figure s1 (a) analysis of purified myeloid dcs and t cells. freshly isolated mdcs were processed for...
DESCRIPTION
control CD3 CD4 HLA-DR CD3CD1c T cell phenotype (freshly isolated) Figure S1 (D) SSCTRANSCRIPT
Figure S1
(A)
Analysis of purified myeloid DCs and T cells.Freshly isolated mDCs were processed for cytospins (A), stained with haematoxylin-eosin (x400 magnification, scale bar represents 50 m). The purity of DCs (B and C) and T cells (D) has been checked by HLA-DR, CD1c, CD11c
(not shown), CD3, CD4, CD20 and CD14. Data are representative of 3 experiments. For ICOSL and PD-L1 analysis, mDCs were stained with Lin-1, HLA-DR and CD11c or their isotype controls freshly (E) or after 18hrs culture (F). After washing, mDCs were analyzed by FACS. Lin-1- HLA-DR+ CD11c+ cells were gated for further analysis (F). Data are representative of 8 experiments.
(B)
(C)
SSC
FSC
HLA-DR
CD1c
HLA-DR
CD3
control
control control
control
control
SSC
control
SSC
SSC SSC
CD14 CD20
Figure S1
DC phenotype (freshly isolated)
control
control
control
control
control
control
CD3
CD4
HLA-DR HLA-DR
CD3 CD1c
T cell phenotype (freshly isolated)
Figure S1
(D)
SSC
SSC
Figure S1
(E)
FSC
SSC
Lin-1Lin-1 CD11c
SSC HLA-DRHLA-DR HLA-DRHLA-DR
control control
control control
DC phenotype (freshly isolated)
FSC Lin-1 CD11c
SSC HLA-DR HLA-DRSSC HLA-DR HLA-DRSSC HLA-DR HLA-DR
(F)
control
control
control
control
Figure S1
Analysis(ICOSL, PD-L1)
DC phenotype (cultured for 18 hrs)
Expression of ICOSL and PD-L1 in freshly isolated and in cultured mDCs.Freshly isolated mDCs or cultured (for 18 hrs) mDCs were stained for ICOSL or PD-L1 (or their isotype controls). Data are from 3 separate experiments.
Figure S2
FreshCultured (18hrs)
ICOSL PD-L10
20
40
60
80
% o
f DC
s
(A) (B)
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Figure S3
mDC viability in culture, upon LPS stimulation.mDCs were isolated and cultured with LPS at different concentrations and time-periods (medium only as control), and stained with propidium iodide (PI) before FACS analysis (A). In addition, mDCs were counted and percentage is shown as reported to baseline (day 0). Data are from 3 experiments (*P < 0.05, ***P < 0.001).
18h 42h 66h0
20
40
60
80 ******PI
% o
f DC
s
18h 42h 66h40
60
80
100
120
cell count
*
% o
f bas
elin
e
Expression of B7 co-stimulatory molecules in LPS-activated mDCs.mDCs were cultured overnight with LPS at different concentrations for 18hrs, and then stained with anti-ICOSL (A), anti-PD-L1 (B), anti-CD80 (C), anti-CD86 (D) or their isotype controls. Data are from 3 experiments (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure S4
(A)
(C) (D)
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
(B)
0
5
10
15
20
25ICOSL
% o
f DC
s
60
80
100
PD-L1
******
% o
f DC
s
0
10
20
30
CD80
****
% o
f DC
s
60
80
100
CD86
***
% o
f DC
s
Expression of ILT3, ILT4 and OX40L in mDCs from allergic rhinitis patients and controls.DCs from allergic rhinitis patients or controls were isolated and stimulated by LPS (1g/ml). Immunostaining and FACS analysis were performed as described in Figure 1. ILT3 (A), ILT4 (B) and OX40L (C) positive cell percentages are shown. Data are mean values ± SEM from 13-14 experiments (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure S5
(A) (B) (C)
RhinitisControlsRhinitisControlsRhinitisControls
ILT3
Medium LPS0
50
100 ****
*
% o
f DC
s
ILT4
Medium LPS0
40
80
*****
% o
f DC
s
OX40L
Medium LPS0
10
20
30
% o
f DC
s
Figure S6
Expression of ICOSL and PD-L1 in LPS-activated mDCs.mDCs were cultured overnight with LPS (1g/ml), and then stained with anti-ICOSL, anti-PD-L1 or isotype controls. ICOSL (A) and PD-L1 (B) expression is shown for allergic asthma patients (n=9) versus controls (n=8). Data are mean values ± SEM (*P < 0.05, **P < 0.01).
ControlsAllergic asthmaControls
(B)(A)
ICOSL
Medium LPS0
20
40**
*
% o
f DC
s
PD-L1
Medium LPS0
25
50
75
100 **
% o
f DC
s
Figure S7
1:51:101:201:1000mDC alone
mDC:T ratio:
1:51:101:201:1000mDC alone
mDC:T ratio:
1:51:101:201:1000mDC alone
1:51:101:201:1000mDC alone
mDC:T ratio:
Cytokine production in co-cultures at different mDC and CD4+ T cell ratios.mDCs were isolated and, after 18hrs, co-cultured for 5 days with allogenic CD4+ T cells. At each indicated ratio, a fixed number of T cells was added to different numbers of mDCs. Supernatants were collected and immuno-assayed for cytokines.
Data are from two separate experiments.
0
300
600
IL-13
pg/m
l
0
100
200
300IL-5
pg/m
l
0
1
2IFN-
ng/m
l
0
80
160
240TNF
pg/m
l
0
250
500IL-10
pg/m
l
Cytokine production by LPS-activated mDCs.mDCs were cultured with LPS at different concentrations and time periods, supernatants were assayed for IL-12p40 (A), TNF (B) and IL-10 (C). Data are mean values ± SEM. Each condition represents 3 experiments (**P < 0.01, ***P < 0.001).
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
Medium 0.01 g/ml 0.1 g/ml 0.5 g/ml 1 g/ml
(A)
(C)
(A)(A)
Figure S8
(B)
18h 42h 66h0
500
1000
TNF
*********
******
*** ******
***
pg/m
l
18h 42h 66h200
250
300
350
IL-12p40
pg/m
l
18h 42h 66h0
5
10
15
IL-10
*** *** *****
ng/m
l
Figure S9
MediumTSLP
ICOSL PD-L10
20
40
60
80%
of D
Cs
Expression of ICOSL and PD-L1 of mDCs in presence of TSLP.mDCs from control donors were cultured with TSLP or IL-33, or both for 18 hrs, before staining with anti-ICOSL (A), anti-PD-L1 (B), or their isotype controls. Data are representative of 3 experiments.