Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the immune response of cord blood-derived T lymphocytes compared with that of adult T lymphocytes. Our findings demonstrate that phospholipase C (PLC) isozymes, which play a pivotal role in the control of protein kinase C activation and Ca2+ mobilization, are differently expressed in cord and adult T lymphocytes. PLCβ1 and δ1 are expressed at higher levels in cord T cells, while PLCβ2 and γ1 expression is higher in adult T lymphocytes. PLCδ2 and γ2 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 PLCγ activation and decreased Lck expression.

The effector activities of T lymphocytes are induced by signals arising from the interaction between the TCR and immunogenic peptides associated with the MHC. The functional TCR complex consists of an αβ heterodimer, which provides T cells with the ability to recognize Ags bound to MHC molecules, the γ-, δ-, and ε-chains of the CD3 complex, and the ζ-chain, elements that transduce the activation signals to the cytoplasm of T lymphocytes. The CD3 complex and ζ-chains are rapidly tyrosine phosphorylated by Src kinases after TCR engagement (1, 2). This leads to phosphorylation and activation of inositol-specific phospholipase Cγ (PLCγ)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 PLCβ occurs following TCR-CD3-mediated signaling (3). As a consequence of either PLCγ or PLCβ 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.

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).

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-PLCβ1, β2, γ1, γ2, δ1, δ2, 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 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).

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-PLCβ1, β2, γ1, γ2, δ1, or δ2 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 ×100/1.3 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 × 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.

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.

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).

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.

Table I.

Phenotypic characterization of mononuclear cells from adult and cord blooda

CD3 (%)CD4 (%)CD8 (%)CD14 (%)CD19 (%)
Adult 70.0 ± 10.0 43.3 ± 7.0 29.3 ± 8.0 5.0 ± 2.6 8.0 ± 3.6 
Cord 74.0 ± 7.0 43.7 ± 10 31.7 ± 4.9 3.3 ± 2.1 10.0 ± 4.4 
CD3 (%)CD4 (%)CD8 (%)CD14 (%)CD19 (%)
Adult 70.0 ± 10.0 43.3 ± 7.0 29.3 ± 8.0 5.0 ± 2.6 8.0 ± 3.6 
Cord 74.0 ± 7.0 43.7 ± 10 31.7 ± 4.9 3.3 ± 2.1 10.0 ± 4.4 
a

Data are the mean ± SD of three different experiments.

FIGURE 1.

Flow cytometry analysis of CD3+ cells before (left) and after (right) purification. FL1, FITC fluorescence of anti-CD3 mAb.

FIGURE 1.

Flow cytometry analysis of CD3+ cells before (left) and after (right) purification. FL1, FITC fluorescence of anti-CD3 mAb.

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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 PLCγ activation.

Table II.

Proliferative response of T lymphocytes from adult and cord blooda

MediumAnti-CD3PHAAnti-CD3 + PMAAnti-CD3 + IonoPMAIonoPMA + Iono
Adult 2,907 38,820 46,691 36,417 31,258 4,156 2,086 33,309 
 (2,492–3,322) (46,677–30,963) (51,832–41,551) (40,918–31,916) (34,708–27,808) (4,650–3,662) (2,700–1,473) (33,640–32,978) 
Cord 1,844 2,761 1,936 3,099 3,640 2,151 3,438 31,916 
 (2,618–1,070) (3,854–1,669) (1,231–2,642) (3,570–2,629) (3,675–3,605) (1,009–3,294) (3,735–3,141) (32,078–31,754) 
MediumAnti-CD3PHAAnti-CD3 + PMAAnti-CD3 + IonoPMAIonoPMA + Iono
Adult 2,907 38,820 46,691 36,417 31,258 4,156 2,086 33,309 
 (2,492–3,322) (46,677–30,963) (51,832–41,551) (40,918–31,916) (34,708–27,808) (4,650–3,662) (2,700–1,473) (33,640–32,978) 
Cord 1,844 2,761 1,936 3,099 3,640 2,151 3,438 31,916 
 (2,618–1,070) (3,854–1,669) (1,231–2,642) (3,570–2,629) (3,675–3,605) (1,009–3,294) (3,735–3,141) (32,078–31,754) 
a

Data (cpm) are the mean of two separate experiments (individual values in parentheses).

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). PLCβ2 and PLCγ1 immunoreactive bands appeared to be very strong in adult T lymphocytes, whereas they were detected at a lower intensity in cord cells. In contrast, PLCβ1 was more represented in cord blood T cells. Cleaved forms of PLCβ1 and/or PLCβ2 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 PLCγ2. The PLCδ1 isoform was difficult to detect and appeared to be slightly higher in cord blood T lymphocytes compared with adult T cells. No difference in PLCδ2 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.

FIGURE 2.

Immunoprecipitation of PLC isoforms from adult and cord T cells. PLC isoforms were immunoprecipitated from an equal amount (400 μg) of proteins from adult and cord T cells and analyzed by Western blot with the respective anti-PLC isoform Abs. Note that the detection of multiple bands in the PLCβ2 blot was due to the formation of cleavage products. The blots are representative of five independent experiments.

FIGURE 2.

Immunoprecipitation of PLC isoforms from adult and cord T cells. PLC isoforms were immunoprecipitated from an equal amount (400 μg) of proteins from adult and cord T cells and analyzed by Western blot with the respective anti-PLC isoform Abs. Note that the detection of multiple bands in the PLCβ2 blot was due to the formation of cleavage products. The blots are representative of five independent experiments.

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Samples depleted of adherent and CD19+ cells were further analyzed for the expression of the PLC isoforms by means of confocal microscopy (Fig. 3). PLCδ2 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, PLCβ1 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. 3,C). In cord cells, the reaction appeared markedly stronger in the cytoplasm, and slightly more intense in the nucleus (Fig. 3,D). The PLCβ2 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). PLCγ1 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. PLCγ2 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.

FIGURE 3.

Confocal microscopy analysis of PLC isoforms expression and distribution in adult and cord T lymphocytes. Macrophage- and B cell-depleted samples were stained for PLC isoforms and analyzed by confocal microscopy. The green fluorescence (FITC) signal localizes the different PLC isoforms, while the red fluorescence (PI) counterstains the nuclei. The panels show the expression of PLCδ2 expression in adult (A) and cord (B) cells; PLCβ1 in adult (C) and cord (D) cells; PLCβ2 in adult (E) and cord (F) cells; PLCγ1 in adult (G) and cord (H) cells; and PLCγ2 in adult (I) and cord (J) cells. The results shown here are representative of at least three different experiments. The arrow in C indicates PLCβ1 staining in the cytoplasm at the external periphery of the nucleus. Bar = 10 μm.

FIGURE 3.

Confocal microscopy analysis of PLC isoforms expression and distribution in adult and cord T lymphocytes. Macrophage- and B cell-depleted samples were stained for PLC isoforms and analyzed by confocal microscopy. The green fluorescence (FITC) signal localizes the different PLC isoforms, while the red fluorescence (PI) counterstains the nuclei. The panels show the expression of PLCδ2 expression in adult (A) and cord (B) cells; PLCβ1 in adult (C) and cord (D) cells; PLCβ2 in adult (E) and cord (F) cells; PLCγ1 in adult (G) and cord (H) cells; and PLCγ2 in adult (I) and cord (J) cells. The results shown here are representative of at least three different experiments. The arrow in C indicates PLCβ1 staining in the cytoplasm at the external periphery of the nucleus. Bar = 10 μm.

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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.

Table III.

IP3 productiona

Time (min)MediumCD3 AbControl AbPervanadAIF4
Adult 185 ± 15 2123 ± 114 167 ± 18 2356 ± 128 591 ± 53 
 15 191 ± 18 2336 ± 120 170 ± 14 2501 ± 113 706 ± 68 
 45 179 ± 14 2328 ± 118 161 ± 13 2490 ± 110 710 ± 63 
       
Cord 96 ± 7 88 ± 9 94 ± 5 157 ± 14 402 ± 41 
 15 93 ± 5 90 ± 5 97 ± 4 178 ± 17 489 ± 45 
 45 90 ± 9 83 ± 7 88 ± 7 175 ± 17 478 ± 35 
Time (min)MediumCD3 AbControl AbPervanadAIF4
Adult 185 ± 15 2123 ± 114 167 ± 18 2356 ± 128 591 ± 53 
 15 191 ± 18 2336 ± 120 170 ± 14 2501 ± 113 706 ± 68 
 45 179 ± 14 2328 ± 118 161 ± 13 2490 ± 110 710 ± 63 
       
Cord 96 ± 7 88 ± 9 94 ± 5 157 ± 14 402 ± 41 
 15 93 ± 5 90 ± 5 97 ± 4 178 ± 17 489 ± 45 
 45 90 ± 9 83 ± 7 88 ± 7 175 ± 17 478 ± 35 
a

Data are expressed as cpm/106 cells ± SD.

The defective response of cord blood T cells could be accounted for by several factors. A defect in TCR/CD3 complex expression was ruled out by the observation that adult and cord blood-derived T cells showed similar CD3 surface density (Table I). 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 PLCγ and β, 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 PLCβ 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.

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.

FIGURE 4.

Tyrosine phosphorylation profiles induced by CD3 ligation or pervanadate treatment in adult and cord T lymphocytes. Adult and cord T lymphocytes were stimulated with anti-CD3 or by pervanadate treatment, as described in Materials and Methods. Cell lysates were resolved by gel electrophoresis, blotted, and probed with anti-phosphotyrosine (PY-99, 0.4 μg/ml; Santa Cruz Biotechnology). The data are representative of three separate experiments. The m.w. of standards are shown on the side.

FIGURE 4.

Tyrosine phosphorylation profiles induced by CD3 ligation or pervanadate treatment in adult and cord T lymphocytes. Adult and cord T lymphocytes were stimulated with anti-CD3 or by pervanadate treatment, as described in Materials and Methods. Cell lysates were resolved by gel electrophoresis, blotted, and probed with anti-phosphotyrosine (PY-99, 0.4 μg/ml; Santa Cruz Biotechnology). The data are representative of three separate experiments. The m.w. of standards are shown on the side.

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T cell activation triggers the phosphorylation cascade of a specific set of intracellular signal-transducing proteins (15, 16, 17, 18). To further confirm the phosphorylation defect observed in the analysis of whole cell lysates, anti-phosphotyrosine immunoprecipitates from anti-CD3- or pervanadate-stimulated cells were immunoblotted with anti-Fyn, Lck, ZAP70, Syk, or PLCγ1 Abs (Fig. 5). To avoid that the lower PLCγ1 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 PLCγ1 underwent tyrosine phosphorylation, while Syk was scarcely phosphorylated. In anti-CD3 or pervanadate-treated cord cells, PLCγ1 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.

FIGURE 5.

Phosphorylation of PLCγ1, Syk, ZAP70, Lck, and Fyn upon anti-CD3 and pervanadate treatment in adult and cord T lymphocytes. Adult and cord T lymphocytes were stimulated with anti-CD3 or by pervanadate treatment, as described in Materials and Methods. Tyrosine-phosphorylated proteins were immunoprecipitated (PY-99; Santa Cruz Biotechnology), resolved by gel electrophoresis, blotted, and simultaneously probed with anti-PLCγ1, Syk, ZAP70, and Lck (Santa Cruz Biotechnology; 1/100). Identity of the different proteins was inferred based on their electrophoretic mobility. The detection with anti-Fyn was performed on a separate membrane because the correspondent Ab was developed in a host different from that of the other Abs. The amount of cord cell lysates from which the immunoprecipitation was performed was 3-fold larger than that of the adult samples to avoid that the lower expression of PLCγ1 could influence the detection of its phosphorylation level. The data are representative of three separate experiments.

FIGURE 5.

Phosphorylation of PLCγ1, Syk, ZAP70, Lck, and Fyn upon anti-CD3 and pervanadate treatment in adult and cord T lymphocytes. Adult and cord T lymphocytes were stimulated with anti-CD3 or by pervanadate treatment, as described in Materials and Methods. Tyrosine-phosphorylated proteins were immunoprecipitated (PY-99; Santa Cruz Biotechnology), resolved by gel electrophoresis, blotted, and simultaneously probed with anti-PLCγ1, Syk, ZAP70, and Lck (Santa Cruz Biotechnology; 1/100). Identity of the different proteins was inferred based on their electrophoretic mobility. The detection with anti-Fyn was performed on a separate membrane because the correspondent Ab was developed in a host different from that of the other Abs. The amount of cord cell lysates from which the immunoprecipitation was performed was 3-fold larger than that of the adult samples to avoid that the lower expression of PLCγ1 could influence the detection of its phosphorylation level. The data are representative of three separate experiments.

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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 PLCγ1 and ZAP70, a near absence of Lck, together with a higher expression of Syk and PLCγ2 compared with adult T lymphocytes.

FIGURE 6.

Western blot analysis of ZAP70, Syk, Lck, and Fyn expression. Equal amounts of adult and cord T cell lysates were electrophoresed, blotted onto nitrocellulose, and probed with anti-ZAP70, anti-Syk, anti-Fyn, and anti-Lck Abs (Santa Cruz Biotechnology; 1/100).

FIGURE 6.

Western blot analysis of ZAP70, Syk, Lck, and Fyn expression. Equal amounts of adult and cord T cell lysates were electrophoresed, blotted onto nitrocellulose, and probed with anti-ZAP70, anti-Syk, anti-Fyn, and anti-Lck Abs (Santa Cruz Biotechnology; 1/100).

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Table IV.

Densitometric analysis of immunoreactive bands related to ZAP-70, Syk, Fyn, and Lcka

ZAP-70SykFynLck
Adult 642 ± 35 31 ± 4 94 ± 10 303 ± 43 
Cord 430 ± 21 171 ± 17 272 ± 22 5 ± 0.2 
ZAP-70SykFynLck
Adult 642 ± 35 31 ± 4 94 ± 10 303 ± 43 
Cord 430 ± 21 171 ± 17 272 ± 22 5 ± 0.2 
a

Data refer to integrated density and represent the mean of three separate experiments ± S.D.

In this study, we have shown that the functional impairment characteristic of umbilical cord blood-derived T lymphocytes is associated with inefficient PLC activation and second messenger generation. Cord T cells displayed lower levels of PLCγ1 together with a decrease in activation-induced protein tyrosine phosphorylation of ZAP70 and PLCγ1 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 PLCγ 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 PLCγ1 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 PLCγ1 levels, increased PLCβ1 levels, and similar amounts of PLCγ2 and PLCδ2. 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 PLCβ1 expression and the decreased representation of PLCγ1 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, PLCγ2 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 PLCβ isoforms through the activation of regulatory G proteins. PLCβ, 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. PLCβ activation, however, could not substitute for defective PLCγ activation. Furthermore, TCR-induced PLCβ 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 PLCβ cannot be fully activated because of impaired tyrosine kinase activation.

TCR perturbation normally results in the phosphorylation of the TCR-associated CD3 and ζ-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 PLCγ1 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 PLCγ1 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.

We thank Drs. S. Kozlowski and M. Shapiro for the critical review of the manuscript.

1

This work was supported by the Italian Ministero dell’Università e della Ricerca Scientifica e Technologica (MURST) 60% Grant 1996/97/98.

3

Abbreviations used in this paper: PLC, phospholipase C; AlF4, aluminum tetrafluoride; InsP, inositol phosphate; PI, propidium iodide; PKC, protein kinase C.

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