TZM-bl cells transduced with FcαRI.

To allow the expression of FcαRI on TZM-bl cells, the cDNAs for the human FcαRI and the γ chain of the FcεR were transduced into TZM-bl cells using lentiviral constructs. The cDNAs for human FcRs were amplified by PCR using primers complementary to regions flanking the receptor open reading frame. PCR products were cloned in the lentiviral vector pLenti6/V5 under the control of a cytomegalovirus (CMV) promoter (Invitrogen). Recombinant retroviral vectors were packed into virions in 293FT cells and used to transduce TZM-bl cells. Transduced cells were selected in blasticidin-containing medium and live-sorted by flow cytometry using a fluorescein isothiocyanate (FITC)-conjugated anti-human CD89 monoclonal antibody (MAb). To detect surface expression of the FcαRI and FcRγε chains on this new TZM-bl cell line, TZM-bl cells expressing FcαR1 were removed from flasks using a nonenzymatic solution (Sigma) and washed twice with PBS. Approximately 2 × 10⁶ cells were stained with a FITC-conjugated mouse monoclonal antibody (MIP8a) to the human CD89 (Abcam, Inc., Cambridge, MA). Cells and antibody were incubated at 4°C for 30 min, washed twice with PBS, and fixed with PBS containing 1% formaldehyde. The levels of CD89 were increased significantly with the coexpression of the human FcεRγ chain. Similarly, phycoerythrin (PE)-conjugated monoclonal antibodies (BD Biosciences/BD Pharmingen, San Diego, CA) against the human CD4, CCR5, and CXCR4 receptors as well as QuantiBrite beads (Becton, Dickinson, San Jose, CA) were used to determine surface expression on the FcαR1 cell line. The cell line was fully susceptible to HIV infection as the levels of CD4, CCR5, and CXCR4 remained comparable to those of the parental cell line. To test IgA binding to TZM-bl/FcαRI-expressing cells, human IgA1(κ), IgA2(κ) (Athens Research and Technology, Athens GA), purified human secretory IgA (MP Biomedicals, LLC, Solon, Ohio), and FITC-conjugated goat anti-human IgA (Sigma) were used. Cells were washed in PBS containing 0.1% bovine serum albumin (BSA) and fixed in PBS containing 1% formaldehyde before analysis.