Donor cells acquire host transmembrane proteins, including MHC class I-SIINFEKL complexes. B6, actOVA, and actOVA.K^b−/− mice were irradiated and reconstituted with C3H.SW BM, 2 × 10⁶ CD8 cells, and 1 × 10⁵ CD4 cells. Mice were killed 5 days after transplantation. (A) Representative flow cytometry of 25-D1 (top row) and K^b staining (bottom row) of donor Ly9.1⁺ CD11c⁺, CD8⁺, and CD4⁺ cells. Data are representative of 2 independent experiments with at least 3 mice per group. (B) actOVA.K^b−/− and actOVA mice were irradiated; and 5 days later, WT CD45.1 B6 or CFSE-labeled K^b−/− splenocytes were infused intravenously. Two days later, mice were killed. Shown is 25-D1 staining of CD45.1⁺ (left panel) and CFSE⁺CD8 cells (right panel). Note the shift in 25-D1 staining, which occurred only in actOVA recipients. (C-D) BALB/c-OVA (CD45.2) mice were irradiated and reconstituted with B6 CD45.1 BM and 10⁶ CD8 and 10⁵ CD4 cells. On day 5, splenocytes were harvested and stained with antibodies against CD11c, K^b, K^d, CD45.1, and surface OVA. Shown is representative flow cytometry gating on CD11c⁺ cells. As controls, nontransplanted B6 and BALB/c mice were analyzed. Note that BALB/c-actOVA recipients of B6 CD45.1 BM had a population of CD11c⁺ cells with a similar intensity of K^b staining as on CD11c⁺ cells from control B6 mice, but with significant staining for K^d. A similar pattern was seen for CD45.2 and surface OVA (histogram). Data are representative of at least 3 independent experiments. For confocal images, mice were transplanted as in panel C, and on day 6 cells were stained with anti–CD11c-PE and CD11c⁺ cell were sorted. Sorted cells were stained with antibodies against K^d (biotin followed by SA-Alexa488) and anti-K^b (APC) and then dropped onto slides. Original magnifications ×400. Note cells that express both K^d and K^b (white circles). There were also cells that stained for K^b or K^d only, which are internal controls for staining specificity. No K^b+ cells were seen when cells from control BALB/c →BALB/c were imaged (not shown). For Amnis image flow analysis (E), BALB/c mice were transplanted as in panels C through D. On day 6, splenocytes were harvested and stained with antibodies against CD11c (Pacific Blue), K^b (PE), and K^d (FITC) in addition to staining with 7-AAD for live/dead exclusion and DRAQ5 to allow isolation of 2N DNA content cells and analyzed on the ImageStream instrument. Shown are representative images from > 200 K^bK^d double-positive CD11c⁺ cells analyzed in B6→BALB/c recipients and more than 200 CD11c⁺ cells in B6→B6 and BALB/c→BALB/c syngeneic controls. Results are representative of 2 independent experiments.