CLINICAL PERSPECTIVE

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(Circulation. 2006;113:2272-2277.)
© 2006 American Heart Association, Inc.

Coronary Heart Disease

Modulation of CD4⁺CD28^null T Lymphocytes by Tumor Necrosis Factor- Blockade in Patients With Unstable Angina

Vittoria Rizzello, MD; Giovanna Liuzzo, MD; Salvatore Brugaletta, MD; Antonio Rebuzzi, MD; Luigi M. Biasucci, MD; Filippo Crea, MD

From the Cardiology Department, Catholic University, Rome, Italy.

Correspondence to Dr Vittoria Rizzello, Cardiology Department, University Hospital "A. Gemelli," Catholic University, Largo "A. Gemelli" 8, 00168 Rome, Italy.

Received September 12, 2005; revision received March 7, 2006; accepted March 15, 2006.

	Abstract

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Background— Tumor necrosis factor- (TNF-) is a proinflammatory cytokine that favors the expansion of CD4⁺CD28^null T cells, an aggressive and unusual proinflammatory lymphocyte subset frequently observed in patients with unstable angina (UA). The purpose of the present ex vivo study was to evaluate whether inflammation in patients with UA may be modulated by selective blockade of TNF-.

Methods and Results— Peripheral blood samples were collected from 17 patients with UA (Braunwald’s class IIIB). CD4⁺CD28^null T cells were assessed by flow cytometry and expressed as a percentage of all CD4⁺ T cells after 24 hours of incubation of whole blood with and without increasing doses (0.1, 1, 10, and 100 µg/mL) of infliximab, an anti–TNF- monoclonal antibody. In addition, CD28 expression was assessed and expressed as mean fluorescence intensity (geometric mean of the CD28 fluorescence value on all CD4⁺ T cells). CD4⁺CD28^null T-cell percentage decreased from a median of 6.2% (range, 1.2% to 23.9%) to 4.9% (range, 1.1% to 21.9%), 4.5% (range, 1.1% to 21.6%), and 4.1% (range, 0.4% to 21.4%) after incubation with 1, 10, and 100 µg/mL of infliximab (P for trend=0.043). Analysis of CD28 mean fluorescence intensity showed that the expression of CD28 on cell surface significantly increased after incubation with increasing doses of infliximab (P for trend=0.03).

Conclusions— The findings of this ex vivo study show that CD4⁺CD28^null T-cell expansion in patients with UA may be reduced by selective TNF- blockade. Further studies are warranted to evaluate the clinical benefit of CD4⁺CD28^null T-cell modulation.

Key Words: angina • inflammation • lymphocytes

	Introduction

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An increasing body of evidence supports the pathogenetic role of inflammation in acute coronary syndromes.^1–3 Inflammatory cells accumulate in coronary atherosclerotic plaques and play a key role in plaque instability.^4–7 In addition, activated circulating monocytes and T lymphocytes^8–12 and increased levels of proinflammatory cytokines have been demonstrated in patients with unstable angina (UA),^13–15 suggesting some similarities with typical immunoinflammatory diseases such as rheumatoid arthritis.¹⁶ Notably, Liuzzo et al¹⁷ demonstrated in patients with UA the expansion of a CD4⁺ T lymphocyte subset that lacks surface expression of CD28 (CD4⁺CD28^null T cells). CD4⁺CD28^null T cells are uncommon (<1%) in healthy subjects but are expanded in patients with rheumatoid arthritis with evidence of vasculitis.¹⁸ Because this T-cell subset shows high proinflammatory and tissue-damaging properties and a deficit in apoptosis, it has been suggested that CD4⁺CD28^null T cells may affect instability in patients with UA.^17,19 CD4⁺CD28^null T-cell expansion is regulated by a complex cytokine network.^18,20 In particular, exposure to tumor necrosis factor- (TNF-) downregulates CD28 expression and favors the expansion of CD4⁺CD28^null T cells.²⁰ Conversely, a decrease in circulating CD4⁺CD28^null T cells may be obtained by inactivation of TNF-.²⁰ Recently, infliximab, a mouse-human chimeric anti–TNF- monoclonal antibody, has been shown to decrease CD4⁺CD28^null T cells in patients with rheumatoid arthritis.^21,22 Infliximab binds to soluble and cell-surface transmembrane TNF- with high affinity (K_a=10¹⁰ [mol/L]^–1) and forms a stable complex that blocks the association of this cytokine with its receptor, neutralizing TNF- effect both in vitro and in vivo.^23–25 In the present ex vivo study, we assessed whether selective blockade of the TNF- by infliximab may modulate CD4⁺CD28^null T-cell expansion in peripheral blood obtained from patients with UA.

Clinical Perspective p 2277

	Methods

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Study Population
Peripheral blood samples were collected from 17 patients (11 men; mean age, 67±8 years) admitted to our Coronary Care Unit with a diagnosis of UA, Braunwald’s class IIIB. Inclusion criteria were as follows: 2 episodes of angina at rest or 1 episode lasting >20 minutes during the preceding 48 hours, ST-segment shift diagnostic for myocardial ischemia during angina, and no elevation of serum creatine kinase levels on admission. Patients with known immunologic, acute, or chronic inflammatory or infectious disease; malignancy; recent (<6 months) myocardial infarction; and moderate to severe left ventricular dysfunction (ejection fraction <40%) were excluded. The study was approved from the local ethics committee. Patients were asked to participate in the study on admission, as soon as the documentation of normal creatine kinase levels was available and after the start of standard medical therapy. All patients gave written informed consent to participate in the study.

Effects of Infliximab on CD4⁺CD28^null T-Cell Percentage
Citrate whole-blood samples were collected from an antecubital vein at the time of patient admission, immediately after medical therapy was started. TNF- levels were measured with a high-sensitivity TNF- ELISA kit (R&D Systems, Minneapolis, Minn). Immediately after sampling, aliquots of 1 mL of blood were incubated for 24 hours under sterile conditions at 37°C in an atmosphere containing 5% CO₂ without and with increasing doses of infliximab (0.1, 1, 10, and 100 µg/mL; Remicade; Centocor, Inc, Horsham, Pa). These doses were chosen on the basis of the observation that comparable plasmatic concentrations have been associated with clinical benefit in patients with rheumatoid arthitis.²⁵ T-cell subsets were assessed by flow cytometry after 24 hours of incubation. In particular, 100 µL blood obtained from these aliquots was stained with fluorescein isothiocyanate–conjugated anti-CD4 (Becton Dickinson, San Jose, Calif) and phycoerytrin-conjugated anti-CD28 monoclonal antibodies (Pharmigen, San Diego, Calif). Stained cells were analyzed on a Coulter flow cytometer. The percentage of CD4⁺CD28^null T cells was obtained with the WinMDI software (Joseph Trotter, Scripps Research Institute, La Jolla, Calif) and expressed as a percentage of the entire population of CD4⁺ T cells. In addition, CD28 expression was expressed as mean fluorescence intensity (MFI; geometric mean of the CD28 fluorescence value on all CD4⁺ T cells). In 8 patients, CD4⁺CD28^null T-cell percentage also was assessed 1 year after discharge.

Statistical Analysis
On the basis of previous studies in patients with UA and in patients with rheumatoid arthritis,^17,19,21,22 we assumed that the percentage of CD4⁺CD28^null T cells likely to be found in our population was 10% (range, 5% to 14%). Moreover, in line with the findings reported by Gerli et al,²¹ we assumed that a 34% reduction in CD4⁺CD28^null T cells after treatment with infliximab would have been significant. From these assumptions, a power calculation (=0.05; power, 0.80) was performed with the GB STAT program. It showed that the number of patients needed to test our working hypothesis was 15.

Because TNF- levels, CD4⁺ T cells, and the percentage of CD4⁺CD28^null T cells were not normally distributed (according to the Kolmogorov-Smirnov normality test), they were expressed as median and range and analyzed with nonparametric methods. Accordingly, the effects of infliximab on the entire population of CD4⁺ T cells and the CD4⁺CD28^null T-cell subset were compared by Friedman ANOVA for repeated measures on ranks with Dunnet’s correction. CD28 MFI values were normally distributed and are expressed as mean±SD. The effects of infliximab on MFI were compared by 1-way ANOVA for repeated measures with the Bonferroni adjustment. Changes in the CD4⁺CD28^null T-cell percentage at the 1-year follow-up were analyzed by the Wilcoxon test. A value of P<0.05 was considered statistically significant.

The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.

	Results

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Study Population
The Table summarizes the clinical characteristics of the study population. Previous myocardial infarction was present in 6 patients (35%). Left ventricular ejection fraction was 53±9%. Serum level of CRP at admission was 3.9 mg/L (range, 1.46 to 20.34 mg/L). Creatine kinase serum levels during the first 24 hours of hospitalization were in the normal range (230 to 460 IU/L) in all patients. All patients at the time of blood sampling were on aspirin (100 mg/d), clopidogrel (loading dose, 300 mg; then 75 mg/d), low-molecular-weight heparin (120 IU/kg SC every 12 hours), statins (atorvastatin 25±14 mg/d), nitrates, and ß-blockers. All patients underwent coronary angiography during hospitalization, and early revascularization was performed in 7 patients.

View this table: [in this window] [in a new window]   Characteristics of the Study Population

TNF- Levels and CD4⁺CD28^null T-Cell Percentage
To evaluate the role of TNF- on CD4⁺CD28^null T-cell expansion, we assessed CD4⁺CD28^null T-cell percentage in relation to TNF- levels. CD4⁺CD28^null T-cell percentage was significantly higher in patients with high levels of TNF- (>3 pg/mL) compared with patients with low (3 pg/mL) levels of TNF- (median, 6.8%; range, 2.4% to 23.9% versus median, 1.75%; range, 1.05% to 6.4%; P=0.013).

CD4⁺CD28^null T-Cell Percentage Is Reduced by Infliximab
As shown in Figure 1, the percentage of CD4⁺CD28^null T cells at baseline was 6.2% (median) (range, 1.2% to 23.9%) and decreased to 5.5% (range, 0.89% to 24.4%), 4.9% (range, 1.1% to 21.9%), 4.5% (range, 1.1% to 21.6%), and 4.1% (range, 0.4% to 21.4%) after incubation with 0.1, 1, 10, and 100 µg/mL infliximab, respectively (P for trend=0.043). CD4⁺CD28^null T-cell decrease was significant starting at 1 µg/mL of infliximab (P=0.02; Figure 1). Analysis of CD28 MFI showed that the expression of CD28 on the cell surface significantly increased after incubation with infliximab (P for trend=0.03; Figure 2). The increase on MFI was significant starting at 10 µg/mL (from 43.0±7.6 to 47.3±7.7; P=0.03). Of note, the percentage of all CD4⁺ T cells was unaffected by incubation with infliximab. CD4⁺ T-cell percentage was 42.0% (median) (range, 37.8% to 61.8%) at baseline and 44.2% (range, 39.3% to 61.9%), 42.3% (range, 37.5% to 62%), 43.1% (range, 39.2% to 65%), and 41.6% (range, 37.2% to 59.4%) after incubation with 0.1, 1, 10, and 100 µg/mL infliximab, respectively (P=0.8).

View larger version (16K): [in this window] [in a new window]   Figure 1. Twenty-four–hour incubation of blood with infliximab results in a significant reduction in CD4+CD28null T cells. Data are presented as box plot with median and 25th to 75th percentiles (box) and 10th to 90th percentiles (whiskers). *P<0.05 vs baseline.

View larger version (13K): [in this window] [in a new window]   Figure 2. Twenty-four–hour incubation of blood with infliximab results in a significant increase in MFI for CD4+CD28+ T cells. Data are expressed as mean±SD. P<0.05 vs baseline and 0.1 and 1 µg/mL.

CD4⁺CD28^null T-Cell Percentage After Stabilization of UA
In 8 patients, the CD4⁺CD28^null T-cell percentage was reassessed at 1 year after discharge. At this time, the CD4⁺CD28^null T-cell percentage was lower than that observed on admission [5.8% (median) (range, 1.2% to 7.71%) versus 2.93% (range, 0.34% to 5.95%); P=0.043].

	Discussion

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The findings in the present study show that in patients with UA, high levels of TNF- are associated with a higher percentage of CD4⁺CD28^null T lymphocytes and that infliximab, an anti–TNF- monoclonal antibody, reduces CD4⁺CD28^null T cells. Reduction of CD4⁺CD28^null T cells was greater after incubation with 100 µg/mL infliximab, a dosage that was associated with maximal responsiveness to treatment in patients with rheumatoid arthritis.²⁵ However, CD4⁺CD28^null T-cell decrease was already significant at lower dosages (1 and 10 µg/mL infliximab). In addition, CD4⁺CD28^null T-cell reduction was paralleled by increased expression of CD28 on CD4⁺ T cells, as indicated by an increase in MFI for the CD28 molecule in the whole population of CD4⁺ T cells.

Blockade of proinflammatory cytokines has become common practice in the treatment of classical inflammatory disease, particularly rheumatoid arthritis.^21,22,25 UA and rheumatoid arthritis share many similarities, including increased systemic levels of TNF-, interleukin-6, C-reactive protein, and serum amyloid A protein and enhanced expression of adhesion molecules, metalloproteinases, and neoangiogenesis at the tissue level.¹⁶ Furthermore, similarly to what previously observed in rheumatoid arthritis, Liuzzo et al¹⁷ showed that CD4⁺CD28^null T cells are increased in the peripheral blood of patients with UA, reflecting a Th1/Th2 lymphocyte imbalance. CD4⁺CD28^null T cells represent an aggressive T-cell subset because of their capabilities to infiltrate tissues, including unstable coronary plaques, and to amplify the inflammatory response by the production of high levels of interferon-.^17–19 Moreover, CD4⁺CD28^null T cells show resistance to apoptosis.^18,20 It has been demonstrated that expansion of CD4⁺CD28^null T cells may have a negative prognostic implication. In patients with ischemic stroke, increased levels of CD4⁺CD28^null T cells are associated with recurrence of stroke and death.²⁶ Furthermore, in patients with rheumatoid arthritis, the expansion of CD4⁺CD28^null T cells is associated with more aggressive features of the disease, including extra-articular manifestations such as vasculitis.²¹

The findings in the present study confirm that a high percentage of CD4⁺CD28^null T cells is present in patients with UA. Higher percentages of CD4⁺CD28^null T cells were associated with higher levels of TNF-, confirming that TNF- plays a key role in the expansion of this unusual T-cell subset. In addition, this study demonstrates for the first time in patients with UA that infliximab, by the selective blockade of TNF-, can significantly decrease CD4⁺CD28^null T cells. The CD4⁺CD28^null T-cell reduction observed in this study (33%) is similar to that reported in the study by Gerli et al,²¹ which showed a 34% reduction in CD4⁺CD28^null T cells in patients with rheumatoid arthritis treated with infliximab. In the study by Pawlik et al,²² a 36% reduction in CD4⁺CD28^null T cells was observed after treatment with infliximab. Because TNF- downregulates the expression of CD28 on T cells at the transcriptional level, inhibition of TNF- is likely to restore the presence of CD28 on cell surface by preventing TNF-–mediated downregulation of CD28 production.²⁷ Accordingly, in the present study, CD4⁺CD28^null T-cell reduction was paralleled by increased expression of CD28 on CD4⁺ T cells, as indicated by an increase in MFI for the CD28 molecule. This finding is in line with a recent study by Gerli et al²¹ showing that in patients with rheumatoid arthritis, treatment with anti–TNF- monoclonal antibodies leads to partial reappearance of CD28 on CD4⁺T cells. Similarly, Bryl et al²⁸ recently showed that treatment with infliximab was associated with normalization of CD28 expression in patients with rheumatoid arthritis.

Clinical Implications
The modulation of CD4⁺CD28^null T cells in patients with UA may be clinically relevant. Liuzzo et al¹⁹ demonstrated CD4⁺CD28^null T-cell infiltration in unstable coronary plaques, suggesting that these cells may be responsible for plaque instability. In addition, Zal et al²⁹ recently suggested that CD4⁺CD28^null T cells reactive to heat shock protein 60 may contribute to vascular damage in patients with acute coronary syndromes. Furthermore, it has been suggested that in patients with rheumatoid arthritis, improvement of endothelial function after treatment with infliximab may be mediated by a reduction in CD4⁺CD28^null T cells.^21,30

From these previous studies, it can be speculated that modulation of CD4⁺CD28^null T cells might represent a new therapeutic target in patients with UA. Indeed, a reduction in CD4⁺CD28^null T-cell percentage has been associated with clinical benefits in patients with rheumatoid arthritis.²² Pawlik et al²² have demonstrated that in patients with rheumatoid arthritis, treatment with infliximab achieved a 36% decrease in CD4⁺CD28^null T cells together, with an improvement in symptoms and functional capacity. Interestingly, the degree of CD4⁺CD28^null T-cell reduction observed by Pawlik et al is similar to that observed in our study, suggesting that our findings may be clinically relevant in patients with UA. It can be speculated that in patients with UA, in vivo reduction in CD4⁺CD28^null T cells by infliximab might translate into lower recurrence of angina and cardiac events. Obviously, because this is an ex vivo study, we cannot draw conclusions on the potential clinical benefits of infliximab; controlled randomized trials with clinical end points are warranted to evaluate whether CD4⁺CD28^null T-cell reduction in patients with UA translates into a clinical benefit. Importantly, trials of anti–TNF- monoclonal antibodies in UA should be limited to patients with normal or nearly normal left ventricular function. Indeed, despite the evidence of a role of TNF- in the evolution and progression of heart failure,³¹ recent data from the Randomized Etanercept Worldwide Evaluation (RENEWAL) and Anti-TNF Therapy Against Congestive Heart failure (ATTACH) trials demonstrated that TNF- blockade in patients with heart failure was associated with a higher prevalence of cardiac events.³² This suggests that the elevation of TNF- in patients with heart failure might play an adaptive role, similar to that conceivable for natriuretic peptides.³³

Previous studies suggest that inflammation may well represent a therapeutic target in patients with UA. In an open-label, randomized trial, meloxicam, an antiinflammatory drug, has been shown to improve the outcome of patients with non–ST-elevation acute coronary syndrome.³⁴ In addition, high doses of statins might improve the outcome of acute coronary syndromes through an antiinflammatory effect.³⁵ Interestingly, in a recent observational study by our group, it has been found that in patients with UA, statin treatment is associated with a 23% reduction in CD4⁺CD28^null T-cell percentages, suggesting that statins also might modulate the CD4⁺CD28^null T-cell expansion.³⁶ This reduction, however, is smaller than that observed in this study through selective blockade of TNF- (33%).

Study Limitations
Some limitations need to be addressed. First, this is an ex vivo study with a small population. Hence, clinical trials are warranted to confirm whether our very early findings are clinically relevant. Nevertheless, the results of the present study show for the first time that in patients with UA, modulation of the T-cell repertoire might be achieved by a selective blockade of cytokines. In addition, it is difficult to establish whether the infliximab concentrations used in this study are effective in vivo. However, similar concentrations have been shown to be associated with clinical benefits in patients with rheumatoid arthritis. Finally, a control group of healthy subjects was not included in the study protocol. This choice was based on the observation that the frequency of CD4⁺CD28^null T cells is <1% in healthy subjects and in patients with stable angina. Therefore, it is difficult to detect downregulation of these cells by infliximab. Nevertheless, CD4⁺CD28^null T-cell percentage was reassessed at 1 year after discharge in 8 stabilized patients. At this time, CD4⁺CD28^null T-cell percentage was lower than that observed in the acute phase of UA, suggesting that the reduction in CD4⁺CD28^null T cells might be associated with clinical stabilization.

Conclusions
The present study shows that in patients with UA, high levels of TNF- are associated with a higher percentages of CD4⁺CD28^null T lymphocytes and that infliximab, an anti–TNF- monoclonal antibody, reduces the percentage of CD4⁺CD28^null T cells. Clinical studies are warranted to evaluate whether modulation of CD4⁺CD28^null T cells in patients with UA might also result in clinical benefits.

	Acknowledgments

 
We give special acknowledgement to Professor Gianfranco Ferraccioli, Rheumatology Department, Catholic University Rome, who kindly provided infliximab.

Disclosures

None.

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CLINICAL PERSPECTIVE

Despite the best treatments currently available, in patients with unstable angina (UA) the rate of adverse cardiac events is still high (15% to 20% at 6 months). This finding highlights the need to explore new therapeutic strategies in these patients. Accumulating data indicate that inflammation plays a key role in the pathogenesis of UA and may represent a therapeutic target. In this ex vivo study, we evaluated whether in patients with UA inflammation may be modulated by infliximab, an anti–tumor necrosis factor- monoclonal antibody currently used in patients with rheumatoid arthritis unresponsive to standard therapy. Patients with UA showed a high percentage (median, 6.2%; range, 1.2% to 23.9%) of circulating CD4⁺CD28^null T lymphocytes, a proinflammatory T-cell subset that infiltrates coronary plaques and might be responsible for plaque instability. CD4⁺CD28^null T-cell percentage was associated with higher levels of tumor necrosis factor- (>3 pg/mL). After incubation of whole blood with infliximab, a significant decrease in CD4⁺CD28^null T-cell percentage was observed. The modulation of CD4⁺CD28^null T cells in patients with UA may have important clinical implications, similar to what was demonstrated in rheumatoid arthritis; interestingly, the degree of CD4⁺CD28^null T-cell reduction observed in our study (33%) was similar to that associated with improvement of symptoms in patients with rheumatoid arthritis. It can be speculated that in patients with UA, in vivo reduction in CD4⁺CD28^null T cells might translate into lower cardiac event recurrence. We hope that our results, if confirmed in other preclinical studies, will stimulate the need for large controlled randomized trials to evaluate whether CD4⁺CD28^null T-cell reduction in UA translates into clinical benefits.

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