How co-stimulatory and co-inhibitory pathways shape atherosclerosis

Abstract

Objective

Immune cells play a critical role in atherosclerosis. Co-stimulatory and co-inhibitory molecules of the tumor necrosis factor receptor and CD28 immunoglobulin superfamilies shape T cell and B cell responses, but also have a major effect on antigen presenting cells and non-immune cells.

Approach and Results

Pharmacological inhibition or activation of co-stimulatory and co-inhibitory molecules as well as genetic deletion demonstrated their involvement in atherosclerosis. This review highlights recent advances in understanding how co-stimulatory and co-inhibitory pathways shape the immune response in atherosclerosis.

Conclusion

Insights gained from co-stimulatory and co-inhibitory molecule function in atherosclerosis may inform future therapeutic approaches.

Graphical Abstract

1. A brief introduction to atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the artery wall. The resulting clinical events are the leading cause of death worldwide.¹ The factors contributing to atherosclerosis are multifaceted, encompassing environmental and genetic risk factors, perturbed cholesterol homeostasis, and a lingering immune response that all influence atherogenesis, plaque progression, vascular dysfunction and ultimately plaque rupture or erosion, the proximal causes of major adverse cardiovascular events (MACE).^2–5 Dyslipidemia and endothelial dysfunction promote the increased influx and retention of lipoprotein particles including low-density lipoprotein (LDL).⁶ Adhesion molecules are expressed by activated endothelial cells (EC) preferentially at sites of disturbed blood flow.^{7, 8} Furthermore, the modification of retained lipoproteins (e.g. oxidation) can induce a low-grade immune response involving activation activating endothelial cells.^{5, 6} Adhering leukocytes, predominately inflammatory monocytes, transmigrate into the subendothelial space, and contribute to the pro-inflammatory micromilieu by secretion of chemokines that further increase recruitment of monocytes, neutrophils, and lymphocytes from the circulation.⁹ Some monocytes differentiate into macrophages that scavenge the trapped lipoprotein particles and transform into foam cells. In addition to their recruitment, macrophages can also undergo local proliferation. Yet, their egress may be prevented by retention signals, thus also contributing to growth of the atherosclerotic lesion.^{10, 11} As the capacity to clear or store lipids is exceeded in these cells, they can undergo apoptosis. When the uptake of apoptotic cells (efferocytosis) fails these cells undergo secondary necrosis and form the acellular necrotic core of atherosclerotic lesions. Retained lipoprotein particles such as LDL undergo oxidation and other modifications. This makes them ligands for scavenger receptors (SR) like CD36, SR-A, SR-B and toll-like receptors (TLRs), which activate various pro-inflammatory signaling pathways that induce co-stimulatory molecules. Modified LDL presents lipid neoepitopes, the role of which in atherogenesis is poorly understood.^{12, 13}

Dendritic cells (DC) are key players in bridging innate and adaptive immunity. Lymph node and spleen DCs are most capable in presenting antigen to naïve T cells. A network of vascular DCs is found in the arterial intima of healthy individuals and the frequency of DCs in arteries increases further during the course of atherosclerosis.^{6, 14} DCs can also take up lipids and contribute to foam cell formation.^{15, 16} Although foam cells seem not to leave the progressive atherosclerotic lesion, monocyte-derived DCs are able to leave atherosclerotic lesions in regression.¹⁷ Most monocyte-derived cells in atherosclerotic lesions express high levels of major histocompatibility complex class II (MHC-II), which is required for presentation of peptide antigens to CD4⁺ T cells. These mechanisms provide the basics for activation of T cells in atherosclerotic lesions and recall responses to both, model antigens and atherosclerosis antigens, which have been demonstrated in mouse arteries using multiphoton imaging.^{18, 19} B cells found in the adventitia also express MHC-II.^{20, 21} In general, B cells can process soluble and membrane-associated antigen after their antigenic activation via the B cell receptor. In vivo imaging demonstrated CCR7-dependent migration of antigen-specific B cells²² to the B cell T cell boundary in lymph nodes after antigen-encounter where these cells engaged in interactions with antigen-specific T cells for up to 60 min.²³ B cells are important antigen-presenting cells, but less able to present antigen to naïve T cells than DCs.²⁴ The reconstitution of μmt^−/− mice lacking B cells with B cells derived from MhcII^−/− mice resulted in an impaired antigen-specific T cell response, demonstrating that antigen presentation by B cells contributes to T cell activation.²⁵ The B cell specific deletion of MHC-II did not alter numbers of T cells, but reduced the frequency of activated CD4⁺ and CD8⁺ T cells in a mouse model of lupus which was accompanied by amelioration of disease and improved kidney function.²⁶ Similarly, mice were protected from experimental autoimmune encephalitis and displayed reduced Th1 and Th17 responses when B cells were devoid of MHC-II expression.²⁷ However, the role of antigen presentation by B cells in atherosclerosis is unknown.

Antibodies specific for plaque-restricted antigens such as oxLDL were detected in human atherosclerotic plaques.¹³ Further antigens detected by antibodies in atherosclerosis are HSP60 and HSP65.²⁸ Lipid peroxidation-derived neoepitopes are found on the surface of oxidized (ox)LDL.²⁹ Spectratyping analysis of the T cell receptor (TCR) repertoire in atherosclerotic lesions revealed a limited variety of TCRs, which is the hallmark of an oligoclonal T cell response.³⁰ Unfortunately, TCR spectratyping allows no conclusions about the nature of the antigenic epitopes. The presence of oligoclonal T cells in atherosclerotic lesions indicates the presence of an adaptive immune response mounted against atherosclerosis-relevant antigens. Such a response usually requires the migration of antigen presenting cells (APC) such as DCs carrying plaque-derived antigens to lymph nodes, although direct proof for such activity in atherosclerosis is still missing. DCs can foster the development of atherosclerosis by modulating the differentiation of effector T cells.¹⁸ Furthermore, ex vivo aorta cultures demonstrated substantial interactions between antigen-loaded DC and antigen-specific T cells within the aortic lesion.¹⁹ Recent evidence suggests peptide moieties of apolipoprotein B (ApoB)-100 as major atherosclerosis-specific antigens that are MHC-II restricted.^31–33 To produce an effective immune response, CD4⁺ T cells must recognize antigens in the presence of co-stimulatory signals, which are the subject of this review.

2. CD4⁺ T cells in atherosclerosis

Most lymphocytes in murine atherosclerotic lesions are CD4⁺ T cells, whereas human atherosclerotic lesions have equal numbers of CD4⁺ and CD8⁺ T cells.^{34, 35} Many lesional CD4⁺ T cells secrete interferon gamma (IFNγ), and mice deficient for the Th1 lineage transcription factor T-bet or the Th1 cytokine IFNγ display reduced atherosclerotic development.^{36, 37} Many of these cells express both T-bet and low levels of the transcription factor Foxp3, suggesting that these cells may derive from regulatory T cells (Tregs).^{38, 39} The contribution of Th2 cells in atherogenesis is unclear. The key cytokines produced by Th2 cells are IL-4, IL-5, IL-10, and IL-13, all of which can contribute to atheroprotection.^{40, 41} However, depending on the atherosclerotic mouse model studied, IL-4 either enhanced or decreased atherosclerotic lesion formation.^{40, 42} Similarly, the role of Th17 cells in atherosclerosis is controversial, as some studies ascribe Th17 cells a pro-atherogenic function whereas others suggest an athero-protective role for IL-17, the hallmark cytokine of Th17 cells.^43–46

Tregs, a subset of CD4⁺ T cells, suppress the proliferation of effector CD4⁺ T cells in response to antigen presentation. Tregs are subdivided into natural Tregs (nTregs) and induced Tregs (iTregs). nTregs are generated in the thymus by selection against self-antigens if the signal strength provided by the T cell receptor is low to intermediate^{47, 48}, whereas iTregs are generated in response to transforming growth factor beta (TGFβ) in the periphery.⁴⁹ Tregs are potent anti-atherogenic cells and exert their function by multiple effector mechanisms⁵⁰, most prominently by secretion of the anti-inflammatory cytokines IL-10 and TGFβ. Deficiency of these cytokines either systemically or in T cells was shown to be pro-atherogenic.^{51, 52} The adoptive transfer of Tregs ameliorated atherosclerosis⁵³ whereas depletion of Tregs exacerbated atherosclerosis.⁵⁴ However, this was accompanied by altered liver lipid metabolism, which makes the data difficult to interpret.⁵⁴ In vitro, both differentiation of Th17 cells and Tregs from naïve T cells requires the cytokine TGFβ. Atherosclerotic lesions of hyperlipidemic Ldlr^−/− mice demonstrated a significant loss of Tregs at advanced stages of atherosclerosis.⁵⁵ Additionally, emerging data suggest that Tregs may convert to Th17 cells in mice and humans as atherosclerosis progresses.^{38, 39, 56–58} Tregs in atherosclerotic lesions also can acquire a Th1-like phenotype associated with IFNγ production and loss of suppressive capacity.^{31, 32} However, more work is needed to provide conclusive evidence and underlying mechanisms of Treg plasticity in atherosclerosis.

3. Co-stimulatory pathways in atherosclerosis

A functional T cell response requires not only recognition of MHC:antigen complexes via the antigen-specific TCR but also the integration of co-stimulatory signals exceeding a certain threshold.⁵⁹ These signals are important during different stages of a T cell response including clonal expansion, skewing towards T cell effector phenotypes and enhancing T cell survival in primary and secondary immune reactions. Two main families of co-stimulatory molecules are instrumental for these processes: the Ig-like CD28 family and the tumor necrosis factor receptor superfamily (TNFRSF). In general, the expression and functionality of co-stimulatory and co-inhibitory is conserved between mice and men. Important examples of functional differences between species will be given below.⁶⁰

3.1 CD28 – CD80/CD86

CD28 on naïve T cells binds to CD80 and CD86 on APCs. Although CD80 is constitutively expressed on many APCs, CD86 is strongly induced by inflammatory stimuli such as TLR agonists derived from pathogens (e.g. LPS).⁶¹ Interactions of CD28 with CD80/CD86 are essential to induce a T cell response including proliferation of effector cells and memory formation. Of note, almost all murine CD4⁺ and CD8⁺ T cells express CD28, whereas only 80% of human CD4⁺ T cells and 50% of CD8⁺ T cells express CD28.⁶² The absence of CD28 stimulation during an antigen-specific stimulus renders T cells anergic, leading to irreversible unresponsiveness to their cognate antigen.⁶³ The activation of T cells occurs physiologically only when the T cell receives a TCR and co-stimulatory signal. Superagonistic CD28 antibodies were thought to have beneficial effects and advanced to clinical trials, but they caused a severe cytokine storm in healthy volunteers, probably by the activation of tissue memory CD4⁺ T cells.⁶⁴ This cytotoxic effect was not apparent in rodents where a Treg response was favored after transient lymphocytosis.⁶⁵ The expression of CD80 and CD86 is increased on monocyte-derived DC of patients with cardiovascular disease.⁶⁶ Expression of CD80 and CD86 strongly correlates with lesional inflammation and plaque vulnerability.^{67, 68} Similarly, atherosclerotic lesions of hypercholesterolemic mice exhibited T cells, DC, and macrophages expressing CD28 and CD80/CD86, respectively.⁶⁹

Combined deficiency of CD80 and CD86 in atherosclerotic Ldlr^−/− mice reduced atherosclerotic burden, which was accompanied by decreased abundance of MHC-II-expressing APC in atherosclerotic lesions and a reduced Th1 response.⁶⁹ However, lethally-irradiated Ldlr^−/− mice transplanted with Cd28^−/− or Cd80^−/−Cd86^−/− bone marrow developed 2-fold larger lesions compared to control mice transplanted with wildtype bone marrow. This accelerated lesion development was likely caused by impaired Treg development and uncontrolled T cell effector response.⁵³ Another costimulatory molecule of the CD28 superfamily is CD83. DC in human atherosclerotic lesions express CD83 and the content of CD83⁺ mature DC increased significantly in unstable plaques as compared to stable ones.^{70, 71} CD83 regulates B cell activation and germinal center responses⁷² as well as CD4⁺ T cell development.^{73, 74} Although largely expressed by activated APCs, the costimulatory function of CD83 seems to be dispensable for murine T cell activation whilst CD83-stimulated human monocytes suppressed T cell responses.^{75, 76} However, the role and function of CD83 in cardiovascular disease is so far unidentified.

3.2 TNFSF and TNFRSF members

3.2.1 CD40 – CD40L

CD40 (TNFRSF5) expression was first discovered on APC, especially B cells.⁷⁷ However, a plethora of immune and non-immune cells expresses CD40 with different functions exerted upon activation by its ligand CD40L (CD154, TNFSF5).^{78, 79} The co-stimulatory CD40-CD40L axis is an important master regulator of immune processes. Among others, germinal center formation and especially immunoglobulin class switching in B cells is highly-dependent on CD40L expression by T cells. DCs receiving CD40L-mediated signals are more potent in antigen presentation and inducing a T cell response.^80–83. CD40 is mediating T cell memory formation and induces the expression of inflammatory cytokines (TNFα, IL-1α, IL-1β, IL-6, IL-8, and IL-12), chemokines (CCL2, CCL3, CCL5) and matrix-degrading enzymes by monocytes and macrophages.^84–88 CD40-CD40L interactions also drive the expression of co-stimulatory molecules such as CD80, CD86, and CD70. Moreover, CD40-CD40L promotes CD8⁺ T cell activation even without the need for CD4⁺ T cell help.^{84, 89}

A genetic polymorphism in the 5′ UTR of CD40 was enriched in a case-control study of Chinese patients with acute coronary syndrome and ischemic stroke.^{90, 91} Furthermore, the rs1535045-T allele of the CD40 locus positively correlated with cardiovascular disease and plasma cholesterol levels in a Chinese Han population.⁹²

Pharmacological inhibition of CD40-CD40L interactions or global deficiency reduced atherosclerotic burden accompanied by stable lesion formation in murine models of atherosclerosis.^93–96 CD40L expressed by activated thrombocytes fosters recruitment of monocytes to the inflamed endothelium and is important for platelet-leukocyte aggregate formation, which can contribute to atheroprogression.^{97, 98} Furthermore, platelet CD40L expression reduced the abundance of atheroprotective Tregs, further contributing to inflammation.⁹⁸ CD40 expression by platelets sustained atherosclerosis by increasing adhesion molecule expression of EC and subsequent increased adhesion of leukocytes.⁹⁹

Pharmacological inhibition of CD40-CD40L interactions is an attractive target, but clinical trials were discontinued because of severe thromboembolic complications as discussed below.

On a cautionary note, transplantation of bone marrow deficient for co-stimulatory or inflammatory molecules into Ldlr^−/− mice often yields results opposing studies using the corresponding compound deficient mice or respective blocking antibodies.^{53, 69, 93, 100, 101}

In particular, lesion size between Ldlr^−/− mice Ldlr^−/− Cd40^−/− mice was comparable¹⁰² whereas transplantation of Cd40^−/− bone marrow into Ldlr^−/− mice reduced lesion formation.¹⁰³ Apoe^−/− mice deficient for CD40 also harbored smaller lesions as compared to Apoe^−/− control mice which is likely based on defective tumor necrosis factor receptor-associated factor (TRAF)6 signaling.¹⁰³ The underlying cause for the observed discrepancies is not clear and might be based on the varying kinetics of atherosclerosis between the mouse models involved. Apoe^−/− mice develop spontaneous atherosclerosis whereas Ldlr^−/− mice need to receive a cholesterol-enriched diet. The comparison of mouse models of atherosclerosis is beyond the scope of this review and has been summarized elsewhere.^{104, 105}

3.2.2 CD27 – CD70

The co-stimulatory molecules CD27 (TNFRSF7) and CD70 (TNFSF7) play important roles during the establishment of long-term T cell immunity.¹⁰⁶ Naïve T cell express CD27, which is increased after TCR engagement with cognate peptide MHC complexes. CD27 is lost from the cell surface by proteolytic shedding, but long-lived central memory cells express CD27 again.¹⁰⁷ Apart from T cells, CD27 is found on NK cells, activated B cells, and hematopoietic stem cells.^{106, 108} CD70, the ligand for CD27, is expressed on T cells, B cells, and activated DC.¹⁰⁹ Whereas antigen-stimulated T cells and B cells transiently increase CD70 expression, APCs in the intestine and medullary thymic epithelial cells constitutively express CD70.¹¹⁰ The development of effector T cells appears independent of the presence of CD27 whereas thymic output of nTregs is reduced in Cd27^−/− mice.^{110, 111} CD27 and CD70 interactions play an important role in mounting fulminant CD4⁺ and CD8⁺ T cell responses including memory formation at effector and priming sites.^{110, 112}

Proper CD27/CD70 signaling is needed for B cell proliferation and plays an important role during the process of Ig synthesis.¹¹³ Insufficient CD70 triggering on B cells leads to an impaired germinal center formation, thereby affecting the humoral immune response.¹¹⁴ However, B cells from Cd27^−/− mice still undergo class switching and Ig maturation in aged mice, thus, other factors contribute and compensate for CD27 defects which are only present during early phases. In contrast, human CD27⁺ B cells produced a higher amount of Ig, IL-10, and displayed enhanced survival.^115–117 In accordance, humans carrying mutations in the CD27 gene suffer from a severe immunodeficiency characterized by hypogammaglobulinemia, dysregulated lymphoproliferation and increased susceptibility for infections with Epstein-Barr virus (EBV).^118–120 Co-stimulation of CD70 via CD27 induced B cell proliferation but impaired terminal differentiation and Ig secretion of human and murine B cells, although stimulation of CD70 via soluble CD27 resulted in increased Ig secretion.^{113, 121, 122} Thus, soluble and membrane bound CD27 interacting with CD70 exert species-specific effects and seem to contribute to a germinal center reaction in mice. In humans, CD27 promotes terminal B cell differentiation and CD70 might downregulate humoral immunity.

Mice deficient for CD27 demonstrated a reduction in the proliferative capacity of antigen-specific T cells which is not dependent on cell cycle entry.¹²³ CD27 acts anti-apoptotic in various manners, thus contributing to T cell survival and memory formation. T cells stimulated in vitro by CD27 signaling increase expression of the anti-apoptotic molecule B-cell lymphoma-extra large (Bcl-x_L).¹²⁴ Moreover, CD27 acts indirect on antigen-experienced CD8⁺ T cells as deficiency for CD27 reduced production of the autocrine growth factor IL-2, thus limiting survival and proliferation of all T cells in non-lymphoid tissue.¹²⁵ Additionally, the pharmacological inhibition of CD27/CD70 interactions with a blocking antibody increased FasL expression on CD4⁺ T cells which in turn induced apoptosis of virus-specific CD8⁺ T cells.¹²⁶ Early work addressing the role of CD27 and CD70 on atherosclerosis demonstrated an atheroprotective role for stimulation of CD70.¹²⁷ Chronic CD70 overexpression in B cells continuously induced CD27 signaling on T cells, leading to a predominant, presumably pro-atherogenic Th1 response. However, this was accompanied by increased rate of apoptosis among pro-atherogenic Ly6C⁺ monocytes, thus reducing macrophage abundance in atherosclerotic lesions. Furthermore, constitutive CD27/CD70 signaling led to immunopathology characterized by the conversion of naïve T cells into IFNγ-producing effector T cells leading to the progressive loss of B cells.^{127, 128} Thus, the model of B cell-restricted CD70 overexpression is of limited use to determine the precise roles of CD27 and CD70 in atherosclerosis.

Macrophages are the main lesional CD70-expressing cells.¹²⁹ Macrophages deficient for CD70 displayed a unique phenotype characterized by enhanced M1 and M2 marker expression, yet were less viable and incompetent in mounting a proper inflammatory response.¹²⁹ Furthermore, Cd70^−/− macrophages were less efficient in scavenging and cholesterol efflux, thereby contributing pathogenically to atherosclerosis progression. Accordingly, deficiency of CD70 resulted in exacerbated atherosclerosis.¹²⁹ Of note, CD70 and Apoe compound mutant mice were protected from hypertension and renal damage due to reduced accumulation of CD4⁺ and CD8⁺ effector memory T cells.¹³⁰

3.2.3. GITR – GITRL

Tregs express significant levels of GITR (TNFRSF18). Stimulation with its ligand (TNFSF18, GITRL) reduces their suppressive capacity.¹³¹ However, naïve T cells also express GITR at low levels, which is increased upon activation.¹³² Moreover, mast cells, APCs, NK cells, and granulocytes express GITR.¹³³ GITR expression on human immune cells is more restricted and expression has been described in Tregs, NK cells and macrophages.¹³⁴ The latter cells also demonstrated GITRL expression upon TLR signaling whereas antigen recognition drives GITRL expression in T cells.¹³³ Functional differences between species-specific GITR-GITRL interactions have been reviewed elsewhere.¹³⁴ Furthermore, EC express GITRL, which is enhanced upon LPS treatment, suggesting potential interaction of T cells and EC via this co-stimulatory axis during inflammation.^{135, 136} In human atherosclerotic lesions, plaque-resident macrophages demonstrate GITR and GITRL expression.¹³⁷ Interestingly, GITR stimulation of macrophages increased expression of matrix metalloproteinase (MMP)-9, which co-localized with GITR expression in atherosclerotic lesions, suggesting that GITR-GITRL interactions exert plaque destabilizing effects.¹³⁷ In mice, chronic GITR stimulation is atheroprotective.¹³⁸ Ldlr^−/− mice transplanted with bone marrow from B cell-restricted GITRL overexpressing mice enhanced thymic generation and lesional abundance of Tregs. Although deficiency for GITR is protective in murine models of asthma, experimental colitis, and collagen-induced arthritis, the exact role of GITR in atherosclerosis needs to be clarified.¹³⁹

3.2.4 OX40 – OX40L

OX40 (CD134/TNFRSRF4) and its ligand (OX40L/CD252/TNFSF4) are predominantly expressed on activated CD4⁺ and CD8⁺ T cells whereas naïve and resting memory T cells express neither OX40 nor OX40L.¹⁴⁰ Besides, other immune cells such as neutrophils and NK cells express OX40 constitutively whereas OX40L is inducible among others on APC, EC, and smooth muscle cells (SMC).¹⁴⁰ OX40 and OX40L expression is important for expansion of antigen-specific T cells to mount a functional T cell response and to form memory.^{141, 142} OX40 also functions as a negative regulator of Tregs, thus further increasing the pro-inflammatory response.¹⁴³ Additionally, OX40 interactions with OX40L drive B cell activation and immunoglobulin production and play a role in macrophage activation.^{144, 145} Plaque-resident macrophages express OX40L in mice and humans, whereas lesional T cells were positive for OX40.^{146, 147} Of note, a single nucleotide polymorphism of the OX40 gene in intron 5 was significantly associated with myocardial infarction.¹⁴⁸ Spontaneous mutations in the Tnfsf4 locus of healthy C57BL/6 mice are associated with susceptibility for atherosclerosis. ¹⁴⁶ The minor allele of the single nucleotid polymorphism rs3850641 of TNFSF4 is associated with an increased risk for women to develop myocardioal infarction.²⁹ However, another group could not confirm associations between theTNFSF4 genotype and an increased risk to develop carotid aretry disease or stroke.¹⁴⁷ Genetic disruption or pharmacological inhibition of this co-stimulatory dyad attenuated atherosclerotic development and even caused regression of established atherosclerotic lesions, including a reduced neovascularization of the vasa vasorum. ^{29,149, 150}

3.2.5 CD30 – CD30L

CD30 (TNFRSF8) and CD30L (TNFSF8, CD153) are expressed by activated T cells and B cells. Furthermore, mature DC, macrophages, and mast cells demonstrate CD30L expression.¹⁵¹ Co-stimulation via CD30-CD30L induces T cell proliferation and activation and is important for long-lived CD8⁺ T cell memory formation.¹⁵² The global absence of CD30 in mice reduced secondary humoral responses by an impaired induction of follicular germinal center responses accompanied by reduced antibody production.^{153, 154} Pharmacological CD30L blockage efficiently prevented development of spontaneous/type I diabetes mellitus in NOD mice.⁸⁰ Furthermore, CD30L blockage reduced atherosclerotic burden in Ldlr^−/− mice presumably by reducing overall T cell proliferation in the spleen and lymph nodes without affecting the humoral response.¹⁵⁵

3.2.6 4-1BB – 4-1BBL

The co-stimulatory molecule 4-1BB (TNFRSF9, CD137) is expressed on activated CD4⁺ T cells, CD8⁺ T cells, NK cells, resting monocytes, and DC whereas its ligand is predominantly expressed on APC.¹⁵⁶ Mice deficient for 4-1BB have less NK and NKT cells whereas T cell development is not affected.^{157, 158} Furthermore, 4-1BB deficiency increased the number of myeloid progenitor cells and mature DCs, yet reduced the survival of the latter cell type.^{159, 160} Of note, while 4-1BBL reduced human NK cell activity in co-cultures with tumor cell lines, it activated murine NK cells and led to enhanced killing activity.¹⁶¹ In addition, Tnfrsf9^−/− mice mounted a stronger antigen-specific T cell response although DC functionality is impaired.¹⁶² Presumably, these effects would contribute to progression of atherosclerosis. However, stimulation of 4-1BB with an agonistic antibody increased atherosclerotic burden accompanied by enhanced lesional inflammation whereas 4-1BB deficiency attenuates atherosclerosis in mice.^{163, 164} Advanced atherosclerotic lesions accumulated more macrophages and T cells when 4-1BB was lacking.¹⁶⁵ Lesions in these mice showed signs of vulnerability, accompanied by reduced SMC survival and collagen production.¹⁶⁵ Interestingly, macrophage glucose metabolism is regulated by the interaction of 4-1BBL with its receptor, leading to increased metabolic activity and cell proliferation.¹⁶⁶ Thus, intervention in 4-1BB-4-BBL interactions might display a valuable therapeutic option in macrophage-driven diseases such as atherosclerosis.

3.2.7 LIGHT – HEVM

HVEM (herpes virus entry mediator, TNFRSF14) is expressed by resting T cells and APCs whereas its main ligand, LIGHT (TNFSF14), is expressed by activated T cells, monocytes, DCs, and NK cells.^{167, 168} HVEM can also interact with lymphotoxin-α, BTLA (B and T lymphocyte attenuator), and CD160.¹⁶⁹ Interactions with LIGHT and lymphotoxin-α contribute to T cell activation and cytokine production whereas ligation of HVEM to BTLA and CD160 promotes co-inhibitory effects.¹⁶⁹ The contribution of HVEM and LIGHT to atherosclerosis is not fully understood. EC and macrophages in atherosclerotic lesions express HVEM and LIGHT and both transcripts were elevated in aortas of atherosclerotic Apoe^−/− mice.¹⁷⁰ HVEM signaling induced the production of MMP-1, -9, and -13 by monocytic cells in vitro and the staining of MMPs overlapped with HVEM in human atherosclerotic lesions suggesting that HVEM signaling contributes to plaque destabilization and rupture.¹⁷¹ Further pro-atherogenic features of this co-stimulatory axis involve the adhesion of platelets to EC and contribute to atheroprogression by guiding leukocyte adhesion to the inflamed endothelium.^{172, 173} Furthermore, LIGHT expressed by platelets induces signals in EC and monocytes that increase the expression of adhesion molecules and chemokines.¹⁷⁴ The increased expression of pro-atherogenic inflammatory mediators such as IL-8 and MCP-1 depends on a LIGHT-mediated induction of proteinase-activated receptor 2.¹⁷⁰ T cell-restricted overexpression of LIGHT induced hyperlipidemia by a substantial reduction of hepatic lipase expression.¹⁷⁵ In the liver hepatic lipase is surface expressed and promotes uptake of lipoproteins containing cholesterol and triglycerides, hydrolyzing the latter. However, the mechanisms underlying how T cell-mediated LIGHT expression alters liver metabolism is not understood.

4. Co-inhibitory pathways shaping atherosclerosis

4.1 PD-1 – PD-L1/PD-L2

The CD28-superfamily also includes the co-inhibitory molecule PD-1 (CD279) which binds to PD-L1 (CD274) and PD-L2 (CD273). Whereas PD-L1 expression is broadly found on APCs and tissue cells of non-hematopoietic origin, especially in the presence of innate inflammatory stimuli, PD-L2 expression is mainly restricted to APCs.¹⁷⁶ Furthermore, PD-L1 can be expressed in vitro by vascular SMC and vascular EC in vitro and in vivo.^177–179 Interestingly, in vitro incubation of vascular ECs with oxLDL increased PD-L1 expression. ¹⁸⁰ This led to a strong induction of anti-inflammatory cytokine production by co-cultured Tregs displaying a potential atherosclerosis counterbalancing mechanism.¹⁸⁰ Furthermore, hypercholesterolemia promoted PD-L1 expression on splenic macrophages and DCs of Ldlr^−/− mice.¹⁸¹ On the other hand, circulating T cells and myeloid DC from patients with coronary artery disease demonstrated reduced PD-1 and PD-L1 expression compared to healthy individuals.¹⁸² Deficiency of PD-1 or its ligands increased CD4⁺ and CD8⁺ T cell activation and their influx into atherosclerotic lesions which accelerated atherosclerosis in Ldlr^−/− mice.^{181, 183} Similarly, the administration of a PD-1 blocking antibody to Ldlr^−/− mice exacerbated atherosclerosis and lesional T cell infiltration.¹⁸³ The overall increased T cell activation in atherosclerotic Pd1^−/− mice did not favor a certain subtype, but enhanced abundance and response of pro- and anti-atherogenic subsets, suggesting that the pro-inflammatory compartment outcompetes immunosuppression by Tregs.¹⁸⁴ PD-1 and Tim-3 expression defines highly exhausted CD8⁺ T cells. In vitro re-stimulation of PD-1⁺Tim-3⁺ CD8⁺ T cells isolated from human atherosclerotic lesions demonstrated skewing towards an anti-inflammatory cytokine profile, which was reverted by applying PD-1- and Tim-3-blocking antibodies, suggesting that these particular CD8⁺ T cells in lesions are of regulatory nature whereas other reports attribute CD8⁺ T cells pro-atherogenic function.^185,149,150 Monoclonal antibodies to PD1 and PD-L1 and PD-L2 are now widely used in immunotherapy of cancer patients.¹⁸⁶ It should be considered that these treatments may increase cardiovascular risk.

4.2 CTLA-4 – CD80/CD86

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) competes with CD28 for binding to CD80 and CD86. However, CTLA-4 decreases immune responses and functions as an immune checkpoint regulator. The clinical targeting of this molecule in tumor malignancies is discussed below. Effector T cells increase surface abundance of CTLA-4 after activation whereas Tregs express CTLA-4 constitutively, which probably represents one of their main immunosuppressive effector mechanisms.¹⁸⁷ The impact of a genetic CTLA-4 deficiency on atherosclerosis is unknown since mice deficient for CTLA-4 succumb to an autoimmune lymphoproliferative disorder.¹⁸⁸ Application of CTLA-4 blocking antibodies resulted in a dramatic increase of atherosclerotic burden in hypercholesterolemic mice.¹⁸⁹ Conversely, the application of a CTLA-4-Ig fusion protein, which mimics CTLA-4 function, prevented CD80/CD86-CD28 interactions accompanied by reduced T cell activation. Such treatment also resulted in limited neointima formation and reduced homocysteine-accelerated atherosclerosis.^{189, 190} In line with these results, transgenic mice constitutively expressing CTLA-4 on T cells were protected from atherosclerosis.¹⁹¹ Similar to the effects of blocking PD1/PDL1/PDL2 in cancer patients, CTLA4 blockade may trigger pro-atherosclerotic effects.

5. Potential therapeutic applications in atherosclerosis

5.1 Targeting CD40/CD40L interactions

The modulation of CD40/CD40L interactions was clinically tested in a variety of chronic inflammatory diseases and cancer.⁷⁸ However, pharmacological interference with antagonistic or agonistic antibodies triggered severe side effects. Patients suffering from lupus glomerulonephritis experienced a marked reduction of hematuria when treated with a blocking anti-CD40L-antibody.¹⁵⁵ However, this trial was prematurely discontinued as anti-CD40L-antibody treatment caused thromboembolic events and myocardial infarctions, likely by the destabilization of platelet aggregates.^{192, 193} Also, agonistic or antagonistic anti-CD40 antibodies failed in diverse clinical disorders as the treatment was not efficient, or side effects including thrombocytopenia, neutropenia, and pleural effusion led to the discontinuation of trials.^194–196 An alternative therapeutic strategy harnesses interactions of CD40L with Mac-1, also known as CD11b/CD18 integrin, which is abundantly expressed of neutrophils, NK cells, monocytes, and macrophages.¹⁹⁷ The intraperitoneal application of a small peptide prevented interaction of CD40L with Mac-1 and reduced atherosclerotic burden in Ldlr^−/− mice, potentially by reducing leukocyte recruitment to the inflammatory site.¹⁹⁸ Disrupting CD40-Mac-1 interactions did not prevent functional CD40-CD40L interactions and spared thrombotic events.¹⁹⁸ A different therapeutic approach targets the interaction of CD40 with its downstream signaling interaction partner TRAF6. Ablation of CD40-TRAF6 interactions led to more stable and smaller atherosclerotic lesions, accompanied by reduced monocyte influx into the arterial wall.¹⁰³ Furthermore, a small compound designed to prevent CD40-TRAF6 interactions increased survival of mice with induced sepsis and improved insulin resistance in obese mice.^{199, 200} However, the efficacy of this compound in atherosclerosis has yet to be tested.

5.2 Anti-CD27/CD70 antibodies in cancer and cardiovascular implications

Hematologic malignancies and solid tumors feature high CD70 expression.^201–205 The constitutive activation of effector T cells by persistent antigen and constitutive signaling via CD27/CD70 interaction leads to exhaustion, demonstrated in patients suffering from B cell non-Hodgkin’s lymphoma.²⁰⁶ Exhausted T cells are less cytotoxic and incapable of attacking the tumor. The pharmacological inhibition of CD27/CD70 interaction by blocking-CD70 antibodies represents a promising therapeutic strategy in the treatment of such malignancies.^{207, 208} Furthermore, the opsonization of CD70 expressing tumor cells by these antibodies could induce antibody-dependent cellular cytotoxicity and phagocytosis, thus directly attacking the tumor cells and contributing to tumor regression. Alternatively, agonistic CD27 modulation by the CDX-1127 (varlilumab) antibody under clinical evaluation in patients suffering from B cell malignancies, melanoma, and renal cell carcinoma.^{209, 210} Such agonistic CD27 stimulation successfully re-activated exhausted effector T cells, leading to a profound anti-tumor immune response and tumor regression in mice with a transgene expressing human CD27.²¹¹ As CD70 deletion exacerbates atherosclerosis, potential side effects on the cardiovascular system by pharmacological modulation of CD27/CD70 interactions require consideration.¹²⁹

5.3 Anti-CTLA-4 and anti-PD-1 antibodies in oncotherapy and their potential cardiovascular effects

The co-inhibitory molecules CTLA-4 and PD-1 regulate T cell activation, however, in different ways. The blockage of both immune checkpoint mediators is desirable in advanced tumor malignancies, since cytotoxic anti-tumor T cells are reactivated and not suppressed anymore. Dual blockage with the monoclonal antibodies ipilimumab and nivolumab, targeting CTLA-4 and PD-1 respectively, has been tested in clinical trials.²¹² Recently, the Checkmate-67 phase III study demonstrated a 55% response rate when both antibodies were used to treat patients suffering from advanced melanoma.²¹³ Furthermore, patients receiving treatment with both antibodies showed a median progression free survival of almost 12 months, 2–4 times higher compared to treatment with one or the other antibody. Data on the overall survival of the dual-treated patients are not yet published. Although an overall enhanced anti-tumor T cell response is highly desirable, uncontrolled T cell activity could exacerbate atherosclerosis and cardiovascular disease. Indeed, as pointed out above, pharmacological blockage or deficiency of CTLA-4 or PD-1 increased atherosclerotic burden. Overall, it has to be considered whether a patient suffering from advanced tumor malignancies can gain expanded lifetime when undergoing immune checkpoint modulating therapy at the potential cost of accepting a higher risk for cardiovascular complications.

6. Open questions and future directions

Past research has identified co-stimulatory or co-inhibitory molecules as important modulators of immune response mediating effects on various cell types. Functional studies by genetic deletions or pharmacological manipulation have shown that these molecules significantly contribute to atherosclerosis. More research is needed to evaluate expression patterns of all these molecules on immune and non-immune cells in health and disease, especially during atherosclerosis progression. Additionally, not much is known about how immune cells integrate signals received by various co-stimulatory and co-inhibitory pathways. Understanding the integration of co-stimulatory pathways in chronic inflammatory conditions such as atherosclerosis will help to develop new, tailored therapeutic approaches. The clinical success of blocking PD1, PD-L1, PD-L2, and/or CTLA-4 in cancer support the general feasibility of manipulating co-inhibitory and co-stimulatory pathways. Whether such approaches will succeed in curbing major adverse cardiovascular events is the subject of ongoing and future studies.

Table 1.

Expression profile and effects of co-stimulatory molecules in atherosclerosis

Co-stimulatory Molecule		Expression on resting cells	Expression on activated cells	Effect on atherosclerosis
Protein	Gene
CD28	Cd28	Naïve T cells, Eosinophils, Basophils, Treg	T cells	Unclear: Increased atherosclerosis in Ldlr−/− mice transplanted with Cd28−/−, Cd80−/−, or Cd86−/− BM53 Reduced atherosclerosis in Cd80−/− Cd86−/− Ldlr−/− mice69
CD80	Cd80	++: APC	+++: APC
CD86	Cd86	+: APC	+++: APC
CTLA-4/CD152	Ctla4	Tregs	Tregs, effector T cells	Clear: CTLA-4 blocking antibodies189 increased atherosclerosis CTLA-4-Ig fusion protein190 reduced atherosclerosis
CD40	Tnfrsf5	+++: B cells, SMC; ++Platelets; +: Macrophages, Neutrophils, EC	+++: T cells, APCs, Platelets, Neutrophils, EC	Clear: Pharmacological inhibition94–96 or global deficiency93 reduced atherosclerosis
CD40L	Tnfsf5	++: B cells; +: Macrophages, DC, Neutrophils, EC, SMC	+++: T cells, B cells, Macrophages, Platelets; +: DC, EC, Neutrophils
CD27	Tnfrsf7	naive T cells, NK cells, murine HSC	human memory B cells, murine centroblasts, memory T cells	Unclear: CD70 deficiency129 and overexpression127 reduced atherosclerosis by limiting macrophage function or survival of Ly6Chi monocytes, respectively
CD70	Tnfsf7	MTEC, APC subset in lamina propria	APC
GITR	Tnfrsf18	+++: Treg; +: naive CD4 T cells, Mast cells, APC	++Macrophages	Clear: Chronic GITRL overexpression is atheroprotective by Treg expansion138
GITRL	Tnfsf18	+: EC	+++: EC, T cells, APC
OX40/CD134	Tnfrsf4	Neutrophils, NK cells	Effector CD4 and CD8 T cells	Clear: Genetic ablation150 or pharmacological inhibition149 attenuates atherosclerosis
OX40L/CD252	Tnfsf4	APC, EC, SMC	Effector CD4 and CD8 T cells, Macrophages
CD30	Tnfrsf8		Activated T and B cells	Insufficient data: Pharmacological CD30L blockage reduced atherosclerosis155
CD30L	Tnfsf8	B cells, MTEC	Activated T and B cells, DC, Macrophages, Mast cells, Granulocytes
4-1BB/CD137	Tnfrsf9	Monocytes, DC, B cells, FDC, NK cells, Granulocytes	Activated CD4 and CD8 T cells, EC	Clear: Agonistic 4-1BB stimulation163 increased atherosclerosis whereas genetic deficiency attenuated atherosclerosis164. Later stage lesions in 4-1BB KO mice show vulnerable lesions165
4-1BBL/CD137L	Tnfsf9		APC
HVEM/CD270	Tnfrsrf14	T cells, APC	EC, Macrophages	Insufficient data: Not fully understood, signaling induces pro-inflammatory mediator expression and altered liver metabolism170, 171
LIGHT/CD258	Tnfsf14	Monocytes, NK cells, DC	T cells, EC, Macrophages
PD-1/CD279	Pdcd1	Myeloid DC, pro-B cells, Treg	Myeloid DC, T cells	Clear: Genetic deficiency of Pd1 or Pd-l1/Pd-l2181 or pharmacological inhibition of Pd-1183 increased atherosclerosis in Ldlr−/− mice
PD-L1/CD274	Pdcd1lg1	+: vascular EC, vascular SMC	T cells, NK cells, Macrophages, Myeloid DC, B cell, Epithelial cells, and vascular EC
PD-L2/CD273	Pdcd1lg2	DC	DC

APC: Antigen Presenting Cell, BM: Bone Marrow, DC: Dendritic Cell, EC: Endothelial Cell, FDC: Follicular DC, HSC: Hematopoietic Stem Cell, SMC: Smooth Muscle Cell, NK cell: Natural Killer cell, +: mild expression, ++: intermediate expression, +++: strong expression

List Of Abbreviations

APC

Antigen Presenting Cell

ApoB

Apolipoprotein B

Bcl-X_L

B-cell lymphoma-extra large

BTLA

B and T Lymphocyte Attenuator

CTLA-4

Cytotoxic T-lymphocyte-associated Protein 4

DC

Dendritic Cell

EC

Endothelial Cell

GITR

Glucocorticoid-induced TNFR-related Protein

HVEM Herpes

Virus Entry Mediator

IFNγ

Interferon gamma

Ig

Immunoglobulin

iTreg

Induced Treg

LDL

Low-density Lipoprotein

LPS

Lipopolysaccharide

MACE

Major Adverse Cardiovascular Events

MHC

Major Histocompatibility Complex

nTreg

Natural Treg

oxLDL

oxidized LDL

SMC

Smooth Muscle Cell

SR

Scavenger Receptor

TCR

T Cell Receptor

TGFβ

Transforming Growth Factor beta

TLR

Toll-like Receptor

TNFRSF

Tumor Necrosis Receptor Superfamily

TNFSF

Tumor Necrosis Factor Superfamily

Treg

Regulatory T cell