ONLINE APPENDIX
Full Description of Study Methods
An apoB-100-related peptide p210 (KTTKQ SFDLS VKAQY KKNKH) was used as
immunogen. As described previously, 100 μg of the peptide (Euro-Diagnostica AB, Malmö,
Sweden) was conjugated to cationic bovine serum albumin (cBSA) as carrier (12, 13). The
conjugated peptide was freshly prepared prior to each immunization with alum as adjuvant in a
1:1 volume ratio.
EXPERIMENTAL ANIMAL MODELS. All male apoE (-/-) mice, C57BL/6J wild-type (WT)
mice, and IL-17A (-/-) mice were on a normal chow diet, housed in a pathogen-free animal
facility, and kept on a 12-hour day/night cycle with ad libitum access to food and water. ApoE (/-) mice were subcutaneously immunized with p210/cBSA/alum conjugate in the dorsal area at 7
weeks of age, followed by a booster at 10 and 12 weeks of age. Mice receiving cBSA/Alum or
phosphate-buffered saline (PBS) served as control groups. At 10 weeks of age, an osmotic pump
(Alzet model 2004, DURECT Corporation, Cupertino, California) was subcutaneously implanted
in the back for a continuous infusion of angiotensin II (AngII, Sigma-Aldrich, St. Louis,
Missouri) at a dose of 1,000 ng/kg/min for 4 weeks to induce AAA (6,8,14,15). Online Figure
1A depicts the protocol and experimental groups. For the CD8+ T cell depletion group, p210
immunized mice were intraperitoneally injected with 150 μg of anti-CD8 antibody (eBioscience,
San Diego, California), clone 53-6.7 (16) or isotype-matched rat IgG as control (Sigma-Aldrich)
3 days before first immunization and then twice a week thereafter. The depletion was assessed
by flow cytometry (Online Figure 1B). C57BL/6 WT mice and IL-17A (-/-) mice were
subcutaneously implanted with an osmotic pump at 10 weeks of age to continuously infuse a
higher dose of Ang II (2,500 ng/kg/min) for 4 weeks to induce AAA (14,17). All animal
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experiments were approved by the Institutional Animal Care and Use Committee of Cedars-Sinai
Medical Center.
QUANTIFICATION, CLASSIFICATION, AND ASSESSMENT. Digital images of the
whole aorta were acquired at euthanasia after removal of loose adipose tissue and maximal aortic
diameter was measured with Image Pro software (Media Cybernetics, Rockville, Maryland).
AAA was defined as aortic size 1.5 times greater than aortas from saline infused mice as
previously reported (18). Aneurysm severity was classified with modification (6) as the
following: type 0: normal aorta; type I: dilated with no thrombus; type II: remodeled aorta that
frequently contains thrombus; type III: a pronounced bulbous form of type II with thrombus;
type IV: multiple aneurysms containing thrombus; type V: aortic dissection or rupture with
death.
Flow cytometry was performed using standard protocols with LSR II (BD Biosciences,
San Jose, California) (12). For intracellular staining, Brefeldin-A (3 μg/ml) was added to the
cultured cells for 2 hours before harvest.
The suprarenal aortic segments were embedded in OCT compound (Tissue-Tek, Sakura,
Alphen aan den Rijn, The Netherlands) and frozen sections fixed in ice-cold acetone and stained
with anti-MOMA-2 antibody (AbD Serote, Kidlington, United Kingdom) for macrophages.
Biotinylated secondary antibody was used for visualization using horseradish peroxidaseconjugated streptavidin and AEC. Blinded computer-assisted analysis was performed to assess
histomorphometric features.
Total RNA was extracted from aorta with TRIzol reagent (Life Technologies, Carlsbad,
California) according to the manufacturer’s instruction. Quantitative real-time PCR was
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performed using β-actin as housekeeping gene and data were analyzed by the delta-delta Ct
method.
Peripheral blood via retro orbital puncture was collected with sodium heparin at
euthanasia. Monocytes were isolated by EasySep monocyte enrichment kit (STEMCELL
Technologies, Vancouver, British Columbia, Canada) following the manufacturer’s instruction.
Harvested aortas from naïve apoE (-/-) mice were opened longitudinally for ex vivo
adhesion assay (19). Aortas were incubated in RPMI-1640 medium containing 10% FBS with
Ang II (10-6 M) overnight at 37° C. Next day, monocytes from peripheral blood of immunized
mice were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life
Technologies) and incubated with aorta for 1 hour at 37° C without Ang II. Unbound monocytes
were washed off with PBS. Aortas were then transferred onto glass slides, mounted with
ProLong Gold Antifade Reagent (Life Technologies), and the number of monocytes adhered to
the aorta counted using fluorescent microscopy. The adhesion fold change was calculated based
on the number of adhered monocytes from p210 immunized mice.
Thioglycollate-elicited peritoneal macrophages were harvested from naïve apoE (-/-)
mice as previously described (19). Peritoneal macrophages as target cells were cultured in 96
well plates in RPMI-1640 medium containing 10% FBS with Ang II (10-6 M) overnight at 37°
C. CD8+ T cells as effector cells were negatively isolated from immunized mice (Life
Technologies). Cells were co-cultured at a 10:1 effector:target ratio for 4 hours at 37° C and
cytotoxicity measured by CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega, Madison,
Wisconsin).
To assess the interaction between CD8+ T cells and CD4+ T cells in the polarization of
CD4+ T cells into Th17 cells, spleen cells were collected from naïve apoE (-/-) mice. For target
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cells, CD8+ T cells were depleted from whole splenocytes using a positive isolation kit (Life
Technologies). For effector cells, CD8+ T cells were isolated using a negative isolation kit.
Target and effector cells were then co-cultured in a 1:1 ratio for 6 hours concomitantly with or
without CD3 and CD28 antibody beads and Ang II (10-6M) as experiments dictated.
To explore whether p210 immunization modified the interaction between CD8+ T cells
and CD4+ T cells in Th17 polarization, spleen cells were collected from naïve or immunized
apoE (-/-) mice with AngII (1,000 ng/kg/min) infusion. For target cells, CD8+ T cells were
depleted from whole splenocytes from naïve mice using a positive isolation kit. For effector
cells, CD8+ T cells were isolated from immunized mice using a negative isolation kit. The target
and effector cells were co-cultured in a 1:1 ratio for 6 hours with CD3 and CD28 antibody beads
and Ang II (10-6M). Brefeldin-A was added to the culture medium 2 hours before harvest.
STATISTICAL ANALYSIS. Data are presented as mean ± SEM. Data were analyzed by
ANOVA followed by Newman-Keuls multiple comparison test, or by t test when appropriate. A
p value <0.05 was considered statistically significant. Incidence of aneurysm formation was
analyzed by chi-square analysis. Kaplan-Meier survival curve was analyzed by log-rank test.
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Supplemental Figure I: Methodological aspects of the study.
(A) The experimental protocol and groups in the study. (B) Monoclonal antibody against CD8
(CD8 Ab) significantly reduced splenic CD8+ T cells compared to isotype-match (Isotype) or no
treatment (None). *P<0.01 vs other groups. CD8 Ab N= 5; Isotype N=11; None N=8. (C)
Antibody response to immunization.
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Supplemental Figure II: Matrix degradation in aortic aneurysm.
Representative photographs of in situ gelatinolytic activity in cross-sections of aortas from the
different groups. Zymography and analysis were limited to the same aortic segments with similar
pathology from the different groups to control for differences in severity between controls (PBS
and cBSA) and p210. In situ gelatinolytic activity was performed with DQ-gelatin (Molecular
Probes, Life Technologies). Briefly, DQ-gelatin solution was prepared as recommended by the
manufacturer. The solution was then applied on air-dried cryo-sections of aortas and incubated
for 3 hours in 370C. Sections were dipped in PBS and visualized using a fluorescent microscope.
Data are expressed as fluorescent units relative to cross section from control mice without AngII
infusion to control for autofluorescence from the elastic lamina and plotted on a bar graph. Some
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sections were incubated with 1mM proteinase inhibitor 1,10 phenanthroline. N=5 each;
*P<0.05.
SUPPLEMENT TABLE: Body Weight and Serum
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