Journal: Frontiers in immunology

Article Title: Genomic Multiple Sclerosis Risk Variants Modulate the Expression of the ANKRD55 - IL6ST Gene Region in Immature Dendritic Cells.

doi: 10.3389/fimmu.2021.816930

Figure Lengend Snippet: FIGURE 5 | Digital droplet PCR (ddPCR) quantification of gene transcript copy numbers in pDC, mDC, CD14+ monocytes, PBMC fraction minus monocytes (F-), and moDC. Analysis was performed in 350 pg cDNA (A, E, F) or 30 ng of cDNA (B–D). (C) ddPCR quantification of ANKRD55 transcript copy numbers in moDC treated with AM-580. (D) ANKRD55 copy numbers in moDC matured or not with IFN-g/LPS measured at 6 h after addition of stimulus. (F) Transcript levels of markers specific for mDC (CD1c) and pDC (IL3RA, CLEC4C).

Article Snippet: To verify the purity of each subpopulation, cells were analyzed by flow cytometry (MACSQuant® flow cytometer, Miltenyi Biotec) using antihuman CD1c (BCDA-1)-APC antibody (Miltenyi Biotec, Ref. 130-110-595) for mDC and CD303 (BDCA-2)-APC antibody (Miltenyi Biotec, Ref. 130-114-177) for pDC, as shown in Supplementary Figure S1.

Techniques:

Journal: Journal for Immunotherapy of Cancer

Article Title: Checkpoint blockade accelerates a novel switch from an NKT-driven TNFα response toward a T cell driven IFN-γ response within the tumor microenvironment

doi: 10.1136/jitc-2020-002269

Figure Lengend Snippet: Functional analysis of CD1d overexpression in MC38 tumors. (A) Detection of NKT cells recognizing the CD1d a-GalCer tetramer among MC38 TIL by flow cytometry. Events were gated so as to exclude aggregates, debris, and non-viable cells, before CD45.2 + cells were selected in a similar manner to , with the exception that only CD3ε + NK1.1 + double-positive events were selected for analysis. An FMO control sample lacking the α-GalCer tetramer is depicted by a dashed black line, while α-GalCer tetramer among MC38 NKT cells is shown is red. (B) CD1d expression as measured by flow cytometry on untransduced (UT) MC38 (black), empty vector (EV) control MC38 (red), or CD1d overexpressed (CD1d over ) MC38 (blue). FMO control samples lacking CD1d staining is depicted by a black dashed line. (C and D) TIL isolated from MC38 tumors were placed in coculture with 51 CR-labeled UT (C) or CD1d over (D) MC38 target cells at increasing effector-to-target ratios, and percent cytotoxicity was determined by 51 CR release after 4 hours in the presence of blocking antibodies against CD1d (gray), H-2K b (red), or control irrelevant IgG antibody (dashed black). Representative of three experiments. (E and F) Subcutaneous MC38 tumor growth (E) and overall survival until endpoint (F) of C57BL/6 mice inoculated in the flank with UT (dashed black), EV (gray), or CD1d over (red) MC38 cells (n=12). (G) Subcutaneous MC38 tumor growth in NSG mice inoculated in the flank with UT (black) or CD1d over (red) MC38 cells. (H) In vitro growth kinetics of UT (dashed black), EV (gray), or CD1d over (red) MC38 cells. (I) MC38 tumor growth in wild-type control mice or in CD1d −/− mice at day 14 postinoculation (n=10). *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001 for indicated comparisons. FMO, fluorescence-minus-one; NKT, natural killer T cell; TIL, tumor infiltrating lymphocyte.

Article Snippet: To generate these derivative lines, lentiviral particles for murine CD1d (Myc-DDK-tagged; NM_007639) or control particles (pLenti-C-Myc-DDK-P2A-Puro) were obtained from OriGene Technologies Inc (Rockville, Maryland, USA) and used for transduction according to the manufacturer’s instructions.

Techniques: Functional Assay, Over Expression, Flow Cytometry, Control, Expressing, Plasmid Preparation, Staining, Isolation, Labeling, Blocking Assay, In Vitro, Fluorescence