Expansion of T-Cell Receptor ζdim Effector T Cells in Acute Coronary Syndromes
Objective— The T-cell receptor zeta (TCRζ)-chain is a master sensor and regulator of lymphocyte responses. Loss of TCRζ-chain expression has been documented during infectious and inflammatory diseases and defines a population of effector T cells (TCRζdim T cells) that migrate to inflamed tissues. We assessed the expression and functional correlates of circulating TCRζdim T cells in coronary artery disease.
Methods and Results— We examined the expression of TCRζ-chain by flow cytometry in 140 subjects. Increased peripheral blood CD4+ TCRζdim T cells were found in patients with acute coronary syndromes (ACS, n=66; median 5.3%, interquartile 2.6 to 9.1% of total CD4+ T cells; P<0.0001) compared to chronic stable angina (CSA, n=32; 1.6%; 1.0 to 4.1%) and controls (n=42; 1.5%; 0.5 to 2.9%). Such increase was significantly greater in ACS patients with elevated levels of C-reactive protein, and it persisted after the acute event. Moreover, TCRζdim cells were also more represented within CD8+ T cell, NK, and CD4+CD28null T cell subsets in ACS compared to CSA and controls. Finally, CD4+ and CD8+ TCRζdim T cells isolated from ACS displayed an enhanced transendothelial migratory capacity.
Conclusions— TCRζdim T cells, an effector T-cell subset with transendothelial migratory ability, are increased in ACS, and may be implicated in coronary instability.
Inflammation plays a key role in the pathogenesis of atherosclerosis with participation of both innate and adaptive immunity.1–3 Upregulation of proinflammatory cytokines is common in acute coronary syndromes (ACS).4 Elevated C-reactive protein (CRP) levels are associated with an adverse prognosis in patients with coronary artery disease (CAD), and healthy subjects.4 The activation of inflammatory pathways in ACS is not confined to coronary lesions but involves the activation of neutrophils, monocytes, and lymphocytes in peripheral blood.5–7
Most T cells found in human atherosclerotic lesions exhibit an effector or memory phenotype with a Th1 bias, and a predominance of CD4+ over CD8+ T cells.8 Different subsets of T cells may drive or regulate inflammation during evolution of the atherosclerotic process, as in other inflammatory diseases.9 An increased prevalence of specific circulating T cell subsets, such as CD4+CD28null T cells, was reported in patients with ACS, as well as in patients with rheumatoid arthritis.10–13
The TCRζ-chain (CD247 or CD3ζ) is a transmembrane protein with a small extracellular domain and an intracellular domain containing 3 immunoreceptor tyrosine-based activation motifs. The TCRζ-chain subunit associates with the TCR-CD3 complex as a homodimer, and couples antigen recognition by the TCR to downstream intracellular signal-transduction pathways through phosphorylation and recruitment of downstream proteins. The TCRζ-chain is also expressed in NK cells, where it is associated with the FcRγIII low affinity IgG receptor (CD16) and other activating receptors.14 TCRζ-chain is therefore a master regulator and sensor of innate as well as adaptive immune responses, and so it follows from this that aberrations in its expression or function should be expected to have profound effects on immune function.15
A subset of T cells expressing low levels of TCRζ-chain (hereafter TCRζdim T cells) has been described in association with infectious, malignant, and inflammatory diseases.14–18
Downregulation of the TCRζ-chain, known to occur after antigen engagement or in response to inflammatory stimuli, may represent an attempt to modulate the immune response.14 However, we previously reported that the TCRζdim T-cell subset, although refractory to TCR-induced proliferation,19 paradoxically displays features of antigen-experienced effector T cells.15 Cell surface markers and tetramer analysis revealed that TCRζdim T-cells are enriched for memory CD45RO+ cells, and for cells that had previously engaged antigen. Moreover, TCRζdim T cells produce high levels of interferon (IFN) γ and tumor necrosis factor (TNF) α, and low levels of interleukin (IL)-10.15 Finally, TCRζdim T-cells display effector functions, eg, they can activate monocytes via cell contact-dependent pathways, and preferentially accumulate in inflamed tissues such as the rheumatoid joint.15
In the present study we report that the frequencies of subsets of T cells and NK cells expressing low levels of TCRζ-chain are increased in patients with ACS when compared to patients with stable atherosclerotic lesions or to controls, particularly in patients with elevated levels of CRP, and remain elevated after the acute coronary event. TCRζdim T cells display an enhanced ability to migrate through activated endothelium in vitro. Our findings suggest the possibility that antigen experienced T cells, defined by the TCRζdim phenotype, may contribute to the inflammatory process that triggers coronary instability by accumulating in coronary plaques.
Institutional Ethics Committees approved the study, and informed consent was obtained from all participating subjects. Venous peripheral blood samples were obtained from all patients on admission to San Raffaele Scientific Institute. Three groups participated in the study. Control group comprised 42 individuals with negative history, clinical, and electrocardiographic signs of CAD. CSA group included 32 patients with effort angina (lasting more than 3 months and without previous history of unstable angina or myocardial infarction) with angiographic evidence of coronary artery stenosis (stenosis >50% diameter). ACS group comprised 66 patients with chest pain accompanied by ischemic electrocardiographic changes (ST-segment changes or T-wave inversions) or ST-segment elevation >2 mm in at least 2 consecutive leads associated to increase of troponin I. All samples were obtained on admission. Evidence of coronary artery stenosis/occlusion was documented by coronary angiography. Exclusion criteria included recent surgery, documented immune, infectious or neoplastic disease, immunosuppressive therapy, ACS attributable to intrastent thrombosis, or occlusion of arterious and venous by-pass grafts (confirmed after angiography). In 17 patients with ACS the levels of TCRζ-chain were also assessed after a 50-day median time (interquartile 39 to 83 days) from admission. Further analysis of TCRζ-chain expression was performed in CD8+ T and NK cells (14 control, 9 CSA and 22 ACS participating in the study) and in CD4+CD28null T cells (19 controls, 18 CSA and 23 ACS).
Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Hypaque (Becton Dickinson) density gradient centrifugation from anticoagulated venous blood samples, before flow cytometry. Alternatively, flow cytometry was performed on fresh whole blood samples. For confocal microscopy and cell culture studies, CD3+ or CD3+CD4+ cells were further enriched by Robosep using EasySep Negative selection Human CD3+ or CD3+CD4+ T cell enrichment kit and procedure (Voden).
TCRζ Expression Analysis
Surface staining of T cell subsets was performed by standard methods. For intracellular staining, cells were fixed with 2% formaldehyde and permeabilized in buffer containing 10 μg/mL saponin. The efficiency of permeabilization was determined by uptake of trypan blue (>99% in all experiments). Isotype-matched controls Abs were used to confirm expression specificity. The following Abs were purchased from Becton Dickinson: mouse IgG1 isotype control conjugated with PE (clone MOPC-21) or with fluorescein isothiocyanate (FITC) (cloneA85-1), anti-CD3-FITC (clone UCHT1), anti–CD4-PE-Cychrome 5 (clone RPA-T4), anti–CD8-Cychrome 5 (clone HIT8a), anti–CD3-PE-Cychrome 5 (clone HIT3a), anti–CD16-FITC (clone 3G8), anti–CD3-Pacific Blue (PB) (clone UCHT1), anti–FoxP3-PB (clone 206D). Two different antibodies were used for TCRζ, expression studies: anti–TCRζ-PE (clone 2H2D9, Immunotech, Coulter), anti–TCRζ-FITC (cloneG3,Dako). The 2 different clones used of anti-TCRζ individuate the same percentage of TCRζdim cells. The CD3−CD56+ NK cell subset was found to be uniformly TCRζ negative, and was not therefore studied further.
We confirmed data also with fresh whole blood analysis (cell viability >99%). Cell viability was assessed using the Molecular Probes Patented LIVE/DEAD Viability (Invitrogen) according to the manufacturer instructions. Moreover, frequencies of TCRζdim within the CD3+CD4+CD28null subset were determined by a 4-color flow cytometry on fresh whole blood performed using anti–CD3-Cascade Yellow (clone UCHT1, Dako), anti–CD4-activated protein C (APC)-Cychrome 7 (clone SK3 Becton Dickinson), anti–CD28-FITC (clone CD28.2, Becton Dickinson), and anti-TCRζ-PE (clone 2H2D9, Immunotech, Coulter).
Cells were analyzed on a Cyan ADP (Dako) flow cytometer. FCS Express version 3 (De Novo Software) was used for analysis. The percentage of TCRζdim was determined within each lymphocyte subset of interest. In addition, we assessed the Median Fluorescent Intensity (MFI) of TCRζ-chain expression as MFI index (MFI TCRζbright/MFI TCRζnegative) as described.18
Cells from 10 subjects randomly chosen among ACS patients (n=3), CSA patients (n=2) and controls (n=5) were fixed and stained for confocal microscopy with anti–CD3-PB (clone UCHT1, Becton Dickinson) or anti–CD3-FITC (clone UCHT1, Becton Dickinson) and, on permeabilization, anti–TCRζ-FITC (clone G3, Dako) or anti–TCRζ-PE (clone 2H2D9, Immunotech, Coulter). Cells were subsequently plated onto glass slides and examined under a Leica TCS SP2 AOBS confocal microscope (Leica Microsystems). Z-series were collected from singles channels, processed to 2D free projection max images, and merged. Single stains either for CD3 and TCRζ served as controls.
Measurement of High Sensitivity CRP (hsCRP)
Peripheral blood samples were centrifuged and serum aliquots were stored at −80°C until assayed in single batch. hsCRP was assessed via nephelometry (BN II–Behring instrument).
Transendothelial Migration Assay
We performed transendothelial migration assays of T cells in 17 study subjects (5 controls, 6 CSA, and 6 ACS), by applying 106 lymphocytes to gelatin-coated transwell upper chambers containing a monolayer of human umbilical vein endothelial cells (HUVECs) previously stimulated with 10 ng/mL TNF–α for 48 hours.15 After 24 hours at 37°C, T cells in the upper and lower chamber were recovered, and the numbers of migrating CD4+ and CD8+ TCRζbright or TCRζdim T cells were determined in each chamber by flow cytometry. Results are expressed as the percentage of cells migrating relative to the total number of each cell subset added to the transwell (TCRζbright in the lower chamber at time=24 hours/ TCRζbright at time=0 versus TCRζdim in the lower chamber time=24 hours/ TCRζdim at time=0).
The datasets did not conform to a normal distribution. Mann–Whitney U test and Kruskall-Wallis test with Dunn multiple comparison test were used as appropriate. Wilcoxon matched paired test was used for repeated measures in the time. Spearman rank test was used to test correlations between variables. GraphPad Prism 4 and GraphPad Instat 3 softwares were used for analysis. A probability value <0.05 was considered significant.
Characteristics of Patients
No significant differences in demographic and risk factors were observed between CSA group and ACS group (Table). However, differences were found in therapeutic regimens at the time of the sampling. Such differences were likely attributable to the fact that for 80% of patients in the ACS group, this hospital admission represented their first clinical manifestation of coronary disease versus only 56% in the CSA group (P=0.02). However, at 50-day follow-up there were no statistical differences regarding therapy between the two groups (P=NS; data not shown). ACS samples were obtained very early after the onset of symptoms when the elevation of troponin I were still minimal (0.4±0.0 to 5.9 ng/mL), thus ruling out the possible confounding effect of myocardial necrosis. The extent of coronary atherosclerosis was similar in patients in CSA and ACS group as documented by the number of diseased vessels in CSA, 2.0±0.8 (expressed as mean±SD), compared to ACS, 1.9±0.9 (P=0.79).
Substantial Downregulation of the TCRζ-Chain in CD4+ in ACS
Flow cytometric analysis (see representative dot-plots and histograms in Figure 1A through 1G) revealed a 3-fold increase in the percentage of CD3+TCRζdim T cells in ACS patients (median; interquartile range: 8.2%; 3.8 to 14.5%, P<0.0001; supplemental Table I, available online at http://atvb.ahajournals.org) compared to controls (2.6; 1.1 to 5.3%) and CSA (3.1; 1.7 to 7.0%). Confocal microscopy images confirmed the presence of CD3+ cells with downregulation of the expression of TCRζ-chain in patients with ACS (Figure 1H through 1J and supplemental Figures I and II). Circulating CD4+ TCRζdim T cells showed a 3-fold increase in patients with ACS (5.3%; 2.6 to 9.1%; P<0.0001, Figure 2A) when compared to controls (1.5; 0.5 to 2.9%) and CSA (1.6; 1.0 to 4.1%). The MFI index of TCRζ-chain was significantly reduced in patients with ACS in comparison to controls and patients with CSA (supplemental Figure III). To exclude that the increased levels of TCRζdim T cells reported in ACS were merely attributable to cell death, we showed that cell viability in CD3+ T cells were >99% out of total CD3+ in fresh blood, and that TCRζdim T cells were not confined to the non viable cells (supplemental Figure IV).
Differences in statin therapy are unlikely to explain the differences in TCRζ expression, as a subgroup analysis in patients without statin therapy (ACS n=54; CSA n=14) confirmed a significant increase of CD4+ TCRζdim T cells in ACS (P=0.002). Moreover, statin treatment itself was unlikely to provoke such changes as by comparing CSA patients with (n=18) or without (n=14) statin therapy (respectively 1.3; 0.9 to 3.7% versus 1.8; 0.9 to 5.1%; P=0.46) no significant differences were found.
Downregulation of TCRζ-Chain in CD8+ and NK Cells in ACS
We further assessed that downregulation of TCRζ-chain was not limited to CD4+ T cells in ACS. Indeed TCRζdim T cells represented 4.0% (2 to 5.7%) of peripheral circulating CD8+ T cells in patients with ACS in comparison to 0.6% (0.2 to 1.9%) in controls, and 1.2% (0.8 to 2.1%) in patients with CSA (P<0.0001; supplemental Figure VA). Also, a 2-fold increase was observed in the levels of TCRζdim within the circulating CD3−CD16+ NK cell subset (5.1%; 2.4 to 7.5%) in patients with ACS, whereas they accounted for 2.5% (1.3 to 3.8%) and 1.6% (0.9 to 2.7%) in controls and in patients with CSA respectively (P= 0.001; supplemental Figure VB). No significant differences were observed between control individuals and CSA patients for either T-cell or NK-cell subsets.
Persistence of Circulating CD4+ TCRζdim T Cells in ACS Patients at Follow-Up
Frequencies of circulating CD4+ TCRζdim T cells were unchanged at a 50-day follow-up after admission (6.4; 3.7 to 10.5% versus 5.5; 3.2 to 7.7%; P=0.95; Figure 2D), despite antiischemic therapy and risk factor management.
Increased Frequencies of TCRζdim T Cells Are Associated With Higher CRP Levels
We observed that the frequency of CD4+ TCRζdim T cells, CD8+ TCRζdim T cells, and TCRζdim NK cells correlated with CRP levels (respectively: r=0.30; P=0.0007, r=0.37; P= 0.02 and r=0.48; P=0.002) (supplemental Figure VIA through VIC). Considering a cut-off of CRP ≥2 mg/L used in previous studies,20 patients with ACS and CRP levels ≥2 mg/L had significantly increased percentages of CD4+ TCRζdim T cells (7.7; 3.8 to 11.3%, P=0.003, n=41) compared to patients with ACS and low CRP levels <2 mg/L (3.3; 1.7 to 7.7%, n=25; Figure 3).
Reduction of TCRζ-Chain in CD4+CD28null T Cells in Patients With ACS
In 60 subjects, blood samples were simultaneously stained for CD3, CD4, CD28, and TCRζ and analyzed by 4-color flow cytometry. We observed a significant correlation between frequencies of CD4+CD28null T cells and CD4+ TCRζdim T cells (r=0.43; P=0.0006; Figure 4A), especially within ACS patients (r=0.62; P=0.002; supplemental Figure VIIA). Interestingly, a reduction of TCRζ-chain expression was observed within the CD4+CD28null subset in ACS (67.2; 39.4 to 82.9%; P<0.0001), as compared to controls (20.9; 11.1 to 33.1%; P<0.001) and CSA (29.2; 23.0 to 46.9%; P<0.05) (Figure 4B and 4C, supplemental Figure VIIB).
Transendothelial Migration of TCRζdim T Cells, Both in CD4+ and CD8+ T Cells
We investigated the potential role of TCRζdim T cells in the immunopathological pathways that drive ACS, by comparing the capacity of this subset to migrate across activated endothelium with their TCRζbright counterparts. An in vitro transendothelial migration assays was performed on 17 individuals enrolled in the study. Increased migration of circulating TCRζdim T cells (CD4+: 62.2; 48.3 to 75.9% and CD8+: 69.0; 50.9 to 84.5%) were observed when compared to the TCRζbright subset (CD4+: 47.8; 46.5 to 50.8%; P=0.005; and CD8+: 49.5; 42.3 to 52.4%; P= 0.004; Figure 5 and supplemental Figure VIII). Similar migratory behavior of TCRζdim T cells in comparison with their TCRζbright counterparts were observed in ACS (n=5), CSA (n=6), and control (n=6) group (supplemental Figure IX). Downregulation was not attributable to the transmigration process per se, becuase the total number of TCRζbright and TCRζdim cells in top and bottom chamber does not change during the 24-hour transmigration assay.15
We report for the first time expansion of circulating T cells expressing reduced levels of the invariant TCRζ-chain in patients with ACS compared to patients with CSA and controls. CD4+ TCRζdim T cells remained elevated for at least 50 days after the acute event. In addition, we also documented that TCRζ-chain downregulation was not limited to T cell subpopulations, but was also present in NK cells. Enrichment of TCRζdim cells correlated with the magnitude of the systemic inflammatory response, raising the possibility that TCRζdim cells might participate to the ongoing inflammatory and immune response in ACS.
Functional Aspects of TCRζdim T Cells in Coronary Artery Disease
Growing similarities are emerging between atherosclerosis and chronic inflammatory diseases, including the expansion of circulating and lesional effector cells such as T-lymphocyte subsets.10 Despite T cells with low levels of TCRζ-chain expression being refractory to TCR signaling,19 we have recently reported that TCRζdim T cells express the hallmarks of memory antigen experienced effector T cells,15 produce abundant inflammatory cytokines such as TNFα and IFNγ, and promote monocyte activation through cell contact dependent pathways.15 Moreover, TCRζdim T cells do not produce antiinflammatory cytokine such as IL-10,15 and do not express the T regulatory cell marker Forkhead Box Protein P3 (FoxP3; supplemental Figure X). Collectively these observations point toward the requirement for intact TCR signaling to maintain immune homeostasis through the generation or function of regulatory T cell subsets.21
Furthermore, in this study we also observed that also in patients with CAD the ability of circulating TCRζdim T cells to migrate through endothelium is enhanced compared to their TCRζbright counterparts. In previous study we showed also enhanced chemotactic migratory ability of TCRζdim T cells in response to chemokines, such as CCL5 and CXCL10.15 Consistent with these in vitro observations, we previously documented that TCRζdim T cells are enriched in synovial fluid and tissue from patients with rheumatoid arthritis.15 Moreover, in 3 patients with ACS enrolled in the study we documented a decrease in the number of CD3+CD4+ TCRζdim T cells in blood acquired from the great cardiac vein before angioplasty compared to levels of CD3+CD4+ TCRζdim T cells sampled from the aorta5 (supplemental Figure XI). These results could suggest an accumulation of this cell population in the coronary circulation during the acute phase of the disease. Thus, antigen experienced effector TCRζdim T cells with enhanced migratory competence have the potential to extravasate and exert local proinflammatory actions in atherosclerotic plaques, thus contributing to plaque destabilization.
Possible Mechanisms of TCRζ-Chain Downregulation
In our study the enrichment of circulating CD4+ TCRζdim T cell in patients with ACS was associated with higher levels of hsCRP, suggesting that the inflammatory milieu in patients with ACS may contribute to TCRζ-chain downregulation. Antigen-dependent and antigen-independent mechanisms have been advocated to explain downregulation of the TCRζ-chain in human and experimental models.14 Both mechanisms may have a role in TCRζ-chain downregulation in ACS. For instance, prolonged exposure to bacterial and viral antigens has been shown to induce TCRζ-chain down-regulation in vivo.14 In the setting of ACS, a variety of infectious and endogenous antigen(s) have been implicated (ie, modified low-density lipoprotein, endogenous and infectious agents-derived heat shock proteins) and could potentially lead to TCRζ-chain downregulation.22–24 Antigen-independent mechanisms, eg, prolonged exposure inflammatory cytokines including IFNγ and TNFα, as well as reactive oxygen intermediates, have also been advocated for TCRζ-chain downregulation.19 In our study, the downregulation of the TCRζ-chain was not limited to CD4+ and CD8+ T cells but was also present in the NK-cell population, in which it may arise through FcR engagement, perhaps via immune complexes or through cytokine stimulation.14
Interestingly, the loss of CD28, as the downregulation of TCRζ-chain, has been linked to both chronic antigen or cytokine exposure.19,25,26 CD4+CD28null T cells, a terminally differentiated effector T cell population, was identified both in patients with rheumatoid arthritis with vascular complications and in patients with ACS.12,13,27 More then 65% of CD4+CD28null T cells from patients with ACS had selectively reduced TCRζ-chain expression. Hence, in the context of ACS, similar mechanisms might explain the reduced TCRζ-chain expression and the loss of CD28, and the generation of T-cell subsets with perturbation of the classical immunologic synapse.
We show that circulating TCRζdim cells are increased in ACS, and such increase is associated with higher levels of hsCRP. TCRζdim cells display an increased ability to cross activated endothelium, and might carry the potential to accumulate in atherosclerotic plaques. Both antigen-dependent and antigen-independent mechanisms may contribute to the emergence of this unusual T-cell surface phenotype. The aberrant expression of TCRζ-chain in circulating cell subsets further supports the role of dysregulation of the immune response in the pathogenesis of ACS. Moreover, our findings may contribute to explain the increased incidence of major cardiovascular events in systemic lupus erythematosus and rheumatoid arthritis.28
We are grateful to Dr Viviana Vecchio, Dr Alessio Palini (Flow Cytometry Core Facility, Service, San Raffaele Scientific Institute, Milan), and Cesare Covino and Maria Carla Panzeri (ALEMBIC, San Raffaele Scientific Institute, Milan), Parisa Amjadi for technical help and guidance. We thank Dr Monica De Metrio, Dr Marco Mussardo who helped us to define patients for recruitment to the study.
Sources of Funding
This work was supported by European Vascular Genomics Network (EVGN), Fondazione Internazionale di Ricerca per il tuo cuore onlus (Italy), the Arthritis Research Campaign UK and AutoCure, an EU Framework 6 funded Integrated Project.
A.P.C. and C.M. share the senior authorship of this article.
Original received April 20, 2008; final version accepted August 30, 2008.
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