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
13
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Supplementary Data, Igyarto et al. - Tel Aviv University - Skin - Resident Mu… · Supplementary Data, Igyarto et al. 2 Supplementary Figure S2, related to Figure 4. Th17 cells co-express
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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.
3
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.
4
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.
5
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.
6
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.
7
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.
8
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-