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Keywords:

  • coronary artery disease;
  • cytomegalovirus;
  • immune system;
  • T lymphocytes

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References

Abstract. Jonasson L, Tompa A, Wikby A (Heart Center, University Hospital, Linköping; Ryhov Hospital, Jönköping; and School of Health Sciences, Jönköping University, Jönköping; Sweden). Expansion of peripheral CD8+ T cells in patients with coronary artery disease: relation to cytomegalovirus infection. J Intern Med 2003; 254: 472–478.

Objectives. The nature of the immune response in coronary artery disease (CAD) is not fully defined. One pathogen that has been linked to atherogenesis, cytomegalovirus (CMV), is known to exert strong and long-lasting effects on peripheral T cells. In the present study, we investigated the effect of prior CMV infection on the immune system in CAD patients.

Subjects. Patients with stable angina and angiographically verified CAD (n = 43) and clinically healthy controls (n = 69) were included.

Methods. The expression of CD57 and CD28 on peripheral CD4+ and CD8+ T cells was evaluated with three-colour flow cytometry. The findings were related to serological markers of inflammation, T-cell activation and CMV seropositivity.

Results. An expansion of CD8+ T cells expressing CD57 but lacking CD28 was seen in the patient group. The numbers of CD8+ CD57+ and CD8+ CD28T-cell subsets were independently related to CMV seropositivity (P < 0.001) but also to CAD per se (P < 0.05). Serum concentrations of C-reactive protein (CRP) and soluble interleukin-2 receptor (sIL-2R) were elevated in the patients but not related to CMV or CD8+ T-cell subsets.

Conclusion. A pronounced shift in peripheral T-cell homeostasis was observed in CAD patients. Primarily CMV infection but also CAD per se contributed to the expansion of CD8+ T-cell subsets. The T-cell changes were not related to a systemic inflammatory response but should rather be considered as markers of a chronic antigen exposure and/or immunosenescence in CAD.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References

There is increasing evidence for a continuous immune activation in coronary artery disease (CAD) characterized by accumulations of immunocompetent cells in the arterial lesion and elevated levels of inflammatory markers in the circulation [1–6]. It is still not clarified as to what extent the augmented T-cell response in CAD is antigen-specific, but both infectious and autoimmune antigens may play a role. A persistent antigen exposure may induce not only short-lived activation markers on T cells but also more permanent alterations in the T-cell phenotype pattern. Enhanced expression of natural killer (NK) cell markers on CD8+ T cells and loss of the co-stimulatory molecule CD28 on CD4+ and CD8+ cells have been reported in patients with chronic antigen exposure, such as autoimmune diseases [7, 8] transplant recipients [9, 10] and human immunodeficiency virus (HIV) infections [11]. In addition, a common human pathogen, cytomegalovirus (CMV), has been shown to strongly exert these effects on the peripheral T-cell homeostasis [12, 13].

Data from experimental and histopathological studies clearly indicate that CMV infection may have a role in atherogenesis [14–16]. However, findings from epidemiological studies are contradictionary and the serological evidence of prior CMV infection associated with CAD and myocardial infarction is a source of controversy [17–20]. In addition, several studies have failed to demonstrate a correlation between CMV antibodies and inflammatory markers in CAD patients [17–19] whilst others have reported that the combination of CMV seropositivity and elevated levels of C-reactive protein (CRP) and IL-6 in CAD patients is an independent predictor of future cardiac mortality [20–22].

In the present study, we investigated whether any change in the peripheral T-cell phenotype distribution, involving surface expression of CD28 and the NK cell marker CD57, was observed in CAD patients and related the findings to CMV seropositivity and inflammatory/T-cell activation markers.

Subjects

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References

Forty-three men (≤60 years of age) with stable angina, having angiographically verified CAD with at least two coronary stenoses (>50% reduction of lumen diameter), were consecutively studied. The diagnosis of stable angina was defined as effort-related angina of Canadian Cardiovascular Society functional class II and III. Patients with myocardial infarction or unstable angina within the last 2 months were excluded. Other exclusion criteria were diabetes, immunological disorders, neoplastic disease, evidence of acute or recent (<2 months) infection, recent major trauma, surgery or coronary revascularization and treatment with antibiotics, immunosuppressive or anti-inflammatory agents. All patients were, however, on low-dose aspirin. Sixty-nine apparently healthy men of equivalent age without symptoms or signs of CAD served as controls. Informed consent was obtained from all subjects. The research protocol was approved by the locally appointed ethical committee.

Flow cytometry analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References

Blood samples were taken from the forearm vein in the morning after a 12-h fast. T-lymphocyte subpopulations were measured by three-colour flow cytometry using fluorescent activated cell sorter scan (FACScan; Becton Dickinson Immunocytometry Systems, Jönköping, Sweden). Monoclonal antibodies against CD3 (all T cells), CD4 (T-helper cells), CD8 (T-cytotoxic/suppressor cells), CD57 and CD28 were purchased from Becton Dickinson. The cells were stained by different triples as follows: CD3/CD4/CD8, CD3/CD4/CD57, CD3/CD4/CD28, CD3/CD8/CD57, CD3/CD8/CD28. The antibodies were marked with one of the three fluorochromes: fluorescein isothiocyanate, phycoerythrin and peridinin chlorofyll protein. A sample of 20 μL of each monoclonal reagent triple was added to 100 μL of whole blood in 12 × 75 test tubes, centrifuged gently and incubated at 4°C for 30 min. Red blood cells were lysed using 3 mL of FACS lysing solution during 2-min incubation at room temperature in the dark. The cells were washed twice with phosphate-buffered saline with 0.1% sodium azide, resuspended in wash buffer, fixed with 1% paraformaldehyde and analysed after 2–3 h. Data were analysed using CELL Quest software (Becton Dickinson). Instrument setups, adjustments and viability controls were performed as described [23].

Serological assays

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References

Serum samples were assayed for CRP by a commercially available highly sensitive latex-enhanced turbidimetric immunoassay with a lower detection limit of 0.03 mg L−1 (Roche Diagnostics GmbH, Mannheim, Germany). Serum samples were assayed for the soluble form of IL-2R by a commercially available sandwich enzyme immunoassay (BioSource Europe S.A, Nivelles, Belgium). Blood samples were tested for anti-CMV IgM and IgG antibodies using an enzyme-linked immunosorbent assay. In brief, microtiter plates were coated with CMV antigens prepared according to an established protocol [24] and incubated with 100 μL diluted serum. Anti-CMV IgM was detected by peroxidase-conjugated goat anti-human IgM (Sigma Chemical Co., St Louis, MO, USA) and anti-CMV IgG was detected by alkaline phosphatase-conjugated goat anti-human IgG (Biosource, Tago immunologicals, Camarillo, CA, USA). A value <10 U was considered negative and a value of ≥10 was considered positive, indicating prior exposure to CMV.

Statistical analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References

All statistics were analysed using the SPSSPC system (1986; Norusis, SPSS Inc., Chicago, IL, USA). Data are presented as mean values and standard deviations. The significance of any difference in mean values between patients and controls was tested by using Student's t-test. Subgroup analyses were performed using one-way anova followed by the post hoc Scheffe's test to evaluate the significance of any difference in mean values between the subgroups. Correlational analysis was performed using Pearson correlation coefficient (r). Multiple logistic regression analysis was performed to assess the independent contribution of different factors to the T-cell changes. Two-tailed P-values <0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References

Patients’ and controls’ characteristics are listed in Table 1. All patients were on low-dose aspirin and various combinations of β-blockers, calcium-antagonists and nitrates. Twenty-five patients were treated with 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) whereas 18 patients were not taking any lipid-lowering drugs. Control subjects were all drug-free. The prevalence of CMV seropositivity was similar in patients and controls (67% vs. 61%, N.S.) as were the serum levels of CMV IgG antibodies (23 (23) vs. 17 (16) units, N.S.). Only two patients and none of the controls had CMV IgM titres above the detectable limit. Patients had significantly higher levels of CRP (4.1 (6.7) vs. 1.6 (1.4) mg L−1, P < 0.01) and the acute T-cell activation marker sIL-2R (578 (295) vs. 397 (148) pg mL−1, P < 0.001).

Table 1.  Characteristics of patients and controls
VariablePatients (n = 43)aControls (n = 69)aP-value
  1. aMean (±SD).

  2. N.S., not significant; BMI, body mass index; HDL, LDL: high- and low-density lipoproteins.

Age (years)55.1 (5.6)49.5 (5.9)<0.05
BMI (kg m−2)27 (4)25 (3)<0.05
Blood pressure (mmHg)
 Systolic133 (15)132 (10)N.S.
 Diastolic80 (8)77 (8)N.S.
Smokers, %2622 
Total cholesterol (mmol L−1)4.4 (1.0)5.3 (1.0)<0.01
 LDL cholesterol2.9 (0.9)3.6 (0.8)<0.01
 HDL cholesterol0.9 (0.2)1.1 (0.3)<0.01
 Triglycerides1.6 (0.5)1.4 (0.9)N.S.

CD57+ and CD28 T-cell subsets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References

The absolute numbers of CD8+ CD57+ and CD8+ CD28 T cells were significantly higher in the patient group (Fig. 1). Similarly, the percentage of CD57+ cells within the CD8+ population was significantly increased in patients compared with controls (47% vs. 37%, P < 0.01) as was the percentage of CD28 T cells (52% vs. 44%, P < 0.05). The fraction of CD4+ T cells expressing CD57 was smaller and did not differ significantly between patients and controls (11.4% vs. 8.4%, N.S.). Similar results were obtained concerning CD4+ CD28 T cells (6.7% vs. 5.8%, N.S.). Correlational analysis, including both patients and controls (n = 112), showed that the correlation coefficient between CD8+ CD57+ and CD8+ CD28 cells was 0.97 (P < 0.001) and between CD4+ CD57+ and CD4+ CD28 cells 0.92 (P < 0.001).

image

Figure 1. CD8+ subsets (cells/mm3) in patients and controls.

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Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References

Table 2 shows the distribution of T-cell subsets in patients and controls related to CMV seropositivity. The CD57+ and CD28 T-cell subsets were increased in all CMV seropositive subjects. However, the increase was most pronounced in the CMV seropositive patients.

Table 2.  CD3+subsets (cells/mm3) in cytomegalovirus (CMV) seropositive (CMV+) and seronegative (CMV–) patients and controls. One-way anova indicated significant differences between the four subgroups
 CMV+CMV–P-value
Patients (n = 29)Controls (n = 38)aPatients (n = 14)aControls (n=24)a
  1. aMean (±SD).

CD8+ CD57+392 (226)a269 (190)167 (183)105 (67)<0.001
CD8+ CD28452 (258)329 (216)172 (174)112 (71)<0.001

The number of CD8+ CD57+ T cells was strongly related to CMV seropositivity (r = 0.52, P < 0.001) but also to CAD per se (r = 0.29, P < 0.01). A linear multiple regression analysis identified CMV seropositivity and CAD as independent predictors of CD57 expression on CD8+ T cells (Table 3). Similar results were obtained when the CD8+ CD28 subsets served as dependent variables (data not shown). No relationship between CAD and CMV seropositivity was found.

Table 3.  Multiple regression statistics for CD8+ CD57+ T cells*
VariableRegression coefficientP-value
BSEBBETAt
  1. B, standardized regression coefficient; CAD, coronary artery disease; CMV, cytomegalovirus; SEB, standard error of regression coefficient; BETA, unstandardized regression coefficient.

  2. *The number of CD8+ CD57+ T cells served as the dependent variable. The CMV antibody titre and the CAD index (0 = no disease, 1 = CAD) served as independent variables.

CMV antibody titre4.970.900.475.50<0.001
CAD82.1735.780.202.30<0.05

The CRP or sIL-2R levels were not correlated with CMV seropositivity or CMV antibody titres. No association was found between CD8+ CD57+ T cells and CRP (r = 0.14, N.S.) or between CD8+ CD57+ T cells and sIL-2R (r = –0.07, N.S.). Furthermore, no association was seen between the CD4+ subsets and inflammatory markers.

Clinical parameters such as age, body mass index, lipid and lipoprotein concentrations or statin medication did not influence the distribution of CD57+ and CD28 T-cell subsets.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References

In this cross-sectional study, a pronounced shift in the peripheral T-cell homeostasis, involving CD8+ T cells, was demonstrated in patients with stable CAD. Parallel to a rise in CD8+ CD57+ T cells a decrease in CD8+ CD28 cells was observed in patients compared with healthy controls. As found in the present study, and also by several others [25, 26], CD57 and CD28 were reciprocally related. CD57 is found on all NK cells but is also normally present on a subset of T cells. CD28 is constitutively expressed by T cells where it acts as a critical co-stimulatory molecule in T-cell activation but following repeated antigen exposure, the cells appear to lose CD28. Long-term persistent expansion of CD57+ CD28 T cells have been described in the elderly and in clinical conditions involving chronic activation of the immune system [11, 12, 27]. In the middle-aged patient group of our study, >50% of the CD8+ cells were CD28, similar to what has been reported in very-old CMV seropositive individuals [28, 29] and in patients with advanced HIV infection [11]. On the contrary, the CAD patients had no increases in CD4+ CD57+ and CD4+ CD28 T-cell subsets which is in agreement with previous studies reporting an expansion of CD4+ CD28 T cells only in unstable but not in stable angina patient [30].

In this context, the proposed link between CMV infection and CAD is of particular interest. Both viral antigen and viral genome has been detected in atheroslerotic tissue [16] and a number of experimental studies have demonstrated that CMV infection may induce potentially atherogenic effects in vascular cells [14, 15]. In addition, CMV is unique amongst Herpes viruses inducing persistent, perhaps permanent, changes on T cells. There is no association between other latent herpes viruses [herpes simplex, varicella-zoster, Epstein–Barr virus] and changes in T-cell phenotype [12, 29, 30]. Previous studies have shown that the most important determinant of CD57+ and CD28 T-cell subsets in elderly individuals as well as in patients with rheumatoid arthritis is CMV seropositivity [28, 29, 31]. In the present study, it was found that CD57+ and CD28 T-cell subsets strongly correlated with CMV seropositivity in all subjects. However, CMV seropositivity alone could not explain the marked expansion of the CD8+ T-cell subset in patients and multiple regression analysis confirmed that CAD per se was an independent determinant. Besides prior CMV infection, overexpression of CD57 and loss of CD28 has been associated with clinical conditions such as transplant recipients and autoimmune diseases. There are close points of similarity between atherosclerosis and autoimmunity. Elevated levels of autoantibodies to endothelial cells and oxidized lipoproteins as well as anti-phospholipid antibodies have been reported to be markers or predictors of cardiovascular disease [32–34] strongly suggesting a role of autoimmune reactivity in atherogenesis.

The expansion of CD8+ T-cell subsets in stable CAD patients was not associated with elevations in CRP or sIL-2R suggesting that these cells are not directly involved in inflammatory actions. Still, this CD8+ phenotype may have a cytotoxic potential, primarily associated with the loss of CD28. It has been shown in vitro that repeated antigen stimulation lead to a state of immunosenescence with irreversible cell cycle arrest, increased resistance to apoptosis and down-regulation of CD28 expression [35, 36]. Both CD4+ CD28 and CD8+ CD28 cells synthesize interferon-γ in culture [27, 37, 38] and in patients with unstable angina, the expansion of CD4+ CD28 T cells appear to be correlated with tissue-damaging effects [39]. Furthermore, the increased interferon-γ production which is normally seen in elderly individuals correlate with expanded CD8+ CD57+ CD28 T-cell subsets [38]. CD8+ CD57+ T cells have otherwise been restricted to suppressive activity, primarily on the basis of several in vitro studies where these cells have been shown to inhibit cytotoxic lymphocytes [40–42] as well as B-cell differentiation [43]. One possibility is that the expansion of CD8+ CD57+ T cells in CAD patients represents a regulatory mechanism to suppress a cytotoxic immune response and as such provides a protective role.

A similar prevalence of CMV seropositivity in CAD patients and controls was found. The lack of association between CMV seropositivity and CAD has been reported by several others [17–19]. An association between CD8+ CD57+ T cells and reactivation of CMV infection has been demonstrated in transplant recipients [9] and it cannot be excluded that the CAD patients in the present study had a higher prevalence of reactivated CMV infection. Two patients were IgM seropositive but only one of them had a high rate of CD8+ CD57+ T cells. However, the determination of CMV serological status (IgM or IgG) is not considered to be a good tool to discriminate between latent or reactivated infection.

To summarize, the present study shows for the first time an altered T-cell homeostasis in patients with stable CAD, mainly determined by CMV seropositivity but also by CAD per se. The CD8+ T-cell expansion was not associated with a systemic inflammatory response but should rather be considered as a marker of excessive antigen load and/or immunosenescence in CAD. Further studies involving patients with acute coronary syndrome as well as in vitro studies are needed to clarify the cause of CD8+ T-cell expansion and its clinical significance in CAD patients.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Subjects
  6. Flow cytometry analysis
  7. Serological assays
  8. Statistical analysis
  9. Results
  10. CD57+ and CD28 T-cell subsets
  11. Relation between T-cell subsets, inflammatory/immune activity and CMV seropositivity
  12. Discussion
  13. Conflict of interest statement
  14. Acknowledgements
  15. References
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