Humanized Mouse Models for Vaccine Developmenti-cubed.org/wp-content/uploads/2010/11/Brehm_Presentation_Vax_Ren... · Humanized Mouse Models for ... Application of Humanized Mice

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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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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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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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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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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

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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

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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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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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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

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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

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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

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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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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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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

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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.

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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)

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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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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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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

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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