Immunity and Inflammation in Atherosclerosis

The atherogenic process starts with the accumulation of several plasma lipoproteins in the subendothelial space at sites of flow perturbation and endothelial dysfunction. This is best documented for LDL, whose accumulation correlates with classical risk factors, such as smoking, hypertension, and metabolic dysregulation in obesity and diabetes mellitus.¹⁵ In the intima, LDL undergoes oxidative modifications by reactive oxygen species, which promote the uptake of oxLDL (oxidized LDL) into macrophages.¹⁶ In addition, oxidized phospholipids per se trigger inflammation of the arterial wall¹⁷ by binding to TLRs (Toll-like receptors), a group of widely expressed PRRs (pattern recognition receptors) that cause proinflammatory signaling.¹⁸ Clinically, oxLDL is a marker of plaque inflammation.¹⁹ Native LDL can also be taken up by macrophages by micropinocytosis or in its aggregated form as cholesterol complexes or crystals by phagocytosis. The sustained influx of cholesterol eventually exceeds the phagocytes’ metabolic capacity and intracellular lipid droplets form. Microscopically, cholesterol-laden macrophages are foam cells. Cholesterol loading is thought to cause a myeloid cell response with proinflammatory cytokine secretion, in situ macrophage proliferation, and further recruitment of myeloid cells.²⁰ A clinically important consequence of cholesterol loading is the formation of intracellular cholesterol microcrystals that activate the inflammasome, a molecular machinery comprising molecules of the cytosolic nucleotide binding domain and leucine-rich repeat gene family (NLRP3) that cleaves pro-IL (interleukin)-1β into its biologically active form.²¹ IL-1β serves as an inflammatory master cytokine that enhances the expression of many proinflammatory cytokines, as well as of CRP (C-reactive protein).²² Notably, attenuating cholesterol storage and enhancing cholesterol efflux pathways may favor the resolution of plaque inflammation and even promote plaque regression.²³ The myeloid response is accompanied by the infiltration of cells of the adaptive immune system, B and T cells.^24,25 Notably, the plaque’s growing content of myeloid cells and lymphocytes correlates with clinical complications and may predispose for future thromboembolic events caused by large cellular infiltrates and a thin fibrous cap (unstable plaque).^26,27

The presence of T and B cells in the plaque²⁸ sparked the hypothesis that atherosclerosis includes an autoimmune response. Adaptive immunity in infection and autoimmunity proceeds by a humoral arm that comprises specific antibodies against the antigen secreted by plasma cells and a cellular arm with T cells that either activate B cells during costimulation or differentiate into effector T cells with pro- or anti-inflammatory cytokine production.²⁹ CD8⁺ and CD4⁺ T cells only initiate immune responses to peptides presented by MHC (major histocompatibility complex)-I on all nucleated cells or MHC-II on antigen-presenting cells (APCs), respectively. Such responses are MHC restricted, that is, they only occur in individuals expressing a specific MHC allele with the ability to bind the relevant peptide epitope. Binding of a specific TCR (T-cell receptor) concomitant with costimulatory events provided by APCs activates T cells and causes their clonal proliferation.³⁰ In mouse atherosclerosis, 2-photon microscopy has revealed an increased rate of APC-CD4⁺ T-helper cell interactions in the plaque specifically in the setting of hypercholesterolemia that resulted in proinflammatory cytokine secretion.³¹ In addition, T-helper cells show an increasing maturation into antigen-experienced effector/memory (T_EM) and central memory (T_CM) T cells in the lymph nodes (Figure 1A) that is also observed in atherosclerotic plaques.^28,31 Sequencing of the TCR revealed an oligoclonal origin of lesional T cells^32,33 suggesting that some (antigen specific) T-cell clones actively expand in the plaque. The enhanced activation of T cells is accompanied by an expansion of lymph nodes draining the atherosclerotic aorta in aged atherosclerotic Apoe^−/− mice (Figure 1B) and a local and systemic proinflammatory response that is further enhanced by a hypercholesterolemia-inducing diet.^34–36 These findings support the concept that specific antigens drive an immune response in the aorta and lymph nodes during atherosclerosis.

Of all candidates that may serve as B- and T-cell–activating antigens, plasma levels of LDL and its core protein ApoB show the strongest clinical and causal link with atherosclerosis in humans.³⁷ ApoB-containing triglyceride-rich remnant particles also show a strong association with cardiovascular disease, inflammation, and immune pathways.⁷ Indeed, LDL as (auto) antigen was first suggested by Gero et al³⁸ in 1959: immunization with LDL protected against atherosclerosis in rabbits, suggesting that autoimmune response against LDL can be atheroprotective.³⁹ Many CD4⁺ T cells in human plaques recognize oxLDL⁴⁰ by binding to MHC-presented peptide epitopes from Apo B₁₀₀.^41,42 A tetramer of recombinant MHC molecules loaded with an ApoB-derived peptide—a tool to detect antigen-specific T-helper cells in vivo⁴³—identified a naturally occurring population of CD4⁺ T cells in the blood that recognizes the human peptide Apo B_3036–3050.⁴² Furthermore, atherosclerosis is accompanied by IgG antibodies against LDL, oxLDL, and ApoB.⁴⁴ Collectively, these findings strongly suggest LDL as a relevant self-antigen that drives an autoimmune response against self-proteins in the atherosclerotic plaque. Besides LDL/ApoB, HSPs (heat shock proteins)^45–47 and some foreign peptides from pathogens such as Cytomegalovirus (CMV), hepatitis C virus (HCV), HIV, human papillomavirus (HPV), and others^48–50 have been proposed as atherosclerosis-relevant antigens.

Early evidence from immunohistochemistry studies,^28,51 more recent single-cell RNA sequencing,^24,52 and Cytometry by Time of Flight (mass cytometry) approaches^24,53 has estimated that ≈25% to 38% of all leukocytes in mouse aortic and human atherosclerotic plaques are CD3⁺ T cells, with CD3⁺CD4⁺ T-helper cells accounting for ≈10%. T cells predominantly populate atherosclerotic lesions with an enrichment in the fibrous cap,^28,51 but are also found in the adventitia of older lesions.^24,54 Their recruitment to the plaque occurs via chemokine receptors CCR5 (C-C chemokine receptor type 5), CXCR6 (C-X-C Motif Chemokine Receptor 6), and others.^55,56 CD4⁺ T cells are critical regulators of the adaptive immune response with the ability to differentiate into distinct T-helper subtypes that can either be immune-dampening or activating to other T cells, exert direct anti- or pro-inflammatory effects on tissue-resident cells, provide B-cell help to induce the production of high-affinity IgG antibodies, or exhibit cytolytic activity²⁹ (Figure 2). Thus, the function of T-helper cells in atherosclerosis is multi-faceted and depends on specific transcriptional programs and patterns of cytokine secretion that can either fuel or attenuate atherosclerosis. Early evidence from Rag-1–deficient mice, which cannot produce mature T and B cells, suggested a pathogenic role for T and B lymphocytes only in early atherosclerosis with moderately enhanced lipid levels, but not in severely hypercholesterolemia Apoe^−/− mice.^57,58 Genetic absence of T cells in athymic nu/nu mice or a depletion of CD4⁺ T cells by anti-CD4 antibodies protected from lesion development.⁵⁹ After antigen presentation by APCs, lesional T cells differentiate into functionally distinct T-helper subtype (T_H) -1, -2, -17, T-regulatory cells (T_reg), T-follicular helper cells (T_FH), and Type 1 regulatory (T_R1) cells.⁶⁰ Atherosclerosis is a known T_H1 disease. Many CD4⁺ T cells in the plaque express the proinflammatory, T_H1-associated cytokines IFN (interferon)-γ, IL-2, IL-3, TNF (tumor necrosis factor), and LT (lymphotoxin), which can activate macrophages, T cells, and other components of the plaque, and thereby aggravate the inflammatory response.⁶¹ T cells that express the plaque-homing chemokine receptor CCR5 in lymph nodes, and T cells from atherosclerotic lesions secrete IFN-γ and express T-bet (T-box transcription factor), the T_H1-lineage–defining transcription factor.^55,62 Knocking out IFN-γ, its receptor, or T-bet protects mice from atherosclerosis.^63–65 IFN-γ may directly reduce plaque stability by inhibiting smooth muscle cell proliferation,⁶⁶ affecting macrophage polarization, and modulating cardiovascular risk factors.⁶⁷ On the other hand, regulatory CD4⁺ T cells (T_regs) that express the transcription factor FoxP3 (forkhead box P3) and the high-affinity IL-2 receptor CD25 protect mice from atherosclerosis.^68,69 T_regs exert their atheroprotective properties by secreting the anti-inflammatory cytokine IL-10,⁷⁰ plaque-stabilizing TGF (transforming growth factor)-β,⁷¹ and by suppressing the proliferation of proinflammatory T-effector cells.⁷² Atheroprotective effects of in vivo treatment with IL-2 complexes⁷³ and anti-CD3 treatment^74,75 have been attributed to a relative increase of T_regs. In addition, T_R1 cells that lack FoxP3 expression but express CD49b and Lag-3 secrete IL-10 and are atheroprotective.^76,77 In the atherosclerotic plaque, a substantial proportion of T cells moreover express transcripts for the T_H2 cytokines IL-4, IL-5, and IL-13.²⁴ In contrast to abdominal aortic aneurysm formation, which is a clear T_H2-dependent disease⁷⁸ and the negative correlation of IL-4 with clinical atherosclerosis,⁷⁹ the relevance of T_H2 immunity in atherosclerosis remains unclear. The T_H2 cytokine IL-4 antagonizes T_H1 responses and diminished lesion formation in 1 study,⁸⁰ although depletion of IL-4 has also been reported to be atheroprotective.⁸¹ Likewise, the role of T_H17 cells in atherosclerosis is controversial: deletion or neutralization of the master cytokine IL-17 protected from atherosclerosis,^82–84 although other studies reported that T_H17 immunity protected from atherosclerosis and induced a stable plaque phenotype.^85–87 T_FH, which are required to costimulate B cells and to induce an immunoglobulin class switch, have also been proposed to be proatherogenic⁸⁸ or to protect from atherosclerosis by secreting LDL-lowering/neutralizing anti-LDL/ApoB secreting antibodies.⁸⁹ The different findings in these studies may reflect different but unknown antigen specificities of the T cells studied.

It is noteworthy that antigen presentation initiates and modulates the CD4⁺ T-helper cell response in atherosclerosis. T-cell activation in an antigen-specific manner is an exclusive consequence of APCs that present antigenic peptides displayed on MHC molecules.²⁹ Blocking MHC-II during costimulation or on APCs abrogates the downstream CD4⁺ T-cell response.^31,90 T-cell immune responses are typically initiated by antigen-loaded dendritic cells migrating to lymph nodes. Several cells in the atherosclerotic plaque act as APCs for recall responses of antigen-experienced effector and memory T cells, including macrophages in the plaque, B cells in the adventitia, along with conventional dendritic cells and plasmacytoid dendritic cells. Depending on costimulatory signals and cytokines provided by these APCs, the immune response can be skewed into a tolerogenic (immune-suppressive) or an immunogenic response.^91,92

The role of other T-cell subsets remains less well defined. It has been suggested that MHC-I–dependent cytotoxic CD8⁺ T cells contribute to plaque inflammation and the build-up of the necrotic core,^93,94 but antigen specificity has not been considered.⁹⁵ Natural killer cells regulate antigen-specific T-cell immunity besides the killing of infected and tumor cells. They are detected at low frequencies in the plaque and may therefore modulate atherosclerosis.⁹⁶ In contrast to earlier studies, a recent report, however, suggested that natural killer cells do not affect atherogenesis.^97–100 In addition, CD1d-restricted natural killer cells can recognize glycolipid antigens. Some NKT-cell subsets were reported to aggravate atherosclerosis, but the atherosclerosis-relevant glycolipids detected by these NKT cells remain unknown.^101–103

It has been challenging to determine the phenotype of ApoB-specific CD4⁺ T cells, that is, the fraction of T cells with a TCR recognizing ApoB peptides presented on MHC-II, within the pool of all lesional T cells. In animal models, ApoB-specific T cells have been expanded in vitro or by vaccination against LDL or ApoB peptides in vivo. A direct transfer of vaccination-induced T cells in 1 study aggravated atherosclerotic disease.¹⁰⁴ T cells restimulated with oxLDL ex vivo promoted atherosclerotic disease after adoptive transfer in a model of immunodeficient scid (severe combined immunodeficiency)/Apoe^−/− mice.¹⁰⁵ Neutralization of T cells that responded to oxLDL stimulation by a monoclonal antibody directed against the TCRBV31 chain protected from atherosclerosis,¹⁰⁶ suggestive of a proatherogenic function of ApoB-reactive T cells. However, more recent technologies to specifically detect ApoB-specific T cells in vivo suggest the opposite: tracking of ApoB-reactive T cells in mice and humans suggests that a majority of antigen-specific T cells are immunosuppressive T_regs.⁴² This is consistent with recent work from Gisterå et al⁸⁹ who transferred ApoB-reactive T cells from a mouse with a transgenic TCR directed against oxLDL/ApoB, which protected from atherosclerosis. These mixed results obscure the exact function of antigen-specific T cells. Likely, their phenotype and function depend on presented peptides, the microenvironment, and cytokine milieu, which potentially affects T-cell polarization. Some proatherogenic antigen-specific T cells^104,106 were isolated and cloned for in vitro assays by a procedure known to predispose and select pathogenic T_H1 and to neglect T_reg clones. It is possible that the population of antigen-specific T cells may be multipotent to give rise to several T_H lineages in-vivo—an idea consistent with the recent observation that MHC-II multimer selected ApoB-reactive T cells can express several T_H-defining transcription factors simultaneously.⁴²

The notion that atherosclerosis has an autoimmune component raised the question whether atherosclerosis is prevented in an antigen-specific manner by ApoB-reactive T_regs³⁹ in healthy individuals. T_regs prevent the onset of autoimmune disease.¹⁰⁷ Naturally occurring T_regs are generated in the thymus (nT_regs) and peripheral T_regs are induced from naive T cells (iT_regs). Despite the proven atheroprotective role of bulk T_regs,^68,69 it has been unclear whether T_regs reactive to ApoB exist and how these may contribute to disease. Interestingly, ample clinical data suggest a strong inverse relationship between T_regs and atherosclerosis: numbers of T_regs and IL-10 expression are lower in patients with myocardial infarction.^108,109 Low T_reg numbers predict cardiovascular events.¹¹⁰ Blood T_reg numbers in established murine atherosclerosis decline in later disease, whereas effector T cells increase³⁶ (Figure 3A). However, in subclinical human atherosclerosis, T_reg numbers correlate positively with LDL.¹¹¹ Likewise, in mice, hypercholesterolemia initially favors the differentiation of T_regs,¹¹² an effect that may be a counter-regulatory response to enhanced inflammation,³⁶ intracellular lipid accumulation,¹¹³ or an antigen-specific response. The latter hypothesis was supported by enhanced T-cell receptor (TCR) downstream signaling events in hypercholesterolemic mice,¹¹⁴ suggesting that a subpopulation of T cells responds to antigens associated with increased LDL levels or to components of LDL particles itself. Thus, these data indirectly suggest the existence of LDL/ApoB-reactive T_regs that bear a TCR specifically responding to ApoB auto-peptides. These cells respond when the corresponding peptides are presented by MHC-II molecules by various APCs. Indeed, we directly demonstrated the existence of such ApoB-reactive T cells by MHC-II tetramers loaded with the human and mouse auto-peptide ApoB_3036–3050. Using this tool, we showed that among all ApoB_3036–3050-reactive CD4⁺ T cells in patients free of cardiovascular disease, two thirds exclusively expressed FoxP3, indicative of a large population of ApoB-reactive T_regs. In patients with subclinical atherosclerosis, the percentage of exclusively FoxP3-positive T cells declined to ≈30%, whereas a substantial proportion of the remaining FoxP3⁺ T cells acquired simultaneous expression of ROR-γT (retinoic acid receptror-related orphan receptor-gammaT) or T-bet, the T_H17 and T_H1-defining transcription factors, respectively.⁴² These observations, along with the diminished pool of T_regs in later mouse and human disease, support the idea that the immunosuppressive phenotype of T_regs disappears as atherosclerosis progresses. Consistent with this hypothesis, T_regs in late atherosclerosis in mice simultaneously express T-bet, lose their ability to regulate and to protect from atherosclerosis, while retaining some phenotypic similarity with T_regs, such as some residual expression of FoxP3.^{36, 55, 62} Gaddis et al⁸⁸ recently proposed that FoxP3 expression may be lost in favor of the transcription factor Bcl-6 (B-cell lymphoma 6), the defining transcription factor for follicular-helper T cells. Adoptively transferred ApoB⁺ T-helper cells turned into T_FH cells after adoptive transfer.⁸⁹ In other autoimmune conditions, such as experimental autoimmune encephalitis and arthritis, an instability of FoxP3 expression triggers the formation of antigen-specific, but dysfunctional, partially nonprotective former T_regs (exT_regs).^115–117 The instability of FoxP3 may be caused by increased methylation of the FoxP3 locus, which is observed in patients with severe cardiovascular disease¹¹⁸ and that may be prevented by modifications of lipid metabolism or anticytokine interventions.^88,115 In addition, the function of FoxP3 may be regulated by alternative splicing favoring pathogenic transcriptional programs.¹¹⁹ These data suggest that the initial protective immune response by T_regs switches into a pathogenic response as atherosclerosis progresses³⁹ (Figure 3B and 3C).

Classically, 2 types of B cells can be distinguished: B1 cells that are part of the innate immune system and secrete germ-line encoded IgM antibodies in a T-cell–independent manner, and B2 cells that need to be activated by T_FH to differentiate into plasma cells that secrete IgG antibodies. In infection and vaccination against pathogens, B-cell–derived plasma cells secrete IgG antibodies that neutralize or opsonize bacteria and viruses.²⁹ In addition, B cells can secrete numerous cytokines that distinctly affect inflammation. Examples include IRA-B (immune response activator B) cells, which are proatherogenic and secrete GM-CSF (granulocyte-monocyte colony stimulating factor) to drive myeloid cell activation and to induce proatherogenic T_H1 immunity.¹²⁰ B-regulatory cells (B_reg) secrete the anti-inflammatory cytokine IL-10 and induce protective T_reg or directly act anti-inflammatory,¹²¹ although the relevance for atherosclerosis is controversial.¹²² The role of other cytokine-producing B-effector (Be) cells is unclear. Only a few B cells are found in the atherosclerotic plaque²⁴; the majority of B cells reside in the adventitia, in particular in aged atherosclerotic animals, where arterial tertiary lymphoid organs form.¹²³ B cells in the spleen respond to a high cholesterol diet,¹²⁴ suggesting local and systemic B-cell responses in atherosclerosis. Global gain- and loss-of-function experiments have suggested an overall protective role of B cells.^125,126 In general, innate B1 responses seem to be atheroprotective and adaptive B2 responses proatherogenic (Figure 4).

B1 cells represent a first-line, innate defense against common pathogens. In mice, they are characterized as CD11b⁺CD43⁺CD23⁻B220^lowCD19⁺ cells and may be subdivided into B1a and B1b cells depending on their location and surface markers.¹²⁷ Typically, most B1 cells reside in the peritoneal cavity. In the atherosclerotic plaque, a few CD11b⁺ B220_negCD19⁺ B1-like cells are found that further decrease in more advanced disease.²⁴ B1 cells secrete germ-line encoded IgM. Typically, B1-derived IgM recognizes phosphocholine head groups of polysaccharides in the wall of bacteria, such as Streptococcus pneumoniae. The same IgMs also bind oxidation-specific neo-epitopes on LDL and epitopes on apoptotic cells.^128–130 Oxidative neo-epitopes also seem to be generated in the spleen during sterile inflammation.¹³¹ In cardiovascular disease, IgM recognizing epitopes on LDL or ApoB are inversely correlated with atherosclerosis, complications, and outcome.^132–138 It has been shown that IgMs directed against oxLDL inhibit its uptake by macrophages and prevent myeloid-cell inflammation.^139,140 Consistently, several studies with gain- and loss-of-function experiments have established an atheroprotective role for B1 cells.^141–145

IgG antibodies originate from plasma cells that have undergone B-cell maturation with the help of T_FH cells in germinal centers, which causes a switch from low-affinity IgM to high-affinity IgG.¹⁴⁶ IgG antibody titers to native and oxidized LDL or ApoB are positively correlated with atherosclerotic disease in mice and humans.^{133,147–149} Inhibiting B2 cells is reportedly atheroprotective,^150–152 whereas specifically interfering with plasma cell functioning seems to be proatherogenic.¹⁵³ The role of IgG antibodies in atherosclerosis is controversial: it was suggested that IgGs against ApoB aggravate¹⁵⁴ or protect from atherosclerosis.^155,156 A clinical phase II study (GLACIER [Goal of Oxidized LDL and Activated Macrophage Inhibition by Exposure to a Recombinant Antibody]) using a monoclonal IgG antibody against a human ApoB peptide failed to show its expected atheroprotective effect.¹⁵⁷ The design of the study with the use of 8F-fluorodeoxyglucose PET (positron emission tomography) imaging as surrogate for plaque inflammation instead of cardiovascular end points, the short observation period of 85 days, and the small study population may have contributed to its lack of efficacy.

The discovery of the autoimmune component of atherosclerosis has sparked the idea of immunizing with LDL or peptides from ApoB to prevent atherosclerosis by inducing or maintaining the traits of protective immunity against ApoB. Almost 60 years ago, it was shown that rabbits develop smaller atherosclerotic lesions after subcutaneous injection of LDL.³⁸ That vaccination with LDL can be atheroprotective was confirmed in a variety of species, LDL preparations, routes, and adjuvants.^158–160 At least 7 MHC-II–restricted peptides from ApoB, which contains the immunodominant epitopes of LDL, are protecting from atherosclerosis when used in vaccines: p3, p6, p101, p102, p103, p18, and p210.^42,161–163 An ongoing challenge is to decipher the mechanism of action, which is critically required to define vaccination protocols translatable to humans. It has been proposed that either T_regs,^42,164–167 IL-10,^{41,42,161,167} or vaccination-induced IgG antibodies may confer atheroprotection, depending on doses, routes, and adjuvants used.⁴⁴ Recent studies, however, suggest that atheroprotection does not require IgG antibodies¹⁶⁸ and primarily proceeds by IL-10⁺ ApoB-specific T_regs.⁴²

Whether vaccination strategies can be translated to humans remains unclear. A first step toward a translatable approach was the identification of human ApoB peptide epitopes accessible to immunomodulation in 2 recent studies.^42,169 In mice, ApoB peptides have been delivered in the nontranslatable classical adjuvants Complete Freund’s Adjuvant, an emulsion of mineral oil supplemented with inactivated mycobacteria, or Incomplete Freund’s Adjuvant, which lacks the mycobacteria component of Complete Freund’s Adjuvant. Subcutaneous or intraperitoneal injections of both, Complete Freund’s Adjuvant and Incomplete Freund’s Adjuvant, were shown to elicit nonspecific inflammation.^170,171 This limitation was recently overcome by the discovery that a squalene oil, a class of adjuvants already used in clinical practice, can be used as an adjuvant for ApoB peptides.¹⁶⁸ In addition, it remains unclear whether vaccination is effective in established atherosclerosis as most studies tested the prevention of de novo atherosclerosis in rodents.

The principles of the cellular and humoral adaptive immune response in experimental murine atherosclerosis have been established. The efficacy of anti-inflammatory therapy in human atherosclerosis has been validated in the CANTOS trial (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study) recently. However, significant challenges remain for the translation of animal studies to humans. First, mice, which represent the most widely used atherosclerosis model, neither develop spontaneous atherosclerosis, nor do atherosclerotic knockout mice develop coronary artery disease. In addition, spontaneous atherothrombotic events resembling heart attacks and strokes do not occur in atherosclerotic mice. Also, blood lipoprotein profiles in mice are unlike those in humans, even in the genetic atherosclerosis models. Second, most atherosclerosis studies are conducted in a single mouse strain (C57BL/6) that cannot capture the genetic diversity seen in humans. Genetic diversity is known to modulate the response against antigens and atherosclerosis-relevant stimuli within a spectrum from pro- to anti-inflammatory.^172,173 Third, some cytokines and immune receptors are not conserved between mice and humans because the immune systems of both species are under intense evolutionary pressure. Fourth, mice represent a simplified model system for antigen presentation and recognition. Unlike humans, mice are housed in specific-pathogen free facilities, which neglects the likely pathogen-driven activation, antigenic repertoire, and differentiation of immune cells.¹⁷⁴ Although C57BL/6 mice bear just 1 MHC-II allele/molecule (I-A^b), humans express several alleles of a large pool of different MHC-II variants that are termed human leukocyte antigens with over 10 000 different human leukocyte antigen allelic forms. This extreme variability renders the direction and amplitude of autoimmunity in humans difficult to predict.

Decreasing LDL levels and attenuating the inflammatory response represent the 2 fundamental therapeutic strategies against atherosclerosis available today. The most successful causal medication as measured by event-free person-years is inhibitors of endogenous cholesterol synthesis by the HMG-CoA (β-hydroxy β-methylglutaryl-coenzyme A) reductase (statins),^175,176 which lower LDL cholesterol and have pleiotropic anti-inflammatory effects beyond what can be expected from the reduction of LDL.¹⁷⁷ Statins can prevent, reduce, and even reverse atherosclerotic plaque burden.¹⁷⁸ Monoclonal antibodies to PCSK9 lower LDL cholesterol even more dramatically by blocking LDL degradation^179,180 without apparent impact on levels of CRP levels,¹⁸¹ a biomarker of systemic inflammation. However, even after LDL lowering with statins and PCSK9 inhibition, a substantial residual inflammatory risk remains.¹⁸² These observations have established the distinct, but overlapping, roles of inflammation- and lipid-associated risk. Low-dose treatment with the antiproliferative and anti-inflammatory drug colchicine prevented cardiovascular events in a small prospective clinical trial.¹⁸³ In addition, the CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study) showed that inhibition of inflammation by the IL-1β antibody canakinumab reduced cardiovascular end points in patients with established atherosclerosis by 15%.¹⁸⁴ Strikingly, these observations have proven the inflammatory hypothesis on a conceptual basis, yet it is unclear, which patients may benefit from novel anti-inflammatory therapies: first, inhibition of IL-1β impaired host defense, which was reflected by an increased incidence of lethal infections.¹⁸⁴ Second, the recent CIRT (Cardiovascular Inflammation Reduction Trial) that tested low-dose anti-inflammatory methotrexate in patients with coronary heart disease did not reach its end points.¹⁸⁵ This lack of efficacy was partially explained by the inclusion of patients at low inflammatory risk and calls for a future personalized risk stratification (inflammatory versus lipid risk) and treatment once anti-inflammatory therapy is available in clinical practice. Whether the autoimmune component of atherosclerosis may already be addressable by unspecific anti-inflammatory therapy is currently unknown. However, vaccination and immunomodulation may provide a future antigen-specific therapy that is unlikely to impair host defense. The first validation of MHC-II tetramers to quantify the ApoB-reactive T-cell responses⁴² and the measurement of autoantibodies¹⁸⁶ in humans may provide feasible risk stratification tools in the challenge to define patients at a high immune risk for atherosclerosis in future.

Atherosclerosis is a chronic inflammatory disease of the vessel wall that is largely driven by an innate immune response through myeloid cells as monocytes and macrophages. Autoimmunity against ApoB and other antigens involves CD4⁺ T-helper cells that instruct myeloid cells and antigen-specific antibodies that may directly modify the pathogenicity of these antigens. This autoimmune response is detectable in humans and animal models with atherosclerosis. Although the classical perception is that autoimmunity is pathogenic per se, recent evidence suggests that ApoB-specific CD4⁺ T-helper cells are already detectable in subjects without clinical atherosclerosis, where many of them show atheroprotective features. As atherosclerosis progresses, the protective autoimmune response converts into a pathogenic one. It is unknown whether this switch in functionality represents a cause or a consequence of atherosclerosis and inflammation. It is clear that the adaptive immune system in atherosclerosis can be pro- or anti-inflammatory and thus pro- or anti-atherogenic. Manipulating the adaptive immune system by immunomodulatory strategies or vaccination is an attractive concept. Limitations in the predictive power of animal models and a lack of a full understanding of the role of autoantibodies, B, and T cells present formidable hurdles to clinical translation.