Original Paper

International Archives of

Allergy
Immunology

and

Int Arch Allergy Immunol 1999;119:612

Received: May 18, 1998

Accepted after revision: November 11, 1998

Expression of the T-Cell Markers CD3, CD4 and

CD8 in Healthy and Atopic Children during the
First 18 Months of Life
Helena Aniansson Zdolsek a Jan Ernerudh b Patrick G. Holt d
Joacim Nilsson c Bengt Bjrkstn a
a Department

of Health and Environment, Division of Paediatrics, b Department of Clinical Immunology and

of Cell Biology, Faculty of Health Sciences, Linkping University, Sweden; d TVW Telethon Institute
for Child Health Research, Perth, Australia
c Department

Abstract
Background: There is little information available about
the development of T-cell immunity in healthy and
atopic children. We have studied prospectively the
mean fluorescence intensity of the T-cell receptor complex-associated CD3, CD4 and CD8 in relation to atopic
family history (AFH) and the development of atopic disease. Methods: Children with a defined AFH (n = 172)
were followed from birth to 18 months and the cumulative history of atopic disease was recorded. Blood samples were obtained at birth and at 18 months, and in a
subgroup of 78 children also at 3, 6 and 12 months. Multicolour flow cytometry was used to analyse pan T-cells
(CD3+CD45+CD14-), T-helper-(CD3+CD4+) and T-cytotoxic-(CD3+CD8+) cells. Results: At 18 months, 31 children were atopic and 118 non-atopic. Children who developed atopic disease had a higher CD4 expression
(mean fluorescence intensity, MFI) on CD4+CD3+ lymphocytes at birth and at 3 months, particularly as compared with non-atopic children without AFH. Furthermore, the CD3 expression on CD3+CD45+CD14

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lymphocytes increased more slowly with age in children with double atopic heredity, as compared with
children with no or only one atopic family member.
Conclusions: The higher expression of the CD4 receptor
in early infancy in children who developed atopic disease compared with non-atopics suggests a delayed expression in T-helper cells. Children with a strong AFH
had a slower increase in the expression of CD3, indicating a delayed T-cell maturation.

Introduction

The tendency to a prolonged IgE antibody response is

genetically determined in both laboratory animals and humans. It is also clear, however, that environmental factors
play a major role in the expression of the genetic potential
[1]. It has been reported that a deviation of the immune
response at the fetomaternal interface towards the Th2
cytokine phenotype is a prerequisite for a successful pregnancy [2]. After birth, the immune system adapts to extrauterine life. Experimental and clinical studies indicate that
the conditions under which primary sensitisation to allergens occur may influence immunity and the development of
allergy several years later [reviewed in ref. 3].

Correspondence to: Dr. Helena Aniansson Zdolsek

Department of Health and Environment, Division of Paediatrics
Faculty of Health Sciences, University Hospital
S581 85 Linkping (Sweden)
Fax +46 13 148 265

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Key Words
T-lymphocyte CD3 CD4 CD8 MFI Newborns
Atopic disease

Materials and Methods

samples were excluded from analysis due to perinatal complications,

leaving 77 cord blood samples, 60 samples obtained at 3 months, 63
at 6 months, 65 at 12 months and 135 at 18 months. All the 77 cord
blood samples used in the statistical analysis were thus from healthy,
full-term babies with normal deliveries, except for 3 cases of caesarian section.
Diagnostic Criteria
Atopic dermatitis was defined as proposed by Hanifin and Rajka
[6], employing the modified criteria for young infants [7]. Asthma
was defined as three or more episodes of bronchial obstruction, each
time verified by a physician [8]. Allergic rhinoconjunctivitis was defined as rhinitis and/or conjunctivitis appearing at least twice after exposure to a particular allergen and not related to infection [8]. Gastrointestinal allergy implied vomiting and/or diarrhoea on at least two
separate occasions after intake of a certain offending food. Urticaria
was defined as allergic if appearing at least twice within 1 h after exposure to a particular antigen.
The classification of the children with regard to atopic disease was
done at the 18-month follow-up based on the results of the physical
examination, interview with one or both parents, and the answers given in the questionnaires. In unclear cases, the records from well-baby
clinics, primary care units, private paediatricians and the Department
of Paediatrics were examined. The results of the laboratory data were
not known at the time of the classification.
Skin Prick Tests
Skin prick tests were performed in duplicate at 18 months, using
fresh hens egg white and cows milk and standardized extracts of cat,
birch, house dust mite (Dermatophagoides farinae), timothy grass,
(Solu-Prick SQ, ALK, Hrsholm, Denmark), and histamine hydrochloride, 10 mg/ml as a reference (ALK). The size of the wheal
was marked with a filter pen after 15 min and transferred onto a micropore tape for measuring. The skin prick tests were regarded as positive when the mean wheal diameter was at least 3 mm.

Study Population
Pregnant mothers at seven maternity-care centres in Linkping
were informed of the study and 172 families accepted to participate.
All children were born at the University Hospital in Linkping. At 3,
6, 12 and 18 months the parents were asked to complete a mailed
questionnaire related to possible allergic symptoms in their children.
At 18 months, a medical examination was done by one of us (H.Z.),
focusing on signs of atopic disease. In addition, a skin prick test was
performed, a venous blood sample was obtained by venipuncture after application of percutaneous anaesthesia. A family history of atopy
in the immediate family, i.e. parents and siblings of the baby, was obtained through two interviews, once at the beginning of the study and
once for rechecking the history at the 18 months follow-up visit. Double (single/no) atopic heredity implied that at least two (one/no) parents and/or siblings had a convincing history of atopic disease.
Venous blood samples were drawn at birth and at 18 months
(range 16.621.0 months). In order to reduce the number of blood
samples, for both ethical and financial reasons, and to prevent drop
outs, venous blood samples were drawn at 3 (2.54.4 months), 6
(5.37.3 months) and 12 months (11.313.5 months) only from 78
children, in addition to the samples at birth and 18 months. Thus,
blood samples were obtained from 141 infants at birth, and from 66,
71, 69 and 137 children respectively at 3, 6, 12 and 18 months. Missing samples were due to technical reasons, e.g. birth during weekends
when research laboratory facilities were unavailable, clotting of the
cord blood, difficulties with drawing the blood sample, and technical
failure in the laboratory.
Eleven samples were discarded as they were obtained from 3 children with diseases which possibly could have interfered with the
study (IgA deficiency, congenital hypothyreosis and mental retardation due to suspected mitochondrial disease). Another 6 cord blood

Flow Cytometry
The following murine IgG monoclonal antibodies were used: T3ECD (CD3), T4-FITC (CD4) and T8-FITC (CD8) (Coulter Co,
Hialeah, Calif., USA), CD45-FITC/CD14-PE (Dako, Copenhagen,
Denmark). The control antibodies included IgG1-FITC/IgG2a-RPE
(Dako), MsIgG2b-ECD and MsIgG1-FITC (Coulter Co). Time-control studies comparing freshly stained and analysed cord blood samples with cord blood samples stained after 24 h and then analysed
yielded similar results in four pilot experiments. The cord blood samples were stained within 24 h (median: 12.3 h, range: 2.024 h), and
then analysed within 6 h. The 3-, 6-, 12- and 18-month samples were
all stained within 3.5 h and then analysed within 4 h.
Twenty-five microlitres EDTA-whole blood was incubated with
5 l monoclonal antibodies for 10 min at ambient room temperature
and erythrocytes were lysed, using 1 ml of Ortho-mune Lysing
Reagent (Ortho Diagnostic Systems Inc., Raritan, N.J., USA), for 15
min, also at room temperature, followed by centrifugation at 400 g for
5 min at 4C. The pellet was resuspended in 1 ml buffer with 0.08%
HSA (Apoteksbolaget, Stockholm, Sweden) and 0.1% paraformaldehyde (Sigma-Aldrich).
Three-colour (FITC, RPE/RD1/PE, and ECD) flow cytometry
was performed on an EPICS Profile cytometer (Coulter). A lymphocyte gate was set according to characteristic forward and side scatter.
Each sample was saved in an ungated version to list mode files, and

Expression of T-Cell Markers

Int Arch Allergy Immunol 1999;119:612

7
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The T-cell maturation process of children is poorly understood, but there is evidence that it may be defective in
atopic children [reviewed in ref. 4]. Therefore, the present
study was designed to prospectively compare the maturation process in normal and atopic children by following the
expression (measured as mean fluorescence intensity; MFI)
of the T-cell surface antigens CD3, CD4 and CD8 during
the first 18 months of life in a large cohort of children. Special reference was made to atopic family history and the development of atopic disease within this period. Threecolour flow cytometry was used to analyse peripheral blood
cells expressing the pan T-cell marker CD3 and the leucocyte marker CD45, excluding cells expressing the monocyte marker CD14. Further, the T-helper- (CD3+CD4+) and
the T-cytotoxic (CD3+CD8+) cells were analysed. Since
there still are no methods available for accurately identifying individuals who later will develop atopic disease in early childhood [5], the levels of MFI expression of CD3, CD4
and CD8 as predictive markers for atopic disease were also
evaluated.

60

]]]
]

50

Fig. 1. CD3 MFI on CD3+CD45+CD14

cells at birth, 3, 6, 12 and 18 months of age.

Significant differences between two adjacent
observations and between birth and 18
months are indicated (Students paired t test).
Number of paired observations: 03 months:
n = 29, 36 months: n = 54, 612 months: n =
58, 1218 months: n = 64, 018months:
n =73. The results are presented as mean B S
for the children independent of allergy.
*p d0.05, ** p d0.01 and *** pd0.001.

CD3 MFI

40

30

20

10

]]]

0
Age, months

12

18

n=

77

60

63

65

135

analysis was performed with Flowmate -software (Dako A/S,

Glostrup, Denmark). MFI was analysed using a 4-decade log scale.
Samples with the CD3/CD45/CD14 mAb, CD3/CD4 mAb and
CD3/CD8 mAb combinations were stepwise gated. The final gate included respectively a population of CD3+CD45+CD14-, CD3+CD4+
and CD3+CD8+ lymphocytes.

Ethical Aspects
The study was approved by the Human Research Ethics Committee of the Medical Faculty at Linkping University.

Results

Int Arch Allergy Immunol 1999;119:612

Atopic disease developed in 31 children within the first

18 months of life and 118 children were non-atopic. Twenty-one children fulfilled most, but not all the criteria for
atopic disease and were excluded from comparisons between atopic and non-atopic, although they were included
in comparisons between children with a different AFH. Two
children were excluded as the families did not comply to
sampling and follow-up during the entire study period. Another 2 children did not attend the follow up at 18 months
and their mothers were thus interviewed over the telephone.
They were both healthy according to the questionnaires and
the interviews. All of the atopic children had eczema, 4 of
them asthma, and 2 had urticaria.
Skin prick tests (SPT) were performed at 18 months in
160 children. A positive SPT was verified in 13 (42%) of the
atopic children, in 2 (2%) of the non-atopic children and in
3 (14%) of the children who did not fulfil the criteria for
atopic disease.
The average levels of CD3 MFI on CD3+CD45+CD14cells (pan T-cells) over the first 18 months of life in all children are shown in figure 1 and the average levels of CD4
MFI on CD3+CD4+ (T-helper) cells and CD8 MFI on
CD8+CD3+ (T-cytotoxic) cells in figure 2. The CD3 ex-

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Statistical Analysis
All data were approximately normally distributed. This was however difficult to decide in some analyses as the number of children in
some groups were low, e.g. for the comparisons between data of children with maternal and paternal atopic family history (AFH). In these
analyses, the Mann-Whitney U test was therefore used. One- and twoway analysis of variance were employed to compare MFI at 0, 3, 6, 12
and 18 months with respect to AFH and the occurrence of atopic disease during the first 18 months of life. Students paired t test was used
to compare changes of MFI on T-cells over time within the groups. In
atopic and non-atopic children, as well as in children with single, double and no AFH, one-way and two-way analysis of variance was performed to compare the changes of data within separate time periods
(03 months, 36 months, 612 months, 1218 months and 018
months). Only paired observations were included.
In addition, one- and two-way analysis of variance was performed
to compare the slopes (changes/month) in atopic and non-atopic children and in children with different AFH. In these analyses, only children with at least two observations during the study period were included. In order to assure that the compared groups did not differ in
age when the blood samples were obtained, the mean ages were individually calculated and the mean value for each group compared with
Students t test. No significant differences were seen. A p value d0.05
was considered to be significant. Where not otherwise indicated,
mean B 1SD are reported. A positive predictive value was defined as
the proportion of atopic children with positive tests to all children
with positive tests. Sensitivity: proportion of atopic children with true
positive test to all atopic children. Specificity: proportion of nonatopic children with true negative test to all non-atopic children.

60

50

]]]

40

Fig. 2. CD4 MFI on CD3+CD4+ cells (D)

and CD8 MFI on CD3+CD8+ cells (K) at 0,
3, 6, 12 and 18 months of age in all children.
Significant differences between two adjacent
observations and between birth and 18
months are indicated (Students paired t test).
Number of paired observations: 03 months:
n = 28, 36 months: n = 53, 612 months: n =
57, 1218 months: n = 64, 018 months: n =
72. The results are presented as mean B SD
for the children independent of allergy.
*pd0.05, ** pd0.01 and *** pd0.001.

MFI

]]]

30

20
s

10

]]]

]]]

s
]]

0
Age, months

12

18

n=

75

59

61

65

134

17

p = 0.032

CD4 MFI

15

cytes at birth, 3, 6, 12 and 18 months of age

in children who developed atopic disease (P)
within the first 18 months of life, as compared with non-atopic children (I). The results are presented as mean B SD for the two
groups. Statistical analysis was performed
with one-way analysis variance of * pd0.05.

NS

NS

NS

11

9
p = 0.046
7
Age, months

12

18

n=

49

40

43

48

92

n=

15

13

12

11

26

pression on pan T-cells increased from birth up to 18

months (p d0.001 fig. 1). In contrast, the CD4 expression
on T-helper and CD8 expression on T-cytotoxic cells decreased from birth up to 18 months of age (p d0.001 for
both, fig. 2).
Two-way analysis of variance including AFH and atopic
disease revealed that children with double AFH had a lower
monthly increase in CD3 expression than children with no
AFH (table 1).
The CD4 expression on T-helper cells was higher at birth
in children who developed atopic manifestations, as com-

pared with non-atopic infants (p = 0.032). This was also the

case at 3 months (p = 0.046, fig. 3). The differences in CD4
expression were more pronounced at birth (14.8B1.3, n =
15, vs. 13.6B1.1, n = 17; p = 0.009) and at 3 months (11.4
B1.1, n = 13 vs. 10.3B1.5, n = 19, p = 0.027) in atopic children regardless of AFH than in non-atopic children without
AFH.
The CD4 expression on T-helper cells decreased more
from 3 to 6 months and from birth to 18 months in the
atopic than in the non-atopic children (data not shown).
One-way (data not shown), as well as two-way variance of

Expression of T-Cell Markers

Int Arch Allergy Immunol 1999;119:612

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Fig. 3. CD4 MFI on CD3+CD4+ lympho-

13

Table 1. Monthly change (mean B SE),

of CD3 expression (measured as MFI) on
CD3+CD45+CD14 lymphocytes and CD4
expression on CD3+CD4+ lymphocytes, in
relation to AFH

p value

No AFH
Single AFH
Double AFH

CD3 MFI change/month

in CD3+CD45+CD14ly
+0.91B0.12
+0.87B0.11
+0.38B0.13

31
26
38

0.003

No AFH
Single AFH
Double AFH

CD4 MFI change/month

in CD3+CD4+ly
0.08B0.02
0.19B0.02
0.16B0.03

AD
No AD

CD4 MFI change/month

in CD3+CD4+ly
0.21B0.03
0.12B0.02

31
25
38

23
72

0.016

0.004

d0.001

0.001

d0.001

0.002

The monthly changes of CD4 expression of CD3+CD4+ lymphocytes, in relation to atopic

disease (AD) are also shown. Two-way analysis of variance including AFH and atopic disease
was performed on data from children with at least two observations during the study period.

Discussion

Our findings indicate that the CD3 expression, reflecting

the T-cell receptor density, is low during the first 6 months
of life and is then upregulated. The low CD3 expression at
birth is supported by studies on cell proliferation responses
to anti-CD3 [9, 10], reporting a reduced response in healthy

10

Int Arch Allergy Immunol 1999;119:612

newborns as compared with adults. A lower CD3 expression

(measured as MFI) on T-cells in cord blood compared with
peripheral blood from adults has been reported [11].
There was a lower monthly increase in CD3 expression
on CD3+CD45+CD14 cells during the first 18 months of
life in children with a double AFH, as compared with children with single or no AFH. This may indicate a delayed upregulation of the CD3 receptor in children with an atopic
family history. The findings were subtle, but they are supported by observations that T-cells from infants with a family history of allergy required higher levels of anti-CD3
stimulation to reach maximum responses than adults, while
low-risk infants did not differ from adults [12].
In contrast, the CD4 and CD8 expression on T-cells were
higher at birth and then decreased until 12 months of age. It
may be speculated that the expressions of CD4 and CD8 are
upregulated during pregnancy and are then downregulated
after birth when the immune system adapts to extra-uterine
life.
Previous comparisons in both cross-sectional and
prospective studies have shown lower proportions of CD8+
cells [13, 14] and CD4+ cells [15], a higher number of
CD3+ and CD4+ cells [16] in atopic compared to nonatopic children, although similar findings [17] have also
been reported. A lower proportion of CD8+ cells [18] or the
combination of a lower number of CD3+ cells and a positive atopic family history [19] has been shown in children
before the onset of atopic disease, as compared to controls.
Higher numbers of CD3+, CD4+ and proportions of CD8+
T-cells have been reported [20], although in other prospec-

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analysis (table 1), indicated that the monthly decrease in

CD4 expression on T-helper cells during the first 18 months
was higher in atopic than in non-atopic children. The
monthly decrease in CD4 expression on T-helper cells was
more pronounced in children with single and double AFH
than in children with no AFH, as observed both in one-way
(data not shown) and in two-way analysis of variance
(table 1).
Children with maternal, as compared to paternal AFH
had a lower CD4 expression on T-helper cells at 6 months
(8.9B1.1, n = 8 vs. 10.5B0.6, n = 6; p = 0.01). The MFI expression of CD3, CD4 and CD8 at any age was not related
to the SPT results at 18 months (data not shown). At 18
months of age, however, the SPT-positive atopic children
had a lower expression of CD3 as compared to non-atopic
children without AFH and a negative SPT 34.0B8.6, n = 12
vs. 39.6B8.2, n = 40, p = 0.043).
The predictive value for atopic disease in this study was
poor and the best value observed was for CD4 expression at
3 months (69% sensitivity and 72% specificity at a cut-off
point at 11).

Expression of T-Cell Markers

atopic disease. One possible explanation might be that children predisposed to atopy are more easily sensitised in early infancy and/or in utero or that there is a transient maturational defect in peripheral blood mononuclear cells with
takes longer to resolve in future atopics [29]. It has also
been suggested [28] that an absence of an effective Th2-inhibitory signal within the first 6 months of life may favour
expansion of Th2-memory cells in children who later on develop atopic disease.
In conclusion, our findings support that the development
of T-cell function differs between atopic and non-atopic infants.

Acknowledgements
This work was supported by grants from the Medical Research
Foundation of the County of stergtland, The National Association
for the Prevention of Asthma and Allergy (RmA), The Swedish Medical Research Council (MFR project No. 07510), The Vrdal Foundation, The Foundation of Queen Silvias Jubilee Fund and the
Samariten Foundation. Assistance by the staff at the Flow Cytometry
Unit, Department of Clinical Immunology and at the Paediatric Research Unit, as well as valuable statistical assistance by Olle Eriksson
is gratefully acknowledged.

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tive studies they were similar in atopic and non-atopic individuals [21, 22]. However, a great deal of biological information is being lost by not determining the fluorescence intensity of the positive cells [23]. Flow cytometry
fluorescence intensity correlates directly with the number of
receptor molecules on the cell surface [24] and variations in
expression may reflect a functional difference [25]. To our
knowledge, this is the first prospective study following the
MFI expression of the CD3, CD4 and CD8 expression in
children.
It has been hypothesised that the balance between Th1
and Th2 memory cells is set during early childhood by a
process of an antigen-driven Th1/Th2 selection [26]. In
non-atopic individuals this will lead to a Th1 dominance,
but when the selection occasionally fails, this will lead to a
dominance for Th2 cells, and eventually atopy. We found
that CD3+CD4+ cells had a higher CD4 expression at birth
and at 3 months in children who subsequently developed
atopic disease, as compared to non-atopics. Since CD4 expression on CD3+CD4+ cells normally decreases during
the first 6 months of life, the elevated levels of expression in
atopic children may be indicative of a delayed functional
maturation. This suggestion is consistent with findings
demonstrating low interferon gamma (IFN-) producing capacity of cord blood mononuclear cells [reviewed in ref. 27]
and CD4+ cells in children [4] with an atopic family history. Further, a recent report has shown a lower IFN- production at birth by cord blood mononuclear cells and purified CD4+ cells stimulated by anti-CD3/CD2/CD28 and
interleukin 2 in children with definite atopy by 2 years compared to non-atopic children [28].
The atopic children in our study had a higher expression
of CD4 on CD3+CD4+ cells at birth and at 3 months, but
not at 6, 12 and 18 months. Miles et al. [29] reported higher
proliferation at birth in response to anti-CD3 or allergens in
infants with a positive family history who developed atopic
eczema, as compared with high-risk infants who did not develop atopic disease. Similar to our observations, these differences had all disappeared at 6 months of age. Our findings are also supported by Prescott et al. [28]. They reported
a consistent fall in cellular interleukin 4 (IL-4) mRNA responses to house dust mite in non-atopic children from birth
to 18 months of age. This appeared to be initiated during the
first 6 months of age, while atopic children continued to
show persistent IL-4 mRNA. They further observed low
IFN- responses at birth in non-atopic infants followed by a
significantly increase to 6 months of age. This was not seen
in the atopic children. Our findings may support the concept
that the first months of life, perhaps even the first 6 months
of life, is a vulnerable period for development of future

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Emory University
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