PMC5347118_01
Chest CT of patient prior to treatment with mycophenolic acid showing multiple infiltrates.
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F1
His CT scan showed multiple nodules throughout the lung (Figure 1).
Patient has decreased T regulatory cell (T reg) numbers and expresses less CD25. (A) Flow cytometric analysis of T reg populations, gated on CD4+ T cells. (B) T regs have decreased CD25 expression, gated on CD4+ FOXP3+ CD127low cells. Dashed line shows naive (CD45RO negative) T cells from a healthy control to show negative CD25 staining. Experiments were repeated at least twice with at least two healthy controls for each experiment.
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F2
T cells with decreased CD25 expression (Figure 2). There were variable responses of phosphorylation of STAT (pSTAT) proteins to cytokine stimuli, which did not lead to a clear pattern (Figure 3).
Phosphorylation of STAT1 and STAT5 in response to stimulus. Peripheral blood mononuclear cells from the patient and a healthy control were stimulated with IL-6 (25 ng/mL) (A) and IL-2 (200 ng/mL) (B) for 20 min. The cells were fixed using 4% paraformaldehyde and permeabilized with methanol, then stained with antibody and analyzed by flow cytometry. Cells were gated on CD4+ (A) or CD4+, pSTAT5+ (B) cells. Dashed line shows unstimulated cells from a healthy control. Experiments were repeated at least twice with at least two healthy controls for each experiment.
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F3
T cells with decreased CD25 expression (Figure 2). There were variable responses of phosphorylation of STAT (pSTAT) proteins to cytokine stimuli, which did not lead to a clear pattern (Figure 3).
p.G419R STAT3 variant shows increased STAT3 activity at baseline. Wild-type (WT) or mutant STAT3 plasmids were co-transfected into STAT3-deficient cells along with a STAT3-responsive luciferase reporter, followed by measurement of luciferase at 48 h. Averages shown are from four independent experiments.
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F4
GOF activity was confirmed using a luciferase assay, as previously reported (Figure 4).