Journal: PLoS ONE

Article Title: Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease

doi: 10.1371/journal.pone.0134518

Figure Lengend Snippet: (A) In vitro treatment of healthy donor neutrophils with 8-methoxypsoralen and UVA induces apoptosis and decreases activation markers. Neutrophils, isolated from blood of healthy donors, were treated with 8-MOP and UVA light and cultured for 24h. Control groups were untreated neutrophils, neutrophils treated with 8-MOP and neutrophils treated with UVA light. Percentage of non-apoptotic cells was determined by the % of Annexin V and 7-AAD double-negative cells via flow cytometry 0, 4, 8 and 24h after irradiation (n = 4). Curves show mean ± standard deviation (SD). ** p≤0.01. (B-D) Viable (= Annexin - / 7-AAD - ) neutrophils of the same groups (n = 4) were stained for activation markers CD11b, CD16 and CD54 at 2, 4, 8 and 24h after irradiation and protein expression was measured by flow cytometry after gating on viable cells. Curves show mean of Δ median of MFI ± SD. MFI = mean fluorescence intensity, Δ median of MFI = MFI of antibody-MFI of isotype shown.

Article Snippet: Antibodies CD11b V450, CD16 PE-Cy7, CD18 PE, CD32PerCP-efluor 710, CD54 APC and CD64 APC-H7 were purchased from BD Bioscience (Heidelberg, Germany) and eBioscience (Frankfurt, Germany).

Techniques: In Vitro, Activation Assay, Isolation, Cell Culture, Control, Flow Cytometry, Irradiation, Standard Deviation, Staining, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease

doi: 10.1371/journal.pone.0134518

Figure Lengend Snippet: (A) Neutrophil granulocytes and mononuclear cells substituted with 8-methoxypsoralen (8-MOP) were obtained from the buffy coat of patients with cGVHD treated with a Therakos UVAR XTS device before (pre-UVA) and after (post-UVA) irradiation. (B) Neutrophils of the buffy coat before and after irradiation with UVA (as described in A) were stained with Annexin V and 7-AAD. Fold change of Annexin V - /7-AAD - double negative (viable) cells determined after 24h of ex vivo culture after treatment normalized to cells assessed directly after treatment (0h). Data show means of 3 patients ± standard deviation (SD). **p≤0.01. (C) Neutrophils from buffy coats of cGVHD patients (n = 3) were stained with antibodies against activation markers CD11b, CD16 and CD54 after 24h in culture. Expression was measured by flow cytometry after gating on viable cells. Data shown as difference of the median of the mean fluorescence intensity (mfi) of the specific antibody and the isotype. (D) Supernatants of neutrophils of the samples described in (A) were generated with and without stimulation with LPS for 24h and concentration of CXCL8 and CCL4 was quantified by ELISA. Mean values ± standard deviation (SD) of 3 patients in pg/ml shown. (E) Arginase activity in the same supernatants as in (D) was determined by enzymatic activity (n = 3). Values shown in U/L. Unit definition: 1 unit of arginase converts 1μmol of L-arginine to ornithine and urea per minute at 37°C and pH 9.5. *p≤0.05

Article Snippet: Antibodies CD11b V450, CD16 PE-Cy7, CD18 PE, CD32PerCP-efluor 710, CD54 APC and CD64 APC-H7 were purchased from BD Bioscience (Heidelberg, Germany) and eBioscience (Frankfurt, Germany).

Techniques: Irradiation, Staining, Ex Vivo, Standard Deviation, Activation Assay, Expressing, Flow Cytometry, Fluorescence, Generated, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Frontiers in Immunology

Article Title: Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins

doi: 10.3389/fimmu.2020.01106

Figure Lengend Snippet: Attenuated IL-2 muteins induce Treg cell expansion with enhanced Treg:NK selectivity in vivo . (A) Study design schematic. One day before the first dose, humanized NSG mice were treated with 10 mg of human IgG. On day 0, wildtype IL-2 or IL-2 mutein was dosed s.c. at 0.5 μg per mouse, followed by a boost on day 7 at 1 μg per mouse. Blood was analyzed 4 days after the second dose, on day 11. Six animals per group were treated. Shown in the graphs, PBS-treated group served as vehicle control, and the muteins are color coded to represent class A (blue), B (green), and C (red). Ordinary one-way ANOVA analysis was performed to determine statistical significance of the comparisons between the wildtype- or IL-2 mutein-treated group and the PBS-treated group. (B) Attenuated IL-2 muteins induce human Treg cell response in vivo . Treg cells are gated based on Foxp3 expression and the representation of Treg cells are shown as percent of Foxp3+ in CD4 T cells. The levels of Ki67 and Foxp3 expression on Treg cells are shown as Ki67 MFI and Foxp3 MFI, respectively. * p < 0.02; ** p < 0.0025; **** p < 0.0001. (C) The sizes of the various non-Treg cell compartments post IL-2 treatment are shown as percent of human CD45+ cells; Tconv (Foxp3– CD4 T), CD8 T, and CD56+ CD3– and CD16+ CD3– NK cell data are shown. * p < 0.04; **** p < 0.0001.

Article Snippet: Whole blood was stained with cell surface markers: CD3 (FITC, BD Biosciences), CD56 (PerCP, BD Biosciences), CD16 (PE-Cy7, BD Biosciences), CD25 (APC, Miltenyi), CD45 (APC-Cy, BD Biosciences), CD4 (V500, BD Biosciences) for 20 min before red blood cells were lysed in BD FACS lysing solution (BD Biosciences).

Techniques: In Vivo, Expressing

Journal: Journal of Translational Medicine

Article Title: First-line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity

doi: 10.1186/1479-5876-8-71

Figure Lengend Snippet: IMP321 increases the numbers of monocytes, NK and activated CD8 T cells in blood (panel A) . Fresh blood samples were collected pre-dosing, at D1, D85 and D170 and directly stained with fluorochrome-conjugated antibodies in tubes containing a precise number of fluorescent control beads. The results show the mean ± sd of the absolute numbers of CD45 + CD14 + (monocytes), CD3 - CD56 + (NK cells) and CD38 + HLA-DR + CD8 + (activated CD8 + cells) cells. The paired non-parametric Wilcoxon signed rank test was used to compare increases observed between D85 or D170 and D1. When significant (< 0.05), p values are indicated. IMP321 increases the percentages of dendritic cells and cytotoxic CD45RA + Effector-Memory CD8 + T cells (EMRA) in PBMCs (panel B). PBMCs cells collected pre-dosing, at D1, D85 and D170 were isolated and stained with fluorochrome-conjugated antibodies and analyzed by flow cytometry. The results show the mean ± sd of the percentages of plasmacytoid dendritic cells (pDC, CD45 + CD14 - CD16 - HLA-DR + CD123 + CD11c - ) and myeloid dendritic cells (mDC, CD45 + CD14 - CD16 - HLA-DR + CD123 - CD11c + ) in CD45 + cells, as well as the percentages of CD45RA + CD45RO - CD62L - in the CD8 + T cell population (CD45RA + EM CD8 T cells or EMRA). The significant Wilcoxon p values are indicated.

Article Snippet: Blood samples were collected pre-dosing at D1, D85 and D170 and directly stained with BD Multitest CD8-FITC/CD38-PE/CD3-PerCP/HLA-DR-APC, with BD Multitest 6-color TBNK (CD3-FITC/CD16-PE+CD56-PE/CD45-PerCPCy5.5/CD4PE-Cy7/CD19-APC/CD8-APC-Cy7), BD Simultest LeucoGate (CD45-FITC/CD14-PE) in tubes containing a precise number of fluorescent control beads (BD Trucount™tubes, BD Biosciences).

Techniques: Staining, Isolation, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: First-line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity

doi: 10.1186/1479-5876-8-71

Figure Lengend Snippet: IMP321 increases the expression of activation markers on blood monocytes . Blood samples were collected pre-dosing, at D1, D85 and D170 and directly stained with fluorochrome-conjugated CD45, CD14, anti-HLA-DR and CD11a, CD11b, CD16, CD35, CD54, CD64, CD80 or CD86 antibodies in tubes containing a precise number of fluorescent control beads. The expression of activation markers on monocytes was directly proportional to the cell-bound fluorescence. The results shown in panel A are the mean ± sd after normalization of the cell-bound fluorescence against the fluorescence of control beads. Statistically significant increases between D85 or D170 and D1 are analyzed using Wilcoxon signed rank test and significant p values (< 0.05) are shown. In panel B, the percentage of patients showing increases in the expression of the indicated numbers of activation markers at D85 or D170 compared to the baseline at D1 was calculated. The number of markers (n) displaying an increase by at least 50% was calculated for each patient in the 1.25 mg (7 patients) and 6.25 mg (12 patients) groups. The pie charts represent the percentages of patients with increases in the indicated number of markers.

Article Snippet: Blood samples were collected pre-dosing at D1, D85 and D170 and directly stained with BD Multitest CD8-FITC/CD38-PE/CD3-PerCP/HLA-DR-APC, with BD Multitest 6-color TBNK (CD3-FITC/CD16-PE+CD56-PE/CD45-PerCPCy5.5/CD4PE-Cy7/CD19-APC/CD8-APC-Cy7), BD Simultest LeucoGate (CD45-FITC/CD14-PE) in tubes containing a precise number of fluorescent control beads (BD Trucount™tubes, BD Biosciences).

Techniques: Expressing, Activation Assay, Staining, Fluorescence