Evaluating CD8+ and CD103+ Dendritic Cells in Tumor Immunotherapy

B6 mice (f, 6–8 wks, 10 per group) were injected subcutaneously (s.c.) in the left flank with GL26 (1x10⁶) tumor cells on day 0. On day 8, tumor-bearing mice were left untreated or they were vaccinated using a GG with XBP1/TRP2hsp70 DNA as previously described (23 (link)). After 3 days, single-cell suspensions of pooled skin/tumor dLN were incubated with anti-CD16/32 (BD Biosciences) on ice for 15 minutes before being stained using anti-CD11c-APC, -CD8α-pacific blue, -CD205-percep-cy5.5, -CD103-PE, and CD11b-FITC Ab (BD Biosciences, eBioscience, Biolegend). CD8α⁺DC (CD11c^hiCD8α⁺CD205⁺) and CD103⁺DC (CD11c^hiCD8α⁻CD103⁺CD11b^lo/−) were then sorted using a BD FACSAria High Speed Cell Sorter (BD Biosciences). Sorted CD8α⁺DC or CD103⁺DC (2x10⁴) were co-cultured with TRP2-specific CD8⁺T cells (1x10⁵, provided kindly by Dr. Hurwitz at NCI) isolated from murine TRP2_180–188 TCR transgenic (Tg) B6 mice (28 (link)) in 200 μl RPMI1640 containing 10% (v/v) FBS for 3 days. IFN-γ in the culture supernatants was measured by ELISA (BD Biosciences).