1
Supplementary Information
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3
Figure S1
CD4+
98.4%
FSC-H
FSC-Hhi cells
86%
FSC-H
Vα2+Vβ5.1,5.2+
75%
Vα2 FITC
PI
FSC-H
PI
Live cells
96%
PI
SSC-H
A
CD4 Pac Blue
Vβ5.1,5.2 PE
Vα2 FITC
CD8+
99%
CD8 AF700
Vβ5.1,5.2 PE
Expression of CD44
% of Max
% of Max
D
Expression of CD69
% of Max
Expression of CD44
% of Max
Expression of CD62L
Expression of CD69
% of Max
C
Vα2+Vβ5.1,5.2+
98.1%
CD4+ T cells
FSC-Hlo cells
Expression of CD62L
% of Max
PI
B
CD8+ T cells
FSC-Hlo cells
4
5
Figure S1: Phenotype of in vitro activated Th2 cells and CTL for in vivo adoptive transfer.
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CD4+ and CD8+ T cells were isolated from LNs of OTII x B6-SJ or OTI mice, respectively, and
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cultured with Ag-loaded DC and cytokines. Cell purity and activation were assessed by flow
8
cytometry before i.v. transfer. (A, B) Gating strategy to identify (A) live, activated CD4+ T cells
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or (B) CD8+ T cells expressing the transgenic Vα2+Vβ5.1,5.2+ TCR. (C, D) Surface expression
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of CD44, CD69 and CD62L on CD4+ and CD8+ T cells gated as in (A) and (B), respectively.
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Figure S2
Total cells
***
1500
1000
500
0
HDM prime
DT depletion
HDM challenge
+
+
+
+
+
+
400
* ns
ns
300
500
250
0
Th2 transfer +
DT depletion OVA challenge +
+
+
+
+
+
+
CD11c-DTR
BMC
ns
+
-
CD11c-DTR
BMC
+
+
+
+
+
C57
BMC
+
-
+
+
C57
150
100
50
** ns
+
+
+
+
ns
+
+
+
+
+
+
CD11c-DTR
BMC
C57
BMC
** ns
200
ns
200
100
+
+
C57
BMC
ns
ns
150
100
50
0
0
+
+
+
***
0
+
+
C57
BMC
***
200
**
800
+
+
Cell number in BAL (x103)
750
Cell number in BAL (x103)
***
T cells
0
+
+
CD11c-DTR
BMC
B
1200
***
Cell number in BAL (x103)
Cell number in BAL (x103)
2000
E osinophils
Cell number in BAL (x103)
A
Cell number in BAL (x103)
1
+
+
+
+
+
+
-
CD11c-DTR
BMC
+
+
+
+
+
C57
BMC
+
-
+
+
C57
+
+
+
+
+
+
-
CD11c-DTR
BMC
+
+
+
+
+
C57
BMC
+
-
+
+
C57
2
3
Figure S2: Airway inflammation in mice primed to HDM, or adoptively transferred with
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OVA-specific Th2 cells, requires CD11c+ cells at the time of airway challenge
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BM chimeras were generated using C57BL/6 recipients and CD11c-DTR or C57BL/6 BM
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donor cells as indicated. Non-chimeric C57BL/6 mice were used as controls. Mice were treated
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at the indicated times with 15 ng DT (Sigma, St Louis, MO) / g body weight to deplete CD11c+
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cells; successful depletion was checked in spleen, LN and lung at the end of experiment. Total
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BAL cells were collected, counted, and the numbers of total cells, eosinophils and CD3+ T cells
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was evaluated by flow cytometry.
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(A) BM chimeric mice (BMC) were rested for 14 weeks after reconstitution, sensitized by i.p.
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injection of 40 µg HDM in alum or alum only as indicated, and challenged i.n. on day 17 with
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100 µg HDM. DT treatment was on day -1, 0 and +2 with respect to i.n. challenge; BAL was on
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d 21. Data are from one experiment with 3-10 mice / group, average + SEM are shown.
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(B) BM chimeric mice (BMC) were rested for 5 weeks after reconstitution, then injected with
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Th2 cells and challenged i.n. with 100 µg OVA as in Figure 1C. DT treatment was on day -2, 0
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and +2 with respect to i.n. challenge. Data are from one experiment with 3 – 7 mice /group,
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average values + SEM are shown.
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*p<0.05; **p<0.01; ***p<0.001; ns, not significant; as determined using a t test or ANOVA
6
with Bonferroni’s correction.
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1
Figure S3
A
SSC-A
FSC-A
FSC-A
AM
72%
FSC-A
T cells
49%
CD3 PE-Cy 7
Siglec F PE
Eosinophils
64%
SSC-A
DAPI
SSC-H
FSC-H
FSC-A
Live cells 57%
CD11c PerCP Cy5.5
SSC-A, SSC-H
96%
FSC-A, FSC-H
96%
SSC-A
MHCII AF488
B
DAPI
50.1%
SSC-A
FSC-H
95%
95%
SSC-H
FSC-A
FSC-A
8.7%
18%
CD4 PAC BLUE
SSC-A
CD8 PERCP CY5.5
Th2 + CTL treated
CD4 PAC BLUE
FSC-A
83.2%
Host
16.8%
Transferred
CD45.1 APC
CD4 PAC BLUE
No cells transferred
99.6%
Host
0.4%
Transferred
CD45.1 APC
Day 1
FSC-H
IL-13 DS Red
CD4 FITC
15.4%
FSC-A
7.9%
IL-4 AmCyan
Day 3
Vα2 APC
IL-13 DS Red
79.1%
CD3 PE-Cy7
SSC-A
C
IL-4 AmCyan
2
4
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Figure S3: Flow cytometric identification of inflammatory cell populations in BAL and
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lung.
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(A) Gating strategy to identify inflammatory cell populations in BAL (Figure 1B and 1D). Cell
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doublets were gated out using FSC-A vs FSC-H and SSC-A vs SSC-H; live cells were identified
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on the basis of DAPI exclusion. Alveolar macrophages were FSC-Ahi and CD11c+. FSC-Alo
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cells were sequentially gated to identify eosinophils (Siglec-F+ SSC-Ahi) and T cells (CD3+
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SSC-Alo).
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(B) Gating strategy to identify adoptively transferred CD4+ Th2 cells in lung cell suspensions
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(Figure 1D). Doublet exclusion and live cell identification were carried out, FSC-A/SSC-Alo-med
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lymphocytes were then gated from live cells, and CD4+ cells further separated into CD45.1+
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adoptively transferred cells or CD45.1- host cells. Staining for a control mouse that did not
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receive adoptively transferred T cells is shown as a comparison.
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(C) Gating strategy to identify reporting of IL-4 and IL-13 production by adoptively transferred
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CD4+ Th2 cells in lung (Figure 2). Doublet exclusion and live cell identification were carried
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out as in (B), FSC-H/SSC-Alo-med CD3+ lymphocytes were then gated from live cells, and
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adoptively transferred OVA-specific V2+ CD4+ T cells identified. AmCyan reports production
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of IL-4 while DsRed reports IL-13; a population of cells expressing both reporters is also
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evident.
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Figure S4
CD11C PE-CY7
2.6%
MHCII APC-CY7
86.3%
2.8%
77.5%
CD103 PE-TEX RED
58.9%
CD11C PECY7
0.6%
CD11C PECY7
19.1%
MHCII APC-CY7
75.2%
46.9%
MHCII APC-CY7
53.1%
SSC-H
96.4% II.
SSC-A
CD11B PERCP CY5.5
CD11C PE-CY7
3.3% I.
CD11B PERCP CY5.5
A
20.7%
CD103 PE-TEX RED
CD64 APC
96.4%
SSC-H
75.7%
19.2%
MHCII PAC BLUE
CD64 APC
CD45.1 APC eFLUOR780
CD103 PE TEX RED
31%
67.8%
C
99.9%
DAPI
SSC-A
CD45.2 PE
MHCII PAC BLUE
SSC-H
49%
3
87.3%
13.1%
40%
CD64 APC
CD103 PE TEX RED
CD45.1 APC eFLUOR780
SSC-H
4.8%
CD11B PERCP CY5.5
99.7%
CD11C PE CY7
EMPTY FITC
79.9%
SSC-H
SSC-H
2
49.1%
50.9%
SSC-H
3.5%
CD11C PE-CY7
EMPTY FL-1
4.1%
CD11B PERCP CY5.5
B
31.7%
66.6%
CD45.2 PE
Figure S4: Identification of DC populations in the lung and MLN
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(A) Gating strategy for lung DC populations in Figure 4. Doublet exclusion and live cell
2
identification were carried out as in Figure S3B. Two populations were identified on the basis of
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SSC and CD11c expression. The CD11c+ SSC-Ahi population (I) contained a clear majority of
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CD11b-CD103- alveolar macrophages with a few CD103+ or CD11b+ DC. The remaining
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population (II) contained CD11c+MHC-IIhigh DC, which were then subtyped into CD103+ and
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CD11b+ populations, with the latter further divided into CD64+ and CD64- cells. DC identified
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in I and II were combined together to calculate total DC numbers.
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(B) Gating strategy for lung DC in Figure 5. Doublet exclusion and live cell identification were
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carried out as in Figure S3B. Autofluorescent cells were gated out on the basis of high FL1
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signal. The remaining cells were gated to identify CD11c+MHC-IIhi DC, which were then
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subtyped into CD103+, CD11b+CD64+ and CD11b+CD64- populations. Each DC subset was
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further separated into CD45.1+ and CD45.2+ cells, of WT and bm-1 origin, respectively.
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(C) Gating strategy for LN DC in Figure 5. Doublets were excluded on the basis of SSC-A/SSC-
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H, and live cells identified by DAPI exclusion. Any autofluorescent cellular debris was
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excluded on the basis of high FL1 signal. The CD11cintMHC-IIhi DC population, which contains
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migratory DC, was identified and then subtyped into CD103+, CD11b+CD64+ and
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CD11b+CD64- populations. Each DC subset was further separated into CD45.1+ and CD45.2+
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cells, of WT and bm-1 origin, respectively.
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