Humanized Mouse Models for Vaccine Development Michael A. Brehm. Why Do We Need Humanized Mouse Models?

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1 Humanized Mouse Models for Vaccine Development Michael A. Brehm Diabetes Center of Excellence Dale Greiner Rita Bortell Philip DiIorio Nancy Phillips The Jackson Laboratory Leonard Shultz Human cells & tissues Why Do We Need Humanized Mouse Models? Most experimental studies done in rodents Outcomes predicted by murine studies are not always representative of actual outcomes in humans Permits study of human-specific infections and therapies Goal Enable clinically relevant in vivo studies of human cells, tissues, and immune systems without putting patients at risk 1

2 Application of Humanized Mice for Biomedical Research -Cancer biology -Regenerative medicine -Human hematopoiesis -Infectious diseases -Transplantation -Immunity and Autoimmunity Humanized Mouse Models to Study Human Immune System Development and Function Development of humanized mouse models Analysis of innate immune cell function Examination of T cell homeostasis Characteristics of the BLT (fetal thymus/fetal liver) mouse model 2

3 Mutations that Abrogate Adaptive Immunity in Mice (T and B Lymphocytes) 1) Severe combined immunodeficiency (Prkdc scid ) -Catalytic domain of DNA-activated protein kinase Required for non-homologous end-joining in immunoglobulin gene and T cell receptor gene rearrangements -Involved in ds-dna repair (radiosensitive) 2) Recombination activation gene-1 and -2 (Rag1 null and Rag2 null ) -Rag1 and Rag2 are required for creating the double stranded breaks required for immunoglobulin gene and TCR gene rearrangements -No role in DNA repair (not radiosensitive) 1983 The Road to Humanized SCID Mice 3

4 CB17-scid mouse Bosma et. al., Nature. -No mature T and B cells, but can be leaky -Engrafted with human PBMC, HSC and fetal tissues Mosier, Nature.; Lapidot, Science; McCune, Science -Very low level of engraftment with human cells: % human cells in the periphery -High level of innate immunity including NK cell activity The Road to Humanized SCID Mice

5 NOD-scid mouse Shultz et.al., J. Immunol. -NOD Strain Defects in Innate Immunity -reduced NK cell number and function -impaired macrophage activation -defective DC maturation -lack of hemolytic complement -NOD Strain Polymorphism in Sirpa gene Signal regulatory protein alpha expressed by NOD mice creates a human-like bone marrow microenvironment for interaction with human HSC (CD47); Takenaka Nat. Immunol. -Increased engraftment with human cells, but still low and variable: % human cells in the periphery -Residual innate immunity and thymic lymphomas The Road to Humanized SCID Mice

6 Receptors Utilizing IL-2r Common Chain for High Affinity Binding and Signaling IL-2r IL-4r IL-7r IL-9r IL-15r IL-21r a a a a a a X-SCID Noguchi, Cell, 73:147 IL-2r mutation leads to severe defects in both adaptive and innate immunity NOD-scid IL2r null (NSG) Mouse Shultz, J. Immunol.; Ishikawa, Blood. Complete absence of IL2rg gene -long life span -further impairment of innate immunity -complete absence of NK cells NOD-scid IL2r null mice engraft at high levels with human cells: 10-90% human cells in periphery -Develop all hematopoietic lineages of cells: T cells B cells NK cells Dendritic cells Macrophages Red blood cells 6

7 Variables For Creating Humanized Mice to Study Human Immune Responses 1. Model system 2. Age of the recipient 3. Strain background 4. Source of human tissues and cell dose 5. Injection route Human Immune System Models Hu-PBL-SCID mice: scid mice injected with human peripheral blood mononuclear cells (PBMC) Hu-SRC-SCID mice: scid mice that have been sublethally irradiated and injected with hematopoietic stem cells (HSC) scid repopulating cells (SRC) = CD34 + cells SCID-Hu mice: scid mice that have been sublethally irradiated and engrafted with human fetal liver and thymus under the renal capsule LD Shultz, et.al., Nat. Rev. Immunol. 7:118 7

8 Engraftment of NOD-scid IL2r null Mice with Human Hematopoietic Stem Cells (HSC) -Low dose whole body irradiation -Human HSC (CD34+) derived from CD3-depleted umbilical cord blood (UCB) -3x10 4 CD34 + cells injected -Screen PBL at weeks using flow cytometry T. Pearson et al (2008) Current Protocols Immunol. 15:21 HSC Engrafted Newborn NOD-scid IL2r null Mice have Higher Levels of CD3 + cells in Blood P=0.07 P= P=0.002 Brehm et al Clin. Immunol, 135:84-98,

9 Major Strain Platforms NSG NOD-scid IL2r null Jackson Lab NOG NOD-scid IL2r Trunc Central Institute for Experimental Animals BRG BALB/c-Rag2 null IL2r null Yale/Univ. Hosp. Zurich H2 d RG Stock-H2 d -Rag2 null IL2r null Pasteur Institute LD Shultz, et.al., Nat. Rev. Immunol. 7:118 Engraftment of Immunodeficient Mice with Human Hematopoietic Stem Cells (HSC) Newborn -Low dose whole body irradiation -Human HSC (CD34+) derived from CD3-depleted UCB -3x10 4 CD34 + cells injected via intracardiac route -Screen PBL at weeks using flow cytometry *Compared CD34 + HSC engraftment in 3 strains: NOD-scid IL2r null, NOD- Rag1 null IL2r null, and BALB/c-Rag1 null IL2r null mice Brehm et al Clin. Immunol, 135:84-98,

10 HSC-Engrafted Newborn NOD Strains have Higher Levels of Human Cells in Peripheral Blood Human Lymphocytes NS P<0.001 Human T cell levels NS P<0.05 Human B cell levels NS P<0.05 Summary-I 1. Immunodeficient mice bearing targeted mutations in the IL2r common -chain are the optimal recipients of human HSC 2. Direct comparison of HSC engraftment in IL2r null strains of mice -Newborn IL2r null mice support higher levels of T cell engraftment as compared to adults -NOD strains support higher levels of human cell engraftment in the periphery -NOD strains support higher levels of T cell engraftment in periphery 10

11 Humanized Mouse Models to Study Human Immune System Development and Function Development of humanized mouse models Analysis of innate immune cell function Examination of T cell homeostasis Characteristics of the BLT (fetal thymus/fetal liver) mouse model Stimulation of Innate Immunity with LPS LPS IFN TLR4 TRAM Mal TNF IL-1 IL-6 IL-8 TRIF MyD88 TRAF3 RIP1 IRAKs TRAF6 IRF3 Nucleus IkB NF-kB IFN-β IRF3 NF-kB Inflammatory cytokines 11

12 Cytokine Production in HSC-Engrafted Newborn NSG-TLR4 null Mice Injected with LPS Unengrafted NSG and NSG-TLR4 null mice were injected with LPS and murine cytokines were measured in the serum 24 hours later -Low dose whole body irradiation -Human HSC (CD34+) derived from CD3-depleted UCB -3x10 4 CD34 + cells injected via intracardiac route into newborn NSG or NSG-TLR4 null mice *Determine levels of inflammatory cytokines (IL1beta, IL-6, IL-8 and TNF) in serum of mice at 6, 12 and 24 hours post LPS injection NSG-TLR4 null Mice Efficiently Engraft with Human HSC (12-14 weeks in peripheral blood) p =

13 HSC-Engrafted NSG-TLR4 null Mice Produce Human Cytokines after LPS Challenge T Cell Turnover in Human HSC-Engrafted Newborn NSG Mice Newborn NSG, NSG-HLA-A2, NSG-HLA-A2-HHD -Low dose whole body irradiation -A2+ HSC (CD34+) derived from CD3-depleted UCB -3x10 4 CD34 + cells injected via intracardiac route *Determine levels of proliferation in T cell populations 13

14 T cells Developing in HSC-Engrafted NSG Mice are Proliferating at a High Level (14-18 weeks in peripheral blood) CD4 T cells CD8 T cells Summary-II 1. Humanized mice will produce human inflammatory cytokines in response to TLR agonists -NSG-TLR4 null mouse reduces the murine response to LPS 2. Human T cells developing in humanized mice are proliferating a high rate -Transgenic expression of HLA does not alter the proliferation -Proliferation might be stimulated by the lymphopenic environment 14

15 Humanized Mouse Models to Study Human Immune System Development and Function Development of humanized mouse models Analysis of innate immune cell function Examination of T cell homeostasis Characteristics of the BLT (fetal thymus/fetal liver) mouse model weeks gestational age BLT Mouse Model Bone Marrow/Liver/Thymus FT Implant thy/liv Organoid 1mm cubes 200 cgy NOD-scid IL2r null FL Isolate CD34+ cells 0.2-1x10 6 -Develops robust immune system comprised of multiple lineages -Sustained, high level T cell development -T cells educated on autologous thymic tissues -Detectable T and B cell responses to viral infection (EBV and HIV) Melkus, Nat. Med.; Sun, 2007, J. Exp. Med.; Brainard, 2009, J. Virol. -Rejection of pig islets: Tonomura, XenoTranspl. -Rejection of non-self human islets: unpublished data, Lafferty et.al. 15

16 Thymus Development in BLT mice at 16 Weeks Post-Implant Thymic Organoid Thymocyte subsets hcd8 hcd4 Human Cell Engraftment is Superior in the BLT Mouse Model (PBL) Total Human Cell Engraftment Human T cell Development Human B cell Development 16

17 Peripheral T cell Development (spleen at 16 weeks) B cells and T cells hcd45 Gate T cell Subsets hcd3 Gate Tregs hcd4 Gate hcd20 hcd3 hcd4 CD4 T cells hcd8 CD25 CD8 T cells FoxP3 CD45RA CD45RA CD45RO CD45RO Dendritic Cell and Monocyte/Macrophage Development (spleen at 16 weeks) Dendritic Cells CD11c BDCA-2 CD123 CD123 Monocyte/ Macrophage CD14 CD33 17

18 CD8 CD8 11/17/2010 Dengue Fever Dengue virus- mosquito-borne Flavivrus Four closely-related serotypes 2.5 billion people at risk 50 million cases/yr worldwide >20,000 deaths Dengue Shock syndrome/hemorrhagic fever high mortality rate in children May accompany secondary infection of a different Dengue virus serotype Associated with Ab-mediated enhancement of Dengue virus in macrophages + DCs Accompanied by elevated TNF-α and other cytokines Anuja Mathew Multifunctional Epitope-Specific CD8 T cell Response Generated by Infection with Dengue Virus Serotype 2 (day 7 from the spleen) Human CD8 T cells Stimulated with NS4a 2148 peptide (known A2 epitope) IFN- TNF Human CD8 T cells Stimulated with NS5-15mer peptide IFN- TNF Anuja Mathew 18

19 Summary-III BLT model allows for robust and consistent engraftment of human cells, including multiple hematopoietic lineages T cells are educated on human thymic epithelium HLA-restricted T cells T cell development is normal, with a low frequency of activated phenotype T cells (RA/RO analysis) Generation of Dengue-specific CD8 T cell responses Limitations of Human Hematopoietic Stem Cell (HSC) Engraftment in NSG Mice Lack of HLA molecules required for appropriate T cell function -HLA-matching is critical for T cell development in the thymus -HLA-matching is important for survival of T cells in the periphery -HLA is an important factor in autoimmune susceptibility Species specificity of growth factors and other molecules Remaining innate immunity -The NOD-scid IL2r null mouse model can be improved upon by introduction of human molecules that enhance engraftment of human HSC and immune cell development 19

20 Biomedical Research with Humanized Mice Acknowledgements UMass-Diabetes Center of Excellence David Harlan Amy Cuthbert Laurence Covassin Waldemar Racki Pam Wooton Jean Leif Phil Durost Linda Paquin Michael Bates UMass Roger Davis JeanMarie Houghton Michelle Kelliher Hardy Kornfeld Anuja Mathew Fumi Urano Liisa Selin Raymond Welsh Michael Czech Katherine Luzuriaga The Jackson Laboratory Leonard Shultz David Serreze USAMRIID Steven Bradfute Sina Bavari NIAID NIDDK 20