| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Istituto di Morfologia Umana Normale, Chieti, Italy;
Cattedra di Neonatologia, Università degli Studi "G. D’Annunzio," Chieti, Italy;
Istituto di Anatomia Umana Normale, Università di Bologna, Bologna, Italy;
§
Dipartimento di Scienze Biomediche e Biotecnologie, Sezione di Anatomia Umana, Università di Brescia, Brescia, Italy; and
¶
Laboratory of Immunobiology, Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Bethesda, MD 20852
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 are expressed at higher levels in
cord T cells, while PLCß2 and 
1 expression is higher in adult T
lymphocytes. PLC2 and 2 appear to be equally expressed in both
cell types. In addition, a functional defect in PLC activation via CD3
ligation or pervanadate treatment, stimuli that activate tyrosine
kinases, was observed in cord blood T cells, whereas treatment with
aluminum tetrafluoride (AlF4-), a G protein
activator, demonstrated a similar degree of PLC activation in cord and
adult T cells. The impaired PLC activation of cord blood-derived T
cells was associated with a a very low expression of the Src kinase,
Lck, along with a reduced level of ZAP70. No mitogenic response to CD3
ligation was observed in cord T cells. However, no signaling defect was
apparent downstream of PLC activation, as demonstrated by the mitogenic
response of cord T cells to the pharmacologic activation of protein
kinase C and Ca2+ by treatment with PMA and ionomycin.
Thus, neonatal cord blood-derived T cells show a signaling immaturity
associated with inadequate PLC activation and decreased Lck
expression. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß heterodimer, which provides T cells with the ability to
recognize Ags bound to MHC molecules, the -, -, and 
-chains of
the CD3 complex, and the 
-chain, elements that transduce the
activation signals to the cytoplasm of T lymphocytes. The CD3 complex
and -chains are rapidly tyrosine phosphorylated by Src kinases after
TCR engagement (1, 2). This leads to phosphorylation and
activation of inositol-specific phospholipase C
(PLC)3 with ensuing
increased cytoplasmic levels of polyphosphoinositide breakdown
products: inositol phosphates and diacylglycerol. Recent studies also
suggest that a G protein-dependent activation of PLCß occurs
following TCR-CD3-mediated signaling (3). As a consequence
of either PLC or PLCß activation, a rapid rise in cytoplasmic
calcium concentration and activation of protein kinase C (PKC) isoforms
occur. These events promote the differentiation and mitotic response of
T cells to Ags, although several additional components, such as Ras
proteins (4), are also involved downstream of the early
tyrosine kinase activation. Contradictory evidences have been reported concerning functional differences between human neonatal and adult immune cells. In general, cord blood T lymphocytes, B lymphocytes, and monocytes show decreased levels of certain cell surface markers and display an inability to elaborate cytokines relative to adult cells (5, 6, 7, 8). It has been described that CD7- T cells, absent in human cord blood lymphocytes, represent a constant portion of PBL progressively increasing during life span (5), and that cord blood contained significantly increased numbers of CD45RA+ cells, but reduced numbers of CD45RO+ cells when compared with the adult. Although such differences associate with the well-known immunological immaturity of newborns, the molecular mechanism underlying the physiologically immature immune response of neonatal T lymphocytes is poorly defined. Given the critical regulatory role of PLC-mediated signaling in the T cell response to Ags, we have studied the activation of phosphoinositide hydrolysis, its correlation with phosphoinositide-specific PLC isozyme expression, and its regulation by upstream events in umbilical cord- and adult peripheral blood-derived T cells. Our results show that, compared with adult T cells, cord blood-derived T lymphocytes display a differential expression of PLC isozymes and a defective induction of phosphoinositide hydrolysis in response to stimuli the action of which depends upon the activation of upstream protein tyrosine kinases. This observation correlates with a selective decrease in Lck expression by cord blood-derived T cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mononuclear fractions were prepared from umbilical cord blood and from peripheral blood of pregnant women immediately before delivery. Each cord sample was compared with T cells derived from the newborn’s own mother. To enrich the population for T lymphocytes, mononuclear fractions were depleted of adherent cells by incubating samples in plastic dishes overnight, followed by B lymphocyte depletion using magnetic beads coated with anti-CD19 mAb (Dynal, Oslo, Norway). Purity of each T cell separation was checked by anti-CD3 fluorescein (FITC) (Becton Dickinson, San Jose, CA) staining and flow cytometry analysis of a small aliquot of cells. Only samples exceeding a purity of 95% were used for the experiments. Stimulation was performed with anti-CD3 Ab (Sigma, St. Louis, MO) or with an isotype control Ab at 10 µg/107 cells. The Ab was allowed to bind for 30 min on ice, and stimulation was initiated by incubation in a 37°C water bath for times up to 45 min. Alternatively, samples were treated with either pervanadate (0.1 mM sodium orthovanadate, 0.3 mM hydrogen peroxide) or aluminum tetrafluoride (10 µM AlCl3, 25 mM NaF).
Immunoprecipitation and immunoblotting
T lymphocytes, washed in PBS, were resuspended in lysis buffer
(10 mM Tris-HCl buffer, pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 1 mg/ml
BSA, 1 mM vanadate, 50 mM sodium fluoride) and left on ice for 30 min.
For anti-phosphotyrosine or anti-PLC immunoprecipitation, cell
lysates (400 µg of proteins) were incubated at 4°C for 60 min with
anti-PLCß1, ß2, 1, 2, 1, 2, or anti-PY-99 Abs
(Santa Cruz Biotechnology, Santa Cruz, CA), previously coupled to
magnetic beads coated with secondary Abs. Immunocomplexes were
collected by a magnet and washed several times with RIPA buffer (PBS
containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) in
the presence of protease inhibitors.
For electrophoretic analysis, whole cell lysates or immunoprecipitated proteins were resuspended in SDS gel sample buffer and resolved on 8% SDS-PAGE, transferred onto nitrocellulose membranes, and incubated for 1 h at room temperature with individual Abs, as specified in the figure legend. Immunoreactive bands were detected by the enhanced chemoluminescence (ECL) system (Amersham, Arlington Heights, IL) using peroxidase-conjugated secondary Abs, according to the manufacturer’s directions. Internal controls, obtained by incubating membranes with the secondary Ab alone, yielded, always, negative results.
Densitometric analysis
Densitometric analyses were conducted by means of a Quantimet 500 Plus (Leica, Cambridge, U.K.) to determine the integrated density levels, using ISO (transmission density standard Kodak 152-3406, Kodak, Rochester, NY).
Confocal laser-scanning microscope analysis
Macrophage- and B lymphocyte-depleted samples were
cytocentrifuged, fixed in 3.7% formaldehyde diluted in PBS, and
permeabilized by immersion in PBS, 0.1% Triton X-100. Slides were then
incubated with anti-PLCß1, ß2, 1, 2, 1, or 2
polyclonal Abs (Santa Cruz Biotechnology), diluted 1/100 in PBS
containing 4 mg/ml normal goat serum and 4 mg/ml human Igs. Slides were
washed and reacted with FITC-conjugated anti-rabbit IgG Ab
(Molecular Probes, Eugene, OR) diluted 1/100 in PBS, 4 mg/ml normal
goat serum, and 4 mg/ml human IgG. After several washes in PBS, samples
were mounted in glycerol containing 1 µg/ml propidium iodide (PI) to
counterstain nuclei. Internal controls, performed omitting the primary
Ab, show no detectable FITC staining (not shown). Confocal analysis was
conducted with a TCS 4D (Leica) mounted on a Leitz DMRB microscope,
equipped with a x100/1.3 NA oil immersion objective. High resolution
fluorescence images were obtained by exciting FITC and PI at 488 and
514 nm, respectively, with an argon ion laser. The laser beam output
energy, the detector voltage, and the pinhole settings were different
for FITC or PI acquisition, but were rigorously maintained constant
during the observation of all samples. Images were acquired with an
averaging function line-by-line, top down, with a scanning mode format
of 512 x 512 pixels. Serial optical sections of FITC signal,
performed in z-axis and merged with the corresponding PI
images, were elaborated by a three-dimensional image processing
system.
Phosphoinositide hydrolysis
T lymphocytes were incubated with [3H]myo-inositol (35 µCi/ml, 10–20 Ci/mmol; Amersham) for 2 h at 37°C in RPMI 1640 with 50% autologous serum. Cells were rinsed twice with HEPES-buffered RPMI 1640 containing 20 mM LiCl and 1 mg/ml BSA and incubated in the same solution at 37°C for 15 min. Samples were then stimulated with anti-CD3, pervanadate, or aluminum tetrafluoride (AlF4-), as described above, for times up to 45 min. To stop the reaction, 5 ml of ice-cold PBS was added and the cells were collected by centrifugation at 4°C. Washed pellets were treated with 0.6 ml of ice-cold 7.5% perchloric acid, and the cell debris were pelleted by centrifugation. Supernatants were diluted 1/15 with 30 mM ammonium formate/2 mM sodium tetraborate and applied to a Bio-Rad (Richmond, CA) AG1-X8 ion-exchange column. The elution of the inositol phosphate esters was performed by stepwise additions of 60 mM ammonium formate/5 mM sodium tetraborate (for glycerophospho[3H]inositol); 0.2 M ammonium formate/0.1 M formic acid (for [3H]InsP); 0.4 M ammonium formate/0.1 M formic acid (for [3H]InsP2); 0.8 M ammonium formate/0.1 M formic acid (for [3H]InsP3); and 1.2 M ammonium formate/0.1 M formic acid (for [3H]InsP4). Eluted fractions were then analyzed by beta scintillation counting.
[3H]Thymidine incorporation
CD19-depleted samples were cultured for 72 h in presence of either 20 µl/ml PHA, 10 µg/ml anti-CD3, 10 µM ionomycin, or 10 nM PMA, and then labeled for 6 h with [3H]thymidine (1 µCi/30,000 cells/well; Amersham) and processed for scintillation counting, as previously described (9).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Before experiments, a cytofluorometric analysis of mononuclear
cells from adult and cord blood was performed to assess the
comparability of the two samples (Table I
). Results evidenced that the proportion
of cells expressing CD3, CD4, CD8, CD14 (monocytes), or CD19 (B cells)
was highly comparable in cord and adult T lymphocytes. After depletion
of adherent cells and of B lymphocytes, purity of each cell separation
was checked by flow cytometry, as shown in Fig. 1. In any case, only samples disclosing
more than 95% of purity were used for experiments.
|
|
We first looked for a signaling deficiency in cord T cells by
culturing the cells in the presence of mitogens and using the
proliferation as an indicator of productive signaling events. We
evaluated the mitogenic response of T cells cultured in medium or in
the presence of PHA, anti-CD3, alone or with different combinations
of PMA and/or ionomycin (Table II). In
particular, the combination of PMA and ionomycin led us to evaluate the
integrity of the signaling pathway downstream the PLC activation by
mimicking the effects of diacylglycerol and Ca2+
mobilization, the end result of PLC-mediated phosphoinositide
breakdown. PMA substitutes for diacylglycerol and activates PKC, while
ionomycin artificially increases the intracellular
Ca2+ concentration in an inositol
Tris-phosphate-independent manner. Compared with the untreated
controls, all of these stimuli, with the obvious exception of PMA or
ionomycin alone, induced proliferation in adult T lymphocytes, as
revealed by an increase in [3H]thymidine
incorporation. In cord cells, treatment with anti-CD3 or PHA gave
rise to negligible rates of DNA synthesis. Furthermore, neither PMA nor
ionomycin alone or in combination with anti-CD3 failed to induce a
proliferative response. The combination of PMA and ionomycin, however,
induced cell proliferation to a level comparable with that observed in
adult T lymphocytes. Therefore, the signaling defect of cord blood T
cells can be bypassed by the direct stimulation of PKC together with
artificially increasing the intracellular Ca2+
concentration, suggesting a substantial integrity of the signaling and
proliferative machinery downstream of PLC activation.
|
Then we studied PLC isoform expression in T lymphocytes from cord
blood or adult peripheral blood. To enrich for the isoform of interest,
equal amounts of detergent-solubilized proteins were subjected to
immunoprecipitation, followed by Western blot analysis (Fig. 2). PLCß2 and PLC1 immunoreactive
bands appeared to be very strong in adult T lymphocytes, whereas they
were detected at a lower intensity in cord cells. In contrast, PLCß1
was more represented in cord blood T cells. Cleaved forms of
PLCß1 and/or PLCß2 isoforms were sometimes detected when calpain
inhibitors were omitted during the experimental procedures. No obvious
difference was found between adult and cord T lymphocytes in the
expression levels of PLC2. The PLC1 isoform was difficult to
detect and appeared to be slightly higher in cord blood T lymphocytes
compared with adult T cells. No difference in PLC2 expression was
detected between adult and cord blood samples. Note that the relative
distribution of the different PLC isoforms varies between cord blood
and adult T cells. This observation rules out the possibility that
differential recovery could account for the differences observed.
|
). PLC2 was
equally expressed both in adult and cord cells, and appeared confined
quite exclusively to the cytoplasm (Fig. 3, A and
B). In adult cells, PLCß1 was faintly detectable in the
cytoplasm at the immediate periphery of the nucleus, while in the
nucleus it showed as a faint, dotlike, or granular distribution (Fig. 3C). In cord cells, the reaction appeared markedly stronger
in the cytoplasm, and slightly more intense in the nucleus (Fig. 3D). The PLCß2 screening evidenced an intense reaction in
adult T lymphocytes, both in the cytoplasmic and nuclear compartment,
whereas cord cells appeared positive, but to a lesser extent (Fig. 3, E and F). PLC1 was homogeneously distributed
in the cytoplasm, appearing slightly more represented in adult than in
cord cells (Fig. 3, G and H). It was weakly
detectable in the nucleus, without any significant difference in its
distribution between the two samples. PLC2 expression was comparable
between adult and cord cells, often showing a compact ring-like
distribution around the nucleus. In the nuclei, it appeared expressed
in dot-like or granular patterns (Fig. 3, I and
J). The immunocytochemical analysis confirms the Western
blot results and provides further evidence that all isoforms were
expressed mainly in the cytoplasmic compartment, with a weaker but
detectable nuclear localization.
|
We next investigated the activity of PLC in intact cells by
measuring the hydrolysis of phosphoinositide in response to CD3
stimulation of adult and cord blood T lymphocytes. The results (Table III) showed that stimulated adult cells
produced high levels of inositol phosphates reaching a plateau after 15
min. In contrast, anti-CD3-stimulated cord blood
T lymphocytes exhibit, along the time course of the treatment, no
detectable inositol phosphate production. Specificity of the
anti-CD3 response was verified by treatment with an isotype-matched
control Ab. No stimulation of phosphoinositide breakdown was found in
response to the control Ab in either adult or cord T cells.
|
). The different array of PLC
isozymes displayed by adult and cord blood T cells suggests the
possibility that the defective phosphoinositide hydrolysis of cord T
cells may be due to inefficient coupling of the isoforms expressed by
cord T cells to the receptor. An alternate possibility is that cord T
cells have an intrinsic TCR signaling defect upstream of PLC
activation.
To first exclude an intrinsic TCR defect, we examined the ability of
pervanadate and AlF4- treatment
to induce PLC activity in adult and cord T cells. Pervanadate and
AlF4- are pharmacological
agents that stimulate PLC and ß, respectively, while bypassing
ligand-receptor interaction (10, 11, 12, 13, 14).
Treatment of [3H]myo-inositol-prelabeled cord
blood T cells with pervanadate induced a modest increase in inositol
phosphate accumulation, while the same treatment of adult T cells led
to a substantial phosphoinositide breakdown (Table III). In contrast,
AlF4- stimulated both cord and
adult cells. These results show that shunting of the TCR by pervanadate
treatment did not rescue the impaired phosphoinositide hydrolysis of
neonatal T cells, and suggest that the signaling block is independent
and downstream of TCR engagement. The ability of
AlF4- to induce equal levels of
phosphoinositide hydrolysis in cord blood and adult T lymphocytes
indicates that there is no impairment in G protein-dependent PLCß
activation. It further indicates that critical experimental conditions
(i.e., labeling efficiency and the availability of the labeled
phosphoinositide pool) were adequate in either cell type to detect a
response.
The signaling defect of cord blood T cells is associated with reduced tyrosine kinase activation
To study the integrity of the TCR-linked signaling pathway, we
analyzed the protein tyrosine phosphorylation events evoked by TCR
engagement and pervanadate treatment. Pervanadate-induced intracellular
signals and cellular responses, while independent of TCR engagement,
are remarkably similar to those observed after TCR stimulation
(10, 11). Detergent-soluble extracts from anti-CD3 or
pervanadate-treated cells were analyzed for the Tyr-P-containing
proteins by immunoblotting. As expected, the anti-CD3 and
pervanadate stimulation of adult T cells induced a substantial increase
above background in protein tyrosine phosphorylation, with a similar
spectrum of tyrosine-phosphorylated substrates. In contrast, in cord
lymphocytes the stimulatory effect was almost undetectable after
anti-CD3 treatment and only marginally above background following
pervanadate treatment (Fig. 4). Thus,
bypassing the TCR with pervanadate treatment failed to significantly
augment protein tyrosine phosphorylation in cord T cells.
|
1 Abs (Fig. 5). To avoid that the lower PLC1
expression in cord cells could erroneously influence the detection of
phosphorylation levels, we performed the immunoprecipitation from a
3-fold greater amount of cord T cell proteins compared with that used
for adult T cells. Consistent with previous reports, in adult T cells
following TCR cross-linking or pervanadate treatment, Lck, Fyn, ZAP70,
and PLC1 underwent tyrosine phosphorylation, while Syk was scarcely
phosphorylated. In anti-CD3 or pervanadate-treated cord cells,
PLC1 was weakly phosphorylated, while all the other signaling
proteins, with the exception of Syk, show no detectable
phosphorylation. Syk was phosphorylated upon anti-CD3 stimulation
and following pervanadate treatment.
|
Next, we sought to determine whether the defective kinase activity
observed in neonatal T lymphocytes correlated with an anomalous
expression of the Src and Syk family kinases. Immunoblotting analysis
of proteins from T cells revealed a discrete reduction of ZAP70
expression in cord T cells, while Syk appeared more represented in cord
than in adult T cells (Fig. 6). The
immunoblot analysis of the expression of the Src kinases, Fyn and Lck,
revealed similar amounts of Fyn, while Lck protein expression was
dramatically reduced in cord T cells (Fig. 6). The differences
evidenced by Western blot were also confirmed through a densitometric
analysis (Table IV). Thus, the signaling
defect of cord blood T cells is characterized by a lower expression of
PLC1 and ZAP70, a near absence of Lck, together with a higher
expression of Syk and PLC2 compared with adult T lymphocytes.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 together
with a decrease in activation-induced protein tyrosine phosphorylation
of ZAP70 and PLC1 compared with that of adult T cells. This
phosphorylation defect was associated with reduced Lck expression,
while impairments downstream of PLC activation were excluded.
Upon Ag recognition, a complex set of signals is transduced from the
plasma membrane to the nucleus of T cells. TCR stimulation activates a
set of protein tyrosine kinases that control different signaling
pathways. One such pathway is centered on the activation of PLC
(19, 20). Two second messengers derived from PLC-mediated
phosphoinositide hydrolysis, diacylglycerol and inositol
1,4,5-triphosphate, control PKC activation and calcium mobilization,
respectively, and are critical for the successful mitogenic progression
of T cells. Compared with adult peripheral T lymphocytes, CD3 ligation
or treatment with pervanadate of cord T cells showed much reduced
inositol phosphate production. Pervanadate treatment bypasses receptor
engagement, but mimics the biochemical responses triggered by TCR
perturbation through the activation of protein tyrosine kinases and
the inhibition of protein tyrosine phosphatases (10, 11, 12, 13, 14).
CD3 ligation or pervanadate treatment preferentially stimulates PLC
isoforms, the activation of which requires tyrosine phosphorylation of
the enzyme (21). Our data are in agreement with those of
Ericsson et al. (22), who failed to detect phosphorylation
of PLC1 in unprimed (naïve) murine T cells after Ag
stimulation. The signaling defect of cord blood T cells is therefore
consistent with that of naive T lymphocytes, a major component of human
umbilical cord blood T cell population (23). The
combination of PMA, which binds and activates PKC, and ionomycin, which
artificially increases intracellular calcium, effectively mimics the
end result of PLC activation. The observation that the
proliferative response of cord T cells to PMA and ionomycin was similar
to that of adult T cells rules out additional impairments downstream of
PLC activation. Therefore, the inability of cord blood-derived
lymphocytes to respond to mitogenic stimuli is attributable to a
signaling defect at the level or upstream of PLC activation.
Compared with adult T cells, cord T lymphocytes exhibit decreased
PLC1 levels, increased PLCß1 levels, and similar amounts of
PLC2 and PLC2. The different distribution of PLC isoforms cannot
be explained by relative differences in T lymphocyte subsets or the
differential presence of contaminating cells between cord and adult
blood. In fact, as shown in Table I, the proportion of cells expressing
CD3, CD4, CD8, CD14 (monocytes), or CD19 (B cells) is virtually
identical in cord and adult T lymphocytes. Such a finding is also in
agreement with previous reports showing that the ratio of CD4 and CD8
subsets and the proportion of NK cells are invariant in cord and adult
blood (23). Therefore, the increase in PLCß1 expression
and the decreased representation of PLC1 are features associated
with the signaling defect of neonatal T cells. All PLC isoforms were
most obvious for their cytoplasmic localization, although they were
also present in the nuclear compartment. This observation is consistent
with data demonstrating the existence of an autonomous nuclear
phosphoinositide signaling route in other cell lineages
(24, 25, 26, 27, 28). The significance of such localization in T
lymphocytes remains to be clarified.
Interestingly, PLC2 was expressed in cord T cells at levels similar
to that of adult T lymphocytes, but did not compensate for the defect
in phosphoinositide hydrolysis. Furthermore, no difference was observed
in inositol phosphate production in response to
AlF4-, a pharmacologic
treatment that stimulates PLCß isoforms through the activation of
regulatory G proteins. PLCß, which also participates in T cell
signal transduction (3), is therefore efficiently coupled
to its regulatory element(s) in both adult and cord blood T cells.
PLCß activation, however, could not substitute for defective PLC
activation. Furthermore, TCR-induced PLCß activation requires the
combined action of G proteins and tyrosine kinase activation
(29). Because cord T cells show an inherent defect in
protein tyrosine phosphorylation after TCR engagement, it is likely
that PLCß cannot be fully activated because of impaired tyrosine
kinase activation.
TCR perturbation normally results in the phosphorylation of the
TCR-associated CD3 and -chains by the Src family kinases, Lck and
Fyn (30). Recruitment, phosphorylation, and activation of
ZAP70 follows (31, 32). Neonatal T cells displayed a
decreased TCR-induced protein tyrosine phosphorylation, suggesting the
existence of a TCR-proximal activation block. Furthermore, cord T cells
exhibited very low Lck levels, and ZAP70, even expressed, did not
undergo significant phosphorylation in response to either CD3 ligation
or pervanadate treatment. Syk, however, was overrepresented in cord T
cells compared with adult T lymphocytes and phosphorylated in response
to CD3 activation or pervanadate treatment of cord T cells. Syk
phosphorylation in cord T cells is consistent with the ability of this
kinase to autophosphorylate in the absence of Src kinase activation
(33). Syk is capable of initiating TCR signal transduction
(34), and may substitute for ZAP70 under certain
conditions (35). Cells expressing a catalytically active
Syk, however, still required the presence of a functional Lck for the
activation of the IL-2 promoter (34), consistent with our
observation that Syk activation in cord T cells was inadequate to
provide a signal sufficient for mitogenesis. Fyn was equally expressed
in adult and neonatal T cells. Fyn signaling function, however,
appears to be primarily related to optimizing signal transduction for
low avidity ligands (35) and cannot fully substitute
for Lck.
Taken together, the low PLC1 and Lck expression levels exhibited by
cord T cells appear to constitute the principal molecular defects
responsible for the inefficient phosphorylation of ZAP70 and the
decreased phosphoinositide hydrolysis. Lck is crucial for the
initiation of the tyrosine kinase cascade (36, 37), while
ZAP70 is required for PLC1 phosphorylation and
Ca2+ mobilization in T lymphocytes
(35). Lck expression, which varies greatly among human T
cell lines, may be dynamically influenced by lymphokine exposure
(38), suggesting that the transition from the low levels
of cord T cells to that of adult T cells be regulated by external
factors. The present study characterizes the molecular defect
associated with the limited signaling ability of human umbilical cord T
cells and provides the basis for future investigation of the mechanism
of maturation of signaling functions in human T cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Sebastiano Miscia, Istituto di Morfologia Umana Normale, Università degli Studi "G. D’Annunzio," Via dei Vestini, 6, 66100 Chieti, Italy. E-mail address:
3 Abbreviations used in this paper: PLC, phospholipase C; AlF4-, aluminum tetrafluoride; InsP, inositol phosphate; PI, propidium iodide; PKC, protein kinase C.
Received for publication April 6, 1999. Accepted for publication June 25, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-interferon by newborn cells in vitro is due to a functionally immature macrophage. J. Immunol. 134:1493.[Abstract]
-subunit-regulated phospholipase C-activating protein. J. Biol. Chem. 266:14217.1 by tyrosine phosphorylation. Science 250:1253.1 and mitogen-activated protein kinase in naive and antigen-primed CD4 T cells by the peptide/MHC ligand. J. Immunol. 156:2045.[Abstract]
stimulates nuclear phospholipase C in human osteosarcoma SaSOS2 cells. J. Biol. Chem. 269:13.This article has been cited by other articles:
|
|
 |
|
  Y. J. Ha, Y.-C. Mun, C.-M. Seong, and J. R. Lee Characterization of phenotypically distinct B-cell subsets and receptor-stimulated mitogen-activated protein kinase activation in human cord blood B cells J. Leukoc. Biol., December 1, 2008; 84(6): 1557 - 1564. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Goldberg, N. Luknar-Gabor, G. Zadik-Mnuhin, P. Koch, J. Tovbin, and Y. Katz Synergy between LPS and immobilized anti-human CD3{epsilon} mAb for activation of cord blood CD3+ T cells Int. Immunol., January 1, 2007; 19(1): 99 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Randolph The Neonatal Adaptive Immune System NeoReviews, October 1, 2005; 6(10): e454 - e462. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kobayashi, K. Ohnuma, M. Uchiyama, K. Iino, S. Iwata, N. H. Dang, and C. Morimoto Association of CD26 with CD45RA outside lipid rafts attenuates cord blood T-cell activation Blood, February 1, 2004; 103(3): 1002 - 1010. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Jullien, R. Q. Cron, K. Dabbagh, A. Cleary, L. Chen, P. Tran, P. Stepick-Biek, and D. B. Lewis Decreased CD154 expression by neonatal CD4+ T cells is due to limitations in both proximal and distal events of T cell activation Int. Immunol., December 1, 2003; 15(12): 1461 - 1472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Canto, J. L. Rodriguez-Sanchez, and S. Vidal Distinctive response of naive lymphocytes from cord blood to primary activation via TCR J. Leukoc. Biol., December 1, 2003; 74(6): 998 - 1007. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. Marchisio, A. Di Baldassarre, D. Angelucci, E. Caramelli, A. Cataldi, S. Castorina, A. Antonucci, L. Di Giovannantonio, C. Schiavone, R. Di Biagio, et al. Phospholipase C {delta}2 Expression Characterizes the Neoplastic Transformation of the Human Gastric Mucosa Am. J. Pathol., September 1, 2001; 159(3): 803 - 808. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
