Journal: Oncoimmunology

Article Title: Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G

doi: 10.1080/2162402X.2016.1250050

Figure Lengend Snippet: In vitro activated NK cells can functionally interact with EwS cells. (A) Flow cytometry analysis of EwS tumor cell lines for expression of DNAM-1 (CD112, CD155) and NKG2D (MICA, MICB, ULBP-1, -2, -3) ligands, and HLA-ABC, HLA-G, and PD-L1. Shown is one representative experiment of two. (B) Intracellular IFNγ and TNF-α expression of CD3−/CD56+ NK cells by flow cytometry following 6 h of co-incubation with EwS cell lines, medium alone, or K562 cells (n = 4). (C) Degranulation responses by CD107a staining of CD3−/CD56+ NK cells after 3 h coincubation with EwS cell lines, medium alone, or K562 cells (n = 4). (D) CD25 upregulation by NK cells after 24 h of co-incubation with tumor cells. Shown are triplicates of the two donors. (E) Viability of EwS cell lines within 24 h of co-culture with NK cells. (F) NK cell lysis of VH-64 spheres. Intact VH-64 spheres on days 8 and 9 were transferred into individual wells of 96-well round bottom plates in sphere culture medium, and maximum perpendicular sphere diameters were quantified. Spheres were co-incubated with NK cells at the indicated effector-to-target (E:T) ratios for 24 h, followed by photodocumentation (left panel). After manual dissociation of spheres, the percentages of viable tumor cells were determined by co-staining of cells with fluorescence-labeled CD56, CD99, and CD45 specific mAbs and subsequent flow cytometry quantification of viable cells in the CD99+CD56−CD45− tumor cell gate. Shown are triplicates of one representative experiment of three.

Article Snippet: NK cells were characterized using mAbs from BD Pharmingen against CD3 (clone SK-7, 557851), CD45 (clone HI30, 555483), CD56 (clone HCD-56, 345811), CD314 (NKG2D, clone 1D11, 558071), CD57 (clone NK-1, 560844) CD226 (DNAM-1, clone DX-11, 559789), from eBioscience against CD244 (clone eBioC1-7, 13-5838-82) and CD85j (ILT2, clone HP-F1, 12-5129-42), and from BioLegend against CD279 (PD-1, clone EH12.2H7, 329905).

Techniques: In Vitro, Flow Cytometry, Expressing, Incubation, Staining, Co-Culture Assay, Lysis, Fluorescence, Labeling

Journal: Scientific Reports

Article Title: Multifunctional CD4 T Cell Responses in Patients with Active Tuberculosis

doi: 10.1038/srep00216

Figure Lengend Snippet: (A) Whole blood from HC, LTBI, and TB patients was stimulated for 6 hrs with PMA/ionomycin and cells were analyzed by flow cytometry for intracellular expression of IFN-γ, IL-2, and TNF-α in CD4 (CD3 + CD8 − ) T cells. (A) Representative dot plots show the gating procedure for analyzing cytokine production of CD4 (CD3 + CD8 − ) T cells. (B) Summary cumulative data show the percentage of CD4 T cells with different patterns of cytokine production in HC (n = 90), LTBI (n = 108), and TB (n = 131). Data were expressed as mean ±SEM. ** p<0.01, ***p<0.001. (C) The pie charts summarize the fractions of single (1+, green), double (2+, blue), and Triple (3+, red) CD4 T cell producers of IFN-γ, IL-2, and TNF-α in HC, LTBI, and TB.

Article Snippet: Cells were washed again in PBS, then stained with surface mAbs against CD3 and CD8 , fixed with FACS Lysing Solution(BD Bioscience, San Jose, USA), permeabilized with permeabilizing solution (BD Bioscience), and intracellularly stained with mAbs against IFN-γ, IL-2, TNF-α, and IL-17 (all from BD Bioscience).

Techniques: Flow Cytometry, Expressing

Journal: Scientific Reports

Article Title: Multifunctional CD4 T Cell Responses in Patients with Active Tuberculosis

doi: 10.1038/srep00216

Figure Lengend Snippet: (A) PBMCs from HC, LTBI, and TB donors were stimulated for 16 h with M. tuberculosis lysate and analyzed by flow cytometry for intracellular expression of IFN-γ, IL-2, and TNF-α in CD4 T cells. (A) Representative dot plots showed the gating procedure for analyzing cytokine production of CD4 (CD3 + CD8 - ) T cells. (B) Summary of cumulative data showing the percentages of CD4 T cells with different patterns of cytokine production in HC (n = 66), LTBI (n = 30), and TB (n = 61). Data are expressed as mean ±SEM. *p<0.05, ** p<0.01, ***p<0.001. (C) The pie charts summarizing the fractions of single (1+, green), double (2+, blue), and triple (3+, red) CD4 T cell producers of IFN-γ, IL-2, and TNF-α in HC, LTBI, and TB.

Article Snippet: Cells were washed again in PBS, then stained with surface mAbs against CD3 and CD8 , fixed with FACS Lysing Solution(BD Bioscience, San Jose, USA), permeabilized with permeabilizing solution (BD Bioscience), and intracellularly stained with mAbs against IFN-γ, IL-2, TNF-α, and IL-17 (all from BD Bioscience).

Techniques: Flow Cytometry, Expressing

Journal: BMC Immunology

Article Title: In vitro analysis of the proliferative capacity and cytotoxic effects of ex vivo induced natural killer cells, cytokine-induced killer cells, and gamma-delta T cells

doi: 10.1186/s12865-015-0124-x

Figure Lengend Snippet: The percentage of NK, CIK, and γδ T cells before and after induction. a The percentage of NK cells (CD3 − CD56 + ) before and after induction. b The percentage of γδ T (CD3 + Vγ9 + ) and CIK (CD3 + CD56 + ) cells before and after induction. γδ T and CIK cells were immunostained with CD4 and CD8. Representative results from a single patient are shown

Article Snippet: The cultures were collected, washed, incubated for 15 min with mouse mAbs against human CD3-PerCP, CD56-FITC, or PE, CD69-APC, CD16-PE (BD Biosciences, USA), and NKG2D-PE (BioLegend, USA).

Techniques:

Journal: BMC Immunology

Article Title: In vitro analysis of the proliferative capacity and cytotoxic effects of ex vivo induced natural killer cells, cytokine-induced killer cells, and gamma-delta T cells

doi: 10.1186/s12865-015-0124-x

Figure Lengend Snippet: Perforin and granzyme B production in expanded NK, CIK, and γδ T cells. Three types of immune cells from twenty cancer patients were harvested after 14 days induction in vitro. NK and CIK cells were stained with mAbs to CD3 and CD56, and γδ T cells were stained with CD3 and Vγ9. After fixation and permeabilization, cells were stained for perforin and granzyme B using specific conjugated anti-human cytokine mAbs. a Perforin-positive cells and mean fluorescence intensity (MFI) of NK, CIK, and γδ T cells. b Granzyme B-positive cells and MFI of NK, CIK, and γδ T cells. Results are expressed as mean ± SD, n = 20. * p < 0.05, ** 0.001 < p < 0.01, *** p < 0.001, ns p > 0.05

Article Snippet: The cultures were collected, washed, incubated for 15 min with mouse mAbs against human CD3-PerCP, CD56-FITC, or PE, CD69-APC, CD16-PE (BD Biosciences, USA), and NKG2D-PE (BioLegend, USA).

Techniques: In Vitro, Staining, Fluorescence