Figure 4.

shRNA construct and ADCC activity validation

HEK293T cells were transduced with lentivirus expressing constructs #2, #5, and #6. Lentiviruses also expressed a GFP tag along with a selectable puromycin resistance gene. 48 h after transduction, puromycin was added to the cell media and allowed to select for positively transduced cells. For an additional 2 weeks, puromycin concentration was gradually increased to select a population of high expressing cells. (A) Flow cytometry was performed on lentivirus-transduced cells to monitor the levels of GFP expression. These levels were expected to correlate with levels of shRNA construct expression. The GFP⁺ gate was set on HEK293T cells not expressing GFP. (B) 10-1074 IgG expressing plasmids were transfected into wild-type HEK293T cells, FUT8 KO cell lines, and lenti-transduced HEK293T cell lines expressing constructs #2, #5, or #6. 10-1074 was purified by protein A column and quantified. ADCC activity was assessed in triplicate. The dashed line indicates 50% RLU or 50% ADCC activity against HIV-1 NL(AD8)-infected target cells. The loss of RLU indicates the loss of virus-infected cells during the 8-h incubation period and represents higher ADCC activity. 10-1074 FUT8 was included as a positive control due to the complete lack of fucose on the purified IgG. Samples were analyzed for statistical significance compared to controls using a two-tailed paired t test.