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(Circulation. 2004;109:2009-2015.)
© 2004 American Heart Association, Inc.

Basic Science Reports

B7-1/B7-2 Costimulation Regulates Plaque Antigen–Specific T-Cell Responses and Atherogenesis in Low-Density Lipoprotein Receptor–Deficient Mice

Chiara Buono, MD; Hong Pang, MD; Yasushi Uchida, MD, PhD; Peter Libby, MD; Arlene H. Sharpe, MD, PhD; Andrew H. Lichtman, MD, PhD

From the Immunology Research Division and the Vascular Research Division, Department of Pathology (C.B., H.P., Y.U., A.H.S., A.H.L.), and Cardiovascular Division, Department of Medicine (P.L.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Andrew H. Lichtman, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail alichtman{at}rics.bwh.harvard.edu

Received August 22, 2003; de novo received November 3, 2003; revision received January 9, 2004; accepted January 15, 2004.

	Abstract

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Background— Several lines of evidence indicate that T-cell responses influence the progression of atherosclerotic disease. Interferon- (IFN-)–producing T cells specific for lesional antigens, including oxidized LDLs and heat shock protein 60 (HSP60), may promote lesion development as well as plaque instability. B7-1 and B7-2 are closely related molecules expressed on antigen-presenting cells that provide costimulatory signals for T-cell activation. This study tested the hypothesis that the ability of T cells to influence atherosclerosis depends on B7-1/B7-2 costimulation.

Methods and Results— B7-1/B7-2/LDL receptor (LDLR)–deficient mice and LDLR-deficient control mice were fed a 1.25% cholesterol or control diet for 8 and 20 weeks. Total serum cholesterol levels and extent and phenotype of atherosclerosis were analyzed. Splenic and lymph node CD4⁺ T cells from the animals were cultured with mouse recombinant HSP60 or media and antigen-presenting cells and analyzed for IFN- and interleukin-4 production. The absence of B7-1 and B7-2 significantly reduced early cholesterol diet–induced atherosclerotic lesion development in LDLR-deficient mice compared with B7-1/B7-2–expressing control mice. Furthermore, CD4⁺ T cells from the cholesterol-fed B7-deficient mice secreted a significantly lower amount of IFN- in response to mouse HSP60 in vitro than did T cells from B7-expressing control mice.

Conclusions— The data show that B7-1 and B7-2 regulated the development of atherosclerotic lesions and the priming of lesional antigen–specific T cells. This study highlights the B7-CD28 pathway as a potentially important target for immunomodulation of atherosclerosis.

Key Words: atherosclerosis • immune system • cytokines • heat-shock proteins • T-lymphocytes

	Introduction

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Atherosclerosis, a chronic inflammatory process,¹ prominently involves interferon- (IFN-)–secreting CD4⁺ T cells,² but the nature of the antigens, antigen-presenting cells, and regulatory pathways that may stimulate plaque-based T-cell responses is incompletely understood.

Heat shock protein 60 (HSP60) is a ubiquitous, highly conserved intracellular chaperone whose expression increases in stressed cells. Adaptive immune responses to microbial HSP60/65 or self-HSP60 are associated with several inflammatory diseases,^3,4 and HSP60 may stimulate both innate and adaptive immune responses in atherosclerosis.⁵ Immunization with HSP65 in New Zealand white rabbits leads to the formation of atherosclerotic lesions; moreover, T cells specific for HSP65 were found to be present in the lesions not only of HSP65-immunized rabbits but also of rabbits fed a cholesterol-rich diet.⁶ Levels of serum antibodies against HSP65 and HSP60 correlate with the progression of carotid atherosclerotic disease in humans.^7,8 Furthermore, immunization of mice prone to atherosclerosis due to a lack of LDL receptor (LDLR) with HSP65 promotes cholesterol diet–induced atherosclerosis,⁹ and mucosal administration of HSP60 to LDLR-null mice leads to immunologic tolerance to this protein and decreased cholesterol diet–induced atherogenesis.¹⁰

The two closely related costimulatory molecules B7-1 (CD80) and B7-2 (CD86) are expressed on antigen-presenting cells and provide signals, in addition to antigen, essential for the full activation of T cells.¹¹ B7-1 and B7-2 both bind to the same receptors on T cells, ie, CD28 and CTLA-4, and they have overlapping functions. This is shown by the finding that mice with deficiencies in just B7-1 or just B7-2 have mild phenotypes, whereas mice with combined B7-1 and B7-2 deficiency have profound defects in T-cell responses.^11,12 B7-1/B7-2 costimulation enhances helper T-cell differentiation and CD40 ligand expression.¹³ Cytokines such as IFN- produced by T helper 1 cells (Th1)^14–16 and CD40 ligand¹⁷ profoundly affect the development of atherosclerosis in murine models.

The present study used B7-1/B7-2, LDLR-null mice with diet-induced atherosclerosis to test the hypothesis that the absence of B7-1 and B7-2 reduces HSP60-specific Th1 responses as well as the development of atherosclerotic lesions.

	Methods

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Mice
All mice were backcrossed at least 10 times on a C57Bl/6 background. Ldlr^–/– mice, purchased from Jackson Laboratories (Bar Harbor, Maine), were crossbred with B7-1^–/–B7-2^–/– mice,¹² and the heterozygous progeny were intercrossed to generate a compound mutant B7-1^–/–B7-2^–/–Ldlr^–/– line. Animal genotype was identified by a polymerase chain reaction–based assay, as described previously.^14,18 All mice were housed and bred in accordance with the institutional guidelines of Brigham and Women’s Hospital and Harvard Medical School.

Study Protocol
At 5 to 6 weeks of age, B7-1^–/–B7-2^–/–Ldlr^–/– or sex-matched Ldlr^–/– mice were started on a semipurified cholate-free diet (No. D12108; Research Diets Inc) containing 40% kcal lipid, 1.25% cholesterol ad libitum.¹⁹ Another group of Ldlr^–/– mice was fed a cholesterol-free "control" diet (No. D12102; Research Diets Inc). After 8 or 20 weeks on the diet, the mice were fasted overnight and killed by halothane inhalation. Blood was collected by vena cava nicking, and the arterial tree was perfused with Dulbecco’s phosphate-buffered saline (Gibco BRL). Perfused aortas were dissected from the aortic valve to the iliac bifurcation: the aortic arches were cut and separated from the remaining aorta, then rapidly frozen in optimal cutting temperature embedding medium (OCT, Tissue-Tek); the remaining thoracic and abdominal aorta (descending aorta) from each mouse was fixed in 10% buffered formalin.

Aortic Atherosclerotic Lesion Analysis
Two types of analyses were performed to quantify atherosclerotic lesions in the aortic arch or the descending aorta, respectively. First, longitudinal 5-µm cryostat sections of the aortic arch (3 per specimen) were stained with oil red O (ORO), and, to ensure uniform analyses, a defined portion of aortic arch wall was defined microscopically in all sections from each mouse, as described.²⁰ This portion included a 2-mm segment of the lesser curvature, defined proximally by the aortic root and distally by a perpendicular axis dropped from the distal side of the innominate artery origin. Medial, intimal, and lipid-positive areas, subtended by this 2-mm stretch of intima, were calculated for each mouse by computerized image analysis, with the use of IMAGEPRO PLUS software (Media Cybernetics). Second, the remaining formalin-fixed thoracic and abdominal aorta from each mouse was stained with ORO, opened longitudinally, pinned out, and photographed with a digital camera to obtain images of lesions en face, as previously described.¹⁴ The percent surface area occupied by ORO-stained lesions viewed en face was determined with the use of IMAGEPRO PLUS software.

Immunohistochemistry, Immunofluorescence, and Histochemistry
For immunohistochemical analysis, serial longitudinal cryostat sections of aortic arch sections from mice fed a high-cholesterol diet were stained with specific primary antibodies from Pharmingen, including anti-CD80 (B7-1, clone 1G10), anti-CD86 (B7-2, clone GL1), anti–I-A/I-E (clone M5/114.15.2), anti-CD4 (clone RM4-5), and anti-CD3 (clone 145-2C11), as described.^14,20

Aortic arch sections from mice fed a high-fat/high-cholesterol diet for 20 weeks were also stained for smooth muscle cells with anti–smooth muscle actin (clone 1A4, Sigma), as described,²⁰ and for collagens type I and III by Picrosirius red, as described.²¹

Quantitative analysis of lesional content of class II major histocompatability complex (MHC)–positive cells, smooth muscle cells, and type I and III collagen was determined by computer-assisted image analysis¹⁴ and expressed as percentage of intimal area to normalize for overall differences between the study groups. Quantification of CD3⁺ or CD4⁺ staining was done by counting individual positively stained lesional cells in the aortic arch sections from 6 mice per group. The total area stained by these antibodies was too small to permit meaningful computer-assisted image analysis.

Serum Cholesterol Analysis
Overnight fasting serum was collected from 6 individual mice for each group of mice at time of euthanasia. Total serum cholesterol levels were measured by enzymatic assays (Roche Diagnostic) and expressed in milligrams per deciliter.

In Vitro Assays of CD4⁺ Cytokine Secretion
Analyses of T-cell cytokine secretion were performed as described.¹⁴ Briefly, spleen and lymph nodes were removed from 6 mice for each study group, and CD4⁺ T cells were isolated by anti-CD4 magnetic beads (Dynal). The cells were stimulated in microwell cultures (5x10⁴ per well) with or without recombinant murine HSP60 (10 µg/mL) produced as described²² plus syngeneic mitomycin C–treated spleen cells as antigen-presenting cells (5x10⁵ per well). Culture supernatants were removed at 48 hours and analyzed by enzyme-linked immunosorbent assay for IFN- and interleukin-4 cytokines with the use of reagents from BD Pharmingen.

Statistical Analysis
All statistical analyses were performed with the use of Prism software. Data were found to be normally distributed and expressed as mean±SEM. Comparisons between 2 groups were analyzed by the Student t test, and comparisons between >2 groups were analyzed by ANOVA followed by the Bonferroni post hoc test. P0.05 was considered significant.

	Results

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Serum Cholesterol Analysis
Total serum cholesterol levels, as determined by enzymatic assay, were significantly elevated in Ldlr^–/– mice fed a cholesterol-enriched diet compared with Ldlr^–/– mice fed a control diet (Table 1), as expected. No significant differences were found in serum cholesterol levels in cholesterol diet–fed B7-1^–/–B7-2^–/–Ldlr^–/– mice versus Ldlr^–/– mice.

View this table: [in this window] [in a new window]   TABLE 1. Total Serum Cholesterol Levels in Ldlr–/– and B7–1–/–B7–2–/– Ldlr–/– Mice

Quantitative Analysis of Atherosclerotic Lesions
Quantitative analysis of aortic lesions indicates that in the absence of B7-1/B7-2, development of atherosclerotic disease is diminished. After 8 weeks of a cholesterol-enriched diet, there was significantly less atherosclerosis in both aortic arch and descending aortas in B7-1^–/–B7-2^–/–Ldlr^–/– mice compared with Ldlr^–/– controls. This was evident from measurements of aortic arch intimal lipid content and intimal area, as well as en face lipid-positive area in the descending aorta. Control diet–fed Ldlr^–/– mice developed very little atherosclerosis (Figure 1).

View larger version (22K): [in this window] [in a new window]   Figure 1. Quantitative analysis of aortic atherosclerosis in B7-1–/–B7-2–/–Ldlr–/– and Ldlr–/– mice. Quantification of ORO-stained aortic lesions was performed on digitally captured images, as described in Methods. Horizontal bars represent means. P values <0.05 are shown for comparisons between Ldlr–/– mice fed the control diet vs cholesterol diet and for Ldlr–/– mice vs B7-1–/–B7-2–/–Ldlr–/– mice, both fed the cholesterol diet.

After 20 weeks of diet, there was progression of atherosclerotic disease in all groups (Figure 1), and quantitative analysis revealed persistent differences between control diet–fed versus high-cholesterol diet–fed Ldlr^–/– mice. B7-1^–/–B7-2^–/–Ldlr^–/– mice fed a cholesterol-enriched diet for 20 weeks developed smaller lesions but with a greater percentage of lipid content compared with Ldlr^–/– controls. Specifically, the mean intimal area of aortic arch lesions was less in B7- 1^–/–B7-2^–/–Ldlr^–/– mice than in Ldlr^–/– mice, but there was no significant difference in the mean lipid positive area (Figure 1). The mean en face lipid-positive area in the descending aorta was not different between the 2 groups at 20 weeks.

Phenotypic Analysis of Atherosclerotic Lesions
Lesional expression of B7-1 and B7-2 was detectable by immunohistochemistry in the intimal area of atherosclerotic plaques of aortic arch sections from Ldlr^–/– mice after 8 and 20 weeks of diet but not from B7-1^–/–B7-2^–/–Ldlr^–/– mice, as expected (Figure 2).

View larger version (66K): [in this window] [in a new window]   Figure 2. Characteristics of aortic lesions of B7-1–/–B7-2–/–Ldlr–/– and Ldlr–/– mice. Frozen sections of aortic arches taken from mice fed a cholesterol-rich diet for 8 weeks were stained with antibodies specific for B7-1, B7-2, and class II MHC, as described in Methods. Frozen sections of aortic arches taken from mice after 20 weeks of a cholesterol-rich diet were stained with antibody specific for CD3 or smooth muscle cell (SMC) actin or with Picrosirius red to detect collagens type I and III, as described in Methods. Sections stained with isotype control antibodies were negative (not shown). See Table 2 for quantification of staining.

View this table: [in this window] [in a new window]   TABLE 2. Lesion Phenotype of Aortic Arch Sections From Ldlr–/– and B7–1–/– B7–2–/– Ldlr–/– Mice

Although there were fewer positively staining CD3⁺ T cells present in lesions from B7-1^–/–B7-2^–/–Ldlr^–/– mice compared with Ldlr^–/– mice (Table 2), these differences were not statistically significant. Anti-CD4 staining was not distinct enough to permit individual cell counting. In contrast, there was significantly less class II MHC expression in the sections from B7-1^–/–B7-2^–/–Ldlr^–/– mice compared with Ldlr^–/– mice after 8 weeks of a cholesterol-enriched diet (Figure 2, Table 2). After 20 weeks of the cholesterol-enriched diet, class II MHC in lesions from B7-1^–/–B7-2^–/–Ldlr^–/– and Ldlr^–/– mice was not different. After 20 weeks of the cholesterol-enriched diet, there was significantly less smooth muscle cell and type I and III collagen content of aortic arch lesions of B7-1^–/–B7-2^–/–Ldlr^–/– mice compared with Ldlr^–/– controls (Figure 2, Table 2). The smooth muscle cell content and collagen content of lesions in all groups after only 8 weeks of diet were too low to be reliably quantified.

In Vitro Assays of CD4⁺ Cytokine Secretion
We also found that B7-1/B7-2 regulates cholesterol diet–induced T-cell responses to the atherosclerosis-associated antigen HSP60. After 8 weeks of diet, CD4⁺ T cells from cholesterol diet–fed Ldlr^–/– mice produced readily detectable amounts of IFN- on restimulation in vitro with mouse HSP60, whereas CD4⁺ T cells from control diet–fed Ldlr^–/– mice did not. This result indicates that priming of self-HSP60 reactive helper T cells occurred in the setting of hypercholesterolemia and early atherogenesis. CD4⁺ T cells from cholesterol diet–fed B7-1^–/–B7-2^–/–Ldlr^–/– mice produced significantly less IFN- compared with T cells from cholesterol diet–fed Ldlr^–/– mice (Figure 3). No interleukin-4 secretion was detected in any of experimental groups (data not shown). After 20 weeks of diet, there was no significant difference in HSP60-inducible IFN- secretion by CD4⁺ T cells from B7-1^–/–B7-2^–/–Ldlr^–/– versus Ldlr^–/– mice (Figure 3).

View larger version (21K): [in this window] [in a new window]   Figure 3. IFN- production by HSP60-stimulated CD4+ T cells from B7-1–/–B7-2–/–Ldlr–/– and Ldlr–/– mice. CD4+ T cells were isolated from spleens of control diet–fed or cholesterol diet–fed mice after 8 (A) or 20 (B) weeks and were stimulated in vitro with HSP60 and antigen-presenting cells and IFN- concentration at 48 hours. Culture supernatants were determined, as described in Methods. Data represent mean±SEM of IFN- determinations for T cells from 6 mice in each group. *P<0.001 (Ldlr–/– mice fed control diet vs cholesterol diet); #P<0.01 (Ldlr–/– mice vs B7-1–/–B7-2–/–Ldlr–/– mice, both fed cholesterol diet).

	Discussion

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In this study, B7-dependent T-cell costimulation of T cells is shown to influence atherosclerosis. After 8 weeks of a cholesterol-enriched diet, B7-1/B7-2–deficient Ldlr^–/– mice developed less atherosclerosis than B7-1/B7-2–expressing control mice. Lesions that developed in costimulator-deficient mice expressed less class II MHC than lesions in control mice. We interpret this finding as a reflection of reduced activation of IFN-–producing T cells in lesions of the B7-1/B7-2–deficient mice, because we also found reduced class II MHC expression in lesions of IFN-–deficient Ldlr^–/– mice.¹⁴ Furthermore, the cholesterol-enriched diet resulted in priming an HSP60-specific Th1 response in control Ldlr^–/– mice but not B7-1/B7-2–deficient Ldlr^–/– mice.

Overall, the differences in lesion development between Ldlr^–/– and B7-1^–/–B7-2^–/–Ldlr^–/– mice diminished after 20 weeks of the cholesterol-enriched diet compared with 8 weeks. This finding might occur because the more prolonged hypercholesterolemia results in a high concentration of plaque antigens, which can effectively activate T cells in the absence of costimulation. This possibility agrees with the accepted concept that costimulators effectively reduce the threshold antigen concentrations that can activate a T cell, but a high antigen dose can override the requirement for costimulation.²³ Nonetheless, even after 20 weeks of the cholesterol diet, there still remained phenotypic differences between B7-1/B7-2/LDLR–deficient mice versus control LDLR-deficient mice. Smooth muscle cell content and collagen content of the lesions were reduced in B7-1/B7-2/LDLR–deficient mice. This finding indicates that the absence of T-cell costimulation causes a delay in evolution of lesions toward a more complex fibrotic phenotype. It is possible that the decreased smooth muscle cell and collagen content seen with costimulatory deficiency may result in a weak fibrous cap prone to rupture.²⁴ This risk may be offset by the overall decrease in amount of lesion development.

The best-characterized role for B7-1 and B7-2 is the costimulation of naive T cells in lymphoid tissues, and in this setting the expression of B7 on dendritic cell antigen–presenting cells is particularly important. However, B7 molecules are expressed on macrophages and other cells within nonlymphoid tissues, and they may contribute to costimulation of effector T cells that are recruited to these sites.²⁵ Our finding that B7-1 and B7-2 are expressed in lesions of Ldlr^–/– mice is consistent with previous reports of B7 expression on macrophages in mouse atherosclerotic lesions²⁶ and supports the hypothesis that costimulation of T cells within lesions may occur. We did not find a statistically significant difference in the number of T cells in lesions from costimulator-deficient versus control mice, suggesting that the role of B7 molecules within the plaque may be more related to activation of T-cell effector functions than proliferation.

The influence of T cells on atherosclerosis remains controversial, in part because of results of studies in which scid or Rag mutations have been bred into the apolipoprotein E– or LDLR-null lines. These mice lack all B and T lymphocytes, including regulatory T cells, and in some cases develop less atherosclerosis compared with immunocompetent controls,^27,28 whereas in other studies they develop equivalent amounts of atherosclerosis compared with controls.^29,30 These inconsistencies may result from differences in the degree of hypercholesterolemia achieved in each study. Additionally, both antibody- and T cell–mediated immune responses are heterogeneous and include regulatory and proinflammatory components. Therefore, global T and B cell deficiency may cause a loss of both antiatherogenic and proatherogenic influences of adaptive immunity. Many other studies with the atherosclerosis-prone apolipoprotein E– and LDLR-deficient mouse lines have demonstrated that molecules involved in activation of effector function of T cells influence lesion development and character. For example, null mutations in genes encoding IFN- receptor¹⁶ or IFN-^14,15 impair atherosclerotic lesion development in atherosclerosis-prone mice, and antibody blockade or genetic disruption of the CD40 ligand/CD40 pathway reduces atherosclerosis.^17,31 The present study establishes that B7 costimulatory molecules also influence atherosclerosis. Because the only well-defined role for B7-1 and B7-2 is the costimulation of T cell, our results lend strong support to the hypothesis that CD4⁺ T-cell responses influence the progression of atherosclerotic disease. Furthermore, our data establish a correlation of B7-1/B7-2 effects on atherosclerosis with B7-1/B7-2 effects on T-cell responses to HSP60, a well-established atherosclerotic disease–associated antigen.

In conclusion, our findings establish that B7-1/B7-2 costimulation contributes to plaque antigen–specific responses and promotes lesion development. Because tolerance to plaque antigens may be a therapeutic approach to reduce atherosclerotic disease¹⁰ and costimulatory blockade is a method for tolerance induction,³² our study highlights the B7-CD28 pathway as a potentially important target for immunomodulation of atherosclerosis.

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health, including HL56985 (Drs Buono and Lichtman), HL48743 (Drs Libby and Lichtman), and AI38310 (Dr Sharpe).

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