Expression of CTLA-4 (CD152) in peripheral blood T cells of children with influenza virus infection including encephalopathy in comparison with respiratory syncytial virus infection

Abstract

Influenza virus and respiratory syncytial virus (RSV) are the most common causes of acute severe respiratory infection in children during the winter. There have been few reports about peripheral blood T cell activation in vivo in influenza virus infection and conflicting results concerning peripheral blood T cells activation in RSV infection. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, CD152) is a receptor present on T cells that plays a critical role in the down-regulation of antigen-activated immune responses. To clarify the status of peripheral blood T cells, we investigated intracellular CTLA-4 expression in T cells in patients with influenza virus and RSV infection. We collected blood samples from 15 patients with influenza virus infection, including three with complications of influenza virus-associated encephalopathy and 18 patients with RSV infection, as well as 44 healthy children. We determined the intracellular expression of CTLA-4 in CD4⁺ and CD8⁺ T cells by flow cytometry. There were no significant differences in the percentages of intracellular CTLA-4-positive CD4⁺ T cells and CD8⁺ T cells by age. The percentages of intracellular CTLA-4-positive CD4⁺ T cells in the patients with influenza virus infection were significantly higher than those in healthy children (P < 0·01). In particular, the patients with influenza virus-associated encephalopathy had sevenfold higher percentages of CTLA-4-positive CD4⁺ T cells than influenza patients without encephalopathy (P < 0·05). The patients with influenza virus-associated encephalopathy had increased percentages of CTLA-4-positive CD8⁺ cells at the acute stage in comparison with the convalescent stage and in control subjects (P < 0·01, respectively). RSV patients showed no increase in CTLA-4-positive CD4⁺ T cells or CD8⁺ T cells. The immunological status of peripheral T cell activation is substantially different in influenza virus infection and RSV infection. The patients with RSV infection did not show any increase in CTLA-4-positive peripheral blood T cells. There was a remarkable increase in intracellular CTLA-4 in CD4⁺ and CD8⁺ T cells in influenza virus-associated encephalopathy. Down-regulation of antigen-activated peripheral blood T cell activation might play an important role in the pathogenesis of influenza virus-associated encephalopathy and host defence against influenza virus infection.

INTRODUCTION

Influenza virus and respiratory syncytial virus (RSV) are the most common causes of acute severe respiratory infection occurring in young children during the winter. Complications of influenza virus infection include viral pneumonia, bronchiolitis and central nervous system (CNS) involvement with encephalopathy (influenza virus-associated encephalopathy) [1–3]. On the other hand, primary RSV infection of infants often results in acute bronchiolitis, leading to obstruction of the airways that is associated with an inflammatory response to the infection [4]. Influenza virus infection causes a more systemic response, while RSV infection causes local inflammation in the respiratory tract. Influenza virus and RS viruses infect primarily upper respiratory tract epithelial cells. Also, macrophages and T cells play an important role in initiating innate and adaptive immune responses against viruses, whereby inflammation is caused. It has been shown in vitro that only monocytes/macrophages among leucocytes are highly susceptible to influenza virus infection, and increased serum levels of tumour necrosis factor (TNF)-α, interleukin (IL)-1 and IL-6 have been found during acute influenza [5]. On the other hand, increases in cytokines and chemokines were noted in nasal secretion and lungs of patients with RSV bronchiolitis, but not in sera [6–8]. Evaluation of protein levels and mRNA levels of cytokines in peripheral blood of RSV infection cases gave conflicting results. Although many studies on the mechanism in vitro and with mice models of both influenza virus and RSV infections are ongoing, the immunology regarding these infections in human peripheral blood is poorly defined [9]. In particular, there have been few studies in vivo on the activation of peripheral blood T cells in these infections.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, CD152) is a receptor present on T cells that plays a critical role in the down-regulation of antigen-activated immune responses. CTLA-4 is a surface molecule on activated T cells exhibiting sequence homology to CD28 [10]. While CD28 is an important co-stimulator of T cell activation, the essential inhibitory function of CTLA-4 is to maintain homeostasis of the immune system [11]. After activation in vitro, CTLA-4 is expressed transiently on both CD4⁺ and CD8⁺ T cells [12]. CTLA-4 is rapidly internalized intracellularly through endocytosis and accumulates in the cytoplasm [13]. T cells expressing intracellular CTLA-4 might reflect the proceeding activation of T cells following antigenic stimulation of the TCR.

We have already reported that the intracellular expression of CTLA-4 is up-regulated in peripheral blood T cells in Kawasaki disease (KD) and Epstein–Barr virus infectious mononucleosis (EBV-IM) at the acute stage [14]. To clarify the down-regulation of peripheral blood T cell activation, we investigated intracellular CTLA-4 expression in T cells in patients with influenza virus infection and RSV infection.

MATERIALS AND METHODS

Subjects

We studied 15 patients with influenza virus infection, including three with the complication of influenza virus-associated encephalopathy, and 18 patients with RSV infection, who were seen at our hospital between December 2001 and February 2004. Forty-four healthy children were also studied as normal controls. All of them were Japanese. Informed consent for participation was obtained from the subjects' parents.

Influenza virus infection

The patients comprised six boys and nine girls (0·6–13 years of age; median 3·5 years). The diagnosis was based on clinical presentation of a sore throat and fever, with virus antigen detection in the throat with the latex agglutination test using a Capilia Flu A/B kit (Becton Dickinson) and/or a fourfold increase in the antibody titre determined with the haemagglutination inhibition test. The criteria for the diagnosis of influenza virus-associated encephalopathy were clinical symptoms and signs compatible with acute encephalopathy, which consisted of a febrile disorder with alteration of consciousness and slow activity on electroencephalography lasting for more than 24 h after an acute onset, and no bacteria or fungi in CSF cultures, with exclusion of all other neurological, vascular, metabolic, endocrine, toxic and drug-induced disorders [2,3]. Three of the 15 patients with influenza virus infection had the complication of influenza virus-associated encephalopathy.

The day of onset of fever was defined as the first day of illness. Samples were taken from the patients at the acute stage, the second to third day of illness (mean: 2·5), and at the convalescent stage (mean: 20·4).

RSV infection

The patients comprised 10 boys and eight girls (0·1–1·2 years of age; median 0·5 years). A definitive diagnosis of RSV infection was dependent on the detection of the viral antigen in the nasopharyngeal fluid using an antigen detection kit, BD Directogen EZ RSV (Becton Dickinson). The patients with RSV infection had severe bronchiolitis and/or pneumonia and thus needed to be hospitalized. The patients who had the complication of a bacterial infection, such as otitis media, were excluded.

Healthy controls

We tested samples from 44 healthy controls consisting of 24 boys and 20 girls (0·2–13·2 years of age; median, 2·5 years) in parallel with the samples from the patients. Twenty-two of them were infants under 2 years of age.

Measurement of intracellular CTLA-4

We detected intracellular CTLA-4 expression in CD4⁺ T and CD8⁺ T cells by flow cytometry, as reported previously [14]. Briefly, whole blood (100 µl) was stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 monoclonal antibody (MoAb) (Becton Dickinson) or anti-CD8 MoAb (Becton Dickinson). Erythrocytes were incubated with 2 ml of a lysing solution (Becton Dickinson) for 10 min. After washing with phosphate-buffered saline containing 0·5% bovine serum albumin and 0·1%NaN₃ (washing buffer), the leucocytes were suspended in FACS permeabilizing solution (Becton Dickinson) for 10 min. The cells were stained with phycoerythrin (PE)-conjugated anti-CD152 (CTLA-4) MoAb (Pharmingen) for 30 min, rinsed with washing buffer and then resuspended in 1%paraformaldehyde. Stained cells were analysed with a FACScan flow cytometer (Becton Dickinson) equipped with a 15-mW argon ion laser, and a filter set for FITC (530 nm) and PE (585 nm). Intracellular CTLA-4 was examined in CD4⁺ and CD8⁺ T cells that had been identified and selected by means of gating. Data were acquired with CELLQuest software, using a fluorescence threshold. All events with 10 000 cells were acquired by setting acquisition gate on CD4⁺ T cells or CD8⁺ T cells in SSC-T cells marker dot plot. The intracellular expression of CTLA-4 was analysed by PE staining, and the number of cells that were positive for CTLA-4 was expressed as a percentage of the CD4⁺ or CD8⁺ T cells. FASTIMMUNE IgG1 PE served as an isotype control (Becton Dickinson).

Statistical analysis

Data were analysed statistically using the Mann–Whitney U-test for comparison of means and Spearman's correlation coefficient by rank test.

RESULTS

Figure 1 shows typical dot plots on flow cytometry by peripheral blood CD4⁺ and CD8⁺ T cells obtained from patients, i.e. a 1·6-year-old girl with influenza virus infection complicated with influenza virus-associated encephalopathy, a 10-year-old boy with influenza virus infection without encephalopathy, a 1-year-old boy with RSV infection and a control (1-year-old healthy boy), respectively. The girl with influenza virus-associated encephalopathy exhibited the highest expression of intracellular CTLA-4 in peripheral blood T cells, the levels being 28·9%in CD4⁺ T cells and 39·8%in CD8⁺ T cells. The 10-year-old boy with influenza virus infection without encephalopathy exhibited a slight increase in CTLA-4-positive CD4⁺ T cells (3·5%), but not in CD8⁺ T cells (2·2%). The patient with RSV infection and the control subject had low percentages of CTLA-4-positive CD4⁺ and CD8⁺ T cells.

Fig. 1.

Typical dot plot of intracellular CTLA-4 expression in peripheral blood CD4⁺ T lymphocytes (a, b, c, d) and CD8⁺ T lymphocytes (e, f, g, h) from a 1·6-year-old girl with influenza virus-associated encephalopathy (a, e), a 10-year-old boy with influenza virus infection without encephalopathy (b, f) and a 1-year-old boy with RSV infection (c, g) during the acute stage, and a 1-year-old healthy boy (d, h). The percentages are of cells expressing CTLA-4 among CD4⁺ or CD8⁺ T lymphocytes. The upper right quadrant shows CTLA-4-positive cells. CTLA-4 expression was more increased in CD4⁺ and CD8⁺ T cells in influenza with encephalopathy, compared with cells without encephalopathy, RSV infection and the healthy control.

To determine the differences, if any, of age, we compared the CTLA-4-positive CD4⁺ cells and CD8⁺ T cells between the infants and children who were older than 2 years as controls. There were no significant differences in the CTLA-4-positive CD4⁺ T cells and CD8⁺ T cells between the two groups (Table 1). The percentages of intracellular CTLA-4 positive CD4⁺ T cells in the patients with influenza virus infection were significantly higher than those in the healthy children (P < 0·01) (Table 1). In particular, the patients with influenza virus-associated encephalopathy had sevenfold higher percentages of CTLA-4-positive CD4⁺ T cells compared with those in the influenza patients without encephalopathy (P < 0·05). Although the percentages of CTLA-4-positive CD8⁺ T cells were not increased in the patients with influenza virus infection without encephalopathy, the patients with influenza virus-associated encephalopathy had significantly increased percentages of CTLA-4-positive CD8⁺ T cells at the acute stage compared to the convalescent stage and in control subjects (P < 0·01, respectively). There were no significant differences in the percentages of the CTLA-4-positive cells among either CD4⁺ T cells or CD8⁺ T cells in the patients with RSV infection in comparison with control subjects.

Table 1.

.Intracellular CTLA-4 expression in T cells obtained from patients with influenza and RSV infection, and control subjects

	n	CD152+CD4+ T cells	CD152+CD8+ T cells
Influenza
Acute stage	15	5·1 ± 7·5*	8·0 ± 14·9
With encephalopathy	3	16·7 ± 11·0**	33·4 ± 14·7**
Without encephalopathy	12	2·2 ± 1·7*	1·1 ± 0·8
Convalescent stage	7	1·1 ± 0·6	1·1 ± 1·0
RSV infection
Acute stage	18	1·6 ± 0·8	1·2 ± 1·2
Controls	44	1·3 ± 0·8	1·0 ± 1·1
Children (older than 2 years)	22	1·1 ± 0·7	0·7 ± 1·2
Infants	22	1·4 ± 1·0	1·0 ± 1·0

Values are expressed as means ± s.d.

^*Significant at P < 0·01 versus convalescent stage and control subjects;

^**significant at P < 0·05 versus patients without encephaopathy. CTLA-4: cytotoxic T lymphocyte-associated antigen 4, CD152. RSV: respiratory syncitial virus.

DISCUSSION

CTLA-4 is responsible for homeostasis of the immune system through regulatory T cells [10–13]. The expression of CTLA-4 on T cells depends on cell activation induced by the CD28–B7 interaction, which is essential for T cell activation caused by contact with antigen-presenting cells. In this study, we investigated intracellular CTLA-4 expression in T cells to clarify the down-regulation of peripheral blood T cell activation in patients with influenza virus and RSV infections as common causes of respiratory infectious diseases in young children. We found that intracellular CTLA-4 expression is up-regulated in peripheral blood CD4⁺ T cells at the acute stage of influenza virus infection by flow cytometry, but not in patients with RSV infection. Because there were no differences in CTLA-4 expression in T cells between infants and young children, the difference in CTLA-4 expression between influenza virus and RSV patients was not caused by the different age distributions. In addition, the patients with the complication of influenza virus-associated encephalopathy exhibited significantly increased expression of intracellular CTLA-4 in both CD4⁺ T and CD8⁺ T cells at the acute stage.

Influenza virus-associated encephalopathy has been reported frequently as a severe complication of influenza virus infection in Japanese children [2,3]. The typical clinical features include a high fever, convulsions and a rapidly progressive coma, and death in approximately 25%cases and neurological sequelae in 25%cases. On pathological examination, vascular occlusion, microthrombus, haemorrhage and oedema around vessels were noted in the brain, without inflammatory monocyte infiltration or detectable viral antigen in brain tissue [15]. The main pathological change may be vascular damage to the CNS and destruction of the blood–brain barrier. We have reported previously that inflammatory cytokines, such as TNF-α and IL-6, are increased in sera and cerebrospinal fluid obtained from the patients with influenza virus-associated encephalopathy [16]. Previous studies have shown that human influenza viruses infect macrophages, and induce the release of TNF-α, IL-6 and IL-1β, chemokines and IL-10 [5,17]. We have shown in this study that massive down-regulation of peripheral blood T cell activation occurs in influenza virus-associated encephalopathy. It has been reported that influenza virus was detected on polymerase chain reaction (PCR) in peripheral blood mononuclear cells obtained only from patients with influenza virus-associated encephalopathy, but not from those without [18]. Taking these reports into consideration, our results suggest that there might be an antigen, such as influenza virus-infected cells, in the peripheral blood of patients with influenza virus-associated encephalopathy, as CTLA-4 is expressed only following stimulation of the TCR on contact with antigen-presenting cells.

Although the expression of CTLA-4 on CD4⁺ T cells is increased in patients with malaria [19], human immunodeficiency virus infection [20] and systemic lupus erythematosus [21], the levels of CTLA-4 expression on CD8⁺ T cells from patients with these diseases are not increased. We have reported previously that the intracellular expression of CTLA-4 in peripheral blood CD8⁺ T cells is increased slightly in KD, and increased markedly in EBV-IM patients [14]. The levels of CTLA-4 positive CD8⁺ T cells were as high in influenza virus-associated encephalopathy as in EBV-IM. EBV-IM is known for the remarkable activation of peripheral blood CD8⁺ T cells. The patients with influenza virus-associated encephalopathy exhibited similar activation of peripheral blood CD8⁺ T cells to those with EBV-IM in terms of CTLA-4 expression. In vitro studies have shown that influenza virus-specific CD8⁺ cytotoxic T lymphocytes are necessary for the clearance of influenza viruses during infection [22]. The down-regulation of peripheral blood CD8⁺ T cell activation might be related to the host defence against influenza virus infection, while the mechanism causing the vascular damage in the CNS of influenza virus-associated encephalopathy is unknown. On the other hand, patients with RSV infection did not show any increase in CTLA-4-positive T cells. It has already been reported that peripheral blood T cell subset analysis, including CD4, CD8, CD16, CD25 and gamma-delta T cells, showed no changes during acute RSV bronchiolitis [23]. We have already reported that analysis of lymphocyte function-associated antigen (LFA)-1α on T cells, which is one of the important adhesion molecules for T cell activation, revealed a significantly higher percentage of strongly LFA-1α positive cells in influenza patients compared to age-matched controls, but not in RSV patients [24]. These data, together with those of our present study of CTLA-4 expression, show that there is little peripheral blood T cell activation in patients with RSV infection. The lack of T cell activation in the peripheral blood during acute RSV infection might be due to redistribution of T cells from the circulation towards the lungs.

In conclusion, the immunological status of peripheral T cell activation is substantially different in influenza virus infection and RSV infection. The patients with RSV infection did not show any increase in CTLA-4-positive T cells. We found that the patients with influenza virus infection without encephalopathy exhibited a slight increase in CTLA-4 positive CD4⁺ T cells. There was a remarkable increase in intracellular CTLA-4 in CD4⁺ and CD8⁺ T cells in influenza virus-associated encephalopathy. Down-regulation of antigen-activated peripheral blood T cell activation might play an important role in the pathogenesis of influenza virus-associated encephalopathy and host defence against influenza virus infection.