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REVIEW TOPIC OF THE WEEK

Adaptive Immunity Dysregulation inAcute Coronary SyndromesFrom Cellular and Molecular Basis to Clinical Implications

Davide Flego, PHD,a Giovanna Liuzzo, MD, PHD,a Cornelia M. Weyand, MD, PHD,b Filippo Crea, MDa

ABSTRACT

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Although the early outcome of acute coronary syndrome (ACS) has considerably improved in the last decade,

cardiovascular diseases still represent the main cause of morbidity and mortality worldwide. This is mainly because

recurrence of ACS eventually leads to the pandemics of heart failure and sudden cardiac death, thus calling for a reap-

praisal of the mechanisms responsible for coronary instability. This review discusses recent advances in our understanding

of how adaptive immunity contributes to the pathogenesis of ACS and the clinical implications that arise from these new

pathogenic concepts. (J Am Coll Cardiol 2016;68:2107–17) © 2016 by the American College of Cardiology Foundation.

I n patients with acute coronary syndrome (ACS)and systemic evidence of inflammation, thehigher frequency of activated T cells compared

with stable angina (SA) (1–4) suggests that the suddenchanges leading to coronary instability might berelated to mechanisms involving T-cell immunity. Inthis review, we discuss recent advances in our under-standing of how adaptive immunity contributes tothe pathogenesis of ACS.

HELPER T-CELL DYSREGULATION IN ACS

Helper T cells (CD4þ lymphocytes) are the key regu-lators of adaptive immunity. After T-cell receptor(TCR) activation by antigen-presenting cells (APCs),T cells differentiate into functionally polarized helperT cells, classified according to cytokine production,surface markers, and the expression of lineage-specifying transcription factors. The cytokine envi-ronment controls the induction of different T-cellsubsets by the amount of antigen and by TCR-mediated signal strength (5,6). The principal helperT-cell subsets are the TH1, TH2, TH17, and follicularhelper (TFH) CD4þ T cells. Moreover, a subpopulation

m the aInstitute of Cardiology, Catholic University, Rome, Italy; and the bD

iversity, Stanford, California. The authors have reported that they have n

disclose.

nuscript received July 14, 2016; revised manuscript received August 9, 20

of TH1 cells, characterized by defective cell surfaceexpression of the costimulatory molecule CD28, isexpanded in different chronic inflammatory condi-tions (7). Emerging subsets of helper T cells are TH22(characterized by interleukin [IL]-22 production) andTH9 (producing IL-9) cells (6), but their role in ACS isstill uncertain.

Patients with ACS have skewed T-cell differentia-tion, oriented toward aggressive effector phenotypesand defective regulatory T cells (Tregs), the lympho-cyte compartment able to suppress the excessiveimmune response (Table 1). Overall, such T-cell ab-normalities characterize about one-half of patientswith ACS (8). In this subset of patients with ACS,helper T-cell dysregulation might affect the biologicaloutcome of the immune response and contribute toplaque destabilization through multiple damagingpathways (Figure 1).

TH1 CELLS. TH1 cells are characterized by the produc-tion of interferon-gamma (IFN-g) and by the expres-sion of the transcription factor T-bet. They contributeto tissue destruction and participate in the patho-physiology of autoimmune diseases, such as

ivision of Immunology and Rheumatology, Stanford

o relationships relevant to the contents of this paper

16, accepted August 16, 2016.

ABBR EV I A T I ON S

AND ACRONYMS

ACS = acute coronary

syndrome

APC = antigen-presenting cell

CREB = cyclic adenosine

monophosphate response

element-binding protein

IFN = interferon

Ig = immunoglobulin

IL = interleukin

ITIM = immunoreceptor

tyrosine-based inhibition motif

LDL = low-density lipoprotein

PTPN22 = protein tyrosine

phosphatase nonreceptor

type 22

RA = rheumatoid arthritis

SA = stable angina

SLE = systemic lupus

erythematosus

TCR = T-cell receptor

TNF = tumor necrosis factor

Treg = regulatory T-cell

Zap70 = zeta-chain–associated

protein kinase of 70 kDa

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rheumatoid arthritis (RA) and inflammatoryvascular diseases (6). The importance of TH1and IFN-g in atherosclerosis progression iswell described in animal models (2), whereastheir role in plaque destabilization is sug-gested by numerous studies, which show amarked increase in TH1 frequency andincreased expression of TH1-related effectors,such as IFN-g, signal transducer and activatorof transcription-4, and T-bet in patients withACS (9–13). IFN-g can contribute to plaquedestabilization in several ways: by therecruitment and activation of macrophages inthe atherosclerotic lesions; by reducingcollagen synthesis; by increasing the produc-tion of extracellular matrix-degrading pro-teins; and by activating APCs (14).Furthermore, the increased IFN-g expressioninduces a positive feed-forward loop of TH1induction, keeping a proinflammatory state.

When activated in the intima, TH1 cellsproduce proinflammatory cytokines andenhance the expression of CD40 ligand.Ligation of CD40 on APCs by CD40 ligandinduces release of extracellular matrix-degrading metalloproteinases and the

expression of tissue factor, the key initiator of thecoagulation cascade, which has been well documentedin experimental models (14).

CD4þCD28null T cells are distinct from classic helperT cells in several respects. This terminally differenti-ated subpopulation shows an increased resistance toapoptosis and a wide range of proinflammatoryproperties (7). The proinflammatory functions ofCD4þCD28null T cells include the production of highlevels of IFN-g, tumor necrosis factor alpha (TNF-a),and IL-2 (9) to direct cytotoxic ability (15). Indeed,they express killer immunoglobulin (Ig)-like receptorsand directly kill endothelial cells in vitro by cytolyticenzymes, such as perforin, granzyme A, and granzymeB, which usually are present in killer T cells and nat-ural killer cells. Several studies showed the associa-tion of CD4þCD28null T cells with infections andchronic inflammatory diseases, particularly RA.CD4þCD28null T cells are present preferentially in un-stable ruptured atherosclerotic plaques (4), and theirfrequency significantly increases the risk of ACS(8,16), particularly in patients with diabetes (17).

In patients with ACS, CD4þCD28null T cells circu-lating in the peripheral blood and infiltrating theunstable atherosclerotic plaque show high levels ofthe costimulatory receptors OX40 and 4-1BB, whichmediate IFN-g and TNF-a production, and modulatecell degranulation (18), as well as increased resistance

to apoptosis (19). Overall, these molecular abnor-malities could enhance the inflammatory and cyto-toxic function of CD4þCD28null T cells and couldcontribute to atherosclerotic plaque destabilization.One study failed to replicate the differences inCD4þCD28null T cells from ACS and controls in pa-tients treated with high-dose statins (20).

TH17 CELLS. TH17 cells are characterized by theexpression of retinoid-related orphan receptor gt, themaster regulator transcription factor responsible forIL-17 production. This T-cell subset, implicated inseveral autoimmune disorders, is important in theimmune responses against fungi and extracellularbacteria (6). The precise role of IL-17 in atheroscle-rosis and ACS remains controversial, as discussed indetail in a recent editorial (21). On the one hand,experimental studies in mouse models have provideddirect evidence that IL-17 is predominantly proa-therogenic. On the other hand, these cells promoteprocollagen expression, which might reduce the riskof fibrous cap fissure (22).

Similarly, in the clinical setting, the TH17 subsetis expanded in patients with ACS (8,23), and theTH17/Treg balance might play a role in the develop-ment of inflammatory disorders, including athero-sclerosis, and autoimmune diseases; however, in acohort of patients with ACS, lower serum levels of IL-17were associated with a higher risk of major cardiovas-cular events (24). The issue of whether TH17 cells areprotective or detrimental in ACS remains unsettled.

REGULATORY T CELLS. Tregs are a subset of CD4þ

T cells capable of suppressing the immune response.These lymphocytes are characterized by expression ofthe transcription factor Foxp3, by high levels of theIL-2 receptor CD25, and by down-regulation of thesurface molecule CD127 (6). Tregs induce immuno-suppression through multiple mechanisms, includingproduction of the anti-inflammatory cytokines IL-10and transforming growth factor-beta, direct cytol-ysis, and inhibition of dendritic cell maturation andfunction (6). In experimental atherosclerosis, the roleof Tregs is well appreciated. Indeed, this T-cell sub-population inhibits atherosclerosis development andprogression by suppressing effector T-cell responses(25). Recently published data have consistentlydemonstrated a defective Treg compartment in theperipheral blood of patients with ACS. Patients withACS show low levels of circulating Tregs, a reducedsuppressive efficiency of Tregs, and increased Tregsusceptibility to apoptosis (26–29).

B CELLS. The contribution of B cells in autoimmunityis well recognized, and their function has been evalu-ated in various animal models of atherosclerosis (30).

TABLE 1 Dysregulation of Helper T-Cell Subsets in Patients With Acute Coronary Syndrome

Helper T-CellSubpopulation Type of Dysregulation Study Populations Experimental Technique(s) Ref. #

TH1 Increased frequency* NSTEMI, STEMI, UA, SA, HC Flow cytometry (9–13)

Increased IFN-g production* NSTEMI, STEMI, UA, SA, HC Flow cytometry, ELISA,real-time PCR

(9–13)

Increased expression ofSTAT-4 and T-bet*

NSTEMI, STEMI, UA, SA, HC Flow cytometry, western blot,real-time PCR

(9,11)

CD28null Increased frequency* NSTEMI, UA, SA, HC Flow cytometry (8,16–18)

Increased cytotoxicity*† UA, STEMI, NSTEMI, SA Cytotoxicity assay, flow cytometry (15,18)

Increased resistance toapoptosis*†

NSTEMI, STEMI, SA Flow cytometry, proliferation andapoptosis assay

(19)

TH17 Increased frequency* NSTEMI, AMI, UA, SA, HC Flow cytometry (8,22,23)

Increased IL-17 production* NSTEMI, AMI, UA, SA, HC Flow cytometry, ELISA (22,23)

Increased STAT-3 phosphorylation* NSTEMI, AMI, UA, SA, HC Flow cytometry, western blot (23)

Treg Reduced frequency* NSTEMI, STEMI, AMI,UA, SA, HC

Flow cytometry, western blot, real-timePCR, multiplex technology

(8,26–29)

Reduced immunosuppressiveefficiency*†

NSTEMI, STEMI, SA, HC Functional suppression assays (26)

Increases susceptibility toapoptosis*†

NSTEMI, SA, CPS Apoptosis assay (29)

Reduced TCR-induced generation*† NSTEMI, SA, HC Stimulation assay, flow cytometry (36)

All evidence comes from observational studies; some are ex vivo, and others are in vitro. *Ex vivo observational study in cohorts. †In vitro study.

AMI ¼ acute myocardial infarction; CPS ¼ chest pain syndrome; ELISA ¼ enzyme-linked immunosorbent assay; HC ¼ healthy controls; IFN-g ¼ interferon gamma; IL ¼interleukin; NSTEMI ¼ non–ST-segment elevation myocardial infarction; PCR ¼ polymerase chain reaction; SA ¼ stable angina; STAT ¼ signal transducer and activator oftranscription; STEMI ¼ ST-segment elevation myocardial infarction; TCR ¼ T-cell receptor; UA¼ unstable angina.

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B1a cells and their secretion of IgM seem tomediate the protective effects of B cells, whereasB2 cells are proatherogenic through modulation ofT-cell–dependent mechanisms. Consistent with thishypothesis, the lack of B cell-activating factor re-ceptor, which is critical for maintaining mature B2cells, substantially mitigates atherosclerosis in low-density lipoprotein (LDL) receptor-deficient (LDLr–/–)mice by altering mature B2-cell–dependent cellularand humoral immune responses while preserving theB1a cell subtype and production of natural IgM anti-bodies (31). The discovery of a new B-cell subtype,the regulatory B cell, which secretes IL-10 andtransforming growth factor beta, and affects Tregdevelopment, has revealed that inhibition of anti-inflammatory activity is another mechanism bywhich B cells can influence autoimmunity andthereby atherosclerosis. Moreover, antibodies againstseveral oxidation-specific epitopes of LDL and otheratherosclerosis-related antigens are found in animalsand humans, both in the circulation and in athero-sclerotic plaques, and are also found in patientswith ACS (32). Although the mechanisms by whichthese antibodies act remain unclear, many experi-mental atherosclerosis studies have shown thatvaccination is protective, and that the protective ef-fects of vaccination can be mediated by the inductionof protective Treg responses and protective anti-bodies (33).

TCR SIGNALING ALTERATIONS IN ACS

The first step in lymphocyte activation is TCR bindingwith specific peptides presented by APCs. TCR trig-gering initiates a cascade of phosphorylation events,culminating in the activation and nuclear trans-location of transcription factors. A complex molecularcoordination is required, including positive andnegative feedback loops necessary to avoid lympho-cyte hyperreactivity and the breaking of immunetolerance. The proper tuning of TCR signaling is alsocritical for T-cell differentiation. Indeed, in additionto the cytokine environment, the induction ofdifferent T-helper cell lineages is driven by TCR-mediated signal strength (5). In patients with ACS,several abnormalities of TCR signaling have beenidentified (Table 2, Figure 2).

CD4þ T cells from patients with ACS show anenhanced response to TCR stimulation (34–36) andhave a lower setting of the T-cell activation threshold,attributable to enhanced amplification of proximalTCR-mediated signals. CD4þ T cells show increasedaccumulation of CD3 complexes and zeta-chain–associated protein kinase of 70 kDa (Zap70) in theimmunologic synapse, higher early tyrosine phos-phorylation after TCR stimulation, and defectivedeactivation of the lymphocyte-specific protein tyro-sine kinase (34–36). These abnormalities involving theupstream TCR signaling pathway strongly contribute

FIGURE 1 Helper T-Cell Dysregulation in ACS

Although no direct evidence exists for some of the pathways proposed, it could be hypothesized that the proinflammatory environment in

patients with acute coronary syndrome (ACS), because of the skewed CD4þ T-cell differentiation oriented toward an aggressive phenotype,

may induce a positive feed-forward loop, causing inflammation and local T-cell recruitment that could damage atherosclerotic plaque through

multiple pathways. APC ¼ antigen-presenting cell; EC ¼ endothelial cell; IFN-g ¼ interferon gamma; IL ¼ interleukin; SMC ¼ smooth muscle

cell; TGF-b ¼ transforming growth factor beta; TNF-a ¼ tumor necrosis factor alpha; Treg ¼ regulatory T cell.

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to the higher cytotoxic ability of helper T cells in pa-tients with ACS. Indeed, CD4þ T cells from patientswith ACS are able to kill endothelial and vascularsmooth muscle cells in vitro (15,34).

Recent studies suggest a defect in the protectivesignals necessary for appropriate T-cell activation.Indeed, CD4þ T cells from patients with ACS exhibitreduced phosphorylation of Zap70 at its inhibitoryresidue Tyr-292 (36). This residue is implicated inTCR/CD3 complex internalization, immunologic syn-apse formation, and down-modulation of the prox-imal events in TCR signaling. Patients with ACS alsoshow enhanced expression of the protein tyrosinephosphatase nonreceptor type 22 (PTPN22). Thisenzyme plays a key role in controlling the intensity ofearly TCR signal transduction acting on lymphocyte-specific protein tyrosine kinase and Zap70 (37).Although PTPN22 does not bind Tyr-292 of Zap70directly, PTPN22 may be brought to phospho-Tyr-292through binding of the SH2 domain of the c-Cblubiquitin ligase complex. Thus, the reduced Zap70

Tyr-292 phosphorylation might be indirectly due toincreased expression of PTPN22 by a c-Cbl–mediatedmechanism. Defective regulation of TCR signalingand increased PTPN22 expression persist in patientswith ACS 1 year after the index event, during a stablephase of the disease, suggesting persistent lympho-cyte hyperreactivity (36).

CD31 is a member of the Ig superfamily of celladhesion molecules expressed on most cells of thehematopoietic lineage, including platelets, mono-cytes, neutrophils, and a small population of T cells.It plays an important role in the inflammatoryresponse through modulation of leukocyte activa-tion, cytokine production, and maintenance ofvascular barrier integrity. CD31 is involved in TCR-signaling immunomodulation by reducing Zap70phosphorylation through the action of protein tyro-sine phosphatases (38). Accordingly, loss of Ig-likedomains 1 to 5 of CD31 prevents homophilic bindinginteractions and consequent activation of the CD31immunoreceptor tyrosine-based inhibition motif

TABLE 2 TCR Signaling Alteration in Patients With ACS

TCR Molecular Pathway Type of Alteration Study Populations Experimental Technique(s) Ref. #

CD3 Increased accumulation in IS* ACS, CAD, HC Confocal microscopy (34,35,41)

Intracellular [Ca2þ]signals

Enhanced* ACS, CAD, HC Confocal microscopy (34,35)

Phosphotyrosinelevels

Enhanced* ACS, SA, NSTEMI,CAD, HC

Pflow experiments, confocalmicroscopy

(35,36)

Zap70 Increased accumulation in IS*Increased phosphorylation of Tyr-319*Reduced phosphorylation of Tyr-292*

ACS, SA, NSTEMI,CAD, HC

Pflow experiments, confocalmicroscopy, ELISA

(34–36,41)

LCK Insufficient deactivation* ACS, CAD, HC Pflow experiments, confocalmicroscopy

(35)

MAPKs Increased p38 activation in naiveT cells*

Reduced ERK activation in memoryT cells*

NSTEMI, SA, HC Pflow experiments (41)

STATs Increased phosphorylation of STAT-3*Reduced phosphorylation of STAT-5*

AMI, SA, HC Pflow experiments (23)

CREB Reduced phosphorylation duringthe acute phase*

Increased phosphorylation at 1-yrfollow-up*

NSTEMI, SA, HC Pflow experiments, immunofluorescencemicroscopy, ChIP assay

(36)

CD31 Reduced expression and signalingduring acute phase*†

NSTEMI, SA, HC Flow cytometry, Pflow experiments (41)

PTPN22 Increased expression*† NSTEMI, SA, HC Flow cytometry, real-time PCR (36)

All evidence comes from observational studies; some are in vitro, and others are ex vivo. *In vitro study. †Ex vivo observational study in cohorts.

ACS¼ acute coronary syndrome; CAD ¼ coronary artery disease; ChIP ¼ chromatin immunoprecipitation; CREB ¼ cyclic adenosine monophosphate response element-bindingprotein; ERK ¼ extracellular signal-related kinase; IS ¼ immunologic synapse; LCK ¼ lymphocyte-specific protein tyrosine kinase; MAPK ¼ mitogen-activated protein kinase;Pflow ¼ phosphoflow cytometry; PTPN22 ¼ protein tyrosine phosphatase nonreceptor type 22; STAT ¼ signal transducer and activator of transcription; Zap70 ¼ zeta-chain–associated protein kinase of 70 kDa; other abbreviations as in Table 1.

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(ITIM)/SHP2 inhibitory pathway, finally leading touncontrolled T-cell activation. CD31 also has beenimplicated in the development of atherosclerosis inanimal models (39) and its clinical complications inhumans (40). Patients with ACS exhibit a transientreduction of CD31 expression and function on CD4þ

T cells (41). In 1 study, the expression and function ofCD31 on CD4þ T-cell subsets was higher in patientswith SA compared with controls and in patients withACS during the acute phase of the disease. Of note,after 1 year of follow-up, CD31 expression and func-tion recovered in ACS, becoming similar to thatobserved in patients with SA and higher than that inhealthy controls. A similar pattern has been observedin the CD14þCD16þ monocyte subset (42). Takentogether, these data suggest an enhanced CD31-mediated protective mechanism operating in pa-tients with SA, which is transiently down-regulatedin patients with ACS during the acute phase of thedisease. Thus, CD31 might have a role in containingthe immune response in patients with SA. In contrast,in patients with ACS, the protective function of CD31is reduced during the acute phase of the disease, thusleading to the uncontrolled lymphocyte activationtypically observed in a subset of patients with ACS.

A critical downstream molecule activated by TCRtriggering is the cyclic adenosine monophosphate

response element-binding protein (CREB), a tran-scription factor believed to be particularly importantfor the generation and maintenance of Tregs and forthe production of IL-2 and IL-10. CD4þ T cells of pa-tients with ACS show reduced activation of CREBduring the acute phase of the disease compared withpatients having SA and controls, which improvesduring follow-up (36). The observed transientexpression of CD31 during ACS might explain thereduced activation of CREB. Indeed, the CD31-ITIM-SH2 phosphatase signaling pathway elicits the activ-ity of extracellular signal-related kinase, which is alsoinvolved in CREB activation. Thus, the transientlyreduced expression of CD31 in the acute setting ofACS might contribute to reduced CREB activation,although CREB phosphorylation is regulated byseveral stimuli in addition to TCR triggering.

Recently, the importance of the TCR signalstrength in the differentiation of helper T-cell subsetshas increasingly been defined (5). A lower setting ofthe T-cell activation threshold could affect the di-rection of T-cell differentiation, leading to the un-balanced expansion of the helper T-cell subsetobserved in patients with ACS. Indeed, strong TCRsignaling in the early stages of lymphocyte activationpromotes TH1 and TH17 differentiation and negativelyregulates Treg induction (5).

FIGURE 2 Schematic Outline of TCR Signaling Pathway Abnormalities Observed in ACS

Proper tuning of T-cell receptor (TCR) activation is critical for the functional regulation of helper T cells, including cytokine production,

lymphocyte differentiation, clonal expansion, and cytotoxic effector functions. The biochemical signal initiated by TCR triggering is dynamically

regulated by protein tyrosine kinases and phosphatases. Fine molecular coordination is required to maintain the feedback loop necessary to

avoid lymphocyte hyperreactivity and the breaking of immune tolerance. Patients with acute coronary syndrome (ACS) have excessive

amplification of proximal TCR-mediated signals and unbalanced downstream molecular phosphorylation. CREB ¼ cyclic adenosine mono-

phosphate response element-binding protein; LCK ¼ lymphocyte-specific protein tyrosine kinase; MAPK ¼ mitogen-activated protein kinase;

PTPN22 ¼ protein tyrosine phosphatase nonreceptor type 22; STAT ¼ signal transducer and activator of transcription; Zap70 ¼ zeta-chain–

associated protein kinase of 70 kDa; other abbreviations as in Figure 1.

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LESSONS FROM AUTOIMMUNE DISEASES

An increasing body of evidence supports the notionthat atherosclerosis shares surprising similaritieswith other inflammatory/autoimmune diseases.Chronic autoimmune disorders, such as systemic

lupus erythematosus (SLE) and RA, characterized bychronic relapsing-remitting systemic inflammation,are recognized as risk factors for accelerated athero-sclerosis. Therapies directed toward inflammatoryprocesses crucially reduce cardiovascular diseasemorbidity and mortality in patients with SLE and

FIGURE 3 Lessons From Autoimmune Diseases

Acute coronary syndrome (ACS) shares surprising similarities with inflammatory/autoimmune diseases, such as rheumatoid arthritis (RA),

characterized by chronic relapsing-remitting systemic inflammation. In particular, helper T-cell dysregulation and T-cell receptor (TCR)

signaling alterations characterize both ACS and RA. Treg ¼ regulatory T cell.

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those with RA (Figure 3). Furthermore, RA and otherinflammatory diseases are associated with increasedACS incidence and poorer outcomes (43).HELPER T-CELL DYSREGULATION. It is tempting tospeculate that CD4þCD28null T cells are likely candi-dates for driving the accelerated atherosclerosis pro-cess observed in autoimmune disorders. Indeed,patients with RA and high frequencies of CD4þCD28null

T cells have increased carotid artery intima-mediathickness and decreased flow-mediated vasodilationcompared with patients with RA in whom this T-cellsubset does not expand. The mechanisms involved inCD4þCD28null T-cell expansion remain poorly under-stood. It has been suggested that CD28 down-regulation is antigen driven (44). An alternative hy-pothesis implicates inflammatory cytokines inCD4þCD28null T-cell expansion, as CD4þCD28null T-cellclones from patients with RA down-regulated CD28transcription after TNF-a treatment in vitro (45).

A large body of evidence supports the concept thatan imbalance between Treg and TH17 cells is a crucialaspect in the pathogenesis of RA. Although oftencontradictory, most studies agree that there is anoverall depletion of Tregs and a parallel increase ofTH17 cells in the peripheral blood and target organsin RA patients. In addition, intrinsic cell abnormalitiesinvolving genetic and epigenetic modificationsmay explain the defective suppressive activity ofTregs in RA.

TCR SIGNALING ALTERATIONS. Abnormalities inproximal TCR signaling have been implicated inautoimmunity, malignancy, and immunodeficiency.T-cell responsiveness in type 1 diabetes, SLE, andRA has been found to be abnormal, with a multitudeof mechanisms involved, ranging from alterations inTCR/CD3 complex composition to excessive expres-sion of costimulatory receptors (46).

A key role in controlling the intensity of TCR acti-vation is played by protein phosphatases, signaltransducing enzymes that dephosphorylate cellularphosphoproteins. PTPN22 is an interesting enzymebecause genetic variants are associated with anincreased risk for several chronic inflammatory dis-eases; therefore, it is considered an importantnon-human leukocyte antigen autoimmunity gene.Interestingly, the R620W polymorphic variant ofPTPN22 has recently emerged as a major risk factorfor the development of several autoimmune diseases,including type 1 diabetes mellitus, RA, and SLE,suggesting that aberrant activity of this phosphatasecan perturb normal lymphocyte functions and ho-meostasis (37), although it has not been specificallyinvestigated in ACS.

The Ig-like immunomodulatory molecule CD31plays an important role in controlling the extent ofT-cell–mediated inflammation in several experi-mental models of immune-mediated disease, suchas experimental autoimmune encephalomyelitis

CENTRAL ILLUSTRATION Responses to Antigen Presentation Observed in Patients With ACS

Reducedregulatory T-cells

(reduced regulatoryimmunity)

Increasedeffector T-cells

(increased immuneactivation)

RegulatoryT-cells

EffectorT-cells

Normal T-cellreceptor activation

Highexpression

of CD31

Highexpressionof PTPN22

Highactivationof CREB

High T-cellreceptor activation

Highexpressionof PTPN22

Lowexpression

of CD31

Lowactivationof CREB

STABLE PHASE OF THE DISEASEACUTE CORONARY SYNDROME

Flego, D. et al. J Am Coll Cardiol. 2016;68(19):2107–17.

Patients with acute coronary syndrome (ACS) show a unique expression pattern of signaling molecules involved in T-cell activation and differentiation. During the

acute phase of the disease, patients with ACS show reduced expression of CD31 and increased expression of protein tyrosine phosphatase nonreceptor type 22 (PTPN22),

2 molecules that play a critical role in T-cell receptor signaling. Patients with ACS also have reduced activation of cyclic adenosine monophosphate response

element-binding protein (CREB), a transcription factor required for regulatory T-cell (Treg) generation and maintenance. After 1 year of follow-up, in a stable phase

of the disease, CD4þ T cells still show increased expression of PTPN22, which is partially counterbalanced by enhanced expression of CD31. Furthermore, at 1-year

follow-up, patients with ACS increased phosphorylation of CREB. Overall, these stable-phase signaling conditions may enhance the functionality of the Treg

compartment, dampening excessive immune activation.

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and collagen-induced arthritis. The progressionand severity of these experimental diseases areincreased in mice lacking CD31 (38). Moreover,CD31-deficient mice show accelerated allograft andtumor rejection. Furthermore, loss of CD31 expres-sion is associated with reduced immunosuppressiveactivity of naturally occurring Tregs, although themolecular mechanism underlying this effect is un-clear (38).

KNOWLEDGE GAPS

The first knowledge gap concerns the associationbetween adaptive immunity alterations in patients

with ACS compared with those with SA, healthycontrols, and patients with coronary thrombosis/myocardial ischemia necrosis. Are the former thecause of the latter, or vice versa? Obviously we donot have an answer. It is tempting to speculate thatalterations persisting after clinical stabilization, suchas enhanced expression of PTPN22, might have acausal role, whereas transient alterations, such asreduced expression of CD31, can be interpreted bothways. In this case, only Mendelian randomization orintervention trials can clarify the direction ofcausality.

The second knowledge gap concerns the causes ofthe adaptive immunity alterations associated with

TABLE 3 Potential Therapeutic Targets Modulating TCR Signaling and

Immune Response in Acute Coronary Syndrome

Drugs Molecular Target and Mechanisms Ref. #

Azathioprine CD28 signaling and purine biosynthesisinhibition*

(51)

Cyclosporine andtacrolimus

NFAT and IL-2 inhibition, increases TGF-bexpression*

(51)

Sirolimus mTOR pathway inhibition* (51)

Imatinib mesylate Early TCR signaling inhibition† (52)

Anti-CD3 (OKT-3) Atherosclerosis inhibition by enhancingTreg responses‡

(25)

CD31-binding peptides CD31 signaling enhancement†‡ (53)

PTPN22 inhibitors PTPN22 signaling inhibition‡ (54)

3-Hydroxyanthranilic acid Atherosclerosis inhibition by regulatinglipid metabolism and T-cell–dependentinflammation‡

(56)

*Human in vivo. †Human in vitro. ‡Mice in vivo.

mTOR ¼ mammalian target of rapamycin; NFAT ¼ nuclear factor of activated T-cells;TGF-b ¼ transforming growth factor beta; Treg ¼ regulatory T-cell; other abbreviations as inTables 1 and 2.

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ACS. Are they caused by genetic alterations leadingto hyperreactive inflammatory cells, by antigenicstimuli, such as oxidized LDL, heat shock proteins,or infectious agents, or by a combination of both?The lessons learned from classic autoimmune dis-eases suggest that both mechanisms probablycooperate.

The third gap concerns the differences betweenACS and classical autoimmune diseases. Which al-terations of autoimmunity are specific to ACS? Theanswer to this critical question might allow theidentification of new diagnostic techniques andtreatments specific for ACS.

The fourth gap is the identification of the subset ofpatients in whom adaptive immunity alterations arelikely to play a causal role in ACS. It is increasinglyclear that the causes of ACS are multiple, and thatsystemic inflammation plays a role in a subset of pa-tients who probably are represented by those exhib-iting systemic evidence of inflammation and plaquerupture (1).

The fifth gap is the correlation between alteration ofadaptive immune markers in the plaque and in pe-ripheral blood. Although at 1 extreme, CD4þCD28null

T-cells are similarly expanded in the unstable plaqueand in peripheral blood (4), this might not be the casefor Treg and TH17 cells (47).

CLINICAL PERSPECTIVE

In addition to the underlying atherosclerotic processitself, which is addressed with cholesterol-loweringdrugs and blood pressure control, coronary throm-bosis is the final common pathway leading to ACS andour main current therapeutic target. More potentantithrombotic regimens have recently been found toincrease the risk of major bleeding (48), whereas therate of death, MI, and recurrent ACS at 1-year follow-up is still very high, approaching about 25% (49).Furthermore, recurrent ACS determines a progressiveimpairment of myocardial function, fueling thechronic heart failure pandemic.

Despite the knowledge gaps highlighted in thepreceding section, the extensive studies reportedin this review strongly support the notion thatadaptive immunity should be seriously addressedin the management of ACS, as is the case in clas-sical autoimmune diseases. A change of focus fromsoluble markers of inflammation to markers ofadaptive immunity regulation, such as the mole-cules involved in TCR signaling, might prove to bemore rewarding in the management of patientspresenting with ACS, although this approach iscurrently limited by the lack of testing for these

biomarkers available at the point of care (CentralIllustration).

Another unmet need is a specific anti-inflamma-tory treatment (50). Expanding pathogenic conceptsin ACS beyond the simplified view of chronicinflammation considerably widens the spectrum oftherapeutic possibilities and enables a more tailoredtherapeutic approach that may avoid unwantedadverse effects of aggressive immunosuppression.Indeed, TCR activation signaling offers several tar-gets for strongly immunosuppressive therapy, such ascyclosporin, tacrolimus, sirolimus, azathioprine, andimatinib mesylate. These drugs prevent T-cell acti-vation, and reduce the hyper-reactivity of the adap-tive immune system and the development ofautoimmunity (51,52) (Table 3). Moreover, a syntheticCD31-derived peptide, able to engage a truncatedextracellular CD31 fragment expressed by T cells thatapparently lack CD31, was recently shown to have animmunosuppressive effect in vivo through restora-tion of the CD31 inhibitory pathway (53). Moreover,the development of a novel series of PTPN22 in-hibitors might contribute to a better understandingits role in immune dysregulation in ACS patients (54).

Another intervention target might be restoring thebalance between effector T-cells and Tregs. Isolationand ex vivo expansion of Tregs might restore thedefective expression and function of this T-cell sub-set in ACS patients (55). Indeed, in mice, CD3 anti-body treatment induced rapid regression ofestablished atherosclerosis via reducing CD4þ T-cellsand increasing the proportion of Tregs. The trypto-phan metabolite 3-hydroxyanthranilic acid wasrecently shown to have immune regulatory properties

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that can be used to decrease atherosclerosis in miceby regulating T-cell dependent inflammation andlowering plasma lipids (56).

Finally, the identification of antigens that triggeradaptive immunity may open the way for specificvaccinations. Of note, the demonstration that vacci-nation against influenza might be associated with a

reduction in cardiovascular events is the firstexample of this approach (57).

REPRINT REQUESTS AND CORRESPONDENCE: Dr.Giovanna Liuzzo, Institute of Cardiology, CatholicUniversity, Largo A. Gemelli, 8, 00168 Rome, Italy.E-mail: giovanna.liuzzo@gmail.com.

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KEY WORDS outcome, pathogenesis,signaling, treatment