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b
d e
DC enriched
Cd11c-Cre
Irf4fl/fl
Cd11c-Cre
MHCIIint
MH
CII
CD
103
CD11c CD11bSpleen
h
MHCIIhi
CD103+
CD11b–
i
2000
4000
6000
MHCIIhi
CD103+
CD11b+
g
CD103+
CD11b–CD103–
CD11b+
MHCIIhi
CD103+
CD11b–
MHCIIhi
CD103–
CD11b+
c
f
# D
Cs
(per
106 ce
lls)
Cd11c-CreIrf4fl/flCd11c-Cre
1000
2000
3000
4000
5000
5000
10000
15000
20000
25000
a
CD103+
CD11b–
IRF4 IRF8
CD103–
CD11b+
IRF4 IRF8
0102 103 104 105
20406080
100
Even
ts (%
of m
ax)
IRF4 IRF8
MHCIIhi
CD103+
CD11b–
MHCIIhi
CD103+
CD11b+
IRF4 IRF8
0102 103 104 105
20406080
100
Even
ts (%
of m
ax)
IRF4 IRF8
MHCIIhi
CD103+
CD11b–
MHCIIhi
CD103–
CD11b+
IRF4 IRF8
0102 103 104 105
20406080
100
Even
ts (%
of m
ax)
0102 103 104 1050
102
103
104105
DC enriched
Cd11c-Cre
Irf4fl/fl
Cd11c-Cre
MHCIIhi
MH
CII
CD
103
CD11c CD11b
CLN
0102 103 104 1050
102
103
104105
NS
*
# D
Cs
(per
106 ce
lls)
Cd11c-CreIrf4fl/flCd11c-Cre
NS
*
DC enriched
Cd11c-Cre
Irf4fl/fl
Cd11c-Cre
MHCIIhi
MH
CII
CD
103
CD11c CD11b
MLN
0102 103 104 1050
102
103
104105
# D
Cs
(per
106 ce
lls)
Cd11c-CreIrf4fl/flCd11c-Cre
NS
*
Supplementary Figure 1. Selective loss of CD11b+ DCs in Irf4fl/fl Cd11c-Cre mice(a) Splenic DC populations of wild-type mice were analyzed by flow cytometry for intracellular expression of IRF4 and IRF8 protein. Filled grey histograms are isotype controls. IRF4 and IRF8 expression was similarly analyzed for MHCIIhi DC populations in cutaneous lymph nodes (CLN) (d) and mesenteric lymph nodes (MLN) (g). (b) Left, DC-enriched cells from spleens of Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were analyzedby flow cytometry for expression of CD11c and MHCII. Numbers indicate percentage of cells within the CD11c+MHCIIint gate. Right, CD11c+MHCIIint DC subpopulations were distinguished by expression of CD11b and CD103. Numbers indicate percentage of cells within each gate. (c) Quantitative analysis of DCs described in (b). Data represent mean ± s.e.m. from 3-5 independent experiments, each with pooled splenic tissue from 5 animals. NS = not significant; * = P < 0.05, two-tailed t-test. (e) Left, DC-enriched cells from CLNs of Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were analyzed by flow cytometry for expression of CD11c and MHCII. Numbers indicate percentage of cells within the CD11c+MHCIIhi gate. Right, CD11c+MHCIIhi migratory DC subpopulations were distinguished by expression of CD11b and CD103. Numbers indicate percentage of cells within each gate. (f) Quantitative analysis of DCs described in (e). Data represent mean ± s.e.m. from at least 3 independent experiments, each with pooled CLN tissue from 5 animals. NS = not significant; * = P < 0.05, two-tailed t-test. (h, i) Migratory MHCIIhi DCs from MLN of Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were analyzed as in panels e and f. Data represent mean ± s.e.m. from 3 independent experiments, each with pooled MLN tissue from 5 animals. NS = not significant; * = P < 0.05, two-tailed t-test.
Spleen Spleen
CLN CLN
MLN MLN
Nature Immunology: doi:10.1038/ni.2795
Irf4fl/fl
Cd11c-Cre
MHCII (OT-II)
MHCI (OT-I)
Cd11c-Cre
MHCII (OT-II)
MHCI (OT-I)
MC38-OVA cells 5x1062.5x1061.25x106
CellTrace Violet0102 103 104 105
8642
12
9
6
3
4
23
5
12
4
6
86
42
8642
10
2
4
6
2
4
6 8
6
42
2
4
68642
8642
# ce
lls (x
102 )
# ce
lls (x
102 )
0102 103 104 105
Supplementary Figure 2. IRF4-dependent CD11b+ DCs preferentially prime helper T cell responses to cell-associated antigen CellTrace violet-labeled OT-I and OT-II T cells were co-transferred into Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice.Recipient mice were subsequently immunized by footpad injection with the indicated numbers of necroticovalbumin-expressing MC38 (MC38-OVA) tumor cells. Histograms show OT-I and OT-II proliferation measuredby dye dilution, 3 days after antigen administration. For each dose, paired OT-II and OT-I data from the same mouse is displayed. Data are representative of 2 independent experiments, n = 2 mice per dose in each experiment.
Nature Immunology: doi:10.1038/ni.2795
a b
e WTUI
Cd11c-CreLCMV
Irf4fl/fl Cd11c-CreLCMV
CD8
IFN
-γ
fVi
ral p
fu/li
ver
Time after immunization (d)
anti-
TNP-
OVA
IgG
1 (μ
g/m
l)
anti-
TNP-
OVA
IgE
(ng/
ml)
CD
4+ T c
ells
(per
lung
)
c d
Neu
troph
ils (p
er lu
ng)
Naive Challenged Naive Challenged
Cd11c-CreIrf4fl/fl Cd11c-Cre
NS
100
101
102
103
–1 7 14 21 28 35100
101
102
103
104
104
105
106
107
104
105
106
107
0102 103 104 1050
102
103
104
105
Time after immunization (d)–1 7 14 21 28 35
Cd11c-CreIrf4fl/fl Cd11c-Cre
Cd11c-CreIrf4fl/fl Cd11c-Cre
Cd11c-CreIrf4fl/fl Cd11c-Cre
UI LCMV LCVM
Cd11c-CreIrf4fl/fl Cd11c-Cre
× WT
Supplementary Figure 3. Selective impairment of TH-dependent immunity in Irf4fl/fl Cd11c-Cre mice(a, b) Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were immunized with TNP-OVA and serum antibody titers forIgG1 (a) or IgE (b) isotypes were determined by ELISA on the indicated days. Data represent antibody titersfrom 4 individual mice per group with horizontal bars indicating mean values. Data are representative of 2independent experiments. (c, d) Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were sensitized with TNP-OVA. 35days after sensitization mice were challenged by administration of nebulized TNP-OVA into the airways for7 consecutive days. 24 h after the last antigen administration lungs were removed. Lungs from naive miceserved as controls. Lung infiltrating CD4+ T cells (c) and neutrophils (d) were enumerated by flowcytometry. Data represent cell counts from individual mice (naive n = 5, challenged n = 8) mean ± s.d.; one experimental repicate. (e, f) Cd11c-Cre and Irf4fl/fl Cd11c-Cre mice were infected with LCMV. Uninfected (UI) wild-type (WT) C57BL/6 mice served as controls. (e) 8 days after infection splenocytes were stimulated in vitro with LCMV gp33 peptide. CD8+ CTLs were analyzed by intracellular flow cytometry for expression of IFN-γ. Representative data from individual mice are shown. Numbers indicate percentage of gated CD8+ IFN-γ+ splenocytes. (f) Viral titers in livers of infected mice were determined at day 8 after infection by pfu assay. Data depict titers determined for individual mice, mean indicated by horizontal bars; NS = not significant; one-tailed Mann-Whitney. Data are representative of 2 independent experiments.
Nature Immunology: doi:10.1038/ni.2795
Mouse Human
Resident MigratoryCD4
CD8CD11b
CD103BDCA1
BDCA3
Blood
5
10
15
20
Y-Ae
(103 g
MFI
)0 0.25 0.5 1.0Eα (mg)
WT MHCIIhiCD103–CD11b+
WT MHCIIhiCD103+CD11b–
*
a b
*
*
Supplementary Figure 4. IRF4 as a regulatory determinant of enhanced pMHCII formation inmurine and human DCs(a) Efficiency of pMHCII formation by MHCIIhiCD11b+CD103– (red) or MHCIIhiCD11b–CD103+ (blue) CLNmigratory DC subsets was assessed after subcutaneous administration of the indicated doses of Eα antigenin wild-type (WT) C57BL/6 mice. DCs were enriched and then stained, 18 h after injection of Eα, with Y-Ae antibody and analyzed by flow cytometry. Data are from two independent experiments, n = 2 mice per dose,and represent geometric mean fluorescence intensity (gMFI) ± s.e.m. * = P < 0.05, one-tailed t-test. (b) Differential gene expression analysis in paired mouse and human DC subsets. Mouse resident DCs are represented by splenic CD4+CD8– (CD4) and CD4–CD8+ (CD8) subsets. Mouse migratory DCs are representedby cutaneous lymph node MHCIIhiCD103–CD11b+ (CD11b) and MHCIIhiCD103+CD11b– (CD103) subsets.Human DCs isolated from blood are represented by BDCA1+ and BDCA3+ subsets. The murine data is fromthe ImmGen database17 whereas the human data is from E-TABM-34 (ref. 36). Expression of indicated genesis displayed as fold-increase (red) or decrease (blue) relative to its average expression in paired DC subsets.
Nature Immunology: doi:10.1038/ni.2795
MH
CII
CD11c
CD
11b
CD11c
Total DCs
IRF4 IRF8
IRF4 IRF8
a
Cd11c-Cre
0102 103 104 105
0102
103
104
105
0102 103 104 105
20
40
60
80
100Even
ts (%
max
)
MH
CII
CD11c
CD
11b
CD11c
Total DCs
IRF4 IRF8
IRF4 IRF8
b
Irf4fl/fl
Cd11c-Cre
0102 103 104 105
0102
103
104
105
0102 103 104 105
20
40
60
80
100Even
ts (%
max
)
Supplementary Figure 5. Flt3L-generated BMDCs express abundant IRF8 but not IRF4 andare not a suitable model for IRF4-dependent CD11b+ DCsBMDCs were differentiated from Cd11c-Cre (a) and Irf4fl/fl Cd11c-Cre (b) hematopoietic progenitorsby culture in the presence of Flt3L. BMDCs were analyzed by flow cytometry for their expressionof CD11c and MHCII. Numbers indicate percentages of cells within the CD11c+MHCII+ DC gate.CD11c+MHCII+ DC subpopulations were distinguised by their expression of CD11b. Numbersindicate percentage of cells within each gate. CD11b+ (red) and CD11b– (blue) DC populations wereanalyzed by flow cytometry for intracellular expression of IRF4 or IRF8 protein. Filled grey histogramsare isotype controls. Data are representative of 2 independent experiments.
Nature Immunology: doi:10.1038/ni.2795
Cd11c-Cre
Irf4fl/fl
Cd11c-Cre
CD80 CD86
Iso
US
LPS
CD11b
a
IRF4 IRF8
b
MHCI
OVA (μg/ml) 1
Cd11c-Cre
Irf4fl/fl
Cd11c-Cre
CellTrace Violet
10 100
c
MHCII
MHCII (OT-II)
15
10
5
108642
0102 103 104 105
201510
5
15
10
5
15
10
5
20
10
30
# ce
lls (x
102 )
0102 103 104 105
10080604020ev
ents
(% m
ax)
0102 103 104 105
10080604020
even
ts (%
max
)
Supplementary Figure 6. IRF4 regulates the DC maturation program and MHCII antigen presentation(a-c) BMDCs were differentiated from Cd11c-Cre hematopoietic progenitors by culture in the presence ofGM-CSF and IL-4. (a) CD11c+ DC populations were analyzed by flow cytometry for intracellular expressionof IRF4 or IRF8 protein. Filled grey histograms are isotype controls. Data are representative of 3 independent experiments. (b) BMDCs derived from Cd11c-Cre or Irf4fl/fl Cd11c-Cre mice were left unstimulated (US, dashed histograms) or stimulated overnight with LPS (solid histograms), then analyzed by flow cytometry for expression of the indicated markers (see also Fig. 4a). Isotype controls are shown in filled grey histograms. Data are representative of at least 3 independent experiments. (c) Cd11c-Cre and Irf4fl/fl Cd11c-Cre BMDCs were loaded with the indicated concentrations of ovalbmin (OVA) then stimulated with LPS. Antigen-loaded CD11c+ DCs were isolated by magnetic separation and used to stimulate OT-II T cells for 3 days. Histograms show OT-II proliferation as measured by dilution of CellTrace violet dye. Data are representative of at least 3 independent experiments.
Nature Immunology: doi:10.1038/ni.2795
c
b
aIRF4
PU.1
BATF
JunB
AICE EICE
IRF4
PU.1
BATF
JunB
IRF4
PU.1
BATF
JunB
Supplementary Figure 7. IRF4 directly targets key genes for MHCII antigen presentation(a-c) Wild-type C57BL/6 BMDCs differentiated with GM-CSF and IL-4 were stimulated for 6 h with LPS.IRF4, PU.1, BATF, or JunB-bound chromatin fragments were immunoprecipitated and subjected to high-throughput sequencing (ChIPseq). Sequence tracks displaying peaks for each of the above transcription factors are shown for Ctss (a), H2-Dmb2 (b), and Ciita (c) genes. Boxed peaks containingEts-IRF (EICE) or AP-1-IRF (AICE) composite motifs are highlighted. Two independent ChIPseq replicates showed greater than 95% overlap in called IRF4 binding sites.
Nature Immunology: doi:10.1038/ni.2795
a
MSCV-IRF4
IRF4 IRF8
MSCV-IRF8
CD80 CD86
MSCVMSCV-IRF4MSCV-IRF8
b
MSCV
MSCV-IRF4
MSCV-IRF8
CellTrace Violet
OVA (μg/ml) 10 1001
MHCI (OT-I)
0 102 103 104 105
20
40
60
80
100
Even
ts (%
max
)
0 102 103 104 105
8
6
4
2
12
9
6
3
2520
15
105
25
20
15
10
5
12
9
6
3
20
15
10
5
12
9
6
3
12
9
6
3
12
9
6
3# ce
lls (x
102 )
Supplementary Figure 8. IRF4 and IRF8 comparably induce aspects of DC maturation(a) Irf4fl/fl Cd11c-Cre hematopoietic progenitors were transduced with MSCV-IRES-huCD4 (MSCV) orits IRF4- or IRF8-expressing derivatives and then differentiated into DCs with GM-CSF and IL-4 (see Fig. 5a).Complemented BMDCs were analyzed by flow cytometry for expression of IRF4, IRF8, CD80, and CD86.Histograms represent staining for MSCV control (black), MSCV-IRF4 (red), and MSCV-IRF8 (blue) complemented BMDCs. Data are representative of at least 3 independent experiments. (b) ComplementedBMDCs were loaded with the indicated concentrations of ovalbumin (OVA) protein and then stimulated overnight with LPS. Transduced CD11c+ cells were purifed by flow cytometry and used to stimulate labeled OT-I T cells for 3 days. T cell proliferation was analysed by dye dilution. Data are representative of 2 independent experiments.
Nature Immunology: doi:10.1038/ni.2795