Supplemental Information Interleukin-4 Production by Follicular … · 2012. 2. 23. ·...

P. Vijayanand, G. Seumois, et al. – HS V is indispensable for Tfh function. 1

Immunity, Volume 36

Supplemental Information

Interleukin-4 Production by Follicular Helper T Cells

Requires the Conserved Il4 Enhancer

Hypersensitivity Site V Pandurangan Vijayanand, Grégory Seumois, Laura J. Simpson, Sarah Abdul-Wajid, Dirk Baumjohann, Marisella Panduro, Xiaozhu Huang, Jeneen Interlandi, Ivana M. Djuretic, Daniel R. Brown, Arlene H. Sharpe, Anjana Rao, and K. Mark Ansel

Inventory of supplemental items 1. Supplemental Figures and Tables

Figure S1 shows the details of the targeting construct used for generating the knockout mice described in Figure 1. Figure S2A shows replicates of experiments shown in Figure 2D and 2E; S2B shows intracellular cytokine staining of restimulated CD4+ T cells that are shown in Figure 2E. Figure S3A and B shows the raw data from independent experiments that are represented as means of normalized data in Figure 7B. Figure S3C shows the raw data from independent experiments in Tfh cells that are represented as means of normalized data in Figure 7D. Figure S3D shows H3K27me3 ChIP-PCR results in Th1 cells for comparison with results shown in Figure 7D. Figure S3E shows H3K4me2 and H3K27me3 ChIP-PCR results in control genes for the experiments shown in Figure 7C and D. Figure S4 shows the gating strategy used for detecting and/or isolating lung infiltrating basophils and eosinophils shown in Figure 6. Table S1 displays the antibody cocktail used for detecting lung infiltrating CD4+ T cells, basophils and eosinophils shown in Figure 6. Table S2 shows the primer sequences used for real-time PCR quantification of mRNA and DNA sequences shown in Figure 2,4 and 7 respectively.

2. Supplemental Experimental Procedures 3. Supplemental References


Figure S1: Generation of HS-V-deficient mice (related to Figure 1).

Figure S1. Generation of HS V-deficient mice. (A) Schematic diagram of wild type Il4 locus (+), HS V targeting construct, targeted allele containing loxP-flanked PGK promoter driven neomycin resistance gene (neo), and HS V-deficient allele used in experiments (–). BamHI (B) and EcoRI (E) cleavage sites and location of probes used in diagnostic Southern blots and DNaseI hypersensitivity (DHS) assays are shown. DHS regions IV, VA, and V are indicated with horizontal bars. Double arrows at top indicate the regions deleted in HS V- deficient mice and the previously reported CNS2 and HS V/VA- deficient mice. (B-C) Southern blot of BamHI-digested genomic DNA from wild type (+/+) and targeted HS V (+/neo) embryonic stem (ES) cells using 3’ probe (B) or 5’ probe (C). (D) Targeted ES cells were transiently transfected with a Cre recombinase expression vector (pMC-CreN; kindly provided by M.E. Keir and G. Freeman, Harvard Medical School) to induce neo cassette excision. Southern blot of EcoRI-digested genomic DNA from untransfected targeted (+/neo), Cre-transfected targeted HS V heterozygous (+/–), and wild type untargeted (+/+) ES cells probed with 3’ probe.

23kb

10 8 6 5

+/+ +/+

+/neo

+/neo

3’ probe

19kb (+)

5.3 (neo)

BamHI digestion

5’ probe

23kb

10 8 6 5

+/+ +/+

+/neo

+/neo

15 (neo) 19kb (+)

BamHI digestion

6kb

5

+/neo

+/– +/+

5.1 (neo) 5.4kb (+)

4.8 (–)

3’ probe

EcoRI digestion

BB

B BB

ex4 ex3

Il4 Kif3a

loxP loxP

VVA IV

Cre BB

5’ probe (DHS probe) 3’ probe

E

EE

E E

E

HSV-TK

VA IV

VA IV

HS V/VA KO (Solymar et al 2002)

HS V KO

Targeting construct

+

neo

–

CNS-2 KO (Yagi et al 2007)

A

B C D

PGK-neor

PGK-neor


Figure S2: Effect of HS V-dependent reduction in IL-4 on Th2 polarization (related to Figure 2D-F).

IL-4

IFN!

KN

2-WT

KN

2-V

51 2.7

43 2.4

0 1 3 11 33 100

IL-4 dose (U/ml)

49 4.4

40 5.3

46 4.5

43 5.3

12 4.3

48 35

13 9.7

35 41

11 4.7

55 28

32 0.6

66 0.3

51 1.2

47 0.6

50 1.3

48 0.6

46 2.8

48 2.8

28 7.9

42 22

15 6.8

51 28

B

Figure S2. Effect of HS V-dependent reduction in IL-4 on Th2 polarization.

(A) CD4+ T cells from the indicated mice were differentiated in vitro under sub-maximal Th2 polarizing conditions. Graphs shows the percentage huCD2+ cells in relation to the concentration of exogenous IL-4 added to the culture. (B) CD4+ T cells were differentiated in vitro under sub-maximal Th2 polarizing conditions by titrating down the concentration of exogenous IL-4 added to the culture. Contour plots showing intracellular staining of cytokines (IL-4 and IFN-γ) in restimulated CD4+ T cells from the above conditions.

80

60

40

20

0

KN2-KN2 KN2-WT

40

30

20

10

0 100 1 3 0 11 33

IL-4 dose (U/ml)

KN2-V KN2-WT

A

huC

D2+

cel

ls (%

)


Figure S3: Chromatin accessibility and NFAT binding in the Th2 locus of HS V deficient T cells.

(related to Figure 7).

0.0

0.6

1.2

0.0

0.4

0.8

0

1

2

3

IL4p IFN!p Va IL4p IFN!p Va0.00

0.03

0.06

Unstimulated Stimulated

H3K27me3

Naive T cells

2.4

1.6

0.8

0.0 Th1 polarized T cells

1.6

0.8

0.0 IL4p Va

Il4

V IV

Kif3a

Chr 11

ChI

P-r

ecov

ery

(% o

f inp

ut)

WT !V

D

IFN!p Gapdhp

H3K4me2 H3K27me3

IFN!p Gapdhp

ChI

P-r

ecov

ery

(% o

f inp

ut)

IFN!p Gapdhp

Follicular helper T cells

Naïve T cells

E

2.4

0.0

0.5

1.0

0

2

40

7

14

0

8

16

IL4p IFN!p0.000

0.008

0.016

0.0240.00

0.03

0.060.00

0.02

0.040.00

0.05

0.10

0.15NAIVE T CELLS

Exp 1

Exp 2

Exp 3

Exp 4

NFA

T C

hIP

-rec

over

y (%

of i

nput

)

B

0.0

0.9

1.8

0.0

0.9

1.8

IL4p IV Va V IFN!p Gapdhp0.0

1.2

2.4

Il4 Kif3a

Chr 11

FOLLICULAR HELPER T CELLS Exp 1

Exp 2

Exp 3

Exp 4

C

H3K

27m

e3 C

hIP

-rec

over

y (%

of i

nput

)

0.00

0.13

0.26

0.00

0.09

0.18

0.00

0.08

0.16

Th2 POLARIZED T CELLS Exp 1

Exp 2

Exp 3

Exp 4

A

WT !V

WT !V

WT !V

WT !V

Th2 polarized T cells


Figure S3. Chromatin accessibility in the Th2 locus of HS V deficient Th1 cells. (A-B) ChIP-PCR analysis: real-time PCR quantification of Il4 promoter (IL-4p), HS VA and Ifng promoter (IFNgp) sequences following ChIP with antibody to NFAT in WT and HS V-deficient Th2 cells (A), either left unstimulated or stimulated for 45 minutes with PMA and ionomycin, and in naïve T cells (B) stimulated under similar conditions. Data are expressed as the percentage of input DNA recovered from 4 independent ChIP experiments.

(C) Real-time PCR quantification of Il4 promoter (IL-4p), HS IV, HS VA and HS V sequences following anti-H3K27me3 ChIP of chromatin extracts obtained from resting Tfh cells derived in vivo from wild type (WT) and HS V-deficient (ΔV) mice following LCMV infection. Data are expressed as the percentage of input DNA recovered from 4 independent ChIP experiments.

(D) ChIP-PCR analysis: real-time PCR quantification of Il4 promoter (IL-4p), HS IV, HS VA and HS V sequences following anti-H3K27me3 ChIP of chromatin extracts obtained from resting CD4+ Th1 cells derived in vitro from wild type (WT) and HS V-deficient (ΔV) mice. Data are expressed as the percentage of input DNA recovered and are representative of at least 3 independent ChIP experiments.

(E) Real-time PCR quantification of Ifng promoter (IFNγp) and Gapdh promoter (Gapdhp) sequences following anti-H3K4me2 and anti-H3K27me3 ChIP of chromatin extracts obtained from resting CD4+ naïve T cells and Th2 cells derived in vitro from wild type (WT) and HS V-deficient (ΔV) mice. Data are expressed as the percentage of input DNA recovered and represent mean and SEM (error bars) of atleast 3 independent ChIP experiments.


Figure S4: Gating strategy for detecting basophils and eosinophils infiltrating the lungs (related to Figure 6).

MH

CII-

eFlu

or 4

50

CD

3-Pe

rCP-

Cy5

.5

Sing

let

CD

45+

MH

CII-

DX5

+ CD

3- C

D13

1+ Ig

E+ FS

C-S

SC

FSC-W

SSC-A

CD11b - Al 700

DX5 – Qdot 605

CD131 - PE

SSC-A FSC

-A

IgE

- FIT

C

CD

45-A

PC-C

y7

FSC

-A

MH

CII-

eFlu

or 4

50

Sing

let

CD

45+

CD

3- C

D11

b+ MH

CII-

Si

glec

F+ C

D11

c- FS

C-S

SC

FSC-W

SSC-A

SSC-A

CD11b-Al 700

Siglec F - PE

SSC-A FSC

-A

CD

11c

- FIT

C

CD

3-Pe

rCP-

Cy5

.5

CD

45-A

PC-C

y7

FSC

-A

SALI

NE

O

VALB

UM

IN

SALI

NE

O

VALB

UM

IN

Siglec F - PE

CD

11c

- FIT

C

huCD2 – PE-Cy7

IL-4

- AP

C

OVA-CHALLENGE MODEL GATING STRATEGY

BASOPHILS EOSINOPHILS

BASO

PHIL

S EO

SIN

OPH

ILS

A B


Figure S4. Gating strategy for detecting basophils and eosinophils infiltrating the lungs. (a) Contour plots outline the gating strategy used for detecting and/or isolating basophils and eosinophils infiltrating the lung of mice that were subjected to the ovalbumin (OVA) model of allergic airway inflammation. (b) Displays contour plots of lung infiltrating basophils and eosinophils following saline or ovalbumin challenge in vivo.


Table S1: FACS staining panels (related to Figure 6).

Antibody Fluorophore Clone Company

LYMPHOCYTE STAINING PANEL - LUNG anti-mouse B220 FITC RA3-6B2 eBiosciences anti-mouse IL-13 PE eBio13A eBiosciences anti-mouse CD3 PerCP- Cy5.5 145-2C11 eBiosciences anti-mouse IL-4 APC 11B11 eBiosciences anti-human CD2 PE-Cy7 L303.1 BD Biosciences anti-mouse CD4 APC-Qdot 750 RM4-5 eBiosciences anti-mouse CD11b Alexa Fluor 700 M1/70 eBiosciences anti-mouse CD8 efluor 450 53-6.7 eBiosciences

BASOPHIL STAINING PANEL - LUNG anti-mouse IgE FITC RME-1 Biolegend anti-mouse CD131 PE JORO50 BD Biosciences anti-mouse CD3 PerCP- Cy5.5 145-2C11 eBiosciences anti-mouse CD49B APC DX-5 eBiosciences anti-human CD2 PE-Cy7 L303.1 BD Biosciences anti-mouse CD45 APC-Cy7 30-F11 Biolegend anti-mouse CD11b Alexa Fluor 700 M1/70 eBiosciences anti-mouse MHCII efluor450 M5/114.15.2 eBiosciences

EOSINOPHIL STAINING PANEL - LUNG anti-mouse CD11c FITC N418 eBiosciences anti-mouse Siglec-F PE E50-2440 BD Biosciences anti-mouse CD3 PerCP- Cy5.5 145-2C11 eBiosciences anti-mouse IL-4 APC 11B11 eBiosciences anti-human CD2 PE-Cy7 L303.1 BD Biosciences anti-mouse CD45 APC-Cy7 30-F11 Biolegend anti-mouse CD11b Alexa Fluor 700 M1/70 eBiosciences anti-mouse MHCII efluor 450 M5/114.15.2 eBiosciences


Table S2: ChIP PCR primers (related to Figure 2 and 7).

mRNA (intron spanning primers) Hprt1 5' CTGGTGAAAAGGACCTCTCG 5' TGAAGTACTCATTATAGTCAAGGGCA Il10 5' GGTTGCCAAGCCTTATCGGA 5' ACCTGCTCCACTGCCTTGCT Il4 5' TTTGGCACATCCATCTCCG 5' AGATCATCGGCATTTTGAACG

Il13 5' GGCCAGGTCCACACTCCATA 5' GCTTATTGAGGAGCTGAGCAACA Il5 5' TGAAAGAGACCTTGACACAGC 5' GGGACAGGAAGCCTCATC ll2 5' CTTTCAATTCTGTGGCCTGC 5' CAGCTGTTGATGGACCTACAG

Ifng 5' GGATGCATTCATGAGTATTGC 5' CCTTTTCCGCTTCCTGAGG Il21 5’ GCTCCACAAGATGTAAAG 5’ CCACGAGGTCAATGATGAATGTC Bcl6 5’ ATGGAGTGTTCCTCGCCCTTTC 5’ CCCACCCCAACTATGATTTGC

Blimp1 5’ ACATAGTGAACGACCACCCCTG 5’ CTTACCACGCCAATAACCTCTTTG Icos 5’ CAGGAGAAATCAATGGCTCGG 5’ TTGGTCTTGGTGAGTTCGCAG Sap 5’ CACATATCGAGTGTCCCAGAC 5’ TGATCCGGCTTCTGAAACG

Promoter regions Gapdh 5' GCGCGAAAGTAAAGAAAGAAGCCC 5' AGCGGCCCGGAGTCTTAAGTATTAG

Ifng 5' GCTTTCAGAGAATCCCACAAGAAT 5' GCTATGGTTTTGTGGCATGTTAGA Il4 5' GGCCCAGAATAACTGACAATCT 5' GCAATGCTGGCAGAGGTC

non coding DNA regions HS IV 5' TGCCCACATGAAATACCAGC 5' GCATACCTTCCCTGATTGGC HS V 5' AAAGTGACTTGAAGGTTGGGTCGC 5' TGTGTGGGAAAGTGATCGTGAGGA HS Va 5' GACTGAGAACCCAACAGAGATGC 5' GCCTTGGTTCCATATATTCATGACT


SUPPLEMENTAL EXPERIMENTAL PROCEDURES Mice and gene targeting.

C57BL/6J and BALB/cJ mice were purchased from The Jackson Laboratory. Unless otherwise

indicated, sex-, age- and strain-matched wild-type control mice were used in all experiments. HS V-

deficient mice were generated using standard gene-targeting techniques. A targeting vector was

constructed in pLNTK to delete a highly conserved 810 bp region marked by HS V (Figure S1A).

Homology arms flanking this region were cloned from a 4.8 kb PsiI restriction fragment and a 2.0 kb

PCR product (primers VKO SA F, TAGTAGCTCGAGCCTCTGCCTGTGATCTCAG and VKO SA R,

TAGTAGCTCGAGTGTGATCTTAAGTCTTGGCTT). C57BL/6 mouse embryonic stem cells (Bruce-

4) were transfected with the linearized targeting vector, selected with G-418 (200 µg/mL), and screened

for homologous recombinants by Southern blot (Figures S1B, S1C and S1D). Successfully targeted ES

clones were injected into BALB/c blastocysts, and the resulting chimeric mice were bred to C57BL/6

mice to generate HS V+/neo offspring. A CMV promoter-driven Cre transgene (B6.C-Tg(CMV-

cre)1Cgn/J) was used to excise the loxP-flanked neomcyin resistance cassette in the germline,

generating HS V+/– mice on a pure C57BL/6 strain background. HS V+/– mice were speed-backcrossed

(The Jackson Laboratory) to a pure BALB/cJ background, interbred to homozygosity, and bred with

KN2 mice (Il4tm1(CD2)Mmrs; kindly provided by R.M. Locksley, UCSF) to produce KN2-V allelic reporter

mice.

Multispecies sequence comparison. Multispecies sequence alignment was done using comparative

genomic tools (based on a phylogenetic hidden Markov model, phastCons; available in the UCSC

Genome Browser). The genome assembly 2006 (NCBI 36/mm8) was used.

DNase I hypersensitivity analysis and Southern blots. Isolation and DNase I digestion of nuclei,

purification of genomic DNA and Southern blots were done as described (Agarwal et al., 2000; Agarwal


and Rao, 1998; Ansel et al., 2004). 5’ DHS probe was described in Agarwal et al., 1998. A 278 bp 3’

probe was PCR amplified using primers GCTAACACCAGCAGCCTGC and

GGTCATGTGCTTCCTAAACCCA.

T cell differentiation and FACS analysis. Purification of CD4+ T cells from spleen and lymph nodes,

in vitro induction of Th1 and Th2 differentiation, and, restimulation for flow cytometric analysis of

intracellular cytokine staining and messenger RNA expression levels were performed as described

previously (Ansel et al., 2004). In brief, purified CD4+ T cells were stimulated with hamster anti-mouse

CD3 (clone 2C11, 0.25 µg/ml) and hamster anti-mouse CD28 (clone 37.51, 1 µg/ml) on plates coated

with goat anti-hamster IgG (MP Biomedicals) for 48–60 h under Th1 (IL-12 and anti–IL-4) and Th2

(IL-4, anti–IFN-γ and anti–IL-12) or nonpolarizing conditions. After 2-3 days, cells were removed from

the plates and expanded in media with 20 U/ml of recombinant human IL-2 (National Cancer Institute)

and analyzed on day 6. For short term stimulation, 5x106 naive T cells were resuspended in media

containing 0.5 µg/ml anti-CD3 and 1 µg/ml anti-CD28 and mixed with 2.5x107 latex beads (5µm

diameter; Interfacial Dynamics Corporation) coated with goat anti-hamster IgG. Unstimulated controls

were cultured with beads but without anti-CD3 and anti-CD28 and were similar to cells held on ice.

Quantification of cytokine messenger RNA (mRNA) expression levels. Total RNA was isolated from

resting or stimulated naive, TH1, TH2 cells and TFH cells, and quantitative RT-PCR was done as

described (Ansel et al., 2004) using a Realplex 2S thermocycler (Eppendorf).

Isolation and flowcytometric analysis of immune cells present in lungs and lymph nodes. Lungs,

well perfused with at least 50 ml of saline, were cut into small pieces and dispersed in Collagenase A

(2mg/ml; Roche), Elastase (0.5mg/ml; Worthington) and DNAse (50U/ml; Sigma) for 30 minutes at 37

°C. Staining for flow cytometry was performed using fluorophore-conjugated antibodies against B220,

CD3, CD4, IL-4, IFN-γ, IL-13, human CD2, CD11b, CD8, IgE, CD131, CD49b, CD45, MHCII, CD11c,


Siglec-F (all BD or Ebioscience), IgE and CD11c (Biolegend). For intracellular cytokine staining, the

dispersed cells were stimulated with 10 nM phorbol 12-myristate 13-acetate (PMA; Sigma), 1 mM

ionomycin (Sigma) and brefeldin A (10 g/ml; Sigma) for 4-6 h. After surface staining with an antibody

cocktail (Table S1), cells were fixed with 4% paraformaldehyde and stained for detecting intracellular

cytokines (IL-4 and IL-13), as described (Ansel et al., 2004). Analysis was performed on LSR II (Becton

Dickinson) and FlowJo software (Treestar). For sorting pure populations of eosinophils, the dispersed

cells were immediately stained with an antibody cocktail (Table S1) and sorted using a FACS Aria

(Becton Dickinson). The gating strategy used for detecting basophils and eosinophils is shown in Figure

S4. Lymph node Tfh cells were stained with anti-CXCR5 (BD) followed by biotinylated anti-rat IgG2a

(BD), and then fluorophore-conjugated streptavidin. Additional fluorophore-conjugated antibodies were

added after blocking with normal mouse and rat sera (Jackson Immunoresearch).

Chromatin immunoprecipitation (ChIP). Cells were fixed for 10 min at room temperature with 1:10

dilution of 11% formaldehyde (in 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA and 0.5 mM

EGTA), neutralized with 1:20 dilution of 2.5 M glycine, washed twice in ice cold phosphate buffered

saline, and cell pellets snap frozen in liquid nitrogen before storing at -80°C. Frozen cells were lysed in

120µl of lysis buffer (Tris 50 mM, pH 8.0, EDTA 10 mM, 1% SDS, 1 mM phenyl methane

sulfonylfluoride, 20 mg/ml sodium butyrate, proteinase inhibitor cocktail; Sigma), and chromatin

sheared by sonication, using BioRuptorTM (Diagenode), to generate 200-800bp DNA fragments. For

immunoprecipitation with anti-H3-K4me2 (clone Y47; Abcam) and anti-H3-K27me3 (Cat. #07-449;

Millipore), chromatin from 1x106 whole-cell equivalents was used. Chromatin was diluted in

radioimmunoprecipitation assay (RIPA) buffer and immunoprecipitation was done for 2 hours at 4°C by

incubating with 1 µg of antibody pre-coated on protein A coated magnetic beads (Invitrogen).

Immunocomplexes were captured and washed for 5 min each with RIPA buffer x3, high-salt buffer (50


mM Tris, pH 8.0, 500 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% Nonidet-P40 and 1 mM EDTA)

x1, LiCl buffer (50 mM Tris, pH 8, 250 mM LiCl, 1 mM EDTA, 1% Nonidet-P40 and 0.5%

deoxycholate) x1, and low salt buffer (Tris 10 mM, EDTA 1 mM and 50 mM NaCl, pH 8) x1. Beads

were resuspended in TE (Tris 10 mM, EDTA 1 mM), transferred to fresh tubes, captured and then

resuspended in elution buffer (Tris 50 mM, pH 8.0, EDTA 10 mM, 1% SDS). Chromatin was detached

from the beads by incubating for 15 minutes at 65°C, and then treated with RNase A (200 mg/ml; ABI)

for 2 hours at 37°C, and, finally, with proteinase K (0.2 mg/ml, Roche) for 30 minutes at 55°C. DNA

was purified using affinity columns (Zymo Research). Selected DNA sequences were quantified by real-

time quantitative PCR. Briefly, in each experiment, three serial dilutions of input DNA purified from

chromatin before immunoprecipitation were analyzed together with experimental samples to create a

standard curve for quantification. ChIP data are presented as the percentage recovery of input, which

was calculated by dividing the number of copies detected in immunoprecipitates by the number of

copies present before immunoprecipitation, assuming two copies per cell. Sequences of primers

(Integrated DNA Technologies) are given in Table S2. For NFAT ChIP, resting and activated T cells

(PMA/Ionomycin treated for 45 minutes) were fixed for 20 minutes. Chromatin from 1x107 whole-cell

equivalents was used for immunoprecipitation with 5µg anti-NFAT1 antibody (clone 67.1) as described

(Ansel et al., 2004).


SUPPLEMENTAL REFERENCES Agarwal, S., Avni, O., and Rao, A. (2000). Cell-type-restricted binding of the transcription factor NFAT to a distal IL-4 enhancer in vivo. Immunity 12, 643-652.

Agarwal, S., and Rao, A. (1998). Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation. Immunity 9, 765-775.

Ansel, K.M., Greenwald, R.J., Agarwal, S., Bassing, C.H., Monticelli, S., Interlandi, J., Djuretic, I.M., Lee, D.U., Sharpe, A.H., Alt, F.W., and Rao, A. (2004). Deletion of a conserved Il4 silencer impairs T helper type 1-mediated immunity. Nat Immunol 5, 1251-1259.

Supplemental Information Interleukin-4 Production by Follicular … · 2012. 2. 23. ·...

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