Comprehensive evaluation ofhuman immune systemreconstitution in NSG andNSG -SGM3 mouse models
toward the development of anovel ONCO-HU xenograft model™

™

™

Aaron Middlebrook,1 Eileen Snowden,2 Warren Porter,2 Friedrich Hahn,2 Mitchell Ferguson,2 Brian Soper,3 James Keck,3 Joan Malcolm,3Shannon Dillmore,2 Smita Ghanekar,1 Rainer Blaesius2.
BD Biosciences, San Jose, CA; 2BD Technologies, Raleigh-Durham, NC; 3The Jackson Laboratory, Bar Harbor, ME

1Abstract

The recent successes of immunotherapeutic approaches in the treatment of melanoma and the promise of similar treatments ina variety of other cancers underscore the importance of the immune system in cancer. Indeed, effective therapeutic design andevaluation require a comprehensive understanding of the interplay between the immune compartment and the proliferating tumor
cells that comprise the tumor microenvironment. A humanized mouse strain engrafted with cancerous tissue from a patient derived
xenograft (PDX) tumor provides researchers with a highly sophisticated tool, ideally suited to facilitate the design of treatment
strategies that prevent tumor evasion of immune cells and improve cytotoxic responses.

Severely combined immunodeficient mice such as NOD scid gamma (NSG™) and triple transgenic NSG™ mice expressing humancytokines KITLG, CSF2 and IL-3 (NSG™-SGM3) are proven hosts for the engraftment of human tumors and establishment of humanimmune system components following hematopoietic stem cell (CD34+) transplantation. The endogenous expression of cytokinesthat support the development of myeloid lineages and regulatory T (Treg) cells potentially represents a substantial improvement overstandard NSG™ mice.
Here, we employ three 14-color flow cytometry panels to perform a comprehensive and detailed analysis of the entire immune system.
The three panels are designed to fully characterize specific branches of the immune system: 1) T cells 2) natural killer (NK) cells/
dendritic cells (DCs)/B cells and 3) myeloid lineages. Blood, spleen and bone marrow tissue from both NSG™ and NSG™-SGM3 mice
were evaluated at 10, 16, 21 and 31 weeks post-engraftment using each of the four phenotyping panels. Our results indicate that thetriple transgenic NSG™-SGM3 mice exhibit a more completely humanized immune system as compared to NSG™ mice, with specificimprovements in the distribution of T-cell subsets and overall representation of the myeloid lineage.
NSG™ mice engrafted with allogeneic human tumors represent a valuable preclinical testing platform for immuno-oncology.

Methods
Mice

NSG™ and NSG™ -SGM3 mice were humanized by transplantation of human cord-blood-derived CD34+ hSC by The Jackson Laboratory(Bar Harbor, ME) and shipped to BD Technologies (Raleigh-Durham, NC) for processing.

Tissue processing

Mice were euthanized and spleen, bone marrow and peripheral blood were harvested. Spleen was placed between two glass slides
and crushed. The slides were then rinsed into a 50 mL conical tube using 10-20 mL PBS, then filtered using a 70-µm sieve to collect asingle cell suspension. Bone marrow was expelled from femurs and broken up using wide-bore pipette tips and trituration. It was thenfiltered through a 70-µm sieve to create a single cell suspension. 200-400 µL of peripheral blood was taken during terminal blooddraw. Peripheral blood samples and single cell suspensions derived from spleen and bone marrow were treated with 4 mL ACK Buffer(Gibco A10492-01) for 7 minutes at room temperature in order to lyse all red blood cells. Samples were then washed once with 45
mL DPBS/2%FBS. Supernatant was aspirated, pellets were incubated in human FcR block, resuspended in PBS and then transferred toFalcon® 96-well plates for staining with antibody cocktails.

Flow cytometry

Cell suspensions from each of the three sample types (blood, spleen and bone marrow) were stained for 30 minutes with each of the14 color panels described in the Results section. Fluorochrome selection for each of the panels was performed using theBD Horizon™ Guided Panel Solution; you can learn more about the tool at bdbiosciences.com/us/tools/s/gps. Antibody cocktails werediluted in 50 µL of BD Horizon™ Brilliant Stain Buffer (BD Biosciences Cat. No. 659611). After staining, cells were washed two times
with PBS and recovered via centrifugation (7 min at 300g). Samples were acquired on a Special Order BD LSRFortessa™ X-20 flow
cytometer (BD Biosciences Cat. No. 658226R1) and data analysis was performed using BD FACSDiva™ software.

Figure 1. NSG™ vs NSG™-SGM3 mice
NSG™ mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, 005557) are devoid of mature B, T and NK cells. NSG™-SGM3 (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ,013062) are NSG™ mice that contain transgenes expressing human Stem Cell Factor (KITLG), GM-CSF (CSF2) and IL-3. Recipient mice were irradiated at 3-4 weeks of age andengrafted with human cord-blood-derived CD34+ hematopoietic stem cells. Prior to co-engraftment of human tumors, mice are validated for human multilineage engraftment andestablishment of human immunity. Figure courtesy of The Jackson Laboratory.

Results

T-cell panel

Sample tissues (bone marrow, spleen and peripheral blood) were stained with a 14-color panel (see table on next page for reagent list).
The panel was designed to enumerate T-cell subsets. Representative flow plots (spleen, 23-week old NSG™ mouse), demonstrate thegating strategy used for analysis. Enumerated populations are plotted as boxplots (next page, bottom). The green bands across eachplot represent the range of each subpopulation in peripheral blood as measured in normal healthy adult donors (n=6) using the samepanel. Not all enumerated population plots are shown. Those that showed little change or were in clear agreement with reference
values (green band) were not shown. The intermediate 16-17 week and 23-week timepoints are not shown.
CD45RA

CD197
CD4 Naive
CD4 Central
MemoryCD4CD45RA

DeadCells

LiveAll
Cells

Hoescht

CD197

HLA-DR

CD38

CD197

HLA-DR

CD38

CD4 Effector
MemoryMouse

Hoescht

CD4 ActivatedCentral MemoryCD45 CD3
HumanCD127
T Cells

CD4 ActivatedEffector Memory

CD25Tregs

Debris

CD45RA

CD8CD197
CD8 Naive
CD8 CentralMemory

CD45RA

CD8 ActivatedCentral Memory

CD197

HLA-DR

CD38

CD197

HLA-DRCD38

CD8 Effector
MemoryCD8 ActivatedEffector Memory

Figure 2. T-cell panel population hierarchy and gating strategy
Cell populations shown in blue (top) were measured across all tissue types and across all strain groups (10 weeks, 16-17 weeks, 23 weeks and 31 weeks). The population hierarchy
was generated using the BD Horizon GPS tool (see Methods). Each population was defined according to the gating strategy shown above. The data shown is a representative setof plots from the spleen of a 23-week old NSG™ mouse. This gating strategy does not include all markers in the panel. Some markers were included to identify specific activatedphenotypes that will be relevant in future comparative studies using tumor bearing mice.

Reagents used in T-cell panel
Laser (nm)
488

561

652

405

355

Fluorchrome

T-Cell Panel

Cat. No.

FITC*Mouse Dump

PerCP-Cy™5.5*Mouse Dump

Antibody

Cat. No.

(at right)

mCD45553079

CD8560662

mH2Kd553592

PE

CD38555480

mTer119561032

PE-CF594

CD197562381

mCD31558738

PE-Cy™7

CD28560684

mCD41561849

APC

CD3555342

mCD71553266

APC-H7

CD45560274

AF700

CD4557922

BV421

CD45RA

562885BV510

FVS Live/Dead564406

BV605

HLA-DR562846

BV711

CD25563159

BV786

CD127

563324

BUV395

Hoescht 33342

561908

NK/DC/B cell panel

Sample tissues (bone marrow, spleen and peripheral blood) were stained with a 14-color panel (see table on next page for reagentlist). The panel was designed to enumerate DC, NK-cell and B-cell subsets. Representative flow plots (spleen, 23-week-old NSG™
mouse) demonstrate the gating strategy used for analysis. Enumerated populations are plotted as boxplots (next page, bottom). The
green bands across each plot represent the range of each subpopulation in peripheral blood as measured in normal healthy adultdonors (n=6) using the same panel. Not all enumerated population plots are shown. Those that showed little change or were in clear
agreement with reference values (green band) were not shown. The intermediate 16-17 week and 23 week timepoints are not shown.
Naive B Cells
CD19

B Cells

DeadHoescht

Hoescht

AllCells
Debris

IgD
IgD
Memory B Cells

MouseLiveCells

HLA-DR

CD3
Human CD45

NKp46

CD16

CD3
NK Cells

CD56

MatureNK Cells

CD56
Immature
NK Cells

CD19

Monocytes
NKp46

pDCs

CD14
CD14

HLA-DR

CD123

DCs

CD11cmDCs

Figure 3. NK/DC/B cell population hierarchy and gating strategy
Cell populations shown in blue (top) were measured across all tissue types and across all strain groups (10 weeks, 16-17 weeks, 23 weeks and 31 weeks). The population hierarchy
was generated using the BD Horizon GPS tool (see Methods). Each population was defined according to the gating strategy shown above. Data shown is a representative set ofplots from the spleen of a 23-week-old NSG™ mouse. This gating strategy does not include all markers in the panel. Some markers were included to identify specific activatedphenotypes that will be relevant in future comparative studies using tumor bearing mice.

Reagents used in NK/DC/B cell panel
Laser (nm)
488

561

652405355

Fluorchrome

NK/DC/B-Cell Panel

Cat. No.

FITC*Mouse Dump

PerCP-Cy™5.5*Mouse Dump

Antibody

Cat. No.

(at right)

mCD45553079

CD3560835

mH2Kd553592

PE

CD19555413

mTer119561032

PE-CF594

CD11c

562393mCD31558738

PE-Cy™7

NKp46562101

mCD41561849

APC

CD16561304

mCD71

553266APC-H7

CD14560180

AF700

CD45560566

BV421

IgD

562518BV510

FVS Live/Dead564406

BV605

HLA-DR562845

BV711

CD123

563161

BV786

CD56564058

BUV395

Hoescht 33342

561908

Myeloid panel

Sample tissues (bone marrow, spleen and peripheral blood) were stained with a 14-color panel (see table on next page for reagentlist). The panel was designed to enumerate myeloid subsets. Representative flow plots (bone marrow, 10-week-old NSG™ mouse),demonstrate the gating strategy used for analysis. Enumerated populations are plotted as boxplots (next page, bottom). The green
bands across each plot represent the range of each subpopulation in peripheral blood as measured in normal healthy adult donors(n=6) using the same panel. Not all enumerated population plots are shown. Those that showed little change or were in clear
agreement with reference values (green band) were not shown. The 16-17-week and 23-week timepoints are not shown.
CD11b

CD16
Neutrophils
CD16
Metamyelocyte

Myelo_1
CD33
CD15
CD33

DebrisAll
Cells

Hoescht
Cells

SSC

Dead
Hoescht

Mouse

CD15

Live
Human

CD11b

CD15+ Myeloid

CD16
Myeloblast

CD33
CD33+ Myeloid

CD45

Monocytes
CD14
SSC

CD11b

CD16Monocytes(Intermediate and Inflammatory)
CD14
CD16
mMDSC
CD11b
Monocytes
CD15(Classical)pMDSC
MDSC

CD14

CD33
CD163

Non-monocytes

M2
Macrophages

CD16
Macrophages

CD206

CD163

Cd206
M1
Macrophages

Figure 4. Myeloid population hierarchy and gating strategy
Cell populations shown in blue (top) were measured across all tissue types and across all strain groups (10 weeks, 16-17 weeks, 23 weeks and 31 weeks). The population hierarchy
was generated using the BD Horizon GPS tool (see Methods). Each population was defined according to the gating strategy shown above. Data shown is a representative set ofplots from the bone marrow of a 10-week-old NSG™ mouse. This gating strategy does not include all markers in the panel. Some markers were included to identify specific activatedphenotypes that will be relevant in future comparative studies using tumor bearing mice.

Reagents used in myeloid cell panel
Laser (nm)
488

561

652

405

355

Fluorochrome

Myeloid Panel

Cat. No.

FITC*Mouse Dump

PerCP-Cy™5.5*Mouse Dump

Antibody

Cat. No.

(at right)

mCD45553079

CD195

560635mH2Kd553592

PECD163560933

mTer119561032

PE-CF594

CD206564063

mCD31558738

PE-Cy™7

CD11b557743

mCD41561849

APC

CD16561306

mCD71553266

APC-H7

CD14560180

AF700

CD45560566

BV421

CD192564067

BV510

FVS Live/Dead564406

BV605

HLA-DR562845

BV711

CD33563171

BV786

CD15563383

BUV395

Hoescht 33342

561908

Conclusions

The expression of three human growth factors (SCF, GM-CSF and IL-3) in the NSG™ mouse model represents a significant
improvement in the development of a suitable xenograft model. BD’s sophisticated and flexible flow cytometry tools facilitated a
deep and comprehensive analysis of the immune system in these mice. NSG™-SGM3 mice exhibit improved reconstitution compared
to NSG™ mice as measured by the frequency of most major immune subsets including T cells, B cells, NK cells and DCs. In termsof percentage, significant deficiencies of the myeloid compartment (most evident in the reduced frequencies of monocytes andneutrophils) persist in both NSG™ and NSG™-SGM3 strains of mice. NSG™-SGM3 mice exhibit increased frequencies of Tregs (notevident in NSG™ mice), and an expanded memory T-cell pool with a concomitant reduction in naïve T cells (CD4+ and CD8+). Whileabsolute cell numbers were not measured, there were qualitative increases in the total numbers of CD45+ cells as a whole in the NSG™SGM3 derived samples. These observations clearly merit a more quantitative investigation into the absolute cell numbers of each of
the target cell populations measured. The overall improvement in immune reconstitution seen in the NSG™-SGM3 mice compared to
NSG™ mice demonstrates the utility of this strain as a potential host for PDX tumors and may improve the ability of researchers tostudy the complex interplay between immune and cancer cells within the tumor microenvironment, which could help in the design anddevelopment of immunotherapeutic approaches to cancer treatment.

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NSG™ and NSG™-SGM3 are trademarks of The Jackson Laboratory.
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