Functionally relevant signaling intermediates contributing to GM-CSF licensing include PI3K, pAKT, pmTOR, pS6, and p4E-BP1. (A-B) Western blot analyses for the indicated markers of whole cell lysates from fresh BM cells or L-Mono optionally treated with IFN-γ, LPS, or IFN-γ/LPS for 1 hour. p, phosphorylated forms of the indicated marker (for quantification and statistics, see supplemental Figure 3). (C) Expression of the indicated phosphorylated markers in Ly-6G+ granulocytes and Ly-6Chigh monocytes from fresh BM or 3-day GM-CSF cultured BM cells analyzed by FACS. Example of 2 independent experiments each with BM from 2 mice, n = 4 or 5. (D-E) Suppression of T-cell proliferation by murine L-Mono generated from days 0 through 3 in the presence of the PI3K inhibitors Wortmannin or Ly294002, or the mTOR inhibitor rapamycin at the indicated concentrations. One representative of 3 (D) or 2 (E) independent experiments is shown. Statistics by comparison of treated vs L-Mono cells with 10 µM inhibitors. (F) Murine BM cells cultured in GM-CSF in the presence of the different STAT inhibitors (inh.) were titrated into CD3/CD28 stimulated T cells for proliferation. Data represent means ± SD of 2 independent experiments. All statistics by unpaired Student t test by comparing STAT3 treatment to the untreated controls (L-Mono) ***P < .001.
