1. Immunity, Volume 33
Supplemental Information
Interferon-Regulatory Factor 4 Is Essential
for the Developmental Program of T Helper 9 Cells
Valérie Staudt, Evita Bothur, Matthias Klein, Karen Lingnau, Sebastian Reuter, Nadine Grebe, Bastian
Gerlitzki, Markus Hoffmann, Alexander Ulges, Christian Taube, Nina Dehzad, Marc Becker, Michael
Stassen, Andrea Steinborn, Michael Lohoff, Hansjörg Schild, Edgar Schmitt, and Tobias Bopp
Figure S1, related to Figure 1: IRF4­deficiency re­directs CD4+ T cells to T helper 1
phenotype
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2. (A) Naïve CD4+ T cells from Irf4+/‐ or Irf4‐/‐ mice were stimulated under Th9‐skewing
conditions. Messenger RNA was prepared 24 h and 72 h upon stimulation and analyzed
for the expression of the house keeping gene HGPRT and the transcription factor T‐bet
by quantitative RT‐PCR. Shown are mean values of two independent experiments +/‐
SEM. (B) To assess IFN‐γ production, naïve CD4+ T cells from Irf4+/+ and Irf4‐/‐ mice were
stimulated under Th9‐promoting conditions. On day 6, cells were re‐stimulated and IFN‐
γ expression was analyzed by FACS analyses.
Figure S2, related to Figure 2: Th9 development is accompanied by a strong
expression of IRF4
Naïve CD4+ T cells were isolated from spleens of C57Bl/6 mice and subsequently
stimulated in vitro under Th1‐, Th2‐ and Th9‐promoting conditions. The expression of
IRF4 was analyzed by FACS analyses directly after preparation (0 h) and 24 h, 48 h and
72 h upon stimulation. Shown is one representative of three independent experiments
+/‐ SEM.
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3.
Figure S3, related to Figure 3: Continuous IRF4 expression is crucial for the
maintenance of a stable Th9 phenotype
Naïve Irf4+/+ CD4+ T cells were stimulated under Th9‐promoting conditions for three
days and subsequently transfected with a specific IRF4 siRNA (IRF4‐siRNA) or the
respective scrambled control (Scr‐siRNA). (A) Immunoblot of IRF4 expression and β‐
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4. actin as loading control. Upon re‐stimulation on day 5 for 24 h the ability to produce IL‐9
(B) and IFN‐γ (C) was assessed. Shown are mean values +/‐SEM. n.d. = not detectable.
Figure S4, related to Figure 6: Experimental design of the adoptive transfer
asthma model
Rag2‐/‐ Mice were adoptively transferred with in vitro differentiated Th9 cells or Th2
cells on day one. Thirty minutes prior to OVA nebulization on days 1‐6 mice were
treated intra nasally with 150µg of an IL‐9 antibody (229.4). Airway function, BAL and
histology were assessed 24 h after the last OVA provocation.
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5. Figure S5, related to Figure 7: Experimental design of the primary challenge
asthma model
C57Bl/6 Wild type, Irf4+/‐ and Irf4‐/‐ mice were sensitized by i.p. injection of Ovalbumin
emulsified in aluminium hydroxide (OVA/ Alum) on day 0 and day 14. On day 27 mice
received either PBS or Th9 cells derived from OT‐II mice via i.v. injection. From day 28
onwards mice were challenged (20min) by Ovalbumin nebulization on three
consecutive days and assays were performed 24 h after the last challenge.
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6. Figure S6, related to Figure 1: The peak of IL­9 mRNA production is accompanied
by a strong expression of IRF4.
Naïve CD4+ T cells from C57Bl/6 mice were stimulated under Th2‐ or Th9‐skewing
conditions. Messenger RNA was prepared prior to stimulation or 24 h, 48 h and 72 h
upon stimulation and analyzed for the expression of IL‐9 by quantitative RT‐PCR (A).
Additionally, IRF4 expression was determined by FACS analyses (B). Shown is one
representative of four independent experiments +/‐ SEM.
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