www.sciencetranslationalmedicine.org/cgi/content/full/6/241/241ra80/DC1

Supplementary Materials for

IL-17 and GM-CSF Expression Are Antagonistically Regulated by Human T Helper Cells

Rebecca Noster, René Riedel, Mir-Farzin Mashreghi, Helena Radbruch, Lutz Harms, Claudia Haftmann, Hyun-Dong Chang, Andreas Radbruch, Christina E. Zielinski*

*Corresponding author. E-mail: christina.zielinski@charite.de

Published 18 June 2014, Sci. Transl. Med. 6, 241ra80 (2014) DOI: 10.1126/scitranslmed.3008706

The PDF file includes:

Fig. S1. GM-CSF+ TH cells coexpress less FOXP3 and GATA-3 compared to GM-CSF− TH cells. Fig. S2. Coexpression of GM-CSF with multiple other effector cytokines on the single-cell level in human TH cells ex vivo. Fig. S3. Existence of GM-CSF–producing TH cells, which are independent of IL-22 production. Fig. S4. The differential expression of CXCR3 and CCR4 distinguishes IFN-γ+ and IFN-γ− GM-CSF–producing TH cells. Fig. S5. Distinction of T-bet+ and T-bet− GM-CSF–producing TH cell subsets by surface markers. Fig. S6. Modulation of GM-CSF expression in distinct TH cell subsets. Fig. S7. TH2 cells are less susceptible to IL-12 stimulation compared to TH1 and TH17 cells as determined by STAT4 phosphorylation. Fig. S8. Stability and flexibility of GM-CSF–only T cell clones. Fig. S9. STAT4-dependent induction of GM-CSF by IL-12. Fig. S10. Evolutionary conserved transcription factor binding sites in the promoter region of the CSF2 gene. Fig. S11. Antagonistic regulation of GM-CSF expression by TH17 and TH1 polarization pathways.

Supplementary figures

Fig. S1. GM-CSF+ TH cells coexpress less FOXP3 and GATA-3 compared to GM-CSF−

TH cells. Flow cytometry of ex vivo isolated CD4+ TH cells. Data are representative of 5

experiments for GATA-3 and 4 experiments for FOXP3 co-staining.

Fig. S2. Coexpression of GM-CSF with multiple other effector cytokines on the single-

cell level in human TH cells ex vivo. Intracellular cytokine staining and flow cytometry

following PMA/ionomycin restimulation of freshly ex vivo isolated human CD4+ T cells.

Data are representative of at least 3 separate experiments for all tested cytokines.

Fig. S3. Existence of GM-CSF–producing TH cells, which are independent of IL-22

production. (A, B) Intracellular cytokine staining and flow cytometry following

PMA/ionomycin restimulation of freshly ex vivo isolated CD4+ T cells (A) and TH22 cells

(CXCR3–CCR4+CCR6+CCR10+). Data are representative of two experiments (B). (C)

Intracellular cytokine staining and flow cytometry following PMA/ionomycin restimulation

of CD4+CD45RA–CCR10+ T cell clones. The frequency of GM-CSF+IL-22+ and GM-

CSF+IL-22– T cell clones within the CD4+CD45RA–CCR10+ T cell population is variable

between different donors. (D) Naïve CD4+CCR7+CD25– TH cells were stimulated with CD3

and CD28 mAbs for 48 h and cultured for 5 days in the presence or absence of FICZ.

Intracellular cytokine staining and flow cytometry on day 5 following PMA/ionomycin

restimulation. Data are representative of two experiments.

Fig. S4. The differential expression of CXCR3 and CCR4 distinguishes IFN-γγγγ+ and

IFN- γγγγ– GM-CSF–producing TH cells. (A, B) Intracellular cytokine staining and flow

cytometry of freshly isolated memory T cells sorted CCR4+CXCR3– versus CCR4–CXCR3+

to demonstrate the existence of IFN-γ+ and IFN-γ– subsets within GM-CSF+ T cells. Cells

were rested for 16 h at 37°C after sorting before PMA/ionomycin restimulation. (B) Shown

are pooled data (mean ± SEM, n=3). *** p < 0.0001 (Student’s t-test).

Fig. S5. Distinction of T-bet+ and T-bet− GM-CSF–producing TH cell subsets by surface

markers. (A) Real-time PCR analysis of the expression of mRNA encoding TBX21 in freshly

isolated GM-CSF-only producing T cells (CXCR3–CCR4+CCR6–CCR10+) versus GM-

CSF+IFN-γ+ T cells (CXCR3+CCR4–) following 3h PMA/ionomycin restimulation (mean ±

SEM, n=3). (B) Intracellular staining of T-bet in the same T cell populations used in (A)

including CD45RO–CCR7+CD25– naïve TH cells. One representative experiment out of three

is shown.

Fig. S6. Modulation of GM-CSF expression in distinct TH cell subsets. (A, B) TH1, TH2

and TH17 cells were sorted as in Fig. 1A, stimulated with anti-CD3 and anti-CD28 for 48h

and cultured in the presence or absence of the indicated cytokines and analyzed by

intracellular staining and flow cytometry on day 5. (A) Gates indicate percentage of GM-

CSF+ cells in the control condition without exogenous cytokines. Shown is one representative

experiment. (B) Cumulative data of 7-9 experiments per TH cell subset. ** p< 0.005, * p<

0.05 (unpaired Student’s t test).

Fig. S7. TH2 cells are less susceptible to IL-12 stimulation compared to TH1 and TH17

cells as determined by STAT4 phosphorylation. TH cell subsets were isolated based on the

differential expression of chemokine receptors (TH1: CCR4–CCR6–CXCR3+, TH2:

CCR4+CCR6–CXCR3–, TH17: CCR4+CCR6+CXCR3–) and stimulated with IL-12 (8ng/ml) or

medium alone for 30 min before intracellular staining for pSTAT4 according to the

manufacturer’s recommendation (BD Biosciences). pSTAT4 (MFI) was determined by

dividing the geometric mean of pSTAT4 fluorescence intensity of IL-12 treated samples by

the geometric mean of pSTAT4 fluorescence intensity of untreated samples for each subset.

Data are presented as mean of three individuals donors, * p = 0.03 (TH1 vs. TH2), * p = 0.02

(TH17 vs. TH2) (unpaired Student’s t test).

Fig. S8. Stability and flexibility of GM-CSF–only T cell clones. Enzyme-linked

immunosorbent assay of cytokine secretion by GM-CSF-only producing T cell clones

following CD3 and CD28 mAb stimulation for 72 h and 3 h PMA/ionomycin stimulation in

the presence or absence of IL-12 (8ng/ml) (mean ± SEM, n=3). n.d., not detectable

Fig. S9. STAT4-dependent induction of GM-CSF by IL-12. Naïve TH cells were rested for

3 h in the presence or absence of the STAT4 inhibitor lisofyllin in serum free media and then

washed three times before stimulation with anti-CD3 and anti-CD28 in complete media for 48

h in the presence or absence of IL-12 (8ng/ml). Supernatant was harvested after a culture

period of 5 days and analyzed by ProcartaPlex (mean ± S.E.M, n=2).

Fig. S10. Evolutionary conserved transcription factor binding sites in the promotor

region of the CSF2 gene. Putative transcription factor binding sites in the Csf2 promotor

(~ 459 bp upstream of transcriptional start) were determined in silico by the rVista 2.0 tool

(http://rvista.decode.org) with a predefined cut-off matrix similarity value of 0.85.

Transcription factor sites are evolutionary conserved between humans and mice.

Fig. S11. Antagonistic regulation of GM-CSF expression by TH17 and TH1 polarization

pathways. Net GM-CSF expression levels by human TH cells are determined by the balance

of competing TH1 (promoting) and TH17 (inhibiting) polarization pathways.