Supplementary online material
Human responses against HER-2-positive cancer cells in Human Immune System (HIS)engrafted mice
Carla De Giovanni1,2, Giordano Nicoletti3, Lorena Landuzzi3, Fabrizio Romani4, Stefania Croci1,
Arianna Palladini1, Annalisa Murgo1, Agnese Antognoli1, Marianna L. Ianzano1, Valeria
Stivani1, Valentina Grosso1, Manuela Iezzi5, Lorenzo Stramucci5, Enza Barbieri2,6, Roberto M.
Lemoli2,7, Patrizia Nanni1,2, Pier-Luigi Lollini2,7
Supplementary Materials and Methods
Generation of HIS mice
Newborn mice (<1-2 days) were sub-lethally irradiated with 2 fractions of 200 cGy with an interval
of 4.5 hours (Roentgen Therapy Machine, 220 kV photon beam with filter (0.8mm 0.25mm Cu + Sn
+ 1.5mm Al), dose rate 56 cGy / min). Then mice received, within 24 h after irradiation, the intraliver injection of 1-2x105 human umbilical cord blood HSC resuspendend in 25 μl of phosphatebuffered saline (PBS).
Immunohistochemistry
Immunohistochemistry for human inflammatory cells, except for anti-CD4, anti-HLA Class I and
anti-Foxp3, was performed on formalin fixed samples, while immunohistochemistry with anti
mouse antibodies was performed on cryosections. Cross reactions between human (h) and mouse
(m) antigens were excluded for all the antibodies (Ab), except for anti-CD3 and anti-Foxp3, by
testing them on formalin fixed and frozen human tonsils and mouse spleens. Anti-CD3 Ab, marker
of T cells and anti-Foxp3 Ab, marker of regulatory T cells, cross react with human and mouse
antigens. Since human CD8 and CD4 T cells were present in tumors while mouse CD8 and CD4 T
cells were not, we consider CD3 and Foxp3 positivity as sign of presence of human T and
regulatory T cells. Sections of formalin-fixed, paraffin- embedded samples (2,5 µm thick) were
heated to 42°C and dehydrated in xylene and graded alcohols. Antigen retrieval was performed in
Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA Solution) pH 9.0 for 10’ at 125°C (hCD8, hCD3)
1
or in 10 mM citric acid pH 6 for 6’ at 125°C (hCD11b, hCD11c, hCD56) using Decloaking
Chamber™ Medical Biocare. Slides were allowed to cool for 5 minutes in ice, followed by repeated
rinsing with distilled water.
Six –micron cryostat section were air- dried and fixed in ice-cold acetone for 10 min (hCD4,
hHLA Class I, mCD4, mCD8, mCD11b, mCD68, mCD11c, mPMN, mCD45R) or in or in 4%
buffered formalin for 8 minutes and then in 99% ethanol for 2 minutes ( -FoxP3) and rinsed twice
in TBS. Antigen retrieval was performed for FoxP3 in 10 mM citric acid pH 6, for 15 minutes by
continuous boiling in a microwave. Endogenous peroxidase activity was quenched with 3%
(hCD8, hCD3, hCD11b, hCD11c) or 0,3% (hCD4, hHLA Class I) hydrogen peroxide. For hCD4 and
hCD56 immunostaining, slides were incubated overnight at 4° C with Rodent Block M (Biocare
Medical, Concord, CA).
Slides were incubated with the following primary antibodies at the indicated dilutions:
1:200 of rat monoclonal anti-mCD4; 1:100 of rat monoclonal anti-mCD8; 1:1500 of rat monoclonal
anti-mCD68; 1:40 dilution of rabbit polyclonal anti-CD3; 1:100 of rabbit monoclonal anti-hCD11b;
1:100 of rabbit monoclonal anti-hCD11c; 1:200 of rat monoclonal anti hHLA Class I (all from
Abcam, Cambridge, UK); 1:40 of rat monoclonal anti-mCD11b; 1:40 of Armenian Hamster antimCD11c; 1:800 of rat monoclonal anti-mGR1; 1:40 of rat monoclonal anti-mCD45R (all from BD
Pharmingen, San Diego, CA); 1:100 of rat monoclonal anti-FoxP3 (eBioscience, Inc. San Diego, CA);
1:50 of mouse monoclonal anti-hCD4 and 1:30 of mouse monoclonal anti-hCD56 (Dako
Corporation, Carpinteria, CA); 1:100 of rabbit monoclonal anti-hCD8 (Biocare Medical, Concord,
CA).
The slides were then incubated with the appropriate peroxidase or biotinylated -conjugated
secondary antibody: anti-rabbit or anti-mouse EnVision System (Dako Corporation, Carpinteria,
CA - USA); MACH 3 Rabbit Probe (Biocare Medical, Concord, CA); goat anti-rabbit IgG or goat
anti-rat IgG or goat anti-Armenian Hamster IgG (Jackson ImmunoResearch Laboratories, Inc.,
West Grove, PA) at 1:500 or 1:750 dilution for 30 minutes.
Immunoreactive antigens were detected using MACH 3 Rabbit HRP - Polymer (Biocare
Medical, Concord, CA) or Streptavidin Peroxidase (Thermo Scientific -Lab Vision Corporation,
Fremont, CA) and DAB Chromogen System (Dako Corporation, Carpinteria, CA) or
NeutrAvidinTM Alkaline Phosphatase Conjugated (Thermo Scientific-Pierce Biotechnology,
Rockford, IL) and Vulcan Fast Red (Biocare Medical, Concord, CA).
2
Immunoprecipitation and western blot
Dynabeads coated with protein A (Invitrogen, Milan, Italy) were used following the indirect
approach. SK-OV-3 cells were lysed with the proteoextract native membrane protein extraction kit
(Calbiochem, Darmstadt, Germany). 120 µg proteins were incubated with plasma samples (a
volume containing 1.5 µg total human IgG) overnight at 4°C. 35 µl dynabeads were then added to
each sample followed by 80 min incubation at 4°C. Dynabead protein A-IgG-antigen complexes
were isolated with the use of a magnet and washed thrice with PBS pH 7.8. Immunoprecipitated
proteins were eluted by boiling in Laemmli sample buffer containing 5% beta-mercaptoethanol,
separated by SDS-PAGE and probed with the anti-HER-2 mouse monoclonal antibody (Ab-8,
clone e2-4001, NeoMarkers, Fremont, CA) diluted 1:600 in PBS containing 0.1% tween 20 + 5%
BSA. After incubation with an anti-mouse IgG-HRP antibody (Santa Cruz Biotechnology, Santa
Cruz, CA) the presence of HER-2 among the immunoprecipitated proteins was detected by a
chemiluminescent reaction (LiteAblot, EuroClone, Milan, Italy).
In vitro effects of sera containing anti-HER-2 human antibodies
To evaluate the effects on cell growth, BT-474 HER-2-positive human breast cancer cells were
seeded at 104/well in tissue-culture multiwell microtiter plates in 110 l DMEM+10% FCS alone or
in the presence of 1 l of serum of non-vaccinated or vaccinated and challenged rCD34 mice.
Control wells with 1-10 g/ml trastuzumab were run in parallel. After a 72 hr culture, 11 l WST-1
Cell Proliferation reagent (Roche) were added to each well and incubated for further 60 min.
Absorbance was determined with a Sunrise Tecan Elisa plate reader with a test wavelength at 450
nm and a reference wavelength at 630 nm. Background (medium with WST-1 alone) was
subtracted and growth inhibition (%) was determined as 100-(absorbance in experimental
well/absorbance in cells alone)*100.
To evaluate effects in antibody-dependent cellular cytotoxicity (ADCC), 104 BT-474 target
cells were incubated 6 hr in 100 l RPMI + 5% FCS in the presence of 1 l of serum of nonvaccinated or vaccinated and challenged rCD34 mice and of Ficoll-isolated human PBMC at 40:1
effector:targed ratio. Experimental controls run in parallel were: medium alone, target alone,
effectors alone, target and effectors plus trastuzumab (2 g/well). Alive BT-474 cells were then
counted and the percentage of cytotoxicity calculated as 100 – (experimental/target alone)*100.
3
Supplementary Figure Legends
Supplementary Figure 1. Engraftment of human HSC in BRG mice at 5 weeks of age. A: all mice;
B: mice grouped according to the numbers of HSC. Cytofluorometric evaluation of the percentage
of human CD45-positive cells in peripheral blood is shown. In panel A, continuous horizontal line
indicates the median value, dashed line indicates the 10% threshold below which mice were
considered not reconstituted. In panel B mean and SEM is reported for each group.
Supplementary Figure 2. Development and maturation of human populations in peripheral blood
of BRG mice engrafted with CD34+ (panels A, B, E, F) or CD133+ (panels C, D, G, H) human cord
blood HSC. Dot plots of individual mice at 5 (panels A-D) and 15 weeks of age (panels E-H).
Supplementary Figure 3. Total cell yield and percentage of human CD45-positive cells in
lymphoid organs of untreated BRG, rCD34 and rCD133 mice at 15 weeks of age. Data from
individual mice and median values (horizontal line) are shown.
Supplementary Figure 4. Decreased serum levels of human VEGF released by human HER-2positive tumors in HIS mice parallel the decreased tumor size. Mean and standard error of the
mean is shown (5-8 mice per group, at 22-23 weeks of age).
Supplementary Figure 5. Total IgG level is correlated to the frequency of human spleen CD38+CD138+ plasma cells. Data include both vaccinated and not-vaccinated rCD34 mice with growing
tumor challenge.
4
Supplementary Figure 1.
B
hCD45 %
hCD45+ (%)
A
100
90
80
70
60
50
40
30
20
10
0
60
40
20
0
rCD34
(105)
rCD34
rCD34 rCD133 rCD133
(2x105) (105) (2x105)
HSC cells (Number of cells injected)
rCD133
5
Supplementary Figure 2.
B
C
D
E
F
G
H
hCD45 
A
hCD19 
hCD3 
6
Supplementary Figure 3.
spleen
100
Cell yield (x10 6)
cell
yield
bone marrow
10
10
10
1
1
0.1
mes. lymphnode
10
1
0.1
1
0.1
0.01
0
0
0.1
100
100
100
100
75
75
75
75
50
50
50
50
25
25
25
25
0
0
0
0
hCD45+ (%)
hCD45
thymus
untreated
rCD34
7
rCD133
Supplementary Figure 4.
Tumor volume
hu-VEGF
5
800
600
3
*
2
pg/ml
cm 3
4
*
200
1
0
400
untreated
0
HIS
hu-IL6
untreated
HIS
hu-IL-8
300
500
400
pg/ml
pg/ml
200
300
200
100
100
0
0
untreated
HIS
8
untreated
HIS
Supplementary Figure 5.
IgG g/ml
600
400
200
p<0.001
0
0
1
2
3
hCD38+ hCD138+ (% total spleen cells)
9
Supplementary Table 1. Human cell engrafting and cytokine production in rCD34 mice,
vaccinated or not, challenged with human HER-2-positive cancer cells, at the time of sacrifice (23
weeks of age). Data from 4-6 mice per group are reported.
Humanized rCD34 mice
no vaccine
Human cells in
peripheral blood
Human
antibodies in
serum (g/ml)
Cellularity of
lymphoid organs
(cell yield x106)
hCD45+ cells in
lymphoid organs
(%)
Human cytokine
production by
hCD45+ spleen
cells
(pg/ml)
Significance
(Wilcoxon
test)
vaccine
median
range
median
range
hCD45
(% over total)
35
6-58
40
22-64
nsa
hCD3
(% over hCD45+)
68
32-95
51
29-80
nsa
hCD19
(% over hCD45+)
28
15-48
33
14-41
nsa
IgG
56
3-561
228
130-519
p = 0,05
IgM
25
1-364
39
11-76
nsa
spleen
5,9
2.1-11.8
7,2
4.7-14.2
nsa
thymus
1,7
0.6-3.6
3,7
2.1-4.8
p = 0,03
bone marrow
4,9
2.7-6.4
4,1
2.9-6.7
nsa
mesentheric
lymph node
0,7
0.1-0.8
1,0
0.2-2.9
nsa
spleen
30
9-56
28
15-34
nsa
thymus
95
87-97
96
91-98
nsa
bone marrow
21
0-32
29
2-62
nsa
mesentheric
lymph node
97
88-98
93
86-97
nsa
hIFN-
5905
52-10458
4835
15-13854
nsa
hIP10
1159
664-1653
523
373-673
nsa
hIL6
11051
8335-13767
27804
1662-53946
nsa
nsa = not significant
10