Defective p56Lck activity in T cells from an adult patient with idiopathic CD4+ lymphocytopenia
Pascale Hubert,
Florence Bergeron,
Valérie Ferreira,
Maxime Seligmann1,
Eric Oksenhendler1,
Patrice Debre and
Brigitte Autran
Laboratoire d'Immunologie Cellulaire, CNRS UMR 7627, CHU Pitié-Salpétrière, 83 Boulevard de l'Hôpital, 75651 Paris Cedex 13, France
1 Service d'Immuno-Hématologie, Hôpital St Louis, 75010 Paris, France
Correspondence to:
P. Hubert
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Abstract
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Idiopathic CD4+ lymphocytopenia (ICL) is defined by a stable loss of CD4+ T cells in the absence of any known cause of immune deficiency. This syndrome is still of undetermined origin. It affects adult patients, some of them displaying opportunistic infections similar to HIV-infected subjects. The hypothesis that the cellular immune defect may be due to biochemical failures of the CD3TCR pathway is investigated here in a patient associating a severe selective CD4+ lymphocytopenia with an increased CD8+ T cell count discovered in the course of a cryptococcal meningitidis. A 40% reduction of T cell proliferation to CD3TCR stimulation is observed only in the CD4+ subpopulation. The early CD3-induced protein tyrosine phosphorylations are conserved in both CD4+ and CD8+ subsets, and the levels of the T cell protein tyrosine kinases p56Lck, p59Fyn and ZAP-70 are normal. However, we find a 50% reduction of p56Lck kinase activity in the patient's T cells compared to a healthy control donor. p59Fyn activity does not appear to be altered. Nevertheless, we do not find any genetic abnormality of p56Lck. These results thus suggest that a defect of an unknown protein regulating p56Lck activity takes place in this patient's T cells. Taken together, these findings reveal p56Lck alteration in ICL and confirm the critical role of this kinase in the maintenance of the peripheral CD4+ T cell subpopulation.
Keywords: BrdU, immunodeficiency, tyrosine phosphorylation
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Introduction
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Idiopathic CD4+ lymphocytopenia (ICL) is a rare immunodeficiency syndrome affecting 0.52% of adults (13). It is defined by a low CD4+ T cell count <300/µl or a percentage of CD4+ T cells <20% on at least two occasions, without HIV infection or any known immune defect (4). The origin of this syndrome is currently not known. Although Laurence et al. (5) and Gupta et al. (6) suggested in 1992 that ICL could be due to a new retrovirus distinct from HIV-1 and HIV-2, epidemiologic studies showed no evidence for a transmissible agent (2,7). ICL differs from HIV infection by stable levels of CD4+ T cell counts in contrast to the progressive loss of this subpopulation observed in the course of HIV disease (79). Moreover, a great heterogeneity in the symptomatology and the T cell phenotypic abnormalities have been reported among patients with ICL. Indeed, ICL can be diagnosed with the onset of opportunistic infections or among autoimmune disorders, whereas it remains asymptomatic in other patients (2,7,8,10). In addition to the CD4+ lymphocytopenia, several patients also display a CD8+ lymphocytopenia while low B or NK cell counts have also been reported in others (2,8,10). The heterogeneity of ICL does not favor the hypothesis of a unique cause (3,11) and some authors have suggested ICL to be classified among common variable immunodeficiency (7). Similarly, we previously suggested that a case of ICL associated with recurrent opportunistic infections could be attributed to a primary immunodeficiency disorder (12).
Recently, Laurence et al. demonstrated that increased spontaneous and activation-induced apoptosis was associated with symptomatic ICL, and might represent one physiopathologic mechanism of the disease (13). Alternatively, other studies reported proliferative T cell defects to mitogens or antigens in patients with ICL and opportunistic infections (68,12). It was thus of interest to evaluate whether the immune deficiency was determined solely by the severity of the lymphocytopenia or whether the residual lymphocytes also displayed molecular abnormalities responsible for defective T cell activation and proliferation. The T cell activation process consists in a cascade of biochemical events triggered by stimulation of the CD3TCR by the MHCpeptide complex or by a mAb specific to CD3
. The earliest events involve the T cell protein tyrosine kinases (PTK) p56Lck, p59Fyn and ZAP-70, which phosphorylate numerous intracellular substrates on tyrosine residues (tyrosine phosphorylation), further leading to cytokine gene expression and cell proliferation (18,19). Supporting this hypothesis, children with severe combined immunodeficiency (SCID) determined by defective expression of the PTK ZAP-70 display a profound depletion of peripheral CD8+ T cells. The absolute counts of CD4+ T cells are normal in these patients, although these cells have a defect of the CD3TCR signal transduction pathway (1416). Moreover, a defective expression of the PTK p56Lck was recently shown in a SCID child who had also a selective loss of CD4+ T cells (17).
In this paper, we analyzed the CD3TCR pathway of the CD4+ and CD8+ T cell subsets from a patient with severe and stable ICL revealed by an opportunistic infection, in order to investigate whether impaired activation events in these cells might explain the T cell depletion and the immune deficiency. We studied T cell proliferation and early protein tyrosine phosphorylations at the cellular level by coupling functional tests with flow cytometric analysis, together with biochemical analysis of T cell PTK.
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Methods
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Patient and controls
The patient described here was a 66-year-old woman, who had a selective CD4+ lymphocytopenia stable for 10 years, discovered in the course of a cryptococcal meningitidis (20). Since then, she remained asymptomatic and did not receive any treatment at time of the study. She was found to be negative for HIV infection by serologic and PCR assays. Informed consent was obtained before the study.
Cells of adult healthy volunteer donors from the blood donor center of the Pitié-Salpêtrière Hospital were used as controls for each assay.
Cell preparation and culture
Whole blood samples were collected on heparinized Vacutainer tubes (Becton Dickinson, San Jose, CA) by venous puncture of patients or control donors. They were used directly for cytometric tests realized on whole blood.
Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll-Hypaque (Eurobio, Les Ulis, France) density-gradient centrifugation. Cells were then cultured for the indicated period of time and with the indicated stimulating agent in complete medium: RPMI 1640 medium (Flow, Irvine, UK) supplemented with 10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine and 1 mM sodium pyruvate.
Phytohemagglutinin (PHA)-induced T cell blasts (PHA-blasts) were obtained by culturing PBMC with PHA (2 µg/ml) for 3 days in complete medium. After washing, T cell blasts were expanded in complete medium containing 20 IU/ml of recombinant IL-2 (Boehringer Mannheim, Meylan, France) for at least 15 days before use.
Purified CD8+ T cells were obtained by negative selection. PHA-blasts were incubated with an anti-CD4 mAb from Coulter-Immunotech (Marseille, France) for 1 h at 4°C. After washing, cells were incubated for 1 h at 4°C with sheep anti-mouse-coated magnetic beads (Dynal, Oslo, Norway). Negative-selected CD8+ T cells were
90% pure.
Antibodies and reagents
The following mAb were used for labeling surface antigens: anti-CD4 (clone Leu3a) and anti-CD8 (clone Leu2a) mAb coupled with PerCP from Becton Dickinson; anti-CD8 mAb coupled with phycoerythrin (PE) from Immunotech; and anti-CD25, anti-HLA DR, anti-CD45RA and anti-CD45RO mAb coupled with FITC from Immunotech.
The anti-CD3
mAb UCHT1 (IOT3) was purchased from Immunotech or was used as an ascitic fluid for T cell stimulation, alone or in combination with a polyclonal rabbit anti-mouse IgG serum (RAM) obtained from Sigma-Aldrich (St Quentin Fallavier, France).
The FITC-conjugated anti-BrdU mAb (clone B44) was provided by Becton Dickinson. The biotinylated and the unlabeled anti-phosphotyrosine mAb (clone 4G10) were obtained from UBI (Lake Placid, NY). Anti-p56Lck, anti-p59Fyn and anti-ZAP-70 mAb used for Western blotting experiments were purchased from Transduction Laboratories (Affiniti Research Products, Nottingham, UK). Polyclonal rabbit serum anti-p56Lck specific for the N-terminus (NT) of the PTK and polyclonal rabbit serum anti-NT-p59Fyn were obtained from UBI and used for immunoprecipitations.
BSA, paraformaldehyde (PFA), saponin, DNase I and BrdU were purchased from Sigma. FACS lysing solution was provided by Becton Dickinson. PBS was from Gibco/BRL (Life Technologies, Cergy-Pontoise, France). [3H]Thymidine (2 Ci/mmol) was obtained from ICN (Orsay, France) and [
-32P]ATP (3000 Ci/mmol) was from Amersham-Pharmacia Biotech (Orsay, France).
Lymphocyte counts and phenotypes
Absolute counts of CD4+ and CD8+ T cells were determined from whole blood samples on the Epics XL flow cytometer using the Flow-Count Fluorospheres system (Coulter, Miami, FL).
Lymphocyte phenotypes were studied by three-color flow cytometry on whole blood samples using PerCPanti-CD4 mAb, PEanti-CD8 mAb and either FITC-conjugated mAb specific for HLA DR, CD25, CD45RA or CD45RO according to standard procedures. Cells were analyzed on a FACSCalibur cytofluorometer (Becton Dickinson) fitted with a 15 mW air-cooled argon-ion laser emitting at 488 nm. Green, orange and red fluorescences were calibrated by using CaliBRITE beads (Becton Dickinson). Forward scatter, side scatter, FITC, PE and PerCP fluorescent signals were detected by using standard procedures, and data were analyzed with CellQuest software (Becton Dickinson).
Cell proliferation assays using [3H]thymidine incorporation
PBMC were cultured at 1x105 cells/well in triplicate in a 96-well microtiter plate with complete medium alone or with coated anti-CD3 mAb (1:2000 dilution of an ascitic fluid) in 200 µl of final volume for 72 h at 37°C. Cells were pulsed with [3H]thymidine (1 µCi/well) during the last 16 h of culture. Cells were then harvested and radioactivity was measured on a ß-counter (Beckman, Palo Alto, CA).
Cell proliferation assays using BrdU labeling
BrdU incorporation assays were performed according to the method of Carayon et al. (21) modified as followed. PBMC from the patient or healthy blood donors were incubated at 106 cells in 1 ml of complete medium in a 24-well flat-bottom microtiter plate either uncoated or coated with the anti-CD3 mAb UCHT1 (5 µg/ml). After 64 h of culture at 37°C, BrdU (30 µg/ml) was added in each well for 1 h. Cells were then harvested, washed in PBS containing 0.5% BSA and 0.02% sodium azide (PBS/BSA/NaN3), and surface labeled with 20 µl of PerCPanti-CD4 or PerCPanti-CD8 mAb. After washing, cells were fixed with 200 µl of PBS, pH 7.5, containing 4% PFA (PBS/PFA 4%) for 20 min at room temperature with gently agitation. Cells were further permeabilized by washing with PBS/BSA/NaN3 containing 0.1% saponin (PBS/BSA/saponin). DNA was denatured with 50 U Kunitz units of DNase I from bovine pancreas (Sigma; DN-25) in 1 ml of PBS containing 5 mM MgCl2. After a wash with PBS/BSA/saponin, cells were labeled with 20 µl of FITCanti-BrdU mAb in 50 µl of PBS/BSA/saponin for 30 min at 4°C. Cells were analyzed on the FACSCalibur cytometer as above. After gating viable lymphocytes on forward and side scatters, CD4+ or CD8+ T cells were gated on the FL3 fluorescence intensity. The percentage of BrdU+ cells was then determined by analyzing the FL1 fluorescence intensity from 2500 events for the CD4+ or the CD8+ subpopulation.
Analysis of CD3-induced protein tyrosine phosphorylation in T cell subsets by flow cytometry
Quantification of cellular tyrosine kinase activities was performed as recently described (22). Briefly, whole blood samples (400 µl/condition for the lymphopenic patient and 200 µl/condition for controls) were stimulated with anti-CD3 mAb UCHT1 (5 µg/ml) and RAM (16 µg/ml) or with medium alone at 37°C for 3 min for CD4+ T cells or for 1 min for CD8+ T cells. Activation was stopped by washing with cold PBS/BSA/NaN3 and rapid centrifugation. Blood pellets were labeled with PerCPanti-CD4 or PerCPanti-CD8 mAb and red blood cells were then lysed with FACS lysing solution. Cells were subsequently permeabilized with PBS/PFA 4% and PBS/BSA/saponin. Intracellular labeling was performed with 10 µg/ml of the biotinylated anti-phosphotyrosine mAb 4G10 for 30 min at 4°C and streptavidinFITC (20 µg/ml). Samples were finally analyzed for FL1 and FL3 fluorescence intensities on the FACSCalibur cytometer. Viable cells, and CD4+ and CD8+ populations were gated as above. In total, 2500 events were analyzed for each CD4+ or CD8+ T cell subset.
Western blotting experiments
For the study of the protein tyrosine phosphorylation process, purified CD8+ PHA-blasts (5x106 per condition of stimulation) were washed in RPMI containing 10 mM HEPES, pH 7.2, and equilibrated for 10 min at 37°C. Cells were then incubated with the UCHT1 mAb (1:500 dilution of the ascitic fluid) or with medium alone for 1 min at 37°C. Activation was stopped by brief centrifugation and transfer on ice and cells were lysed in lysis buffer (TrisHCl 20 mM, pH 7.5, EDTA 1 mM, NaCl 140 mM, 1% NP-40, aprotinin 50 U/ml, sodium orthovanadate 1 mM and PMSF 1 mM) at 4°C for 15 min. For the study of PTK expression, cells were lysed immediately after washing. Nuclei and cellular debris were removed by centrifugation. After dilution in Laemmli sample buffer (TrisHCl 500 mM, pH 6.8, 10% SDS, 10% glycerol, 5% 2-mercaptoethanol and 10% bromophenol blue) and boiling for 5 min, the same amount of proteins per sample determined by using the Bradford assay was loaded on an 8% SDSpolyacrylamide minigel (Novex, San Diego, CA). Proteins were electrophoretically transferred to nitrocellulose sheet (Schleicher & Schuell, Dassel, Germany) for 1 h at 65 V. Blots were then hybridized for 2 h with 0.2 µg/ml anti-phosphotyrosine mAb 4G10, 1 µg/ml anti-p56Lck mAb, 1 µg/ml anti-p59Fyn mAb or 1 µg/ml anti-ZAP-70 mAb, before addition of goat anti-mouse antiserum labeled with peroxidase (BioRad, Richmond, CA). Reaction was revealed with an enhanced chemiluminescence system (Amersham-Pharmacia Biotech) according to the supplier's instructions.
In vitro kinase assays
CD8+ PHA-blasts were lysed as above and samples were normalized for the amount of proteins. Lysates were incubated for 2 h with 2.5 µl of rabbit anti-NT-p56Lck or 10 µl of rabbit anti-NT-p59Fyn and immune complexes were then recovered with 20 µl of Protein ASepharose beads for 1 h at 4°C. Immunoprecipitates were washed 3 times with lysis buffer and once with kinase buffer (50 mM PIPES, pH 6.7, 10 mM MgCl2, 10 mM MnCl2, 1 mM PMSF, 50 µM sodium orthovanadate and 2% aprotinin). Kinase assay was started by addition of 30 µl of kinase buffer containing 5 µM unlabeled ATP, 5 µCi of [
-32P]ATP and 2.5 µg enolase as a specific exogenous substrate. Samples were incubated 5 min at 30°C and kinase reaction was stopped by addition of 30 µl of Laemmli sample buffer. After boiling at 95°C for 5 min, radiolabeled proteins were resolved by 8% SDSPAGE and transferred to nitrocellulose. Membranes were exposed for autoradiography during the indicated period of time. Radioactivity of the bands was quantified using a ß-Imager 1200 (Biospace, Paris, France). Gel Bands were also scanned using a laser densitometer (BioRad).
RT-PCR, cloning and sequencing p56Lck
RNA extraction was performed by using the RNA isolation kit (Stratagene, La Jolla, CA). RNA (10 µg) was reverse transcribed using the MMLV reverse transcriptase (RT-PCR kit from Stratagene). p56Lck cDNA (2.5 µl) was amplified using the primers 5'-GGAATTCCGGGACCATGGGCTGTGGCTGC-3' and 5'-GCTCTAGAGCTCTCAAGGCCTCCTCTCAAG-3' under the following conditions: 94°C for 90 s, 60°C for 90 s, 72°C for 90 s, 30 cycles. PCR products (1500 bp) were digested using EcoRI and XbaI restriction endonucleases (Boehringer Mannheim), gel purified, and cloned into the pcDNA3 vector. Sequences were performed on a ABI Prism 377 automated DNA sequencer (Perkin-Elmer, Foster City, CA).
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Results
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Phenotypic characteristics of T lymphocytes
A severe selective CD4+ lymphocytopenia (100156/µl) was associated with increased CD8+ T cell counts (~1000/µl), both remaining stable during the 10-year follow-up (Table 1
) (20). The expression levels of CD4 and CD8 antigens were comparable in the patient's and control cells (data not shown). The B cell (CD19+) and NK cell (CD3CD16+CD56+) percentages were also stable and within the normal ranges.
Examination of ex vivo T cell activation status over a 6-year follow-up showed that high percentages of CD4+ T cells displayed the activation markers CD25 (2947%) and/or HLA-DR (1335%) (Table 1
). In contrast, CD8+ T cells were weakly activated, 26% of them displaying CD25 and the HLA-DR marker being expressed at normal levels. Moreover, most of the CD4+ T cells were differentiated into memory cells since 7886% of them displayed the CD45RO isoform (23), while 82100% of CD8+ T cells were CD45RA+. Co-expression of the CD45RO and CD45RA isoforms was however detectable on 1455% of CD4+ and 3031% of CD8+ T cells, suggesting a recent activation of those circulating T cells (24). The cell surface expression of these activation and memory markers on both T cell subsets also remained stable over time (Table 1
).
T cell proliferation to CD3TCR stimulation was altered only in the CD4+ T cell subset
To test the hypothesis of additional T cell dysfunctions, we first studied the ability of T cells to proliferate in response to CD3TCR stimulation first by using the [3H]thymidine incorporation assay. As shown in Table 1
, the patient's PBMC proliferative responses to CD3TCR cross-linking were similar to those of normal controls and remained stable during the 6-year follow-up.
Since patient's T cells were composed mainly by the CD8+ subpopulation (see Table 1
), this assay did not discriminate between the proliferation of CD4+ and CD8+ T cells. We thus studied the proliferation of each T cell subset by using the BrdU incorporation assay coupled to flow cytometric analysis as described in Methods. As shown in Fig. 1
, after 64 h of CD3 stimulation, cells from healthy donors incorporated BrdU in a narrow range of values similar to those previously described by Carayon et al. (21): CD4+ = 46 ± 5% and CD8+ = 54 ± 6% (n = 16). Interestingly, a significant 40% decrease in the patient's CD4+ T cell proliferation was observed, only 28% of them incorporating BrdU (Fig. 1
). In contrast, CD8+ T cells proliferated normally compared to controls, thus explaining the normal c.p.m. values obtained in the total PBMC proliferation assay using [3H]thymidine (see Table 1
). These results were stable in three experiments performed over the last 3 years (mean ± SD was 27.6 ± 1.5% for CD4+ T cells and 44.6 ± 2.5% for CD8+ T cells), suggesting that a specific failure to proliferate of the CD4+ T cell subset might participate in the process of CD4+ T cell depletion.

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Fig. 1. BrdU incorporation by CD4+ and CD8+ T cells after stimulation of the CD3TCR complex. PBMC from a normal blood donor and from the patient (1x106 per condition of stimulation) were stimulated with coated anti-CD3 mAb UCHT1 or with medium alone, for 64 h at 37°C. BrdU was added in the last hour of culture. After surface labeling with PerCPanti-CD4 or PerCPanti-CD8 mAb, cells were permeabilized and labeled with FITCanti-BrdU mAb. Samples were analyzed by FACS. After gating live lymphocytes, CD4+ or CD8+ T cells were then gated on a dot-plot (not shown) and the percentage of BrdU+ cells was determined for each subset on a histogram plot. Percentages of CD4+ and CD8+ cells positive for BrdU staining are indicated.
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CD3-induced protein tyrosine phosphorylations were conserved in CD4+ and CD8+ T cell subsets
To determine whether alterations in early biochemical events of the CD3TCR pathway might explain the defective CD4+ T cell proliferative response, we studied the CD3-induced tyrosine phosphorylation process of cellular proteins. To analyze this phenomenon in each CD4+ and CD8+ T cell subset, we used the previously described phosphotyrosine assay coupled to flow cytometric analysis (22). This technique allows us to quantify the tyrosine phosphorylation of whole cellular proteins at the cell subset level and to detect PTK activity deficiency (22). After intracellular labeling with the anti-phosphotyrosine mAb 4G10, the amounts of spontaneous and CD3-induced protein tyrosine phosphorylations were quantified by the median of the phosphotyrosine fluorescence intensity (Ptyr MFI). For each subset, results were expressed as the ratio (R) between CD3-induced Ptyr MFI and spontaneous Ptyr MFI. One of two independent experiments is shown in Fig. 2
. No major differences were identified in the intensity of the tyrosine phosphorylation process between the patient's and control's CD4+ T cells (R = 1.79 for the patient's CD4+ T cells; normal CD4+ T cells R = 2.3 ± 0.27, range: 1.92.8). In addition, the tyrosine phosphorylation process was normally induced by CD3 triggering in CD8+ cells with R = 1.86 (normal CD8+ T cells R = 1.83 ± 0.43, range: 1.42.5).

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Fig. 2. CD3-induced protein tyrosine phosphorylation in T cell subsets. Whole blood samples were stimulated with UCHT1 and RAM for 3 min for CD4+ T cells, for 1 min for CD8+ T cells or with medium. Samples were cell surface labeled with PerCPanti-CD4 or PerCPanti-CD8 mAb. After lysis of red blood cells and permeabilization, samples were labeled intracellularly with the biotinylated anti-phosphotyrosine mAb 4G10 and streptavidinFITC. Cells were then analyzed by flow cytometry. Among living lymphocytes, CD4+ and CD8+ cells were gated, and the intensity of the phosphotyrosine fluorescent signal was determined on a histogram plot for each stimulated and unstimulated sample. The ratio: R = Ptyr MFI of stimulated CD4+ or CD8+ T cells/Ptyr MFI of unstimulated CD4+ or CD8+ T cells is indicated in each case. Solid lines, unstimulated samples. Dotted lines, CD3-stimulated samples.
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Non-altered CD3-induced protein tyrosine phosphorylation pattern in purified CD8+ T cell blasts
We then determined whether qualitative alterations of the CD3TCR-induced tyrosine phosphorylation pattern could be detected in the patient's T cells by using classical Western blotting experiments. Because of the severe CD4+ lymphocytopenia, only CD8+ T cells could be examined, after negative purification from a PHA-derived cell line. The CD8+ T cell blasts from the patient and a healthy control were stimulated with the anti-CD3 mAb UCHT1 or left unstimulated, and lysed. The same amounts of proteins for each sample (30 µg) were migrated on a mini-SDSPAGE and analyzed by Western blotting using the anti-phosphotyrosine mAb 4G10. As shown in Fig. 3
, CD3TCR stimulation induced significant protein tyrosine phosphorylation in the patient's CD8+ T cell blasts, with an intensity similar to that observed in control CD8+ T cell blasts. Moreover, the tyrosine phosphorylation pattern appeared identical in the patient's and control cells. Thus, this early biochemical event of the CD3TCR pathway seemed qualitatively conserved in CD8+ T cell blasts from this patient.

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Fig. 3. Analysis of CD3-induced protein tyrosine phosphorylation by Western blotting experiments in CD8+ T cell blasts. Purified CD8+ T cell blasts (2.5x106/condition of stimulation) from the patient (P) and controls (C) were stimulated with soluble UCHT1 mAb or with medium alone for 1 min, and lysed. Lysates (30 µg/sample) were migrated on a 8% SDSpolyacrylamide minigel and transferred to nitrocellulose. Blots were hybridized with the anti-phosphotyrosine mAb 4G10 and goat anti-mouse coupled to peroxidase.
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Normal expression but defective p56Lck activity in CD8+ T cell blasts
p56Lck is thought to be the earliest PTK to be activated after CD3TCR stimulation (18,19) and to play a critical role in CD3TCR signaling pathway (25) as well as in T cell development (26). Moreover, defective p56Lck expression has been recently described in a child with SCID presenting a selective CD4+ lymphocytopenia (17). We thus examined the expression of this kinase by Western blotting experiments in the patient's and control's CD8+ T cell blasts. As shown in Fig. 4
(a), the patient's cells displayed the same amount of p56Lck as control cells. Similarly, the expression of each of the other T cell PTK p59Fyn (Fig. 4b
) and ZAP-70 (Fig. 4c
) appeared to be identical in the patient's and control CD8+ T cell blasts.

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Fig. 4. Expression of PTK in CD8+ T cell blasts. For both patient (P) and control (C), CD8+ T cell blast lysates (30 µg/sample) were migrated on a 8% mini-SDSPAGE, transferred to nitrocellulose and revealed with the anti-p56Lck mAb (a), the anti-p59Fyn mAb (b) or the anti-ZAP-70 mAb (c) and goat anti-mouse coupled to peroxidase.
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We then analyzed the spontaneous p56Lck activity by immunoprecipitating the kinase from unstimulated CD8+ T cell blast lysates. Immunoprecipitates were then subjected to in vitro kinase assay, migration on SDSPAGE and transfer to nitrocellulose sheet. As shown in Fig. 5
(a), both p56Lck autophosphorylation and activity toward the exogenous substrate enolase were reduced by 50% in patient's compared to control's CD8+ T cell blasts, although the same quantity of the enzyme was precipitated in each case (Fig. 5b
). The intensity of the p56Lck band was quantified using a ß-Imager at 76,640 counts for the control and 37,853 counts for the patient. Similarly, 105,731 and 50,349 counts were found for the band corresponding to the exogenous substrate for the control and the patient respectively. The same result was obtained by scanning the bands with a laser densitometer. In addition, analysis of p56Lck activity in five healthy subjects revealed that kinase autophosphorylation and enolase phosphorylation were in a narrow range of values (see Fig. 6
) with maximal variations of 8.8 and 6.3% respectively. In contrast, p59Fyn autophosphorylation and kinase activity were identical in the patient's and control CD8+ T cell blasts (Fig. 5a and b
), showing that the kinase activity deficiency specifically affected p56Lck. All these results were confirmed in three separate experiments and were also found in purified CD8+ T cells isolated from fresh PBMC (data not shown), suggesting that this deficiency in p56Lck activity was a stable phenomenon and was not secondary to PHA activation.

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Fig. 5. p56Lck kinase activity in CD8+ T cell blasts from the patient and control. (a) CD8+ PHA-blasts lysates from the patient (P) and a control subject (C) were immunoprecipitated with anti-NT-p56Lck rabbit serum or anti-NT-p59Fyn rabbit serum and subjected to in vitro kinase assay. After migration on a 8% SDSPAGE, immunoprecipitates were transferred to nitrocellulose. The blot was first exposed on a X-ray film for 2 days. (b) The same blot was hybridized with an anti-p56Lck mAb or an anti-p59Fyn mAb and goat anti-mouse serum coupled to peroxidase.
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Fig. 6. p56Lck activity in CD8+ T cell blasts from healthy blood donors. (a) CD8+ PHA-blasts from five control donors were lysed in NP-40 lysis buffer. The same amounts of proteins per sample were immunoprecipitated with the anti-NT-p56Lck rabbit serum and subjected to in vitro kinase assay in the presence of the exogenous substrate enolase. Samples were migrated on 8% SDSPAGE, transferred to nitrocellulose and the blot was first exposed for 2 days of autoradiography. Positions of p56Lck and enolase are indicated by an arrow. A properly exposed autoradiograph was then scanned using a laser densitometer and the relative values of each band are indicated. (b) This same blot was next incubated with the anti-p56Lck mAb and goat anti-mouse serum coupled to peroxidase. p56Lck and Ig heavy chains (Ig) are indicated by an arrow. The intensity of each p56Lck band was quantified as above and relative values are indicated, showing that the same quantity of the enzyme was immunoprecipitated in each sample.
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The kinase domain of p56Lck has been shown to be responsible for the phosphorylation of exogenous substrates and also for the autophosphorylation of the PTK (27,28). Since a kinase-defective p56Lck could be obtained either by deletion of the kinase domain or by mutation of the Lys273 (29,30), we finally attempted to determine whether a sequence abnormality could be evidenced in the patient's p56Lck cDNA. The p56Lck RNA was extracted from the patient's PBMC and from the same control as above, and was amplified by RT-PCR. The 1500 bp band corresponding to the size of p56Lck cDNA was gel-purified and cloned into the pcDNA3 vector. Ten clones for each patient and control were sequenced. For both subjects, we found two types of p56Lck cDNA, one corresponding to the full-length p56Lck sequence (31) and the other corresponding to the previously described p56Lck cDNA deleted of exon 7 (17,25). However, no sequence disparity was found in each case between the patient's and control p56Lck sequences.
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Discussion
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ICL defined by the criteria of the Centers for Disease Control and Prevention (4) appears to be a heterogeneous entity, when considering either the clinical symptoms or the level of CD4+ T cell depletion (2). Hence, the mechanism(s) and the etiology(ies) of this syndrome are currently unknown and are likely of multiple origins (3). Here we studied a patient with a stable and severe loss of CD4+ T cells associated with a history of clinical symptoms of cellular immune deficiency. Since alterations of the CD3TCR signal transduction pathway have been described in several cases of children with SCID associated with selective lymphocytopenia (1416), we postulated that the immune deficiency of this patient could be related to molecular defects in the T cell activation process.
We found an abnormality of the PTK p56Lck in CD8+ T cells from our patient that might play a role in this case of ICL. Similarly, one SCID child presented a selective CD4+ lymphocytopenia associated with an absence of p56Lck expression in the remaining CD8+ T cells (17). However, in our case, a full-length p56Lck was expressed in T lymphocytes which rather displayed a 50% decrease in autophosphorylation and in in vitro kinase activity. This observation contrasted with the conserved protein tyrosine phosphorylation process induced by CD3 triggering in the patient's CD4+ and CD8+ T cells (see Fig. 2
). p56Lck is linked to CD4 and CD8 cell-surface receptors (32), and has been demonstrated to be critical for early CD3TCR signaling pathway. Indeed, no protein tyrosine phosphorylation can be induced by CD3 stimulation in the Jurkat variant JCam1 defective for Lck expression (25). Moreover, tyrosine phosphorylation of the major in vitro autophosphorylation site of p56Lck (tyrosine 394) correlates with an enhanced kinase activity (27). However, protein tyrosine phosphorylations are similarly detected after CD3
triggering in cells expressing a CD4p56Lck chimera deleted of the kinase domain or containing the entire kinase domain (30). In the same way, the p56Lck SH2 domain, rather than the kinase domain, has been shown to be critical for CD3-induced intracellular protein tyrosine phosphorylation (33,34). Finally, our patient's finding is in accordance with the observation of the SCID child (17) who displayed a normal protein tyrosine phosphorylation process in CD8+ T cells despite the absence of p56Lck expression. These findings support the hypothesis of an indirect role for the kinase domain of p56Lck in the early tyrosine phosphorylation of whole cellular proteins. Alternatively, one could argue that a 50% reduction in p56Lck kinase activity was not sufficient to alter the protein tyrosine phosphorylation pattern. Another hypothesis would be that p59Fyn might complement p56Lck as previously proposed (35), although no increased amount and activity of this PTK was evidenced in our patient's T cells.
Defective T cell proliferation to mitogens have been described for several patients with ICL (7). The BrdU assay allowed us to clearly demonstrate that the impaired proliferative response was observed only in the depleted CD4+ T cell subpopulation. Since only live lymphocytes were gated in this assay, this proliferation defect was not due to increased activation-induced cell death (13) but rather to a true defect in the renewal capacity of peripheral CD4+ T lymphocytes which might participate in the selective CD4+ lymphocytopenia. The degree of decreased proliferative responses of the patient's CD4+ T cells correlated here with the level of deficiency in p56Lck activity found in CD8+ T cells (4050% inhibition), rather than with the weak protein tyrosine phosphorylation defect (only 10% decrease) found in the CD4+ T cell subset. This finding suggested that the defective Lck kinase activity might also be present in CD4+ T cells and be responsible for the proliferation defect of this subset, perhaps by inhibition of downstream activation events. The reason why the CD8+ subpopulation was not depleted but was rather increased and was still able to proliferate normally to anti-CD3 mAb despite the defect of p56Lck activity remains unsolved. In fact, previous reports suggested that p56Lck is preferentially involved in CD4+ T cell rather than in CD8+ T cell differentiation and activation (3637). Importantly, the numbers of CD4+ single-positive cells are reduced in mice expressing a Lck-dead kinase transgene, whereas the CD8+ T cell counts remain unaltered (37). Moreover, the CD8+ T cell numbers are also increased in the SCID child who lacks Lck expression (17) and these CD8+ cells proliferate normally to CD3 triggering at least during the first 6 months of life. Thus, the results reported here reinforced the idea that p56Lck might have a preferential role for CD4+ T cell development and/or activation compared to CD8+ T cells.
However, the precise origin of the abnormal CD4+ T cell activation and p56Lck activity described in this case of ICL remains to be determined. One hypothesis would be an indirect origin due to T cell anergy, to the CD4+ T cell hyperactivation status or to an unknown infectious agent similar to HIV despite the lack of any evidence for epidemic transmission of ICL (2,7). However, in these cases, a CD4+ T cell proliferation deficiency is associated with an impaired CD3-induced tyrosine phosphorylation process, and altered levels of p56Lck and p59Fyn (3942). On the other hand, the patient described here might rather display a primary T cell deficiency caused by structural abnormalities of transducing molecules, similar to those found in children with inherited immunodeficiency (1416). Indeed, the deficiency in p56Lck activity was observed in both freshly isolated PBMC and in cultured PHA-blasts, suggesting that this defect was not due to extracellular factors, but rather to an intrinsic T cell abnormality. The best candidate was p56Lck itself. However, in >10 p56Lck cDNA clone sequences examined, none were found to be different from control sequences (31). This suggested that a defect of a yet undetermined protein kinase or alternatively an enhanced level of a cellular protein phosphatase regulating p56Lck kinase activity might take place in T cells from the patient. Since p56Lck tyrosine phosphorylation was similar in the patient's and control CD8+ T cell blasts (data not shown), this altered protein might be a serine kinase or phosphatase. This unknown defect might thus represent a link between p56Lck and downstream activation events, and might also account for the loss of CD4+ T cell proliferation capacity and CD4+ T cell depletion. Contrasting with the recent study describing a SCID child with Lck deficiency and CD4+ lymphopenia who displayed clinical signs of immune deficiency as soon as the first months of life, our patient has reached the adult stage without any serious illness. The loss of the entire Lck expression in the child's case might elicit more dramatic clinical consequences than a 50% deficiency in Lck kinase activity. Nevertheless, this report showing PTK alterations in an adult ICL supports a preferential role for p56Lck in human CD4+ T cell activation and renewal.
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Acknowledgments
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We thanks Dr G. Bismuth for critical reading of the manuscript, and E. Lefranc and P. Grenot for technical assistance. This work was supported by grants from the Délégation à la Recherche Clinique de l'AP-HP.
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Abbreviations
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ICL idiopathic CD4+ lymphocytopenia |
NT N-terminus |
PBMC peripheral blood mononuclear cells |
PE phycoerythrin |
PFA paraformaldehyde |
PHA phytohemagglutinin |
PTK protein tyrosine kinase |
SCID severe combined immunodeficiency |
MFI mean fluorescence intensity |
Ptyr phosphotyrosine |
RAM rabbit anti-mouse IgG serum |
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Notes
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Transmitting editor: A. Fischer
Received 5 July 1999,
accepted 3 December 1999.
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