Journal: Cells

Article Title: Extensive Phenotype of Human Inflammatory Monocyte-Derived Dendritic Cells

doi: 10.3390/cells10071663

Figure Lengend Snippet: Phenotype of monocyte-derived inflammatory dendritic cells.

Article Snippet: The following monoclonal antibodies were used: anti-CD14-FITC (clone MφP9, BD Biosciences), anti-CD1c-AF488 (clone AD5–8E7, Miltenyi-Biotec, Bergisch-Gladbach, Germany), anti-CD1a-AF647 (clone 214A9, Dendritics), anti-CD40-AF647 (clone G28-5, Dendritics), anti-CD80-AF647 (clone Mab104, Dendritics), anti-CD208-AF647 (clone 109G3, Dendritics), anti-CD209 like-AF647 (clone 118A8, Dendritics), anti-CD206-AF647 (clone 122D2, Dendritics), anti-CD367-AF647 (clone 111F8, Dendritics), anti-CD207-AF647 (clone 808E10, Dendritics), anti-CD303-AF647 (clone 104C12, Dendritics), CD123-AF647 (clone 107D2, Dendritics), anti-CD304-AF647 (clone 211H6, Dendritics), anti-TLR2-AF647 (clone 1308F10, Dendritics), anti-TLR3-AF647 (clone 1213F10, Dendritics), anti-TLR7-AF647 (clone 66H3, Dendritics), anti-TLR8-AF647 (clone 112H7, Dendritics), and anti-FDFO3-AF647 (clone 36H2, Dendritics).

Techniques:

Journal: Cells

Article Title: Extensive Phenotype of Human Inflammatory Monocyte-Derived Dendritic Cells

doi: 10.3390/cells10071663

Figure Lengend Snippet: Inflammatory Mo-DCs share markers of plasmacytoid DCs and markers of activation. Monocytes were cultured with the cocktail of differentiation (Mo-DCs), or in the presence of TNFα (Control). Surface expression of plasmacytoid DCs markers (CD303, CD123, and CD304) was evaluated by flow cytometry ( A ), as well as the expression of costimulatory molecules (CD40, CD80) and the expression of the maturation marker CD208 ( B ). Grey shaded histograms are control stainings with an irrelevant antibody. Representative results of eight independent experiments for Mo-DCs, and four independent experiments for control cells.

Article Snippet: The following monoclonal antibodies were used: anti-CD14-FITC (clone MφP9, BD Biosciences), anti-CD1c-AF488 (clone AD5–8E7, Miltenyi-Biotec, Bergisch-Gladbach, Germany), anti-CD1a-AF647 (clone 214A9, Dendritics), anti-CD40-AF647 (clone G28-5, Dendritics), anti-CD80-AF647 (clone Mab104, Dendritics), anti-CD208-AF647 (clone 109G3, Dendritics), anti-CD209 like-AF647 (clone 118A8, Dendritics), anti-CD206-AF647 (clone 122D2, Dendritics), anti-CD367-AF647 (clone 111F8, Dendritics), anti-CD207-AF647 (clone 808E10, Dendritics), anti-CD303-AF647 (clone 104C12, Dendritics), CD123-AF647 (clone 107D2, Dendritics), anti-CD304-AF647 (clone 211H6, Dendritics), anti-TLR2-AF647 (clone 1308F10, Dendritics), anti-TLR3-AF647 (clone 1213F10, Dendritics), anti-TLR7-AF647 (clone 66H3, Dendritics), anti-TLR8-AF647 (clone 112H7, Dendritics), and anti-FDFO3-AF647 (clone 36H2, Dendritics).

Techniques: Activation Assay, Cell Culture, Expressing, Flow Cytometry, Marker

Journal: Nature Communications

Article Title: The neurotransmitter calcitonin gene-related peptide shapes an immunosuppressive microenvironment in medullary thyroid cancer

doi: 10.1038/s41467-024-49824-7

Figure Lengend Snippet: A Heatmap showing expression correlation of gene programs in DCs across tumor and normal tissue samples from PTC and MTC patients and clustered by column. Based on the correlated-expression relationship, modules consisting of gene programs were defined and characterized by immune pathways (enriched by the top gene signature of each module). Here, PTC and MTC represent tumors of PTC and MTC respectively, as above. PTC_P represents normal thyroid tissue of PTC and MTC_P represents normal thyroid tissue of MTC. B Heatmap showing the distribution ratio of DC modules in PTC and MTC. Calculated by Chi-square test, * p < 0.01. C Heatmap showing the correlation coefficient between module gene signature scores and DC antigen-presenting and co-stimulatory scores. Calculated by Spearman or Pearson’s correlation test, # p < 0.05. D The gene scores of antigen-presenting, co-stimulatory signatures were calculated in DCs between PTC and MTC and shown in violin plots. The box inside illustrates the interquartile range in relation to the median, while the middle lines represent the median, and the lower and upper hinges denote the 25-75% interquartile range (IQR), with whiskers extending up to a maximum of 1.5 times IQR. Calculated by two-sided Wilcoxon rank-sum test. E Multiplex immunofluorescence (mIHC) image showing HLA-DR and CD86 expression on CD11c + cells in PTC ( n = 9) and MTC ( n = 9) tumor regions (left panel). Bar graph shows the proportion of HLA-DR + and CD86 + DCs in PTC and MTC calculated by two-sided Student’s t -test. F Left panel shows the pseudotime trajectory of CD14 + monocytes and DCs derived from Monocle2. The right panel showing DCs from PTC and MTC along the trajectory, respectively. G The trajectory of DCs was colored by the score of the co-stimulatory gene signature. Monocytes are shown in gray without score calculation. H The trajectory was colored by cell state. I Pseudo-time expression of the genes CD80 and CD40 along the trajectories 1 and 2. Source data are provided as a Source Data file.

Article Snippet: DCs were stained in PBS containing Zombie Live-Dead dye or Live-Dead dye (FVS-780) for 20 min and then in FACS buffer with antibody cocktails including CD45 (cat#563792, BUV395, BD), CD11c (cat#561356, PE-Cy7, BD), CD86 (cat#566131, BV480, BD), CD83 (cat#305336, BUV605, BD), CD80 (cat#305216, AF647, BD), CD40 (cat#334305, FITC, BD), HLA-DR (cat#748338, BUV805, BD) on ice for 20 min. CD8 + T cells were stained in PBS containing Live-Dead dye (FVS-780), CD3 (cat#612895, BUV805, BD), CD8 (cat# 563823, BV786, BD Pharmingen) for 20 min and then stained for IFN-γ (cat# 554700, FITC, BD) and GZMB (cat# 562462, PE-CF594, BD) for an hour.

Techniques: Expressing, Multiplex Assay, Immunofluorescence, Derivative Assay

Journal: Nature Communications

Article Title: The neurotransmitter calcitonin gene-related peptide shapes an immunosuppressive microenvironment in medullary thyroid cancer

doi: 10.1038/s41467-024-49824-7

Figure Lengend Snippet: A Workflow of in vitro experiments. Monocytes isolated from PBMC were cultured and induced into immature DCs by specific cytokines. During induction, DCs were treated with 400 nM CGRP. After inducing maturation by cytokine cocktail for 24 h, DCs were harvested for RNA extraction and flow cytometry analysis. B Real-time PCR analysis of KLF2 transcripts at day 0,2,4 and 6 during DC induction ( n = 3 for each timepoint). The box illustrates the interquartile range in relation to the median, while the middle lines represent the median, and the lower and upper hinges denote the 25–75% interquartile range (IQR), with whiskers extending up to a maximum of 1.5 times IQR. P value between groups in one day was calculated by unpaired student’s t test. C Real-time PCR analysis of KLF2 transcripts at day 6 during DC in different groups ( n = 3 for each group). The data was presented as mean ± Standard deviation (SD). P values between groups were calculated by one-way ANNOVAR. D Representative flow cytometry histogram and increasing degrees of mean fluorescence intensity (MFI) of co-stimulatory markers CD80, CD40, CD86 and HLA-DR expressed on DCs ( n = 3 for each group). P values between groups were calculated by one-way ANNOVAR. The data was presented as mean ± SD. All experiments were performed independently for three times. Source data are provided as a Source Data file.

Article Snippet: DCs were stained in PBS containing Zombie Live-Dead dye or Live-Dead dye (FVS-780) for 20 min and then in FACS buffer with antibody cocktails including CD45 (cat#563792, BUV395, BD), CD11c (cat#561356, PE-Cy7, BD), CD86 (cat#566131, BV480, BD), CD83 (cat#305336, BUV605, BD), CD80 (cat#305216, AF647, BD), CD40 (cat#334305, FITC, BD), HLA-DR (cat#748338, BUV805, BD) on ice for 20 min. CD8 + T cells were stained in PBS containing Live-Dead dye (FVS-780), CD3 (cat#612895, BUV805, BD), CD8 (cat# 563823, BV786, BD Pharmingen) for 20 min and then stained for IFN-γ (cat# 554700, FITC, BD) and GZMB (cat# 562462, PE-CF594, BD) for an hour.

Techniques: In Vitro, Isolation, Cell Culture, RNA Extraction, Flow Cytometry, Real-time Polymerase Chain Reaction, Standard Deviation, Fluorescence

Journal: Nature Communications

Article Title: The neurotransmitter calcitonin gene-related peptide shapes an immunosuppressive microenvironment in medullary thyroid cancer

doi: 10.1038/s41467-024-49824-7

Figure Lengend Snippet: A Circle plot showing the cell-cell interaction strength of CD86 (left panel) and CD80 (right panel) between T cells and other immune cell types in PTC (green) and MTC (red). B Scatter plot showing the correlation relationship between CGRP expression and the Immune score, the cytotoxic score of CD8 + T cell, the dysfunctional score of CD8 + T cell and the co-stimulatory score of DCs in tumor bulk-RNA data ( n = 14). r indicates the correlation coefficient calculated by Spearman correlation test. C Schematic representation of CGRP-induced immunosuppressive tumor microenvironment in MTC. Source data are provided as a Source Data file. Created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: DCs were stained in PBS containing Zombie Live-Dead dye or Live-Dead dye (FVS-780) for 20 min and then in FACS buffer with antibody cocktails including CD45 (cat#563792, BUV395, BD), CD11c (cat#561356, PE-Cy7, BD), CD86 (cat#566131, BV480, BD), CD83 (cat#305336, BUV605, BD), CD80 (cat#305216, AF647, BD), CD40 (cat#334305, FITC, BD), HLA-DR (cat#748338, BUV805, BD) on ice for 20 min. CD8 + T cells were stained in PBS containing Live-Dead dye (FVS-780), CD3 (cat#612895, BUV805, BD), CD8 (cat# 563823, BV786, BD Pharmingen) for 20 min and then stained for IFN-γ (cat# 554700, FITC, BD) and GZMB (cat# 562462, PE-CF594, BD) for an hour.

Techniques: Expressing

Journal: Genes and immunity

Article Title: Monocyte polarization: the relationship of genome-wide changes in H4 acetylation with polarization

doi: 10.1038/gene.2011.17

Figure Lengend Snippet: Flow cytometric identification of polarization-induced cell surface molecules. Primary monocytes were polarized with the indicated cytokines for 18 h. The cells were studied using flow cytometry immediately and after a 3-day washout period. The cells were gated on physical parameters and CD14. CD16, CD11c, CD23, CD32, CD64, CD80, CD197, CD206 and CCR2 were studied with the graphs demonstrating the statistically significant changes after polarization. The first set of points represents the changes immediately after cytokine treatment. The arrows denote the statistically significant changes due to polarization (P<0.05). The second set of points reflects the cell surface staining after the 3-day washout. The graphs represent an average of four experiments with four separate donors. Representative flow plots are shown below.

Article Snippet: Isotype controls were used to define background staining, and the following antibodies were used in combination with PE-CD14 as an anchor: CD16, CD32, CD64, CD80, CD206 (all fluorescein isothiocyanate and all from BD Biosciences, San Jose, CA, USA) and AlexaFluor 647 CCR2 (BD Biosciences).

Techniques: Flow Cytometry, Staining

Journal: Gene Therapy

Article Title: Lentivirus-induced ‘Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma

doi: 10.1038/gt.2015.43

Figure Lengend Snippet: Pre-clinical validation of SmartDC-TRP2 in melanoma patients ( n =5). Human SmartDC-TRP2 generated from CD14 + monocytes from melanoma patients show DC immunophenotye and are potential stimulators of T cells. CD14 + monocytes were isolated from PBMNCs of melanoma patients, preconditioned with cytokines for 8 h and transduced with 2.5 μg p24 equivalents of LV-G242T for 16 h. Post transduction, cells were washed and cultured for 7 days in medium without cytokines. ( a ) Tricistronic LV used for generating the SmartDC-TRP2 showing the transgenes and 2 A elements. ( b ) Representative example of immunophenotypic analyses performed on day 7 SmartDC and SmartDC-TRP2. Filled and unfilled histograms indicate FACS analyses with isotype control or relevant mAbs; numbers indicate percentages of positive cells. ( c ) Immunophenotypic analyses of SmartDC-TRP2 generated from five independent melanoma patients. Bar graphs indicate percentage positive cells. ( d ) Secreted cytokines in supernatants of SmartDC and SmartDC-TRP2 analyzed at day 7. White bars indicate GM-CSF and black bars indicate IL-4. ( e ) Baseline TRP2 reactivity analyzed by quantifying the CD8 + T cells reactive against TRP2 tetramers: TRP2 180-188 and TRP2 360-368 in five different melanoma patients used in the study. An irrelevant tetramer was used as negative control. ( f ) SmartDC, SmartDC/pep or SmartDC-TRP2 were used in stimulation of autologous CD8 + T cells from the respective melanoma patients. Relative fold expansion of T cells per group determined with trypan blue exclusion staining (relative to T-cell input). Line graph indicate the average values of independent experiments and error bars indicate mean±s.e.m. * P <0.05. ( g ) IFN-γ ELISPOT analyses after two stimulations. 30 000 CD8+ T cells were co-cultured in the presence of KA2 cells or KA2 cells endogenously expressing TRP2 (KA2/TRP2) on IFN-γ coated ELISPOT plate. T cells without any were used as controls. Average number of spots from duplicate wells was quantified. Histograms represent average number of spots from pooled microculture wells ( n =3) per experimental condition. Bar graphs indicate average values of experiments performed and error bars indicate mean±s.e.m. *** P <0.001 and ** P <0.01. ( h ) Analyses of TRP2-specificity by tetramer analyses. Dots indicate the frequencies of non-specific (control tetramer, circles), TRP2 180-188 (squares) and TRP2 360-368 (triangles) reactive CD8 + T cells after three cycles of stimulation with SmartDC, SmartDC/pep or SmartDC-TRP2. Results for three melanoma patients are shown.

Article Snippet: We used the following monoclonal antibodies reactive against DC markers: CD86-PE, HLA-DR-PerCP, CD80-PE, CCR2-AlexaFlour 647, CCR5-PerCP (Becton Dickinson, Heidelberg, Germany) and respective monoclonal antibody isotype controls.

Techniques: Biomarker Discovery, Generated, Isolation, Transduction, Cell Culture, Control, Negative Control, Staining, Enzyme-linked Immunospot, Expressing

Journal: Gene Therapy

Article Title: Lentivirus-induced ‘Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma

doi: 10.1038/gt.2015.43

Figure Lengend Snippet: Pre-clinical validation of SmartDC-TRP2 potency generated from melanoma patients. CTLs stimulated with SmartDC or SmartDC/pep or SmartDC-TRP2 were analyzed for their ability to specifically lyse the target cells expressing TRP2. CTLs were co-cultured with TRP2 expressing KA2/TRP2 or FEMX-I cells at various effector to target (E:T) ratios and the cytotoxic ability was determined by CFSE-based assay. ( a ) Scheme of cytotoxicity assays. ( b ) TRP2 expression in KA2, KA2/TRP2 and FEMX-I target cells analyzed by intracellular staining. Filled and unfilled histograms indicate FACS analyses with isotype control or relevant mAbs; numbers indicate percentages of positive cells. ( c ) CFSE staining of FEMX-I targets used in the cytotoxicity assays. CFSE staining of FEMX-I targets before co-culture with the effector cells in the cytotoxicity assay and CFSE staining of FEMX-I targets after co-culture with CTL generated with SmartDC and CTL generated with SmartDC-TRP2. The right peak in the histograms show the live CFSE + targets after the cytotoxicity. Filled histogram in the first panel indicates isotype control for CFSE staining. ( d– f ) CFSE-based cytotoxicity assays. The line graph shows the percentage-specific lyses of T cells obtained from the microcultures of melanoma patients ( n =3). The data represents the average of triplicate wells performed with cells obtained from microcultures after three stimulations. ( d ) KA2 as targets, ( e ) KA2/TRP2 as targets and ( f ) FEMX-I as targets. T cells stimulated with SmartDC without any antigen is shown in open circles; T cells stimulated with SmartDC-TRP2 are shown in black triangles. Error bars indicate mean±s.e.m.; *** P <0.001, ** P <0.01 and * P <0.05.

Article Snippet: We used the following monoclonal antibodies reactive against DC markers: CD86-PE, HLA-DR-PerCP, CD80-PE, CCR2-AlexaFlour 647, CCR5-PerCP (Becton Dickinson, Heidelberg, Germany) and respective monoclonal antibody isotype controls.

Techniques: Biomarker Discovery, Generated, Expressing, Cell Culture, CFSE Assay, Staining, Control, Co-Culture Assay, Cytotoxicity Assay