STAT1 and STAT3 phosphorylation in hematological patients with IMI.

<p>(a-d) IFN-γ-induced STAT1 phosphorylation and IL-6-induced STAT3 phosphorylation were measured in peripheral blood mononuclear cells using phospho-flow. (a) On the <i>left</i>, representative dot plots for gating of naïve and memory CD4+ T cells are shown; gating of T cells (CD3+) or monocytes (CD33+, not shown) was done on CD45^high cells in order to avoid interference of the analysis by potential blasts in leukemic patients with residual disease. On the <i>right</i>, representative histograms of the levels of phosphorylated STAT (pSTAT) proteins in non-stimulated (NS) and cytokine-stimulated cells are shown; baseline pSTAT levels are shown in grey, IFN-γ-induced pSTAT1 is shown in purple and IL-6-induced pSTAT3 is shown in orange. (b) Representative histograms of IL-6 induced pSTAT3 (top row) and IFN-γ-induced pSTAT1 (bottom row) in different patient groups. Y axis corresponds to number of events (i.e. number of naïve CD4+ T cells) and X axis corresponds to fluorescence intensity (i.e. pSTAT-Alexa Fluor 488). (c) Percentage of monocytes (CD45^highCD33+ cells), naïve T helper (Th) cells (CD45^highCD3+CD4+CD45RO- cells) and memory Th cells (CD45^highCD3+CD4+CD45RO+ cells) expressing pSTAT3 (top row) and pSTAT1 (bottom row) in response to IL-6 and IFN-γ, respectively, in healthy controls (blue circles; n = 7), hematological patients with IA (red squares; n = 13) or mucormycosis (black inverted triangles; n = 4) and non-IFI hematological controls (green triangles; n = 15). *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.005 using the unpaired two-tailed Student’s <i>t</i>-test. Data are shown as mean ± s.e.m. (d) Heat map for log2 scale of mean fluorescence intensity (MFI) fold change. Fold change was calculated by dividing the MFI of the cytokine-stimulated sample by that of the unstimulated sample. Heat map color scale is showed in the bottom. Each row on the heat map corresponds to an individual patient or control as indicated by the study ID number on the left. Each column on the heat map corresponds to specific cell type/pSTAT as indicated on the top. Note reduced IL-6-induced pSTAT3 in monocytes (<i>p</i> = 0.01 for IA vs. healthy controls and <i>p</i><0.05 for IA vs. non-IFI) and naïve CD4+ T cells (<i>p</i> = 0.006 for IA vs. healthy controls and <i>p</i> = 0.04 for IA vs. non-IFI) from patients with IA, using unpaired two-tailed Student’s <i>t</i>-test.