Figure 2.

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.