CD4-Specific Designed Ankyrin Repeat Proteins Are Novel Potent HIV Entry Inhibitors with Unique Characteristics
Figure 5
Interaction of DARPins with CD4 has no detectable effect on cell viability and stimulation.
(A) PBMC stimulation with IL-2 and OKT3 to induce proliferation was not altered in presence of the CD4-specific DARPin 55.2 (red), the non-binding DARPin E3_5 (blue) or absence of DARPin (gray) over a 4 day period. Proliferation was monitored by flow cytometry by determining CFSE dilution as a result of cell division. One representative experiment out of two is shown. (B) Activation of dendritic cells (DC) as determined by CD80 expression. Neither addition of DARPin 55.2 (red), nor of control DARPin E3_5 (blue), resulted in detectable upregulation of CD80 on DC over a 24 h period. One out of two independent experiments is depicted. (C) Prolonged treatment of T lymphocytes (4 days) and immature monocyte derived DC (24 h) with DARPin 55.2 (CD4 specific) and E3_5 (unselected control DARPin) has no effect on cell viability. Viability was determined by propidium iodide staining. DARPin concentration in the lymphocytes and DC cultures were 500 nM and 375 nM, respectively. (D) Interaction of DARPins with CD4 does not result in downregulation of surface CD4. Untouched peripheral blood CD4+ T cells were cultured in presence or absence of the indicated DARPins for 1 h, 3 h or 18 h at either 37°C (red line) or 4°C (blue line). Cells were stained for CD4 and the expression of surface CD4 was analyzed by flow cytometry. Shown is one representative experiment out of four.