Derivation and functional characterization of distinct DC subsets from hematopoietic

stem cells.

Ofer M. Wellisch MD MPHDavid E. Levy PhD

NYU School of Medicine June 24th, 2008

Introduction

Mouse HSC are conditionally immortalized by transduction with a Hox oncoprotein that will enforce self-renewal of factor-dependent progenitors

We initially utilized both Hoxb8 and Hoxa9

Hoxb8 displayed better efficiency thus it has been used for all recent immortalizations

Hoxb8 was conjugated to an estrogen receptor ligand binding domain (ERBD) that rendered it dependent on estradiol for activity

Cells transduced in this manner proliferate in the presence of estradiol as factor-dependent progenitors

Cells terminally differentiate following estradiol withdrawal and concomitant Hoxb8 inactivation.

Introduction

Dendritic cells (DCs):

Rare cells of hematopoietic origin

Heterogeneous in nature

Essential sentinels and mediators of innate and adaptive immunity and immune homeostasis and tolerance

Reside lymphoid and peripheral tissues forming a network critical for pathogen detection, antigen presentation, as well as lymphocyte stimulation and suppression

Current and future applications include DC-targeted vaccines and immune suppressors to control allergy, autoimmunity, and transplant rejection

Introduction

We have adapted a method for immortalizing lineage-committed hematopoietic progenitors in a conditional manner, allowing us to derive replicating populations of cells capable of undergoing functional differentiation in culture.

HA Hoxa9ERBD

HA Hoxa9ERBD

Exterior

Cytosol

Nucleus

hsp90

Estradiol

HA

Hoxa9

ERBD

hsp90

HA Hoxa9ERBD

HA Hoxa9ERBD

Introduction

We proposed this method to:

analyze the progenitor populations of distinct DC subsets

characterize the pathway to functionally differentiated cells and to obtain of differentiated DC subsets in sufficient quantities to facilitate further biochemical and functional characterization

Harvest HSCs from bone marrow

Spinoculation

Virus production in Pheonix Ampho cells (Hoxb8-ERBD fusion construct)

Titering

Cells grown in presence of cytokines (FLT3L and SCF or GM-CSF) for 48 hours

Cytokine supplemented

cultureGM-CSF FLT3L and SCF

cDC pDC

Remove estradiol

(6-10 days)

Methods

(Remove SCF)

Results

cDC

(CD11b+CD11c+Siglec-H-)

pDC

(CD11b-CD11c+Siglec-H+120G8+)

Results

Loss of STAT1 impairs DC function.DC lines were derived from bone marrow of wild type and STAT1-/- mice, expanded in Flt3 or GM-CSF, and differentiated by E2 withdrawal prior to analysis for mRNA expression by qRT-PCR. Cultures were analyzed under basal conditions (Ctl) or after stimulation with IFNβ or infection with NDV for 8h, as indicated. Flt3 cultures expressed basal IFN and IRF7, which were enhanced by stimulation, while GM cultures were mostly dependent on stimulation. Both types of DCs were affected by loss of the STAT1 gene.

Results

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Control FLT3L f/fNDV FLT3L f/f

INF beta FLT3L f/f

Control FLT3L stat1 ko

NDV FLT3L stat1INF beta FLT3L stat1

Control GM-CSF f/fNDV GM-CSF f/f

INF beta GM-CSF f/f

Control GM-CSF stat1 koNDV GM-CSF stat1 ko

INF beta GM-CSF stat1 ko

estradiol FLT3L f/festradiol GM-CSF f/f

Normalized to GAPDH

RIG-I

IRF7 new

Real Time PCR on cell lines

RIG-I a well characterized ISG and IRF7 are up-regulated in cDC f/f line following infection with NDV

RIG-I and IRF7 constitutively expressed in pDC

This phenomenon is not present in stat1 ko pDC suggesting an interferon autocrine loop is required for constitutive IRF7 expression in the plasmacytoid DC.

Pre-differentiated pDC (estradiol) does not express constitutive RIG-I or IRF7

IFN feed-forward loop demonstrated in ex vivo cell lines

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3.00E+00

4.00E+00

5.00E+00

6.00E+00

7.00E+00

Control

FLT3L

f /f

NDV

FLT3L

f /f

INF beta

FLT3L

f /f

Control

FLT3L

stat1 ko

NDV

FLT3L

stat1

INF beta

FLT3L

stat1

Control

GM-CSF

f /f

NDV

GM-CSF

f /f

INF beta

GM-CSF

f /f

Control

GM-CSF

stat1 ko

NDV

GM-CSF

stat1 ko

INF beta

GM-CSF

stat1 ko

estradiol

FLT3L

f /f

estradiol

GM-CSF

f /f

Normalized to GAPDH

Siglec-H

OAS2

MX-1

RIG-I

IRF7

`

Ex Vivo Cell Lines: pDC specific markers and 4E-BP

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2.00E+00

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6.00E+00

8.00E+00

1.00E+01

1.20E+01

Control

FLT3L f /f

NDV

FLT3L f /f

INF beta

FLT3L f /f

Control

FLT3L

stat1 ko

NDV

FLT3L

stat1

INF beta

FLT3L

stat1

Control

GM-CSF

f /f

NDV

GM-CSF

f /f

INF beta

GM-CSF

f /f

Control

GM-CSF

stat1 ko

NDV

GM-CSF

stat1 ko

INF beta

GM-CSF

stat1 ko

estradiol

FLT3L f /f

estradiol

GM-CSF

f /f

Normalized to GAPDH

4E-BP

Bst2

Siglec-H

`

* * * * **

**

* *

The mammalian target of rapamycin (mTOR) or an undefined protein kinase may lead to hyperphosphorylation of 4E-BP, allowing release of the eukaryotic translation initiation factor (eIF)4E, which is now free to bind to eIF4G, forming the eIF4F translation initiation complex.

This increase in the pool of functional eIF4F allows the translation of mRNAs, such as IRF-7 mRNA, that were only inefficiently translated or translationally silent under conditions of 4E-BP hypophosphorylation.

Functional elF4F levels are high in resting pDCs due to low levels of 4E-BP1 and 4E-BP2; thereby, always allowing for efficient translation of IRF-7

4E-BP

MACS separation of splenocytes: Demonstration of secreted IFN in WT

pDC

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1.00E+01

1.20E+01

1.40E+01

1.60E+01

f lox/f lox

120G8

Control

f lox/f lox

120G8 INF

beta

f lox/f lox

CD11c

Control

f lox/f lox

CD11c INF

beta

f lox/f lox

Eluted

Control

f lox/f lox

Eluted INF

beta

f /f Stat1ko

120G8

Control

f /f Stat1ko

120G8 INF

beta

f /f Stat1ko

CD11c

Control

f /f Stat1ko

CD11c INF

beta

f /f Stat1ko

Eluted

Control

f /f Stat1ko

Eluted INF

beta

Cell line

Normalized to GAPDH

IRF7

INFa

MX-1

OAS2

MACS separation of splenocytes with analysis of pDC specific expression

markers

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4.00E+00

6.00E+00

8.00E+00

1.00E+01

1.20E+01

1.40E+01

1.60E+01

f lox/f lox

120G8

Control

f lox/f lox

120G8 INF

beta

f lox/f lox

CD11c

Control

f lox/f lox

CD11c INF

beta

f lox/f lox

Eluted

Control

f lox/f lox

Eluted INF

beta

f /f Stat1ko

120G8

Control

f /f Stat1ko

120G8 INF

beta

f /f Stat1ko

CD11c

Control

f /f Stat1ko

CD11c INF

beta

f /f Stat1ko

Eluted

Control

f /f Stat1ko

Eluted INF

beta

Cell line

Normalized to GAPDH

Siglec-H

Bst2

IRF7

IRF7 mRNA and Stat1

Arun Prakash 2004

Flu Flu

**

IRF-7

α-Tubulin

Positive Feedback Induction of IFN Production

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2.50E+00

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5.00E -01

1.00E+00

1.50E+00

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2.50E+00

Relative Gene Expression

IFNalphaIRF7IFNalphaIRF7

NDV Ctl IFN NDV Ctl IFN NDV Ctl IFN NDV Ctl IFN

Flt3 GM-CSF GM-CSF Stat1 -/-Flt3 STAT1 -/-

Positive Feedback Induction of IFN Production

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2.50E+00

0.00E+00

5.00E -01

1.00E+00

1.50E+00

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2.50E+00

Relative Gene Expression

IFNalphaIRF7IFNalphaIRF7

NDV Ctl IFN NDV Ctl IFN NDV Ctl IFN NDV Ctl IFN

Flt3 GM-CSF GM-CSF Stat1 -/-Flt3 STAT1 -/-

IRF7 Protein

Positive Feedback Induction of IFN Production

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5.00E -01

1.00E+00

1.50E+00

2.00E+00

2.50E+00

0.00E+00

5.00E -01

1.00E+00

1.50E+00

2.00E+00

2.50E+00

Relative Gene Expression

IFNalphaIRF7IFNalphaIRF7

NDV Ctl IFN NDV Ctl IFN NDV Ctl IFN NDV Ctl IFN

Flt3 GM-CSF GM-CSF Stat1 -/-Flt3 STAT1 -/-

Whole spleens (B6)

Ex vivo cell lines FLT3L (Balb/c)

IRF-7

α-Tubulin

50 kD

IRF7 Protein

Wild type flox/flox flox/flox stat1 ko

IFNAGR

Stat1 and IRF7 Protein

Arun Prakash 2004

Acute application of Ab will provide further proof of an IFN feed-forward phenomenon in pDCs

Analyze subsequent gene regulation; notably, IRF7

Comparative analysis of acute neutralizing Ab applied to wt pDCs versus resting mutant (IFNAGR -/-) pDCs should bypass any potential developmental roles of IFN.

IFN may be required for phenotypic and/or functional pDC

IFN neutralizing antibody

Even further proof of an IFN feed-forward phenomenon may be demonstrated by incubating MEFs (or IFN ultra-sensitive cell line) with conditioned media of terminally differentiated pDCs or cDCs.

Proper controls must be in place excluding roles of Flt3L and GM-CSF respectively.

Perform in conjunction with or w/o IFN neutralizing Ab

Readout by real-time PCR for ISGs (OAS2, MX-1,RIG-I etc.)

Additionally, IRF7 may be up-regulated in treated cells as well.

Positive result would suggest role in vivo of IFN secreted by pDCs in “priming” other cells of hematopoietic or non-hematopoietic origin.

Conditioned Media

Whole spleens (B6)

Ex vivo cell lines FLT3L (Balb/c)

Wild type flox/flox flox/flox stat1 ko

IFNAGR

IRF-7

α-Tubulin

4E-BP1

50 kD

10 kD

IRF7 and 4E-BP Protein

4E-BP Protein

Colina, R. Nature, 2008

Cytokine supplemented

cultureGM-CSF FLT3L and SCF

cDC pDC

Remove estradiol

(6-10 days)

Additional transgenic

mouse derived DCs

(Remove SCF)

B6 f/f stat3B6 Stat1 -/-B6 IFNAGR -/-MyD88 -/-MyD88 -/+

Additional transgenic mouse derived DCs

TLR signaling has been suggested to influence DC behavior. All TLR isoforms associate with MyD88, except TLR3 which uses the TRIF adapter molecule. By deriving lines lacking MyD88 or TLR3, we will be in a position to assess the importance of all TLR responses to ligands and viruses.

IFNAGR -/- will provide an additional model (in addition to whole spleen protein data) to support a maintained feed-forward IFN loop in pDCs under resting conditions.

IRF7 -/- DCs are planned for future once obtain mouse.

“Near” future directions

Infect new ex vivo cell lines (including MyD88) with a battery of viruses.

This may demonstrate unique ways in which pDCs function in the face of viral infection.

Compare B6 Stat1 -/- with IFNAGR -/- pDCs to further confirm an IFN dependent lack of function as opposed to a non-conventional stat1 related mechanism.

Cre introduction to abrogate STAT3 and define role in development and/or maturation

Retro/lenti

Cell-permeable protein. Dowdy and colleagues have developed a recombinant form of Cre fused to the cell permeability peptide of HIV TAT, which efficiently mediates entry of the fusion protein into many cell types in an enzymatically active form

“Near” future directions Phenotypic characterization by antibody fingerprinting

using the BD mouse CD antibody library (200 Abs) of antibodies

may provide a more precise definition of DC subsets

may yield novel cell type specific biomarkers for further characterization of DC heterogeneity

define markers for the progenitor stage define and distinguish committed progenitors from ones that still retain plasticity to switch lineages. (re-introduce estrogen at various stages)

“not exactly near” future directions Generation of peripheral DCs ex vivo (lung, skin)

Generation of human DCs. HSC could be obtained through BM biopsies routinely performed at NYUMC. Human cells would provide efficient working models for HIV infection of pDCs, a relatively enigmatic phenomenon.

Provide a model for autoimmune diseases such as SLE

considering to harvest HSCs from individuals with SNPs at IRF5 (among others), a known risk factor for lupus

Special Thank You…

• David • Isabelle• Yaming• Matt• Valentina• And everyone else in the lab!

The End ;)