CD8⁺DCs are required for cross-presentation of TSAs and generation of TSA donor T-cell responses. (A) The representative confocal microscopy images revealed efficient B6 WT CD8⁺ DC uptaking tumor cells. DCs and tumor cells are shown labeled with green (Cell Tracker Green CMFDA) and red (mCherry) fluorescent protein, respectively. Yellow arrows represent overlap of both fluorescent signals. (B) The percentage of engulfed DCs per high-power field (HPF) in either CD8⁺ DCs or CD8⁻ DCs (n = 7, pooled from 2 experiments). (C) Representative data from donor-derived Gag-specific CD8⁺ T cells of either allogeneic WT B6 animals (upper panels) or Batf3^−/− animals (lower panels) on day 14 after allo-BMT. Negative control hemagglutinin-tetramer staining (left) and Gag-tetramer staining (right). (D) The frequency and absolute number of donor-type Gag⁺CD8⁺ T cells (n = 7 to 11, pooled from 3 experiments). The bars represent mean ± standard deviation.