Figure 5

Fludarabine eliminates IDO1 induced by MV-Edm and/or CD8⁺NKG2D⁺ cells leading to improved immune activation. (a) Tumour tissues were isolated from mice that received treatments as in Fig. 4. Cell lysates were obtained and IDO1 expression was detected by Western blot analysis. The average IDO1/GAPDH ratio was quantified by densitometric analysis. (b,c) LM3 cells were cultured in the presence or absence of 300 nM fludarabine for 24 h, and then (b) cells were infected with MV-Edm at a MOI of 1 for another 24 h, or (c) cells were treated with CD8⁺NKG2D⁺ cells at the ratio of 2:1 for another 24 h before cell lysates were harvested. Results are representative of three independent experiments. (d–f) 1 × 10⁷ LM3 cells were implanted subcutaneously into the flank of Balb/c nude mice. When tumours reached an average volume of 40 mm³, mice were randomized to five groups (n = 3 each group). Then mice were received intratumoural injection of MV-Edm (5 × 10⁶ PFU per mouse) on day 0, 1, 7 and were injected intravenously with CD8⁺NKG2D⁺ (1 × 10⁷ per mouse) on day 2 and 8, or injected with CD8⁺NKG2D⁺ cells on day 2 and 8, combined with or without intraperitoneal injection of fludarabine (0.75 mg per mouse) on day 2 and 8. Untreated mice were used as control. 3 days after the last treatment, mice were sacrificed and tumours were dissected. (d) Tumour lysates were obtained for IDO1 Western blot. The average IDO1/GAPDH ratio was quantified by densitometric analysis. (e) Single cell suspension obtained from tumours or (f) spleens was subjected to ELISPOT assay to quantify the human INF-γ-producing cells. Means + SD of three mice are shown. (g,h) LM3 cells were cultured in the presence or absence of (g) fludarabine (300 nM) or (h) 1-MT (100 μM) for 24 h followed by infection with or without MV-Edm (MOI = 1) for another 24 h, then cells were washed and harvested and mixed with CD8⁺NKG2D⁺ cells at a ratio of 2:1 (E:T) for 24 h. The number of INF-γ-producing cells was determined by ELISPOT assay. Means + SD of triplicates from two independent experiments are shown. * P < 0.05, ** P < 0.01.