Membrane and transmembrane signaling in Herpesvirus saimiri-transformed human CD4+ and CD8+ T lymphocytes is ATM-independent.

Miguel Rivero-Carmena, Oscar Porras1, Blondineth Pelaez, Alberto Pacheco-Castro, Richard A. Gatti2 and José R. Regueiro

Inmunología, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain
1 Inmunología, Hospital Nacional de Niños, San José, Costa Rica
2 Department of Pathology, University of California at Los Angeles School of Medicine, Los Angeles, CA 90095-1732, USA

Correspondence to: J. R. Regueiro


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the genetic disorder ataxia telangiectasia (AT), humoral (B) and cellular (T) immunological abnormalities are frequently observed. As a consequence, AT patients are predisposed to life-threatening sinopulmonary infections. The pathogenic mechanisms remain unknown, but a role for ATM in signal transduction from membrane receptors has been proposed. We have explored the effects of a defective ATMgene on isolated human T-lineage cells from 13 AT patients with proven T cell dysfunction by transforming their CD4+ and CD8+ T lymphocytes with Herpesvirus saimiri, and analyzing their signaling behavior as compared to normal controls. Several functional parameters were assayed in response to both membrane (anti-CD3 and IL-2) and transmembrane (phorbol myristate acetate plus the calcium ionophore ionomycin) stimuli: (i) calcium mobilization, (ii) induction of activation molecules (CD25, CD40 ligand, CD69 and CD71), (iii) cytokine synthesis (IL-2 and tumor necrosis factor-{alpha}) and (iv) proliferation. All these early and late activation events were found to be normal in the transformed ATM–/–T cells, indicating that ATM is not necessary for their induction. As expected, ATM–/– transformed T cells showed an increased radiosensitivity by both radioresistant DNA synthesis and cell survival assays. In contrast to an earlier report testing transformed B lymphocytes, our results indicate that transformed mature peripheral T lymphocytes from AT patients do not have intrinsic immune function defects. Rather, the described T-lineage signaling impairments observed in patients may be secondary in vivo to extrinsic ATM-dependent suppressive factors and/or to a developmental defect. These transformed T cells may help to understand the distinct biological role of ATM in different cell types and to develop rational therapies for the immunological dysfunction of AT patients.

Keywords: ATM protein, ataxia telangiectasia, Herpesvirus saimiri-transformed human T cells, T cell signaling


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ataxia telangiectasia (AT) is an autosomal recessive multisystemic disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, radiosensitivity, high incidence of lymphoid malignancies, and combined cellular (T) and humoral (B) immunodeficiency (1). The major debilitating features of AT are the progressive neurological abnormalities and a variable immunodeficiency that predisposes patients to recurrent sinopulmonary infections. Mutations in the ATM gene are responsible for the defect in AT (2), although the pathogenic mechanisms remain unclear. ATM is a member of a family of high mol. wt proteins that share the phosphatidyl- inositol 3-kinase-like domain. Phosphatidylinositol 3-kinase is capable of phosphorylating several substrates and plays a key role in linking surface receptors to signal transduction events within the cell. This family includes several proteins that appear to be required for the G1 to S transition, the S/G2 and G2/M cell cycle checkpoints, and for sensing double-strand breaks in damaged DNA (3,4). ATM is present in the nucleus as well as in cytoplasmic vesicles (5).

Mice lacking ATM are profoundly T cell deficient, thus providing an explanation for their combined immunodeficiency (6). In contrast, AT patients have substantial or even normal numbers of peripheral blood lymphocytes (PBL) which, however, show impaired functional responses in vivo (poor skin tests and graft rejection) and in vitro (low responses to mitogens) (1). Despite normal or even increased numbers of B lymphocytes, AT patients frequently show hypogammaglobulinemia (absence or marked deficiency of serum IgA, IgE and subtypes of IgG), which may be due to intrinsic B lymphocyte switch defects or to the lack of T cell-derived switch factors. Prior to the cloning of the ATM gene, Kondo et al. reported that T lymphocytes from AT patients (ATM–/– hereafter) showed reduced calcium flux, IL-2 production and proliferation after TCR triggering (7), despite normal mitogen internalization (8). Intrinsic defects in signal transduction have also been described in ATM–/– B lymphocytes. Recently, Khanna et al. reported defective signaling through the BCR in Epstein–Barr virus (EBV)-transformed ATM–/– cells (9). Thus, a general role for ATM in signal transduction from membrane receptors is widely accepted (10), but poorly defined.

Herpesvirus saimiri (HVS) is a common lymphotropic virus of squirrel monkeys. It is known to transform both CD4+ and CD8+ human T lymphocytes into stable growth by still undefined mechanisms (11). HVS-transformed T lymphocytes remain IL-2 dependent, but become antigen and mitogen independent for their continued growth (12). These cells display normal downstream functional responses (proliferation, cytokine synthesis, induction of activation markers, cytotoxicity, apoptosis induction, etc.) to membrane (anti-TCR–CD3, anti-CD95 ligand and IL-2) and transmembrane [phorbol myristate acetate (PMA) + calcium ionophore] stimuli (1317). Previously, T lymphocytes from several immunodeficiencies have been transformed using HVS, and in all cases these models have been shown to faithfully preserve the phenotypical and functional features of the original T cells (1621).

We have thus explored the role of the ATM protein in T lymphocyte signaling by transforming T cells from AT patients with proven T cell dysfunctions [low phytohemagglutinin (PHA) response]. The results indicate that, in contrast to what has been reported in transformed B cells, membrane and transmembrane signaling to the cytoplasm and nucleus is ATM independent, as it is not defective in HVS-transformed ATM–/– T lymphocytes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patients and controls
HVS-transformed T cells were obtained from PBL of 15 patients with AT (ATM–/–) from Costa Rica (CRAT1–13) or Spain (SPAT1 and 2; Hospital La Paz, Madrid) and four normal donors named N1–4 (ATM+/+) from the Blood Bank of Hospital San Carlos, Madrid. All patients were diagnosed with progressive cerebelar ataxia and telangiectasias. The ages of the AT patients were between 8 and 17 years. In addition, all patients showed defects in one or more Ig isotypes. Ten out of 13 patients showed low levels of IgM. In most cases, they showed a decrease in the total number of CD4+ T lymphocytes (12 out of 15), an increase of B lymphocytes (13 out for 15), a severely reduced response to PHA, pokeweed and concanavalin (seven out of eight, CRAT1, CRAT2 and CRAT4 among them), and reduced response to anti-CD3 (one, SPAT1, out of two SPAT donors). Only one patient developed cancer (non-Hodgkin lymphoma), but samples were obtained before chemotherapy was started. AT haplotypes and ATM mutations from these patients were determined on DNA or RNA from the HVS-transformed T cell lines and have been previously published (Table 1Go).


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Table 1. Phenotype/genotype comparison of HVS-transformed T cell lines from AT patients (ATM–/–)
 
Immortalization procedures
HVS-transformed T lymphocytes were obtained as previously described (11). Briefly, PBL were resuspended (2x106 cells/ml) in a mixture (1:1 proportion) of two culture media [RPMI 1640 and cell growth medium from Vitromex, Vilshofen, Germany] supplemented with 10% FCS, 2 mM L-glutamine and 50 IU/ml human rIL-2 (Hoffman La Roche, Nutley, NJ). They were subsequently exposed to 1 ml of HVS supernatant in 24-well plates in the presence or absence of 1 µg/ml PHA (Difco, Detroit, MI). Deprivation of PHA at day 1 is believed to increase the chance of obtaining immortalized CD4+ cells. Thereafter, medium was replaced every 3–4 days by fresh mixed medium containing rIL-2, but not PHA or HVS. A transformed phenotype was indicated by the death of control cultures (i.e. non-HVS exposed) as compared to the sustained growth and T lymphoblast cell morphology of HVS-exposed cultures as described (12). HVS-exposed T cells had been maintained in long-term culture for >10 months when the reported experiments were performed.

Phenotypical analyses
The following mAb were used for cytofluorometric analyses: Leu4 (anti-CD3), Leu2a (anti-CD8{alpha}) and Leu3a (anti-CD4) from Becton Dickinson (MountainView, CA); MsIgG (used as negative control) and BMA031 (anti-TCR{alpha}ß) from Caltag (San Francisco, CA); and TCR{delta}1 (anti-TCR{gamma}{delta}) from T Cell Science (Cambridge, MA). Briefly, for single- and two-color immunofluorescence, 1x106 cells were incubated for 30 min at 4°C with appropriate FITC- or phycoerythrin-conjugated mAb in PBS/EDTA buffer containing 1% FCS. After two washes with PBS buffer, the cells were analyzed in an Epics Elite Analyzer cytofluorometer (Coulter, Hialeah, FL). Data were collected on 2–5x104 viable cells as determined by electronic gating on forward and side light scatter parameters. For intracellular staining using anti-IL-2 or anti-tumor necrosis factor (TNF)-{alpha}, the cells were first permeabilized and fixed as described below (cytokine synthesis assays).

Radioresistant DNA synthesis assay
To measure radioresistant DNA synthesis we used standard [3H]thymidine uptake assays (17). Briefly, transformed cells were adjusted to a final concentration of 8x105 cells/ml. All irradiations (5 and 10 Gy doses; dose rate 4.098 Gy/min) were performed at room temperature using a 60Co source (Centro de Investigación Energética Medioambiental y Tecnológica, Madrid, Spain). After irradiation, the cells were washed and resuspended in fresh mixed medium. Then the cells were plated in a round-bottom 96-well plate (180 µl of cells/well), pulsed with [3H]thymidine (1 µCi/well; Amersham, Little Chalfont, UK) for 2.5 h and harvested onto glass fiber filters. Thymidine incorporation into cellular DNA was evaluated as mean c.p.m. of triplicate wells in a scintillation ß-counter (Packard, Meriden, CT). The results were expressed as DNA synthesis of irradiated cells relative to nonirradiated cells.

Survival assay
To measure survival of HVS-transformed cells after irradiation, cells were adjusted to a final concentration of 1.5x 105 cells/ml. The 5 and 10 Gy irradiations were done at room temperature using a 60Co source, dose rate 4.098 Gy/min (Centro de Investigacion Energética Medioambiental y Tecnológica, Spain). After irradiation, the cells were washed and resuspended in fresh mixed medium, plated in 24-well plates and cultured at 37°C in a humidified atmosphere of 5% CO2 for 48 h. Aliquots of 50 µl of cells were stained and mixed with 50 µl of Trypan blue 0.3%. Viable cells were scored under a phase-contrast microscope (Nikon SE). The results were expressed as a percentage of unirradiated cells.

Intracellular Ca2+ mobilization assays
Intracellular calcium release was induced in cells loaded with the fluorescent dye Fluo-3AM (Sigma, St Louis, MO) according to a standard procedure (16). Briefly, 2x106 cells were washed twice and resuspended in Ca2+-free medium (Sigma) at a final concentration of 1x106 cells/ml. Then, the cells were incubated in a stirring water bath at 37°C for 30 min with 4 mM Fluo-3AM, washed again with Ca2+-free medium, resuspended at a final concentration of 0.5–1x106cells/ml and analyzed by flow cytometry. Changes in relative fluorescence intensity were recorded as a function of time, before and after the sequential addition of the following reagents: (i) anti-CD3 mAb (IOT3b from Immunotech, Marseilles, France; 20 µl at 12.5 µg/ml) and (ii) cross-linking reagent [human-adsorbed goat anti-mouse IgG (H + L) from Caltag; 40 µl at 1.25 mg/ml].

Induction of activation molecules
To measure CD69, CD25, CD154 (CD40 ligand) and CD71 induction after stimulation, the transformed cells were starved in cell growth/RPMI medium without recombinant human IL-2 for 7 days, resuspended at 5x105 cells/ml in a 96-well plate in the absence or presence of 1 µg/ml plastic-bound anti-CD3 mAb (IOT3b) or PMA (10 ng/ml; Sigma) plus ionomycin (750 ng/ml; Sigma) for 6 h (or 24 h for CD25) at 37°C. Then, the cells were washed twice in PBS, stained with antiCD69, anti-CD71 and anti-CD25 (Caltag) or anti-CD154 (PharMingen, San Diego, CA), for 30 min at 4°C, washed twice in PBS, and analyzed by flow cytometry as described above.

Cytokine synthesis assays
To analyze intracellular cytokine induction, IL-2-starved cells were resuspended at 5x105 cells/ml in a 96-well plate and stimulated for 6 h with or without 1 µg/ml plastic-bound anti-CD3 mAb (IOT3b) or PMA (10 ng/ml; Sigma) plus ionomycin (750 ng/ml; Sigma). After 2 h, 10 µg/ml Brefeldin A (Sigma) were added to the cultures in order to block secretion. Cells were harvested, washed twice in PBS buffer and fixed with 500 µl of PBS/4% formaldehyde for 20 min at room temperature. Then the cells were stained intracellularly for cytokine content using a modified method based on that described by Assenmacher et al. (24). Briefly, the cells were washed twice in PBS containing 0.1% saponin (Sigma), incubated with FITC-conjugated anti-IL-2 or anti-TNF-{alpha} from R & D systems (Abingdon, UK) in 100 µl of PBS containing 1% saponin for 30 min at room temperature and washed with PBS/0.1% saponin buffer. The cytometric analyses were performed in an Epics Elite Analyzer as described above.

Cell proliferation
Proliferation was measured by standard [3H]thymidine uptake assays (16). HVS-transformed T cells were starved for 7 days in the absence of IL-2, adjusted to a final concentration of 5x105 cells/ml in fresh mixed medium and incubated for 48 h in round-bottom 96-well plates (180 µl of cells/ml), previously coated with different concentrations of anti-CD3 (IOT3b; Immunotech), containing rIL-2 (50 IU/ml human rIL-2; Hoffmann La Roche) or PMA (10 ng/ml; Sigma) plus ionomycin (750 ng/ml; Sigma). Cells were then pulsed with [3H]thymidine (1 µCi/well; Amersham) for an additional period of 16–18 h and harvested onto glass fiber filters. Thymidine incorporation into cellular DNA was evaluated as c.p.m. in a scintillation ß-counter (Packard). All experiments were performed in triplicate wells and the mean was calculated for each stimulus.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T lymphocyte transformation by HVS is ATM independent
At 1–2 months after a single exposure to HVS, PBL from 13 of 15 AT patients became cell lines with a stable growth rate that was undistinguishable from that of controls (cf. empty with filled triangles in Fig. 1Go). HVS-unexposed cells, in contrast, became non-viable by day 30 (circles in Fig. 1Go). Most HVS-transformed ATM–/– T cell lines were TCR{alpha}ß+CD3+CD8+ (n = 11), but two were TCR{alpha}ß+CD3+CD4+ (Table 1Go), which in our hands is a normal distribution (18). The immunophenotype of transformed ATM–/– T cells by immunofluorescence indicated that the percentage and intensity of surface expression of TCR–/–D3 and CD4 or CD8 was comparable to that of ATM+/+ T cells (Table 1Go and data not shown). Four Costa Rican mutations were included (haplotypes [A], [C], [D] and [E]); all truncated ATM at different sites. One Spanish T cell line (SPAT1) had a missense mutation. Mutation detection is pending in a second Spanish donor (SPAT2). The [A] and [D] mutations were substitutions at positions 5908C -> T in exon 41 and 4507C -> T in exon 32 respectively; this resulted in a termination codon in both cases. The [C] mutation was also a substitution at position 7449G -> A in exon 52; this mutation created a false splice acceptor site so that the last 70 nucleotides of exon 52 were deleted starting from codon 2481. The [E] mutation was a deletion at position 8264del5 in exon 58; this mutation resulted in the deletion of exon 58 (for further details, see 23). The Spanish mutation was a substitution at position 2572T -> C in exon 19; this putative mutation results in a substitution of Phe to Leu. A variety of ATM genotypes, by combination of these five mutations was studied: in all, seven homozygotes (CRAT1, CRAT2, CRAT3, CRAT4, CRAT7, CRAT10 and CRAT12), five compound heterozygotes (CRAT6, CRAT8, CRAT9, CRAT13 and SPAT1) and an uncharacterized donor (SPAT2). No obvious genotype/phenotype correlation could be discerned from these data.



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Fig. 1. Efficiency of HVS transformation of ATM–/– versus ATM+/+ PBL. Cell recovery from ATM+/+ PBL did not differ significantly from that of ATM–/– PBL in the absence (circles) or presence (triangles) of HVS supernatant. In some cases, the cells were frozen in part, so data points represent total number estimates based on the growth kinetics of the remaining cells. Representative data from cell lines that became CD4+ (ATM+/+, N3 and ATM–/–, CRAT13) or CD8+ (ATM+/+, N2 and ATM–/–, CRAT6) are included. All other cell lines showed similar profiles.

 
ATM–/– transformed T cells show characteristically increased radiosensitivity
AT fibroblasts and lymphoblastoid B cell lines have been shown to be unusually sensitive to ionizing radiation. A consistent feature of AT cells is their failure to arrest DNA synthesis and a reduced cell survival in response to irradiation. To study the effect of radiation in HVS-transformed ATM–/– T cells, we first measured DNA synthesis after 5 and 10 Gy irradiation. As illustrated in Fig. 2Go(A), three different ATM–/– T cell lines, but not the ATM+/+ control cell line, showed a markedly radioresistant DNA synthesis. The recovery of viable cells in the absence of radiation was similar in both ATM–/– and ATM+/+ cells (see also basal levels of DNA synthesis below, Fig. 6Go). Second, we measured cell survival after irradiation (Fig. 2BGo). Cell survival was comparatively more reduced in ATM–/– T cells than in ATM+/+ T cells.



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Fig. 2. Dose response to {gamma}-irradiation. Transformed T cells, both ATM+/+ (black symbols) and ATM–/– (white symbols), were exposed to increasing amounts of ionizing radiation, as indicated. (A) The DNA synthesis relative to unirradiated cells is plotted as a function of {gamma}-irradiation. The average of three assays ± SD is shown. The following HVS-transformed cell lines were used: ATM–/–, CRAT1, CRAT4 and CRAT6; ATM+/+, N2. (B) The cell count of surviving cells relative to unirradiated cells is plotted as a function of {gamma}-irradiation. The following HVS-transformed cell lines were used: ATM–/–, CRAT1, CRAT4, CRAT6 and SPAT2; ATM+/+, N1

 


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Fig. 6. Induction of proliferation. Proliferative responses of ATM+/+ and ATM–/– HVS-transformed CD4+ or CD8+ T cell lines to membrane stimuli (anti-CD3, IL-2). The results are given as mean c.p.m. values ± SD of three independent experiments. In several instances, transmembrane stimuli (PMA + ionomycin) were also tested, and no differences were observed between ATM+/+ and ATM–/– T cell lines (data not shown). See Methods for the proliferative responses of fresh PBL from the same donors to several mitogens.

 
Taken together, the data of radioresistant DNA synthesis and cell survival indicated that HVS transformation preserves the characteristic radiosensitivity of AT cells. This thereby validates HVS-transformed T cells as a model system for studying the role of ATM protein in T lymphocyte function.

Membrane and transmembrane T cell activation events are ATM independent
We next explored the role of ATM protein in several immediate, early and late T cell activation events using TCR–CD3-dependent (anti-CD3 mAb) and TCR–CD3-independent (PMA + ionomycin) stimuli, most of which have been described to be impaired in many AT patients, including ours (1). PHA does not stimulate HVS-transformed T cells (data not shown), as reported also for purified primary T cells (25). Therefore, it was not used as a stimulus.

First, calcium flux, an immediate activation event after TCR–CD3 engagement (it takes place in seconds), was tested and found to be undistinguishable in ATM–/– versus ATM+/+ T cell lines, whether CD4+ or CD8+ (Fig. 3Go).



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Fig. 3. Calcium mobilization. Representative calcium mobilization (induced by cross-linking the TCR–CD3 complex) in ATM+/+ and ATM–/– HVS-transformed CD4+ or CD8+ T cell lines is shown as time (x-axis) versus relative florescence intensity (y-axis) versus number of cells (z-axis), ranging from white (no cells) to black (most cells). Calcium levels were measured consecutively: (i) baseline control, (ii) after addition of IOT3b mAb (M) and (iii) after cross-linking the mAb (X). The calcium levels in each of these conditions are separated by a white vertical lane. The following HVS-transformed cell lines were used: ATM–/– CD4+, CRAT13 and ATM–/– CD8+, CRAT6; ATM+/+ CD4+, N4 and ATM+/+ CD8+, N1. Similar results were found in all tested ATM–/– and ATM+/+ T cell lines (data not shown).

 
Second, the induction of several cell surface molecules (CD25, CD69, CD71 and CD154), an early activation event (measurable a few hours after triggering), was tested using membrane (anti-CD3) and transmembrane (PMA + ionomycin) stimuli, and no significant differences were found between ATM–/– and ATM+/+ cells, either CD4+ or CD8+ (Fig. 4Go).



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Fig. 4. Induction of cell surface molecules (CD154, CD71, CD69 and CD25). Representative logarithmic induction of cell surface activation molecules in ATM+/+ and ATM–/– HVS-transformed CD4+ or CD8+ T cell lines. HVS T cells were incubated with medium (black histograms), with plastic-bound anti-CD3 mAb or with PMA + ionomycin (ION) (left or right white histograms respectively), washed and stained with anti-CD154, anti-CD69, anti-CD71 or anti-CD25. The bold vertical lines indicate the upper limit of background fluorescence using isotype-matched irrelevant mAb. In our hands, CD25 induction is negligible in transformed CD4+ cells and thus not analyzable. The following HVS-transformed cell lines were used: ATM–/– CD4+, CRAT13 and ATM–/– CD8+, CRAT4; ATM+/+ CD4+, N3 and ATM+/+ CD8+, N1. Similar results were found in all tested ATM–/– and ATM+/+ T cell lines (data not shown).

 
Third, a further early activation event, i.e. the induction of two cytokines (IL-2 and TNF-{alpha}), was assayed in response to both membrane and transmembrane stimuli. As shown in Fig. 5Go, both ATM–/– and ATM+/+ cells, either CD4+ or CD8+, showed comparable inductions of such soluble molecules.



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Fig. 5. Induction of soluble molecules. Representative logarithmic intracellular cytokine induction in ATM+/+ and ATM–/– HVS-transformed CD4+ or CD8+ T cell lines. HVS-transformed T cells were incubated with medium (black histograms), with plastic-bound anti-CD3 mAb or with PMA + ionomycin (ION) (left or right white histograms respectively), blocked with Brefeldin A, fixed, permeabilized and stained for intracellular IL-2 or TNF-{alpha}. The bold vertical lines indicate the upper limit of background fluorescence using isotype-matched irrelevant mAb. The following HVS-transformed cell lines were used: ATM–/– CD4+, SPAT1 and ATM–/– CD8+, CRAT6; ATM+/+ CD4+, N4 and ATM+/+ CD8+, N2. Similar results were found in all tested ATM–/– and ATM+/+ T cell lines (data not shown).

 
Fourth, proliferation, a comparatively late event as it is measured 2 days after activation, was tested using two independent membrane stimuli: IL-2 and anti-CD3. Again, no significant differences were observed between ATM–/– and ATM+/+ T cell lines, either CD4+ or CD8+ (Fig. 6Go).

In summary, we found that HVS-transformed ATM–/– T cells, irrespectively of their CD4/CD8 phenotype, showed a normal profile of functional responses to both TCR–CD3-dependent and TCR–CD3-independent stimuli. Cell proliferation in response to rIL-2 was also ATM independent (Fig. 6Go).

Taken together, these results strongly suggest that the ATM protein is not required for the membrane or transmembrane induction of several activation events in HVS-transformed T lymphocytes. These findings differ from an earlier report of defective signaling in EBV-transformed B cells (9).


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
ATM is an ubiquitous protein. However, not all tissues or cell types are equally affected by a deficiency of ATM. Available in vitro human model systems for AT are presently limited to fibroblasts and B-lineage cells. Our data add CD4+ and CD8+ T-lineage cells to this list, and this may be important for therapeutic purposes, e.g. as a model system to test new drugs. This also may be of some practical interest for the treatment of T cell versus B cell malignancies in AT patients. Indeed, certain chemotherapeutic agents have been shown to be more effective on B cells than on T cells (26). Our data also indicate that the radiosensitivity of AT patients extends to HVS-transformed CD4+ and CD8+ T cells.

It is believed that HVS transforms T lymphocytes by targeting specific signal transduction pathways (11). The viral proteins StpC and Tip may influence T cell growth by associating with cellular Ras and Lck respectively (27,28). The details of this important aspect of HVS biology are, however, still unclear. Our ability to transform T cells from AT patients implies that the signal transducing role of ATM (if any) is distinct and separate from the transduction pathway(s) leading to the transformation of human T lymphocytes.

Our data may modify present notions on the role of ATM protein in T lymphocyte biology, although it must be stressed that they were produced using transformed T cells, not primary T cells. It is generally accepted that AT patients show defective T cell responses (1,29). While this could be due to a primary role for ATM in signal transduction (10), it now appears less likely that this is true (see below). Rather, the T cell dysfunction in AT may be secondary to extrinsic ATM-dependent suppresive factors in vivo and/or to an impaired generation of mature T cells. Indeed, absence or abnormal development of the thymus is a consistent feature of AT (3033). Perhaps as a consequence, T lymphocytopenia is also very common (but not universal), particularly in the naive (CD45RA) CD4+ subset (34), which is highly thymus dependent (35). The murine immunodeficient phenotype can be more easily attributed to a primary developmental T cell defect, because mice lacking ATM show a severe T lymphopenia (6), with normal numbers of T precursor cells (36). AT patients, in contrast, have substantial or even normal numbers of peripheral blood T cells.

Alternatively, a defect in the function of mature T cells might underlie the impaired cellular responses of AT patients. Skin tests to common antigens are frequently weak, and proliferation and cytokine synthesis in response to antigens (e.g. tetanus toxoid), mitogens, allogeneic cells and EBV-infected autologous B cells are typically decreased or absent (7,30,33,34,37,38). Delayed rejection of allogeneic skin grafts is very common (39) and defects in T cell-mediated cytolysis have also been described (40). It has been proposed that an ATM-dependent failure in T cell signaling may cause these functional impairments in mature T cells (10). In support of this hypothesis, intrinsic defects of signal transduction have been reported in isolated AT T and B lymphocytes (7,9), and the ATM protein has been localized recently to vesicular structures in the microsomes, as well as to the nucleus (5,41). However, our results using transformed ATM–/– and ATM+/+ T lymphocytes indicate that ATM is not required for the induction of: (i) proliferation (Fig. 6Go), (ii) cytokines (Fig. 5Go), (iii) cell surface activation molecules (Fig. 4Go) or (iv) calcium flux (Fig. 3Go), using both membrane (anti-CD3 and IL-2) and transmembrane (PMA + ionomycin) stimuli. A normal profile of functional responses was observed despite severe PHA response deficiency in fresh PBL from the same donors (see Methods).

It is unlikely that our results are an artifact of viral infection, unless HVS complements the membrane signaling but not the DNA-sensor activity of ATM (42). The transformed T cells of AT patients, but not of healthy controls, showed increased radiosensitivity as measured by two parameters, radioresistant DNA synthesis and cell survival (Fig. 2Go). Also, we have shown previously that HVS-transformed T cells from other immunodeficient patients preserve functional defects such as proliferation (16) or cytokine induction (17). Thus, we conclude that mature transformed AT T cells do not have intrinsic immune function signaling defects. Rather, the T-lineage impairments described in AT patients are most likely secondary to a (partial) developmental defect (i.e. quantitative rather than qualitative) and/or to the existence of extrinsic (non-T) ATM-dependent suppressive factors in vivo [e.g. {alpha}-fetoprotein (43)], both of which would be absent in our long-term cultures. Taken together, our results suggest that the substantial or even normal numbers of T cells that survive the thymic microenvironment in AT patients will be intrinsically normal, although subjected to (non-T) ATM-dependent suppression. Very recently, by genetic analysis of the mouse model of AT, it has been shown that ATM is required for normal V(D)J recombination and thymocyte expansion, but dispensable for normal mature T cell function when a transgenic TCR is provided (44). Thus, human and murine AT would differ (i) because T cell development is more impaired in mice than in humans and (ii) because the lack of ATM secondarily alters T cell functions in humans, but not in mice.

Our results are in contrast with those reported recently using EBV-transformed B cell lines from AT patients (9). The authors found several defective early and late BCR-mediated activation events. However, EBV transformation, but not HVS transformation, selectively blocks antigen-receptor-mediated signaling with the EBV-encoded LMP2A protein (45). Moreover, no signaling defect has been described in B cells from ATM–/– mice using anti-IgM, lipopolysaccharide and CD40 ligand (46). The analysis of BCR-mediated signaling in human fresh peripheral blood ATM–/– and ATM+/+ B lymphocytes or the establishment of LMP2 EBV-transformed B cells from AT patients may be able to determine whether AT B cells are indeed defective in BCR-induced signaling or not.

The immunological deficit in AT is probably the main cause of morbidity in certain AT populations (e.g. Costa Rica). Therefore, increasing our understanding of the differential effect of ATM deficiency on T- and B-lineage cells may help to develop rational therapies to increase the quality of life of AT patients.


    Acknowledgments
 
This work was supported in part by grants from the Fondo de Investigaciones Sanitarias (00/936), Ministerio de Educación (Programa de Cooperación con Iberoamérica, PR25/95 and PR112/96) and the Comunidad Autónoma de Madrid (8.3/13/97) to J. R. R., and from the US PHS (ND# 223482) to R. A. G. We thank G. Fontán (Inmunología, Hospital La Paz) for the SPAT samples, and the Centro de Técnicas Inmunológicas (Universidad Complutense de Madrid), Philip Chen (Queensland Institute of Medical Research, Brisbane, Australia), Juan García Cabanillas (Inmunología, Facultad de Medicina, Universidad Complutense de Madrid) and J. Fuentes (Centro de Investigación Energética Medioambiental y Tecnológica, Madrid, Spain) for technical support and advice. Eduardo Martínez Naves critically reviewed the manuscript. Hoffmann-La Roche is gratefully acknowledged for continuous supply of rIL-2.


    Abbreviations
 
AT ataxia telangiectasia
ATM ataxia telangiectasia mutated
EBV Epstein–Barr virus
HVS Herpesvirus saimiri
PBL peripheral blood lymphocyte
PHA phytohemagglutinin
PMA phorbol myristate acetate
TNF tumor necrosis factor

    Notes
 
Transmitting editor: T. Saito

Received 22 November 1999, accepted 25 February 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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