Naïve OT-I CD8+ T cells (>95% purity) were obtained from LN cell suspensions by depletion of CD19+, CD4+, CD16/32+, and GR1+ cells using corresponding purified antibodies (BioXcell) and sheep anti-rat IgG magnetic beads (Dynabeads) following manufacturer’s instruction. Activated OT-I and Pmel-1 TCR transgenic CD8+ T cells were expanded in vitro from spleen cell suspensions. Spleen cells were incubated for three days at 37°C in RPMI medium containing recombinant human IL-2 (100IU/ml, TECIN) and OVA254-262 (20ng/ml, Genscript) or gp10025-33 peptide (100ng/ml, Genscript). Cells were washed and incubated for two more days in RPMI medium containing IL-2 (100IU/ml). OT-I and Pmel-1 in vitro expansion typically yielded >95% purity. Activated TCR transgenic CD8+ T cells were labeled with CellTrace Violet (Life Technologies) and transferred (5×106 cells) i.v. into naïve C57BL/6 mice.
For in vivo proliferation assay, cells were stained with APC-conjugated H-2Kb/OVA257-264 tetramers (NIH Tetramer facility) and 24G2 antibody for 20min at 4°C. Then stained with CD8-PE (53-6.7), CD45.1-PE.Cy7 (A20), CD4-APC/Cy7 (RM4-5), CD103-FITC (1E7) antibodies (Biolegend) for 20min at 4°C.
For in vivo proliferation assay, cells were stained with APC-conjugated H-2Kb/OVA257-264 tetramers (NIH Tetramer facility) and 24G2 antibody for 20min at 4°C. Then stained with CD8-PE (53-6.7), CD45.1-PE.Cy7 (A20), CD4-APC/Cy7 (RM4-5), CD103-FITC (1E7) antibodies (Biolegend) for 20min at 4°C.