Figure 6

Decreased proliferation and cytotoxicity of T-cells stimulated with RBP-J deficient SPDCs. (A) FACS analysis. The CFSE-labeled T-cells (1 × 10⁶) were co-cultured with SPDCs (2 × 10⁵) pre-incubated with B16 tumor antigens for 5 days, and the T-cell proliferation was analyzed. (B) SPDCs (1 × 10⁶) pre-incubated with the B16 tumor antigens were injected subcutaneously at the lower left abdomen of normal mice. The draining lymph nodes were dissected 7 days later, and the total cell number in the lymph nodes was compared between the RBP-J^-/- and RBP-J^+/- groups. (C) BrdU incorporation assay. SPDCs (1 × 10⁶) pre-incubated with the B16 tumor antigens were injected subcutaneously at the left lower abdomen of normal mice. BrdU was injected intraperitoneally twice per day from the 5th day for 3 days. BrdU⁺CD3⁺ cells in the draining lymph nodes were detected by flow cytometry 7 days later. (D-G) Cytokine production. Naïve T-cells were co-cultured with SPDCs pre-loaded with B16 tumor antigens for 72 h. Cytoplasmic staining was performed to analyze IFN-γ(D) and IL-4 (F) production of T-cells by flow cytometry. ELISA was carried out to detect IFN-γ (E) and IL-4 (G) secretion in the supernatants. (H) CTL assay. Normal T-cells were co-cultured with SPDCs pre-loaded with B16 tumor antigens for 3 days. Live B16 melanoma cells were then co-cultured with the activated T-cells at different ratios of 1:10, 1:20, 1:40, 1:80 for 36 h. LDH in the supernatants was detected by using an LDH kit. (I) CTL assay. DCs (1 × 10⁶) pre-loaded B16 tumor antigens were injected subcutaneously at the left lower abdomen of normal mice. Seven days later, the draining lymph nodes were dissected and single-cell suspensions were obtained. T-cells were magnetically sorted and were co-cultured with live B16 melanoma cells at different ratios of 10:1, 20:1, 40:1, 80:1 for 36 h. LDH activity in supernatants was detected. Bars, means ± SD. n = 3.