S5: Expression of CD101 and TREM1 by lymphoid and non-lymphoid DC ... plots show the frequency of CD101+, TREM1+ or CD101+TREM1+ DC in the ...
Description of Supplementary Files File Name: Supplementary Information Description: Supplementary figures, supplementary tables
Supplementary Figure 1 a
Stomach
Liver
Lungs
Cre– Cre–
80 60 40 Cre 5
10
15
Cre
→
+
→ →
0
+
20
→
20
→
Survival (%)
100
b
Age (weeks)
CD8+ T cells Cre– Cre+
CD69
d
**
6100 4100
*
2100
** **
100 50
**
25 0 Il
1b
Il6
Events (% of max)
CD62L
mRNA (relative expression)
f CD44
e
Stomach 8100
mRNA (relative expression)
CD4+ T cells
mRNA (relative expression)
c
g Ifn
fa Tn
fb Tg
Liver 500
***
400
***
200 150 100
**
50 0 Il
1b
Il6
** fa Tn
If
ng
g
Lungs
600
*
500
bp 500
400 240
Cre–
Cre+
Cre+
Cre+
fb Tg
Transferred T cells (Cre+)
400 300
Tgfbr1 Deleted ‘Floxed’
120
0 Il1
b
Il6
fa Tn
g
Ifn
fb
Tg
Fig. S1: CD11c-Cre.Tgfbr1fl/fl Mice Develop a Lethal Systemic Inflammatory Disease (a) Survival of lymphocyte-sufficient CD11c-Cre.Tgfbr1fl/fl (Cre+) or Tgfbr1fl/fl (Cre–) mice with 5 (Cre+) or 6 (Cre–) mice/group and data are representative of 2 experiments. (b) H&E staining of stomach, liver and lungs from Cre– (upper panels) or Cre+ (lower panels) mice. Images are representative of at least 4 mice/group. Scale bars 500µm. (c) Expression of CD69, CD44 and CD62L by splenic CD4+ and CD8+ T cells from Cre– (dashed line) or Cre+ mice (solid line). Shaded histograms represent staining with appropriate isotype controls. (d-f) qRT-PCR analysis of IL1β, IL6, TNFα, IFNγ and TGFβ mRNA in stomach (d), liver (e) and lungs (f) of Cre– or Cre+ mice. (g) Genomic PCR for Tgfbr1 expression by splenic CD3+ T cells from intact lymphocyte-sufficient Cre– or Cre+ mice, or by donor CD3+ T cells purified from the recipients of Cre+ splenic T cells (‘Transferred T cells’). Flow cytometric analysis from one of two independent experiments with n=4-8/group.
Supplementary Figure 2 a
Gating strategy for identification of DC in small intestinal and colonic LP 25.1
FSC-H
FSC-A
b
7-AAD
20.1
CD11c
Assessment of CD103/CD11bdefined DC subsets and CD64+ Mφ (Figure 3a-d)
CD11c
55.4
SSC-A
94.1
SSC-A
79.8
FSC-A
SSC-A
60.4
CD45
CD64
MHCII
SI LP Live CD45+ CD11c+ MHCII+ CD64– Cre+
CD103
Tgfbr1+/fl Rag+/– 27.5 34.2
Tgfbr1fl/fl Rag+/– 33.5 16.7
25.8
31.7
CD11b Gating strategy for identification of DC in spleen (Figure 3e)
d
FSC-H
7-AAD/Ly6G
Assessment of CD11b-defined subsets (Figure 3e)
CD11c
SSC-A
93.8
SSC-A
FSC-A
SSC-A
45.1 FSC-A
8.04
96.7
70.2
MHCII
CD45
Gating strategy for identification of DC in small intestinal LP of mixed BM chimeric mice (Figure 4b & c) 24.9
FSC-A
FSC-H
7-AAD
48.3
‘NOT’ gate
Assessment of contribution of CD45.2 (Tgfbr1fl/fl) BM to each subset (Figure 4b)
CD11c
CD45.1+ (WT)
94.4
SSC-A
78
FSC-A
SSC-A
49.4
CD103
c
MHCII
CD45.2+ (Tgfbr1fl/fl)
CD11b
CD45.1 (WT) +
CD11c
CD64
e
Assessment of CD103/CD11bdefined subsets (Figure 4c)
CD45.2+ (Tgfbr1fl/fl) Gating strategy for identification of migratory DC in mesenteric lymph node (MLN) of mixed BM chimeric mice
2.5
FSC-A
MHCII
CD45.1+ (WT)
45.4 15.1
CD11c
93.8
FSC-H
CD3/CD19/7AAD
56.4
FSC-A
FSC-A
SSC-A
3.43
Assessment of CD103/CD11bdefined subsets (Figure 4d) 50.1
CD45.2+ (Tgfbr1fl/fl)
Fig. S2: Gating strategies used throughout study (a) Gating strategy to identify DC and macrophages in the small intestine and colonic lamina propria in Figure 3 and Figure 6. (b) Representative expression of CD103 and CD11b by DC subsets from the small intestine of CD11c-Cre.Tgfbr1+/fl Rag1+/– or CD11c-Cre.Tgfbr1fl/fl Rag1+/– mice. Plots are from one experiment of two independent experiments performed with 3 mice per group. (c) Gating strategy to identify DC in the spleen in Figure 3. (d) Gating strategy to identify DC subsets in the small intestinal lamina propria of mixed BM chimeric mice in Figure 4. (e) Gating strategy to identify DC subsets in the mesenteric lymph node (MLN) of mixed BM chimeric mice in Figure 4.
Supplementary Figure 3 b
Small Intestinal mononuclear phagocytes - CD11c-Cre.Rosa26-LSL-eYFP mice
40 20
80
20
60 40 20
D
II +
CD19+ MHCII + B cells CD3+ MHCII – T cells Total CD45+ leukocytes
80 60 40 20 0102 103 104 105 CD11c
C
C
D
C
10
100
0 II –
010 10 10 10 CD11c
0
5
C
4
H
3
H
2
19
3+ C D D 11 10 b– 3+ C C D D 11 10 b+ 3– F4 C D /8 11 0+ b+ M H C II + M φ
0
C
20
0
Events (% of max)
40
eYFP+ (%)
60
80
3+ M
20
100
CD103+ CD11b – CD103+ CD11b + DC CD103 – CD11b+ Subset F4/80+ MHCII+ Mφ Total CD45+ leukocytes
80
0102 103 104 105 eYFP
M
40
100
13.2
40
+
60
35.3
60
0
Events (% of max)
80
eYFP+ (%)
100
0102 103 104 105 YFP eYFP
100
CD3+ MHCII –
CD19+ MHCII +
82.5
60
0
Small Intestinal lymphocytes - CD11c-Cre.Rosa26-LSL-eYFP mice
Ly6Cint CD11b+ neutrophils Ly6Chi monocytes Ly6Clo monocytes CD19+ MHCII + B cells CD3+ MHCII – T cells
100 80 60 40 20 0
0102 103 104 105 CD11c
Ly 6
eYFP
0
Events e ts (% of o max) a )
Ly6Chi monocytes
eYFP+ (%)
Events (% ( of max))
CD11b+ Ly6Cint SSChi neutrophils 100
C in St S Ch i Ly neu 6C tro hi p Ly mo hils 6 C no cy lo C D 1 9 mo tes + no M H cyte C C D II + s 3+ B M ce H C II – lls T ce lls
c
Fig. S3: Efficiency of CD11c-Driven Cre Recombinase Activity (a) Representative expression of eYFP by DC subsets and F4/80+MHCII+ macrophages from the small intestine of CD11c-Cre.Rosa26-LSL-eYFP mice or CD11c-Cre mice (shaded histograms). Scatter plots show the frequency of eYFP+ cells in each subset. Each symbol represents an individual animal and the horizontal bar represents the mean of n=7 mice from a single experiment. The lower histogram shows CD11c expression by DC subsets, F4/80+MHCII+ macrophages and total CD45+ leukocytes from the small intestine of 6 week old CD11c-Cre.Rosa26-LSL-eYFP mice. (b) Representative expression of eYFP by CD19+ MHCII+ B cells and CD3+ MHCII– T cells from the small intestine of CD11cCre.Rosa26-LSL-eYFP mice or CD11c-Cre mice (shaded histograms). Scatter plots show the frequency of eYFP+ cells in each lymphocyte subset. Each symbol represents an individual animal and the horizontal bar represents the mean n=7 mice from a single experiment. The lower histogram shows CD11c expression by CD19+ MHCII+ B cells, CD3+ MHCII– T cells and total CD45+ leukocytes from the small intestine of CD11c-Cre.Rosa26-LSL-eYFP mice. (c) Representative expression of eYFP by neutrophils, classical Ly6Chi monocytes, non-classical Ly6Clo monocytes, CD19+ MHCII+ B cells and CD3+ MHCII– T cells from the blood of CD11c-Cre.Rosa26-LSL-eYFP mice or CD11c-Cre mice (shaded histograms). Scatter plots show the frequency of eYFP+ cells in each leukocyte subset. Each symbol represents an individual animal and the horizontal bar represents the mean n=7 mice from a single experiment. The histogram (right) shows CD11c expression by each blood leukocyte subset from blood of CD11c-Cre.Rosa26-LSL-eYFP mice.
Supplementary Figure 4 Events (% of max)
a
b
100
c
F4/80+ MHCII+ M CD103+ CD11b– CD103+ CD11b+ CD103– CD11b+
80 60 40 20 0
0102 103 104 105 SiglecF
EpCAM
Gp2
Fig. S4: Assessment of markers identified by microarray analysis at protein level Representative expression of SiglecF (a), EpCAM (b) and GP2 (c) by CD103/CD11b-defined DC subsets and by F4/80+MHCII+ macrophages from the small intestinal LP of unmanipulated WT mice. Data are from a single experiment.
c
r
ey
ve
ng
dn Ki
Li
on
Lu
ol C
e
en le
tin nt lI al Sm
TREM1+ of CD103+ CD11b– DC (%)
r
ey
ve
dn
Li
Ki
ng Lu
on ol
lI
C
le
e tin es nt
en
0 Sp
r
20
al Sm
60 40 20
r
ey dn
Ki
ng
ve Li
Lu
ol C
es
CD101
on
0
2.43
en
97.5
0.0293
e
3.47 7
40
80 CD103– CD11b– 0.0293
tin
1.7 7 96.2
0.201 1
le
0 0.101
60
Sm
al
lI
CD11b
98.3
3.73
CD103– CD11b+
nt
CD103
TREM1
0
96.1
80
ey
Sm 0.319 9
CD103+ CD11b–
CD101+ TREM1+ of CD11b+ DC (%)
99.7
0
dn
al
‘Resident’ DC CD11c+ MHCIIhi
20
Ki
ng
ve Li
Lu
on ol C
le
lI
nt
en
0
0
e
CD103– CD11b+
0.182
20
tin
0
40
es
CD103+ CD11b–
60
Sp
0
‘Migratory’ DC CD11c+ MHCIIhi
r
lI al
Inguinal LN
80
Sp
b
40
ey
35.4
60
dn
54.7
CD101
80
Ki
21
Sm CD11b
TREM1+ of CD11b+ DC (%)
TREM1
CD103
‘Migratory’ DC CD11c+ MHCIIhi
ng
3.58
4.72
ve
1.89 9
5.51
Li
96.6
74.7 7
on
0.767
ol
1.13 3
nt
0.567
0
CD103– CD11b+
Lu
CD103+ CD11b+
20
C
18.5
e
CD103+ CD11b–
81.7
40
en
0.926
60
tin
99.1
CD101+ of CD103+ CD11b– DC (%)
80
‘Resident’ DC CD11c+ MHCIIhi
Sp
0
es
CD103– CD11b+ 0.381
le
0
es
CD103+ CD11b–
Sp
0
CD101+ of CD11b+ DC (%)
Supplementary Figure 5 a Mesenteric LN
Fig. S5: Expression of CD101 and TREM1 by lymphoid and non-lymphoid DC subsets (a-b) Representative expression of CD103 and CD11b by ‘resident’ (CD11c+ MHCII+) and ‘migratory’ (CD11c+MHCIIhi) DC in the mesenteric (a) or inguinal (b) lymph nodes (left panels), and representative expression of CD101 and TREM1 by CD103- and CD11b-defined DC subsets (right panels). (c) Scatter plots show the frequency of CD101+, TREM1+ or CD101+TREM1+ DC in the tissues indicated. Each symbol represents an individual animal and the horizontal bar represents the mean of 4 mice from one of two representative experiments performed.
Table S5: Genes shared by SILP CD103+CD11b+ and CD103–CD11b+ DC subsets and highly significantly differentially expressed compared with CD103+CD11b– DC.
