Activation of CD4⁺ T cells by antigen-incorporated BMMCs is mediated by other APCs. (A) Anti-DNP IgE pretreated BMMCs were incubated with (bottom) or without (top) DNP-OVA (50 μg/mL) for 48 hours. Cell surface and (B) intracellular MHC class II expression was measured on CD117⁺ BMMCs by flow cytometry. (C) Anti-OVA IgE pretreated BMMCs from wild-type (top) or MHC class II^−/− (bottom) mice were incubated for 48 hours with OVA (50 μg/mL). The BMMCs were washed and cocultured with MACS-purified CD4⁺ T cells from OT-II mice. (D) Anti-OVA IgE pretreated MHC class II^−/− BMMCs were incubated for 48 hours with OVA (50 μg/mL), washed, and cocultured with FACS-purified CD4⁺ T cells (top left), MACS-purified CD4⁺ T cells (top right), unsorted splenocytes (bottom left), or MACS-purified CD4⁺ T cells mixed with BMDCs (bottom right) from OT-II mice. (E) OVA-incorporated BMMCs were prepared as described here and cocultured with unsorted OT-II splenocytes (top) or CD11c⁺ cell-depleted OT-II splenocytes (bottom). CD69 was detected 48 hours later on CD4⁺ T cells by flow cytometry. The number in the upper right corner indicates the percentage of CD69⁺CD4⁺ T cells of total CD4⁺ T cells.