Age-Related Increase in the Frequency of CD4+ T Cells That ...

IFN-g Production and Staphylococcal Enterotoxin B • JID 2009:200 (15 December) • 1921

M A J O R A R T I C L E

Age-Related Increase in the Frequency of CD4+

T Cells That Produce Interferon-g in Responseto Staphylococcal Enterotoxin B during Childhood

Rima Hanna-Wakim,1,a Linda L. Yasukawa,1 Phillip Sung,1 Mimi Fang,1 Barbara Sullivan,1 Mary Rinki,1

Ross DeHovitz,2 Ann M. Arvin,1 and Hayley A. Gans1

1Department of Pediatrics, Stanford University School of Medicine, Stanford, and 2Palo Alto Medical Foundation, Palo Alto, California

Background. The susceptibility of infants to infections is well defined clinically, and immunologic abnormalitieshave been described. Immune maturation is complex, however, and the interval during which changes occurduring childhood has not been identified.

Methods. To assess age-related differences in the CD4+ T cell responses, we evaluated the frequency of CD4+

T cells that produced interferon (IFN) g in response to staphylococcal enterotoxin B (SEB) stimulation in 382healthy 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-g production by CD4+ and CD45RO+CD4+ T cells.

Results. CD69 and CD40-L expression by CD4+ and CD45RO+CD4+ T cells were similar to adult levels frominfancy, but the frequency of activated T cells that produced IFN-g remained lower than adult responses untilchildren were 10 years of age.

Conclusions. These observations indicate that the IFN-g response of CD4+ T cells to SEB remains limited fora much longer interval than was reported elsewhere, extending to the second decade of life. Observed differencesin CD45RO+CD4+ T cell function indicate that CD4+ T cells with the same phenotypes do not possess equivalentfunctional 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 path-

ogens, and relative deficiencies of T-helper functions,

including interferon (IFN) g production by infant T

cells, have been consistent findings [2, 3]. Through its

Received 29 March 2009; accepted 1 July 2009; electronically published 12November 2009.

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.Reprints or correspondence: Dr Gans, 300 Pasteur Dr, Rm G 312, Stanford

University School of Medicine, Stanford, CA 94305-5208 ([email protected]).

The Journal of Infectious Diseases 2009; 200:1921–7� 2009 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2009/20012-0016$15.00DOI: 10.1086/648375

immunoregulatory functions, IFN-g acts as a potent T-

helper 1 (Th1) cytokine, playing an important role in

the control of viral, bacterial, mycobacterial, and par-

asitic infections [4, 5]. IFN-g is secreted primarily by

activated T lymphocytes and natural killer cells [6];

thus, deficiencies are associated with increased suscep-

tibility to viral infections and delayed clearance of in-

tracellular pathogens [3].

T cells from healthy newborns produce significantly

less IFN-g in response to various stimuli than do adult

T cells [7, 8]. After the neonatal period, CD4+ and CD8+

T cell production of IFN-g increases over time in re-

sponse to nonspecific stimulation with the mitogens

phorbol 12-myristate 13-acetate and phytohemagglu-

tinin [9–12]. In addition, our studies of antigen-specific

CD4+ T cell production of IFN-g 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-g

production by activated CD4+ T cells induced by staph-

ylococcal enterotoxin B (SEB). SEB, like other super-

antigens, triggers T cell activation and leads to pref-

erential expansion of T cells bearing particular T cell

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1922 • JID 2009:200 (15 December) • Hanna-Wakim et al

receptor (TCR) Vb elements [15–17]. SEB binds sequentially

to the TCR on the responder T cell and to major histocom-

patibility complex class II molecules expressed in antigen-pre-

senting 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 ex-

pressing the specific Vb motifs [18, 19]. Large amounts of Th1

cytokines, including IFN-g, 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-g

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]. Expres-

sion 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-g–producing lym-

phocytes in peripheral blood and CD45RO surface antigen ex-

pression [9]. Therefore, we evaluated IFN-g production by both

total CD4+ T cells and the CD45RO+CD4+ T cell subset after

SEB stimulation. Our study evaluated healthy individuals rang-

ing in age from infancy to adulthood to determine when IFN-g

production by SEB-stimulated CD4+ T cells reaches levels ob-

served in adults.

METHODS

Study population. Three 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 Foun-

dation, 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 Foun-

dation and by the Stanford University Committee for the Pro-

tection 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 assay. Intracellular

cytokine flow cytometry assay was performed on whole blood

samples [18, 29, 30]. After 200-mL aliquots of heparinized whole

blood were placed into 1.5-mL microcentrifuge tubes, costim-

ulatory 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 compar-

ative results, later specimens were tested with longer incubation

time (data not shown). Brefeldin A (Sigma), at a final concen-

tration of 10 mg/mL, was added to each tube for the final 4 h of

the incubation period. After incubation, 20 mL of 20 mmol/L

ethylenediaminetetraacetic acid (Sigma) was added to each sam-

ple, 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 staining.

Frozen samples were thawed, washed with a wash buffer (Dul-

becco’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-g–fluorescein isothi-

ocyanate (FITC), CD45RO-phycoerythrin (PE), and CD40-L–

allophycocyanin. For CD69 experiments, these were CD4-

PerCPCy5.5, IFN-g–FITC, CD45RO-PE, and CD69-allophy-

cocyanin. Except for CD45RO-PE (Invitrogen), all monoclonal

antibodies and conjugates were purchased from BD Biosciences.

Staining reactions were incubated for 30 min at room tem-

perature. The stained cells were washed with fluorescence-ac-

tivated cell sorter wash buffer and then fixed with 1%

paraformaldehyde.

Flow cytometric analysis. Samples 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 Bio-

sciences). Lymphocytes were gated using forward versus side

scatter, and gates were then set to analyze CD4+ T cells (side



Figure 1. Representative flow cytometric analysis of staphylococcal enterotoxin B (SEB) stimulation of CD4+ T cells from infancy to adulthood. Dataare shown as representative plots of SEB-stimulated CD4+ T cells that express CD69 (upper quadrants) and interferon (IFN) g (right quadrants) ininfants, children, and adults, by age. Numbers in histograms represent percentages of SEB-stimulated CD4+CD69+ T cells that also express IFN-g(upper right quadrants), as demonstrated by flow cytometry of 50,000 CD4+ T cells.

Table 1. CD69 Expression and Interferon (IFN) g Production by CD4+ and CD45RO+CD4+ T Cells from Infants,Children, and Adults, as Determined by Flow Cytometry

Age

CD4+ T cells,mean � standard error, %

CD45RO+CD4+ T cells,mean � standard error, %

No. ofindividuals CD69+ CD69+IFN-g+

No. ofindividuals CD69+ CD69+IFN-g+

2 months 46 33.37 � 1.7 0.48 � 0.06 46 57.74 � 2.52 1.36 � 0.196 months 65 37.07 � 1.51 0.40 � 0.05 22 56.12 � 4.69 1.50 � 0.299 months 60 37.84 � 1.51 0.23 � 0.03 22 41.99 � 3.29 1.71 � 0.2512 months 102 45.33 � 1.20 0.39 � 0.04 48 51.59 � 2.58 1. 92 � 0.2218 months 54 44.69 � 1.69 0.44 � 0.05 22 46.98 � 2.93 2.14 � 0.3324 months 16 54.56 � 1.38 0.32 � 0.08 6 61.91 � 3.26 1.10 � 0.163–4 years 11 49.34 � 2.87 0.53 � 0.12 9 62.59 � 2.83 1.51 � 0.295–9 years 28 47.11 � 2.30 0.71 � 0.10 21 59.29 � 2.55 1.56 � 0.1810–11 years 7 48.04 � 2.55 1.38 � 0.42 6 59.20 � 4.34 3.24 � 0.89Adult 66 34.72 � 1.50 2.43 � 0.24 38 37.00 � 2.29 3.79 � 0.51

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-

g+ or CD69+IFN-g+ events (Figure 1).

Statistics. Student’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 . BecauseP � .05

no significant differences were found when responses of chil-

dren 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-g production by CD4+ T cells. CD69

was expressed equally on CD4+ T cells after stimulation with

SEB in infants aged 2 ( ), 6 ( ), and 9 ( )n p 46 n p 65 n p 60

months, compared with responses in the adult group ( ;n p 66

for all comparisons) (Table 1). In contrast, the meanP 1 .05

frequency of CD69+CD4+ T cells was significantly higher when

the 12-month-old infants ( ) were compared with then p 102

younger infants and remained so for comparisons with all other

age groups tested: 18 months ( ), 24 months ( ), 3–n p 54 n p 16

4 years ( ), 5–9 years ( ), and 10–11 years ( ;n p 11 n p 28 n p 7

for all comparisons) (Table 1).P � .05

This pattern was different when the mean frequencies (�

standard errors [SEs]) of CD4+ T cells that expressed CD69

and produced IFN-g in response to SEB stimulation were com-

pared. Among infants and children !10 years of age, the mean

CD69+IFN-g+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 and1.38% � 0.42% 2.43% � 0.24%

in children aged 10–11 years and adults, respectively (P � .05



Figure 2. CD69 activation and interferon (IFN) g production inCD4+CD45RO+ T cells from infancy to adulthood, compared with CD4+

responses. Data are shown as mean percentages and standard errors ofstaphylococcal enterotoxin B–stimulated CD4+ and CD4+CD45RO+ T cellsexpressing CD69 and producing IFN-g in infants, children, and adults, asdemonstrated by flow cytometry of 50,000 CD4+ T cells after stimulationof whole blood samples.

Table 2. CD40 Ligand (CD40-L) Expression and Interferon (IFN) g Production by CD4+ and CD45RO+CD4+ T Cellsfrom Infants and Adults, as Determined by Flow Cytometry

Age

CD4+ T cells,mean � standard error, %

CD45RO+CD4+ T cells,mean � standard error, %

No. ofindividuals CD40-L+ CD40-L+IFN-g+

No. ofindividuals CD40-L+ CD40-L+IFN-g+

6 months 26 15.42 � 1.84 0.06 � 0.01 6 12.16 � 3.41 0.68 � 0.259 months 47 18.01 � 1.55 0.12 � 0.01 28 14.83 � 1.39 1.19 � 0.1512 months 63 17.29 � 1.35 0.18 � 0.04 37 13.71 � 1.32 1.41 � 0.2418 months 30 21.61 � 2.24 0.34 � 0.08 14 16.78 � 3.39 1.36 � 0.22Adults 17 15.48 � 3.05 2.98 � 0.53 17 19.07 � 3.44 5.41 � 0.82

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.

CD69 expression and IFN-g production by CD45RO+CD4+

T cells. As shown in Table 1, the frequency of memory CD4+

T cells (CD45RO+) expressing CD69 and producing IFN-g 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-g than children aged 10–11 years and adults

( for all comparisons of infants and children vs childrenP � .05

aged 10–11 years and adults). The only exception was the dif-

ference between children aged 18 months and children aged

10–11 years; however, this difference was not statistically sig-

nificant ( ), unlike the difference between children agedP p .2

18 months and adults ( ). When the subgroups of in-P p .03

fants and children aged !10 years were compared, there were

no statistically significant differences. The frequencies of

CD45RO+CD4+ T cells that produced IFN-g 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-g production by CD4+ T cells.

CD40-L expression and IFN-g production by CD4+ T cells

stimulated with SEB were measured in infants at age 6 months

( ), 9 months ( ), 12 months ( ), and 18n p 26 n p 47 n p 63

months ( ). No age-related differences in the frequencyn p 30

of cells with these markers were observed among the groups,

except when infants aged 6 months were compared with in-

fants aged 18 months ( ). In addition, frequencies ofP p .04

CD4+CD40-L+ T cells were equivalent in infants 6–18 months

of age and adult CD4+ T cells after SEB stimulation ( )n p 17

(Table 2).

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-g after SEB stimulation, compared

with adults ( , , ,0.06% � 0.01% 0.12% � 0.02% 0.18% � 0.03%

, and among infants aged 6, 9,0.34% � 0.07% 2.98% � 0.53%

12, and 18 months and among adults, respectively; forP � .05

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-g after SEB stimulation were observed

with increasing age ( ), except in the comparison of infantsP � .05

aged 9 months with infants aged 12 months ( ).P p .08

CD40-L expression and IFN-g production by CD45RO+CD4+

T cells. The frequency of memory CD4+ T cells (CD45RO+)

expressing CD40-L and producing IFN-g in response to SEB was

also assessed in subgroups of infants aged 6 months ( ), 9n p 6

months ( ), 12 months ( ), and 18 months (n p 28 n p 37 n p

) and in adults ( ). The frequency of CD45RO+CD4+ T14 n p 17

cells that produced IFN-g was significantly lower in infants up

to age 18 months than in adults ( ,0.68% � 0.25% 1.19% �



Figure 3. CD40 ligand (CD40-L) activation and interferon (IFN) g pro-duction in CD4+CD45RO+ T cells in infants and adults, compared withCD4+ responses. Data are shown as mean percentages and standarderrors of staphylococcal enterotoxin B–stimulated CD4+ and CD4+CD45RO+

T cells expressing CD40-L and producing IFN-g in infants and adults, asdemonstrated by flow cytometry of 50,000 CD4+ T cells after stimulationof whole blood samples.

, , , and0.15% 1.41% � 0.40% 1.36% � 0.22% 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 sus-

ceptibility 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-

g in response to a potent stimulus, SEB, remains lower than

the adult response for a prolonged interval after birth. A ma-

turational transition was identified at ∼10 years of age, when

the frequencies of CD4+ T cells that produced IFN-g 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-g 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 19 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-g, the major Th1

cytokine, in response to SEB, a potent T cell stimulatory pro-

tein, 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 112 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 pop-

ulation 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-g 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-g [36]. When first exposed, naive

CD4+CD45RA+ T cells must mature into effector CD45RO+ T

cells [37]. However, differences in IFN-g–producing capacity

have not been definitely shown, because it is hard to differ-

entiate 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 re-

sponse 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 demon-

strating 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-g as a marker and comparing SEB responses in

infants and children with those in adults.

Several mechanisms might account for this relative defi-

ciency. One possibility points to the function of the APCs. It



has been shown that, after the initial interaction between ac-

tivated T cells and APCs via CD40-L-CD40 binding, a matu-

ration that produces professional APCs must occur. These pro-

fessional APCs produce cytokines, such as IL-12, which in turn

induce IFN-g 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 acti-

vation 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 devel-

opment 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 re-

sponse to SEB in vitro because of a relative insensitivity to this

stimulus, with plasmacytoid DCs not recognizing this signal ow-

ing 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 clin-

ical 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 superan-

tigens, 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-g, a pivotal cytokine for generating a robust adaptive im-

mune 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 an-

tigenic stimuli continues long after the newborn period. Use

of SEB as a potent CD4+ T cell activator demonstrated limi-

tations that are not as easily measurable with antigen-specific

stimulation because of the low number of T cells that are in-

duced to produce IFN-g. 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 chil-

dren. 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 staffat the Palo Alto Medical Foundation for their assistance with this study.

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