Experimental procedures

2-1 Mice

Six- to eight-week old Wild type C57BL/6 mice from National Taiwan University

College of Medicine Laboratory Animal Center or from national laboratory animal

center were used as the source of CD4+ T cells.

2-2 Generation of the reconstituted c-Maf deficient mice

The donor cells for hematopoietic reconstitution were collected from fetal livers of

c-Maf deficient fetuses (C57BL/6J-CD45.1) on embryonic 14.5 day and were injected

into X-ray irradiated C57BL/6J-CD45.2 mice (7 Gy) by tail vein injection (5 x 106 cells

in 200 μl PBS per mouse). After two months of transplantation, the chimerisms of

recipient mice were analyzed by flow cytometry. The recipient mice which are

reconstituted by transplanted cells showed more than 95% hematopoietic cells from

donor cells.

2-3 Purification and differentiation of Th cells

CD4+ T cell were purified from spleen and peripheral lymph nodes of WT

CH57BL/6 mice by EasySep CD4 selection kit (STEM CELL CO) according to

manufacturer's instruction. On day 0, CD4 T cell were harvested and purified by the

selection kit, Purified T cells (2x10⁶ cells/ml) were cultured in RPMI complete medium

containing 10% FBS, 1X L-glutamine, 1X NEAA, 1X sodium-pyruvate, 1X

penicillin/streptomycin, 10 mM HEPES and beta-Mercaptoethanol, and stimulated with

plate-bond anti-CD3 (2 μg/ml), soluble anti-CD28 (2 μg/ml) for all cells condition, and

additionally with anti-IL-4 (10 μg/ml), IL-12 (1 ng/ml), IL-2 (100 U/ml) for Th1

condition, and anti-IFN-gamma (10 μg/ml), IL-4 (10 ng/ml), IL-2 (200 U/ml) for Th2

condition, and anti-IFN-gamma (10 μg/ml), anti-IL-4 (10 μg/ml), TGF-beta (2.5 ng/ml),

IL-6 (20 ng/ml), IL-23 (20 ng/ml) for Th17 condition, For Th2 differentiation,

additional IL-2 (200 U/ml) were added in Day2, for Th17 differentiation, additional

IL-23 (20 ng/ml), TGF-beta (2.5 ng/ml) , IL-6 (20 ng/ml), were added.

2-4 Retrovirus Preparation from HEK293T cells.

The retroviral plasmids including Gag-pol, Env, and expression vector were

co-transfected into HEK 293T cells by Meastrofectin (Omics Bio) transfection reagent

according to the manufacturer’s instructions. After 48, 72, and 96 hr, the

virus-containing medium was collected, and filtered with 0.22 μM filer, then PEG6000

and NaCl were added to the final concentration of 8.5% and 0.3 M respectively. After

shaking at 4^oC for 1 hr the virus containing medium was centrifuge at 7,000 X g for 10

min at 4^oC, the virus pellet was precipitated and re-suspend in PBS, than stored at -80℃

for future use.

2-5 Retroviral transduction

Beads-isolated CD4⁺ T cells were stimulated with plated-bound anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) antibodies with polarizing conditions for 48 hrs. Cells

were then infected with GFP-RV Mock, and GFP-RV WT, Y3F c-Maf retrovirus by

spin infection. Polybrene (Sigma) were added to the medium at final concentration of 8 μg/ml and centrifuged 2000 RPM for 1 hr at room temperature. After additional 30 to

60 min incubation at 37℃, the virus/polybrene containing medium were removed and

replaced by fresh complete RPMI medium with IL-2 200 U/ml for Th2 cells, or IL-23

(20 ng/ml) for Th17 cells. After 48 hours incubation, the transduced cells were sorted

by cell sorter according to the GFP expression. GFP⁺ cells were re-stimulated at 1x10⁶

cells/ml with plate-bound anti-CD3 antibody (1 μg/ml) for 24 hours. IL-21, IL-17

mRNA level and cytokine production in the supernatant were measured by real time

PCR or ELISA. The total cells and sorted cells can also use for ChIP assay.

2-6 ChIP assay

Formaldehyde was added directly to cell culture medium at a final concentration of

1% after PMA (50 ng/ml) and ionomycin (1 mM) stimulation to in vitro skewed

retroviral transduced Th2 and Th17 cells. Fixation proceeded at room temperature for

10-15 min on rocking platform shaker. Glycine was added to a final concentration of

0.125M to stop the reaction. The fixed cell were washed with PBS twice then

re-suspended in nuclei lysis buffer containing PMSF and protease inhibitor (50 mM

Tris-HCl pH 8.1, 10 mM EDTA, and 1% SDS) and incubated on ice for 10 min, then

subjected to sonication. The sheared DNA fragments should range in size from 200-600

base pairs. The sheared chromatin extract was then frozen in aliquots at –80 ℃ until

required. After sonication, the samples were centrifuged at 15,000 g for 10 min at 4℃

and transfer the supernatant into a new eppendorf. Use 50 μl sample as input, and 200 μl

for IP or control. Normal mouse IgG (Santa Cruz) and Monoclonal anti-Flag antibody

(Sigma), were add to sample to immunoprecipitate the chromatin and incubated

overnight at 4℃. After incubation, 60 μl washed 50% protein G-sepharose beads were

added to sample for additional 1-2 hours at 4℃. The sepharose beads were washed four

times with IP wash buffer (100 mM Tris pH9.0, 500 mM LiCl, 1% NP-40 and 1%

deoxycholic acid), then eluted twice with 150 μl IP elution buffer (100 mM NaHCO3

1% SDS) by vortex for 15 min each time. Adjusted NaCl to 0.3 M final concentration

with 1 μl RNase A (10mg/ml, Sigma) per sample, then incubated overnight at 65℃.

After incubation, 2 μl proteinase K (20mg/ml) were added to each sample, and

incubated at least 2 hours at 50℃. After proteinase K digestion, DNA was extracted by

phenol/chloroform/isoamyl alchol or Small DNA Fragments Extraction Kit (Geneaid

Co.), and then analyzed by real-time PCR analysis.

2-7 Western Blotting

Beads-isolated CD4⁺ T cells were stimulated with plated-bound anti-CD3 and

anti-CD28 antibodies with polarizing conditions for 48 hours, then add additional

cytokine for 48 hours. The cells were harvested on day 4 followed by re-stimulation

with 50 ng/ml PMA and 1 μM ionomycin for indicted hours. Whole cell extracts were

fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane

using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad). After

incubation with 4% nonfat milk or BSA in TBST (10 mM Tris, pH 8.0, 150 mM NaCl,

0.5% Tween 20) for 1 hour, the membrane was washed three times with TBST and

incubated with antibodies against c-Maf (1:2000 Santa Cruz) or Tubulin (1:5000), at

4°C for 12-16 hours. Membranes were washed three times for 5-10 min and incubated

with a 1:5000-1:10000 dilution of horseradish peroxidase-conjugated anti-mouse or

anti-rabbit light chain antibodies for 1 hour. Blots were washed with TBST three times

and developed at ECL system (Omics Biotechnology) according to the manufacturer’s

protocols.

2-8 Immunoprecipitation and Immunoblot Analyses

For immunoprecipitation, Th2 and Th17 cells were harvested on day4 followed by

re-stimulated with 50 ng/ml PMA and 1 μM ionomycin for 4 hours. The total cell

extract was prepared by lysing the cell pellet with RIPA buffer (50 mM Tris-HCl, pH

8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium

deoxycholate, and 0.1% sodium dodecyl sulfate). After incubation, cell lysate was

centrifuged at 15,000 g for 10 min. Cell lysate concentration were detected by

PierceTM BCA Protein Assay Kit according to the manufacturer’s protocols (Thermo).

500 μl of cell lysate containing 500 μg were precleared with 40μl of protein

A-sepharose beads (50% slurry) for 2h. The supernatant was then incubated with 0.8 μg

of c-Maf antibody or control antibody at 4°C for 12-16 hours followed by incubation

with 30 μl protein A-sepharose beads with gentle rocking. Beads were washed with

RIPA lysis buffer for three times and target proteins were eluted with 2x SDS loading

dye, boiled at 100°C for 5 min, and analyzed by Western blotting. The blot were

detected by anti-c-Maf antibody (Santa Cruz.) or HRP conjugated 4G10

(anti-phosphotyrosine antibody). The signals were detected by ECL system according to

the manufacturer’s protocols.

2-9 ELISA assay

For the ELISA assay, CD4⁺ T cells were sorted form WT B6 mice, and skewed to

Th17 conditions. 48 hours later, the primary cells were transduced with GFP-RV Mock,

or either WT and Y3F c-Maf. After additional 48 hours, retroviral transduced cells were

sorted according to the GFP expression, and re-stimulated with plated bond anti-CD3 1 μg/ml for 24 hours. IL-21 and IL-17 production were measured by eBioscience ELISA

Ready-SET-Go kit according to manufacturer's instruction.