Journal: Thoracic Cancer

Article Title: Experimental model of micronodular thymic neoplasm with lymphoid stroma

doi: 10.1111/1759-7714.14716

Figure Lengend Snippet: Representative immunofluorescent staining of cultured lymphoid cells. (a) Lymphoid cells exhibited dull staining on the cell surface with anti‐CD3. Major lymphoid cells did not exhibit immunoreactivity with anti‐CD4 or anti‐CD8 antibody. Approximately 20% of lymphoid cells demonstrated CD4 immunoreactivity, while approximately 10% lymphoid cells showed CD8 immunoreactivity. (b) Fixed lymphoid cells were stained without or with anti‐CD3 antibody conjugated with Alexa Fluor 488 (right or left, respectively). Note the green CD3 immunostaining in the cytoplasm of lymphoid cells. DAPI (4′, 6‐diamidino‐2‐phenylindole) blue staining was also used to visualize the nuclei.

Article Snippet: They were then incubated with or without 1:100 diluted hamster antimouse CD3 conjugated with Alexa Fluor 488 (MCA2690A488) (Bio‐Rad) at room temperature for 30 min and washed with PBS.

Techniques: Staining, Cell Culture, Immunostaining

Journal:

Article Title: Dermal Microvascular Injury in the Human Peripheral Blood Lymphocyte Reconstituted-Severe Combined Immunodeficient (HuPBL-SCID) Mouse/Skin Allograft Model Is T Cell Mediated and Inhibited by a Combination of Cyclosporine and Rapamycin

doi:

Figure Lengend Snippet: The phenotype of T cells infiltrating the skin grafts. Skin graft specimens were harvested between days 16 and 21 after i.p. injection of whole PBMCs (A and B; magnification ×250) or CD8+ lymphocyte-depleted PBMCs (C and D; magnification ×150) and immunostained for CD4 or CD8 antigens as described in Materials and Methods (C, inset : Magnification ×150). A and C, CD4 stain; B and D, CD8 stain. Some lymphocytes infiltrating skin grafts from mice injected with CD8-depleted PBMC stain for both CD3 (blue) and perforin (red), D, inset.

Article Snippet: The following antibodies were used for immunohistochemistry: rabbit polyclonal anti-human T cell (anti-CD3), mouse anti-human monocyte (anti-CD68), mouse anti-human platelet endothelial cell adhesion molecule (anti-CD31, all from DAKO, Carpinteria, CA), mouse anti-human human leukocyte antigen-DR (LB3.1, a gift from J. Strominger, Harvard University, Cambridge, MA), OKT4 mouse anti-human CD4 and OKT8 mouse anti-human CD8 (American Type Culture Collection, Manassas, VA), mouse anti-human VCAM-1, (E1/6, a gift from M. P. Bevilacqua, Amgen, Denver, CO), mouse anti-human perforin (T Cell Diagnostics, Cambridge, MA), hamster anti-mouse CD3, rat anti-mouse NK cell clone DX5 (Pharmingen, San Diego, CA), rat anti-mouse macrophage (clone F4/80, American Type Culture Collection), and peroxidase-conjugated rabbit anti-mouse C3 (Cedarlane, Hornby, Ontario, Canada).

Techniques: Injection, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Myeloid-derived suppressor cells impair CD4+ T cell responses during chronic Staphylococcus aureus infection via lactate metabolism

doi: 10.1007/s00018-023-04875-9

Figure Lengend Snippet: Suppression of CD4+ T cell responses by MDSC. a Flow cytometry histograms showing the kinetic of CD4+ T cells proliferation in spleen cells isolated from either uninfected (upper panels) or S. aureus -infected mice (middle panels) upon stimulation with anti-CD3/anti-CD28 antibodies. Proliferation of stimulated CD4+ T cells in the spleen cell population from S. aureus -infected mice that has been depleted of MDSC prior to stimulation is shown in the lower panels. The gating strategy is described in Supplementary Fig. S1. The percentage of divided CD4+ T cells in each group is shown in b . c Flow cytometry contour plots showing the intracellular staining of IL-2 (upper panels) and IFN-γ (lower panels) in CD4+ T cells within the spleen cell population isolated from uninfected (left panels) or S. aureus -infected (middle panels) mice as well as in MDSC-depleted spleen cells isolated from S. aureus -infected mice (lower panels) cultured for 72 h in the presence (green) or absence (red) of anti-CD3/anti-CD28 antibodies. The frequencies of CD4+ T cells expressing IL-2 (upper panel) or IFN-γ (lower panel) are shown in d . Each bar shows the mean ± SD of three independent experiments. *** p < 0.001, **** p < 0.0001

Article Snippet: Spleen cells were seeded in 96-well plates at a concentration of 5 × 10 6 /ml and incubated in the presence of 2 μg/ml of Armenian hamster anti-mouse CD3 plus 2 μg/ml of Syrian hamster anti-mouse CD28 antibodies (BD Pharmingen) for the specified time periods.

Techniques: Flow Cytometry, Isolation, Infection, Staining, Cell Culture, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: Myeloid-derived suppressor cells impair CD4+ T cell responses during chronic Staphylococcus aureus infection via lactate metabolism

doi: 10.1007/s00018-023-04875-9

Figure Lengend Snippet: Metabolic activity of spleen CD4+ T cells from uninfected or from S. aureus -infected mice upon stimulation with anti-CD3/anti-CD28 antibodies determined by SCENITH. Flow cytometry histograms showing the levels of protein translation (puromycin MFI) in CD4+ T cells within spleen cells isolated from either S. aureus -infected ( a ) or from uninfected ( b ) mice in the presence or absence of anti-CD3/anti-CD28 antibodies. Unstained CD4+ T cells (without anti-puromycin antibodies) were used as control. The gating strategy is described in Supplementary Fig. S3. Quantification of protein translation levels (puromycin MFI) in the different groups is shown in c . Each bar shows the mean ± SD of five independent experiments. *** p < 0.001, **** p < 0.0001

Article Snippet: Spleen cells were seeded in 96-well plates at a concentration of 5 × 10 6 /ml and incubated in the presence of 2 μg/ml of Armenian hamster anti-mouse CD3 plus 2 μg/ml of Syrian hamster anti-mouse CD28 antibodies (BD Pharmingen) for the specified time periods.

Techniques: Activity Assay, Infection, Flow Cytometry, Isolation

Journal: Cellular and Molecular Life Sciences

Article Title: Myeloid-derived suppressor cells impair CD4+ T cell responses during chronic Staphylococcus aureus infection via lactate metabolism

doi: 10.1007/s00018-023-04875-9

Figure Lengend Snippet: Expression of glucose transporter Glut-1 and glucose uptake by spleen CD4+ T cells from uninfected or from S. aureus -infected mice upon stimulation with anti-CD3/anti-CD28 antibodies. a Flow cytometry histograms showing the levels of intracellular Glut-1 expression in CD4+ T cells in spleen cells isolated from either uninfected (left panel) or S. aureus -infected (right panel) mice cultured for 24 h in the presence (red histogram) or absence (black histogram) of anti-CD3/anti-CD28 antibodies. Quantification of intracellular Glut-1 expression levels in the different groups is shown in b . c Flow cytometry histograms showing the kinetic of glucose uptake in CD4+ T cells in spleen cells isolated from either uninfected (upper panel) or S. aureus -infected (lower panel) mice unstimulated (black histograms) or stimulated for 24 h (pink histograms), 48 h (green histograms) or 72 h (blue histograms) with anti-CD3/anti-CD28 antibodies. Glucose uptake was determined using 2-NBDG. Quantification of glucose uptake in the different groups is shown in d . e percentage of divided CD4+ T cells in spleen cells from either uninfected (black bars) or S. aureus -infected (white bars) mice at 72 h upon stimulation with anti-CD3/anti-CD28 antibodies in the presence of either 10 mM, 50 mM or 100 mM glucose. f Frequencies of CD4+ T cells expressing IL-2 (left panel) or IFN-γ (right panel) within the spleen cell population isolated from uninfected (black bars) or S. aureus -infected (white bars) mice at 72 h upon stimulation with anti-CD3/anti-CD28 antibodies in the presence of either 10 mM, 50 mM or 100 mM glucose. Each bar shows the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Spleen cells were seeded in 96-well plates at a concentration of 5 × 10 6 /ml and incubated in the presence of 2 μg/ml of Armenian hamster anti-mouse CD3 plus 2 μg/ml of Syrian hamster anti-mouse CD28 antibodies (BD Pharmingen) for the specified time periods.

Techniques: Expressing, Infection, Flow Cytometry, Isolation, Cell Culture

Journal: Cellular and Molecular Life Sciences

Article Title: Myeloid-derived suppressor cells impair CD4+ T cell responses during chronic Staphylococcus aureus infection via lactate metabolism

doi: 10.1007/s00018-023-04875-9

Figure Lengend Snippet: Metabolic profile of spleen CD4+ T cells from either uninfected or S. aureus- infected mice upon stimulation with anti-CD3/anti-CD28 antibodies. Flow cytometry histograms showing the levels of protein translation (puromycin MFI) in CD4+ T cells in spleen cells from uninfected ( a ) or S. aureus- infected ( b ) mice at the indicated times of stimulation with anti-CD3/anti-CD28 antibodies and treated with either the glycolysis inhibitor 2-DG (upper histograms) or the oxidative phosphorylation inhibitor oligomycin (lower histograms). Quantification of protein translation levels (puromycin MFI) in the different conditions is shown in the lower panels in a and b . * p < 0.05, **** p < 0.0001

Article Snippet: Spleen cells were seeded in 96-well plates at a concentration of 5 × 10 6 /ml and incubated in the presence of 2 μg/ml of Armenian hamster anti-mouse CD3 plus 2 μg/ml of Syrian hamster anti-mouse CD28 antibodies (BD Pharmingen) for the specified time periods.

Techniques: Infection, Flow Cytometry

Journal: Cellular and Molecular Life Sciences

Article Title: Myeloid-derived suppressor cells impair CD4+ T cell responses during chronic Staphylococcus aureus infection via lactate metabolism

doi: 10.1007/s00018-023-04875-9

Figure Lengend Snippet: Contribution of glycolysis and oxidative phosphorylation to the metabolic activity of spleen CD4+ T cells from either uninfected or S. aureus- infected mice upon stimulation with anti-CD3/anti-CD28 antibodies. Metabolic dependence on glycolysis (cyan bars) or oxidative phosphorylation (purple bars) of spleen CD4+ T cells isolated from uninfected ( a ) or S. aureus- infected ( b ) mice upon stimulation with anti-CD3/anti-CD28 antibodies. Metabolic dependence was determined as described in the “Materials and methods” section. Each bar shows the mean ± SD of five independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Spleen cells were seeded in 96-well plates at a concentration of 5 × 10 6 /ml and incubated in the presence of 2 μg/ml of Armenian hamster anti-mouse CD3 plus 2 μg/ml of Syrian hamster anti-mouse CD28 antibodies (BD Pharmingen) for the specified time periods.

Techniques: Activity Assay, Infection, Isolation

Journal: Cellular and Molecular Life Sciences

Article Title: Myeloid-derived suppressor cells impair CD4+ T cell responses during chronic Staphylococcus aureus infection via lactate metabolism

doi: 10.1007/s00018-023-04875-9

Figure Lengend Snippet: Lactate levels in spleen tissue and in the supernatant of cultured spleen cells from uninfected or S. aureus- infected mice a Scheme showing the potential role of lactate in the suppression of CD4+ T cell responses by MDSC in the spleen of S. aureus- infected mice. LDH (lactate dehydrogenase), NAD+ (oxidized nicotinamide adenine dinucleotide), NADH (reduced nicotinamide adenine dinucleotide), MCT1 (monocarboxylate transporter 1), MDSC (myeloid-derived suppressor cells). b Lactate concentration in spleen tissue of uninfected (black bar) or S. aureus -infected (white bar) mice. c Lactate concentration in the supernatant of spleen cells isolated from either uninfected (black symbols) or S. aureus -infected (white symbols) mice at progressing times of incubation in either medium alone (left panel) or with anti-CD3/anti-CD28 antibodies (right panel). d Flow cytometry histograms showing proliferation of CD4+ T cells in spleen cells isolated from S. aureus -infected mice at 72 h upon stimulation with anti-CD3/anti-CD28 antibodies without changing (upper panel) or after changing the medium every 24 h (lower panel). The percentage of divided CD4+ T cells in each group is shown in e . f Flow cytometry histograms showing proliferation of CD4+ T cell in spleen cells isolated either from uninfected (left histograms) or from S. aureus -infected (right histograms) mice at 72 h upon stimulation with anti-CD3/anti-CD28 antibodies in the presence of either the MCT1 inhibitor AZD3965 (100 nM) (lower histograms) or vehicle control DMSO (upper histograms). The percentage of divided CD4+ T cells in each group is shown in g . Each bar shows the mean ± SD of five independent experiments. * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001

Article Snippet: Spleen cells were seeded in 96-well plates at a concentration of 5 × 10 6 /ml and incubated in the presence of 2 μg/ml of Armenian hamster anti-mouse CD3 plus 2 μg/ml of Syrian hamster anti-mouse CD28 antibodies (BD Pharmingen) for the specified time periods.

Techniques: Cell Culture, Infection, Derivative Assay, Concentration Assay, Isolation, Incubation, Flow Cytometry

Journal: Cellular and Molecular Life Sciences

Article Title: Myeloid-derived suppressor cells impair CD4+ T cell responses during chronic Staphylococcus aureus infection via lactate metabolism

doi: 10.1007/s00018-023-04875-9

Figure Lengend Snippet: NAD+/NADH ratio in stimulated spleen CD4+ T cells from uninfected or S. aureus -infected mice. a NAD+/NADH ratio in CD4+ T cell isolated from cultured spleen cells from either uninfected (black symbols) or S. aureus -infected mice in the presence (white symbols) or absence (grey symbols) of 200 µM NR and stimulated with anti-CD3/anti-CD28 antibodies. Each symbol shows the mean ± SD of three independent experiments. b Flow cytometry histograms showing proliferation of CD4+ T cell in spleen cells isolated from either uninfected (left histograms) or S. aureus -infected (right histograms) mice at 72 h upon stimulation with anti-CD3/anti-CD28 antibodies in the absence (upper histogram) or presence (lower histograms) of NR (200 µM). The percentage of divided CD4+ T cells in each group is shown in c . Each bar shows the mean ± SD of five independent experiments. * p < 0.05, ** p < 0.005, *** p < 0.001

Article Snippet: Spleen cells were seeded in 96-well plates at a concentration of 5 × 10 6 /ml and incubated in the presence of 2 μg/ml of Armenian hamster anti-mouse CD3 plus 2 μg/ml of Syrian hamster anti-mouse CD28 antibodies (BD Pharmingen) for the specified time periods.

Techniques: Infection, Isolation, Cell Culture, Flow Cytometry

Journal: eLife

Article Title: Neutrophil infiltration regulates clock-gene expression to organize daily hepatic metabolism

doi: 10.7554/eLife.59258

Figure Lengend Snippet:

Article Snippet: Antibody , Biotinylated monoclonal hamster anti-mouse CD3 (Clone 145–2 C11) , BD Pharmingen , Cat# 553057 RRID: AB_394590 , 1:20.

Techniques: Transfection, Construct, Synthesized, Plasmid Preparation, shRNA, Flow Cytometry, Cytometry, Sequencing, Recombinant, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Western Blot, Staining, Software

Journal: Immunology

Article Title: Function and expression of CD1d and invariant natural killer T‐cell receptor in the cotton rat ( Sigmodon hispidus )

doi: 10.1111/imm.12532

Figure Lengend Snippet: WTH‐2 and 1B1 but not WTH‐1 monoclonal CD1d antibodies can be used to stain cotton rat CD1d. Representative data from one of two experiments in total are shown. The biotinylated monoclonal mouse anti‐rat antibodies WTH‐1 and WTH‐2 (each 125 ng/ml), detected with streptavidin‐allophycocyanin, and rat anti‐mouse 1B1 phycoerythrin (2 μg/ml) were used to stain Raji cells transduced with rat, mouse, or cotton rat CD1d. Staining intensities are compared to isotype controls.

Article Snippet: CD1d dimer stainings were carried out as previously described 31 and a biotinylated hamster anti‐mouse CD3 ε antibody (145‐2C11; BD Pharmingen) was used to identify TCR expression of TCR transductants.

Techniques: Staining, Transduction

Journal: Immunology

Article Title: Function and expression of CD1d and invariant natural killer T‐cell receptor in the cotton rat ( Sigmodon hispidus )

doi: 10.1111/imm.12532

Figure Lengend Snippet: Organ‐specific CD1d expression in cotton rat primary cells. (a) Representative data for a total of three experiments are shown. Cells (5 × 105) isolated from mouse tissue or cotton rat tissues were stained with the monoclonal antibody (mAb) WTH‐2 FITC (2 μg/ml) or an isotype control. Geometric means of isotype control (tinted grey) or CD1d staining (black) are indicated in each graph. Geometric means of different expression levels (cotton rat lymph node and spleen) are additionally indicated below the histogram. (b) Primary cells (5 × 105) where stained with biotinylated WTH‐2 (3·6 μg/ml) and antibodies against CD3 (2 μg/ml), MHC class II (5 μg/ml, GαM R‐PE) and cotton rat IgG (10 μg/ml, GαR FITC). WTH‐2 positive cells from cotton rat spleen and lymph node were divided into low and high expression and CD3, MHC class II and cotton rat IgG expression of those two populations are compared in histograms. Representative data for two independent experiments are shown.

Article Snippet: CD1d dimer stainings were carried out as previously described 31 and a biotinylated hamster anti‐mouse CD3 ε antibody (145‐2C11; BD Pharmingen) was used to identify TCR expression of TCR transductants.

Techniques: Expressing, Isolation, Staining