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TRANSCRIPT
Humanized NSG™ Mice
for Innovative
Preclinical Research
Technical Information Services
August 10, 2017
The Jackson Laboratory’s Mission
2
Performing Research
Investigating genetics and
biology of human disease
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JAX® Mice Clinical & Research
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more…
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2
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Others, including:
JAX-Clinical Knowledgebase
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3 JAX® Technical Support |
JAX® Mice The Gold Standard for Biomedical Research
JAX® Mice The Gold Standard for Biomedical Research
4
NIH funded resource
>9,000 strains and growing
o 2.7 million mice shipped annually
Unsurpassed genetic quality & animal health
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Common inbred strains (C57BL/6J, BALB/cJ, DBA/2J) support
development/collection of specialty strains and other valuable
community research resources
JAX® Mice | 4
Presentation Outline
5 JAX® Mice |
Overview of the
immunodeficiency of NSG™ mice
Humanization of NSG™ &
NSG™-SGM3
o Hu-CD34-NSG™ & Hu-CD34-SGM3
o Hu-PBMC-NSG™
Human immune function in
humanized mice
Research applications of
humanized NSG™
Humanized NSG™ resources
What Makes NSG™ Such a Good Host? NSG™, NOD scid gamma
6
NOD background contributes:
Absence of hemolytic complement
Reduced dendritic cell function
Defective macrophages
Optimal human hematopoietic stem cell engraftment (Sirpα allele)
Scid mutation prevents development of mature T and B cells
Il2rg deficiency
Eliminates signaling from 6 distinct interleukins (IL-2, IL-4, IL-7, IL-9, IL-15,
& IL-21)
Blocks NK cell development
Considerations: scid side effects: radiation sensitivity; genotoxic drugs may have higher toxicity
JAX® Mice |
The Immune System in Mice
JAX® Mice | 7
NSG™: A Highly Immunodeficient
Mouse
8 JAX® Mice |
Humanizing NSG™ Mice
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Hu-CD34-NSG™ = enriched CD34+ hematopoietic stem cell (HSC)
Hu-BLT-NSG™ = enriched CD34+ HSC, liver, thymus
Hu-PBMC-NSG™ = peripheral blood mononuclear cells
Shultz et al. 2012. Nat Rev Immunol. 12(11):786-98. PMID:23059428
In Vivo Pharmacology Services |
Tips for Creating “Humanized”
NSG™ Mice
10
Can be injected as newborns (intracardiac) or adults (intravenous)
Higher engraftment levels via intracardiac route, but lower survival
Females engraft more efficiently than males
Human HSCs can be sourced from different tissue
o human cord blood
o human fetal liver
o G-CSF mobilized peripheral blood
Pre-conditioning treatment necessary for engraftment
Choi, et al. J Clin Immunol. 2010 Oct 28. [Epub] PMID:20981478
In Vivo Pharmacology Services |
Experimental Timeline for JAX® Hu-NSG™
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CD34+ cells engrafted in 3 week old female mice
Human
T cells
appear
Human
B cells
appear
Whole body irradiation
Tail vein injection
3 weeks 12 weeks 15 weeks Mouse Age
In Vivo Pharmacology Services |
CD34 +
CD56+ CD3+, CD4+ & CD8+
CD19+
CD14+
&
CD33+
© 2001 Terese Winslow, Lydia Kibiuk
In Vivo Pharmacology Services | 12
NSG™ is a Superior Host for Engraftment of
Human HSC & Multi-lineage Differentiation
NSG™ is a Superior Host for Engraftment of
Human HSC & Multi-lineage Differentiation
13
Most efficient engraftment of human HSCs to date
Permits differentiation of all major cell types
o Myeloid lineage Erythrocytes, megakaryocytes
Monocytes, macrophages, dendritic cells, granulocytes
o Lymphoid lineage T cells (CD4+ and CD8+)
B cells
Natural Killer (NK) cells
Some mucosal and adaptive immune functions
Ishikawa F et al. 2005. Blood 106(5):1565-73. PMID:15920010
In Vivo Pharmacology Services |
Successful Multilineage
Engraftment in Hu-CD34-NSG™
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Multilineage engraftment in bone marrow, spleen & blood
CD3+ T cells, CD19+ B cells, CD56+ NK cells, CD33+ myeloid cells
Tanaka S et al. 2012. J Immunol 188(12):6145-55. PMID:22611244
In Vivo Pharmacology Services |
T Cell Homing and Maturation
in the Thymus JAX® Hu-CD34-NSG™ (16 weeks)
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Gate on hCD45+, mCD45- human donor derived cells
Immature CD4 & CD8 double positive cells mature to single positive
oCD4+, CD8- helper T cells
oCD4-, CD8+ cytotoxic T cells
0 102
103
104
105
anti-human CD4
0
102
103
104
105
anti
-hu
man
CD
88.38
16.6 54.9
CD4
CD
8
16.6%
8.4%
54.9%
0 100 1000 10000 1x105
0
100
1000
10000
1x105 5.79
mCD45
hC
D4
5
5.8%
Human cells in mouse thymus
In Vivo Pharmacology Services |
Bone Marrow in Hu-CD34-NSG™
16 In Vivo Pharmacology Services |
Humanization Donor Comparison - Comprehensive Lineage Analysis (BM-12 Weeks)
hCD45+
hCD3+ T
cell
hCD3-C
D20+ B
cell
hCD3- C
D19+ B c
ell
hCD3+CD
4+ T h
elper
hCD3+CD
8+ Cyto
toxic
T c
ell
hCD3-C
D56+ N
K c
ells
hCD33+ m
yeloid
hCD15+ M
onocytes
hCD68b+ M
acrophages
hCD123+/C
D11c Dendrit
ic c
ells
hCD235ab+(h
CD45-)
RBC
0
10
20
30
40
50
60
70
80
90
100 Donor#1
Donor#2
Donor#3
Pe
rce
nta
ge
Lineage Analysis of Human Cells in Bone Marrow (12 weeks)
Spleen in Hu-CD34-NSG™
17 In Vivo Pharmacology Services |
Humanization Donor Comparison - Comprehensive Lineage Analysis (Spleen-12
Weeks)
hCD45+
hCD3+ Tcell
hCD3-CD20+ B
cell
hCD3- CD19+ B
cell
hCD3+CD4+ T h
elper
hCD3+CD8+ Cyto
toxic
T c
ell
hCD3-CD56+ N
K cells
hCD33+ myelo
id
hCD15+ Monocyte
s
hCD68b+ Macro
phages
hCD123+/CD11c D
endritic
cells
hCD235ab+(hCD45-)
RBC
0
10
20
30
40
50
60
70
80
90
100 Donor#1
Donor#2
Donor#3
Pe
rce
nta
ge
Lineage Analysis of Human Cells in Spleen (12 weeks)
Peripheral Blood in Hu-CD34-NSG™
18 In Vivo Pharmacology Services |
Humanization Donor Comparison - Comprehensive Lineage Analysis (Blood-12
Weeks)
hCD45+
hCD3+ T
cell
hCD3-C
D20+ B c
ell
hCD3- C
D19+ B c
ell
hCD3+CD4+ T
help
er
hCD3+CD8+ C
ytoto
xic T
cell
hCD3-C
D56+ NK c
ells
hCD33+ m
yeloid
hCD15+ M
onocytes
hCD68b+ M
acrophages
hCD123+/C
D11c Dendrit
ic c
ells
hCD235ab+(h
CD45-) RBC
0
10
20
30
40
50
60
70
80
90
100 Donor#1
Donor #2
Donor # 3
Pe
rce
nta
ge
Lineage Analysis of Human Cells in Peripheral Blood (12 weeks)
NSG™ vs. NSG™-SGM3
NOD scid gamma (NSG™)
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (005557)
The current gold standard for reconstitution of the human immune system
Excellent functional human T cell responses
Limited human myeloid lineage development etc.
NSG™-SGM3
NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (013062)
Promotes improved AML engraftment efficiency
Improves normal human myeloid cell development after HSC transplantation
Wunderlich M. et al. 2010. Leukemia 24(10):1785-8. PMID: 20686503
Billerbeck E. et al. 2011. Blood 117(11):3076-86. PMID: 21252091
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Humanization of NSG™ vs. NSG™-SGM3
Experimental Design
Mice
o Female NSG™, 3 weeks of age, 140 cGy (N=20)
o Female NSG™-SGM3, 4 weeks of age, 100 cGy (N=9)
Donor Cells
o Hu CD34+ HSC, ~130,000 cells/mouse
o All mice transplanted with cells from the same donor
Blood Collection
o PBL collected 4, 6, 9, 12, 15 & 18 weeks post Tx
o Flow for huCD45, huCD33, huCD19, huCD3, huCD4, huCD8, huTreg
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Strain: NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ
Common name: NSG-SGM3 (013062)
Expression of human IL-3, GM-CSF, & Stem Cell Factor (KIT Ligand)
Improves normal human myeloid cell development after HSC transplantation
Promotes AML xenograft efficiency
Human Immune Cells in Peripheral Blood of Hu-
NSG™ vs. Hu-NSG™-SGM3: Absolute Counts
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Total Human Donor (cells/µl)
HuCD19 B Cells (cells/µl) HuCD3 T Cells (cells/µl)
HuCD33 Myeloid Cells (cells/µl)
In Vivo Pharmacology Services |
Greater total cell numbers of huCD45 in
NSG™-SGM3
Greater numbers of myeloid cells in
NSG™-SGM3
Equivalent B cells, but higher numbers of
T cells in NSG™-SGM3
Human Immune Cells in Peripheral Blood of Hu-
NSG™ vs. Hu-NSG™-SGM3: Absolute Counts
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HuCD3 T Cells (cells/µl)
HuCD8 Cytotoxic T Cells (cells/µl)
HuCD4 Helper T Cells (cells/µl)
In Vivo Pharmacology Services |
Greater numbers of huCD3 T cells in
NSG™-SGM3
Greater expansion of huCD4 T cells in
NSG™-SGM3
Greater expansion of huCD8 T cells in
NSG™-SGM3
Improved PBMC Engraftment in NSG™
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Higher donor engraftment compared to NOD-scid
IV delivery better than IP
Donor cells expand through 4 weeks (20 x 106 donor cells, no irradiation, adult mice)
King M et al. 2008. Clin Immunol 126(3):303-14. PMID:18096436
JAX® Mice |
Weeks Post Engraftment
1 2 3 4
Immune Cell Function
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Innate Immunity
Myeloid phagocytosis
NK cell activity
Adaptive Immunity
T cell cytotoxicity
B cell Immunoglobulins
Delayed Type Hypersensitivity (DTH)
JAX® Mice |
Hu-CD34-NSG™ Have Functional
Myeloid Cells
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Engrafted Human Myeloid Cells
Phagocytosis fluorescent beads
LPS induced cytokine release
Express TLR2 & TLR4
INF-ɣ induced phagocytosis & killing
of Salmonella typhimurium
Tanaka S et al. 2012. J Immunol 188(12):6145-55. PMID:22611244
Bead phagocytosis
LPS induced cytokine release
JAX® Mice |
JAX® Hu-CD34-NSG™ Release of Proinflammatory Cytokines
Following Immune Challenge
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Human proinflammatory cytokines (pg/ml) in mouse blood following LPS challenge
Hu-CD34-NSG™ Have Functional
NK Cells
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NK cells (CD3- NKp46+) constitute
1-3% of spleen & 7% of lung
Majority of splenic NK cells are
immature (NKp46+CD56-)
They can mature (NKp46+CD56+)
o Directly with IL-15 exposure
o Indirectly (poly I:C treatment)
Immature NK cells from spleen
display degranulation and
cytokine production
Strowig T et al. 2010. Blood 116(20): 4158-67. PMID:20671122
JAX® Mice | Confidential, not intended for distribution
Hu-CD34-NSG™ Have Functional
T Cells
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Hu-CD4 and hu-CD8 T cell clones derived from hu-CD34 NSG™ spleen
Clones co-cultured with allogeneic or syngeneic target cells o Hu-CD4 cytotoxicity of allogeneic targets, activity blocked by class II mAb
o Hu-CD8 cytotoxicity of allogeneic targets, activity blocked by class I mAb
Ishikawa F et al. 2005.
Blood 106(5):1565-73. PMID:15920010
Allo target cell
Allo target cell + class I mAb
Allo target cell + class II mAb
Syngeneic (matched) target cell
JAX® Mice |
JAX® Hu-CD34-NSG™ Preclinical Model of DTH Response
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Sensitization Sensitization Challenge +/- treatment
Day -1 Day 1 Day 2 Day 7 Day 8 Day 9 Day 14
Ear thickness Ear thickness Ear thickness Ear thickness Ear thickness Ear thickness
Sensitization Sensitization Challenge+/- treatment
In Vivo Pharmacology Services |
Delayed-Type Hypersensitivity (DTH)
Immune sensitization and challenge with dinitrofluorobenzene (DNFB)
Hu-CD34-NSG™ Have Some
B Cell Functions
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Humanized NSG™ mice immunized with ovalbumin
Sera analyzed for IgM and IgG by ELISA
Ishikawa F et al. 2005. Blood 106(5):1564-73. PMID:1895228
Nature Reviews, Immunology
IgG
IgM
JAX® Mice |
Research Applications for NSG™ Mice
31 JAX® Mice |
(Adequate small animal models did not exist prior to NSG™)
Basic Biology & Preclinical Research
Human hematopoiesis
Stem cells/regenerative medicine
Graft versus Host Disease (GvHD)
Infectious disease
Immuno-oncology
Regenerative Medicine Creating Blood Progenitors from Human Fibroblasts
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Human fibroblasts transduced to express Oct4 (Pouf5f1)
Express hu-CD45 (CD45+ FibsOCT4)
Culture with Flt3 and Stem Cell Factor (SCF)
o Monocytic progenitors
o Granulocytic progenitors
Culture with Erythropoietin (EPO)
o Erythroid progenitors
o Megakaryocyte progenitors
Szabo E et al. 2010. Nature 468(7323):521-26. PMID:21057492
JAX® Mice |
Modeling GvHD in NSG™ Understanding the Biology & Developing Treatment
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Adult sublethally irradiated NSG™ injected IV with 5x106 hu-PBMC
Weight loss, anemia, & decreased platelet count in 100% of recipients
GvHD develops irrespective of allogenic donor
Rate of disease development is dose dependent
o 20x106 donor cells = MST of 12 days
o 5x106 donor cells = MST of 22 days
Etanercept treatment (Enbrel®)
Soluble TNF-α decoy receptor
Greater survival with repeated treatment
o Neutralization of TNF-α
o Decreases donor engraftment
King M et al. 2009. Clin Exp Immunol Jul;157(1):104-18. PubMed:19659776
JAX® Mice |
Hu-PBMC-NSG™ for HIV Research
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Kumar P et al. 2008. Cell 134(4):577-86. PMID:18691745
CD3
CD
4
CD
8
No HIV
HIV
HIV + Control
HIV + Therapy
JAX® Mice |
Other applications of humanized NSG™ include:
Epstein Barr Virus, Ebola, CMV, Malaria, Tuberculosis,
Typhoid and more…
52%
30%
Mosquito Bite Delivery Improves
Humanized Dengue Model
Cox 2012 J Virol PMID: 22573866
35
Viremia Erythema
Humanized NSG™ (hu-CD34+ engrafted) were infected with DENV-
2 strain via mosquito bite or intradermal injection
EBV detected in
different organs at 4
weeks
EBV co-localizes
with human T cells
Strowig T et al. 2009. J Exp Med. 206(6): 1423-34. PMID:19487422
36
(EBER; Epstein Barr Encoded RNA)
EBV Infected Hu-CD34-NSG™ Mice Human T Cells Help Control Infection
EBV Infected Hu-CD34-NSG™ Mice
Have Effector Memory T Cells
Strowig T et al. 2009. J Exp Med. 206(6): 1423-34. PMID:19487422
37
Human T cells:
Expand in spleens of
infected mice
Have an activated,
memory phenotype
Expansion is significant
T cell depleted, EBV infected, Hu-NSG™ mice develop tumors
(OKT-4 and OKT-8 mAb’s = ADCC depletion)
T Cells Control Tumor Growth in
Humanized EBV Models
Strowig T et al. 2009. J Exp Med 206(6): 1423-34. PMID:19487422
38
Hu-CD34-NSG™ Have Adaptive T
Cell Function In Vivo
B6 Rag -/- (H2b) skin is accepted on non-
humanized NSG™ (H2g7) recipients
B6 Rag -/- skin is rejected on humanized NSG™
Syngeneic Hu-NSG™ skin is accepted by Hu-
NSG™ recipients
Waldron-Lynch F et al. 2012. Am J Transplant 1Oct;12(10):2652-62. PMID:229007175
39
B6 Rag -/- skin on NSG™ vs. Hu-NSG™
Hu-NSG™ skin on Hu-NSG™ (Day 35)
Hu-CD34-NSG™ T Cells Respond
to Immuno-Modulatory Biologics
Anti-CD3 mAb (Teplizumab) depletion of T cells blocked rejection
Anti-CTLA-4 mAb (Ipilimumab) accelerated rejection
40
Waldron-Lynch F et al. 2012. Am J Transplant 1Oct;12(10):2652-62. PMID:22900717
B6 Rag -/- skin onto Hu-NSG™
Immuno-Oncology Onco-Hu™ for Immune Modulation Studies
41
hu-CD34 HSC NSG™
Humanized NSG™
PDX Tumor or Cell Line
(Selected for PD-L1+)
Humanized NSG™ with Tumor (When tumors reach 70-120 mm3 mice are treated
with therapeutics for 21 to 28 days)
Irradiation
Schilbach, et al. 2015. OncoImmunology. Mar 19;4(7):e1014760. eCollection PMID: 26140238
42
Tumor-Bearing Humanized Mice IL-12/IL-2 Stimulated Innate & Adaptive Immunity
Rhabdomyosarcoma cell line A204
43
Innate & Adaptive Immune Cells
Infiltrate Tumors
44 In Vivo Pharmacology Services |
JAX® Data Humanization of NSG™ Mice has No Significant Impact on PDX
Growth Kinetics
• Non HLA matched
• 100% take rate in NSG™ and Hu-NSG™
Breast Lung
Soft Tissue Carcinoma
45
JAX® Data Human Lymphocytes Infiltrate Human Tumors in
Onco-Hu™ Mice
In Vivo Pharmacology Services |
46 In Vivo Pharmacology Services |
JAX® Data Keytruda & Cisplatin Inhibit Tumor growth of TNBC
BR1126 PDX in Hu-CD34-NSG™ Mice
Tumor PD-L1 surface expression: 56.9%
Hu-CD45 engraftment in Hu-CD34-NSG™
>25%
Keytruda (Pembrolizumab); anti-PD-1 mAb
Immuno-Oncology Summary
NSG™ and NSG™-SGM3 are a proven platform for
engraftment of the human immune system
PDX growth is not grossly effected by HLA-type matching
o Tumor growth kinetics are similar in humanized and non-humanized
hosts
Human immune effector cells infiltrate human tumors in
both Hu-CD34-NSG™ and Hu-CD34-SGM3
o CD4 and CD8 T cells and CD19 B cells
Hu-CD34-NSG™ and Hu-CD34-SGM3 PDX respond to
anti-tumor agent Keytruda (Pembrolizumab)
47 In Vivo Pharmacology Services |
Online NSG™ Resources
48
https://www.jax.org/jax-mice-and-services/find-and-order-jax-mice/nsg-portfolio
49
CRISPR/Cas9 Mouse Model Generation
Common Inbred and Specialty JAX® Mice
Aged C57BL/6J Mice (25-78 wks)
Inbred, Outbred and B6J Nude Mice
Syngeneic Mice Studies for Cancer Research
(i.e., Allograft Mouse Tumor Studies)
Mouse Genome Scanning
NSG™ & NRG Mouse Model Variants
Mouse Cryopreservation and Recovery
Basic and Complex Mouse Breeding,
Speed Congenics, and Rederivation
Humanized Mice, Patient-Derived
Xenograft Preclinical Models and
Therapeutic Drug Evaluation
Neurobiology Models and Resources
JAX® Mice & Services: Leading Experts in Mouse Modeling
Upcoming JAX Webinars™
46
Achieving Reproducible Mouse Studies
o Aug. 17, 2017, 1:00 pm ET; 5:00 pm GMT
Genetic Enhancement of NSG™/NRG Mice for Improved Human Disease Modeling
o Aug. 23, 2017 in the USA: 5:00 pm PT; 8:00 pm ET
o Aug. 24, 2017 in Australasia and Asia: 12:00 am GMT; 8:00 am AWST & China
Standard Time; 9:00 am JST & KST; 10:00 am AEST; 12:00 pm NZST
Essential Tips for New Mouse Researchers
o Sep. 7, 2017, 1:00 pm ET; 5:00 pm GMT
Generating Mouse Models Using CRISPR/Cas Technology
o Sep. 14, 2017, 1:00 pm ET; 5:00 pm GMT
Genetic Enhancement of NSG™ & NRG Mice for improved Human Disease Modeling
o Sep. 21, 2017, 1:00 pm ET; 5:00 pm GMT
Introduction to Mice for Cancer Immunotherapy
o Sep. 26, 2017, 9:00 am ET; 1:00 pm GMT
www.jax.org/education-and-learning/webinars |
Subscribe to the monthly webinar announcements email list: https://subscribe.jax.org/
Thank you!
51
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