5 15 25 20 10 Ct value Log concentration miR-375 miR-221 Supplementary Figure S1.
Supplementary Data, Igyarto et al. - Tel Aviv University - Skin - Resident Mu… · Supplementary...
Transcript of Supplementary Data, Igyarto et al. - Tel Aviv University - Skin - Resident Mu… · Supplementary...
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
Grz
B
CFSE58.5
37.7
59
31.5
IFN
CFSE
Igyarto et al. Supplemental Figure 1
Supplementary Data, Igyarto et al.
2
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
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.
Supplementary Data, Igyarto et al.
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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
Supplementary Data, Igyarto et al.
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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
Supplementary Data, Igyarto et al.
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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
Supplementary Data, Igyarto et al.
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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
Supplementary Data, Igyarto et al.
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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
Supplementary Data, Igyarto et al.
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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).
Supplementary Data, Igyarto et al.
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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)
Supplementary Data, Igyarto et al.
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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
Supplementary Data, Igyarto et al.
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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
Supplementary Data, Igyarto et al.
13
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).