Development of In Vivo Models to Detect the Immune Cell Infiltration Response To Treatment: Humanized Mice

Michael A. Brehm

Diabetes Center of Excellence Dale Greiner David Harlan Rita Bortell

The Jackson Laboratory Leonard Shultz

James Keck

Regenerative

Medicine

Hematological

Disorders

Immunology

Autoimmunity

Cancer

Beta-Cell

Biology

Infectious

Diseases

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

• Seok et al, 2013. PNAS, Vol. 110, p3507

– 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

The Road to Humanized SCID Mice

1983

1995

2005

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) Mouse Shultz, 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-Rag1null IL2rnull (NRG)

•NSG and NRG mice engraft at high levels with

human cells, tissues and immune systems: -Tumors

-Skin

-Islets

-Hepatocytes

-ES cells and iPS cells

-Adipose Tissue

-Hematopoietic stem cells (HSC)

-Mature immune cells

Human Immune System Models • Hu-PBL-SCID mice: immunodeficient mice injected with

human peripheral blood mononuclear cells (PBMC) – Mosier, 1988. Nature, 335:256

• Hu-SRC-SCID mice: immunodeficient mice that have been

sublethally irradiated and injected with hematopoietic stem cells (HSC) – scid repopulating cells (SRC) = CD34+ cells – Lapidot, 1992. Science, 255:1137

• SCID-Hu mice: immunodeficient mice that have been

engrafted with human fetal liver and thymus under the renal capsule – BLT (bone marrow/liver/thymus) with autologous CD34+ cells

from liver – McCune, 1988. Science, 241:1632

LD Shultz, et.al., 2007. Nat. Rev. Immunol. 7:118

Model of Xenogeneic-GVHD Mediated by Human PBMC

Intravenous Injection of 10x106

Human PBMC

NSG Mice

+ or - radiation

Follow for engraftment

and disease

-hair loss/erythema -hunched posture -weight loss -death

Hu

man

CD

45

+ ce

lls (

% in

blo

od

)

2 Weeks 4 Weeks 6 Weeks

At 8 weeks of age NSG mice were injected IP with 1x107 human PBMC

Human Cell Chimerism in PBMC-Engrafted NSG Mice Human CD45+ cells in blood

0

20

40

60

NSG Mice Succumb to Xeno-GVHD Mediated by Human PBMC

Cu

mu

lati

ve S

urv

ival

(%

)

King et al., Clin Exp Immunol 157:104, 2009

Days

Reduction of Xeno-GVHD in the Hu-PBL-SCID Model

• Study human T cell function in the absence xeno-GVHD

• Majority of engrafted cells recovered from blood and spleen of NSG injected with human PBMC are T cells

– Our hypothesis is that mice lacking murine MHC will have reduced susceptibility to xeno-GVHD

• NOD-scid IL2rnull Ab0

• NOD-scid IL2rnull 2Mnull

• NOD-scid IL2rnull KbDbnull

King et al., Clin Exp Immunol 157:104, 2009

Covassin et al., Clin Exp Immunol. 2011

NSG Mice Lacking Expression of Murine MHC Class I (2Mnull) have a Delayed Xeno-GVHD

0 20 40 60 800

50

100

Days

Su

rviv

al (%

)

NSG PBS

NSG PBMC

B2Mnull PBS

B2Mnull PBMC

EAnull PBMC

EAnull PBS

Groups N=5

NSG mice were injected IP with 10 million human PBMC (No irradiation)

p<0.001

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 (BLT) LD Shultz, et.al., 2007. Nat. Rev. Immunol. 7:118

Hematopoietic Stem Cells and Hematopoiesis

L. Shultz, 2012

LD Shultz, et al (2007) Nat. Rev. Immunol. 7:118 T Pearson et al (2008) Current Protocols Immunol. 15:21

X-Ray dose 100cGy 100cGy

Engraftment of NSG Mice with Human Hematopoietic Stem Cells (HSC)

-Low dose whole body irradiation

-Human HSC (CD34+) derived from CD3-depleted umbilical cord

blood (UCB)

-1x105 CD34+ cells injected

-Screen PBL at 6-18 weeks using flow cytometry

21 to 28 Days 1 to 3 Days

0

20

40

60

80

100

At 3 weeks of age NSG mice were irradiated (100 rads), injected IV with 100K cord

blood derived HSC

Human Cell Chimerism in HSC-Engrafted NSG Mice Human CD45+ cells in blood

Hu

man

CD

45

+ ce

lls (

% in

blo

od

)

6 Weeks 9 Weeks 12 Weeks 15 Weeks 18 Weeks

0

20

40

60

80

At 3 weeks of age NSG mice were irradiated (100 rads), injected IV with 100K cord

blood derived HSC

Human T Cell Development in HSC-Engrafted NSG Mice Human CD3+ cells in blood

Hu

man

CD

3+

cells

(%

of

CD

45

in b

loo

d)

6 Weeks 9 Weeks 12 Weeks 15 Weeks 18 Weeks

Human Immune System Models • Hu-PBL-SCID mice: immunodeficient mice injected with

human peripheral blood mononuclear cells (PBMC) – Mosier, 1988. Nature, 335:256

• Hu-SRC-SCID mice: immunodeficient mice that have been

sublethally irradiated and injected with hematopoietic stem cells (HSC) – scid repopulating cells (SRC) = CD34+ cells – Lapidot, 1992. Science, 255:1137

• SCID-Hu mice: immunodeficient mice that have been

engrafted with human fetal liver and thymus under the renal capsule – BLT (bone marrow/liver/thymus) with autologous CD34+ cells

from liver – McCune, 1988. Science, 241:1632

LD Shultz, et.al., 2007. Nat. Rev. Immunol. 7:118

BLT Mouse Model: Bone Marrow/Liver/Thymus

17.3

8.4667.9

Thymic Organoid, 16 Weeks

HuCD4

Hu

CD

8

16-22 weeks gestational age

FL

FT

Implant thy/liv 1mm cubes

200cGy

Isolate CD34+ HSC 1-2x105

NOD-scid IL2rγnull mouse

5% 0.9%

0.2%

Mac/Mono mDC and pDC

HuCD33

Hu

CD

14

HuCD123

Hu

CD

11

c

Human Immune Cells, Blood 16 Weeks

Human CD45

Mo

us

e C

D4

5

72%

Total Human

HuCD20

Hu

CD

3

60%

32%

T & B cells

HuCD8

Hu

CD

4

81%

16%

T cell Subsets

Phenotypic Characterization of Human Tregs from NSG-BLT mice (spleen, 12 weeks)

0 102

103

104

105

<Alexa Fluor 405-A>: CD4

0

102

103

104

105

<P

E-A

>: F

oxp3

11.111%

0 102

103

104

105

<FITC-A>: Helios

0

102

103

104

105

<P

E-A

>: F

oxp3

10.110%

0 102

103

104

105

<PE-Cy7-A>: CD127

0

102

103

104

105

<P

E-A

>: F

oxp3

10.3 0.347

43.445.9

11%

CD4

FoxP3

Helios

FoxP3

CD127

FoxP3

T cell Subsets hCD3 Gate

71 0.3

271.6

hCD8

hCD4

8

Tregs hCD4 Gate

FoxP3

CD25

Modeling Human Tumor Immunotherapy in NSG Mice

NSG

Humanized Mouse Models to Study Human Tumor/Immune Interactions

1. Model 1: Tumor Infiltrating Lymphocytes

-study the tumor infiltrating lymphocytes from primary tumor in NSG mice

-Short term model

2. Model 2: Primary Tumor and Autologous PBMC

-Recover tumor and PBMC from same patient and implant both into NSG mice

-Problem with GVHD?

3. Model 3: Primary Tumor and Allogeneic Hematopoietic and Immune System

-Implant tumors into NSG mice engrafted with human immune system

-Will allogeneic immune system reject the tumor?

0 10 20 30 400

100

200

300

400

500

600

Time (Days)

Tu

mo

r S

ize (

mm

3)

Hu-SRC-SCID-NSG+ Tumor

NSG+Tumor

Growth Kinetics of a Patient Derived Melanoma into HSC-Engrafted NSG Mice

Immune tumor interaction during disease progression

PDL-1

Melanoma

CD8 T cells

CD16+ NK cells

CD4 T cells M1 Macrophages

Mature DCs

CD56+ NK cells

M2 Macrophages

Immature DCs

Anergic CD4 T cells

Immune surveillance (Tumor elimination)

Immune escape (Tumor tumor growth)

Anergic CD8 T cells

PD-1

MDSC

Treg

Human Immune Cells Infiltrate Melanomas Implanted into HSC-Engrafted NSG Mice

Total Human Cells Human T Cells CD4/CD8 Ratio

CD8

CD8 CD4

High Levels of Human T cells Recovered from Melanomas Implanted into HSC-Engrafted NSG Mice PD1

Spleen

Tumor

Enrichment of Human Treg in Melanomas Implanted into HSC-Engrafted NSG

FOXP3+ CD27+/CD127-

Spleen

Tumor

Enrichment of Human Myeloid Cells in Melanomas Implanted into HSC-Engrafted NSG

Spleen Tumor0

10

20

30

40

50

CD

33+

Myelo

id C

ells (

% o

f C

D45+

)

p<0.001

The Future of Personalized Medicine

Acknowledgements

•UMass-Diabetes Center

of Excellence

–Ken-Edwin Aryee

–Nicole Walsh

– Sonal Jangalwe

– Laurie Kenney

– Phil Durost

–Darcy Reil

– Linda Paquin

– Laurence Covassin

– Jamie Kady

–Meghan Dolan

– Pam St. Louis

–Mike Bates

•The Jackson Laboratory

– Leonard Shultz

– James Keck

NIDDK NIAID

•UMass

– Roger Davis

– Jeremy Luban

– John Harris

– Jack Leonard

– JeanMarie Houghton

– Michelle Kelliher

– Hardy Kornfeld

– Michael Czech

– Katherine Luzuriaga

– Eric Huseby

– Egil Lien

– Giles Whalen

– Alan Rosmarin