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- Cancer Immunol Immunother
- v.52(11); 2003 Nov
- PMC11032921
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Cancer Immunol Immunother. 2003 Nov; 52(11): 708–714.
Published online 2003 Jun 26. doi:10.1007/s00262-003-0413-8
PMCID: PMC11032921
PMID: 12830324
A. P. Valeri,1 M. Pérez-Blas,1 A. Gutiérrez,2 M. López-Santalla,1 N. Aguilera,1 C. Rodríguez-Juan,1 L. Sala-Silveira,1 J. Martín,2 I. Lasa,2 J. M. Mugüerza,2 A. López,2 L. García-Sancho,2 J. Granell,2 and J. M. Martín-Villa
1
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Abstract
We have taken advantage of a recently described technique of transformation and immortalization of T lymphocytes using the lymphotropic Herpesvirus saimiri, to achieve long-lasting T-cell lines from gastric cancer patients and healthy volunteers. Blood samples were drawn and T lymphocytes were transformed. Once sustained growth was observed, lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. Cytofluorometric analysis revealed that CD3 and CD45 were found at lower proportion in primary cells from patients than from control individuals (54% vs 75%, p<0.001, 90% vs 96%, p<0.05, respectively), and in HVS-derived T-cell lines (90% vs 98%, p<0.05, 97% vs 100%, p<0.05, respectively). Proliferative analyses showed that primary isolated cells were unable to respond adequately to CD3-, CD2-, and PHA-mediated stimulation, as compared to controls. Similarly, T-cell lines from patients proliferated to a lesser extent when CD3- and CD2-mediated stimuli were considered, especially when simultaneous stimulation via CD3 and CD2 molecules was carried out (47,824 counts per minute [cpm] vs 121,478cpm, p<0.05). Altogether these results show that the defects reported in T cells from patients with cancer are not exclusively due to tumour-derived factors, since the alterations persist in long-lasting, HVS-transformed, T-cell lines, suggesting that this model seems a suitable one to disclose them.
Keywords: T Lymphocytes, Gastric adenocarcinoma, Herpesvirus saimiri, CD3, CD2
Introduction
Recent research carried out in cancer patients or animal models tries to delineate the delicate network established between cells of the immune system and tumour cells. The term "immune surveillance" [1] describes the ability of the immune system to detect tumour cells and destroy them. Yet, tumours are able to escape this control and expand, despite the expression of tumoural antigens on neoplastic cells. This progression might be due either to poor antigenicity of tumoural cells (absence of costimulatory molecules, loss of MHC molecules, antigenic modulation, tumour-induced immune suppression) or to a defective response of T lymphocytes from patients [2]. Alterations in the function of T lymphocytes, including dysfunctions in the cytokine production, inability to proliferate to antigenic stimuli and poor cytotoxic capacity, have been long described in patients with cancer and in tumour-bearing mice [3, 4, 5].
The aetiology of gastric cancer is complex, and among several risk factors, infection with H. pylori shows a close association with tumour appearance and progression. It has been proposed that upon initial infection with the pathogen, an inflammatory response follows that, finally, contributes to mucosal damage and to gastric cancer pathogenesis [6]. In this respect, it seems then that the cells of the immune system may be involved either in the pathogenesis, sustaining the inflammatory events [7], or in the dissemination, failing to mount a specific response to antigens expressed by newly arising neoplastic cells, of the tumoural process [8].
To finely dissect the functionality of T cells in patients, repetitive isolation from blood or tissue samples is required. This is a tedious and cumbersome task, especially if cancerous tissue samples, instead of peripheral blood, are the source from which T lymphocytes are to be purified. Alternatively, the use of long-lasting T-cell lines may provide a useful tool.
To circumvent the inherent difficulties of growing primary T cells, Herpesvirus saimiri (HVS), a common lymphotropic virus of squirrel monkeys, has been used to immortalize T lymphocytes. It is known that the virus is able to transform both CD4+ and CD8+ human T lymphocytes into stable growth by still undefined mechanisms [9]. HVS-transformed T lymphocytes remain IL-2–dependent but become antigen- and mitogen-independent for their continuous growth [10]. These cells display normal downstream functional responses (proliferation, cytokine synthesis, induction of activation markers, cytotoxicity) to membrane (anti-TcR-CD3, anti-CD95 ligand, and anti-IL-2) and transmembrane (phorbol myristate acetate, PMA, or calcium ionophores) stimuli [11]. Moreover, this methodology has been proven to successfully attain durable cell lines either from peripheral blood or tissue samples [12]. Following this methodology, long-lasting T-cell lines from blood samples of patients with gastric cancer and healthy volunteers were established. The phenotype and proliferative responses of these lines were compared with those of peripheral blood mononuclear cells (PBMC). Results obtained are reported in the present work.
Materials and methods
Preparation of blood samples
Samples were drawn at surgery of 16 adult patients (7 women, 9 men) with gastric adenocarcinoma. All the experiments were implemented with the approval of the Ethical Committee of the institution. Control population consisted of adult healthy volunteers (n=94). Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation with Lymphoprep (Axis Shield PoC AS, Oslo, Norway) and split in two aliquots. One of them was used to carry out a basic phenotypic study by cytometry and a functional study assessed by cell proliferation. Cells of the remaining aliquot were subjected to transformation with Herpesvirus saimiri (HVS).
Transformation of lymphocytes using HVS
The isolated lymphocytes were resuspended (1–3×106cells/ml) in a mixture (1:1 proportions) of two culture media: RPMI 1640 (Gibco BRL, Life Technologies, Paisley, UK) and Panserin 401 medium (Pan, Hamburg, Germany) with 10% FCS (Bio Whittaker, Verviers, Belgium) and 1% l-glutamine (Gibco BRL, Life Technologies), supplemented with 100U/ml recombinant human IL-2 (rhIL-2, TECIN, kindly provided by Hoffmann-La Roche, Nutley, NJ, USA) and seeded into 24-well microplates (Sarstedt, Newton, NC, USA).
Test cultures were exposed once (day0) to infectious doses of HVS subgroup C-488 strain. Control cultures were treated identically but HVS was omitted. The source of the infectious virus was supernatant from cultures of a lytically infected owl monkey kidney cell line (OMK). After infection, cells were regularly fed with medium containing 50U/mlrhIL-2. An immortalized phenotype was suggested by death of control cultures versus sustained growth and T-lymphoblast cell morphology of test cultures, as described [9]. Two months after inoculation of HVS, the cell lines were established with a stable morphology and surface phenotype. To provide hom*ogeneity to the results, and to ensure that differences detected could not be attributed to the age of the cell line, experiments herein shown were carried out with lines grown in culture for a period of time of between 6 and 9months.
Flow cytometry analysis
Cytofluorometric analysis of PBMC and HVS-derived T-cell lines was carried out using standard procedures. Cells (2×105) were incubated for 30min at 4°C using the following monoclonal antibodies: αCD2 (T11, Coulter, Miami, FL, USA), αCD3 (UCTH1, Immunotech, Marseille, France), αCD4 (13B8.2, Immunotech), αCD8 (B9.11, Immunotech), αCD16 (Leu-11a-NKP15, Becton Dickinson, San Jose, CA, USA), αCD19 (SJ25-C1, Caltag Laboratories, Burlingame, CA, USA), αCD25 (CD25–3G10, Caltag Laboratories), αCD28 (IOT28, Immunotech), αCD45 (J33, Immunotech), αCD45RA (ALB11, Immunotech), αCD45RO (UCHL-1, Becton Dickinson), αCD56 (N901-NKH-1, Immunotech), αCD80 (L307.4, Becton Dickinson), αCD86 (BU63, Caltag Laboratories), αCD103 (LF61, Caltag Laboratories), αHLA-DR (Immu-357, Immunotech), and αTcRαβ (WT31, Becton Dickinson). All antibodies were directly conjugated with fluorescein isothiocianate (FITC), phycoerythrin (PE), or cyanine 5-R-phycoerythrin (Cy5-PE). Negative control used was Opticlone (Immunotech), containing a mixture of mouse IgG1-FITC, IgG1-PE, and IgG1-Cy5-PE-conjugated monoclonal antibodies. Cells were washed twice with PBS (Gibco BRL, Life Technologies) and analysed in a Coulter EPICS Elite ESP flow cytometer (Coulter). A minimum of 5,000 cells was analysed per sample, gated to exclude nonviable cells. Fluorescence intensities above the upper limit of the negative control distribution were considered positive.
Proliferative assays
PBMC or HVS-derived cell lines were stimulated with monoclonal antibodies to CD3, CD2, or CD28, alone or in combination with other mitogenic substances such as IL-2, phorbol esters (PMA), or ionomycin. PBMC, additionally, were stimulated with phytohemagglutinin (PHA).
Cells were seeded (9×104 cells/well) in triplicate in a 96-well plate (Sarstedt) and stimulated 72 hours (PBMC) or 48 hours (T-cell lines), and then pulsed with 1-μCi 3H-thymidine (Moravek Biochemicals, Brea, CA, USA), left for a further 18h, and harvested to evaluate incorporation in cellular DNA in a beta counter (Matrix 96 TM, Packard, Canberra Company, Canberra, Australia). Results are expressed as mean counts per minute (cpm). The concentrations of the mitogens used are shown in Table1, and the stimuli applied to PBMC or cell lines, in Table2.
Table1.
Mitogens and concentrations used throughout the experiments
| Mitogen | Concentration |
|---|---|
| PHA (PHA-L, Sigma Aldrich, St Louis, MO, USA) | 5µg/ml |
| αCD3 (Orthoclon OKT3, Ortho Biotech Products, Raritan, NJ, USA) | 12.5ng/ml (soluble) |
| αCD3 (Orthoclon OKT3, Ortho Biotech Products, Raritan, NJ, USA) | 1µg/ml (plastic-bound) |
| αCD2 (T11 1/1, clone 6G4, and 2/1, clone 4B2) (CLB, Amsterdam, The Netherlands) | 2.5µg/ml |
| αCD28 (KOLT-2) (CLB, Amsterdam, The Netherlands) | 50ng/ml |
| PMA (Sigma Aldrich) | 10ng/ml when used alone, 1.2 ng/ml when combined with ionomycin |
| Ionomycin (Calbiochem, La Jolla, CA, USA) | 1µM |
| IL-2 (rhIL-2, TECIN, Hoffman-La Roche, Newton, NJ, USA) | 50U/ml |
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Table2.
Stimuli applied to PBMC or T-cell lines
| Stimulus | PBMC | T-cell lines |
|---|---|---|
| IL-2 | + | + |
| PMA | + | + |
| αCD3 (soluble) | + | − |
| αCD3 (plastic-bound) | − | + |
| αCD3+IL-2 | + | + |
| αCD3+PMA | + | + |
| αCD3+αCD28 | + | + |
| αCD3+αCD2 | − | + |
| αCD2 | + | + |
| αCD2+IL-2 | + | + |
| αCD2+PMA | + | + |
| αCD2+αCD28 | + | + |
| αCD28 | + | + |
| αCD28+PMA | + | + |
| Ionomycin | + | − |
| Ionomycin+PMA | + | + |
| PMA+IL-2 | + | + |
| PHA | + | − |
| PHA+IL-2 | + | − |
| PHA+PMA | + | − |
| PHA+αCD28 | + | − |
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Different conditions with regard to mitogen concentrations, number of cells per well (45,000 or 90,000) and length of stimulation (48h or 72h), were tested when setting up the culture experiments. The conditions finally chosen and used in the present work, were those that rendered the most consistent and repeatable results.
Statistical analysis
Results obtained (either in the phenotype or proliferative assays) are shown as mean value±standard error of the mean (SEM). Two-tailed Mann-Whitney two-sample test was used to compare results between cancer and volunteers group. Significance was reached when a p value less than 0.05 was obtained.
Results
Peripheral blood mononuclear cells
Phenotypic analysis
Differences were observed for CD3 (54% vs 75%, p<0.001), CD4 (37% vs 47%, p<0.05), CD8 (25% vs 31%, p<0.05), CD28 (38 % vs 51%, p<0.05), CD45 (90% vs 96%, p<0.05) and CD86 (10% vs 3%, p<0.05) when cancer patients were compared with healthy controls (Table3). Double-staining analysis carried out revealed that the CD3+CD4+ and CD3+CD8+ populations were under-represented in patients (34% vs 45%, p<0.01, and 15% vs 25%, p<0.001, respectively). CD4:CD8 ratio showed no difference between patients and control group (1.48 vs 1.51). The low percentage of CD3 and CD28 positive cells in our group of patients matches with the findings previously described in different types of carcinoma patients, though none of them had gastric carcinoma [8].
Table3.
Expression (%) of CD markers on the surface of freshly isolated PMBC from gastric cancer patients and healthy volunteer controls; n number of patients or volunteers tested. Only relevant results are shown. See text for a complete list of markers studied
| Surface marker | Cancer patients (mean±SEM) | Controls (mean±SEM) |
|---|---|---|
| CD3+ | 54±5 (n=16)*** | 75±2 (n=34) |
| CD4+ | 37±5 (n=15)* | 47±2 (n=33) |
| CD8+ | 25±3 (n=15)* | 31±1 (n=33) |
| CD28+ | 38±5 (n=16)* | 51±4 (n=29) |
| CD45+ | 90±3 (n=16)* | 96±1 (n=28) |
| CD86+ | 10±4 (n=14)* | 3±0 (n=24) |
| CD3+CD4+ | 34±5 (n=14)** | 45±2 (n=34) |
| CD3+CD8+ | 15±2 (n=15)*** | 25±1 (n=34) |
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*p<0.05; **p<0.01; ***p<0.001
When comparing mean fluorescence intensity (MFI) values, no discrepancies were observed in any of the markers tested in the present study.
Finally, the frequency of B cell, NK cell, or monocyte markers, as assessed with the monoclonal antibodies used, was equally distributed in both groups.
Proliferative assays
When the CD3 pathway was tested (Fig.1), defective responses were found in diseased individuals, and a significantly lower response was obtained upon stimulation with antibodies against the CD3 molecule (30,600 vs 42,417cpm, p<0.05). The CD2-mediated signalling pathway also revealed dysfunctions in the group of patients, as they responded poorly when antibodies to CD2 were used in combination with IL-2 (29,219 vs 51,183cpm, p<0.01) or PMA (48,232 vs 83,892cpm, p<0.01), or antibodies to CD28 (31,355 vs 39,144cpm, p<0.05). Finally, stimulation with PHA, whether alone (72,394 vs 87,092cpm, p<0.05) or in combination with IL-2 (81,577 vs 110,895cpm, p<0.05) or PMA (58,533 vs 91,308cpm, p<0.05), rendered fewer counts in cancer patients.
Fig.1.
Proliferative response in counts per minute (cpm) to mitogens of freshly isolated PBMC. Results are given as mean+SEM. Only relevant stimuli are shown. See text for a complete list of stimuli used; n number of patients and controls tested in each stimulus (*p<0.05, **p<0.01, ***p<0.001)
HVS-derived T-cell lines
Phenotypic analysis
All cell lines obtained, whether from patients or controls, were CD8 positive. A feature of HVS transformation is that either CD8 or CD4 cell lines are obtained, CD8 being the phenotype most frequently obtained. Previous PCR analysis of Vβ family genes carried out by our group, revealed that the HVS cell lines obtained were oligoclonal [12].
CD3 (90% vs 98%, p<0.05), CD45 (97% vs 100%, p<0.05), and HLA-DR (37% vs 69%, p<0.01) were found diminished in the lines from cancer patients (Table4). Thus, they maintain the low percentage of CD3 and CD45 expression found in PBMC.
Table4.
Expression (%) of CD markers on the surface of HVS-transformed T-cell lines from gastric cancer patients and healthy volunteer controls; n number of patients or volunteers tested. Only relevant results are shown. See text for a complete list of markers studied
| Surface marker | Cancer patients (mean±SEM) | Controls (mean±SEM) |
|---|---|---|
| CD3+ | 90±5 (n=7)* | 98±1 (n=15) |
| CD45+ | 97±2 (n=7)* | 100±0 (n=10) |
| HLA-DR+ | 37±6 (n=7)** | 69±6 (n=13) |
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*p<0.05; **p<0.01; ***p<0.001
Proliferative assays
Figure2 reveals that HVS-derived T cells from cancer patients showed, in general, defective responses in several membrane and transmembrane signalling pathways, although significance is only reached in a few instances, probably due to the size of the samples so far achieved. When monoclonal antibodies to CD3 plus CD2 were used as stimuli, cell lines from patients showed a significantly diminished proliferation when compared with control lines (47,824 vs 121,478cpm, p<0.05), a defect also found upon stimulation with antibodies to CD2 plus PMA (17,830 vs 46,238cpm, p<0.05), or with PMA alone (10,812 vs 24,454cpm, p<0.05).
Fig.2.
Proliferative response in counts per minute (cpm) to mitogens of HVS-transformed T-cell lines. Results are given as mean+SEM. See text for a complete list of stimuli used; n number of patients and controls tested in each stimulus (*p<0.05)
This indicates that alterations in different signalling pathways are present in the HVS-derived cell lines, making them an adequate tool to analyse the dysfunctions present in primary T cells from cancer patients.
Discussion
Peripheral blood mononuclear cells
Phenotypic analysis
The decrease in the percentage of CD3+ cells in peripheral blood is noteworthy and may reflect the existence of an altered T-cell population in cancer patients, a finding which might explain the lower proliferative response found upon stimulation through this pathway (see below). CD4+ and CD8+ cells are diminished in the diseased group, the CD3+CD8+ subpopulation being the most divergent when compared with control individuals (15% vs 25%, p<0.001). These results clearly indicate that T cells show an altered profile in patients, and suggests that the lack of adequate levels of CD4+ and CD8+ cells renders them unable to carry out an effective response (either helper or cytotoxic) against tumour cells. This is further stressed by the finding of a diminished CD28+ population in patients (see Table3). CD28 is a costimulatory molecule required for activation of T cells. If scarcely present on cells from diseased individuals, they would be less readily activated than cells from healthy individuals, hampering their ability to mount a response against tumour cells. In fact, it is known that gastric epithelial cells express B7.1 or B7.2 molecules, the cognate receptors of CD28 and required to induce activation signals in T lymphocytes. However, if the latter fail to express CD28, they would not establish adequate interactions with gastric cells, allowing tumour to progress freely. Concordant with our results, an increase in the CD8+CD28− population has been reported in patients with gastric cancer [2].
CD4:CD8 ratio shows no difference between the patients and control group. This is at odds with some published data on gastric cancer which refer either to a decreased [13] or increased [14] value. These discrepancies may reflect the heterogeneity of the patients studied in each cohort. Carefully selected groups of patients, matched according to clinical stage, are required to achieve consistent results.
Proliferative assays
Upon stimulation with mitogenic substances at the membrane (αCD3, αCD2, IL-2, or PHA) level, PBMC from cancer individuals showed clear defective responses. Engagement of the T-cell antigen receptor (TcR) triggers signal transduction pathways that regulate the activation of T lymphocytes. This process depends upon the activity of protein tyrosine kinases (PTKs), which induce the phosphorylation of a number of proteins. PTKs mediating those functions include the Src family members, Lck and Fyn, and the Syk family members, ZAP 70 and Syk [15].
Such activation processes may take place whenever a T lymphocyte encounters a tumoural antigen, a step tending toward the elimination of neoplastic cells. However, and according to our results, this ability may be hampered in patients with gastric adenocarcinoma. Several membrane stimuli used (see Fig.1) were unable to induce proliferation at the levels achieved with control cells. Some of the PTKs mentioned earlier, such as Lck, contribute to signalling by the membrane molecules stimulated, locating the defect early in the signal transduction pathway. Interestingly, several reports have shown a defective Lck function in PBMC from patients with gastric adenocarcinoma [3, 4, 5], a finding that matches our present data. Moreover, a decreased expression of the TCRζ subunit of the TCR complex, has been found in T lymphocytes from patients with gastric cancer [16]. This decreased expression correlates with reduced proliferative responses after antigenic challenge.
The suggestion that a tumour-derived factor [17] is the only one responsible for the defect observed in patients, is not sustainable according to our results. It is not clear that a single tumour-derived factor might be able to simultaneously alter several membrane-based stimuli, such as those mediated by CD3, CD2, PHA, or IL-2. Finally, the fact that HVS-derived T-cell lines, grown in vitro for several months and thus without the influence of any tumour-derived factor, maintain the low CD3 expression present in PBMC (see Table2), and also poor proliferative responses (see below), soundly discards the effect of any tumour-derived factor as the exclusive causing agent.
Increased apoptosis rates have been described in T cells from patients with gastric [16] or head and neck [18] cancer, and it may provide an alternative explanation for the proliferative defects in our patients. However, the fact that HVS-derived T-cell lines from patients revealed comparable growth rates to that of controls, and yet lower responses when stimulated, strongly supports defective signalling pathways as the major explanation.
HVS-derived T-cell lines
Phenotypic analysis
HVS-transformed T lymphocytes show differences in the expression of some surface markers, also found in primary cells. CD3 and CD45 appear in fewer cells in patients than in healthy individuals (90% vs 98%, p<0.05, and 97% vs 100%, p<0.05). The implications of this finding are two-fold. On the one hand, it shows that the HVS model is a suitable one to study the phenotypic defects found in the PBMC of patients. On the other, it indicates that this defect (low CD3 and CD45 expression) is inherent to the patient's cells, rather than induced by a tumour-secreted factor. Recently published work revealed that CD3 expression was lost on T cells from cancer patients, due to a 14 kD factor secreted by tumoural cells [16]. If this were the case, the CD3 expression would appear at normal levels in our T-cell lines, some of which were grown for more than 8months. Evidently, any tumour-derived factor would run out of the culture media we used in such a long time, and would no longer exert such a suppressive effect.
It is presently unclear what the CD3− population does represent. Assuming that they are T lymphocytes (nonlymphoid cells such as epithelial cells or fibroblasts are clearly discarded), these may represent either cells expressing an unusual form of the CD3 complex not readily recognized by the antibody used, or cells in which the complex is not exported to the surface.
Some other markers, whose expression was found altered in fresh cells, show no differences in HVS lines. This is not striking since it is known that upon transformation cells may up- or down-regulate the expression of some markers [12].
Proliferative assays
Stimulation through CD3 or CD2 molecules always yielded fewer counts in patient-derived cell lines (Fig.2), although significance was only reached in some instances, probably due to sample size.
The CD2-based defect in cell lines is intriguing. It is known that transformation with HVS activates the Lck tyrosine kinase by binding of the Tip protein of the virus, and antigen-independent growth of the lines is mediated by CD2 interaction with its cognate ligand CD58 on the cell's surface [19]. Yet, cancer cells respond poorly and either viral-induced Lck activity is not adequate, or another signalling kinase is altered in patients. In this sense, our model is useful for dissecting the CD2 alterations found in PBMC. Thus, a significant lower response is observed in cancer lines when stimulated with antibodies to CD2 in combination with PMA (17,830 vs 46,238cpm), a combination that also produced an impaired response in primary cells from cancer patients (see Fig.1). Likewise, when the CD3 and CD2 signalling pathways are analysed, transformed cells from patients show lower responses than control-derived ones (see Fig.2), in keeping with results of PBMC, although significance is not reached in all instances.
The fact that costimulation via CD3 and CD2 shows significantly lower counts in patients, suggests that either the defective CD3 or CD2 pathways are unable to render diseased cells to a normal state upon costimulation or, alternatively, that assembly of an as-yet-unidentified functional signalling complex, specific for CD2 and CD3 costimulation, fails to take place in lines from patients but not from healthy volunteers. Biochemical studies are required to confirm this hypothesis. CD3 and CD2-mediated costimulation in primary cells produced lower counts than when any of the two stimuli were considered independently (data not shown), as if an apoptotic phenomenon took place.
In summary, we show here the establishment of long-lasting T-cell lines of blood origin from gastric cancer patients, taking advantage of the transformation process with a lymphotropic virus, the HVS. Such cells maintain phenotypic (low CD3 and CD45 expression) and functional (altered CD3- and CD2-mediated signalling) dysfunctions also found in freshly isolated cells. The fact that the cell lines maintain these defects after several months in culture, indicates that they are not exclusively due to a tumour-derived factor and suggests the existence of an intrinsic alteration in the lymphocytes of patients. This alteration would allow tumour progression and dissemination.
The model herein shown can be exploited to obtain cells lines derived from gastric tumoural tissue which, once fully characterized, may be used to carry out immunotherapy assays tending to increase the T-cell response against tumoural antigens. Preliminary data obtained in our laboratory with such lines reveal that a deep CD3- and CD2-mediated stimulation defect is present.
Acknowledgements
This work was supported by a FIS grant (99/0999). We thank the Centro de Técnicas Inmunológicas (Universidad Complutense de Madrid) for technical support, Pilar Lucea, Paloma del Pico, and Genoveva Vallejo for help in the shipment of samples, and Ángeles Mencía for help in growing cell lines.
Footnotes
APV and MP-B should be considered as joint first authors
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