Flt3 receptor expression and mTOR signaling in pDC and cDC subsets. (A) Effect of increasing rapamycin dose on pDC and cDC numbers. DO11.10-tg x Rag-2^−/− BALB/c mice (n = 4 per group) were treated three times per week for 3 weeks IP with Flt3L/OVA_323-339/increasing doses of rapamycin: 4 mg/kg (1× rapa), 8 mg/kg (2× rapa) and 16 mg/kg (3× rapa). Total numbers of pDCs and cDCs were enumerated and statistical differences compared by 1-way ANOVA with Dunnett’s multiple comparison posttest using naïve animals as the control group, against which all other treatment groups were tested. (B) Histogram overlays showing differential p-mTOR^Ser2448 expression in splenic pDCs and cDCs of naïve DO11.10-tg x Rag-2^−/− BALB/c mice on in vitro incubation for 60 minutes with Flt3L (red histogram), rapamycin (blue histogram), or a combination of Flt3L/rapamycin (purple histogram). Shown is 1 representative of 2 independent experiments. (C) Graphical representation of p-mTOR^Ser2448 expression in pDC and cDC populations from panel B. Data show showing percent positive cells for each treatment, with histogram subtraction applied against the control group, which represents unstimulated cells. Histogram subtraction was applied using FCS express 4.0 software. The graphs represent data from 2 independent experiments. One-way ANOVA with Tukey’s multiple comparison test was used to calculate significance. (D) Representative western blot images of splenic pDCs and cDCs probed for p-mTOR^Ser2448 (upper) and actin (lower). Flow-sorted pDCs and cDCs from spleens of naïve, non-pretreated DO11.10-tg x Rag-2^−/− mice (top) or mice that received repeated Flt3L injections for 10 days (bottom), were serum starved for 2 hours and treated for 60 minutes with 2 μg/mL Flt3l, 100 nM rapamycin, or a combination of Flt3L/rapamycin. Images for pmTOR^Ser2448 and β-actin were analyzed by the ImageJ densitometric software. Normalized relative density of pmTOR to β-actin is represented for both sets of western blot images.