Macs depletion

Nonadherent cells were cultured in AIM-V medium. On days 3, 5, and 7, recombinant human IL-2 (Shionogi Pharmaceutical Co., Tokyo, Japan) was added to the cultures to a final concentration of 10 IU/mL. The plates were incubated in a 5% CO₂ atmosphere at 37°C. On day 10, cultured cells were collected and washed 3 times with phosphate buffered saline (PBS). T cells were then isolated by MACS depletion (Miltenyi Biotec, Bergisch Gladbach, Germany). Cultured cells were labeled with microbeads using a CD8 T-cell isolation kit and separated on magnetic columns in a VarioMACS separator, according to the manufacturer’s recommendations (Miltenyi Biotec). CD8⁺ T cells were suspended at 5×10⁶ cells/mL.

Rag1^−/− or Rag1^−/−/I-A^b−/− mice at 6–10 weeks of age were irradiated with two split doses of 400 cGy from a 137Cs source with a 4-h interval, and BM cells were transferred within 24 h of the second irradiation. The BM cells were flushed from the femurs and tibiae of TCR^mini Tg or TCR^mini Tg Op, Plck-CIITA^Tg/CIITA^−/−/Cα^−/− and TCRα^−/− mice. A single-cell suspension of BM cells was filtered through a sterile nylon mesh, incubated with CD4 and CD8 magnetic beads (Miltenyi Biotec, Auburn, CA, USA), and subjected to MACS depletion according to the manufacturer's protocols (Miltenyi Biotec). The T-cell-depleted BM cells (3.0 × 10⁶) were mixed at a ratio of 1:1 and prepared in a volume of 300 μl of phosphate-buffered saline. Subsequently, these cells were injected intravenously into the lateral tail vein of the irradiated recipient mice. At 6–8 weeks post transplantation, the chimeras were killed for flow cytometric analysis of the thymocytes and splenocytes and subjected to single-cell sorting.