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Age-Related Decrease in Adenovirus-Specific T Cell Responses

  1. Martina Sester1,
  2. Urban Sester1,
  3. Susana Alarcon Salvador1,
  4. Gunnar Heine1,
  5. Sabine Lipfert2,
  6. Matthias Girndt1,
  7. Barbara Gärtner3 and
  8. Hans Köhler1
  1. 1Medical Department IV, Institute of Medical Microbiology and Hygiene, University of the Saarland, Homburg, Germany
  2. 2Departments of Pediatrics, Institute of Medical Microbiology and Hygiene, University of the Saarland, Homburg, Germany
  3. 3Departments of Virology, Institute of Medical Microbiology and Hygiene, University of the Saarland, Homburg, Germany
  1. Reprints or correspondence: Dr. Hans Köhler, University of the Saarland, Medical Dept. IV, Nephrology, D-66421 Homburg, Germany (inhkoe{at}uniklinik-saarland.de).
 
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Abstract

Infections with persistent viruses, such as cytomegalovirus (CMV) or adenovirus, are not, in general, clinically apparent but may cause serious complications in the immunocompromised host. As has been shown for CMV, the cellular arm of the immune response is essential in controlling viral replication. However, cellular immunity toward adenoviruses has not been well characterized in humans. The aim of the present study was the quantitative and functional analysis of adenovirus-specific T cell responses from 171 healthy individuals and 59 long-term renal transplant recipients by use of flow-cytometric, as well as standard proliferation and enzyme-linked immunosorbant, assays. Adenovirus-specific immunity is dominated by CD4 T cells with memory/effector phenotype.Of interest, the frequency of adenovirus-specific T cells decreases significantly with age. This age-related decline indicates the eventual elimination of adenoviruses within a lifetime that may explain the well-known clinical observation of a predominant incidence of adenoviral complications in children and young adults, compared with older adults, after transplantation.

Adenoviruses are nonenveloped viruses with a double-stranded DNAgenome of ∼36,000 bp. As yet, 51 different human serotypes have been identified and are classified into 6 subgenera, A–F [1, 2]. Adenoviruses can establish acute, as well as persistent, infections [3] that are, in general, mild or even subclinical in the healthy population but can be severe or even fatal in immunocompromised hosts [3–5]. Depending on the serotype, adenoviruses have tropism for the respiratory, gastrointestinal, or urinary tracts or the eye and may cause different disease manifestations, such as viral interstitial pneumonia, colitis, gastroenteritis, hemorrhagic cystitis, hepatitis, conjunctivitis, and disseminated disease [5–7].

Persistent viruses have established strategies to counteract the host immune system. The early transcription unit 3 (E3) of the adenoviral genome has been implicated in the establishment of a persistent infection. Although the E3 region is dispensable for adenovirus replication in vitro, it is present in all human adenoviruses and encodes proteins with immunomodulatory functions [8–10]. Various molecularmechanisms of E3 proteins have been identified that mediate escape from recognition and elimination by the host immune system. Binding of the E3 protein 19K to major histocompatibility class (MHC) I molecules in the endoplasmic reticulum of infected cells prevents the presentation of viral peptides to cytotoxic T lymphocytes and cell lysis [11, 12]. The E3 protein 14.7K, as well as the complex of the E3 proteins 10.4K and 14.5K, mediate resistance against tumor necrosis factor (TNF)-α [8–10, 13], and the expression of 10.4K and 14.5K induces the internalization and degradation of the apoptosis receptor Fas from the cell surface of infected cells [14–16].

Similar strategies have been used by other persistent viruses, such as cytomegalovirus (CMV).All these immune evasion mechanisms target the cellular arm of immune responses and result in a well-balanced equilibrium between viral replication and the T cell responses in the immunocompetent host [17]. As a consequence, any kind of immunosuppression may alter this equilibrium, thereby facilitating viral replication and the eventual progression to symptomatic disease. Hence, in transplant recipients who are taking systemic immunosuppressive medication, infections with persistent viruses such as CMV are among the viral complications most frequently observed [18–20]. The importance of adenovirus as a cause of disseminated disease has remained rather underappreciated. More recently, however, the overall importance of this virus has been emphasized mainly in pediatric patients after both solid-organ and stemcell transplantation [21–24]. In a large study among cardiac allograft recipients, adenovirus not only was the pathogen isolated most often from various clinical specimens but also was associated with reduced graft survival and with acute rejection [23].

A hallmark of successful containment of viral replication is the generation of antigen-specific T cells [25, 26]. In response to an acute infection, the frequencies of these T cells may dramatically increase and normally decline after successful control of the virus [25, 27–29]. In the case of persistent viruses of both humans and animals, however, the continuous antigenic challenge has been shown to be associated with the sustained presence of antiviral T cells [30–34]. As a consequence, the presence of detectable virus-specific T cells can be considered as an indicator for the presence of a particular virus. In contrast, the absence of detectable virus-specific immunitymay reflect either the complete absence of that particular virus or a pathological situation in which that virus is present and left uncontrolled. The former holds true for either uninfected individuals or individuals who have successfully cleared the virus. The latter situation may apply for immunocompromised patients who have viral complications. In the case of renal transplant recipients, a medication-induced decrease in CMV-specific immunity indeed correlates with an uncontrolled viral replication and progression to symptomatic disease [30].

Although both the frequencies and the function of virus-specific T cell responses have been studied extensively for human pathogens such as CMV, little is known about the role of cellular immune responses in the control of human adenoviruses in vivo. Because both viruses share similar immune evasion strategies and increased rates of infectious complications after transplantation, comparable immunologic control mechanisms may apply to limit viral replication. Thus, the present study was carried out to characterize both the frequency and the functional properties of adenovirus-specific T cells in healthy and immunosuppressed individuals.

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Materials and Methods

Subjects. The study was conducted among 171 healthy individuals (mean age [±SD], 48.3 ± 21.2 years; range, 19.3–96.8 years) and 59 long-term renal transplant recipients (mean age [±SD], 49.3 ±13:8 years; range, 21.3–75.7 years; mean time posttransplantation [±SD], 5.9 ± 4.2 years; range, 7.1 months–14.9 years).All transplant recipients had stable graft function and received a maintenance immunosuppressive double- or triple-drug regimen that consisted of a calcineurin inhibitor, steroids, and/or azathioprine. Blood was drawn in the morning at drug trough levels before intake of immunosuppressive drugs.

Stimulation of adenovirus-specific CD4 and CD8 T cells within whole blood. Stimulation of adenovirus-specific CD4 T cells was performed in heparinized whole blood, essentially as described elsewhere for CMV- or human immunodeficiency virus (HIV)-specific T cells [30, 31, 35].As a stimulus, titered amounts of adenovirus antigen with a broad specificity for all serotypes of adenoviruses (complement fixation reagent; BioWhittaker) were used in the presence of 1 μg/mL αCD28 and αCD49d (clones L293 and 9F10; BD PharMingen), respectively. The antigen was derived from a whole lysate of infected KB cells. As negative controls, blood cells were stimulated with control antigen that was derived from mock-infected cells (BioWhittaker). In selected individuals, additional stimulations were carried out by use of 2.5 μg/mL Staphylococcus aureus enterotoxin B (SEB; Sigma). Cells were incubated in polypropylene tubes at 37°C for a total of 6 h. During the last 4 h, 10 μg/mL of Brefeldin A (Sigma) was added to block extracellular secretion of cytokines. Thereafter, the blood was treatedwith 2mMEDTA for 15 min. Subsequently, erythrocytes were lysed, and leukocytes were fixed for 10 min by use ofBDlysing solution (BD PharMingen), according to the manufacturer's instructions. Cells were washed once with FACS buffer (PBS, 5% filtered fetal calf serum, 0.5% bovine serum albumin, and 0.07% NaN3) and either were processed immediately for flow-cytometric analysis or were left overnight at 4°C.

Determination of the frequency and characterization of adenovirus-specific T cells by flow cytometry. Flow-cytometric staining was done essentially as described elsewhere [30, 31]. In brief, fixed leukocytes were permeabilized with 2 mL FACS buffer that contained 0.1%saponin (Sigma) for 10min at room temperature. Thereafter, theywere immunostained for 30min at room temperature in the dark, using the saturating conditions of the following antibodies: anti- CD4 or anti-CD8 (clones SK3 or SK1), anti-interferon (IFN)-γ (clone 4S.B3), and anti-CD69 (clone L78; all from BD PharMingen). In selected individuals, adenovirus-specific T cellswere further characterized by use of antibodies directed against the following antigens: CD45RO (clone UCHL1), interleukin (IL)-2 (clone MQ1-17H12), IL-4 (clone 8D3-8), IL-5 (clone JES1-39D10; all from BD Phar-Mingen), TNF-α (clone 6402.3; R&D Systems), and perforin (clone δG9; Ancell). After staining, cells were washed once with 3 mL of FACS buffer and fixed with 1% paraformaldehyde. At least 10,000 CD4+ or CD8+ lymphocytes were analyzed on a FACScan with Cellquest software (both from Becton Dickinson). Although control antigens did usually not stimulate any IFN-γ production, the percentage of specific T cells was calculated by subtraction of the frequency obtained by the respective control stimulations. Duplicate determinations of single specimens varied by <5%.

Determination of antigen-specific proliferation. Peripheral blood mononuclear cells (PBMC) were isolated by use of a Ficoll density gradient (d = 1.077; Linaris); 1 × 105 PBMC per well were stimulated in triplicate, as described above, by use of adenovirus antigen, control antigens, or SEB.Moreover, samples were also stimulated with 62.5 ng/mL phytohemagglutinin (PHA; Biochrom). Cells were pulsed with 9.25 kBq/well 3H-thymidine after 3 and 5 days, respectively, and incubated for a further 20 h. Thereafter, counts per minute were determined by use of a standard harvesting apparatus (Wallac). The stimulation index (SI) is defined as the counts per minute after antigen-specific stimulation divided by the counts per minute of the respective control stimulations.

Determination of antigen-specific IFN-γ secretion. PBMC were isolated by use of density gradient centrifugation; 1 × 105 PBMC per well were stimulated as described above with use of adenovirus antigen, control antigen, SEB, or PHA. Supernatants were harvested after 3 and 5 days, respectively, and analyzed for the presence of IFN-γ by use of a standard ELISA (BD PharMingen). The lower limit of cytokine detection was 7.8 pg/mL. The amount of specific cytokine production was calculated by subtraction of the amount obtained by the respective control stimulations.

Determination of adenovirus serostatus. The adenovirus serostatus was determined by a commercial adenovirus IgG test (Serion ELISA classic; Virion/Serion). According to the manufacturer, low titers are <14 U/mL, moderately high titers are 14–19 U/mL, and high titers are >19 U/mL.

Statistical analysis. Statistical analysis was performed by use of Prism V3.02 software (Graphpad). Significant differences among 2 or 3 groups were determined by use of the Mann-Whitney U or Kruskal-Wallis test, respectively. The correlation between T cell frequencies and either proliferative responses or IFN-γ secretion was calculated according to Pearson's correlation coefficient. The χ2 test was used to address the association between age and T cell responses below or above the detection limit or serological responses <14 or ⩾14 U/mL, respectively.

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Results

Adenovirus-specific T cell responses are dominated by CD4 T cells. Heparinized blood of healthy donors was stimulated for 6 h with use of whole adenovirus antigen.During this time, effector and memory CD4 and CD8 T cells were specifically stimulated, which resulted in the up-regulation of CD69 and the production of cytokines [35–37]. Thereafter, the frequency of responding T cells was determined by the combined flow-cytometric analysis of the early T cell activation marker CD69 and intracellular IFN-γ induction in both CD4 and CD8 T cells. As shown in a typical example (figure 1A), adenovirus antigen induced IFN-γ production in 0.43% of CD4 T cells, whereas respective CD8 T cells were not detectable (data not shown). The lack of detectable CD8 T cells could be due to a less efficient generation of MHC class I peptides from soluble antigens. However, when soluble CMV or HIV gag antigen is used, specific CD8 T cells can be detected readily [30, 31]. Stimulations with control antigen and SEB were carried out as negative and positive controls, respectively. In neither CD4 nor CD8 T cells did control antigen induce any relevant cytokine induction, whereas SEB readily did (figure 1A) [30, 31].

Figure 1
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Figure 1

Adenovirus-specific T cells detected directly from whole blood by use of flow cytometry. Cells were gated on CD4+ lymphocytes. A, Specifically activated cells were identified as being positive for CD69 and interferon (IFN)-γ, interleukin (IL)-2, or tumor necrosis factor (TNF)- α. Typical dot plots of T cells are shown after stimulation with control antigen (Co), adenovirus antigen (Ad), and Staphylococcus aureus enterotoxin B (SEB). The frequency of TNF-α-positive CD4+ T cells was 1.3-fold higher than that of cells positive for IFN-γ. However, because there is a highly significant correlation between the percentage of IFN-γ- and TNF-α-positive T cells (r = 0.97; P < .0001, Pearson's correlation coefficient), the percentage of IFN-γ-positive cells is a representative measure for the total virus-specific T cell immune response in each individual. B, Adenovirus-specific CD4+ T cells are positive for CD45RO and negative for the expression of perforin. The percentages of CD45RO-positive and perforin-negative cells, respectively, among IFN-γ-positive CD4+ T cells are indicated. A representative example among 15 individuals is shown.

Adenovirus-specific CD4 T cells were further characterized by staining a variety of cytokines, memory markers, and perforin. Adenovirus-specific T cells were of the CD45 ROphenotype (figure 1B), with expression of Th1 cytokines such as IFN-γ, IL-2, or TNF-α (figure 1A). Although all Th1 cytokine-positive T cells were of an activated phenotype (figure 1A), the typical Th2 cytokine IL-4 was not detectable in CD69+ T cells. IL-4, however, was readily induced after SEB stimulation (median, 1.42%; range, 0.34%–2.43%; n = 10; data not shown). Similarly, there was no adenovirus-specific IL-5 production, whereas up to 0.46% of SEB-reactive CD4+ T cells were positive for IL-5 (median, 0.13%; range,⩽0.05%–0.46%; n = 7, data not shown).Although a proportion of CD4+ T cells may have the capacity to produce perforin to an individually different extent (figure 1B, upper left quadrant ), adenovirus-specific T cells were always perforin negative (figure 1B, lower right quadrant ). Taken together, adenovirus- specific T cells can be identified directly from whole blood and detectable T cells are of effector/memory phenotype with the capacity to be rapidly activated. Moreover, specifically activated T cells were shown to produce Th1 cytokines.

Adenovirus-specific T cell responses decrease with age. The frequency of adenovirus-specific T cells was characterized in a cohort of both healthy control individuals (n = 171) and long-term renal transplant recipients (n = 59; figure 2A). Although absolute frequencies showed interindividual variations from ⩽0.05% up to 0.9%, measurable frequencies within a single individual were considerably stable (data not shown). Despite immunosuppressive medication, median frequencies of adenovirus- specific T cells did not differ between transplant recipients and control subjects (P = .66, Mann-Whitney U test). Of interest, however, in both groups, only ∼50% of all individuals had adenovirus-specific T cell frequencies above the detection limit of 0.05% (figure 2A and table 1). Further analysis of adenovirus-specific T cell frequencies in relation to the age of the respective blood donor clearly revealed that both the absolute frequencies and the percentage of individuals with detectable virus-specific T cells were significantly higher in younger individuals, compared with those of the older individuals (figure 2B and table 1). This was analyzed by separating both healthy control subjects and transplant recipients into 3 different groups: 18–39, 40–60, and >60 years old, respectively. Although 88% of both control subjects and transplant recipients in the youngest age group had T cell frequencies above the detection limit, the respective percentage steadily decreased with increasing age (table 1; P < .0001 for control subjects; P = .0094 for transplant recipients; both by the χ2 test). Similarly, the mean percentage of adenovirus-specific T cells was highest in the group of individuals 18–39 years old, compared with the older age groups (figure 2B; P < .0001 for control subjects; P = .006 for transplant recipients; both by the Kruskal-Wallis test). The progressive decline in T cell responses was adenovirus specific and not due to a general loss of IFN-γ-producing cells, because frequencies of T cells reactive toward SEB did not show any age-related differences in either control subjects (P = .54) or transplant recipients (P = .29; figure 2C). Taken together, adenovirus- specific T cell immunity shows a significant decline with increasing age. This applies both to the general population and to long-term transplant recipients under immunosuppressive medication.

Figure 2
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Figure 2

Frequencies of adenovirus-specific CD4+ T cells in healthy seropositive individuals (n = 171) and long-term transplant (Tx) recipients (n = 59). A, Frequencies of adenovirus-specific CD4+ T cells may range from ⩽0.05% (detection limit, dashed line) to 0.62% in healthy control subjects and from ⩽0.05% to 0.90% in transplant recipients. Median T cell frequencies did not differ between both groups (P = .66, Mann- Whitney U test). B, Mean T cell frequencies showed a significant decrease with age in both healthy control subjects (18–39 years old, 0.22% ± 0.14% [n = 74]; 40–60 years old, 0:09%±0:06% [n = 30]; and >60 years old, 0:06% ± 0:02% [n = 67]; P < .0001, Kruskal-Wallis test) and transplant recipients (18–39 years old, 0:17%±0:21% [n = 16]; 40–60 years old, 0:08%±0:05% [n = 27]; and >60 years old, 0:06%±0:02% [n = 16]; P = .006, Kruskal-Wallis test). C, T cells reactive to Staphylococcus aureus enterotoxin B (SEB) did not show any age-related changes in either control subjects (P = .54) or transplant recipients (P = .29; both by the Kruskal-Wallis test).

Adenovirus-specific antibody titers decrease with age. A major function of CD4 T cells is to provide help to B cells to secrete specific antibodies. Because adenovirus-specific CD4 T cell frequencies showed an age-dependent decline, adenovirusspecific IgG antibody titers were analyzed in healthy individuals 18–39, 40–60, and >60 years old (figure 3 and table 2). In line with results obtained for adenovirus-specific CD4+ T cells (figure 2B), the percentage of individuals with high adenovirusspecific IgG titers (⩾14 U/mL) is highest in the youngest age group and decreases with increasing age (table 2). Similarly, although all individuals had detectable adenovirus-specific IgG, mean titers significantly decreased with increasing age (figure 3; P < .0001).

Figure 3
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Figure 3

Relationship between titers of adenovirus-specific IgG antibodies and increasing age. Adenovirus-specific IgG was detectable in all healthy individuals. Mean titers showed a significant decrease with age (18–39 years old, 11.4±6:6 U/mL [n = 59]; 40–60 years old, 9.7±5:3 U/mL [n = 43]; and >60 years old, 6.5±4:8 U/mL [n = 50]; P .0001, Kruskal-Wallis test).

Figure 4
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Figure 4

Adenovirus-specific T cell frequencies and proliferative responses. A, The frequencies of adenovirus-specific T cells correlate with proliferative responses (stimulation index [SI]) after 5 days of stimulation; r = .59; P = .0002, Pearson's correlation coefficient). B, Proliferative responses after 3 and 5 days of antigenic stimulation in healthy individuals >50 years old (n = 17) are significantly lower, compared with those of their younger counterparts (n = 19; left panel; day 3, P = .0061; day 5, P = .0006). This difference was not found after polyclonal stimulation with either Staphylococcus aureus enterotoxin B (right panel; day 3, P = .31; day 5, P = .35) or phytohemagglutinin (data not shown; day 3, P = .45; day 5, P = .93). Because of the lower total number of individuals, 2 instead of 3 groups were chosen. The mean age in the 2 groups was statistically different (31.6±7.7 vs. 67.4±18.6 years; P < .0001, Mann-Whitney U test).

Figure 5
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Figure 5

Correlation of interferon (IFN)-γ secretion after stimulation with adenovirus antigen and age. Healthy individuals were separated into 2 age groups ⩽50 (n = 13) and >50 years old (n = 8). IFN-γ was analyzed from supernatants of peripheral blood mononuclear cells after stimulation with adenovirus antigen (A) or Staphylococcus aureus enterotoxin B (SEB; B) for 3 and 5 days. Adenovirus-specific IFN-γ secretion was significantly lower in the older group (day 5, P = .0008; day 3, P = .0004; Mann-Whitney U test for all comparisons). In contrast, IFN-γ secretion after 5 days of SEB stimulation was not significantly different in both age groups (P = .88). The amount of specific cytokine production that was calculated by subtraction of the amount obtained by the respective control stimulations is given.

Table 1
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Table 1

Adenovirus-specific T cell frequencies, by control subject or transplant patient status and age group.

Table 2
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Table 2

Adenovirus-specific antibody titers in healthy individuals, by age group.

The frequency of adenovirus-specific T cells correlates with proliferative responses. Adenovirus-specific T cells were characterized by their capacity to rapidly produce cytokines within 6 h of antigenic stimulation. To prove the reliability of the method and to further assess the functional properties of adenovirusspecific T cells, flow-cytometrically determined T cell frequencies were compared with responses measured by standard T cell proliferation assays. PBMC from healthy control subjects were stimulated with adenovirus antigen and control antigen for 3 and 5 days, respectively, and pulsed with 3H-thymidine for a further 20 h. Frequencies determined by flow cytometry showed a strong correlation with proliferative responses after both 3 (r = 0.43; P = .0091) and 5 days (figure 4A; r = 0.59; P = .0002).

The age-dependent changes in proliferative responses were analyzed in individualswho were divided into 2 groups,⩽50 and >50 years old. As expected from the increased frequencies of adenovirus-specific T cells in the younger age group, SIs were significantly higher, compared with those of the older group (figure 4B, left panel; day 3, P = .0061; P = .0006). Increasing age, however, is not generally associated with a decrease in T cell reactivity, because proliferative responses toward a polyclonal stimulus such as SEB (figure 4B, right panel ) or PHA (data not shown) did not differ in both groups. Thus, the specific decrease in proliferative responses after adenovirus- specific stimulation results from significantly lower frequencies of adenovirus-specific T cells in the older subjects (figure 2B).

The frequency of adenovirus-specific T cells correlates with cytokine secretion. The age-related decline in adenovirus-specific CD4 T cell frequencies was confirmed by use of another well-established long-term assay system for the characterization of antigen-specific T cells. PBMC were stimulated as described above, and IFN-γ secretion into the supernatant was analyzed after 3 and 5 days, respectively. As was shown for proliferative responses, the frequency of flow-cytometrically determined T cell frequencies correlated with the level of IFN-γ secretion into the supernatant (r = 0.55; P = .01, Pearson's correlation coefficient). Similarly, adenovirus-specific IFN-γ secretion was significantly lower in individuals >50 years old, compared with that of younger control subjects (figure 5A). In contrast, no differences were found after stimulation with polyclonal SEB (figure 5B) or PHA (data not shown) for 5 days, which indicates that older individuals did not show any strong generalized defect in IFN-γ secretion. Although there was a 2-fold lower SEB-induced IFN-γ secretion after 3 days in the older individuals, this decrease was far less pronounced, compared with the 22-fold difference in adenovirus-specific IFN-g secretion. Taken together, the use of 3 assay systems demonstrated the existence of adenovirus-specific CD4 T cells with both IFN-γ-secreting and proliferative capacity in young individuals and their progressive decrease with increasing age.

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Discussion

In the present study, an analysis of the frequencies and phenotypic characteristics of adenovirus-specific CD4+ T cells in healthy control persons and immunosuppressed long-term renal transplant recipients was performed.When a rapid, 6-h assay was used, adenovirus-specific T cells were detectable directly from whole blood without the need for any in vitro expansion. The determined frequencies of T cells correlated with proliferative responses and cytokine secretion capacities, as evaluated by standard long-term proliferation and IFN-γ release assay. Despite immunosuppression, adenovirus-specific T cell frequencies do not differ in healthy control subjects and transplant recipients. Responding T cells were CD4+ T helper cells that showed a considerably homogenous expression of markers characteristic of antigen-experienced memory/effector cells of the Th1 phenotype.

Of interest, the frequency of adenovirus-specific CD4+ T cells in the older group was markedly decreased, compared with that in the younger age group, and this decrease was accompanied by significantly lower proliferative responses, IFN-γ secretion, and specific IgG titers. Because frequencies and responses toward polyclonal stimuli were unaffected, this decline in immune responses was not a sign of a generalized immunosenescence in older individuals. Obviously, a decrease in detectable specific immunity in the older group may, on the one hand, reflect the eventual elimination of the pathogen and the concomitant lack of necessity to maintain high-level immunity. On the other hand, it may indicate an increased susceptibility toward that particular virus. Recent observations obtained from immune responses toward varicella zoster virus (VZV) showed that waning VZV-specific cellular immunity with age correlates with the clinical observation of an increased susceptibility to VZV reactivation and morbidity in older persons [38–40]. In the case of adenoviruses, however, there is no direct clinical evidence to support an increased incidence of infectious complications with age. Thus, the specific decline in adenovirus-specific T cell immunity may instead be indicative of a successful elimination of the pathogen over a lifetime.

Indirect evidence for this hypothesis is provided by studies of other persistent viruses. These studies have indicated that the presence or absence of virus-specific T cells may indeed be triggered by the presence or absence of that particular virus. In support of this view, a therapy-induced decline in HIV load leads to a progressive loss of both virus-specific CD4+ and CD8+ T cells in HIV-infected individuals [31, 41]. On the other hand, the fact thatCMV-specific T cell responses are detected within each seropositive individual irrespective of age [30] is indicative of a continued antigenic stimulation and lifelong persistence of CMV. Although, in general, it is difficult to directly prove the actual presence of CMV in healthy seropositive subjects by use of standard approaches, CMV load can be detected with the use of highly sensitive assay systems [42].

More direct evidence toward a limited persistence of adenovirus during a lifetime is given by both epidemiological studies [43] and observations in patients after transplantation. Infectious complications due to adenoviruses are far more frequent in pediatric or young adult transplant recipients than in older age groups [21–23, 44]. This fact has been explained largely by a higher rate of primary infections in young individuals [5]. However, because adenovirus infections and the induction of a specific T cell immunity occur early in life ([43] and authors' unpublished observations), and because many patients are seropositive for adenovirus before transplantation [4, 45], primary infections alone can hardly account for all infectious episodes observed after transplantation. Instead, they may represent reactivations or symptomatic reinfections due to a disruption in the well-balanced equilibrium between adenovirus-specific T cell responses and viral replication. This disruption of the immunologic control of viral replication is reminiscent of our recent findings in CMV infection, where the immunosuppression-induced decline in CMV-specific T cells after transplantation correlated with an uncontrolled viral replication and symptomatic disease, whereas patients with preserved specific immunity did not suffer from infectious complications [30]. Therefore, similar immunologic control mechanisms might apply in the younger population to minimize adenoviral complications after transplantation.

In view of the decreased adenovirus-specific immune response in the older group, the low incidence of adenoviral complications both among patients after transplantation and healthy subjects can most conceivably be explained by an elimination of the pathogen over lifetime. Thus, in contrast to their young counterparts, reactivations presumably do not occur in older persons. On the other hand, how are reinfections controlled in the face of a decreased cellular immune effector response? Most probably innate defence mechanisms (e.g., NK cells), the persistence of humoral immune responses (figure 3), and/or the existence of low-frequency memory T cells might be sufficient to protect the older group host from symptomatic reinfections.

Until recently, the lack of rapid and sensitive diagnostic assay systems has limited the widespread recognition of adenoviral complications in transplant recipients. Moreover, the clinical management of adenovirus disease is complicated by the fact that some patients manage to control their infection, whereas others die from disseminated infection with multiorgan failure. In addition, not all patients with detectable virus load develop symptoms that are attributable to adenovirus infection [21, 23, 46]. Diagnostic tests include standard culture techniques and, more recently, the use of polymerase chain reaction to detect adenoviral DNA in biopsy specimens [23] or in serum as a marker for an early stage of viral dissemination [24]. Along with the establishment of reliable diagnostic procedures, therapeutic options have to be developed. Current treatment options include the application of intravenous immune globulins and/or ribavirin, but the overall benefits of these regimens have not yet been proved in controlled clinical trials [47–49]. The present study and those on CMV have indicated that adenovirus-specific T cells may open avenues toward both diagnostic and therapeutic options in the management of adenoviral infections. Similar to CMV [30], the combined monitoring of adenovirus-specific CD4+ T cell responses and virus load in children and young adults after transplantationmay help identify patientswho are at risk for uncontrolled viral replication. The underlying assay can be performed within 1 day, and the small sample volumes (<1 mL of whole blood) make it particularly well suited for use in children. In addition, as has been shown for CMV or Epstein-Barr virus [50, 51], the adoptive transfer of adenovirus-specific T cells may be used as a therapeutic option to prevent uncontrolled viral replication and progression to symptomatic disease. Clearly, an increase in understanding of this pathogen and the underlying immune responses will lead to an improvement in the development of novel diagnostic and therapeutic strategies.

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Acknowledgements

We thank Candida Guckelmus and Ulrike Thamke, for excellent technical assistance, and Barbara Niemeyer-Hoth, Hans-Gerhard Burgert, and Andreas Meyerhans, for critical review of the manuscript.

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Footnotes

  • Presented in part: joint annual meeting of the German and Dutch Societies of Immunology, Düsseldorf, Germany, 29 November–2 December 2000 (abstract O.1).

  • Informed consent was obtained from all individuals, and human experimentation guidelines were followed in the conduct of clinical research.

  • Received September 28, 2001.
  • Revision received December 20, 2001.
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  1. J Infect Dis. (2002) 185 (10): 1379-1387. doi: 10.1086/340502
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