Figure 4.

Anti-CD134-mediated targeting does not lead to liposome internalization by activated CD4⁺ T cells in vitro. A2b T cells were cultured with antigen-presenting cells and Con A to induce CD134 expression; CD25 is expressed constitutively on these cells. (a) Viable T cells were incubated with isotype control liposomes (filled histogram) or with anti-CD134 liposomes (black line). As a control, the binding of anti-CD134 liposomes or anti-CD134 monoclonal antibodies to resting T cells was assessed (gray lines). Cell-associated fluorescence was analyzed by flow cytometry, with live cells gated on the basis of forward scatter (FSC) and side scatter (SSC) profiles. One representative experiment of three is shown. (b) Viable T cells were incubated for 30 min with anti-CD134 liposomes on ice. After the removal of non-bound liposomes by washing, cells were cultured subsequently at 37°C. Samples were taken at the indicated time points and analyzed for the cellular localization of the liposomal fluorescence with the use of confocal microscopy. A representative cell from each time point is shown. As a positive control for cellular internalization of liposomes, cells incubated with anti-CD25 liposomes are shown. One of two experiments, yielding similar results, is shown.