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*
Department of Cellular Biology, Faculty of Biology, and
Department of Cellular Biology, Faculty of Medicine, Universidad Complutense de Madrid, Madrid, Spain; and
Immunology and
§
Pediatric Services, Hospital Gregorio Marañón, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ßhighCD69-CD1-CD45RO+/RAdull
and CD45ROdull/RA+ cells, which presumably
correspond to recirculating T lymphocytes into the thymus. A notable
reinforcement of the subcapsular epithelial cell layer as well as an
increase in the intralobular extracellular matrix network accompanied
modifications in the thymocyte population. Additionally neonatal thymic
dendritic cells were found to be more effective than dendritic cells
isolated from children’s thymuses at stimulating proliferative
responses in allogeneic T cells. All these findings can account for
several alterations affecting the peripheral pool of T lymphocytes in
the perinatal period. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among peripheral T lymphocytes, most neonatal T cells exhibit an
antigenically naive cell surface phenotype (i.e.,
CD45RA+) and a correspondingly naive functional
program (16). The cytokine profile of neonatal T cells is
unique, and it has been reported that T cells from newborns produce
less IFN-
, IL-2, IL-4, IL-10, and TNF- than adult T cells in
response to various stimuli (17, 18, 19). The CD40 ligand
activation marker, which is involved in T-B cell interactions, is
reduced on neonatal T cell activation (20), and the
expression of a number of cytokine receptors, including the , ß,
and chains of the IL-2 receptor, is also diminished compared with
adult cells (21, 22). Normal responses to alloantigens and
improvement of both proliferative responses and cytokine production
after enhancement of costimulatory signals suggest that neonatal T
cells have altered thresholds of responsiveness (1).
Despite the large number of results showing the impairment of newborn
immunity, very little information is available about the neonatal
condition of the thymus gland, a key organ for the maturation of an
efficient immune system. In this regard, a decrease in the proportion
of
CD4+CD8+TCRßlow
thymocytes has been reported in the neonatal rat and mouse thymus,
concomitant with the start of differentiation of a new wave of T cell
precursors colonizing the organ (23, 24, 25). In the present
report, we analyze the different cellular components of the neonatal
human thymus, demonstrating that a transient severe depletion of
immature CD4+CD8+
thymocytes occurs after birth, along with remarkable phenotypic and
functional modifications of the thymic microenvironment components.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human thymus samples were obtained from neonates (age range, 1–20 days; n = 9) and children (age range, 1 month to 10 years; n = 11) undergoing corrective cardiovascular surgery at the Hospital Gregorio Marañón (Madrid, Spain). All donors were born at term after either spontaneous onset labor or cesarean section in the absence of labor; and at the time of sampling, donors were free of treatment that may influence immune functions, such as irradiation, steroids, glucocorticoids, or other immunosuppressive treatments.
Thymuses were dissected free of surrounding connective tissue and then gently disrupted with a Potter homogenizer until completely disaggregated. Thymocyte suspensions were washed and resuspended in RPMI 1640 supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100 UI/ml penicillin, 100 µg/ml streptomycin (all products from Life Technologies, Eragny, France), and 5% FCS (Biosys, Compiègne, France), hereafter referred to as complete medium.
Immunofluorescence and flow cytometry
mAbs of the following specificities were used in our study: CD4
(RPA-T4-FITC or -PE), CD8 (RPA-T8-PE), CD1 (HI149-biotin or -FITC),
CD45RO (UCHL1-FITC), CD45RA (HI100-PE), CD11c (B-ly6-PE), CD69
(FN50-FITC), CD34 (581-FITC), CD80 (BB1-FITC), CD86 (2331-biotin), CD40
(5C3-FITC), and HLA-ABC (G46-2.6-FITC) were obtained from PharMingen
(San Diego, CA); TCRß (WT31-biotin), CD3 (SK7-PE), and HLA-DR
(L243-FITC or -PE or -PerCP) from Becton Dickinson (San Jose, CA); CD54
(15.2-FITC) from Southern Biotechnology (Birmingham, AL); and CD49d
(HP2/1-FITC) from Immunotech (Marseille, France).
Two- and three-color immunofluorescence stainings were performed by incubating the cells in PBS containing 1% FCS and 0.1% NaN3 in the presence of saturating amounts of FITC-, PE-, PerCP-, and biotin-labeled mAbs for 30 min at 4°C. The biotinylated Abs were revealed with second-step streptavidin-CyChrome (PharMingen). Isotype-matched irrelevant Abs were used as negative controls to define background fluorescence. Stained cells were analyzed in a FACScan flow cytometer (Becton Dickinson) from the Servicio Común de Investigación, Faculty of Biology, Complutense University of Madrid (Madrid, Spain). Debris and dead cells were excluded from the analysis by forward light scatter (FSC), side light scatter (SSC), and propidium iodide gatings, and analysis was performed on at least 10,000 events. The data were analyzed using PC-lysis research software (Becton Dickinson).
Cell cycle analysis
To determine the proportion of proliferating cells, 2–3 x 105 cells were stained with anti-CD4-FITC and anti-CD8-PE for 30 min at 4°C. Cells were washed twice with PBS and fixed in 30% ethanol for a minimum of 30 min, but usually overnight at 4°C. The cells were then washed, resuspended in a solution of 25 µg/ml 7-amino actinomycin D (Sigma España) in PBS with 0.025% Nonidet P-40 (Sigma), and incubated in the dark at 4°C for 2 h. Analysis was conducted in a FACScan, using Cell Fit and PC-lysis software (Becton Dickinson).
Apoptosis assay
After cells were stained with anti-CD3-PE and washed twice with PBS containing 1% FCS, cells were stained with annexin V-FITC (Boehringer Mannheim, Mannheim, Germany) according to the instructions of the manufacturer. Cells were analyzed on a FACScan and gated according to FSC, SSC, and their ability to exclude propidium iodide. Apoptotic cells were considered as those annexin V positive and propidium iodide negative.
Serum cortisol levels
Blood samples were collected before surgery in nonheparinized tubes and, after four h at room temperature, centrifuged at 2,200 rpm for 15 min at 4°C. Sera were stored at -70°C until assayed. A fluorescence polarization immunoassay kit (TDx/TDx Cortisol, Abbott Laboratories, Abbott Park, IL) was used for the determination of serum cortisol levels according to the commercial supplier’s instructions.
Immunohistological staining
Thymic cryosections 7 µm thick were air-dried for 2 h at room temperature and fixed in acetone for 10 min. Sections were incubated for 1 h at room temperature in the presence of the following unlabeled Abs: anti-cytokeratin (NCL-Pan-CK) from Novocastra (Newcastle, U.K.); anti-laminin (LAM-89), anti-fibronectin (IST-4), and anti-type I collagen (COL-1) from Sigma; anti-HLA-DR (L243) from Becton Dickinson; anti-CD1a (HI149) and anti-CD11c (B-ly6) from PharMingen; and anti-cortical epithelial cells (TE-3) and anti-medullary/subcapsullary epithelial cells (TE-4) kindly provided by Dr. B. F. Haynes (Duke University Medical Center, Durham, NC). Control slides were incubated with anti-thyroglobulin Abs or normal mouse IgG (Dako, Glostrup, Denmark).
For immunohistochemistry stainings, endogenous peroxidase activity was inhibited with 1% H2O2 in methanol for 20 min, and sections were then incubated for 45 min with a 1/40 solution of peroxidase-conjugated rabbit anti-mouse Igs in PBS (Dako). The peroxidase reaction was developed with 0.05% 3,3'-diaminobenzidine (Sigma) in PBS with 0.1% H2O2 for 10 min. Sections were counterstained with methylene blue, dehydrated, and mounted in DePeX.
For immunofluorescence stainings, binding of primary mAbs was detected by incubation with a 1/100 solution of FITC-conjugated F(ab')2 fragment of rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and then examined using a Zeiss Axioplan-2 microscope.
Histological study
Thymic fragments were fixed by immersion in 4% cold glutaraldehyde, buffered to pH 7.3 with Millonig’s fluid, postfixed in 1% osmium tetroxide in the same buffer, and dehydrated in acetone for embedding in Araldite (Fluka, Buchs, Switzerland). Semithin sections (1–2 µm) were obtained with a Reichert OM-U3 ultramicrotome (Reichert-Jung, Wien, Austria) and stained with alkaline toluidine blue for the histological studies.
MLR assay
Thymic DC were isolated according to a modified procedure described by Beaulieu et al. (26). Briefly, unfractionated human thymic cells were first depleted of CD2+ thymocytes by incubation with neuraminidase-treated sheep RBC followed by Ficoll separation. Recovered cells were then depleted of T, B, myeloid, and NK cells by treatment with anti-CD3 (UCHT1), anti-CD7 (M-T701), anti-CD19 (B43), anti-CD14 (M5E2), and anti-CD56 (B159) (all from PharMingen) bound to sheep anti-mouse Ig-coated magnetic beads (Dynal, Oslo, Norway). Subsequently, thymic DC were used at different numbers (10 cells to 5 x 104 cells) as stimulators for resting allogeneic T cells (2 x 105) isolated from peripheral blood. The cultures were performed in 96-well flat-bottom culture plates, using 0.2 ml complete medium. After 5 days at 37°C in a 5% CO2-in-air incubator, the cultures were pulsed for 12 h with 10 µM 5-bromo-2'-deoxyuridine (BrdU). A specific kit from Boehringer Mannheim (BrdU Labeling and Detection Kit III) was used to measure BrdU incorporation into newly synthesized DNA. Briefly, the labeling medium was removed, and cells were dried (2 h at 60°C), fixed in ethanol in HCl (0.5 M) for 30 min at -20°C, treated with nucleases (30 min at 37°C), and then incubated with peroxidase-conjugated Fab fragments of mouse anti-BrdU (30 min at 37°C). The peroxidase reaction was developed with ABTS substrate, and the sample absorbance was measured using an ELISA reader at 405 nm with a reference wavelength at 492 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The flow cytometric analysis of CD4 and CD8 Ag expression by
thymocytes from newborns and children showed that the proportion of
double-positive (DP)
CD4+CD8+ thymocytes was
dramatically reduced in neonatal thymuses. On the contrary, the
percentages of single-positive (SP)
CD4+CD8-/CD4-CD8+
thymocytes and, mainly, double-negative (DN)
CD4-CD8- cells were
notably higher than those found in children (Fig. 1
A). Furthermore, although the
total proportion of TCRß+ thymocytes hardly
changed, the subset of TCRßlow cells dropped
from 35–45% to 5–20% whereas mature
TCRßhigh thymocytes accumulated in newborns
(Fig. 1B). Within each CD4:CD8-defined cell population, the
relative distribution of TCRß expression remained unaltered,
except for a 2- to 3-fold increase in the proportion of
TCRßhigh cells detected within the
CD4+CD8+ subset (Fig. 1B). No important variations in CD4:CD8 cell ratio were
observed (newborns, 2.2; children, 2.7).
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,
thymic glands from children showed a well-developed cortex, filled by
CD1+ cells. In contrast, neonatal thymuses
exhibited a reduction in the size of thymic cortex, where only a few
scattered CD1+ cells appeared. This neonatal
depletion of cortical DP thymocytes was evident in all samples from
donors with ages ranging from 1 to 20–30 days. From the second
month of postnatal life, all donors exhibited normal proportions of
thymocyte subsets (Fig. 1
C).
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). Therefore, variations in
proliferation cannot account for the differences found in the
proportion of the thymocyte subsets.
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). When cells were gated according to
their CD3 expression, the highest proportion of cells undergoing
apoptosis was found in the CD3- and, mainly,
CD3low cell subsets, where almost one-half of the
population stained with annexin V (Fig. 3). The incidence of cells
undergoing apoptosis in the mature CD3hi cell
subset was low in both newborns and children (Fig. 3). These data show
that it is predominantly the
CD4+CD8+CD3-/low
cell population that is undergoing apoptosis, therefore largely
accounting for the decreased proportion of DP thymocytes found in the
neonatal thymus.
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Thymocyte depletion observed in newborns could be reflecting an
acute stress as a consequence of the complex congenital cardiac defects
exhibited by these neonatal donors. We then determined the levels of
cortisol in the serum of newborns and children, just before cardiac
surgery. Our results demonstrate that there is no correlation between
the levels of circulating cortisol and the percentage of DP thymocytes
(Fig. 4). In addition, serum cortisol
levels from all donors used in this study were in the same range of
values than those obtained from healthy newborns (range, 2–11 µg/dl)
and children (3–21 µg/dl) (28). We can therefore
conclude that the transient thymic involution is a physiological rather
than a stress-associated pathological event occurring in the
neonates.
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The nature of mature TCRßhigh
thymocytes which increased in neonatal thymuses was investigated by
analyzing the expression of different cell markers, including CD69,
CD1, CD45RO, and CD45RA, which define phenotypically distinct stages
during terminal differentiation of human thymocytes (29, 30). Three parameter analysis of the neonatal
TCRßhigh thymocyte subset showed an
accumulation of
CD69-CD1-CD45RO-/dullCD45RA+/CD45RO+CD45RAdull
cells (Fig. 5), the phenotype of these
cells being identical with that found in T cells from cord blood and
neonatal peripheral blood (Fig. 5).
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The earliest T cell precursors in the thymus have been shown to be cortical thymocytes contained within the CD3-CD4-CD8- triple negative (TN) population that express the human pluripotent stem cell marker CD34 (31). Using three-color flow cytometric analysis, we found that TN CD34+ cells represented 1.1% (range, 0.8–1.5%) of total thymocytes from children, whereas their proportion was increased 3–4 times in neonatal thymuses (mean, 3.9%; range, 2.9–6.1%). In addition, a higher proportion of TN CD34+ cells did not express CD1 in newborns (70–75%) compared with children (40–50%), indicating that the most immature intrathymic precursors accumulate in the newborn thymus. This increased levels of primitive precursor cells could be related with the regeneration of the thymus gland which occurs by the end of the first month of postnatal life.
Analysis of neonatal thymic microenvironment components
An immunohistochemical analysis of thymic samples was conducted to assess whether the modifications found in neonatal thymocytes correlate with changes in the thymic stroma, including thymic epithelial cells (TEC) and extracellular matrix (ECM) components.
When comparing thymuses from children and newborns, no significant
differences could be observed in the keratin-positive epithelial
reticulum appearing in both thymic cortex and medulla (Fig. 6, a and b), by
using an anti-pan cytokeratin Ab defining all epithelial cell
subsets. Similarly, the pattern of distribution of
TE-3+ and TE-4+ cells,
which corresponded to cortical and medullary TEC, respectively, was not
modified in neonatal thymuses (data not shown). It is important,
however, to emphasize the notable reinforcement of the subcapsular
epithelial cell layer occurring in the thymus of newborns, as evidenced
by staining with anti-cytokeratin and TE-4 Abs (Fig. 6, c–f). By contrast, important changes occurred in the
pattern of immunostaining of class II MHC molecules on TEC. Whereas in
children’s thymuses a regular network of class II-positive epithelial
cell processes occupied the whole thymic cortex (Fig. 6g),
in neonatal thymuses the cortical areas exhibited a confluent pattern
of staining (Fig. 6h), similar to that observed in the
thymic medulla from children (Fig. 6g).
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a). Fibronectin and laminin
were detected in the basement membranes, as well as forming a fine
network within the medulla of the thymic lobes (Fig. 7, c
and e). On the contrary, a remarkable ECM pattern was
consistently found in neonatal thymuses. A thickening of the basement
membrane bordering the capsule and septa was observed after staining
with anti-laminin and anti-fibronectin Abs (Fig. 7d). The cortical areas showed an increased expression of
any ECM component (Fig. 7, b and d), partially
associated with numerous enlarged blood vessels (Fig. 7, g
and h). In addition, there was an increase in the number of
fibronectin-positive thin fibers localized in the thymic medulla (Fig. 7f).
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Thymic DC constitute a relative sparse cell population which is
thought to be involved in intrathymic negative selection of
autoreactive thymocytes. Then, we first analyzed the pattern of
expression of several surface markers on thymic DC from children and
newborns. Virtually all thymic DC isolated from children and newborns
were positive for CD11c, class II and I MHC molecules, CD86, ICAM-1,
and VLA-4, although they lacked CD80 and CD1 expression (Fig. 8A). The level of expression
of these molecules was, however, modified when both DC populations were
compared, being that the levels of CD86 and MHC Class II molecules
increased and those of the adhesion molecules ICAM-1 and VLA-4
decreased in neonatal DC (Fig. 8A). Likewise, both the
proportion of positive cells and the level of expression of CD40 were
increased in the neonatal DC population (Fig. 8A).
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B,
neonatal thymic DC were more efficient than thymic DC isolated from
children at stimulating the proliferation of alloreactive T cells. Finally, the intrathymic localization of DC was also investigated using immunohistochemistry on tissue sections. CD11c+ DC were mainly found in the medulla and the corticomedullary border in both newborns and children (data not shown), which shows that the distribution of DC is not altered in the neonatal thymus.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To our knowledge, a similar thymocyte depletion in the neonatal period
has not been previously reported in humans, although Bertho et al.
(34) have described a reduced thymic cellular density
during the first weeks after birth. Decreased proportions of
CD4+CD8+TCRßlow
thymocytes have been also pointed out in the thymus of neonatal mice
and rats (24, 25). The underlying mechanisms to this
process have not been formally proved, but two distinct mechanisms are
known to be involved in the intrathymic cell death of DP thymocytes.
One seems to be dependent of endogenous glucocorticoids
(35), and the other operates via TCR and involves thymic
DCs (36). The elevation of endogenous glucocorticoid
levels has been demonstrated to result in a massive apoptosis of
thymocytes, largely immature DP cells, and loss of thymic cellularity
(37, 38). In this regard, it has been reported that fetal
levels of corticosteroids rise very markedly by 3- to 12-fold
throughout late gestation (from weeks 24 to 26 until the onset of
labor), and sharply decline under adult levels immediately after birth
(39, 40, 41). This physiological increase in fetal
corticosteroid levels taking place by the end of gestation has a
critical and unique function in the fetus in inducing a wide range of
enzyme systems before birth that have little or no function during
fetal life, but on which survival after birth is dependent. These
maturation events have been reviewed by Liggins (42) and
include stimulation of enzymes responsible for glycogen accumulation
and gluconeogenesis in the liver; maturation of ß cells of the
pancreas; induction of cytodifferentiation of type II alveolar cells
and synthesis and release of pulmonary surfactant; induction of
epithelial maturation and alkaline phosphatase activity of the small
intestine; and increase in the production of triiodothyronine and
catecholamines.
Then, the prolonged exposure of fetal thymus to the increased levels of corticosteroids could explain the cellular depletion affecting the neonatal thymus. The finding that reduced numbers of DP cells can be detected in thymus samples from 1-day-old donors strongly suggests that the depletion of DP thymocytes begins to occur some time before birth. The normal proportions of thymocyte subsets are, however, recovered by the end of the first month of postnatal life, which indicates that the perinatal thymus recovers slowly after glucocorticoid exposure. In agreement with this point, Bakker et al. (43) described that the prenatal exposure to glucocorticoids induce long-lasting effects on the neonatal rat thymus, which exhibited decreased T cell numbers.
Neonatal thymic DC could also contribute to the decrease in the thymic DP cell numbers. Supporting this notion, we demonstrate that neonatal thymic DC exhibit an enhanced allostimulatory activity compared with DC isolated from children thymuses. This was in complete agreement with the up-regulated expression of CD86, CD40, and MHC class II molecules shown in the neonatal thymic DC population. The functional importance of MHC Class II Ags and CD86 and CD40 costimulatory molecules in the allogeneic MLR stimulated by human DC has been repeatedly reported (44, 45, 46). In contrast, cord blood DC have been shown to be poor stimulators of the allogeneic MLR compared with adult DC (10). The differential function of these two types of DC populations, as well as their putative distinct origin (36), could explain the different results obtained.
An important issue which this study raises is the impact that the transient neonatal thymic involution could have on the peripheral T cell population. Interestingly, several authors have described a 20–70% reduction in the proportion of T cells in cord blood and neonatal peripheral blood when compared with adult blood (our unpublished observations and Refs. 47, 48, 49). The drastic reduction in the numbers of immature CD4+CD8+ thymocytes and the subsequent depletion of their progeny, which includes the mature SP thymocytes population that will exit the thymus to the periphery, could account for the diminished numbers of peripheral T cell occurring in the perinatal period.
Another finding of this study is that the alterations in thymocyte
subpopulations are accompanied by modifications of the thymic stromal
cell components. Some of these alterations, such as the change in the
pattern of expression of Class II MHC molecules, could just be
reflecting the loss of cortical thymocytes. However, other
modifications of the nonlymphoid thymic components described here would
represent an unique situation of the perinatal period rather than a
direct influence of the cortical thymocyte depletion. Our data
demonstrate a remarkable reinforcement of the subcapsular epithelial
cell layer, as well as the existence of numerous enlarged blood vessels
throughout the neonatal thymic parenchyma, which suggests the
occurrence of important changes in thymic permeability. Similarly,
enlarged perivascular spaces appear throughout rat thymic parenchyma
during the perinatal period (25). Also Martín et
al. (50) described, after increasing sex steroid levels,
an increase of thymic cortical and corticomedullary vascular
permeability, along with an almost total disappearance of the
transcapsular route, caused by the reinforcement of the subcapsular
epithelial cell layer. High levels of circulating sex steroids occur in
the perinatal period (51) and, therefore, could explain
the changes in neonatal thymic permeability, which could favor the
entry of cells from the periphery. In fact, the analysis of
TCRßhigh thymocyte subset from newborn
thymuses showed a preferential accumulation of
CD69-CD1-CD45RA+
cells. These cells exhibit the same phenotype as T cells from cord
blood as well as peripheral blood from neonates and children, and would
correspond to recirculating T lymphocytes that enter the thymus
from the periphery. Supporting this, it has been pointed out that
increased numbers of peripheral T cells with a resting/naive phenotype
readily enter the mouse thymic gland during the neonatal period
(52).
Additionally, the emigration of thymocytes from the thymus could be also altered by the increased thymic vascular permeability. We and other authors (27, 47, 49) have shown the presence of immature T cell subpopulations in neonatal blood, including CD4+CD8+ and CD1+ cells. A phenotypic characterization of human peripheral DP cells has been conducted by Res et al. (27), who conclude that these cells correspond to a mature stage of CD1- DP thymocytes that have been submitted to positive selection. With regard to CD1+ cells, they must include SP thymocytes that have not conclude the maturational process by which functionally mature SP T cells are generated (29, 30).
Although most functionally immature thymocyte subsets are stationary, their migration activity has been shown to be stimulated by high concentrations of ECM proteins, such as fibronectin (53). Our results show an increased expression of different ECM components in the neonatal thymus, and mainly of fibronectin in the thymic medulla. Therefore, the increase in the intrathymic ECM network could explain the presence of immature T cell subsets in peripheral blood during the perinatal period.
In conclusion, our results provide evidence that a profound involution transiently affects the human neonatal thymus; therefore, this is another event to incorporate in the ever-increasing list of immunological alterations occurring in the perinatal period.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alberto Varas, Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain.
3 Abbreviations used in this paper: DC, dendritic cells; DP, CD4+CD8+ double positive; DN, CD4-CD8- double negative; ECM, extracellular matrix; SP, single positive; TEC, thymic epithelial cells; TN, CD3-CD4-CD8- triple negative; FSC, forward light scatter; SSC, side light scatter; BrdU, 5-bromo-2'-deoxyuridine.
Received for publication January 3, 2000. Accepted for publication March 27, 2000.
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and its receptors. Pediatr. Res. 40:475.[Medline]
chain on cord blood lymphocytes: relationship to functional immaturity of the neonatal immune response. Immunology 87:86.[Medline]
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, newborns) represent the percentages of positive cells
for the indicated phenotype, for each sample. B, CD4 vs
CD8 expression is represented in the dot plots. The percentages of the
thymocyte subsets were: for children, CD8 SP, 4.89%; DP, 76.97%; DN,
4.87%; CD4 SP, 13.26%; and for newborns, CD8 SP, 17.27%; DP,
29.55%; DN, 33.63%; CD4 SP, 19.55%. TCR
