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Derivation and functional characterization of distinct dc subsets

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Ofer Wellisch
Ofer Wellisch

1) Mouse hematopoietic stem cells were conditionally immortalized using a Hox oncoprotein to derive distinct dendritic cell (DC) subsets for analysis. 2) The method derived conventional DCs (cDCs) and plasmacytoid DCs (pDCs) that could be terminally differentiated in culture and analyzed functionally. 3) Preliminary results showed constitutive expression of antiviral genes in pDCs but not cDCs, suggesting an interferon autocrine loop in pDCs.

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Derivation and functional characterization of distinct DC subsets from hematopoietic stem cells. Ofer M. Wellisch MD MPH David E. Levy PhD NYU School of Medicine June 24 th , 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.
Exterior Cytosol Nucleus hsp90 Estradiol hsp90
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 culture GM-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 IFN  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
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
* * * * ** ** * *
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
IRF7 mRNA and Stat1 Arun Prakash 2004 Flu Flu * *
IRF-7 α -Tubulin IRF7 Protein
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 culture GM-CSF FLT3L and SCF cDC pDC Remove estradiol (6-10 days) Additional transgenic mouse derived DCs (Remove SCF) B6 f/f stat3 B6 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!
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Derivation and functional characterization of distinct dc subsets

  • 1. Derivation and functional characterization of distinct DC subsets from hematopoietic stem cells. Ofer M. Wellisch MD MPH David E. Levy PhD NYU School of Medicine June 24 th , 2008
  • 2. 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.
  • 3. 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
  • 4. 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.
  • 5. Exterior Cytosol Nucleus hsp90 Estradiol hsp90
  • 6. 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
  • 7. 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 culture GM-CSF FLT3L and SCF cDC pDC Remove estradiol (6-10 days) Methods (Remove SCF)
  • 8. Results cDC (CD11b+CD11c+Siglec-H-) pDC (CD11b-CD11c+Siglec-H+120G8+)
  • 9. 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 IFN  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.
  • 11. 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
  • 12.
  • 13. * * * * ** ** * *
  • 14. 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
  • 15.
  • 16.
  • 17. IRF7 mRNA and Stat1 Arun Prakash 2004 Flu Flu * *
  • 18. IRF-7 α -Tubulin IRF7 Protein
  • 19. 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
  • 20. Stat1 and IRF7 Protein Arun Prakash 2004
  • 21. 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
  • 22. 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
  • 23. 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
  • 24. 4E-BP Protein Colina, R. Nature , 2008
  • 25. Cytokine supplemented culture GM-CSF FLT3L and SCF cDC pDC Remove estradiol (6-10 days) Additional transgenic mouse derived DCs (Remove SCF) B6 f/f stat3 B6 Stat1 -/- B6 IFNAGR -/- MyD88 -/- MyD88 -/+
  • 26. 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.
  • 27. “ 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
  • 28. “ 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)
  • 29. “ 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
  • 30. Special Thank You… David Isabelle Yaming Matt Valentina And everyone else in the lab!