mc38-ova cells 1 - nature research · (a, b) cd11c-cre and irf4fl/fl cd11c-cre mice were immunized...

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)


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)


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

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12

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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.


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

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ml)

CD

4+ T c

ells

(per

lung

)

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Neu

troph

ils (p

er lu

ng)

Naive Challenged Naive Challenged

Cd11c-CreIrf4fl/fl Cd11c-Cre

NS

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–1 7 14 21 28 35100

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0102 103 104 1050

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Time after immunization (d)–1 7 14 21 28 35




UI LCMV LCVM


× 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.


Mouse Human

Resident MigratoryCD4

CD8CD11b

CD103BDCA1

BDCA3

Blood

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Y-Ae

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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.


MH

CII

CD11c

CD

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CD11c

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IRF4 IRF8

IRF4 IRF8

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Cd11c-Cre

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Irf4fl/fl

Cd11c-Cre

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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.


Cd11c-Cre

Irf4fl/fl

Cd11c-Cre

CD80 CD86

Iso

US

LPS

CD11b

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IRF4 IRF8

b

MHCI

OVA (μg/ml) 1

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Irf4fl/fl

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MHCII (OT-II)

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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.


c

b

aIRF4

PU.1

BATF

JunB

AICE EICE

IRF4

PU.1

BATF

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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.


a

MSCV-IRF4

IRF4 IRF8

MSCV-IRF8

CD80 CD86

MSCVMSCV-IRF4MSCV-IRF8

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MSCV

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OVA (μg/ml) 10 1001

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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.


mc38-ova cells 1 - nature research · (a, b) cd11c-cre and irf4fl/fl cd11c-cre mice were immunized...

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