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11/17/2010
1
Humanized Mouse Models for Vaccine Development
Michael A. Brehm
Human cells
& tissues
Diabetes Center of Excellence Dale GreinerRita Bortell
Philip DiIorioNancy Phillips
The Jackson LaboratoryLeonard Shultz
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
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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
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3
Mutations that Abrogate Adaptive Immunity
in Mice (T and B Lymphocytes)
1) Severe combined immunodeficiency (Prkdcscid)
-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 (Rag1null and
Rag2null)
-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)
The Road to Humanized SCID Mice
1983
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4
CB17-scid mouseBosma et. al., 1983. Nature.
-No mature T and B cells, but can be leaky
-Engrafted with human PBMC, HSC and fetal tissues
Mosier, 1988. Nature.; Lapidot, 1992. Science; McCune, 1988.
Science
-Very low level of engraftment with human cells: 0.01-
0.1% human cells in the periphery
-High level of innate immunity including NK cell
activity
The Road to Humanized SCID Mice
1995
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5
NOD-scid mouseShultz et.al., 1995. 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 2007. Nat. Immunol.
-Increased engraftment with human cells, but still
low and variable:
0.1-10% human cells in the periphery
-Residual innate immunity and thymic lymphomas
The Road to Humanized SCID Mice
2005
11/17/2010
6
Receptors Utilizing IL-2r Common Chain
for High Affinity Binding and Signaling
Noguchi, 1993.
Cell, 73:147
a
IL-2r
a
IL-4r
a
IL-7r
a
IL-21r
a
IL-15r
a
IL-9r
X-SCID
IL-2r mutation leads to severe defects
in both adaptive and innate immunity
NOD-scid IL2rnull (NSG) MouseShultz, 2005. J. Immunol.; Ishikawa, 2005. Blood.
•Complete absence of IL2rg gene-long life span
-further impairment of innate immunity
-complete absence of NK cells
•NOD-scid IL2rnull mice engraft at high levels with
human cells: 10-90% human cells in periphery
-Develop all hematopoietic lineages of cells: T cellsB cellsNK cellsDendritic cellsMacrophagesRed blood cells
11/17/2010
7
Variables For Creating Humanized Miceto 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 sublethallyirradiated and engrafted with human fetal liver and thymus under the renal capsule
LD Shultz, et.al., 2007. Nat. Rev. Immunol. 7:118
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8
Engraftment of NOD-scid IL2rnull Mice with Human Hematopoietic Stem Cells (HSC)
T. Pearson et al (2008) Current Protocols Immunol. 15:21
-Low dose whole body irradiation
-Human HSC (CD34+) derived from CD3-depleted umbilical cord
blood (UCB)
-3x104 CD34+ cells injected
-Screen PBL at 12-16 weeks using flow cytometry
HSC Engrafted Newborn NOD-scid IL2r null Mice have Higher Levels of CD3+ cells in Blood
P=0.07
P=0.0009 P=0.002
Brehm et al Clin. Immunol, 135:84-98, 2010
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9
Major Strain Platforms
NSG NOD-scid IL2rnull Jackson Lab
NOG NOD-scid IL2rTrunc Central Institute for
Experimental Animals
BRG BALB/c-Rag2null IL2rnull Yale/Univ. Hosp. Zurich
H2dRG Stock-H2d-Rag2null IL2rnull Pasteur Institute
LD Shultz, et.al., 2007. 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
-3x104 CD34+ cells injected via intracardiac route
-Screen PBL at 12-16 weeks using flow cytometry
*Compared CD34+ HSC engraftment in 3 strains: NOD-scid IL2rnull, NOD-Rag1null IL2rnull, and BALB/c-Rag1null IL2rnull mice
Brehm et al Clin. Immunol, 135:84-98, 2010
11/17/2010
10
HSC-Engrafted Newborn NOD Strains have Higher Levels of Human Cells in Peripheral Blood
Human Lymphocytes
P<0.001
NS
Human T cell levels Human B cell levels
P<0.05
NS 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 IL2rnull strains of mice
-Newborn IL2rnull 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
11/17/2010
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
Nucleus
IFN-β IRF3
NF-kB
IkB
Inflammatory cytokines
TLR4
TRAF6
IFN
TRAF3 RIP1
NF-kB
LPS
IL-1
IL-6
IL-8
TNF
Stimulation of Innate Immunity with LPS
TRAM Mal
IRAKs
IRF3
TRIF MyD88
11/17/2010
12
Cytokine Production in HSC-Engrafted Newborn NSG-TLR4null Mice Injected with LPS
-Low dose whole body irradiation
-Human HSC (CD34+) derived from CD3-depleted UCB
-3x104 CD34+ cells injected via intracardiac route into
newborn NSG or NSG-TLR4null 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
Unengrafted NSG and NSG-TLR4null
mice were injected with LPS and
murine cytokines were measured in
the serum 24 hours later
p = 0.003
NSG-TLR4null Mice Efficiently Engraft with Human HSC (12-14 weeks in peripheral blood)
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13
HSC-Engrafted NSG-TLR4null 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
-3x104 CD34+ cells injected via intracardiac route
*Determine levels of proliferation in T cell populations
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14
CD4 T cells CD8 T cells
T cells Developing in HSC-Engrafted NSG Mice are Proliferating at a High Level(14-18 weeks in peripheral blood)
Summary-II
1. Humanized mice will produce human inflammatory cytokines in response to TLR agonists
-NSG-TLR4null 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
11/17/2010
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
BLT Mouse ModelBone Marrow/Liver/Thymus
16-22 weeks
gestational age
FL
FT
Implant thy/liv
“Organoid”
1mm cubes
200cGy
Isolate CD34+
cells
0.2-1x106
NOD-scid IL2rnull
-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, 2006. Nat. Med.; Sun, 2007, J. Exp. Med.; Brainard, 2009, J. Virol.
-Rejection of pig islets: Tonomura, 2008. XenoTranspl.
-Rejection of non-self human islets: unpublished data, Lafferty et.al.
11/17/2010
16
Thymus Development in BLT mice at 16
Weeks Post-Implant
Thymic Organoid Thymocyte subsets
hCD4
hCD8
Total Human Cell Engraftment
Human T cell Development Human B cell Development
Human Cell Engraftment is Superior in the BLT Mouse Model (PBL)
11/17/2010
17
B cells and T cellshCD45 Gate
T cell SubsetshCD3 Gate
Peripheral T cell Development
(spleen at 16 weeks)
hCD3
hCD20
hCD8
hCD4
TregshCD4 Gate
FoxP3
CD25
CD45RO
CD45RA
CD4 T cells CD8 T cells
CD45RO
CD45RA
Dendritic Cell and Monocyte/Macrophage
Development (spleen at 16 weeks)
Dendritic Cells
CD123
CD11c
CD123
BDCA-2
Monocyte/Macrophage
CD33
CD14
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18
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
IFN-
Multifunctional Epitope-Specific CD8 T cell Response Generated by Infection with Dengue Virus Serotype 2 (day 7 from the spleen)
1.56 1.50
1.031.50
TNF
Human CD8 T cells Stimulated with NS5-15mer peptide
Human CD8 T cells Stimulated with NS4a 2148 peptide (known A2 epitope)
CD
8
IFN- TNF
CD
8
Anuja Mathew
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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 HematopoieticStem 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 IL2rnull mouse model can be improved upon by introduction of human molecules that enhance engraftment of human HSC and immune cell development
11/17/2010
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
•The Jackson Laboratory
– Leonard Shultz
– David Serreze
•USAMRIID
– Steven Bradfute
– Sina Bavari
NIDDKNIAID
•UMass
– Roger Davis
– JeanMarie Houghton
– Michelle Kelliher
– Hardy Kornfeld
– Anuja Mathew
– Fumi Urano
– Liisa Selin
– Raymond Welsh
– Michael Czech
– Katherine Luzuriaga