In vivo targeting of conjugates and T-cell proliferation mediated by anti–CD19-OVA or isotype mAb-OVA conjugates. (A) Anti–CD19-OVA or isotype mAb-OVA conjugates (10 μg) were intravenously injected into mice. Peripheral blood (PB) was drawn at 30 minutes after injection. Cells were stained with B220 and anti-OVA Ab. Mice were then killed after 2 hours of injection. Splenocytes (Sp's) were stained with B220, CD11c, CD11b, and anti-OVA Ab. Data suggest that anti–CD19-OVA conjugates predominately bind to B cells. For in vivo T-cell proliferation assay, 10⁶ CFSE-labeled naive CD4 OT-II T cells (B) or CD8 OT-I T cells (C) were intravenously adoptively transferred into naive C57Bl/6 mice. The next day, mice were injected with a single dose of anti–CD19-OVA or isotype mAb-OVA conjugates or PBS. Recipient mice were killed after 3 days and turnover of T cells from splenic cells was examined by flow cytometry. Cells were gated on CFSE-positive population. T-cell proliferation shown in overlay histogram was from PBS, 2.5 μg isotype-OVA, or 2.5 μg anti–CD19-OVA. (D) Naive C57Bl/6 mice were immunized with isotype mAb-OVA or anti–CD19-OVA conjugates (10 μg). Mice injected with PBS were used as controls. Five days after immunization, mice were killed and splenocytes were stained with anti-CD8, anti-CD19, and H-2K^b-OVA/SIINFEKL pentamer. Cells were gated on CD19⁻ lymphocytes. Representative of 3 or more experiments. Error bars represent SD.