Supplementary Data, Igyarto et al. - Tel Aviv University - Skin - Resident Mu… · Supplementary...

13
Supplementary Data, Igyarto et al. 1 Supplementary Figure S1, related to Figure 3. The absence of LC does not alter CTL phenotype. CFSE-labeled, naive OT-I cells were adoptively transferred into WT and huLangerin-DTA mice one day prior to skin infection with Calb-Ag. Expression of IFN-γ and granzyme B was analyzed on OT-I cells (CD8+, CD90.1+) four days after infection. Data are representative of 3 individual experiments. 63.5 31.6 70.2 25.2 GrzB CFSE 58.5 37.7 59 31.5 IFN CFSE

Transcript of Supplementary Data, Igyarto et al. - Tel Aviv University - Skin - Resident Mu… · Supplementary...

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Supplementary Data, Igyarto et al.

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Supplementary Figure S1, related to Figure 3. The absence of LC does not

alter CTL phenotype.

CFSE-labeled, naive OT-I cells were adoptively transferred into WT and

huLangerin-DTA mice one day prior to skin infection with Calb-Ag. Expression of

IFN-γ and granzyme B was analyzed on OT-I cells (CD8+, CD90.1+) four days

after infection. Data are representative of 3 individual experiments.

63.5

31.6

70.2

25.2

Grz

B

CFSE58.5

37.7

59

31.5

IFN

CFSE

Igyarto et al. Supplemental Figure 1

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Supplementary Figure S2, related to Figure 4. Th17 cells co-express IL-22

but not IFN-γ .

CD90.1+ TEα cells isolated from adoptively transferred WT mice infected with

Calb-Ag were stimulated in vitro with PMA and onomycin. Co-expression of IL-

17F and IL-22 was confirmed. Cells expressing IL-17F and IL-22 do not express

IFN-γ. Data are representative of 3 individual experiments.

5.8 16.2

2.46

IL-1

7F

IL-22

8.05 1.46

9.02

IFN

IL-22

Igyarto et al. Supplemental Figure 2

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Supplementary Data, Igyarto et al.

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Supplementary Figure S3, related to Figure 6. Anti-human Langerin 2G3

binding to human and mouse Langerin ectodomains.

a) Elisa plates were coated with (left)human Langerin ectodomain-IgGFc fusion

protein or (right) mouse Langerin ectodomain-IgGFc fusion protein and incubated

with serial dilutions of mAb 2G3, then washed, incubated with anti-mouse IgG-

HRP, then developed with TMB reagent.

Igyarto et al. Supplemental Figure 3

a.

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Supplementary Figure S4, related to Figure 6. Antigen presentation by LC

is sufficient to drive proliferation and Th17 differentiation of endogenous

CD4 T cells.

a) As in figure 6e, WT and huLangerin-DTR mice were injectioned i.p with 1.0 ug

of 2G3-2W1S. Mice were then infected with Calb-WT on their skin. Skin-

draining LN were harvested on day+7 and the binding of the I-Aβ-2W1S tetramer

vs. CD44 is shown. The total number of tetramer positive cells is shown above

the gate. WT mice have a low number of naïve (i.e. CD44lo) tetramer positive

cells compared with huLangerin-DTR mice in which there is a 10 fold expansion

of I-Aβ-2W1S tetramer positive CD44high cells. b) Cells were also stimulated ex

vivo with PMA and Ionomycin. The expression of IL-17A in gated I-Aβ-2W1S

tetramer positive cells is shown. Data are representative of 3 individual

experiments.

Igyarto et al. Supplemental Figure 4

WT huLang-DTR

CD

44

2W1S

CD

44

IL-17A

0.0 7.75

b.

a.

665±99 5074±1245

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Supplementary Figure S5, related to Figure 7. RT qPCR of skin-resident DC

populations.

a) To isolated DC, skin-draining LN from muLangerin-EGFP mice were enriched

by CD11c MACS positive selection and sorted as follows: Singlets were first

gated on MHC-IIbright. GFP-, CD8- cells were isolated as Langerin- dDC (red).

Gated GFP+ cells were collected as LC (CD103-, CD11b+, green) or Langerin+

dDC (CD103+, CD11b-, blue). b) As in Figure 7b, LC (open bars), CD103+

Langerin+ dDC (black bars) and Langerin- dDC (gray bars) were FACS sorted

from S. aureus infected muLangerin-EGFP mice. Relative expression of mRNA

of the indicated cytokines are shown. n.d.=not detected, * p<0.01

CD

8

GFP

CD

11b

CD103

MH

CII

GFP

Langerin- dDCLangerin+ dDC

LC

Igyarto et al. Supplemental Figure 5

b.

a.

0.1

1

10

100

TGFIL-1 IL-6 IL-12 IL-23 IL-27

Rel

ativ

e ex

pres

sion

LCLangerin+ dDCLangerin- DC

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Supplementary Figure S6, related to Figure 7. Proliferation of TEα cells

unaffected in Batf3-/- and muLangerin-DTR mice.

As in Figures 7c and 7e, the total number of TEα cells 4 days after infection with

Calb-Ag was determined based on the number of Thy 1.1 congenic CD4+. a)

Numbers of TEα cells isolated from individual a) WT and Batf3-/- mice or b) PBS

injected and DT injected muLangerin-DTR mice are shown.

muDTR -DT muDTR +DT1000

10000

100000

WT Batf3 -/-1000

10000

100000

Num

ber T

E

Num

ber T

E

a. b.

Igyarto et al. Supplemental Figure 6

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Supplemental Methods

Construction of recombinant C. albicans

All Candida albicans strains used in this study were derived from SC5314 (Calb-

WT)(Fonzi and Irwin, 1993) and are listed in Table 1. C. albicans cells were

grown at 30°C in rich medium(YPAD) or in synthetic complete medium(Sherman,

1991). Escherichia coli strain XLI-blue (Stratagene, La Jolla, CA), E. coli growth

conditions, DNA manipulations and primer design and synthesis were essentially

as described previously(Gerami-Nejad et al., 2004). Transformants were

selected on YPAD medium containing 400 ug/ml nourseothericin and were

screened by polymerase chain reaction (PCR) using oligonucleotide primers

listed in Table 2. Construction of plasmid pMG2278 (pENO1-ENO1-GFP-2W1S-

OVA323-339-OVA257-264-I-Eα50-66-NAT1) was performed by site-directed

mutagenesis (see supplemental methods). Wild-type strain SC5314 was

transformed using standard methods(Wilson et al., 1999). Transformants were

screened for GFP expression by detection of fluorescence in the colonies that

grew on YPAD medium containing 400 ug/ml nourseothericin. Integration at the

Eno11 locus in recombinant clone YJB11522 (Calb-Ag) was confirmed using 2

primer sets (4150/503) and (3790/795).

Construction of recombinant C. albicans plasmids

Construction of plasmid pMG2278 (pENO1-ENO1-GFP-2W1S-OVA323-339-

OVA257-264-I-Eα50-66-NAT1) involved several steps. First, we performed site-

directed mutagenesis of plasmid pMG1416 (Gerami-Nejad et al., 2001) using

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primer 1386 to change the TAA stop codon to GGT, resulting in pMG2070. Next,

we annealed two primers (3179 and 3180) that encode 2W1S-OVA323-339-

OVA257-264 (LQEAWGALANWAVDSAGGISQAVHAAHAEINEAGRGGSIINFEKL )

with Pst1 and BamH1 recognition site overhangs and ligated them to BamH1 and

Pst1 digested pMG2070 to yield plasmid pMG2249.

Plasmid pMG2120 (GFP-NAT1)(Selmecki et al., 2008) was digested with Nde1

and BglII and the large fragment containing a portion of the GFP and NAT1

marker was gel-purified. To insert I-Eα50-66(ASFEAQGALANIAVDKA) into this

construct, a PCR product amplified from plasmid pMG2249 with primer 3637 and

primer 503 was digested with BglII and Nde1 and then ligated to BglII and Nde1

digested pMG2249. This yielded pMG2268, which includes GFP fused to the 4

peptides followed by the ADH1 terminator and NAT1 marker. Next, the ENO1-

GFP fusion was amplified from strain YJB5957 (ura3Δ::λimm434/ura3Δimm434

his1::hisG/his1::hisG arg4::hisG/arg4::hisG ENO1/ ENO1::GFP-URA3) with

primers 480 and 3651, designed to have Nae1 and Not1 restriction sites,

respectively. The amplified fragment was digested with Nae1and Nde1 and

ligated to pMG2268 that had been digested with Msc1 and Nde1, yielding

pMG2271 (ENO1-GFP-2W1S-OVA323-339-OVA257-264-I-Eα50-66-NAT1). Sequence

from the RPS10 locus then was isolated by digesting p1535 (Care et al., 1999)

with Nar1 and AlwN1. The 2.6 kbp RP10 band was purified from the gel and

ligated to pMG2271 digested with Nar1 and lwN1 to produce plasmid pMG2272.

Finally, pMG2278 (pENO1-ENO1-GFP-2W1S-OVA323-339-OVA257-264-I-Eα50-66-

NAT1) was constructed by digesting pMG2272 with Not1, treating the ends with

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alkaline phosphatase and then inserting a Not1 digested fragment of the wild-

type ENO1 promoter that had been amplified from strain BWP17 (Wilson et al.,

1999).

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Supplemental Methods Table 1.

SC5314 CaURA3/CaURA3 (Fonzi and Irwin, 1993)

YJB11522 CaURA3/CaURA3

pENO1-ENO1-GFP-2W1S-OVA323-339-OVA257-264-I-Eα50-66, Green colony

This study

BWP17 ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG

(Wilson et al., 1999)

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Supplemental Methods Table 2

Primer Number Sequence Reference

1386 CCTCTAGAGTCGACCTGCAGaccTTTGTACAATTCATCCATAC

This study

503 TGTGGAATTGTGAGCGGATA This study

3179 GAAGCTTGGGGTGCTTTGGCTAACTGGGCCGTTGATTCTGCTGGTGGTATTTCTCAAGCTGTTCACGCTGCTCATGCCGAAATTAACGAAGCTGGTAGAGGTGGTTCTATCATCAACTTCGAGAAGTTG

This study

3180 GATCCAACTTCTCGAAGTTGATGATAGAACCACCTCTACCAGCTTCGTTAATTTCGGCATGAGCAGCGTGAACAGCTTGAGAAATACCACCAGCAGAATCAACGGCCCAGTTAGCCAAAGCACCCCAAGCTTCTGCA

This study

3637 ggaagatctAGCTTTATCAACAGCAATGTTAGCCAAAGCACCTTGAGCTTCGAAAGAAGCCAACTTCTCGAAGTTGATGATAG

This study

480 ggATAAggCAgATTggTAggATAAgTAATggTTgTC This study

3651 catggccggcgcggccgcATGTCTTACGCCACTAAAATCCACGCC This study

3703 AAGGAAAAAAGCGGCCGCGCCGGCACTTGTGGGTTTAACATCATTTGTATCTTTAG

This study

3704 AAGGAAAAAAGCGGCCGCTGTTGTAATATTCCTGAATTATCAATTGATG

This study

4150 GTCTCCGTTCATCTCTTCACCAGG

3790 GATATATCGAATCCTCTAGCGTGG

795 CCATCTAATTCAACCAAAATTGG

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Recombinant antibody fusion protein production

DNAs encoding the 2G3 hybridoma H (GenBank HQ724328) and L chain

(GenBank HQ724329) variable regions were isolated and characterized using

methods already described(Klechevsky et al., 2010). An anti-human Langerin

2G3 H chain UCOE cetHS puro vector (Millipore, CA) directed the synthesis of

the 175 N-terminal residues from the 2G3 hybridoma IgG1 H chain fused to the

301 C-terminal residues of anti-DEC-205 mIgG2b (kindly provided by Ralph

Steinman, Rockefeller University, NY) with residues

ASQTPTNTISVTPTNNSTPTNNSNPKPNPAS (a flexible linker sequence) and

FEAQGALANIAVDKAGGAS (containing the Eα52-68 epitope), SIINFEKL

(containing the OVA257-264 epitope) or EAWGALANWAVDSA (containing the

2W1S epitope) appended to the C-terminus. A anti-human Langerin 2G3 L chain

cetHS puro vector directed the synthesis of the 125 N-terminal residues of the

2G3 hybridoma lambda chain fused to the 110 C-terminal residues of mouse

kappa chain. Stable CHO-S cell lines co-expressing the H and L chains were

isolated, cultured, and the anti-human Langerin 2G3-Eα52-68 epitope fusion

protein was purified from the culture supernatant as described(Benton et al.,

2002).

ELISA

Anti–human Langerin interaction with Langerin was determined by ELISA using

plates coated with 2 µg/ml recombinant human Langerin ectodomain-Fc or

human IgG-Fc fusion proteins. Goat anti-human IgG (Fab)-HRP (Jackson

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ImmunoResearch Laboratories, West Grove, PA) was used as the detection

reagent.

DC-sorting by flow cytometry and qPCR

Single cell suspension of LN cells were enriched by CD11c MACS positive

selection (Miltenyi Biotec, Auburn, CA). LC, Langerin+ dDC and Langerin- DC

were sorted using a FACSAria cell sorter based as follows: Langerhans cells

(GFP+, MHCII+, CD11c+, CD11b+, CD103-, CD8-), Langerin+ dDC (GFP+,

MHCII+, CD11c+, CD11b-, CD103+, CD8-) and Langerin- DC (GFP-, MHCII+,

CD11c+, CD8-). RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia,

CA) and quantified from Nanodrop readings (NanoDrop, Wilmington, DE). cDNA

was generated using a High Capacity cDNA Reverse Transcription Kit (Applied

Biosystems, Carlsbad, CA). TaqMan Gene Expression Master Mix, TaqMan

Gene Expression Assays for IL-1β, IL-6, IL-12p35, IL-23p19, IL-27p28, and TGF-

β and ABI Prism 7900HT(Applied Biosystems) were used to complete the qPCR.

All kits were completed according the manufacturer’s instructions. All Ct values

were normalized to HPRT expression and are shown either as 2-∆Ct (Figure 7a

and 7b).