michael a. brehm - tumor models 2018 | hanson...
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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
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
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
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
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
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