letter to the editors · 48 supplementary figures supplementary figure 1. injection of liver...
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48
Supplementary Figures
Supplementary Figure 1. Injection of liver purified iNKT cells, depleted of iNKT cells, failed
to increase the survival of PR8 infected Jα18-/- mice. WT and Jα18-/- mice (n=5/ group) were
injected i.n. with PR8. Liver purified iNKT cells were simultaneously stained with anti CD5, anti
TCRβ specific antibodies and CD1d/α-GalCer tetramers and triple positive cells, representing
75% of the total population, were removed. Mice were injected 1 day post infection either with
the total population sorted with anti CD5 and anti TCRβ specific antibodies (CD5+ TCRβ+
sorted cells) or with CD5+ TCRβ+ cells from which the CD1d/α-GalCer tetramer+ cells were
depleted (CD5+ TCRβ+ sorted cells depleted of iNKT cells). Survival rate is shown as the
percentage of live mice at different time points after the infection.
Supplementary Figure 2. Phenotypic and functional analysis of MDSC.
(A) Phenotypic analysis of BM derived MDSC (gated on Gr-1+ and CD11b+ cells) stained with
CD40, CD1d and TLR9 antibodies (open histograms). Grey histograms indicate no antibody
staining.
(B) MDSC suppress CFSE labelled OT-I proliferation induced by matured DC. DC from BM
culture were matured with LPS (10µg/ml) or α-GalCer in the presence of iNKT cells and then
pulsed with SIINFEKL peptide for 2 h. OT-I splenocytes were cultured with matured DC
(5x104) and in the presence or absence of MDSC (3x104). CFSE labelled OT-I splenocytes alone
are shown in green histograms.
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(C) Staining with CD1d/α-GalCer tetramers and anti CD3 antibody of iNKT cells in CD11c and
Gr-1 depleted BM cultures.
Supplementary Figure 3. Lack of iNKT cell dependent lysis of α-GalCer pulsed MDSC.
CFSE labelled BM derived MDSC were either pulsed or not pulsed with α−GalCer and
incubated with CD11c and Gr-1 depleted BM cultures, which contain iNKT cells. The
percentage of Bisbenzimide negative CFSE labelled MDSC is very similar in the α−GalCer
pulsed and unpulsed cultures, indicating that MDSC pulsed with 100 ng of α−GalCer were not
sensitive to killing by iNKT cells. As a positive control for iNKT cell activity, the right panel
shows CD11c expression on CFSE positive and Bisbenzimide negative MDSC, 48 h after
incubation with iNKT cells. Blue histogram shows CD11c expression on α-GalCer pulsed CFSE
labelled MDSC in the presence of iNKT cells, while green histogram shows CD11c expression
on unpulsed CFSE labelled MDSC in the presence of iNKT cells. Red histogram shows CD11c
expression on unpulsed CFSE labelled MDSC in absence of iNKT.
Supplementary Figure 4. MDSC can be infected by PR8 virus.
(A) MDSC from lungs of WT, Jα18-/- and CD1d-/- mice infected i.n. with PR8 (3x104 pfu/106
cells) were purified with anti-CD11b-coated magnetic beads. The cells were analysed using
semi-quantitative PCR.
(B) BM derived MDSC from WT mice were infected with PR8 (2.5x104 pfu/106 cells) for 1 h.
Cells were analysed for expression of PR8 nucleoprotein (NP) 12 hrs and 24 hrs later using semi-
quantitative PCR.
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Supplementary Figure 5. Phenotypic changes of CpG treated MDSC.
(A) CpG can induce IL-12p40 secretion from hexβ-/- and iGb3S-/- MDSC in the presence or
absence of anti CD1d antibody. Supernatants were harvested at 48 h.
(B) MDSC derived from BM of hexβ-/-, hexβ-/+, iGb3S-/- and iGb3S-/- mice were treated with CpG
for 24 h. Treated and untreated MDSC were stained with anti CD1d-PE antibody.
Supplementary Figure 6. H17 IAV (H3N2) infection induces a greater expansion of MDSC
in Jα18-/- mouse than in WT mice. Expansion of lung infiltrating CD11b+ Gr-1+ cells in Jα18-/-
and in WT mice injected i.n. with H17 IAV (20 HAU), which peaked at approximately day 9
after the infection. Data represents the average of n= 5 mice/ group ± S.D.
Supplementary Figure 7. Inhibition of T cell proliferation by human MDSC can be rescued
by iNKT cells.
(A) CD11b+ cells, purified from PBL either from individuals shortly after an acute respiratory
illness (associated with the presence of high IAV specific Ab titer shown in Table 1) or from
healthy donors were added to PBL and incubated with allogeneic irradiated DC. Purified CD11b+
cells were either untreated (), or treated with either α-GalCer (100 ng/ml) in presence of iNKT
at a MDSC:iNKT cell ratio of 1:0.25 () or L-NMMA and NOHA ( ). The data are expressed
as described in Methods. Addition of either iNKT cells or L-NMMA and NOHA to the
alloreactive T cells in the absence of CD11b+ cells did not affect T cell proliferation (data not
shown). The ratio of irradiated DC to purified irradiated CD11b+ cells was 1:1.
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(B) Conditions to prevent lysis of α-GalCer pulsed human MDSC by iNKT cells. Human MDSC
(0.5x106) were treated with α−GalCer (100 ng/ml) and co-cultured with increasing numbers of
iNKT cells for 24 h. MDSC were stained with Propidium Iodide to calculate the percentage of
iNKT cell dependent MDSC killing.
(C) iNKT cells are activated by α-GalCer pulsed CD11b+ cells from IAV infected patients. IFN-
γ release from iNKT cells incubated for 24 h with α-GalCer pulsed CD11b+ cells, derived from
the IAV infected patients and healthy controls described in Figure 7. Data represent the average
of 3 replicates ± S.D.
0
20
40
60
80
100
0 2 4 6 8 10 12
WTJα18-/-
Jα18-/-+TCRβ+CD5+sorted cells Jα18-/-+TCRβ+CD5+sorted cells depleted of iNKT cells
Surv
ival
Rat
e (%
)
Days after infection
Supplementary Figure 1
R6
CD40 CD1dGr-1
CD11b-PE
80.46%C
D11
b
CD3-APC
2.5%
CD1d
/αGa
lCer
-tetra
mer
A
C
TLR9
CFSE
coun
ts
DC+α-GalCer DC+LPS
-MDSC
+MDSC
B
Supplementary Figure 2
0 10K 20K 30K
100
101
102
103
104
<FL 1 Log>: FITC
91%
9.9%
10K 20K 30K
100
101
102
103
104
FL 6 Log: Violet 1
32%
0 10K 20K 30K
100
101
102
103
104
FL 6 Log: Violet 1
30%
0 10K 20K 30K
100
101
102
103
104
<FL 1 Log>: FITC
90%
10%
100 101 102 103 1040
20
40
60
80
100
% of Max
0 10K 20K 30K
100
101
102
103
104
<FL 1 Log>: FITC
99%
0 10K 20K 30K
100
101
102
103
104
FL 6 Log: Violet 1
31.7%
57.6%
7.89%
CD11c
10
coun
ts
FSC-HFSC-H
Bis
benz
imid
e
CFS
EMDSC
MDSC +iNKT
α−GalCer pulsed MDSC +iNKT
0.03%
Supplementary Figure 3
12 h 24 h PR8 infected
12 h 24 h uninfected
WT WT Jα18-/- CD1d-/-
NP
GAPDH
PR8 infected mice
NP
GAPDH
A
B
Supplementary Figure 4
0
50
100
150
200
250IL
12p4
0 re
leas
e (ng
/ml) α−GalCer
CpG CpG+anti-CD1d
hexβ+/- hexβ-/- iGbS3+/- iGbS3-/-
untreated
100 101 102 103 1040
20
40
60
80
100
% of Max
100 101 102 103 1040
20
40
60
80
100
% of Max
100 101 102 103 1040
20
40
60
80
100
% of Max
100 101 102 103 1040
20
40
60
80
100
% of Max
CD1d
coun
ts
hexβ iGbS3
A
B
+/-
-/-
Supplementary Figure 5
0
1
2
3
4
5
6
7
T
otal
num
ber o
f M
DSC
(
x106 )
WT WT Jα18-/-
H17 infected mice
Supplementary Figure 6
A
B
Supplementary Figure 7
+ MDSC from healthy donors + MDSC donors with acute respiratory illness
untreated
L-NMMA+NOHA
α−GalCer+iNKT
% P
rolif
erat
ion
0
2
4
6
8
10
12
no cd11b4.08.061.5.0721.10.0624.04.0706.10.0625.01.079.12.0521.07.0615.03.0631.10.07
180
160
140
120
100
80
60
40
20
0
IFN
-γ re
leas
e (n
g/m
l)
04.0
8.06
01.0
5.07
24.1
0.06
24.0
4.07
06.1
0.06
25.0
1.07
09.1
2.05
21.0
7.06
15.0
3.06
31.1
0.07
− C
D11
b
+ CD11b from IAV infected patients and heathy donors
C
Donor 5 Donor 4Donor 3Donor 2Donor 1
0 200 400 600 800 1000
100
101
102
103
104
FL3-H
400 600 800 1000
100
101
102
103
104
FL3-H
0 200 400 600 800 1000
100
101
102
103
104
FL3-H
50%
0 200 400 600 800 1000
100
101
102
103
104
FL3-H
0 200 400 600 800 1000
0
200
400
600
800
1000
SSC-H
0 200 400 600 800 1000
0
200
400
600
800
1000
SSC-H
0 200 400 600 800 1000
0
200
400
600
800
1000
SSC-H
0 200 400 600 800 1000
0
200
400
600
800
1000
SSC-H
FSC-H
SSC
-HPr
opid
ium
Iodi
de
FSC-H
44.2% 35.2%51.9%
0 200
α-GalCer-pulsed MDSC:iNKT
1:0 1:11:0.51:0.25
Donor 6 Donor 7 Donor 8 Donor 9 Donor 10