ratio of 1:20 for 96 h. 1 μCi/well of 3H-TdR was pulsed for 16 h. The values of counts per minute (cpm) were measured with a LS6500 liquid scintillation counter. Data were expressed as the stimulating indices (SIs). The results were obtained from five independent experiments. b IL-2 production of T cells. The IL-2 concentrations in the supernatants of co-cultured DCs and T cells were measured by ELISA. The results were obtained from six independent experiments. c The T-bet expression in T cells. The T-bet mRNA levels in T cells co-incubated with DCs were measured by semi-quantitative RT-PCR. The data were obtained from five separate experiments. d One of the representive experiments of RT-PCR. *P < 0.05 when compared with Ad-LacZ-DCs and NTDCs; **P < 0.05 when compared with any of other four groups

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Incubation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Cytotoxic function of T cells stimulated with Ad-6Ckine/IFNγ-DCs (C, Ad-6Ckine-DC group; I, Ad-IFNγ-DC group; F, Ad-6Ckine/IFNγ-DC group; L, Ad-LacZ-DC group; N, NTDC group). Autologous T cells were co-cultured with antigen-pulsed DCs at a DC:T ratio of 1:20 for 5 days. The cytotoxicity of activated T cells was analyzed with a CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, USA), HepG2 or LoVo cells were used as the target cells (E:T = 20:1 or 50:1). The data were obtained from five separate experiments. *P < 0.05 when compared with Ad-LacZ-DCs and NTDCs groups; **P < 0.05 when compared with other four groups, respectively

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Cell Culture, Cytotoxicity Assay

Journal: Frontiers in Immunology

Article Title: Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7

doi: 10.3389/fimmu.2019.02156

Figure Lengend Snippet: Endogenous CCL21 and CCL19 and chimeric N-terminal swap ligands. (A) Sequences of CCL21 and CCL19. The secondary structures are marked below with beta strands identified as arrows and C-terminally alpha helix identified as a sheet. N-term, core domain , and C-terminal domain refer to domains within the tertiary structure. The 16 N-terminal residues are swapped within (B) . Black refers to identical residues in the two sequences, blue refers to positively charged and red refers to negatively charged. (B) Schematic overview of CCL21, CCL19, and N-terminally swapped chimeras employed in this study, CCL19 CCL21N−term and CCL21 CCL19N−term . Blue refers to CCL21-derived residues and red to CCL19-derived residues not to be confused with red and blue in (A) . (C) Dose-response curves of CCL19, CCL21, CCL19 CCL21N−term , and CCL21 CCL19N−term in G protein signaling (left) and β-arrestin-2 recruitment (right) . The signals are obtained by co-transfecting CHO cells with CCR7 and reporter constructs able to measure intracellular cAMP changes (G protein) or β-arrestin-2 recruitment. Data are measured as the arbitrary unit BRET ratio and normalized to that of CCL19 within each separate experiment to compensate for inter-assay variations. Signaling in response to wild type CCL21 and CCL19 are shown as dotted lines, while the curves of chimeric ligands are shown with solid lines and symbols representing mean values (±SEM). Data are represented as mean values for several independent experiments performed in duplicates ( n = 7 for G protein signaling and n = 6 for β-arrestin-2 recruitment). ANOVA has been used to compare the curves of CCL19 and CCL19NCCL21N−term or the curves of CCL21 and CCL21CCL19N−term and asterisks identify significant differences, while ns refers to no significant changes. (D) Illustrative overview of wild type and chimeric ligand employed in this study with colors as described in (B) .

Article Snippet: The human chemokines CCL19 (catalog No. 361-MI) and CCL21 (catalog No. 366-6C) were purchased from R&D Systems (Minneapolis, MN, USA) or PeproTech (LuBioScience, Luzern, Switzerland).

Techniques: Derivative Assay, Construct, Inter Assay

Journal: Frontiers in Immunology

Article Title: Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7

doi: 10.3389/fimmu.2019.02156

Figure Lengend Snippet: Mutagenesis study of CCR7 showing changes of CCL21 and CCL19 G protein signaling by CCR7 mutations. (A) Barplot displaying change of CCL21- or CCL19-signaling by CCR7 mutations evaluated in a cAMP accumulation assay. Changes are displayed as relative change of efficacy at 100 nM CCL21, or fold change of potency for CCL19. Colors correspond to colors in (B) , where purple identifies mutations impairing both ligands, blue refers to mutations only affecting CCL21, red refers to mutations only affecting CCL19, and gray identifies mutations with no impact. Three mutations are highlighted, which are also highlighted in (B) and presented with their dose-response curves in (D) . (B) Scatterplot comparing the effect on CCL21 signaling to that of CCL19. Mutations are plotted with their values from (A) and colored according to the description in (A) . (C) Helical wheel of CCR7 with mutations identified according to effect on CCR7 signaling in (A,B) . (D) Dose-response curve of CCR7 W114A , CCR7 E193A , CCR7 R209A , and CCR7 WT stimulated with CCL21 or CCL19 in a cAMP accumulation assay. Significant differences between mutant and WT curve analyzed by two-way ANOVA are identified with colored asterisks corresponding to the color of the signaling curve. Data are represented as mean values (±SEM) of at least three independent experiments performed in duplicates. To compensate for inter-assay variations data have been normalized to wildtype within each separate experiment before the collection of data. The n value of independent experiments for each mutation can be found in .

Article Snippet: The human chemokines CCL19 (catalog No. 361-MI) and CCL21 (catalog No. 366-6C) were purchased from R&D Systems (Minneapolis, MN, USA) or PeproTech (LuBioScience, Luzern, Switzerland).

Techniques: Mutagenesis, Inter Assay

Journal: Frontiers in Immunology

Article Title: Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7

doi: 10.3389/fimmu.2019.02156

Figure Lengend Snippet: Functional analysis of CCR7 mutations.

Article Snippet: The human chemokines CCL19 (catalog No. 361-MI) and CCL21 (catalog No. 366-6C) were purchased from R&D Systems (Minneapolis, MN, USA) or PeproTech (LuBioScience, Luzern, Switzerland).

Techniques: Functional Assay, Residue

Journal: Frontiers in Immunology

Article Title: Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7

doi: 10.3389/fimmu.2019.02156

Figure Lengend Snippet: Overview of mutagenesis study, surface expression, and change of CCL21- and CCL19-directed migration by CCR7 mutations. (A) Serpentine structure of CCR7 with mutations from identified according to effect on CCR7 signaling, purple identifies mutations impairing both ligands, blue refers to mutations only affecting CCL21, and red refers to mutations only affecting CCL19. Gray shows residue where alanine substitution did not affect signaling. The predicted 24-residues N-terminal signal sequence of CCR7 which is cleaved of from the mature protein is displayed as faded and predicted cleavage site is indicated by a dotted line. (B) Surface expression levels of CCR7 were analyzed by flow cytometry in 300–19 pre-B-cells stably transfected with CCR7 WT , CCR7 R54A , CCR7 L61A , CCR7 W114A , or CCR7 R209A constructs using anti-human CCR7-APC (gated on the live cell population). Data show mean APC fluorescence intensity values (±SEM) derived from all migration assays performed in (C,D) . (C,D) Transwell chemotaxis in response to CCL21 and CCL19 by R54A N−term , L61A 1.35 , and W114A 2.60 (C) or R209A ECL−2 (D) . 300–19 cells were allowed to migrate in response to gradient concentrations of chemokines for 180 min. Migrated cells were counted and percentages of specifically migrated cells relative to the input were calculated. Mean values (±SEM) derived from four independent experiments are shown. Asterisks identify significant differences between WT and the mutant at 10 nM chemokine calculated performing two-way ANOVA. In (C) significance levels are positioned with L61A at top, followed by W114A (middle), and R54A (bottom).

Article Snippet: The human chemokines CCL19 (catalog No. 361-MI) and CCL21 (catalog No. 366-6C) were purchased from R&D Systems (Minneapolis, MN, USA) or PeproTech (LuBioScience, Luzern, Switzerland).

Techniques: Mutagenesis, Expressing, Migration, Residue, Sequencing, Flow Cytometry, Stable Transfection, Transfection, Construct, Fluorescence, Derivative Assay, Chemotaxis Assay

Journal: Frontiers in Immunology

Article Title: Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7

doi: 10.3389/fimmu.2019.02156

Figure Lengend Snippet: Migratory capacity of CCR7 mutations.

Article Snippet: The human chemokines CCL19 (catalog No. 361-MI) and CCL21 (catalog No. 366-6C) were purchased from R&D Systems (Minneapolis, MN, USA) or PeproTech (LuBioScience, Luzern, Switzerland).

Techniques: Mutagenesis, Migration

Journal: Frontiers in Immunology

Article Title: Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7

doi: 10.3389/fimmu.2019.02156

Figure Lengend Snippet: Signaling events at CCR7 rely on core domain interactions and ECL-2 of CCR7. (A) Chemokine cores determine differential signaling by CCL21 and CCL19. Chemokines containing the core of CCL19 display G protein signaling and β-arrestin-2 recruitment, whereas chemokines containing the core of CCL21 display G protein signaling but fail to recruit β-arrestin-2. (B) The current mutagenesis study of CCR7 shows that especially the extracellular events are important for chemokine signaling at CCR7, and the figure highlights ECL-2, which was found to play an important but differential role during interactions with the two chemokines. (C) The current study also found a lysine in TM3, K130 3.26 , to be important for regulating signaling, as the alanine substitution of this residue selectively impaired G protein signaling while not showing an effect on β-arrestin-2 recruitment upon CCR7 stimulation.

Article Snippet: The human chemokines CCL19 (catalog No. 361-MI) and CCL21 (catalog No. 366-6C) were purchased from R&D Systems (Minneapolis, MN, USA) or PeproTech (LuBioScience, Luzern, Switzerland).

Techniques: Mutagenesis, Residue

Journal: Cell reports

Article Title: Regulatory T Cells Condition Lymphatic Endothelia for Enhanced Transendothelial Migration

doi: 10.1016/j.celrep.2019.12.083

Figure Lengend Snippet: (A) A total of 1 × 105 wild-type (WT) or LTα−/−nTregs (Foxp3-GFP+CD25+CD4+) or naive CD4 (Foxp3-GFP-CD25-CD4+) were incubated with LEC layers in Boyden chambers for 16 h, T cells were removed by washing, and chambers were then loaded with 2 × 105 naive CD4 (i) or CD8 (ii) T cells or 1 × 105 BMDCs (iii) and migrated toward 50 ng/mL CCL19 (CD4 and CD8) or 200 ng/mL CCL21 (BMDC) for 3 h.

Article Snippet: Immunohistochemistry Murine or human LEC monolayers were stained for surface VCAM-1, VE-Cadherin or Lyve-1 with rat anti-mouse VCAM-1 (Clone 429, eBioscience) or mouse anti-human VCAM-1 (Clone STA, Biolegend), rat anti-mouse VE-Cadherin (Clone 11D4.1, BD Biosciences) or mouse anti-human VE-Cadherin (Clone BV9, Biolegend), goat-anti-mouse or anti-human CCL21 polyclonal antibody (R&D System), or rabbit anti-mouse Lyve-1 (70R-LR003, Fitzgerald, Acton, MA), then were fixed for 20 min at 4 °C with 4% (w/v) paraformaldehyde (Affymetrix, Santa Clara, CA), Cells were permeablized with PBS 0.2% (v/v) Triton X-100 (Sigma-Aldrich), and treated with 5% donkey serum for 30 min then incubated with listed primary antibodies for overnight at 4 °C.

Techniques: Incubation

Journal: Cell reports

Article Title: Regulatory T Cells Condition Lymphatic Endothelia for Enhanced Transendothelial Migration

doi: 10.1016/j.celrep.2019.12.083

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Immunohistochemistry Murine or human LEC monolayers were stained for surface VCAM-1, VE-Cadherin or Lyve-1 with rat anti-mouse VCAM-1 (Clone 429, eBioscience) or mouse anti-human VCAM-1 (Clone STA, Biolegend), rat anti-mouse VE-Cadherin (Clone 11D4.1, BD Biosciences) or mouse anti-human VE-Cadherin (Clone BV9, Biolegend), goat-anti-mouse or anti-human CCL21 polyclonal antibody (R&D System), or rabbit anti-mouse Lyve-1 (70R-LR003, Fitzgerald, Acton, MA), then were fixed for 20 min at 4 °C with 4% (w/v) paraformaldehyde (Affymetrix, Santa Clara, CA), Cells were permeablized with PBS 0.2% (v/v) Triton X-100 (Sigma-Aldrich), and treated with 5% donkey serum for 30 min then incubated with listed primary antibodies for overnight at 4 °C.

Techniques: Recombinant, Selection, Cell Isolation, Software