In Vivo CD8+ T Cell Proliferation Assay

To detect antigen-specific T cell proliferation in vivo, CD8⁺ T cells (1 × 10⁷) from OT-I transgenic mice were labeled with 10 µM cell tracker red dye (CellTracker™ Cell Proliferation Kit, Thermo Fisher Scientific) for 20 min at 37°C and 10 µM CFSE for 30 min at 37°C, as previously described (24, 25). Cell tracker red dye was used before transfer of donor T cells to track the administered T cells in recipient mice, because it has been reported that the cell tracker dye crosses the plasma membrane of cells, where the stable and well-retained fluorescent label offers a consistent fluorescent signal (24). Fluorescence-labeled OT-I CD8⁺ T cells (5 × 10⁶ per recipient) were injected into the tail veins of C57BL/6 recipient mice (n = 3). In vivo stimulation of administered OT-I T cells was performed 16 h after cell transfer by intravenous injection of 20 µg OVA protein (Sigma Aldrich) with DMSO (0.01%), Inotodiol (6.5 mg/kg), and LPS (250 µg/kg) as a positive control in C57BL/6 recipient mice. In parallel, we also tested the in vivo effects of inotodiol-treated BMDCs on T cell proliferation. BMDCs (2 × 10⁵) were stimulated with inotodiol or LPS for 24 h, and the stimulated cells were pulsed with 10 µM OVA_257-264 peptide for 1 h at 37°C. OVA-pulsed cells were then injected into the foot pad 16 h after fluorescence-labeled OT-I CD8⁺ T cells (2 × 10⁶⁾ were injected into recipient mice (n = 3). After four days, the spleen of recipient mice was recovered and the extent of proliferating T cells was measured as the percentage of CFSE-diluted cells among the cell tracker red dye-positive cells. In parallel with the T cell proliferation assay, blood was collected by cardiac puncture to quantify cytokines in the serum.