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

Article Title: Ligation of the CD44 Glycoform HCELL on Culture-Expanded Human Monocyte-derived Dendritic Cells Programs Transendothelial Migration

doi: 10.4049/jimmunol.1800188

Figure Lengend Snippet: (A) Transendothelial migration (TEM) assay of CD14-S and PA-S mo-DCs on HUVEC monolayers. The relative TEM value is the ratio of mo-DCs transmigrated on TNFα-stimulated HUVECs compared with mo-DCs transmigrated on non-stimulated HUVECs and in the absence of chemokines. TEM was assessed using mo-DCs (untreated), mo-DCs preincubated with isotype control or with function blocking anti-LFA-1 mAb or with function blocking anti-VLA-4 mAb, HUVECs preincubated with function blocking anti-E-selectin mAb, and mo-DCs treated with sialidase or pertussis toxin (PTX). (B) Adhesiveness to CS-1 fibronectin fragment of CD14-S or PA-S mo-DCs. Cells were first incubated on uncoated or E-selectin-coated plates and then tested for binding to CS-1 peptide coated plates. Mo-DCs were stained with crystal violet and adherence was quantified by measuring light absorbance at 595 nm. Function blocking anti-E-selectin mAb was used as negative control and PMA treatment used as a positive control. (C) Flow cytometry analysis of activated β1-integrin expression by CD14-S and PA-S mo-DCs. Mo-DCs were incubated on uncoated plates or on E-selectin-coated plates, or cells were stimulated with PMA, and then stained with mAb HUTS-21 that identifies an activation-dependent epitope of β1-integrin (CD29*). Graph values represent the mean fluorescence intensity of HUTS-21 staining (n= 3, mean ± SD) subtracted from the values obtained using isotype control. (D) E-selectin ligand engagement on CD14-S mo-DCs does not lead to direct LFA-1 activation. Mo-DCs were incubated on uncoated or on E-selectin coated plates, or stimulated with PMA, and then stained with mAb 24 that recognizes an activation-dependent epitope of CD11/CD18 β2-integrin (CD11/CD18*). Graph values represent the mean fluorescence intensity of mAb 24 staining (n= 3, mean ± SD), subtracted from the values obtained using isotype control.

Article Snippet: Blood monocytes were enriched using anti-CD14 mAb-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) (CD14-S) or by plastic adherence (PA-S), and differentiated by culturing them in 1000 U/ml IL-4 and 1000 U/ml GM-CSF for 5 days as previously described ( 18 , 19 ).

Techniques: Migration, Blocking Assay, Incubation, Binding Assay, Staining, Negative Control, Positive Control, Flow Cytometry, Expressing, Activation Assay, Fluorescence

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

Article Title: Ligation of the CD44 Glycoform HCELL on Culture-Expanded Human Monocyte-derived Dendritic Cells Programs Transendothelial Migration

doi: 10.4049/jimmunol.1800188

Figure Lengend Snippet: (A) Representative flow cytometry analysis of mo-DCs staining using mAbs to CD49d (VLA-4), PSGL-1 and CD44, and HECA-452 mAb and E-Ig. Grey lines represent isotype control (or, for E-Ig, staining in the absence of Ca2+), dotted black line represents PA-S mo-DCs and solid black lines are CD14-S mo-DCs. (B–D) Analysis of glycoprotein E-selectin ligands expressed by mo-DCs: (B) Equivalent amounts of cell lysates of CD14-S and PA-S mo-DCs were resolved by SDS-PAGE electrophoresis, and immunoblotted with E-Ig chimera. Two E-selectin-reactive bands were visible in lysates of CD14-S mo-DCs (~130 kDa and ~80 kDa), whereas only one band (~130 kDa) was reactive on PA-S mo-DCs. (C) Equivalent amounts of cell lysates of CD14-S and PA-S mo-DCs were immunoprecipitated with E-Ig, and immunoprecipitates were then electrophoresed and immunoblotted with anti-PSGL-1 mAb. (D) CD44 immunoprecipitates (CD44 IP) from equivalent amounts of cell lysates of CD14-S and PA-S mo-DCs were immunoblotted with E-Ig chimera or anti-CD44 mAb. (E) Blot rolling assay of CD44 immunoprecipated from CD14-S mo-DCs. CD44 immunoprecipitates were resolved by SDS-PAGE, blotted and stained with anti-CD44 mAb (blot below the graph). E-selectin-transfected CHO cells (CHO-E) were perfused over blots at 1.4 dyn/cm2 and then shear stress was increased to 4.2 dynes/cm2. E-selectin-dependent tethering and rolling was observed at the CD44 band (arrow). Assays were performed in the presence or absence of 5 mM EDTA, or following preincubation of CHO-E with isotype control mAb or function blocking anti-human E-selectin mAb (clone 68-5H11).

Article Snippet: Blood monocytes were enriched using anti-CD14 mAb-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) (CD14-S) or by plastic adherence (PA-S), and differentiated by culturing them in 1000 U/ml IL-4 and 1000 U/ml GM-CSF for 5 days as previously described ( 18 , 19 ).

Techniques: Flow Cytometry, Staining, SDS Page, Electrophoresis, Immunoprecipitation, Transfection, Shear, Blocking Assay

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

Article Title: Ligation of the CD44 Glycoform HCELL on Culture-Expanded Human Monocyte-derived Dendritic Cells Programs Transendothelial Migration

doi: 10.4049/jimmunol.1800188

Figure Lengend Snippet: (A) Flow cytometry analysis of activated β1-integrin expression. Expression of the activation-dependent epitope of CD29 (HUTS-21) was assessed on CD14-S mo-DCs (white bars) and PA-S mo-DCs (black bars) incubated on plates coated with either E-selectin, P-selectin, or HA, or stimulated with PMA. Graph values represent the mean fluorescence intensity of HUTS-21 mAb staining (CD29*) of mo-DCs (n= 3, mean ± SD). (B) Adhesion of CD14-S and PA-S mo-DCs to CS-1 fibronectin fragment. Mo-DCs were incubated on uncoated plates or plates coated with HA, and then collected for analysis of binding to CS-1 peptide coated on plates. Integrin activation by treatment with PMA was used as positive control; the numbers of CS-1-adherent cells were quantified by light absorbance (595 nm) following crystal violet staining. As shown in the Figure, binding to HA by CD14-S mo-DCs, but not by PA-S mo-DCs, induced VLA-4 adhesion to CS-1 peptide, which was abrogated by treatment with anti-VLA-4 blocking mAb (HP2/1). Values are means ± SD (n= 3). Statistically significant differences (P ≤ 0.05) related to HA engagement are indicated by brackets and asterisks.

Article Snippet: Blood monocytes were enriched using anti-CD14 mAb-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) (CD14-S) or by plastic adherence (PA-S), and differentiated by culturing them in 1000 U/ml IL-4 and 1000 U/ml GM-CSF for 5 days as previously described ( 18 , 19 ).

Techniques: Flow Cytometry, Expressing, Activation Assay, Incubation, Fluorescence, Staining, Binding Assay, Positive Control, Blocking Assay

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

Article Title: Ligation of the CD44 Glycoform HCELL on Culture-Expanded Human Monocyte-derived Dendritic Cells Programs Transendothelial Migration

doi: 10.4049/jimmunol.1800188

Figure Lengend Snippet: (A) Transendothelial migration (TEM) assay of untreated (Unt) and TNFα-treated CD14-S and PA-S mo-DCs. Both CD14-S and PA-S mo-DCs were left untreated or treated with TNFα for 2 days (after 5 days of differentiation). The relative TEM value was calculated as the ratio of transmigrated cells on TNFα-stimulated HUVECs compared with cells transmigrated on non-stimulated HUVECs. TEM values were analyzed for mo-DCs preincubated with isotype mAb, for HUVECs preincubated with function blocking anti-E-selectin mAb clone 68-5H1, for mo-DCs preincubated with function blocking anti-VLA-4 mAb HP2/1, and for mo-DCs treated with sialidase or PTX. (B) Flow cytometry analysis of cell surface expression of integrins VLA-4 and LFA-1 on CD14-S mo-DCs. Histograms show the staining in untreated (dotted black line) and TNFα-treated (solid black line) mo-DCs. Grey lines represent isotype control. (C) Analysis of CD14-S mo-DCs binding to CS-1 fibronectin fragment. Adhesion of untreated (Unt) or TNFα-treated CD14-S mo-DCs (TNFα) to CS-1 peptide was assessed following incubation of mo-DCs on plates containing monolayers of E-selectin-transfected CHO cells (CHO-E), mock transfected CHO (CHO-mock), or plates containing no CHO cells (uncoated). After incubation, cells were collected for binding to CS-1 peptide coated on plates. The number of CS-1-adherent cells was quantified by light absorbance (595 nm) following crystal violet staining. Cells activated with PMA (positive control) bound avidly to CS-1, and preincubation of cells on CHO-E markedly augmented binding to CS-1. Incubation with anti-VLA-4 blocking antibody (HP2/1) for the last 15 min of CHO-E engagement (CHO-E+anti-VLA-4 mAb) abrogated binding to CS-1. Values are mean ± SD (minimum of n=4). Statistically significant differences (P ≤ 0.05) related to CHO-E engagement are indicated by brackets and asterisks.

Article Snippet: Blood monocytes were enriched using anti-CD14 mAb-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) (CD14-S) or by plastic adherence (PA-S), and differentiated by culturing them in 1000 U/ml IL-4 and 1000 U/ml GM-CSF for 5 days as previously described ( 18 , 19 ).

Techniques: Migration, Blocking Assay, Flow Cytometry, Expressing, Staining, Binding Assay, Incubation, Transfection, Positive Control

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

Article Title: Ligation of the CD44 Glycoform HCELL on Culture-Expanded Human Monocyte-derived Dendritic Cells Programs Transendothelial Migration

doi: 10.4049/jimmunol.1800188

Figure Lengend Snippet: (A) Flow cytometry analysis of E-Ig reactivity on CD14-S mo-DCs. Representative histograms of E-Ig staining of untreated mo-DCs (dotted black line) and mo-DCs treated with TNFα (solid black line) collected after 3 days of culture. Grey line represents E-Ig staining in absence of Ca2+. (B) Rolling adhesive interactions on TNFα-stimulated HUVECs of cultured CD14-S mo-DCs untreated (Unt) or treated with TNFα (TNFα) for 3 days. Rolling velocities were measured at shear stress of 2.0 dyn/cm2. The velocity was calculated by measuring the displacement of the centroid of the cell over 1 sec.; lower rolling velocity of TNFα mo-DCs indicates higher avidity to E-selectin. (C) Western blot analysis of E-Ig reactivity of whole cell lysates of CD14-S mo-DCs. Cells were left untreated (−) or treated (+) with TNFα (at Day 0) and collected at Day 3 after treatment. Representative western blot analysis (left) and the average quantifications of E-Ig reactivity obtained by densitometric analysis of three independent experiments (right) show that lysates obtained from TNFα-treated mo-DCs display higher E-Ig reactivity at bands at ~130 kDa and ~80 kDa, than those obtained from untreated DCs. Values are shown in arbitrary density units as the density ratio of the ~130 kDa (Top plot) and ~80 kDa (Bottom plot) bands obtained from DCs collected at Day 3 in relation to those obtained from DCs collected at Day 0. Statistically significant differences (P ≤ 0.05) are indicated by asterisks. (D) Western blot analysis of CD44 expression of CD14-S mo-DCs. Cells were collected at Day 0 and Day 3 with (+) or without (−) TNFα treatment. As shown in the figure, TNFα treatment sustains the expression of HCELL in vitro. (E) Schematic of sLeX biosynthesis on N- and O-glycans. Enzymes that were analyzed in this study are indicated in grey next to the arrows: core 2-synthase (C2GnT1), sialyltransferases (ST3GalI, ST3GalIII, ST3GalIV and ST3GalVI, ST6GalNAcII), fucosyltransferases (FTIV, VI and VII), and sialidases (Neu1, Neu3). (F) Real-time PCR analysis showing relative expression of selected genes. The relative mRNA level was computed as the permillage fraction (‰, i.e., the proportion of target mRNA per thousand of the reference GAPDH mRNA expression) of mo-DCs treated for 3 days with TNFα (grey bars) or left untreated (white bars). Values are means ± SD (n= 3). Statistical significant differences (P ≤ 0.05) are indicated by asterisks.

Article Snippet: Blood monocytes were enriched using anti-CD14 mAb-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) (CD14-S) or by plastic adherence (PA-S), and differentiated by culturing them in 1000 U/ml IL-4 and 1000 U/ml GM-CSF for 5 days as previously described ( 18 , 19 ).

Techniques: Flow Cytometry, Staining, Adhesive, Cell Culture, Shear, Western Blot, Expressing, In Vitro, Real-time Polymerase Chain Reaction

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

Article Title: Ligation of the CD44 Glycoform HCELL on Culture-Expanded Human Monocyte-derived Dendritic Cells Programs Transendothelial Migration

doi: 10.4049/jimmunol.1800188

Figure Lengend Snippet: (A) Western blot analysis comparing E-selectin binding (E-Ig reactivity) of KG1a and CD14-S mo-DCs. Input lysates on each blot were normalized to equivalent cell numbers. (B) Effects of exofucosylation on E-selectin ligand expression of mo-DCs. CD14-S mo-DCs were buffer treated (BT) or FTVI-treated, and lysates (of equivalent cell numbers) were analyzed by Western blot for reactivity with E-Ig. (C) Transendothelial migration (TEM) assay of buffer treated (BT) and FTVI-treated CD14-S mo-DCs. The relative TEM value is the ratio of mo-DCs transmigrated on TNFα-stimulated HUVECs compared with mo-DCs transmigrated on non-stimulated HUVECs. TEM was assessed using mo-DCs (untreated), HUVECs preincubated with function blocking anti-E-selectin mAb clone 68-5H11, mo-DCs preincubated with function blocking anti-VLA-4 mAb HP2/1 or isotype mAb, and mo-DCs treated with sialidase or PTX. (D) Analysis of mo-DCs tethering/rolling interactions and firm adherence on TNFα-stimulated HUVEC monolayers under flow conditions. Left: Shear stress was increased stepwise every 30 seconds, and the number of tethering/rolling cells per cm2 of HUVEC was counted, during each 30 second segment. Right: At the end of each time segment, the number of cells that became firmly adhering per cm2 area was measured. (E) Average rolling velocity of BT- and FTVI-treated mo-DCs perfused over TNFα-stimulated HUVECs. The velocity was estimated at 2.0 dyn/cm2 flow, as the displacement of the centroid of the cell over 1 sec. Values are means ± SD (n= 3). Statistically significant differences are indicated by asterisks. (F) FTVI treatment does not affect cell surface protein expression. Flow cytometry analysis of E-Ig reactivity, MHC-II, CD44, PSGL-1, CD29, CD49d, CD18 and CD11a staining on buffer treated (BT, white bars) and FTVI-treated CD14-S mo-DCs (dark grey bars). Statistically significant differences are indicated by asterisks. (G) FTVI treatment of CD14-S mo-DCs improves their homing to bone marrow. FTVI-treated or buffer-treated (Unt) mo-DCs were labeled reciprocally with either CFSE or Far Red and mixed at 1:1 ratio. CFSE and Far Red labeled cells were retro-orbitally co-injected into NSG mice, with dye combination swapped between the mice. 24h after injection, bone marrow was harvested and the presence of labeled cells in BM was assessed by flow cytometry. At left, representative flow cytometry plots show mo-DCs migration to BM. At right, FTVI-treated mo-DCs show significantly increased BM homing compared to buffer treated-mo-DCs (baseline). Data are shown as mean ± SEM. P-values calculated by unpaired 2-tailed student's t-test (*, P <0.05). Data are representative of 6 mice (3 mice for each dye combination).

Article Snippet: Blood monocytes were enriched using anti-CD14 mAb-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) (CD14-S) or by plastic adherence (PA-S), and differentiated by culturing them in 1000 U/ml IL-4 and 1000 U/ml GM-CSF for 5 days as previously described ( 18 , 19 ).

Techniques: Western Blot, Binding Assay, Expressing, Migration, Blocking Assay, Shear, Flow Cytometry, Staining, Labeling, Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: Rapid tumor vaccine using Toll-like receptor-activated ovarian cancer ascites monocytes

doi: 10.1136/jitc-2020-000875

Figure Lengend Snippet: CD14 + cells retrieved from human OC ascites express low levels of costimulatory and antigen-presentation markers and present TAAs in situ. (A) Malignant ascites were collected via paracentesis from chemotherapy-naïve patients with stage III or IV epithelial OC prior to primary debulking surgery. (B) Flow cytometry characterization revealed different percentages of immune cells representing CD14 + cells, T, B, natural killer and tumor cells in the malignant ascites of four OC patients’ samples. (C) Representative dotplots of ascites-derived CD14 + cells showing the expression of myeloid-lineage markers and D) costimulatory markers. (E) Principal component analysis showing clustering of ascites CD14 + cells with endometrial cancer macrophages. (F) Left panel in blue and white, immunopeptidomic analysis with mass spectrophotometry highlighting the MHC Class I and II presentation of TAAs on CD14 + cells and tumor cells from two OC patients. The data was presented as relative MS signal (log2). Right panel with red and light pink, comparing the transcriptional levels of expression of TAAs in CD14 + cells and tumor cells from the same two OC patients. The data were presented as relative expression (transcripts per million (TPM)/TPM average). OC, ovarian cancer; TAAs, tumor-associated antigens.

Article Snippet: Adherent cells were considered candidate APCs and were further enriched using magnetic CD14 beads (Miltenyi Biotec, California USA).

Techniques: In Situ, Flow Cytometry, Derivative Assay, Expressing, Spectrophotometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Rapid tumor vaccine using Toll-like receptor-activated ovarian cancer ascites monocytes

doi: 10.1136/jitc-2020-000875

Figure Lengend Snippet: CD14 + cells retrieved from human ascites can be matured ex vivo on stimulation with LPS, CpG and IL-10RAb and are capable of activating autologous TALs. (A) CD14 + cells from ascites were isolated by magnetic cell sorting and stimulated in vitro with LPS, CpG and/or IL-10R Ab for 48 hours. Activated CD14 + cells were subsequently cocultured with autologous TALs for 72 hours to assess T cell activation, and TALs were further expanded to assess proliferation and reactivity. (B) Fold-increase in cytokine secretion on TLR stimulation and IL-10R Ab treatment, and relative to untreated ascites monocytes as measured by multiparameter analyte profiling (Myriad Rules Based Medicine Luminex multianalyte profile). (C) Flow cytometry analysis depicting upregulation of maturation markers expression on CD14 + cells on TLR agonist stimulation and/or IL-10R Ab treatment. (D) activated CD14 + cells were cocultured with autologous TALs, and IFN-γ, granzyme A (GzmA) and B (GzmB) levels in the supernatant were measured after 72 hours coculture by CBA. (E) After 72 hours of coculture, media was changed and IL-2 (3000 U/mL) added for TAL expansion over 21 days. The expansion was compared with that of TALs expanded in the absence of DCs or following conventional expansion protocol. (F) T cell repertoire analysis at the end of the 21 days expansion. (G) IFN-γ ELISPOT assay was used to evaluate tumor-associated antigens TALs reactivity following 21 days of expansion. Data was representative of three independent experiments and were presented as mean±SEM *P<0.05, **P<0.001, ****P<0.0001. CBA, Cytometric Bead Array; DC, dendritic cell; IFN-γ, interferon-γ; IL-10R Ab, antibody to interleukin-10 receptor; LPS, lipopolysaccharide; TAA, tumor-associated antigens; TALs, tumor-associated lymphocytes; TLR, Toll-like receptor.

Article Snippet: Adherent cells were considered candidate APCs and were further enriched using magnetic CD14 beads (Miltenyi Biotec, California USA).

Techniques: Ex Vivo, Isolation, FACS, In Vitro, Activation Assay, Luminex, Flow Cytometry, Expressing, Enzyme-linked Immunospot