Figure 4.

Inhibition of PMA-stimulated cell adhesion as a function of aptamer expression. (A) The CD18cyt binder TR-D20 reduces phorbol ester-activated Jurkat cell adhesion to ICAM-1. Jurkat E6 cells infected with recombinant vaccinia viruses were allowed to adhere to plastic dishes coated with a recombinant ICAM-1 chimera as described in Material and Methods. Jurkat E6 cell adhesion to ICAM-1 was superinducible by PMA in the presence of several control viruses (vT7/vTR, wild-type vaccinia virus, vT7, and vTR-D20). However, induction of CD18cyt-specific aptamer expression (vT7/vTR-D20, Right) reduced PMA-stimulated Jurkat E6 cell adhesion but not basal adhesion. These results were independently reproduced at least three times. Percentage reflects values normalized to stimulated vT7/vTR double infection, which was set to 100%. (B) Human PBMC are good targets for protein overexpression by recombinant vaccinia viruses. Cytoplasmic Ig (cIg) fusions of cytohesin-1 subdomains were detected with the help of an FITC-conjugated anti-human Ig antibody preparation in permeabilized PBMC. Upper Left, control infection, no recombinant molecules expressed; Upper Right, cIg-domains alone; Lower Left, cIg-PH; Lower Right, cIg-PH+c-domain. (C) TR-D20 specifically reduces PMA-stimulated adhesion of PBMC to ICAM-1. Aptamers or control proteins were expressed in PBMC by recombinant vaccinia viruses as described above. PMA-stimulated adhesion of these cells to ICAM-1 was equivalent when the TR-D42 aptamer, cIg, or cIg-PH control proteins were expressed, but stimulated cell adhesion was dramatically reduced after expression of the intact PH + c-domain of cytohesin-1 (cIg-PH+c) or of the TR-D20 aptamer.