Figure 3

CD134⁺ T cells in joint-draining lymph nodes are targeted by subcutaneous injection of anti-CD134 liposomes. On day 7 after immunization with Mycobacterium tuberculosis (Mt), rats were injected subcutaneously with fluorescent isotype control liposomes or anti-CD134 liposomes. Rats were killed 30 min later, and popliteal lymph nodes (PLN), inguinal lymph nodes, and spleens were isolated. (a) Cells were stained for CD4 and T-cell antigen receptor (TCR)-αβ and cell-associated fluorescence was analyzed by flow cytometry. Dot plots show cell-associated fluorescence due to in vitro monoclonal antibody (mAb) staining (left panels) or in vivo liposome binding (right panels). Cells were gated for live TCR-αβ⁺ CD4⁺ cells. The numbers in the dot plots indicate the percentage of cells above the cut-off line, which was set by using non-stained cells from sham-injected animals. Three rats were analyzed per group; representative stainings of one rat per group were selected and are shown here. (b) PLN cells of anti-CD134 liposome-injected rats were stained with anti-CD4 and anti-CD134 or its isotype control. Cells were gated for live CD4⁺liposome⁺ cells. Histograms show cell-associated fluorescence due to the binding of anti-CD134 (filled) or isotype control mAb (open). Representative stainings of one rat of three are shown. (c) PLN cells were stained with anti-TCR-αβ and anti-CD45RA (rat B cells). Cells were gated for live, TCR-αβ^-, and liposome⁺ cells. Histograms show cell-associated fluorescence due to ex vivo CD45RA (filled histogram) or isotype control mAb staining (thin line) on anti-CD134 liposome⁺ cells, or CD45RA (thick line) mAb staining on isotype control liposome⁺ cells. Representative stainings of one rat of three are shown.