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Clinical Cancer Research Vol. 5, 329-334, February 1999
© 1999 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Clinical Significance of Decreased {zeta} Chain Expression in Peripheral Blood Lymphocytes of Patients with Head and Neck Cancer1

Iris Kuss, Takao Saito, Jonas T. Johnson and Theresa L. Whiteside2

University of Pittsburgh Cancer Institute [I. K., T. S., 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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Patients with squamous cell carcinoma of the head and neck (SCCHN) frequently have impaired immune responses. Alterations in T-cell receptor-associated signaling molecules in tumor-infiltrating as well as circulating lymphocytes have been reported in these patients. Using quantitative flow cytometry analysis, we have demonstrated that expression of the {zeta} chain is significantly decreased relative to normal controls in both CD8+ and CD4+ T cells as well as CD3-CD56+CD16+ natural killer cells in the peripheral blood of patients with SCCHN who, as a result of previous therapies, have no evident disease. Patients with a more aggressive type of SCCHN and those who experienced a recurrence or had a second primary cancer within the last 2 years of the study had the lowest {zeta} chain expression. In addition, SCCHN patients showed a significantly greater spontaneous ex vivo apoptosis, as measured by a terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay, in PBMCs, compared to normal controls. The observed decreased expression of {zeta} in T and natural killer cells coincided but did not directly correlate with significantly increased spontaneous apoptosis of lymphocytes obtained from treated patients with no evident disease. The results suggest that in patients with SCCHN, {zeta} chain defects and lymphocyte apoptosis are manifestations of long-lasting negative effects of tumor on the immune system.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Patients with advanced malignancy, including those with SCCHN,3 have been reported to have impaired immune responses (1, 2, 3) . Thus, proliferative or cytolytic activities as well as cytokine production by immune cells may be depressed in patients with cancer (3 , 4) . The impairments are often subtle, as reflected by variably decreased but not absent functions of lymphocytes, and are more pronounced at the tumor site than in the peripheral circulation (4) . These observations, reported from many different laboratories in the last 20 years, have been interpreted to indicate that tumor-induced suppression of immune cell functions occurs in patients with cancer (reviewed in Ref. 5 ).

Recently, we demonstrated that, in patients with SCCHN, lymphocytes isolated from tumor-involved lymph nodes or PBLs consistently show a number of functional abnormalities, such as defective Ca2+ mobilization, impaired tyrosine kinase activity, and decreased expression of TCR-associated {zeta} and {epsilon} chains (3 , 5) . These and other signaling defects in TILs or tumor-associated lymphocytes or PBMCs have been observed in patients with ovarian, renal cell, cervical, and colorectal carcinoma, prostate cancer, and melanoma (6, 7, 8, 9, 10, 11, 12, 13) as well as in lymphocytes obtained from tumor-bearing mice (14 , 15) . More importantly, decreased expression of the {zeta} chain in patients’ T cells was biologically significant because it was found to be normalized in patients who responded to interleukin 2 biotherapy (13) and also to be a significant predictor of better survival in patients with melanoma (16) .

To further examine potential clinical significance of the {zeta}-chain abnormalities, we studied its expression by flow cytometry in PBLs of patients with SCCHN. Our findings suggest that a correlation exists between low expression of {zeta} and the tumor site and tumor aggressiveness. We also show that these patients’ PBLs are significantly more likely to undergo spontaneous apoptosis than are those of normal controls. These observations imply that decreased {zeta}-chain expression may be linked to the systemic apoptosis program initiated in vivo in patients with cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Cells.
Venous blood samples were obtained from 17 patients with histologically proven SCCHN, who were consecutively seen at the Eye and Ear Institute Clinic, University of Pittsburgh Medical Center, between January and June 1998. All 17 patients with SCCHN underwent treatment 1–10 years prior to their enrollment in this study. The patients (11 males and 6 females) ranged in age from 34 to 83 years, with a mean age of 65.1 ± 12.3 years. All patients signed an informed consent. At the time of specimen collection, the patients were not receiving any tumor-specific treatment, and all were classified as no evident disease. In addition to 17 healthy volunteers (8 males and 9 females) whose median age was 38 years old, a group of 8 volunteers matched in age (mean age = 61 ± 1.5 years) with the patients was studied. All patients and controls were asked to donate from 20–30 ml of venous blood for this study.

PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation, as described previously (13) , and they were washed in medium, counted, and tested immediately after separation. In each experiment, PBMCs obtained from patients were evaluated together with PBMCs obtained from at least one normal control.

Flow Cytometry.
Expression of the {zeta} chain was investigated in permeabilized cells using a mAb to CD3 {zeta} (TIA-2, IgG1; Coulter Immunology, Hialeah, FL), which recognizes the cytoplasmic domain of the {zeta} subunit of the TCR. PBMCs (0.5 x 106 cells) were fixed in 1% (w/v) paraformaldehyde in PBS for 20 min on ice, and after two washes, the cells were permeabilized using 0.1% saponin solution in PBS. The cells were stained with mouse anti-{zeta} mAb (TIA-2) or mouse IgG isotype control (DAKO Corp., Carpinteria, CA) for 30 min at 4°C, washed three times, and incubated with pretitered FITC-conjugated goat antimouse IgG antibody (Caltag, San Francisco, CA) for 30 min at 4°C. After three washes in PBS, the cells were resuspended in 0.2 ml of PBS containing 0.1% sodium azide. Next, an excess of mouse IgG1 was added for 30 min at 4°C to saturate free binding sites on the FITC-conjugated secondary antibody and prevent nonspecific staining. The cells were then stained with one of the following phycoerythrin-conjugated antihuman mAbs: anti-CD3 {epsilon}, anti-CD4, anti-CD8, anti-CD56/16, or IgG1 isotype control (all purchased from Becton Dickinson, Mountain View, CA). All mAbs were pretitered on normal PBMCs to determine their optimal dilutions.

The cells were analyzed using a FACScan (Becton Dickinson). For each sample, 10,000 events were acquired, and gates were set on lymphocytes. MFI of the {zeta} chain was measured in CD3+, CD4+, and CD8+ T cells. In selected cases, {zeta}-chain expression in CD56+/CD16+ cells was determined. The proportion of CD3+ cells with low {zeta} expression was determined for each patient by establishing the MFI of CD3 {zeta} values minus 2 SD for all 17 normal controls as the benchmark. A lymphocyte population was considered to have low {zeta} expression when the MFI of the population was found to be below the benchmark MFI.

TUNEL Assay.
A TUNEL assay was performed to study lymphocyte apoptosis by labeling DNA breaks at a single cell level, using reagents purchased from Boehringer Mannheim Corp. (Indianapolis, IN). PBMCs (5 x 105 cells) were washed twice with PBS and incubated with phycoerythrin-labeled CD3 mAb or IgG isotype control for 30 min on ice. Following incubation, cells were washed twice with PBS containing 0.1% sodium azide and 0.1% BSA and fixed for 30 min with 2% paraformaldehyde at room temperature. Cells were washed again and permeabilized for 3 min on ice in 0.1% sodium citrate in PBS containing 0.1% Triton X-100. After washing, cells were incubated for 1 h with FITC-conjugated-dUTP in a terminal deoxynucleotidyl transferase enzyme solution or enzyme solution alone as a negative control. Following washing, the cells were analyzed by flow cytometry.

Statistical Analysis.
The significance of observed differences was calculated using the Mann-Whitney U test. All differences with a P < 0.05 were considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics.
Among 17 patients with SCCHN whose PBLs were studied for CD3 {zeta}-chain expression, 7 patients were pathologically diagnosed to have stage I disease, 3 patients had stage II, 3 patients had stage III, and 4 patients had stage IV disease. Eight of the patients were diagnosed with laryngeal cancer that is considered to have a better prognosis than tumors at other head and neck sites (17) . Tumor sites in the other nine patients included the oral cavity and pharynx. The histological grades of tumors were: well-differentiated carcinoma in 6 patients versus moderately differentiated carcinoma in 11 patients. One patient was diagnosed with a synchronous second primary tumor of the lung, which was diagnosed at the same time as the SCCHN; another patient developed a new primary SCCHN 10 years after the first diagnosis. Among the 3 patients with cancer recurrence, one showed a recurrence in lymph nodes, whereas the other 2 had local recurrences. Two patients received radiation therapy and chemotherapy, and three patients were treated with radiation therapy alone at least 1 year prior to blood draws. At the time of blood draw, no patient was receiving a tumor-specific therapy.

Lymphocyte Subsets in Patients with SCCHN and Controls.
The phenotypic characteristics of T-lymphocyte subsets in the peripheral blood of patients with SCCHN and of healthy donors were determined by flow cytometry (Table 1)Citation . In comparison to normal volunteers, the proportion of CD3+ T cells was found to be significantly lower in patients with SCCHN (P = 0.01). The observed decrease was mainly due to a lower proportion of CD4+ T cells in patients’ PBLs, as compared to controls. These changes appeared to be of borderline significance, however, because the CD4:CD8 ratio was not significantly decreased in the PBLs of patients compared to that in normal controls.


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  		Table 1 T-lymphocyte subsets in PBMCs of patients with SCCHNa

 
Expression of TCR-associated {zeta} or {epsilon} Chains in PBLs.
To determine expression of the {zeta} chain in CD3+, CD4+, and CD8+ T cells in patients with SCCHN, double immunostaining with mAbs and flow cytometry analysis were performed. Both MFI of CD3+{zeta}+ cells (Fig. 1)Citation and the percentage of CD3+ cells with low {zeta} (Fig. 2)Citation were determined. The results were compared to the {zeta}-chain expression in PBLs obtained from 17 normal controls. As shown in Fig. 1Citation , the MFI for the {zeta} chain staining was significantly reduced in CD3+ cells of patients compared to healthy donors, with a mean ± SE of 174 ± 26 for patients and 450 ± 34 for normal controls (P <= 0.0001). The decrease in {zeta}-chain expression in patients was not related to the age because both younger and age-matched control groups had very similar MFI values for {zeta}, which were significantly higher from those in patients. The MFI values for {zeta} in CD4+ cells were: 184 ± 28 for patients and 497 ± 146 for normal controls (P <= 0.0001), and those for CD8+ cells were 141 ± 21 for patients and 367 ± 33 for normal controls (P <= 0.0001). Although these values were all significantly lower for patients than controls, the range of CD3+ low {zeta} cells was broad for patients, as shown in Fig. 2Citation .



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  		Fig. 1. Expression of the {zeta} chain determined by flow cytometry in CD3+ T cells as well as CD4+ and CD8+ T-cell subsets of patients with SCCHN (PT) or normal controls (NC). Horizontal bars, means; all differences between patients and normal controls were significant (P <= 0.0001).

 


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  		Fig. 2. The proportion of CD3+ cells with low {zeta} expression in patients (PT) and normal controls (NC) was determined as described in "Materials and Methods." Horizontal bars, means; the mean for NC is 8.5%, whereas that for patients is 53.6%. The difference between NC and patients is significant (P <= 0.0001).

 
Fig. 3aCitation illustrates the shift observed in the MFI of CD3{zeta} in PBLs of a representative patient, compared to a normal control analyzed on the same day. The entire CD3{zeta} peak is shifted to the left, indicating that in this patient with SCCHN, expression of the CD3 {zeta} chain is considerably decreased relative to that in the normal control. No significant differences were found in expression of the CD3 {epsilon} chain or the CD4 {epsilon} chain and CD8 {epsilon} chain between patients and normal controls. In addition to the CD3+{zeta}+ population, a smaller population of CD3-{zeta}+ cells was analyzed by flow cytometry. CD3- cells that expressed the {zeta} chain were NK cells, as confirmed by flow cytometry analysis of selected samples stained with CD56/16 mAbs (data not shown). The entire population of NK cells (CD3-{zeta}+) showed low {zeta}-chain expression in patients with SCCHN, as compared to normal controls (note a shift to the left in Fig. 3bCitation ).



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  		Fig. 3. a, example of the shift in MFI of CD3{zeta} in PBLs of a patient with SCCHN relative to MFI of CD3{zeta} in PBLs of a normal control. A representative experiment (n = 17) performed is shown. b, flow cytometry analysis of the CD3+{zeta}+ and CD3-{zeta}+ subsets in PBLs of a patient with SCCHN and of a normal control. Both X and Y axis are log fluorescence intensity. Note that fluorescence intensity for {zeta} is shifted to the left in the patient’s CD3+ as well as in CD3- cells, relative to fluorescence intensity for {zeta} in the normal control. A representative experiment (n = 8) is shown.

 
Correlation between {zeta}-Chain Expression and Disease.
When percentages of CD3+ cells with low {zeta}-chain expression were compared in patients versus normal donors, a significant difference was detected (Fig. 2)Citation , with means ± SE of 53.6 ± 7.5% for patients and 8.5 ± 2.4% for normal controls (P <= 0.0001). Four patients showed an almost absent expression of CD3{zeta} (Fig. 2)Citation . Two of these patients had new primary tumors within the last year, and one patient has developed a metastatic disease in the neck in January 1998. All three were diagnosed to have oral cancer. The fourth patient with low {zeta}-chain expression was diagnosed with laryngeal carcinoma in 1992 and has been free of tumor since then. Nevertheless, it is important to emphasize that the three patients who had either new primaries or a recurrence of their disease during the last year had an almost complete loss of the {zeta} chain in their peripheral T cells. On the other end of the spectrum (Fig. 2)Citation are four patients with normal {zeta}-chain expression, three of whom have laryngeal carcinoma and one who has oral cancer. It appears that down-regulation of the {zeta} chain correlates with the aggressiveness of disease in SCCHN. Also, when we compared the group of eight patients with laryngeal carcinoma to that of nine patients with primary tumors at sites other than larynx, a significant difference in CD3 {zeta}-chain expression was observed. Although both groups had decreased MFI for {zeta} relative to normal controls, mean MFIs ± SE were 227 ± 36.8 for the patients with laryngeal carcinoma and 126 ± 29.8 for patients with primary sites other than larynx (P = 0.03). As laryngeal cancer has an overall better prognosis than other SCCHN (17) , these data suggest that expression of {zeta} may have some prognostic value.

Detection of Low {zeta}-Chain Expression and Spontaneous Apoptosis in PBL.
In addition to assessing expression of the {zeta} chain in lymphocytes, we also looked for evidence of spontaneous lymphocyte apoptosis at the time of blood collection, using a TUNEL assay. We consistently observed that the proportion of PBLs with spontaneous DNA fragmentation was significantly higher in patients than in controls (P < 0.001). At the same time, there were also significantly more T cells with low {zeta} in patients’ peripheral blood than in that of controls. However, statistical analysis of these data indicated no correlation between decreased expression of the {zeta} chain in PBLs and a higher apoptosis rate seen in the patient than control lymphocytes in vivo. Nevertheless, the concomitant presence of these abnormalities in PBLs of the patients suggests that they are both cancer related.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The TCR-associated CD3 {zeta} chain is involved in transduction of signals delivered via the receptor, and therefore, its expression is important for activation of T cells (3 , 4 , 15) . The absence or reduced expression of the {zeta} chain impairs T-cell signaling and, thus, contributes to immune cell dysfunction (3 , 18) . Over the last few years, several laboratories have reported the presence of signaling defects in T cells from patients with cancer (6, 7, 8, 9, 10, 11, 12, 13) . These defects appear to be consistently present in lymphoid cells derived from the tumor microenvironment but are less frequent or less prominent in the PBLs (3) . We previously reported that expression and function of the signaling molecules, which are components of the TCR/CD3 or Fc{gamma}RIII pathways in T cells or NK cells, were decreased in tumor-associated lymphocytes obtained from patients with ovarian cancer, as compared to T or NK cells purified from normal peripheral blood (19) . Several other groups have recently found similar changes in the {zeta}-chain expression in TILs and/or PBL from patients with renal cell (7 , 11) colon (9 , 10) , or prostate (8) carcinomas or myeloid malignancies (20) . On the other hand, Cardi et al. (21) found that expression of the {zeta} chain was normal in CD3+ PBLs of patients with renal cell carcinoma (21) . Also, in some tumor-bearing mice, degradation of the TCR-associated {zeta} chain either was not observed (22) or was ascribed to artifacts induced during splenocyte in vitro separation (23 , 24) . Thus, considerable controversy has developed over the presence and in vivo significance of the {zeta}-chain degradation in tumor-bearing hosts.

It has been suggested that methodological differences account for disparate results reported for the {zeta}-chain expression from different laboratories. This may be the case because the quantitative multicolor flow cytometry method we use is more sensitive than Western blots. Also, the possibility has to be considered that activation of proteases in cell lysates or induction of proteolysis during cell separation may contribute to an artifactual decrease in the {zeta}-chain expression (23) . Nevertheless, the fact remains that in situ analysis confirms deficiency of {zeta} in TILs or lymphocytes isolated from tumor-involved lymph nodes of patients with cancer (3 , 13 , 25 , 26) . As immunostaining of fixed or snap-frozen tissue sections does not require cell isolation, it is highly unlikely that artifacts account for the {zeta} deficiency seen in lymphocytes in tumor tissues (25 , 26) .

The existing controversy can be readily explained by considerable variability in {zeta}-chain as well as {epsilon}-chain expression in lymphocytes obtained from different patients. This variability is well illustrated in Figs. 1Citation and 2Citation . When cells from small numbers of patients are studied, e.g.,n = 8 in a study by Cardi et al. (21) , the {zeta} or {epsilon} defects might be missed altogether. In an earlier study of 26 patients with advanced (mostly stage III and IV) SCCHN, we observed significantly lower expression of both {zeta} and {epsilon} chains in peripheral blood CD3+ cells, as compared to normal controls (3) . In contrast, no abnormalities in the {epsilon} chain were detected in the present group of 17 patients, all of whom had no evident disease. We showed, however, that patients who had new primary tumors or recurrences or those with more aggressive types of SCCHN had significantly lower {zeta}-chain expression in T cells than patients without recurrences and with a better prognostic profile. These observations and other data in our laboratory indicate that, far from being an in vitro artifact, low or absent {zeta} expression in immune cells is biologically significant, and it appears to be a marker of advanced or aggressive disease. In a very recent retrospective immunohistological study of 131 SCCHN biopsy samples, low or absent {zeta}-chain expression in TILs was found to be significantly correlated with poor 5-year survival in stage IV disease (26) . Importantly, Ochoa and colleagues (16) reported that overall survival of patients with melanoma who had decreased {zeta}-chain expression in peripheral blood T lymphocytes was significantly shorter than that of patients with normal {zeta}-chain expression.

Another important observation made in this study is the presence of CD3-/low {zeta} population in the peripheral blood of patients with SCCHN. Not only T cells but also NK cells (CD16+/CD56+) showed decreased expression of {zeta} associated with Fc{gamma}RIII. This finding is consistent with our earlier data in ovarian carcinoma (6 , 19) and those of others (27 , 28) . Thus, in patients with cancer, the two main effector cell populations that could mount an antitumor immune response, T and NK cells, show decreased expression of {zeta} and are likely not to function normally (28) .

Finally, the mechanism(s) responsible for the {zeta}-chain defects are currently unknown but appear to be related to apoptosis, which might be tumor-induced and which leads to DNA fragmentation in lymphocytes at the tumor site (3 , 25) as well as in a subset of activated circulating T or NK cells. We found that down-regulation of {zeta}-chain expression in lymphocytes of patients with SCCHN is accompanied by spontaneous apoptosis of these cells ex vivo. No such apoptosis is seen in normal PBMCs handled in a parallel manner. Our earlier studies showed that coincubation of tumor cells with activated lymphocytes induces both {zeta} degradation and DNA fragmentation in these lymphocytes and that both events can be blocked by caspase-3 inhibitors (25) . Here and in a submitted manuscript,4 we demonstrate that, compared to normal controls, T cells of patients with SCCHN are preprogrammed to undergo apoptosis. Even at the time of blood collection, a small fraction of TUNEL+ cells is significantly higher in patients with SCCHN than that in normal PBMCs. It is likely that activated effector cells in the peripheral circulation of patients with SCCHN are preprogrammed to die in vivo. We observed no correlation between the percentages of lymphocytes with low {zeta} and apoptotic (TUNEL+) cells. Nevertheless, both phenomena may contribute to tumor-induced lymphocyte dysfunction and, in case of apoptosis, perhaps to a rapid turnover of T cells in patients with advanced malignancies.

The findings reported here are important for the future of immunotherapy of cancer. If the {zeta}-chain defects are confirmed to be a consequence of tumor-induced immunosuppression, then they could serve as a biomarker, not only of the extent of immune dysfunction in patients with cancer but also of prognosis and of response to immunotherapy. Preliminary observations from our laboratory, although not from others (29) , suggest that the {zeta}-chain expression might be a long sought-after laboratory measure that correlates with in vivo responses to biotherapy (13) . For this reason and because of the possibility that {zeta} expression might also predict survival (16) , additional studies of this biologically important phenomenon are necessary.


   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 an NIH Grant PO-1 DE 12321-01 (to T. L. W.). Back

2 To whom requests for reprints should be addressed, at University of Pittsburgh Cancer Institute, W1041 Biomedical Science Tower, 211 Lothrop Street, Pittsburgh, PA 15213-2582. Phone: (412) 624-0096; E-mail: Whitesidetl{at}msx.upmc.edu Back

3 The abbreviations used are: SCCHN, squamous cell carcinoma of the head and neck; PBL, peripheral blood lymphocyte; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte; PBMC, peripheral blood mononuclear cell; mAb, monoclonal antibody; MFI, mean fluorescence intensity; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick end labeling; NK, natural killer. Back

4 T. Saito, I. Kuss, G. Dworacki, J. T. Johnson, and T. L. Whiteside. Spontaneous ex vivo apoptosis of periphereal blood mononuclear cells in patients with head and neck cancer, submitted for publication. Back

Received 7/20/98; revised 11/30/98; accepted 2/12/98.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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