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Spontaneous ex Vivo Apoptosis of Peripheral Blood Mononuclear Cells in Patients with Head and Neck Cancer¹

University of Pittsburgh Cancer Institute [T. S., I. K., G. D., W. G., J. T. J., T. L. W.] and Departments of Pathology [T. L. W.] and Otolaryngology [J. T. J., T. L. W.], University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

	ABSTRACT

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Proportions of apoptotic (TUNEL⁺) peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry in patients with head and neck cancer and normal controls at the time of blood draws (0 time) and after 24-h incubation. PBMCs were incubated at 37°C in medium (spontaneous apoptosis) and in the presence of CH-11 antibody (anti-Fas) or tumor necrosis factor (TNF)-, both capable of inducing DNA fragmentation in activated T cells expressing the TNF family of receptors. PBMCs obtained from the patients had significantly higher (P < 0.0001) proportion of apoptotic cells than PBMCs of controls at 0 time as well as after 24-h incubation. Ex vivo apoptosis included all subsets of PBMCs: CD3⁺ T cells, CD16⁺CD56⁺ natural killer cells, CD19⁺ B cells, and CD14⁺ monocytes, as determined by two-color flow cytometry. However, T cells represented the largest PBMC subset undergoing apoptosis, and lymphocytes rather than monocytes were the major TUNEL⁺ PBMC population. Among T cells, the level of spontaneous ex vivo apoptosis was nearly as high as that of CH-11 antibody-induced or TNF--induced apoptosis, indicating that activated Fas⁺ and TNFR1⁺ T cells were preprogrammed in vivo to die. Also, elevated levels of spontaneous apoptosis at time 0 in patients with head and neck cancer (P < 0.0001) indicated that a higher fraction of PBMCs was undergoing apoptosis in vivo in patients than controls. Together, the data suggest that an increased rate of turnover of lymphocytes is associated with cancer and may be responsible for functional lymphocyte imbalance, even in treated patients who have no evident disease.

	Introduction

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The tumor microenvironment may influence the functional potential of immune cells that accumulate at the site of tumor growth or metastasis. Evidence has accumulated that TILs³ isolated from human tumors and tested for phenotypic and functional characteristics express activation markers (1, 2, 3) and yet are functionally impaired (1, 2, 3, 4, 5, 6) . In addition to impairments in cell proliferation in response to mitogens as well as tumor-associated antigens, fresh TILs or tumor-associated lymphocytes are poorly cytotoxic against tumor targets, have an altered cytokine profile, including deficiencies in interleukin 2 and IFN- and do not signal normally upon T-cell receptor engagement (1 , 2 , 7, 8, 9, 10, 11, 12) . More recent data from our and other laboratories indicate that lymphocytes undergo apoptosis at the tumor site (5 , 13, 14, 15, 16) . The ability of human tumors to induce apoptosis in activated normal lymphocytes has been confirmed by in vitro experiments in which fresh or cultured tumor cells were shown to induce caspase activation and DNA fragmentation in lymphocytes coincubated with fresh or cultured tumor cells for 24 h or less in the absence of any other apoptosis-inducing agent (13) .

Immune effector cells obtained from the peripheral blood of patients with cancer, including HNC, have also been reported to have a variety of functional abnormalities, which may vary in magnitude from patient to patient and which may be related to the extent of disease (1 , 5 , 9 , 17) . Thus, it appears that immunoinhibitory influence of the tumor might extend far beyond the tumor microenvironment and might become a systemic effect, especially in patients with advanced metastatic disease. The possibility that immune cells may be actively dying in patients with cancer and that a rapid turnover of these cells occurs as a consequence of this process has profound implications for the development of adequate antitumor immune responses and for immunotherapy of cancer. Clearly, the ability to assess the level of this turnover might be viewed as an important component of or indicator for therapeutic interventions aimed at the protection of immune cells from apoptosis.

We have observed recently that mononuclear cells obtained from the peripheral blood of patients with HNC spontaneously undergo DNA cleavage upon their incubation in culture medium for several hours. In contrast, PBMCs of normal individuals have very low levels of ex vivo apoptosis under the same experimental conditions. This observation has prompted us to investigate the nature of this ex vivo phenomenon and to provide a rationale and evidence for the hypothesis that a substantial number of mononuclear cells in the peripheral circulation of patients with HNC are preprogrammed in vivo to undergo apoptosis.

	Materials and Methods

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Patients and Controls.
Twenty-two patients with histologically proven SCCHN, who were consecutively seen at the University of Pittsburgh Medical Center (Pittsburgh, PA) between January 1998 and June 1998, were included in this study. All patients signed the informed consent. The patients underwent surgery and, when indicated, radio- or chemotherapy for their tumors at various time periods (1 month to 17 years; median, 28 months) prior to the visit during which they donated peripheral blood for the study. At the time of blood draws, all of the patients had NED. There were eight patients with stage I, four with stage II, four with stage III, and six with stage IV disease. Comorbidity was retrospectively graded according to the method of Kaplin-Finestein. No patients had grade 2 or 3 comorbidity, and no patient was on ß blockers or calcium channel inhibitors. One patient was taking oral prednisone. Table 1 lists patient characteristics, including sex (16 males and 6 females) and age (median, 65 years), tumor site and stage, the presence and number of recurrent or second primary tumors, and clinical status of at the time of a blood donation for this study.

PBMCs.
Venous blood was obtained from patients and controls (20 mls) in the morning and collected into heparinized tubes. Blood samples were hand carried to the laboratory and processed by Ficoll-Hypaque gradient centrifugation. PBMCs were recovered from the gradient interface, washed in DPBS, counted in trypan blue, and immediately used for experiments.

The experimental design for ex vivo apoptosis of PBMCs is outlined in Fig. 1 . To determine the proportion of PBMCs with evidence of DNA fragmentation, a TUNEL assay (see below) was performed on an aliquot (3.5 x10⁶ cells) of patients’ or control cells immediately after their isolation (i.e., at time 0). The remaining PBMCs were divided into aliquots (3.5 x 10⁶ cells), and each aliquot was incubated for 24 h at 37°C under the following conditions: (a) medium alone; (b) medium plus CH-11 Ab (agonistic anti-Fas Ab; UBI Biotechnology, Lake Placid, NY) at the concentration of 400 ng/ml; and (c) medium plus recombinant TNF- (Knoll Pharmaceuticals, Whippany, NJ) used at the concentration of 50 ng/ml. After incubation, cells were harvested and immediately tested in TUNEL assays for evidence of apoptosis. The proportions of PBMCs that underwent spontaneous apoptosis (medium alone) or induced apoptosis (medium plus CH-11 Ab or medium plus TNF-) during the 24-h incubation period were determined by flow cytometry. The medium used for all experiments was RPMI 1640; it was supplemented with 1 mML-glutamine, 10% (v/v) of FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin. All media reagents were purchased from Life Technologies, Inc. (Grand Island, NY).

View larger version (14K): [in this window] [in a new window]   Fig. 1. A diagram of the experimental design used to study ex vivo spontaneous and induced apoptosis of PBMCs obtained from patients with HNC.

TUNEL Assay.
The TUNEL assay was performed to study DNA fragmentation in freshly harvested or ex vivo-incubated cells. PBMCs were washed after fixation with paraformaldehyde and permeabilized with 0.1% (w/v) sodium citrate in phosphate saline buffer containing 0.1% (v/v) Triton X-100 for 3 min on ice. After washing, cells were incubated with FITC-conjugated dUTP in the presence of terminal deoxynucleotidyl transferase enzyme solution for 1 h at 37°C, using reagents purchased from Boehringer Mannheim Corp. (Indianapolis, IN). After incubation, the cells were washed, and 10,000 events were acquired and analyzed by two-color analysis, using FACScan. Controls included cells incubated without the enzyme in labeling buffer. To determine the assay reliability, intra- and interassay correlations were established based on blood samples collected from six normal individuals on different days and tested after splitting each sample into three aliquots. The analysis of this data set indicated that the assay was highly reliable, with the interassay correlation of 0.909.

Flow Cytometry.
Two-color flow cytometry analysis was performed on a FACScan (Becton Dickinson) equipped with a single 488-nm argon ion laser. To reliably detect small subpopulations of cells, at least 10,000 events were acquired for each sample. The following settings were used: 640 and 615 V on photomultiplier tubes for FL1 (FITC-TUNEL) and FL2 (PE), respectively. FITC and PE fluorescence were measured through 530/30 and 585/42 bandpass filters. Compensation for FL1-% FL2 and FL2-%FL1 was 0.7% and 29%, respectively. Threshold was set on 30 for forward scatter.

The percentages of apoptotic cells were calculated by scoring TUNEL⁺ cells in two-color gated subpopulations: CD3⁺/TUNEL⁺, CD3^-/TUNEL⁺ in comparison to CD3⁺/TUNEL^-, and CD3^-/TUNEL^-. All of the gated mononuclear cell subpopulations were visualized on FCS/SSC dot-plots. To include all apoptotic cells and avoid debris with a high SSC signal, the gate was set to include a wide boundary of mononuclear cells ("open gate"), because apoptotic cells accumulated mainly in the lower FSC/SSC channels (18) . The strategy of double gating eliminated a majority of debris, which usually overlaps with apoptotic cells. The level of debris within the gated populations did not exceed 10%, and it was not significantly higher in samples with high than low (control) apoptotic values. MFI was determined for all gated cell populations.

The percentages or MFI of Fas⁺ or TNFR1⁺ PBMCs were determined after staining of the cells with anti-Fas Ab (ZB4; Upstate Technology, Lake Placid, NY) or with Ab to TNFR1 (R&D Systems, Minneapolis, MN). For these determinations, we individually gated on lymphocytes and monocytes, based on the FCS/SSC dot plots.

Cytospin Preparation.
PBMCs obtained from patients and normal control samples were doubly stained for TUNEL and CD3 surface marker, as described above. Stained cells were centrifuged onto glass slides, using a cytocentrifuge (Cytospin 2; Shandon) and directly mounted with fluorescent mounting medium (Dako). The slides were examined independently by two observers, using a fluorescent microscope (Microphot-FX; Nikon), and photographs of representative fields were taken, using appropriate filters.

Data Analysis and Statistics.
The probability values for differences among the TUNEL⁺ cell population between the normal volunteers with no known cancer and SCCHN patients were calculated by Mann-Whitney U test using StatView software version 4.54 (Abacus Concepts, Inc. Berkley, CA). Twenty-four-h assays were compared by subtracting least squares means derived from a mixed linear model for percentage of TUNEL⁺ cells. The model independent variables were group (patient or control), assay time 0, 24-h spontaneous, 24-h CH-11 Ab, 24-h TNF-, and group by time interactions. Differences in least squares means were tested for significance, using a pooled within-person estimate of SE.

	Results

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Spontaneous and Induced Apoptosis in Patients’ PBMCs.
When PBMCs obtained from patients and normal controls were examined at the time of blood draws (time 0) for the presence of cells with the evidence of DNA fragmentation (i.e., TUNEL⁺ cells), only a very low proportion of PBMCs was found to be TUNEL⁺ (Table 2 ). Nevertheless, even at time 0, a small but significantly higher proportion of PBMCs were TUNEL⁺ in patients (mean ± SD, 4 ± 2%) than in controls (mean ± SD, 1 ± 0.5%). After 24-h incubation of patients’ PBMCs in medium alone at 37°C, a significantly higher proportion (P < 0.0001) of these cells became TUNEL⁺. As shown in Table 2 , the proportion of total TUNEL⁺ PBMCs with spontaneous apoptosis after 24-h incubation ranged from 9 to 36% in patients with SCCHN, and it was significantly higher (P < 0.0001) than that in PBMCs obtained from normal controls and tested in parallel with patients’ cells in the same assays. It was also significantly higher than spontaneous apoptosis measured among PBMCs of age-matched controls (data not shown). In these and all other experiments, the data obtained for age-matched controls were not significantly different from those obtained for laboratory controls.

The data shown in Fig. 2 indicate that after 24 h of incubation in medium or medium supplemented with anti-Fas CH-11 Ab or TNF-, the patients’ PBMCs contained a significantly higher proportion (P < 0.0001) of TUNEL⁺ cells than control PBMCs. The PBMCs susceptible to agonistic CH-11 Ab or to TNF- (presumably, cells expressing Fas or TNFR1, respectively) were significantly more numerous among PBMCs of patients with SCCHN than of controls (Fig. 2) . The mean interassay differences among percentages of apoptotic PBMCs are listed in Table 3 . Despite closeness of the means for results of 24-h assays among controls as well as patient cells and a high degree of correlation found for these assays in individual subjects, statistical analysis indicated significant differences (Table 3) .

View larger version (34K): [in this window] [in a new window]   Fig. 2. Percentages of TUNEL+ PBMCs detected by flow cytometry at time 0 or after 24-h incubation of PBMCs obtained from patients with SCCHN or normal controls in the presence of medium (MED), CH-11 Ab, or TNF-. Horizontal bars, means. *, Ps for differences between patients and controls.

Spontaneous or Induced Apoptosis of CD3⁺ T Cells.
To determine the fate of T cells after 24-h incubation of PBMCs with medium, CH-11 Ab, or TNF-, we also measured the percentage of TUNEL⁺ cells among CD3⁺ lymphocytes in patients and normal controls (Fig. 3) . Among CD3⁺ lymphocytes, a significantly higher proportion of cells was TUNEL⁺ in patients than in controls, and this was observed for spontaneous apoptosis as well as apoptosis induced by incubation of these cells with CH-11 Ab or TNF- for 24 h (Fig. 3) . A high degree of correlation was observed between the three assays for apoptosis in the individual control or patients’ cells incubated with medium, CH-11 Ab, or TNF- (Fig. 4) . In aggregate, however, CH-11 Ab or TNF- induced a somewhat higher proportion (borderline significance) of T cells to undergo apoptosis in control PBMCs than that observed after lymphocyte incubation with medium alone. In the patients’ PBMCs, both CH-11 Ab and TNF- induced significantly higher (P < 0.0001 and 0.0004, respectively) apoptosis in CD3⁺ T cells than medium alone (Table 4) . These observations suggest that the proportion of all T cells expressing Fas or TNFR and undergoing ex vivo apoptosis is significantly higher in patients with SCCHN than in controls.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Percentages of TUNEL+ cells among CD3+ T lymphocytes for all patients and controls were determined by two-color flow cytometry at the time of blood draw (time 0) or after 24-h incubation in the presence of medium, CH-11 Ab, or TNF-. Horizontal bars, means. *, Ps for differences between patients and controls.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Scatterplot matrix showing correlations between the three assays for apoptosis among CD3+ cells performed after 24-h incubation of the patients’ PBMCs in medium +CH-11 Ab or +TNF-. In each rectangle, percentages of CD3+/TUNEL+ cells detected in a pair of assays are plotted. The straight lines are the fit from a linear regression model. All correlations were 0.96 or greater (P < 0.0001).

Flow Cytometry Analysis of CD3⁺/TUNEL⁺ Lymphocytes.
To quantitate the proportions of CD3⁺/TUNEL⁺ T lymphocytes among patients’ and normal PBMCs, two-color flow cytometry was performed (Fig. 5) . An open gate was used, and FSC versus SSC indicated the presence of a heterogeneous population of TUNEL⁺ cells (Fig. 5 , green).

View larger version (53K): [in this window] [in a new window]   Fig. 5. Representative flow cytometry data for a patient (A and B) and a control (C and D) illustrating the wide gate used (A and C) and the proportion of TUNEL+ cells (green in B and D) among the CD3+ (red) or CD3- subsets.

As shown in Fig. 6 , a considerable proportion of CD3⁺ cells in the patients’ PBMCs were TUNEL⁺ (Fig. 6A , blue). This fraction contained cells with lowest FSC (red) and higher FSC (Fig. 6B , green). The red cells were strongly TUNEL⁺ and had the weakest expression of CD3, as indicated by their fluorescence intensity (Fig. 6C) . The CD3^- fraction also contained strongly TUNEL⁺ cells (Fig. 6C) . In addition, when we examined PBMCs undergoing ex vivo spontaneous apoptosis in medium at 2, 6, and 24 h of incubation by two-color (CD3⁺/TUNEL⁺) flow cytometry, it became apparent that the fraction of weakly CD3⁺ but strongly TUNEL⁺ T cells was increasing with the incubation time (data not shown). This experiment indicated that T cells gradually lose CD3 expression in the course of ex vivo incubation. Surface density of CD3 expression decreases (hence, the appearance of cells that are weakly CD3⁺ or CD3^- and still TUNEL^-). By FSC, it was possible to identify a subset of small cells, which were most intensely TUNEL⁺ (Fig. 6, B and C , red) and which did not express CD3 or were weakly CD3⁺. These cells most likely represent a fraction of T cells at the fairly advanced stage of apoptosis that have lost surface markers, resulting in the increased proportion of CD3^- TUNEL⁺ cells at 24 h of incubation (Fig. 6C) .

View larger version (48K): [in this window] [in a new window]   Fig. 6. Representative flow cytometry data showing a bimodal distribution of TUNEL+ cells (from weak to strong intensity) in A. The strongly TUNEL+ cells (red in B and C) have lower FSC than weakly stained TUNEL+ cells (green in C). In C, strongly TUNEL+ (red) cells are present both in the CD3+ and CD3- subsets. Note that within the CD3+ subset, red cells express less CD3 surface antigen than green cells (C). Cytocentrifuge smears of PBMCs obtained from a control in D and a patient in E. The cells were incubated in medium for 24 h and then stained with PE-conjugated anti-CD3 Ab and TUNEL reagents to visualize apoptotic T cells (green and red), nonapoptotic T cells (red), and apoptotic CD3- mononuclear cells (green or yellow). Note a lower frequency of apoptotic cells in E relative to panel D.

Flow Cytometry Analysis of CD3^- TUNEL⁺ Cells.
Because we observed that DNA fragmentation occurred not only in CD3⁺ T cells but also in PBMCs that were CD3^-, two-color flow cytometry was used to discriminate between these two populations and to identify the CD3^- mononuclear cell subsets, which become TUNEL⁺ during 24-h incubation of PBMCs in medium alone. The data shown in Table 5 indicated that TUNEL⁺ cells were detected in the CD16⁺CD56⁺ (NK) cell, CD19⁺ (B) cell, and CD14⁺ (monocyte) cell populations. Thus, TUNEL⁺ cells were present among each of the subsets of PBMCs. The proportions of TUNEL⁺ among all of the PBMC subset cells were higher in patients than the accompanying controls. Together, the CD3^- cell subpopulations accounted for 60%, and CD3⁺ T cells accounted for the other 40% of TUNEL⁺ cells. No preferential spontaneous or induced apoptosis was observed in any of the PBMC subsets in patients or controls. However, lymphocytes were the major PBMC subset undergoing apoptosis ex vivo.

	Discussion

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A characteristic feature of ex vivo apoptosis we observed was that it included all subsets of PBMCs: T, NK, and B cells as well as monocytes. Lymphocytes accounted for a great majority of apoptotic cells. We initially focused on CD3⁺ T cells as a target for apoptosis, because of the previous data indicating that: (a) a substantial proportion of T lymphocytes present at the tumor site showed evidence of apoptosis (5 , 13) ; (b) functional impairments were demonstrable in TILs as well as circulating T cells in patients with HNC and other cancers (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) ; (c) decreased expression of the and chains associated with TcR was linked to functional defects and perhaps apoptosis in CD3⁺ TILs or PBMCs of patients with cancer (1 , 5 , 7, 8, 9, 10, 11, 12 , 20) ; and (d) coincubation of activated T cells with tumor cells induced the apoptotic pathway in these T cells, culminating in their death (13) . Cumulatively, these data suggested that T lymphocytes, especially those accumulating at the tumor site, were activated in patients with cancer (1, 2, 3) and were thus susceptible to AICD or TICD or both (3 , 13) . However, our present data indicate that in addition to CD3⁺ T cells, other subsets of PBMCs were undergoing apoptosis at a significantly higher level than that observed in PBMCs of normal controls. Thus, apoptosis was observed within all of the mononuclear cell subsets examined in this study.

The second interesting characteristic of the process of ex vivo apoptosis was that the level of spontaneous apoptosis at 24 h was nearly as high as that induced by agonistic anti-Fas Abs (CH-11) or by exogenous TNF-. This finding suggested that most of PBMCs expressing Fas or TNFR1 were undergoing spontaneous apoptosis. Furthermore, the level of CH-11 Ab-induced apoptosis was higher than that of spontaneous apoptosis, indicating that not all Fas⁺ T cells had died upon incubation with medium alone. In all cases, the proportion of PBMCs sensitive to Fas or TNFR1 engagement was significantly higher in patients with HNC than in normal controls. Also, the actual percentages and/or MFI of Fas+ or TNFR1+ cells were found to be significantly higher in PBMCs of the patients than of controls. However, no attempts were made in this study to correlate expression of these receptors with the level of apoptosis. Nevertheless, these data suggest that more PBMCs in patients than in controls were activated and thus presumably sensitive to AICD. However, AICD by definition involves TcR-mediated signaling (21 , 22) , whereas in our study, non-T cells constituted a considerable proportion of PBMCs undergoing apoptosis (Table 5) . Therefore, a possibility of FasL-mediated or TNF--mediated cell death not involving the TcR pathway has to be considered as a mechanism responsible in vivo for apoptosis of PBMCs in patients, and to a much smaller extent, in normal individuals. The question remains as to why PBMCs expressing Fas or TNFR1 spontaneously undergo apoptosis ex vivo. We suggest that these PBMCs are preprogrammed in vivo to die, and that the proportion of such preprogrammed PBMCs is significantly higher in patients than in controls.

The third aspect of our studies that, in part, helps to explain spontaneous ex vivo apoptosis concerns the proportion of TUNEL⁺ PBMCs at time 0, i.e., at the time of blood draws. Spontaneous apoptosis was observed at that time in a small but significantly higher proportion of PBMCs in patients than in controls (4% versus 1% in Fig. 2 ). Because in vivo apoptotic PBMCs are cleared rapidly from the circulation by the reticuloendothelial system, this observation suggests that: (a) a higher fraction of PBMCs is undergoing apoptosis in vivo in patients than in controls; and (b) a more rapid turnover of PBMCs must be occurring in patients than in controls. This means that PBMCs are dying and are replaced at a much higher rate in patients than in controls. If this speculation were correct, it could explain why lymphocytes are often found to be functionally impaired in the circulation of patients with cancer (7, 8, 9, 10, 11, 12) . In particular, our earlier results, indicating that the proportion of lymphocytes with low or absent expression of the chain is significantly increased in the circulation of patients with HNC, could be explained by the rapid rate of apoptosis of such cells (1) . Our in vitro and in situ data have indicated that a link might exist between the chain degradation and apoptosis in cancer (23 , 24) , and more recent results indicate that protein contains caspase-sensitive amino acid motifs.⁴ Taken together, the findings of decreased expression of in T and NK cells in patients with HNC (5 , 23 , 24) and other cancers (6 , 8 , 11 , 9 , 17 , 25, 26, 27, 28) , functional defects observed in lymphocytes (1 , 20) , and the increased rate of PBMC turnover might all represent events related to tumor-induced and/or activation-induced apoptosis.

The distinction between the two mechanisms of apoptosis (AICD versus TICD) operative in patients with cancer is not clear at present. It is highly likely that the presence of tumor or even of occult metastases induces activation not only of TILs but also of a fraction of PBMCs and thus predisposes these cells to AICD. On the other hand, we and others have provided evidence that human tumors, including HNC, can directly induce apoptosis in activated lymphocytes via the Fas-FasL, TRAILR-TRAIL, or other tumor-derived signals (13 , 29) . These mechanisms of TICD are under extensive examination in our laboratory.⁵

The biological and clinical significance of the findings reported here is of considerable interest. Certainly, not all patients with HNC show elevated spontaneous apoptosis ex vivo. Thus, as illustrated in Fig. 2 , a broad range of values for spontaneous or induced ex vivo apoptosis can be observed in patients relative to a narrow range in controls. On the basis of the proportions of TUNEL⁺ cells among CD3⁺ T lymphocytes, the patients with HNC can be divided into two groups, one which is similar to normal controls (i.e., with TUNEL⁺/CD3⁺ cells <9% which is less than the normal mean + 2SD) and second, which is significantly higher than normal controls (i.e., with TUNEL⁺/CD3⁺ cells >9%), as shown in Table 4 . However, we could not discern any apparent clinical differences between the two groups, except that patients with a lower percentage of apoptotic T cells in PBMCs tended to have well-differentiated tumors.

We could not detect an association between signaling defects present in T cells at time 0 (e.g., decreased chain expression) and levels of spontaneous or induced apoptosis of these cells after 24-h incubation (data not shown). We expected to find such association based on the new data in our laboratory, suggesting a link between abnormalities in T lymphocytes and apoptosis (13) .⁶ Finally, the extent of ex vivo spontaneous apoptosis in SCCHN patients with NED is surprising. It suggests that tumor-induced effects on the hematopoietic system are long lived. It is possible that in SCCHN patients with large tumor burden, i.e., in patients studied before surgery, ex vivo apoptosis might be even more pronounced. When confirmed and extended, this finding could provide a basis for estimating the degree of immunoincompetence in patients with SCCHN and other cancers. The presence of extensive ex vivo apoptosis of T lymphocytes in HIV has been well documented in the literature (30, 31, 32) , suggesting that in infections as well as cancer, circulating activated lymphocytes are preprogrammed to die. The mechanisms responsible for this phenomenon remain to be elucidated.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by NIH Grant PO1-DE 12321 (to T. L. W.).

² To whom requests for reprints should be addressed, at University of Pittsburgh Cancer Institute, W1041 BST, 211 Lothrop Street, Pittsburgh, PA 15213-2582. Phone: (412) 624-0080; Fax: (412) 624-0264; E-mail: whitesidetl{at}msx.upmc.edu

³ The abbreviations used are: TIL, tumor-infiltrating lymphocyte; HNC, head and neck cancer; PBMC, peripheral blood mononuclear cell; SCCHN, squamous cell carcinoma of the head and neck; NED, no evident disease; DPBS, Dulbecco’s PBS; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; Ab, antibody; TNF, tumor necrosis factor; TNFR, TNF receptor; PE, phycoerythrin; FSC, forward angle scatter; SSC, side angle scatter; MFI, mean fluorescence intensity; NK, natural killer; TcR, T-cell receptor; AICD, activation-induced cell death; TICD, tumor-induced cell death.

⁴ H. Rabinowich, unpublished data.

⁵ B. R. Gastman, D. E. Johnson, T. L. Whiteside, and H. Rabinowich. Activation of caspase-3 cleavage of Bcl-2 in tumor-induced apoptosis of T lymphocytes, submitted for publication.

⁶ H. Rabinowich, personal communication.

Received 10/12/98; revised 2/25/99; accepted 3/ 2/99.

	REFERENCES

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