Age-Related Increase in the Frequency of CD4⁺ T Cells That Produce Interferon-γ in Response to Staphylococcal Enterotoxin B during Childhood

Abstract

BackgroundThe susceptibility of infants to infections is well defined clinically, and immunologic abnormalities have been described. Immune maturation is complex, however, and the interval during which changes occur during childhood has not been identified

MethodsTo assess age-related differences in the CD4⁺ T cell responses, we evaluated the frequency of CD4⁺ T cells that produced interferon (IFN) γ in response to staphylococcal enterotoxin B (SEB) stimulation in 382 healthy infants and children (2 months to 11 years of age) and 66 adults. Flow cytometry was used to assess SEB-induced CD69 and CD40 ligand (CD40-L) expression and IFN-γ production by CD4⁺ and CD45RO⁺CD4⁺ T cells

ResultsCD69 and CD40-L expression by CD4⁺ and CD45RO⁺CD4⁺ T cells were similar to adult levels from infancy, but the frequency of activated T cells that produced IFN-γ remained lower than adult responses until children were 10 years of age

ConclusionsThese observations indicate that the IFN-γ response of CD4⁺ T cells to SEB remains limited for a much longer interval than was reported elsewhere, extending to the second decade of life. Observed differences in CD45RO⁺CD4⁺ T cell function indicate that CD4⁺ T cells with the same phenotypes do not possess equivalent functional capabilities

Infections pose a significant risk to healthy infants and young children. Studies examining the development of the immune system have revealed limitations in many components of both innate and adaptive immunity in infancy, demonstrating both phenotypic and functional restrictions that resolve over time [1]. Vulnerability to infections is most pronounced for intracellular pathogens, and relative deficiencies of T-helper functions, including interferon (IFN) γ production by infant T cells, have been consistent findings [2, 3]. Through its immunoregulatory functions, IFN-γ acts as a potent T-helper 1 (Th1) cytokine, playing an important role in the control of viral, bacterial, mycobacterial, and parasitic infections [4, 5]. IFN-γ is secreted primarily by activated T lymphocytes and natural killer cells [6]; thus, deficiencies are associated with increased susceptibility to viral infections and delayed clearance of intracellular pathogens [3]

T cells from healthy newborns produce significantly less IFN-γ in response to various stimuli than do adult T cells [7, 8]. After the neonatal period, CD4⁺ and CD8⁺ T cell production of IFN-γ increases over time in response to nonspecific stimulation with the mitogens phorbol 12-myristate 13-acetate and phytohemagglutinin [9–12]. In addition, our studies of antigen-specific CD4⁺ T cell production of IFN-γ in response to measles and mumps in infants 6–12 months of age have revealed significant limitations, compared with adults [13, 14]

In this study, we evaluated the ontogeny of IFN-γ production by activated CD4⁺ T cells induced by staphylococcal enterotoxin B (SEB). SEB, like other superantigens, triggers T cell activation and leads to preferential expansion of T cells bearing particular T cell receptor (TCR) Vβ elements [15–17]. SEB binds sequentially to the TCR on the responder T cell and to major histocompatibility complex class II molecules expressed in antigen-presenting cells (APCs), bypassing the peptide groove by which conventional antigens are presented [16, 17]. This leads to a vigorous induction of the cellular immune system, because there is a polyclonal expansion of all T cell populations expressing the specific Vβ motifs [18, 19]. Large amounts of Th1 cytokines, including IFN-γ, are produced from T cells activated in this manner [16, 17]. By circumventing the need for antigen-specific TCR engagement, our goal was to determine whether T cells from infants and children have a capacity equivalent to that of adults for activation without the confounding difficulties of measuring CD4⁺ T cell responses seen after antigen-specific recognition, which are typically much lower. Limited data have suggested that the TCR repertoire in infants is similar to that in adults and, thus, should allow for similar activation of T cells after superantigen stimulation [20, 21]

To evaluate the activation of CD4⁺ T cells, we investigated 2 cellular markers, CD40 ligand (CD40-L) and CD69, both of which are expressed on activated CD4⁺ T cells. Through binding with other cell types, CD40-L is a key APC-activating factor, resulting in secretion of interleukin (IL) 12 [22, 23], and it plays an important role in priming the production of IFN-γ by T cells [22, 23]. CD40-L expression by neonatal T cells has been reported both as reduced and as equivalent to expression in adults [24, 25], and if CD40-L levels are low, it is not known when they mature. CD69 is an early T cell activation marker that contributes to signal transduction, Ca²⁺ influx, cytokine production, and cytokine receptor synthesis [26, 27]. Expression of CD69 is reported to be equivalent on activated neonatal and adult T cells [28]

Other studies have shown a strong correlation between the progressive increase in the frequency of IFN-γ–producing lymphocytes in peripheral blood and CD45RO surface antigen expression [9]. Therefore, we evaluated IFN-γ production by both total CD4⁺ T cells and the CD45RO⁺CD4⁺ T cell subset after SEB stimulation. Our study evaluated healthy individuals ranging in age from infancy to adulthood to determine when IFN-γ production by SEB-stimulated CD4⁺ T cells reaches levels observed in adults

Methods

Study populationThree hundred eighty-nine healthy infants and children (aged 2 months to 11 years) were recruited among patients receiving well-child care at the Palo Alto Medical Foundation, Palo Alto, California. Sixty-six healthy adults (aged 18–40 years) were also evaluated. Participants had no chronic illness and no known immunosuppressive conditions. Written informed consent was obtained from each child’s parent or guardian and from each adult before study entry. The study was approved by the institutional review board at the Palo Alto Medical Foundation and by the Stanford University Committee for the Protection of Human Subjects. Blood samples were collected from each participant, but not every sample was tested for all markers of T cell activation and cytokine production

Intracellular cytokine flow cytometry assayIntracellular cytokine flow cytometry assay was performed on whole blood samples [18, 29, 30]. After 200-μL aliquots of heparinized whole blood were placed into 1.5-mL microcentrifuge tubes, costimulatory factors CD28 and CD49d (BD Biosciences) were added. Stimulation with SEB (Sigma) at a concentration of 0.5 mg/mL was compared with a negative control, phosphate-buffered saline (PBS). Initially, samples were incubated with SEB for 6 h at 37°C and 5% carbon dioxide, based on published protocols. Because comparative analysis of samples from the same study participants showed that overnight stimulation gave comparative results, later specimens were tested with longer incubation time (data not shown). Brefeldin A (Sigma), at a final concentration of 10 μg/mL, was added to each tube for the final 4 h of the incubation period. After incubation, 20 μL of 20 mmol/L ethylenediaminetetraacetic acid (Sigma) was added to each sample, followed by FACS Lysing Solution ×1 (BD Biosciences), and tubes were incubated for 15 min at room temperature. The tubes were then centrifuged for 5 min, the supernatant was discarded, and the cell pellet was resuspended in freezing solution (PBS, 1% bovine serum albumin [BSA], and 10% dimethyl sulfoxide), frozen at −80°C, and analyzed by flow cytometry within 2 weeks

Cell surface markers and intracellular cytokine stainingFrozen samples were thawed, washed with a wash buffer (Dulbecco’s PBS ×1, 0.5% BSA [Sigma], and 0.5 g of 0.1% sodium azide [Sigma]), and permeabilized with FACS Permeabilizing Solution (BD Biosciences). A mixture of fluorescent mouse anti-human monoclonal antibodies was added to each sample. For CD40-L experiments, these were CD4–peridinin chlorophyl protein (PerCP)–cyanin 5.5 (Cy5.5), IFN-γ–fluorescein isothiocyanate (FITC), CD45RO-phycoerythrin (PE), and CD40-L–allophycocyanin. For CD69 experiments, these were CD4-PerCPCy5.5, IFN-γ–FITC, CD45RO-PE, and CD69-allophycocyanin. Except for CD45RO-PE (Invitrogen), all monoclonal antibodies and conjugates were purchased from BD Biosciences. Staining reactions were incubated for 30 min at room temperature. The stained cells were washed with fluorescence-activated cell sorter wash buffer and then fixed with 1% paraformaldehyde

Flow cytometric analysisSamples were analyzed with a FACSCalibur flow cytometer (BD Biosciences); ∼100,000 events were collected for each sample. Acquisition and analysis were performed with CellQuest Pro software (version 4.0.1; BD Biosciences). Lymphocytes were gated using forward versus side scatter, and gates were then set to analyze CD4⁺ T cells (side scatter vs CD4). Memory CD4⁺ T cells were determined by gating on CD4⁺ T cells that were CD45RO⁺. Frequencies of responding cells were reported as percentages of CD40-L⁺IFN-γ⁺ or CD69⁺IFN-γ⁺ events (Figure 1)

Figure 1

Representative flow cytometric analysis of staphylococcal enterotoxin B (SEB) stimulation of CD4⁺ T cells from infancy to adulthood. Data are shown as representative plots of SEB-stimulated CD4⁺ T cells that express CD69 (upper quadrants) and interferon (IFN) γ (right quadrants) in infants, children, and adults, by age. Numbers in histograms represent percentages of SEB-stimulated CD4⁺CD69⁺ T cells that also express IFN-γ (upper right quadrants) as demonstrated by flow cytometry of 50,000 CD4⁺ T cells

StatisticsStudent’s unpaired t test for mean differences was used to analyze data between the different age groups. Differences were considered to be statistically significant at P⩽.05. Because no significant differences were found when responses of children were compared using each year separately or when these ages were combined into age cohorts of 3–4, 5–9, and 10–11 years, data are reported using the grouped ages

Results

CD69 expression and IFN-γ production by CD4⁺T cellsCD69 was expressed equally on CD4⁺ T cells after stimulation with SEB in infants aged 2 (n=46), 6 (n=65), and 9 (n=60) months, compared with responses in the adult group (n=66; P>.05 for all comparisons) (Table 1). In contrast, the mean frequency of CD69⁺CD4⁺ T cells was significantly higher when the 12-month-old infants (n=102) were compared with the younger infants and remained so for comparisons with all other age groups tested: 18 months (n=54), 24 months (n=16), 3–4 years (n=11), 5–9 years (n=28), and 10–11 years (n=7; P⩽.05 for all comparisons) (Table 1)

Table 1

CD69 Expression and Interferon (IFN) γ Production by CD4+ and CD45RO+CD4+ T Cells from Infants, Children, and Adults, as Determined by Flow Cytometry

This pattern was different when the mean frequencies (± standard errors [SEs]) of CD4⁺ T cells that expressed CD69 and produced IFN-γ in response to SEB stimulation were compared. Among infants and children <10 years of age, the mean CD69⁺IFN-γ⁺CD4⁺ T cell frequencies (± SEs) were 0.48% ± 0.06%, 0.40% ± 0.05%, 0.23% ± 0.03%, 0.39% ± 0.04%, 0.44% ± 0.05%, 0.32% ± 0.08%, 0.53% ± 0.12%, and 0.71% ± 0.10% at 2, 6–7, 9, 12, 18, and 24 months and 3–4 and 5–9 years of age, respectively (Table 1 and Figure 2). These mean frequencies of CD4⁺CD69⁺ T cells were significantly lower than the mean frequencies of 1.38%±0.42% and 2.43%±0.24% in children aged 10–11 years and adults, respectively (P⩽.05 for all comparisons of infants and children aged <10 years vs children aged 10–11 years and adults). With use of these CD4⁺ T cell markers after SEB stimulation, no differences were found between responder cell frequencies in children aged 10–11 years and adults

Figure 2

CD69 activation and interferon (IFN) γ production in CD4⁺CD45RO⁺ T cells from infancy to adulthood, compared with CD4⁺ responses. Data are shown as mean percentages and standard errors of staphylococcal enterotoxin B–stimulated CD4⁺ and CD4⁺CD45RO⁺ T cells expressing CD69 and producing IFN-γ in infants, children, and adults, as demonstrated by flow cytometry of 50,000 CD4⁺ T cells after stimulation of whole blood samples

CD69 expression and IFN-γ production by CD45RO⁺CD4⁺T cellsAs shown in Table 1, the frequency of memory CD4⁺ T cells (CD45RO⁺) expressing CD69 and producing IFN-γ in response to SEB was also evaluated in infants, children, and adults. Even in this subset of the CD4⁺ T cell population, infants and children <10 years of age had significantly fewer cells that produced IFN-γ than children aged 10–11 years and adults (P⩽.05 for all comparisons of infants and children vs children aged 10–11 years and adults). The only exception was the difference between children aged 18 months and children aged 10–11 years; however, this difference was not statistically significant (P=.2), unlike the difference between children aged 18 months and adults (P=.03). When the subgroups of infants and children aged <10 years were compared, there were no statistically significant differences. The frequencies of CD45RO⁺CD4⁺ T cells that produced IFN-γ in response to SEB did not differ between children aged 10–11 years and adults (Table 1 and Figure 2)

CD40-L expression and IFN-γ production by CD4⁺T cellsCD40-L expression and IFN-γ production by CD4⁺ T cells stimulated with SEB were measured in infants at age 6 months (n=26), 9 months (n=47), 12 months (n=63), and 18 months (n=30). No age-related differences in the frequency of cells with these markers were observed among the groups, except when infants aged 6 months were compared with infants aged 18 months (P=.04). In addition, frequencies of CD4⁺CD40-L⁺ T cells were equivalent in infants 6–18 months of age and adult CD4⁺ T cells after SEB stimulation (n=17) (Table 2)

Table 2

CD40 Ligand (CD40-L) Expression and Interferon (IFN) γ Production by CD4+ and CD45RO+CD4+ T Cells from Infants and Adults, as Determined by Flow Cytometry

However, all of the infant age groups (6–18 months) had significantly lower frequencies of CD4⁺ T cells that expressed CD40-L and produced IFN-γ after SEB stimulation, compared with adults (0.06%±0.01%, 0.12%±0.02%, 0.18%±0.03%, 0.34%±0.07%, and 2.98%±0.53% among infants aged 6, 9, 12, and 18 months and among adults, respectively; P⩽.05 for all comparisons) (Table 2 and Figure 3). Nevertheless, there was a gradual maturation of these responses with increasing age in the infant cohorts. Significant increases in the CD4⁺CD40-L⁺ T cells that produced IFN-γ after SEB stimulation were observed with increasing age (P⩽.05), except in the comparison of infants aged 9 months with infants aged 12 months (P=.08)

Figure 3

CD40 ligand (CD40-L) activation and interferon (IFN) γ production in CD4⁺CD45RO⁺ T cells in infants and adults, compared with CD4⁺ responses. Data are shown as mean percentages and standard errors of staphylococcal enterotoxin B–stimulated CD4⁺ and CD4⁺CD45RO⁺ T cells expressing CD40-L and producing IFN-γ in infants and adults, as demonstrated by flow cytometry of 50,000 CD4⁺ T cells after stimulation of whole blood samples

CD40-L expression and IFN-γ production by CD45RO⁺CD4⁺T cellsThe frequency of memory CD4⁺ T cells (CD45RO⁺) expressing CD40-L and producing IFN-γ in response to SEB was also assessed in subgroups of infants aged 6 months (n=6), 9 months (n=28), 12 months (n=37), and 18 months (n=14) and in adults (n=17). The frequency of CD45RO⁺CD4⁺ T cells that produced IFN-γ was significantly lower in infants up to age 18 months than in adults (0.68%±0.25%, 1.19%±0.15%, 1.41%±0.40%, 1.36%±0.22%, and 5.41%±0.82% among infants aged 6, 9, 12, and 18 months and among adults, respectively) (Table 2). No statistically significant differences were detected in comparisons among the infant age groups (Figure 3)

Discussion

Understanding the kinetics of the maturation of the CD4⁺ T cell response is important to account for the well-known susceptibility of infants and young children to serious bacterial, viral, and fungal infections. Several studies have defined both phenotypic and functional limitations in the immune responses of infants that become less prominent with age [9–12, 31–33]. However, it is not clear how long these differences persist during childhood, and an age threshold by which their maturation can be expected has not been established. Our experiments indicate that the frequency of activated CD4⁺ T cells that produce IFN-γ in response to a potent stimulus, SEB, remains lower than the adult response for a prolonged interval after birth. A maturational transition was identified at ∼10 years of age, when the frequencies of CD4⁺ T cells that produced IFN-γ after exposure to SEB reached levels equivalent to those seen in CD4⁺ T cell populations in peripheral blood samples from adults. This pattern of diminished frequencies of CD4⁺ T cells with the capacity to produce IFN-γ as elicited by SEB was observed in the memory CD45RO⁺CD4⁺ T cell population, as well as in the total CD4⁺ T cell population, when children were evaluated during the first decade of life. These observations support the concept that immune maturation to SEB continues well beyond infancy, extending into late childhood, in contrast to some previous assumptions. Other studies have shown reduced Th1 cytokine responses by T cells from neonates and children up to 1 year of age, compared with responses in children >9 years of age and adults [9, 10]; however, because children were not studied in cohorts from the intervening age groups, the time course of maturation was not defined. Our analysis indicates that maturation of the capacity to make IFN-γ, the major Th1 cytokine, in response to SEB, a potent T cell stimulatory protein, does not occur until the second decade of life. This is consistent with findings showing that IL-12 production from peripheral blood mononuclear cells did not reach adult levels until children were >12 years of age [34]

Limitations in the immune response of infants and young children reflect the naive state of most of their circulating T cells, which have not yet encountered antigens produced by infectious agents in the host. The major CD4⁺ T cell population at birth is composed of CD45RA⁺ T cells; however, this population progresses over time and with exposure to a gradual predominance of antigen-specific CD45RO⁺CD4⁺ T cells, which are functionally mature and considered to be effector cells [35, 36]. Our experiments using SEB to induce IFN-γ production by CD45RO⁺CD4⁺ T cells as evidence of an effector cytokine response indicate that effector T cells in infants and young children have significantly decreased capacity. This difference may reflect a primary response of CD4⁺ T cells from infants and younger children to antigenic stimulation with SEB, as opposed to a secondary memory T cell response elicited in adults, which would be expected to include an enhanced capacity to produce IFN-γ [36]. When first exposed, naive CD4⁺CD45RA⁺ T cells must mature into effector CD45RO⁺ T cells [37]. However, differences in IFN-γ–producing capacity have not been definitely shown, because it is hard to differentiate primary from secondary CD45RO⁺CD4⁺ T cells

The capacity of CD4⁺ T cells in infants and young children to express the activation markers CD69 and CD40-L in response to SEB was not impaired, compared with that in older children and adults. Instead, expression of these activation markers are enhanced in young children, with a plateau in late childhood. This observation is in contrast to other reports of progressive maturation of CD40-L expression in stimulated CD4⁺ T cells [38] but parallels more recent findings demonstrating that CD40-L levels in activated CD4⁺ T cells are intact even in early infancy [39]. Nonetheless, despite our findings of equivalent initial activation, pathways to full effector function of the CD4⁺ T cells did not mature until ∼10 years of age, with use of IFN-γ as a marker and comparing SEB responses in infants and children with those in adults

Several mechanisms might account for this relative deficiency. One possibility points to the function of the APCs. It has been shown that, after the initial interaction between activated T cells and APCs via CD40-L-CD40 binding, a maturation that produces professional APCs must occur. These professional APCs produce cytokines, such as IL-12, which in turn induce IFN-γ production from natural killer cells that promote the development of effector T cells [40]. On the basis of the findings in our analysis of SEB responses and those published elsewhere, there appears to be a lack of additional feedback from the professional APCs to the CD4⁺ T cells, through key cytokines produced by dendritic cells (DCs) or monocytes, whereas initial activation of infant CD4⁺ T cells is intact [39]. In support of this explanation, IL-12 production in infants and children has been shown to be reduced [13, 34], and reduced IL-12 production in stimulated cord blood DCs was recently shown to result from a defect in the transcription of the IL-12p35 subunit [41, 42]

Alternative explanations may include differences in the activation of cells of the innate immune pathway that inform T cell maturation by different cytokine profiles [43]. Work focusing on the role of Toll-like receptors (TLRs) in instructing the development of adaptive immunity has revealed that different DC populations express distinct TLRs, which in turn will recognize specific stimuli [44]. Activation through a given TLR biases the cytokine profile expressed by the DCs, which will determine the T cell response [43–45]. Plasmacytoid DCs, the major DC subset in circulation in neonates, have a different activation pattern than do myeloid DCs, which represent the main population circulating later in life [43, 44]. SEB uses TLR2 to activate DCs [46], and plasmacytoid DCs preferentially express TLR7 and TLR9 [44]. Thus, neonates, infants, and children may have a decreased response to SEB in vitro because of a relative insensitivity to this stimulus, with plasmacytoid DCs not recognizing this signal owing to a lack of expression of TLR2 and, thus, not fully triggering a T cell response [44, 46]

Clinically relevant syndromes in children that are caused by Staphylococcus aureus especially via toxin production [47–50], support the findings in our study. Of interest, diseases such as staphylococcal scalded fever and Kawasaki syndrome have been associated with SEB and occur predominantly in children, in particular those <10 years of age [48, 49]. In addition, age <2 years is a risk factor for colonization and subsequent diseases related to methicillin-resistant S. aureus [50]. The unique clinical susceptibility of children to disease caused by S. aureus which is often toxin mediated, may be secondary to a decreased immune response. Because these toxins function as superantigens, they are likely to follow patterns of immune activation similar to that described here

In summary, several points can be made about the ontogeny of CD4⁺ T cell immunity to SEB in infants, children, and adults. Maturation of the critical function of these cells to produce IFN-γ, a pivotal cytokine for generating a robust adaptive immune response, is a prolonged process that is established only in the second decade of life. Thus, the transition from the protected environment of the fetus to the world of varied antigenic stimuli continues long after the newborn period. Use of SEB as a potent CD4⁺ T cell activator demonstrated limitations that are not as easily measurable with antigen-specific stimulation because of the low number of T cells that are induced to produce IFN-γ. The assessment of both CD4⁺ and CD45RO⁺CD4⁺ T cells revealed that the latter population does not achieve full memory effector function in infants and children. This important distinction between phenotypic markers and the functional capacity of T cell populations needs to be considered in future studies of CD4⁺ T cell responses in children and adults. Further analysis is necessary to fully define events in the ontogeny of the immune response of infants and children and the time course during which maturation occurs, especially to identify antigen-specific kinetics. The present analysis will serve as background that may aid in developing new vaccine strategies for these susceptible hosts

Acknowledgments

We thank the families, pediatricians, nursing staff, and laboratory staff at the Palo Alto Medical Foundation for their assistance with this study

Financial support: National Institute of Allergy and Infectious Diseases (AI37127)

Potential conflicts of interest: none reported

Presented in part: Pediatric Academic Societies Annual Meeting 2006, San Francisco, California, 29 April–2 May 2006 (abstract 172)

Current affiliation: American University of Beirut, Beirut, Lebanon