Immunity to Low-Density Lipoprotein 21G€oran K. Hansson

21.1 Low-Density Lipoprotein Initiates VascularInflammation

Animal experiments, epidemiological studies and clinical investigations all show

that high levels of plasma low-density lipoprotein (LDL) promote atherosclerotic

cardiovascular disease [1]. LDL particles contain epitopes that trigger cellular and

humoral immune responses. Autoimmunization during the course of atherosclerosis

generates proinflammatory T cell responses whereas vaccination with LDL

components can lead to atheroprotective immunity. Mechanisms underlying these

responses are discussed in the present review.

As a consequence of its subendothelial retention, LDL particles are trapped

within the intima where they may undergo oxidative modifications due to enzy-

matic attack by myeloperoxidase and lipoxygenases, or by reactive oxygen species

such as HOCl, phenoxyl radical intermediates or peroxynitrite (ONOO�) generatedwithin the intima in inflammation and atherosclerosis [4]. Lipid peroxidation

generates reactive aldehydes as well as modified phospholipids such as

lysophosphatidylcholine and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-

phosphocholine (ox-PAPC), which can initiate innate inflammatory responses [5].

These lipids activate endothelial cells and macrophages to produce adhesion

molecules and chemokines. The mechanisms mediating this response involve

activation of the early growth response-1 (Egr-1) [6] and Jak-STAT [7] pathways

and the unfolded protein response [8].

G.K. Hansson (*)

Department of Medicine, Karolinska Institute, Stockholm, Sweden

Center for Molecular Medicine, Karolinska University Hospital L8:03, SE-17176 Stockholm,

Sweden

e-mail: Goran.Hansson@ki.se

G. Wick and C. Grundtman (eds.), Inflammation and Atherosclerosis,DOI 10.1007/978-3-7091-0338-8_21, # Springer-Verlag/Wien 2012

423

Oxidized LDL is found in several other tissues in addition to the arterial intima

and also in peripheral blood. For instance, oxLDL particles have been identified in

synovial fluid and peripheral blood of patients with rheumatoid arthritis [9, 10].

Their presence in blood is surprising since blood contains powerful antioxidants

and cells with scavenger receptors rapidly internalize these particles. However,

several studies report detectable levels in blood and have suggested that oxLDL

levels may reflect increased risk for cardiovascular disease [11].

OxLDL and components thereof have also been reported to activate innate

immunity by binding to Toll-like receptors (TLR) [12], and cholesterol crystals

were recently reported to trigger inflammasome activation, leading to secretion

of interleukin-1b [13]. Activation of innate immunity through any of these

mechanisms may indirectly impact also on adaptive immunity, for example by

modulating the function of antigen-presenting cells or T cells.

21.2 T Cells: Key Components of Adaptive Immunityin Atherosclerosis

Components of adaptive immunity are present in human lesions throughout the

course of atherosclerosis, and several studies point to an important role for antigen-

specific adaptive immune responses in the atherogenic process [14]. Studies

performed in mouse models of atherosclerosis, such as Apoe�/� or Ldlr�/� mice,

in combination with mice deficient in both B and T cells have shown a substantial

role for the adaptive arm of immunity in atherosclerosis. Apoe�/�mice crossed with

lymphocyte-deficient Rag1�/�, Rag2�/� or SCID mice, show dramatically reduced

atherosclerosis (Fig. 21.1) [15–17].

Fig. 21.1 Drastically reduced atherosclerosis in mice lacking adaptive immunity. Hypercholes-

terolemic Apoe�/� mice that develop spontaneous atherosclerosis were bred with mice carrying

the SCID mutation. This mutation in the gene for an enzyme involved in VDJ recombination of

immunoglobulin and T cell receptor genes results in severe combined immunodeficiency with lack

of B and T cells. The bar graph shows that Apoe�/� mice carrying the SCID mutation exhibited a

70% reduction of aortic atherosclerosis (Data from [17])

424 G.K. Hansson

T cells are recruited in parallel with macrophages, by similar mechanisms

involving adhesion molecules and chemokines [14]. They are not as abundant,

with a macrophage:T cell ratio of between approximately 4:1 and 10:1 in human

lesions [18]. However, T cells are activated in lesions, produce proatherogenic

mediators, and contribute very substantially to lesion growth and disease aggrava-

tion [14, 17, 19, 20].

T cells of the atherosclerotic plaque are of the memory-effector phenotype and

mostly T cell receptor ab+ (TCRab+) CD4+ cells, although many CD8+ T cells can

also be found, as well as a small population of TCRgd+ cells [21]. Clonal expansionof T cells has been demonstrated in lesions from humans and Apoe�/�mice [22, 23]

and this suggests that antigen-specific reactions take place within the lesion.

Reconstitution of Apoe�/� SCID mice with CD4+ T cells from atherosclerotic

Apoe�/� mice accelerates atherosclerosis, with homing of T cells to the lesions

[17]. CD8+ T cells stimulated with an agonist to the tumor necrosis factor (TNF)-

like surface protein, CD137, or activated towards an artificial antigen expressed

by smooth muscle cells cause significantly increased atherosclerosis in Apoe�/�

mice [24, 25]. Ldlr�/� mice deficient in the inhibitory molecules PD-L1 and PD-L2

have increased plaque size with massive lesional infiltration of CD8+ T

cells, indicating that these cells might be controlled by the PD-1 molecule in

atherosclerosis [26].

Among the different CD4+ T cell subsets, Th1 is clearly proatherosclerotic, Treg

atheroprotective, while the effects of other subsets (Th2, Th17) are less well

characterized [14]. These aspects are dealt with in detail in other chapters of this

volume.

21.3 Infiltration of Antigen-Presenting Dendritic Cells

Adaptive immune responses are initiated by dendritic cells (DC). These cells are

professional antigen-presenting cells that take up and process antigen, leading to

display of antigenic peptide fragments bound to MHC molecules present at high

density together with co-stimulatory factors on the surface of the DC. Several years

ago, DC were detected in human atherosclerotic lesions [27].

DC that patrol arteries may take up LDL components for subsequent antigen

presentation in regional lymph nodes [28]. It is likely that uptake of oxLDL as well

as native LDL occurs in these cells. In addition to classical myeloid DC, type I

interferon-producing plasmacytoid DC are also present in the artery wall [29, 30].

In the normal artery wall, resident DC are thought to promote tolerization against

antigen by silencing T cells. Danger signals generated during atherogenesis may

activate DC, leading to a switch from tolerance to activation of adaptive immunity

[29, 31]. The properties of the adaptive immune response elicited by antigen

presentation therefore depend on the DC phenotype.

21 Immunity to Low-Density Lipoprotein 425

21.4 B Cells and Humoral Immunity

B cells are infrequent in lesions but abundant on the abluminal, adventitial side of

the atherosclerotic artery [32, 33]. Indeed, tertiary lymphoid structures are often

associated with regions of advanced atherosclerosis [33].

A series of experiments suggest that B cells play a protective role, however,

different subsets also among this cell population may exert different effects.

Transfer of splenic B cells from aged atherosclerotic Apoe�/� mice protected

young Apoe�/� recipients against disease, whereas splenectomy aggravated it

[34]. Bone marrow transfer from B cell deficient mMT mice into Ldlr�/� mice

clarified that these cells are protective in late as well as early atherosclerosis [35].

Surprisingly, anti-CD20 antibodies, which mainly deplete the B2 subtype of B

cells, reduced disease in hypercholesterolemic mice [36], suggesting that subsets of

B cells exert contrasting effects on disease. This notion also received support from

the finding that transfer of B2 but not B1 cells aggravated atherosclerosis in

immunodeficient Apoe�/� mice [37].

Whereas the mechanisms by which B cell subsets may promote atherosclerosis

remain unclear, several mechanistic studies underpin our understanding of B-cell

dependent protection. Transfer of polyclonal immunoglobulin preparations (IVIG)

reduces atherosclerosis in Apoe�/� mice [38–40] and also intimal thickening after

mechanical injury in wildtype C57BL/6 mice [41]. The atheroprotective effect is at

least partly mediated via Fc receptors [39, 40] and also depends on a functioning

complement system [42].

21.5 Antigens of Atherosclerosis

The clonal expansion of T cells and their clustering in close proximity to DC and

macrophages point to a local immune response in the plaque [43, 44]. Autoantigens

as well as microbial molecules have been implicated. Two antigens have emerged

as potentially important: heat shock protein-60 (HSP60) and LDL. For both,

experiments in hypercholesterolemic mice and rabbits show substantial effects on

promoting disease development, and seroepidemiological studies support a role

also in human cardiovascular disease [45]. The role HSP60 is discussed in other

parts of this volume.

LDL elicits both cellular and humoral immune responses in the course of

atherosclerosis [14]. It is a complex particle that contains several B and T cell

epitopes. Circulating antibodies in patients and experimental animals recognize

oxidation-induced epitopes of LDL particles [46, 47]. Malondialdehyde (MDA),

4-hydroxynonenal, and other molecular species generated through lipid peroxida-

tion can form adducts on lysyl residues of ApoB100 [46]. Proteins with such

modified lysyl residues can be immunogenic, as are modified phospholipid species

[48]. Antibodies to such phospholipids inhibit the binding of oxLDL to

macrophages and have shown atheroprotective effects in animal experiments

[49–51]. Such antibodies recognize oxidatively-modified phospholipids in LDL

426 G.K. Hansson

(oxLDL) and apoptotic cell membranes but also phosphocholine in the cell wall of

Staphylococcus aureus (pneumococcus) [49]. The finding of immunological cross-

reactions between oxLDL and the pneumococcal cell wall raises the question of

whether molecular mimicry between pathogens and LDL could lead to

atheroprotective immune activity.

T cell clones reactive to LDL have been isolated from human plaques [52] and

antibodies to LDL are abundant in patients with atherosclerosis. Adoptive transfer

of LDL reactive T cells accelerates atherosclerosis in hypercholesterolemic mice

[53], while immunization against oxLDL particles reduces it [54, 55]. Interestingly,

parenteral immunization with native LDL [55], parenteral immunization with

phosphocholine, and mucosal immunization to native LDL peptides also show

atheroprotective effects [50, 56]. As discussed above, OxLDL is readily taken up

by antigen-presenting macrophages and DC. Scavenger receptors on these cells

internalize oxLDL – and other antigens – for degradation [57], but also for antigen

processing and presentation to T cells [58]. DC loaded with oxidized LDL and

injected into Apoe�/� mice induce a T cell response to components of LDL; this

response is associated with increased atherosclerosis [59].

21.6 Tolerance and Reactivity to LDL

LDL is a major circulating plasma component, with a concentration of approxi-

mately 2–3 mmol/L, therefore immunological tolerance to this particle is necessary

for survival. LDL-reactive T cells were thought to be eliminated by negative

selection, leading to central tolerance. Oxidation of LDL was thought to generate

neoantigens and T cell clones reactive to these would thus not be removed during

thymic education. Recent data have challenged this hypothesis by showing that

peripheral T cells in atherosclerotic mice recognize peptide motifs of native LDL

particles and its protein moiety, apoB100 (Fig. 21.2) [60]. Surprisingly, oxidation

extinguished rather than promoted LDL-dependent T cell activation [60]. Interest-

ingly, all T cell clones reactive to apoB100 carried a certain T cell receptor type

characterized by the b chain TRBV31. Immunization against this T cell receptor not

only induced blocking antibodies that reduced T cell responses to apoB100 and

(native) LDL, it also reduced the extent of disease [60]. This implies that cellular

immunity towards native LDL protein involves a limited set of T cell clones.

Furthermore, it shows that these T cell clones are involved in the development of

atherosclerosis.

In line with this notion, immunization with certain native, non-modified

apoB100 peptides protects hypercholesterolemic mice against atherosclerosis

[61]. The existence of peripheral T cells recognizing native LDL implies that

central tolerance to this autoantigen is far from complete. T cells capable of

recognizing epitopes of native LDL are present in the adult organism but probably

kept in check by peripheral tolerance mechanisms.

LDL is produced as very low density lipoprotein (VLDL) in the liver, converted

to LDL by enzymatic action on its triglycerides in peripheral tissues, and finally

21 Immunity to Low-Density Lipoprotein 427

taken up by LDL receptors in the liver and elsewhere. It was recently shown that

expression of tissue specific autoantigens under a liver-specific promoter leads to

inhibition of autoimmunity [62]; this and other findings identify a major immuno-

regulatory role of liver-associated immunity [63].

We speculate that VLDL/LDL immunoreactivity is controlled by such

mechanisms (Fig. 21.3). According to this hypothesis, VLDL would induce a

regulatory/inhibitory immune response when secreted by hepatocytes. Therefore,

circulating lipoprotein particles would not trigger any autoimmune reactions.

Modification of LDL in the inflamed artery wall may change the situation and

permit a local autoimmune attack.

21.7 Regulatory T Cells: Mediators of Tolerance

Several studies demonstrate a protective effect of different subsets of T regulatory

(Treg) cells in models of atherosclerosis. FoxP3+ cells have been found in the

plaques of mice as well as humans, although in low numbers [64, 65]. The Treg

cell cytokine products, TGFb1 and IL-10, have profound atheroprotective effects inmouse models but it should be kept in mind that these cytokines are produced also

by several other cell types. Further evidence for the atheroprotective effect of Treg

cells were obtained in studies of CD28 deficient mice that have reduced numbers of

LDL oxLDL ApoB100 Negative0

100

200

300

400

500

600

700IL

-2 (

pg/m

l)

Fig. 21.2 T cells recognize native, not oxidized LDL. T cell hybridoma clones generated from

mice immunized with oxLDL particles were challenged with oxLDL, native LDL, apoB100

protein isolated in the presence of antioxidants, or medium alone. The graph shows T cell

activation as revealed by IL-2 secretion of hybridomas. Native LDL and apoB100 induced

activation of a set of T cell hybridomas, whereas oxLDL did not trigger activation (From [64].

With permission of the Journal of Experimental Medicine)

428 G.K. Hansson

Treg cells. Reconstitution of atherosclerotic mice with CD28 deficient bone marrow

led to increased disease [66]. Transfer of natural FoxP3+ T cells has also been

shown to be protective against experimental atherosclerosis [66, 67].

Peripheral Treg cells can be induced by mucosal administration of antigen or

anti-CD3 antibodies. Nasal immunization of Apoe�/� mice with an ApoB100

peptide fused to the B subunit of cholera toxin that binds to mucosal gangliosides

led to induction of ApoB100-specific regulatory Tr1 cells that produced IL-10 and

reduced atherosclerosis [56].

A remarkable extent of atheroprotection was also achieved when hypercholes-

terolemic mice were injected with antigen-loaded, tolerogenic DC [68]. Isolated

DC were loaded with apoB100, made tolerogenic by treatment with IL-10, and

injected into hypercholesterolemic ApoB100tg � Ldlr�/� mice. A single injection

of such tolerogenic DC not only induced Treg and anti-LDL antibodies, it reduced

atherosclerosis by 70% [68].

Inhibition ofautoreactiveT cell clones

Immunosuppressive /regulatory signals

Activation of autoreactiveT cell clones

Suppression of immuneresponses in the liver

Tolerance

Inflammation

Nascent proteinsincluding apoB Proinflammatory

activating signals

Activation of immuneresponse in the artery

Accumulationof LDL

Fig. 21.3 Lipoprotein secretion in the liver is accompanied by peripheral tolerance that can be

broken when particles accumulate in the artery wall. Hypothetical mechanisms showing that ApoB

containing lipoproteins produced in the liver are accompanied by immunosuppressive signals that

may include immunoregulatory cytokines and autacoids, death signals for activated T cells, and

non-activating costimulatory factors. This leads to peripheral tolerance against plasma

lipoproteins, although apoB reactive T cell clones are present and thus not deleted by central

tolerance. When lipoproteins are retended in the artery wall, they activate innate immunity,

leading to local inflammation. Antigen presentation in this milieu, in the presence of costimulatory

factors, IL-1b, IL-12 and other cytokines, stimulates the activation of effector T cells that

accelerate vascular inflammation and promote the formation of an atherosclerotic lesion

(Drawings in the figure were taken from [1]. With permission from the New England Journal of

Medicine)

21 Immunity to Low-Density Lipoprotein 429

21.8 Protective Effects of Anti-oxLDL Antibodies

A series of studies have demonstrated atheroprotective effects of antibodies to

oxLDL. In many experimental studies on rabbits and mice where oxLDL was

used for immunization, there was a correlation between high titers of anti-oxLDL

and degree of protection against atherosclerosis [54, 55, 69]. In line with this,

infusion of anti-LDL antibodies reduces atherosclerosis in hypercholesterolemic

mice [51]. As is often the case, the situation is more complex in humans, with

various studies showing positive, negative or no correlation between anti-LDL

titers and atherosclerosis or its manifestations [70–73].

21.9 Conclusion

In conclusion, immunization with components of LDL can confer protection

against atherosclerosis, although autoimmunization during the course of disease

leads to proatherosclerotic immune responses. The latter type of response may be

promoted by the presence of proinflammatory signals during antigen presentation

and T effector cell differentiation. Analogously, atheroprotection may develop

when LDL derived antigens are presented in immunoregulatory contexts. This

would take place concomitantly with VLDL secretion in the liver but could also

be elicited when apoB100 is presented by tolerogenic DC, or together with immu-

noregulatory molecules. Tolerogenic vaccination may represent a therapeutic

approach to achieving atheroprotection. It can be achieved by immunization with

apoB100 or peptides thereof, packaged with appropriate adjuvants or loaded onto

tolerogenic DC. Future studies will clarify whether such approaches are beneficial

for atherosclerotic cardiovascular disease in man.

References

1. Hansson GK (2005) Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med

352(16):1685–1695

2. Tabas I, Williams KJ, Boren J (2007) Subendothelial lipoprotein retention as the initiating

process in atherosclerosis: update and therapeutic implications. Circulation 116(16):

1832–1844

3. Skalen K, Gustafsson M, Rydberg EK, Hulten LM, Wiklund O, Innerarity TL, Boren J (2002)

Subendothelial retention of atherogenic lipoproteins in early atherosclerosis. Nature 417

(6890):750–754

4. Steinberg D (2009) The LDL modification hypothesis of atherogenesis: an update. J Lipid Res

50(Suppl):S376–S381

5. Navab M, Ananthramaiah GM, Reddy ST, Van Lenten BJ, Ansell BJ, Fonarow GC,

Vahabzadeh K, Hama S, Hough G, Kamranpour N, Berliner JA, Lusis AJ, Fogelman AM

(2004) The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and

HDL. J Lipid Res 45(6):993–1007

6. Bochkov VN, Mechtcheriakova D, Lucerna M, Huber J, Malli R, Graier WF, Hofer E,

Binder BR, Leitinger N (2002) Oxidized phospholipids stimulate tissue factor expression

430 G.K. Hansson

in human endothelial cells via activation of ERK/EGR-1 and Ca(++)/NFAT. Blood

99(1):199–206

7. Gharavi NM, Alva JA, Mouillesseaux KP, Lai C, Yeh M, Yeung W, Johnson J, Szeto WL,

Hong L, Fishbein M, Wei L, Pfeffer LM, Berliner JA (2007) Role of the Jak/STAT pathway in

the regulation of interleukin-8 transcription by oxidized phospholipids in vitro and in athero-

sclerosis in vivo. J Biol Chem 282(43):31460–31468

8. Gargalovic PS, Gharavi NM, Clark MJ, Pagnon J, Yang WP, He A, Truong A, Baruch-Oren T,

Berliner JA, Kirchgessner TG, Lusis AJ (2006) The unfolded protein response is an important

regulator of inflammatory genes in endothelial cells. Arterioscler Thromb Vasc Biol 26

(11):2490–2496

9. Dai L, Zhang Z, Winyard PG, Gaffney K, Jones H, Blake DR, Morris CJ (1997) A modified

form of low-density lipoprotein with increased electronegative charge is present in rheumatoid

arthritis synovial fluid. Free Radic Biol Med 22(4):705–710

10. Frostegard J, Svenungsson E, Wu R, Gunnarsson I, Lundberg IE, Klareskog L, Horkko S,

Witztum JL (2005) Lipid peroxidation is enhanced in patients with systemic lupus

erythematosus and is associated with arterial and renal disease manifestations. Arthritis

Rheum 52(1):192–200

11. Holvoet P, Perez G, Zhao Z, Brouwers E, Bernar H, Collen D (1995) Malondialdehyde-

modified low density lipoprotein in patients with atherosclerotic disease. J Clin Invest

95:2611–2619

12. Miller YI, Choi SH, Wiesner P, Fang L, Harkewicz R, Hartvigsen K, Boullier A, Gonen A,

Diehl CJ, Que X, Montano E, Shaw PX, Tsimikas S, Binder CJ, Witztum JL (2011) Oxidation-

specific epitopes are danger-associated molecular patterns recognized by pattern recognition

receptors of innate immunity. Circ Res 108(2):235–248

13. [a] Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG, Abela GS,

Franchi L, Nunez G, Schnurr M, Espevik T, Lien E, Fitzgerald KA, Rock KL, Moore KJ,

Wright SD, Hornung V, Latz E (2010) NLRP3 inflammasomes are required for atherogenesis

and activated by cholesterol crystals. Nature 464(7293):1357–1361; [b] Rajamaki K,

Lappalainen J, Oorni K, Valimaki E, Matikainen S, Kovanen PT, Eklund KK (2010) Choles-

terol crystals activate the nlrp3 inflammasome in human macrophages: A novel link between

cholesterol metabolism and inflammation. PLoS ONE 5:e11765

14. Hansson GK, Hermansson A (2011) Immunity in atherosclerosis. Nat Immunol 12:204–212

15. Dansky HM, Charlton SA, Harper MM, Smith JD (1997) T and B lymphocytes play a minor

role in atherosclerotic plaque formation in the apolipoprotein E-deficient mouse. Proc Natl

Acad Sci USA 94:4642–4646

16. Reardon CA, Blachowicz L, White T, Cabana V, Wang Y, Lukens J, Bluestone J, Getz GS

(2001) Effect of immune deficiency on lipoproteins and atherosclerosis in male apolipoprotein

E-deficient mice. Arterioscler Thromb Vasc Biol 21(6):1011–1016

17. Zhou X, Nicoletti A, Elhage R, Hansson GK (2000) Transfer of CD4(+) T cells aggravates

atherosclerosis in immunodeficient apolipoprotein E knockout mice. Circulation 102

(24):2919–2922

18. Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK (1986) Regional accumulations of T

cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arterioscle-

rosis 6(2):131–138

19. Jonasson L, Holm J, Skalli O, Gabbiani G, Hansson GK (1985) Expression of class II

transplantation antigen on vascular smooth muscle cells in human atherosclerosis. J Clin

Invest 76(1):125–131

20. Hansson GK, Holm J, Jonasson L (1989) Detection of activated T lymphocytes in the human

atherosclerotic plaque. Am J Pathol 135(1):169–175

21. Hansson GK, Robertson AKL, S€oderberg-Naucler C (2006) Inflammation and atherosclerosis.

Annu Rev Pathol 1:297–329

21 Immunity to Low-Density Lipoprotein 431

22. Paulsson G, Zhou X, Tornquist E, Hansson GK (2000) Oligoclonal T cell expansions in

atherosclerotic lesions of apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 20

(1):10–17

23. Liuzzo G, Goronzy JJ, Yang H, Kopecky SL, Holmes DR, Frye RL, Weyand CM (2000)

Monoclonal T-cell proliferation and plaque instability in acute coronary syndromes. Circula-

tion 101(25):2883–2888

24. Olofsson PS, Soderstrom LA, Wagsater D, Sheikine Y, Ocaya P, Lang F, Rabu C, Chen L,

Rudling M, Aukrust P, Hedin U, Paulsson-Berne G, Sirsjo A, Hansson GK (2008) CD137 is

expressed in human atherosclerosis and promotes development of plaque inflammation in

hypercholesterolemic mice. Circulation 117(10):1292–1301

25. Ludewig B, Freigang S, Jaggi M, Kurrer MO, Pei YC, Vlk L, Odermatt B, Zinkernagel RM,

Hengartner H (2000) Linking immune-mediated arterial inflammation and cholesterol-induced

atherosclerosis in a transgenic mouse model [In process citation]. Proc Natl Acad Sci USA 97

(23):12752–12757

26. Gotsman I, Sharpe AH, Lichtman AH (2008) T-cell costimulation and coinhibition in athero-

sclerosis. Circ Res 103:1220–1231

27. Bobryshev YV, Lord RS (1995) S-100 positive cells in human arterial intima and in athero-

sclerotic lesions. Cardiovasc Res 29(5):689–696

28. Angeli V, Llodra J, Rong JX, Satoh K, Ishii S, Shimizu T, Fisher EA, Randolph GJ (2004)

Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobili-

zation. Immunity 21(4):561–574

29. Niessner A, Sato K, Chaikof EL, Colmegna I, Goronzy JJ, Weyand CM (2006) Pathogen-

sensing plasmacytoid dendritic cells stimulate cytotoxic T-cell function in the atherosclerotic

plaque through interferon-alpha. Circulation 114(23):2482–2489

30. Choi JH, Do Y, Cheong C, Koh H, Boscardin SB, Oh YS, Bozzacco L, Trumpfheller C,

Park CG, Steinman RM (2009) Identification of antigen-presenting dendritic cells in mouse

aorta and cardiac valves. J Exp Med 206(3):497–505

31. Niessner A, Weyand CM (2010) Dendritic cells in atherosclerotic disease. Clin Immunol 134

(1):25–32

32. Kovanen PT (2007) Mast cells: multipotent local effector cells in atherothrombosis. Immunol

Rev 217:105–122

33. Grabner R, Lotzer K, Dopping S, Hildner M, Radke D, Beer M, Spanbroek R, Lippert B,

Reardon CA, Getz GS, Fu YX, Hehlgans T, Mebius RE, van der Wall M, Kruspe D, Englert C,

Lovas A, Hu D, Randolph GJ, Weih F, Habenicht AJ (2009) Lymphotoxin beta receptor

signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE�/�

mice. J Exp Med 206(1):233–248

34. Caligiuri G, Nicoletti A, Poirier B, Hansson GK (2002) Protective immunity against athero-

sclerosis carried by B cells of hypercholesterolemic mice. J Clin Invest 109(6):745–753

35. Major AS, Fazio S, Linton MF (2002) B-lymphocyte deficiency increases atherosclerosis in

LDL receptor-null mice. Arterioscler Thromb Vasc Biol 22(11):1892–1898

36. Ait-Oufella H, Herbin O, Bouaziz JD, Binder CJ, Uyttenhove C, Laurans L, Taleb S,

Van Vre E, Esposito B, Vilar J, Sirvent J, Van Snick J, Tedgui A, Tedder TF, Mallat Z

(2010) B cell depletion reduces the development of atherosclerosis in mice. J Exp Med

207(8):1579–1587

37. Kyaw T, Tay C, Khan A, Dumouchel V, Cao A, To K, Kehry M, Dunn R, Agrotis A, Tipping

P, Bobik A, Toh BH (2010) Conventional B2 B cell depletion ameliorates whereas its adoptive

transfer aggravates atherosclerosis. J Immunol 185(7):4410–4419

38. Nicoletti A, Kaveri S, Caligiuri G, Bariety J, Hansson GK (1998) Immunoglobulin treatment

reduces atherosclerosis in apo E knockout mice. J Clin Invest 102(5):910–918

39. Yuan Z, Kishimoto C, Sano H, Shioji K, Xu Y, Yokode M (2003) Immunoglobulin treatment

suppresses atherosclerosis in apolipoprotein E-deficient mice via the Fc portion. Am J Physiol

Heart Circ Physiol 285(2):H899–H906

432 G.K. Hansson

40. Okabe TA, Kishimoto C, Shimada K, Murayama T, Yokode M, Kita T (2005) Effects of

late administration of immunoglobulin on experimental atherosclerosis in apolipoprotein

E-deficient mice. Circ J 69(12):1543–1546

41. Keren G, Keren P, Barshack I, Pri-Chen S, George J (2001) The effect of intravenous

immunoglobulins on intimal thickening in a mouse model of arterial injury. Atherosclerosis

159(1):77–83

42. Persson L, Boren J, Nicoletti A, Hansson GK, Pekna M (2005) Immunoglobulin treatment

reduces atherosclerosis in apolipoprotein E�/� low-density lipoprotein receptor�/� mice via

the complement system. Clin Exp Immunol 142(3):441–445

43. Hansson GK, Holm J, Jonasson L (1989) Detection of activated T lymphocytes in the human

atherosclerotic plaque. Am J Pathol 135(169):169–175

44. Paulsson G, Zhou X, T€ornquist E, Hansson GK (2000) Oligoclonal T cell expansions in

atherosclerotic lesions of apoE-deficient mice. Arterioscler Thromb Vasc Biol 20:10–17

45. Hansson GK, Libby P (2006) The immune response in atherosclerosis: a double-edged sword.

Nat Rev Immunol 6(7):508–519

46. Palinski W, Rosenfeld ME, Yl:a-Herttuala S, Gurtner GC, Socher SS, Butler SW,

Parthasarathy S, Carew TE, Steinberg D, Witztum JL (1989) Low density lipoprotein

undergoes oxidative modification in vivo. Proc Natl Acad Sci USA 86(4):1372–1376

47. Palinski W, Yla-Herttuala S, Rosenfeld ME, Butler SW, Socher SA, Parthasarathy S,

Curtiss LK, Witztum JL (1990) Antisera and monoclonal antibodies specific for epitopes

generated during oxidative modification of low density lipoprotein. Arteriosclerosis

10(3):325–335

48. Palinski W, H€orkk€o S, Miller E, Steinbrecher UP, Powell HC, Curtiss LK, Witztum JL (1996)

Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipopro-

tein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human

plasma. J Clin Invest 98:800–814

49. Binder CJ, Horkko S, Dewan A, Chang MK, Kieu EP, Goodyear CS, Shaw PX, Palinski W,

Witztum JL, Silverman GJ (2003) Pneumococcal vaccination decreases atherosclerotic lesion

formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat

Med 9(6):736–743

50. Caligiuri G, Khallou-Laschet J, Vandaele M, Gaston AT, Delignat S, Mandet C, Kohler HV,

Kaveri SV, Nicoletti A (2007) Phosphorylcholine-targeting immunization reduces atheroscle-

rosis. J Am Coll Cardiol 50(6):540–546

51. Schiopu A, Bengtsson J, Soderberg I, Janciauskiene S, Lindgren S, Ares MP, Shah PK,

Carlsson R, Nilsson J, Fredrikson GN (2004) Recombinant human antibodies against

aldehyde-modified apolipoprotein B-100 peptide sequences inhibit atherosclerosis. Circulation

110(14):2047–2052

52. Stemme S, Faber B, Holm J, Wiklund O, Witztum JL, Hansson GK (1995) T lymphocytes

from human atherosclerotic plaques recognize oxidized low density lipoprotein. Proc Natl

Acad Sci USA 92(9):3893–3897

53. Zhou X, Robertson AK, Hjerpe C, Hansson GK (2006) Adoptive transfer of CD4+ T cells

reactive to modified low-density lipoprotein aggravates atherosclerosis. Arterioscler Thromb

Vasc Biol 26(4):864–870

54. Palinski W, Miller E, Witztum JL (1995) Immunization of low density lipoprotein (LDL)

receptor-deficient rabbits with homologous malondialdehyde-modified LDL reduces athero-

genesis. Proc Natl Acad Sci USA 92:821–825

55. Ameli S, Hultgardh-Nilsson A, Regnstrom J, Calara F, Yano J, Cercek B, Shah PK, Nilsson J

(1996) Effect of immunization with homologous LDL and oxidized LDL on early atheroscle-

rosis in hypercholesterolemic rabbits. Arterioscler Thromb Vasc Biol 16(8):1074–1079

56. Klingenberg R, Lebens M, Hermansson A, Fredrikson GN, Strodthoff D, Rudling M,

Ketelhuth DF, Gerdes N, Holmgren J, Nilsson J, Hansson GK (2010) Intranasal immunization

with an apolipoprotein B-100 fusion protein induces antigen-specific regulatory T cells and

reduces atherosclerosis. Arterioscler Thromb Vasc Biol 30(5):946–952

21 Immunity to Low-Density Lipoprotein 433

57. Greaves DR, Gordon S (2009) The macrophage scavenger receptor at 30 years of age: current

knowledge and future challenges. J Lipid Res 50(Suppl):S282–S286

58. Nicoletti A, Caligiuri G, T€ornberg I, Kodama T, Stemme S, Hansson GK (1999) The

macrophage scavenger receptor type A directs modified proteins to antigen presentation. Eur

J Immunol 29:512–521

59. Hjerpe C, Johansson D, Hermansson A, Hansson GK, Zhou X (2010) Dendritic cells

pulsed with malondialdehyde modified low density lipoprotein aggravate atherosclerosis in

Apoe(�/�) mice. Atherosclerosis 209(2):436–441

60. Hermansson A, Ketelhuth DF, Strodthoff D, Wurm M, Hansson EM, Nicoletti A, Paulsson-

Berne G, Hansson GK (2010) Inhibition of T cell response to native low-density lipoprotein

reduces atherosclerosis. J Exp Med 207(5):1081–1093

61. Fredrikson GN, Hedblad B, Berglund G, Alm R, Ares M, Cercek B, Chyu KY, Shah PK,

Nilsson J (2003) Identification of immune responses against aldehyde-modified peptide

sequences in apoB associated with cardiovascular disease. Arterioscler Thromb Vasc Biol

23(5):872–878

62. Luth S, Huber S, Schramm C, Buch T, Zander S, Stadelmann C, Bruck W, Wraith DC,

Herkel J, Lohse AW (2008) Ectopic expression of neural autoantigen in mouse liver

suppresses experimental autoimmune neuroinflammation by inducing antigen-specific Tregs.

J Clin Invest 118(10):3403–3410

63. Thomson AW, Knolle PA (2010) Antigen-presenting cell function in the tolerogenic liver

environment. Nat Rev Immunol 10(11):753–766

64. Veillard NR, Steffens S, Burger F, Pelli G, Mach F (2004) Differential expression patterns of

proinflammatory and antiinflammatory mediators during atherogenesis in mice. Arterioscler

Thromb Vasc Biol 24(12):2339–2344

65. de Boer OJ, van der Meer JJ, Teeling P, van der Loos CM, van der Wal AC (2007) Low

numbers of FOXP3 positive regulatory T cells are present in all developmental stages of

human atherosclerotic lesions. PLoS One 2(1):e779

66. Ait-Oufella H, Salomon BL, Potteaux S, Robertson AK, Gourdy P, Zoll J, Merval R,

Esposito B, Cohen JL, Fisson S, Flavell RA, Hansson GK, Klatzmann D, Tedgui A,

Mallat Z (2006) Natural regulatory T cells control the development of atherosclerosis in

mice. Nat Med 12(2):178–180

67. Mor A, Planer D, Luboshits G, Afek A, Metzger S, Chajek-Shaul T, Keren G, George J (2007)

Role of naturally occurring CD4+ CD25+ regulatory T cells in experimental atherosclerosis.

Arterioscler Thromb Vasc Biol 27(4):893–900

68. Hermansson A, Johansson DK, Ketelhuth DF, Andersson J, Zhou X, Hansson GK (2011)

Immunotherapy with tolerogenic apolipoprotein B-100 loaded dendritic cells attenuates ath-

erosclerosis in hypercholesterolemic mice. Circulation 123:1083–1091

69. Nilsson J, Hansson GK, Shah PK (2005) Immunomodulation of atherosclerosis: implications

for vaccine development. Arterioscler Thromb Vasc Biol 25(1):18–28

70. Hulthe J, Wikstrand J, Lidell A, Wendelhag I, Hansson GK, Wiklund O (1998) Antibody titers

against oxidized LDL are not elevated in patients with familial hypercholesterolemia.

Arterioscler Thromb Vasc Biol 18:1203–1211

71. Tornvall P, Waeg G, Nilsson J, Hamsten A, Regnstrom J (2003) Autoantibodies against

modified low-density lipoproteins in coronary artery disease. Atherosclerosis 167(2):347–353

72. Fredrikson GN, Hedblad B, Berglund G, Alm R, Nilsson JA, Schiopu A, Shah PK, Nilsson J

(2007) Association between IgM against an aldehyde-modified peptide in apolipoprotein B-

100 and progression of carotid disease. Stroke 38(5):1495–1500

73. Sjogren P, Fredrikson GN, Samnegard A, Ericsson CG, Ohrvik J, Fisher RM, Nilsson J,

Hamsten A (2008) High plasma concentrations of autoantibodies against native peptide 210

of apoB-100 are related to less coronary atherosclerosis and lower risk of myocardial infarc-

tion. Eur Heart J 29(18):2218–2226

434 G.K. Hansson