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Blood, Vol. 112, Issue 5, 1784-1793, September 1, 2008
STAT3 is required for IL-21–induced secretion of IgE from human naive B cells
Blood Avery et al.
112: 1784
Supplemental materials for: Avery et al
Files in this Data Supplement:
- Figure S1. Effect of IL-4 and IL-21 on the expression of cytokine receptors by CB B
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Left hand panels: CB B cells were labeled with mAb specific for CD20 and either (a) IL-4Rα or (b) IL- 21R. The solid outline histograms represent expression of IL-4R or IL-21R on CB B cells; the dashed histograms is the fluorescence intensity of cells labeled with an isotype control mAb. Middle and right hand panels: CB B cells were either unstimulated or cultured with CD40L alone or in the presence of IL-4 or IL-21. Expression of IL-4Rα and IL-21R were determined before culture (nil) or after 24 (middle) or 48 hours (right panel) of stimulation. The solid symbols represent the change in mean fluorescence intensity (ΔMFI; ie MFI specific mAb- MFI isotype control mAb) of expression of each cytokine receptor from two independent experiments; the shaded column is the mean.
- Figure S2. Kinetic of IL-21-induced phosphorylation of STAT3 (JPG, 80.9 KB)
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(A) CB B cells were cultured with CD40L alone (outline black histogram) or together with IL-21 (solid grey histogram). After different times, the cells were harvested, fixed and permeabilised and expression of phospho-STAT1, 3, and 5 determined by flow cytometry. (B) Controls for the anti-phosphoSTAT mAbs were: stimulation of U937 cells for 30 minutes with IFN-# (STAT1) or IL-6 (STAT3), or stimulation of CB MNC with PHA/IL-2 for 16 hours (STAT5). The solid histograms represent expression of phosphorylated STAT1, 3 or 5 in the presence of the appropriate stimulus; the outline black histograms represents expression of phosphorylated STATs in cells cultured without exogenous stimulus.
- Figure S3. Differential induction of expression of Igε germline transcripts, AICDA, and IgE secretion by CD40L, cytokines and anti-CD40 mAb (JPG, 53.4 KB)
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(A) CB B cells were cultured in medium alone (lanes 1-4) or with CD40L (lanes 5-8) in the absence or presence of IL-4, IL-21 or IL-4 and IL-21. After 5 days, RNA was extracted from the B cells, transcribed into cDNA, normalised for expression of the house-keeping gene GAPDH, and then used as template for the amplification of Igε germline transcripts and AICDA (encoding AID). Primers used were Igε GLT 5′: GAC GGG CCA CAC CAT CCA CAG GCA CC; 3′: CAG GAC GAC TGT AAG ATC TTC ACG; AICDA 5′: CCT GTG CTA CGT AGT GAA GAG GC; 3′: AAA GGA TGC GCC GAA GC. (B) B cells were cultured with CD40L, anti-CD40 mAb or anti-CD40 mAb plus CpG 2006 alone or in the presence of IL-4, IL-21 or IL-4 plus IL-21. IgE secretion was determined after 12 days by ELISA. The anti-CD40 mAb only induced IgE secretion when used in conjunction with IL-4 and CpG 2006.
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