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Blood, Vol. 110, Issue 1, 251-258, July 1, 2007
CD4 engagement by CD1d potentiates activation of CD4+ invariant NKT cells
Blood Thedrez et al.
110: 251
Supplemental materials for: Thedrez et al, Vol 110, Issue 1, 251-258
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 114 KB)
- Figure S1. CD1d surface expression levels on human LCL C1R and mouse T lymphoma RMA/S cells (JPG, 63.1 KB)
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Cell lines were stained with PE-conjugate human CD1d-specific mAb (Becton Dickinson), and analyzed by flow cytometry. Stainings of cells incubated with isotype antibody (Becton Dickinson) are used as negative controls (filled).
- Figure S2. Phenotypic characterization of human iNKT-cell clones isolated from a single healthy donor (JPG, 80.4 KB)
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(A) Nucleotide sequence and predicted amino acid sequence of V 24-J18 (TCR) and V 11-D1-J2 (TCR) junctional transcripts derived from the human iNKT-cell clones 20.22 and 20.49. CDR3 amino acid sequences are underlined. “N” refers to nongermline nucleotides. (B) V24/V11 TCR (left panel) and CD4 coreceptor (right panel) expression levels of human iNKT-cell clones 20.49 and 20.22. Results are representative of at least 5 independent experiments.
- Figure S3. Cytokine secretion profiles of human iNKT-cell clones 20.22 (DN) and 20.49 (CD4+) (JPG, 80.5 KB)
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(A) IFN- and IL-4 production following nonspecific stimulation. Human iNKT-cell clones 20.22 (open histograms) and 20.49 (filled histograms) were stimulated for 5 hours in the following conditions: no stimulation (Non Stim.), PMA and ionomycin (PMA/Iono) and anti-CD3 mAb (T3 clone; -CD3). Cytokine expression was then evaluated by flow cytometry following cell permeabilization. Fluorescence intensity is indicated in the y axis and the percentage of positive cells is indicated on the top of each histogram. (B) IFN- and IL-4 production following -GalCer/CD1d stimulation. HeLa CD1d cells were pulsed with 10−5 µg/mL -GalCer and used to stimulate NKT cells, which were further permeabilized and stained with antibodies to CD4, IFN-, and IL-4. The percentage of cytokine-producing cells and the cytokine mean fluorescence intensity value (mfi) are shown in the quadrants. The results are representative of 3 independent experiments.
- Figure S4. Anti-CD4 mAbs specifically modulate the reactivity of CD4+ iNKT-cell clone 20.49 (JPG, 87.7 KB)
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Effect of anti-CD4 mAbs on cytolytic activity of iNKT-cell clones. Four hours’ 51Cr release assays: effector-target ratios of 1:1 and 10:1 were used, and control IgG (diamonds) or anti-CD4 mAbs (squares: 6D10 and triangles: 13B8) were added at 5 µg/mL to show their effect on HeLa CD1d lysis pulsed with either DMSO (open symbols), 10−3 and 10−5 µg/mL (black symbols) or -GalCer. Results are representative of 3 independent experiments.
- Figure S5. Anti-CD4 mAb modulates α-GalCer-loaded CD1d tetramer binding on human iNKT-cell polyclonal populations (JPG, 96.1 KB)
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(A) B7 and B9 polyclonal NKT-cell populations (> 95% purity) were stained with anti-CD4 mAb (BL4; 5 µg/mL). Percentage of CD4+ cells is indicated (left panel). Cells were then preincubated with either control IgG or anti-CD4 mAb (BL4; 5 µg/mL) and further incubated with either anti-CD3 mAb (middle panel; dotted line: CD3 staining isotype; light line: control IgG; solid line: anti-CD4) or 10 nM -GalCer–loaded CD1d tetramer (light line: control IgG; solid line: anti-CD4). (B) MAD11 polyclonal invNKT-cell population (> 95% purity) isolated from a healthy donor was stained with either anti-CD4 mAb (top left panel) or -GalCer–loaded CD1d tetramer at 10 nM (top right panel). Sorted CD4– and CD4+ cell subsets were isolated by immunomagnetic sort (left panel: CD4 staining) and incubated with -GalCer–loaded CD1d tetramer (right panel) in the presence of control IgG (filled histograms) or anti-CD4 mAb (BL4 at 5 µg/mL) (open histograms).
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