Journal:

Article Title: CD46-induced Immunomodulatory CD4 + T-cells Express The Adhesion Molecule And Chemokine Receptor Pattern of Intestinal T-cells

doi:

Figure Lengend Snippet: CD3/CD46-activated, α4β7-expressing T cells form conjugates with MAdCAM-1-expressing HeLa cells but not with HeLa cells in which MAd-CAM-1 expression had been silenced. (A) siRNA transfection efficiency of HeLa cells. HeLa cells were transfected with either Cy3-labeled siRNA targeting MAd-CAM-1, negative control scramble siRNA or buffer (mock, shaded histrogram) and transfection efficiency determined via FACS 24 hrs post transfection. (B) Analysis of MAd-CAM-1 protein expression/knockdown post siRNA transfection. HeLa cells were transfected with the indicated siRNAs and MAd-CAM-1 expression analyzed 24 hrs later via FACS. The shaded histrogram shows staining with an isotype control Ab. (C and D) CD3/CD46-activated CD4+ PBL form increased numbers of conjugates with MAd-CAM-1-expressing HeLa cells. HeLa cells were transfected with Cy3-labeled control scramble siRNA or MAd-CAM-1 siRNA, grown to confluence in culture plates and incubated with CFSE-labeled PBL that had been activated for 72 hrs under the depicted activation conditions. Non-attached cells were removed, remaining cells detached and analyzed by FACS analysis for the amount of retained PBL and HeLa/PBL conjugates. Freshly isolated LPL were used as positive control and non-activated PBL as negative control. Shown is one representative FACS analysis of three similarly performed experiments. (E) Statistical analysis of three separate experiments performed as described under ‘C’ (mean ± SD). (F) The addition of an anti-β7 antibody decreases binding of CD3/CD46-activated PBL and LPL to HeLa cells. Experiments were performed as described under ‘C’ using the control siRNA transfected HeLa cells but with the addition of a blocking anti-β7 antibody. Shown is the statistical analysis of β7-mediated blocking of the PBL or LPL/HeLa interaction of three separate experiments (mean ± SD). Statistical differences in panels ‘D’ and ‘E’ were determined using the paired Student's t test.

Article Snippet: Cells were then transfected with either 15 nM siRNA targeting MAdCAM-1 (Santa Cruz, sc-43037) or 15 nM negative control scramble siRNA (Santa Cruz, sc-37007) + 2.5 nM Cy3-labeled negative control siRNA (Ambion, AM4621) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and cultured.

Techniques: Expressing, Transfection, Labeling, Negative Control, Staining, Incubation, Activation Assay, Isolation, Positive Control, Binding Assay, Blocking Assay

Journal:

Article Title: CD46-induced Immunomodulatory CD4 + T-cells Express The Adhesion Molecule And Chemokine Receptor Pattern of Intestinal T-cells

doi:

Figure Lengend Snippet: Analysis of the adherence properties of CD3/CD46-activated T cells mediated by the α4β7/MAdCAM-1 interaction using a parallel plate flow chamber assay. (A) Microscopical comparison of adherence behavior between differently activated PBL and freshly isolated LPL. CD3 or CD3/CD46-activated CD4+ PBL or non-activated LPL were CSFE-labeled, injected into the flow chamber containing a monolayer of HeLa cells and incubated for 15 min prior to the start of flow. Time-lapse microscopy was used to record differential interference contrast (DIC) and fluorescent images of adherent T-cells as the flow rate of the medium was increased. Representative DIC/CFSE-merged images are shown for indicated wall shear stress levels. Bar is equal to 150 μm. (B) CD3/CD46-activated T cells adhere strongly to HeLa cells and this interaction is significantly decreased by pre-incubation with an anti-β7 antibody. Experiments were performed as described under ‘A’. At depicted wall shear stress levels, the number of adherent cells was assessed. Shown is the statistical analysis of β7-mediated blocking of the PBL/HeLa interaction of three separate experiments (mean ± SD). *Statistical difference in adherence between CD3 and CD3/CD46-activated T cells and between cell populations with and without Ab addition was p < 0.05 in all cases as determined by ANOVA using the Student-Newman Keuls post-hoc test. (C) LPL display similar adherence behavior as CD3/CD46 activated T cells. Experiments were performed as described under ‘A’ and the number of adherent LPL plotted against the wall shear stress as described under ‘B’. Shown is the result of one experiment. (D) siRNA knockdown of MAd-CAM-1 expression in HeLa cells abrogates adherence of CD3/CD46-activated T cells. HeLa cells were transfected with either control scramble siRNA or MAd-CAM-1 siRNA. Flow experiments were then performed as described under ‘A’ using CD3 or CD3/CD46-activated T cells and analyzed as described under ‘B’. Shown is the result of one experiment.

Article Snippet: Cells were then transfected with either 15 nM siRNA targeting MAdCAM-1 (Santa Cruz, sc-43037) or 15 nM negative control scramble siRNA (Santa Cruz, sc-37007) + 2.5 nM Cy3-labeled negative control siRNA (Ambion, AM4621) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and cultured.

Techniques: Boyden Chamber Assay, Isolation, Labeling, Injection, Incubation, Time-lapse Microscopy, Blocking Assay, Expressing, Transfection