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Subjects affected by DS have an increased susceptibility to infections and are at increased risk for leukemia(1–4). Although the antigen exposure due to institutionalization may contribute to frequent infections in these subjects, an increased susceptibility to infections has also been documented in non-institutionalized children, thus indicating that an intrinsic immune defect is associated with the syndrome. In spite of a large number of studies that document a derangement of several immunologic functions, the knowledge of the pathogenetic mechanism responsible for the immune deficiency is still poor. Abnormalities of either cellular or humoral immune response have been observed in DS children, but without much consistency(5–9). Based on the observations of numerical abnormalities of T cell populations in DS and thymic hypoplasia, functional T and B cell impairment in this syndrome has been thought to be due to abnormal T cell ontogeny, resulting in the release of phenotypically immature T cells into the peripheral blood(10,11). However, peripheral blood lymphocytes from DS subjects express high levels of CD3 molecules and α/β chains of the TCR(12), indicating the presence of circulating mature-type T lymphocytes. This suggests that there is a functional defect in the immune response. The intracellular calcium response is reduced in T lymphocytes from DS subjects(13). Moreover, a selective abnormal regulation of IL-2 has been documented, supporting the hypothesis that the defect may also be qualitative in nature(14). Evidence indicates that several intracytoplasmic molecules in either T or B lymphocytes are involved in signal transduction from membrane receptors to nuclei during the activation process(15). Triggering of the TCR/CD3 complex results in a cascade of events, whose hallmarks are the phosphorylation of the ζ chain of the receptor itself, the activation of the src family tyrosine kinases fyn and lck, and recruitment to the receptor of ZAP-70(16–18). Of note, it has been shown that the integrity of the intracellular communication network is also required for cell development and differentiation(19–22). Genetic alterations of transducing elements involved in either the T cell or B cell activation/differentiation process, such as the common γ element of IL-2, IL-4, IL-7, and IL-15 receptors(23–26), the Janus family JAK-3 kinase and the B cell-specific tyrosine kinase may cause different forms of congenital immunodeficiencies(20,22,27). However, alterations in signal transduction through the TCR/CD3 complex may occur even in the absence of a genetic alteration of molecules involved in the signaling pathways. Experimental evidence indicates that anergy or a productive immune response is associated with the differential involvement of protein tyrosine kinases, thus suggesting a correlation between functional and biochemical arrays(28,29). This study was undertaken to define whether the immune defect in DS was qualitative in nature and associated with abnormalities of signal transduction via the TCR/CD3 complex.

METHODS

Subjects and controls. Twenty noninstitutionalized subjects with cytogenetically documented trisomy 21, and 20 age-matched healthy control subjects were included in the study after informed consent was obtained from their parents. Their ages ranged from 4 to 16 y (mean age, 7.1 y). All patients were free of infections at the time of the study. Eleven DS subjects had an autoreaction, as revealed by the presence of one of the following antibodies to nuclear antigen, double strand DNA, thyroglobulin, and smooth muscle.

Phenotypic analysis. The expression of surface-membrane antigens on PBMC was examined by flow cytometry (Becton Dickinson, San Jose, CA) using the following MAbs in two-color immunofluorescence: anti-CD3(Leu-4), anti-CD4 (Leu-3a), anti-CD8 (Leu-2a), anti-CD19 (Leu-12), and anti-HLA-DR (Becton Dickinson, San Jose, CA).

Cell preparation and proliferation assays. PBMC were isolated by Ficoll-Hypaque (Biochrom, Berlin, Germany) density gradient centrifugation by the standard procedure. PBMC (2 × 105) were cultured, in triplicate, in RPMI 1640 medium containing 10% FCS, 10 mg/mL gentamicin sulfate (GIBCO Laboratories, Grand Island, NY), and 2 mM glutamine for 72 h at 37°C in 5% CO2 in air. Cells were stimulated by 10 µg/mL PHA (Difco Laboratories, Detroit, MI), 8.25 µg/mL concanavalin A (Difco Laboratories, Detroit, MI), 10 µg/mL pokeweed mitogen (Life Technologies Ltd, Paisley, Scotland), 20 ng/mL PMA, and 0.5 mM ionomycin (Sigma Chemical Co., St. Louis, MO). CD3 cross-linking was performed by precoating tissue culture plates with 10 and 1 ng/mL anti-CD3 MAb (4B6, gift of Dr. C. Morimoto, Dana Farber Cancer Institute, Boston, MA). Cultures were pulsed with 1µCi per well of [3H]thymidine (Amersham International, Buckinghamshire, England) for the last 16 h. Cells were harvested, and the[3H]thymidine incorporation was measured by standard liquid scintillation techniques. Human recombinant IL-2 (Amgen, Thousand Oaks, CA) was used at a concentration of 100 U/mL.

Immunoblotting and immunoprecipitations. After appropriate stimuli, 3-5 × 106 cells were incubated on ice with lysis buffer containing 20 mmol/L Tris, pH 8, 10% glycerol, 137 mmol/L NaCl, 1% Nonidet P-40, 10 mmol/L EDTA, 1 mmol/L phenylmethanesulfonyl fluoride, 1 mmol/L sodium orthovanadate (Na3VO4), 5 µg/mL leupeptin, and 5 µg/mL aprotinin. Insoluble material was removed by centrifuging the samples at 14 000 × g for 20 min at 4°C; supernatants were boiled in 50 µL of SDS sample buffer. In immunoprecipitation experiments, cell lysates, obtained from 7-10 × 106 cells in glycerol-free lysis buffer, were incubated for 2 h with protein A-Sepharose precoated with 10 µg of anti-P-tyr (4G10, kindly provided by Dr. Brian Druker, Dana Farber Cancer Institute, Boston, MA), with 7 µL of anti-ZAP 70 rabbit serum (Amersham International, Buckinghamshire, England), with preimmune serum, or with anti-CD45 MAb (GAP 8.3, gift of Dr. C. Morimoto, Dana Farber Cancer Institute, Boston, MA), as control antibodies. Proteins were resolved by 10% or 5 to 16% continuous-gradient SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then blocked with a buffer containing 10 mmol/L Tris, pH 8, 137 mmol/L NaCl, and 3% BSA. Immunoblotting was performed by a 2-4-h incubation with antibodies as follows: MAb to P-tyr, diluted 1:3200; MAb to γ chain of the IL-2 receptor, diluted 1:1500 (3B5, gift of Dr. Jerome Ritz, Dana Farber Cancer Institute, Boston, MA)(30); and rabbit serum against ZAP-70, diluted 1:4000. The membrane was washed four times, incubated with the AP-conjugated secondary antibody, and then, after three additional washings, with the developing mixture containing nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Pro-mega, Madison, WI). Alternatively, the IL-2R γ and ZAP-70 were detected by incubating with horse radish peroxidase secondary antibody and enhanced chemiluminescence (Amersham International, Buckinghamshire, England).

Statistical analysis. Significance of the differences was calculated using the Wilcoxon rank sum test by the Epistat program. Correlation analysis was performed by the single regression line.

RESULTS

Proliferative response and cell populations. Low responder DS subjects were selected on the basis of a persistent low proliferative response after CD3 cross-linking, that mimics in vivo antigen exposure. Fifty percent of DS subjects had absent or very low proliferative response after CD3 cross-linking. Figure 1A shows the proliferative response in these low responder DS subjects and in age-matched control subjects to anti-CD3 MAb. Proliferation to CD3 cross-linking, expressed as median of counts/min, was 2333 (range, 227-5806)versus 18 760 (range, 11 659-26 810) in the control group(p < 0.01). In contrast, PMA and ionomycin stimulation induced a normal proliferation in all low responder DS subjects, with median values in DS subjects and controls being 91 353 (range: 22 549-150 464) and 103-342(range: 23 016-141 226), respectively. Because PMA by-passes the very early events and directly activates protein kinase C, the normal proliferative response to phorbol esters in DS subjects suggests a blockage upstream to protein kinase C activation. As shown in Figure 1B in low responder DS subjects the decreased low proliferation to CD3 cross-linking was observed using either optimal or sub-optimal stimulation. No correlation between low and normal responder DS subjects with regard to age, sex, and history of recent infections was found. Moreover, no difference between low responder to CD3 cross-linking, normal responder DS subjects, and healthy control subjects in the proliferative assays after stimulations with potent mitogens, as PHA and concanavalin A, was found.

Figure 1
figure 1

Proliferative responses in children affected by DS and age-matched control subjects. (A) PBMC from low responder DS subjects and controls were stimulated through CD3 triggering(CD3 X-L; left panel), or after stimulation with PMA and ionomycin (IONO; right panel), and [3H]thymidine incorporation was evaluated. Each point represents an individual value. Median values are indicated. (*p < 0.01 vs control subjects). (B) Dose-response curve using optimal (10 ng/mL) or suboptimal (1 ng/mL) anti-CD3 MAb concentrations in controls, normal responder, and low responder DS subjects. Proliferation assays were performed as indicated in “Methods" (low responder DS vs controls p. < 0.01).

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In previous studies a decreased number of CD3+CD4+ cells had been reported(12). Therefore, we evaluated whether the decreased proliferative response was related to a decrease of the cell number. Figure 2 shows the percentages of CD3+, CD4+, CD8+, CD19+, and activated T cells(CD3+HLA-DR+) in DS subjects with the functional T cell defect compared with DS subjects with normal proliferative response and healthy controls. No statistically significant difference was appreciable among the three groups in either percentage values or absolute numbers. A positive trend of increased percentage of CD3+ cells bearing HLA-DR molecules was noted in the group of DS subjects with the T cell immune defect, indicating an increase of constitutively activated T cells (data not shown). However, there was no correlation between the increase of CD3+HLA-DR+ cells and the presence of serum autoantibodies. Moreover, no correlation was found when the proliferative response after CD3 perturbation was compared with the absolute number of CD4+ cells(r = 0.44). To determine whether the low responsiveness of PBMC after the CD3 cross-linking was related to a decreased expression of the surface CD3 molecule, the intensity of fluorescence was compared in DS subjects with a functional T cell defect and in healthy age-matched control subjects. No difference was found in the mean fluorescence intensity between the two groups (209.56 ± 82 versus 213.19 ± 84).

Figure 2
figure 2

Surface expression of T and B cell markers in controls and children affected by DS with or without functional immune deficiency. PBMC were stained with specific MAb as indicated, and analyzed by flow cytometry. Each column represents the mean value ± SEM.

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Protein tyrosine phosphorylation induced through TCR/CD3 complex triggering. To determine whether an abnormality of the early events during T cell activation may be responsible for the defective cell proliferation, the number and the timing of intracytoplasmic protein tyrosine phosphorylation events after CD3 cross-linking were evaluated. In normal subjects CD3 cross-linking induces tyrosine phosphorylation of several proteins of 21, 42-44, 60, 70, 85, and 110 kD. This phenomenon occurs early during cell activation, being appreciable as soon as 30 s after the stimulation, with maximal intensity at 5-10 min. In all DS subjects who had a low proliferative response to CD3 cross-linking an abnormal pattern of protein tyrosine phosphorylation was observed.Figure 3A illustrates a representative experiment showing that signaling after CD3 cross-linking was partial and characterized by increased tyrosine phosphorylation of proteins of 42-44, 85, and 110 kD, and absence of tyrosine phosphorylation of proteins migrating in the area of 70 and 21 kD. Although PMA stimulation was able to induce protein tyrosine phosphorylation, a few differences with controls were appreciable. In particular, the 21-kD protein was not tyrosine-phosphorylated, whereas the signal of a 44-45-kD protein was much stronger in the patient than in the control. Figure 3B illustrates the densitometric analysis of the tyrosine phosphorylation of the 70-kD protein, that follows CD3 cross-linking or PMA stimulation, in 10 low responder DS subjects and in 10 age-matched control subjects. In all experiments the tyrosine phosphorylation of the 70-kD protein did not significantly increase after CD3 cross-linking. Mean values ± SD, expressed as arbitrary units, were 16± 9 and 13 ± 6 after CD3 cross-linking for 5 and 10 min in DS subjects, respectively, compared with 42 ± 10 and 39 ± 15 in control subjects (p < 0.01). In this area migrates the ZAP-70 protein tyrosine kinase that is associated with the ζ chain of the CD3 complex, and which is promptly tyrosine-phosphorylated during cell activation. In contrast, in DS subjects who had a normal proliferation to CD3 cross-linking, the pattern of protein tyrosine phosphorylation occurred normally. Figure 4A shows a representative experiment in this group of normal responder DS subjects.Figure 4B depicts the densitometric analysis of the 70-kD protein phosphorylation in five distinct assays that is comparable to healthy control subjects. Immunoprecipitates containing ZAP-70, obtained from PBMC unstimulated or stimulated through CD3 cross-linking for 5 min, were immunoblotted with anti-P-tyr. Figure 5A shows that no tyrosine phosphorylation of ZAP-70 occurred in two low responder DS subjects. Western blot experiments aimed to detect the ZAP-70 protein in cell lysates showed that the protein was present in a comparable amount in controls and DS subjects (data not shown). Again, ZAP-70 tyrosine phosphorylation was normal in a DS subject with normal proliferation, as shown in Figure 5B. In addition, ZAP-70 was regularly phosphorylated on tyrosine residues after PHA stimulation in DS subjects who poorly responded to CD3 X-L. To define whether signal transduction through other receptors implicated in cell activation was abnormal, tyrosine phosphorylation of theγ element of IL-2, IL-4, IL-7, and IL-15 receptors was studied.Figure 6 shows that exogenous IL-2 promptly induces tyrosine phosphorylation of the γ element, suggesting that the abnormal signaling selectively affects the transducing apparatus of the TCR/CD3 complex.

Figure 3
figure 3

Tyrosine phosphorylation after CD3 cross-linking (CD3 X-L) or PMA stimulation of PBMC from controls and subjects with DS and low proliferative response to CD3 X-L. PBMC were stimulated for the times indicated using anti-CD3 (1:500) and rabbit anti-mouse Ig or 20 ng/mL PMA. Proteins were resolved by SDS-PAGE using a 10% polyacrylamide gel under reducing conditions and detected using anti-Ptyr MAb and AP-conjugated anti-mouse Ig. (A) Representative experiment indicating a partial signaling in the DS subjects. Molecular mass markers are indicated. (B) Densitometric analysis of the tyrosine phosphorylated 70-kD protein in PBMC unstimulated or after CD3 triggering or PMA stimulation in 10 control and 10 DS subjects.

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Figure 4
figure 4

Tyrosine phosphorylation after CD3 cross-linking (CD3 X-L) or PMA stimulation of PBMC from subjects with DS and normal proliferative response to CD3 X-L. PBMC stimulations and protein detection were performed under the same conditions indicated in Figure 3. (A) Representative experiment indicating a normal signaling in the DS subject. Molecular mass markers are indicated. (B) Densitometric analysis of the tyrosine-phosphorylated 70-kD protein in PBMC unstimulated or after CD3 triggering or PMA stimulation in five normal responder DS subjects.

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Figure 5
figure 5

Tyrosine phosphorylation of ZAP-70 protein tyrosine kinase in control subjects and subjects affected by DS. PBMC from control or DS subjects with low proliferative response to CD3 cross-linking (CD3 X-L) (A) or normal proliferation(B) were stimulated for 5 min using anti-CD3 (1:500) and rabbit anti-mouse Ig (CD3 X-L). Representative experiments of three distinct assays. ZAP-70 was immunoprecipitated using protein A-Sepharose and anti-ZAP-70 rabbit serum. Tyrosine-phosphorylated ZAP-70 was detected by incubation with anti-Ptyr MAb, and then with horseradish peroxidase secondary antibody. Signals were obtained by enhanced chemiluminescence. Molecular mass markers are indicated.

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Figure 6
figure 6

Tyrosine phosphorylation of the γ common chain of IL-2, IL-4, IL-7, and IL-15 receptors. PBMC were stimulated for 5 min using exogenous recombinant IL-2. Phosphotyrosyl proteins were immunoprecipitated from cell lysates using protein A-Sepharose precoated with an anti-P-tyr MAb. The γ chain was detected by western blot using the MAb anti-γ chain, and horse radish peroxidase secondary antibody. Signals were obtained by chemiluminescence. Molecular mass markers are indicated.

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DISCUSSION

Although numerous studies have previously documented a derangement of several immune functions and, in particular, of cell-mediated immunity in subjects affected by DS, the mechanism underlying the immunodeficiency is poorly understood(5–8). Most of the peripheral blood T cells in DS subjects have a mature immunologic phenotype. Circulating T cells in DS express high levels of TCR-α/β and CD3 molecules(12). These mature-type T cells are expected to appropriately proliferate after CD3 stimulation(31). In contrast, cell proliferation after CD3 perturbation is depressed in DS individuals(32), suggesting the presence of a T cell activation defect. The low proliferative response was not correlated to either the number of CD4+ cells or the presence in the individual subject of a high number of constitutively activated T cells, supporting the notion that the defect is qualitative in nature.

We have shown that DS individuals, who have an impaired cell proliferation after CD3 cross-linking, have a normal proliferative response to phorbol esters and ionomycin. This suggests a defect in an early stage of the activation process, upstream to protein kinase C activation and translocation.

TCR stimulation induces a complex cascade of biochemical events, eventually resulting in the activation of nuclear factors that regulate gene transcription and lead to cell proliferation(15,33). Most of the early events are phosphorylation/dephosphorylation processes(17,34). Intracytoplasmic tyrosine kinases are associated with surface receptors and play a major role in signal transduction(16,35–37). Analysis of intracellular proteins phosphorylated on tyrosine residues after CD3 cross-linking in DS showed an aberrant pattern, characterized by the absence of tyrosine phosphorylation of proteins migrating in the area of 21 and 60-70 kD. In contrast, proteins of 42-44 kD were appropriately phosphorylated, indicating that a partial signaling occurred after TCR/CD3 triggering.

Previous evidence suggested that the immunodeficiency in DS was associated with aberrations of the intrathymic maturation process(10,12). The role of the cell signaling apparatus in lymphocyte ontogeny and development is still unclear. The inactivation of a src family proto-oncogene, encoding for p56lck protein tyrosine kinase, led to a block of intrathymic T cell differentiation, resulting in the absence of CD4 and CD8 positive cells(19). Most of the primary immunodeficiencies are associated with alterations of molecules involved in the intracellular communication network. Mutations of the common γ chain for IL-2, IL-4, IL-7, and IL-15 receptors(23–26) result in the X-linked form of severe combined immunodeficiency(20). Alterations of the ZAP-70 tyrosine kinase, which associates with the TCR/CD3 complex, also result in a clinical phenotype of combined immunodeficiency inherited as an autosomal disorder(21,38).

In this study we observed that the γ chain of the IL-2 receptor is normally expressed and properly phosphorylated during cell activation, whereas the CD3-associated ZAP-70 kinase is not phosphorylated. It is noteworthy that signaling through the TCR/CD3 complex is only partial in DS, in that protein tyrosine phosphorylation of some proteins is preserved. This indicates that signaling pathways can be dissociated; which is perhaps not surprising because over 100 intracytoplasmic elements are involved(16,37,39).

As for the relationship between the overall clinical and laboratory features of DS and the cytogenetic abnormality, no explanation is available(40,41). Perhaps overexpression of an unidentified molecule involved in the T cell differentiation and activation process may dysregulate the fine tuning of the intracellular communication network. However, none of the already known signaling molecules that directly participate in the process is encoded by genes on chromosome 21. An abnormality in the extracellular microenvironment might down-regulate the signal transduction through the TCR/CD3 complex in DS. Cytokines participate in the activation process by providing accessory signals(42). Chromosome 21 contains genes encoding for the interferon α receptor, and the β chain of the interferon γ receptor(43,44). Overexpression of one or all of these genes may interfere with the CD3 signaling. This hypothesis may also explain the differences between the inherited ZAP-70 deficiency, and the immune defect in DS with regard to either the severity of the defect or the lack of CD8+ lymphocytes observed in congenital ZAP-70 defect. Presumably, the mutated ZAP-70 leads to a more profound alteration of intrathymic T cell ontogeny, which is in some how preserved in DS. On the chromosome 21 is also located the gene encoding the β2 integrin adhesion molecule, which is overexpressed in lymphoblastoid cells from DS subjects, resulting in increased homotypic adhesion(45). However, overexpression of CD18 does not seem to be responsible for the defect we have described, in that engagement of this molecule results in a potentiation of CD3 signaling, resulting in enhanced transcription of IL-2(46). Our finding is in keeping with previously reported experimental observations, indicating that a partial signaling may occur after TCR triggering. Engagement of TCR by altered major histocompatibility complex-peptide ligands results in the phosphorylation of the ζ chain, but not of ZAP-70(47). In this system, TCR ligands were able to increase lck activity, despite the failure to activate ZAP-70(48). Moreover, the pattern of physical and biochemical interactions of TCR may change in relationship with the functional outcome(28). TCR ligation of anergic T cells results in the tyrosine phosphorylation of a few TCR/CD3 signaling proteins that differ from those phosphorylated in responsive T cells after the same stimulation(28). Appropriate costimulation, mediated through the CD28 accessory molecule, facilitates phosphorylation of CD3 ζ and ε chains, and the subsequent recruitment of lck and ZAP-70, thus resulting in a productive immunity(28). Taken together, these observations strongly support the concept that a proper immune response results from the integration in a finely regulated network of different signaling pathways(29). At the best of our knowledge, this is the first identification of a partial signaling through TCR associated with a human immune defect. New drugs may be able to activate signaling pathways in a selective fashion to restore immune functions in DS patients and in others with T cell activation deficiencies.

In conclusion, we provide evidence that the immunodeficiency in DS is, in part, functional in nature and associated with an aberrant pattern of protein tyrosine phosphorylation induced by TCR/CD3 triggering.