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Molecular Oncology, Markers, Clinical Correlates |
Department of Oral and Maxillofacial Surgery, University of Mainz, 55131 Mainz, Germany [T. E. R., L. S., E. M. W.], and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213 [W. G., T. L. W.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chain expression and anti-CD3 antibody-induced proliferation of the PBL obtained from each patient. In addition, expression of CD3, CD4, and CD8 molecules on TIL and Fas ligand (FasL) on the tumor was studied by immunohistochemistry. Soluble FasL was measured in the patients’ sera. PBL obtained from 20 age-matched normal donors was used as a control. Reduced -chain expression was observed in TIL-T of 9 of 28 patients and in PBL-T of 12 of 28 patients. Low expression in autologous TIL-T and PBL-T was correlated (P < 0.0012), and it was associated with high levels of expression of FasL on the tumor (P = 0.0002 and P < 0.0013, respectively). Low expression in PBL-T was also associated with the poor ability of these cells to proliferate in response to anti-CD3 antibodies (P = 0.0012). Increased proportions of apoptotic cells were detected in PBL of 6 of 28 (21%) patients versus 13 of 28 patients (46%) in TIL. Apoptosis in autologous PBL and TIL was found to correlate (P = 0.0322) and was significantly associated with reduced -chain expression. Serum levels of soluble FasL were decreased in patients relative to normal controls but did not correlate with PBL apoptosis or FasL expression on the tumor. Decreased expression of TcR-associated chain, depressed immune function, and apoptosis of T cells were observed to occur concomitantly in TIL and circulating PBL-T of a subset of patients with oral carcinoma. These alterations correlated with high levels of FasL expression on the tumor but not with the disease stage. The results suggest that tumor exerts systemic suppressive effects on immune cells, which may be, in part, mediated via the Fas/FasL pathway. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Freshly isolated TIL or circulating T lymphocytes of patients with cancer were evaluated in proliferation, cytotoxicity, or cytokine production assays and observed to show various levels of dysfunction (reviewed in Ref. 14 ). Signaling defects in T cells were observed at the tumor site and also in the peripheral circulation of patients with cancer (reviewed in Ref. 15 ). To date, it remains unclear whether these defects in T-cell functions are tumor induced or are a manifestation of endogenous cell response to activation (i.e., activation induced cell death). Most of the available data have been obtained in cross-sectional studies that examined either peripheral blood mononuclear cell or TIL in cohorts of patients with a particular type of cancer and compared their phenotype and/or functions to those of PBLs obtained from normal donors. These studies also attempted to relate the degree of immune dysfunction to tumor burden or disease activity (16 , 17) . In general, the consensus has developed that TIL, localized to the tumor, was more functionally impaired than PBL, which were distant from the tumor, and that the greatest immune dysfunction was associated with a large tumor burden and advanced disease. However, studies in which functions of TIL were directly compared with those of autologous PBL are few and limited to a small number of patients (e.g., Ref. 18 ).
We reasoned that if the tumor in some way contributes to dysfunction and perhaps death of T lymphocytes, then a gradient of functional impairments extending from TIL to PBL in individual patients with cancer should be demonstrable. To test this hypothesis, we examined expression of , a TcR-associated signaling molecule, ability to proliferate ex vivo and the frequency of apoptosis in paired TIL and PBL of a cohort of patients with oral cancer. The results clearly show that such gradient exists, thus reinforcing the conclusion that the presence of the tumor has a significant negative and generalized effect on functions of the immune cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In all cases, the tumor tissues were snap-frozen immediately after surgical removal, embedded in the ornithine carbamyl transferase compound, and maintained at -80°C until analyzed.
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Determination of Spontaneous Apoptosis in PBL.
Aliquots of 5 x 106 cells were incubated for 24 h at 37°C in 10 ml of medium. The medium used was RPMI 1640 supplemented with 1 mM L-glutamine, 10% (v/v) of FCS, 100 µg/ml penicillin, and 100 µg/ml streptomycin. These reagents were purchased from Life Technologies, Inc. (Karlsruhe, Germany). After incubation, cells were harvested and immediately spun down onto glass slides. The slides were examined for evidence of apoptosis in PBL using a TUNEL assay.
Antibodies.
Monoclonal Abs to the chain (6B10.2) and FasL (Q-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany); monoclonal Abs to CD4 (clone DK25), CD8 (clone MT 310), CD3 (clone UCHT-1), and polyclonal Abs to CD3 were all purchased from DAKO (Hamburg, Germany). The LSAB2 kit from DAKO, containing biotinylated secondary antibodies, was used to perform the labeled streptavidin-biotin technique. Isotype control Abs were from DAKO and Sigma (Taufkirchen, Germany). The optimal working dilutions were determined in preliminary titration experiments performed with human tonsils and activated PBL.
Immunohistochemistry.
Cytospins were prepared using 100 µl aliquots of cell suspensions (2 x 104 cells/ml) in PBS. Cells were centrifuged onto glass slides using a Labofuge (Hareus, Osterode, Germany) for 5 min at 500 rpm. Slides were air dried, fixed with precooled acetone for 10 min, and stored at -80°C before staining.
Cryostat sections of tumor tissues (5 µm) were cut consecutively from each ornithine carbamyl transferase-embedded specimen in a Leitz cryostat and mounted on electrostatically precharged slides (Superfrost Plus; Menzel-Gläser, Frankfurt, Germany). The slides were air dried, fixed with precooled acetone for 10 min, and stained by an indirect immunoperoxidase method. After endogenous peroxidase quenching (0.3% H2O2 in PBS for 30 min), the labeled streptavidin-biotin technique was performed. Optimal working dilutions of the primary antibodies were: 1:50 for CD3, CD4, and CD8; 1:800 for -chain; and 1:100 for FasL. The triple staining was performed using Ab Q-20 (Santa Cruz Biotechnology), anti-CD3 Ab (DAKO), and the TUNEL kit purchased from Roche Diagnostics.
Staining was developed with peroxidase, diaminobenzidine, and nitroblue tetrazolium when three-color immunostaining was performed. In all experiments, isotype antibodies were used as controls.
All immunostained slides were examined by light microscopy using a Leitz microscope (Wetzlar, Germany) at x400 magnification. To enumerate CD3+, CD4+, and CD8+ cells, at least 10 HPFs were randomly selected at the border of the tumor and within the peritumoral tissue. The numbers of positive T cells were estimated semiquantitatively, and the following groups were created: group 1 contained <10 positive cells/HPF; group 2 had from 10–30 positive cells/HPF; and group 3 had a dense infiltration of T cells (>30 cells/HPF). The density of CD4+ and CD8+ cells in the tumor was compared with that of CD3+ cells. Expression of the chain in TILs was determined as described previously (19)
. Briefly, staining intensity for in TILs was graded as either reduced or normal relative to the staining of T lymphocytes infiltrating the adjacent nontumor squamous epithelium or of T lymphocytes present in normal tissues or cytospins used as controls. In cytospins, -chain expression in T lymphocytes was scored by counting the number of + and CD3+ T cells and by comparing -staining intensity of patients’ T cells with that of T cells obtained from normal controls. To determine FasL expression in the tumor cells, both the staining intensity and the number of positive cells were determined. The tumor was considered to have low FasL expression if <30% of tumor cells were positive for FasL and/or the staining intensity was weak compared with the expression of a FasL+ human squamous cell carcinoma of the head and neck cell line PCI-13 (8)
, established and maintained in our laboratory as described previously (20)
. The tumor was considered to express high levels of FasL when >30% and <80% of tumor cells showed a strong expression of FasL, comparable with FasL expression on the control PCI-13 cells stained in parallel. Tumor was considered to express a very high level of FasL when nearly all cells showed a strong staining, comparable with staining intensity of the control PCI-13 cells.
TUNEL Assay.
To identify fragmented DNA in sections of tumor tissue and in PBL on cytospins, the TUNEL assay was performed. The In Situ Cell Death Detection kit (Roche Diagnostics, Mannheim, Germany) was used as specified by the manufacturer. Control tissue sections or cells were treated with the TUNEL reaction mixture containing fluorescein-dUTP without terminal deoxynucleotidyl transferase. Stained cells or sections were analyzed under a light microscope. The number of TUNEL+ cells was counted at x400 magnification and related to the total number of cells in the examined area or to the size of 1 HPF. In tissue sections, at least 10 HPF/section were analyzed. In cytospins, the percentages of TUNEL+ cells were determined. In tissue sections, the following three groups were distinguished: group 1, with no apoptotic TIL; group 2, with low numbers of apoptotic TIL (<5 apoptotic lymphocytes/HPF); and group 3, with high numbers of apoptotic TIL (
5 apoptotic lymphocytes/HPF).
WST Proliferation Assay.
PBL obtained from normal donors or cancer patients were stimulated by anti-CD3 or isotype control Abs used at two different concentrations (0.5 µg/ml and 5 µg/ml). The cells (1 x 106 cells/100 µl/well) were incubated in flat-bottomed microtiter plates in RPMI medium at 37°C in a humidified atmosphere of 5% CO2 in air for 24 h. After the incubation period, 10 µl of proliferation reagent WST-1 (Roche Diagnostics) were added and incubated for 1 h. Absorbance was measured at 450 nm against a reference wavelength of 655 nm using a microplate reader (Model 550; Bio-Rad, München, Germany).
ELISA for FasL.
Patients sera were diluted 1:2 with PBS and tested for the presence of soluble FasL by ELISA (Oncogene, Boston, MA). The samples were tested against a reconstituted lyophilized standard, and absorbance (450/540 nm) was measured using a spectrophotometric plate reader (Bio-Rad). Supernatants of PCI-13 cells transduced with the human FasL gene and secreting FasL (21)
were used as a positive control.
Statistical Analysis.
Several exact two-tailed nonparametric tests were used for data analysis, depending upon the level of measurement of variables. For two variables measured on a continuous scale, the Wilcoxon test was used to compare the medians of two populations, and the Spearman rank correlation test was used to test the correlation. If more than two populations were considered (e.g., populations expressing no, low, or high level of a given marker) the Jonckheere-Terpstra test was used to detect a trend within the considered categories. For categorical data with unordered categories, Fisher’s exact test was used. For 2xC contingency tables with C-ordered binomial populations, the Cochran-Armitage Trend test was used, whereas for doubly ordered RxC contingency tables, the linear x linear association test was used. The statistical procedures used have all been described previously (22
, 23)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We evaluated a limited number of immunological end points ( expression, proliferation, and apoptosis in T cells) and correlated them with biological and pathologic characteristics of the tumor in each of the patients, including FasL expression, density, and phenotype of TIL, T, and N stage of the tumor, as well as disease stage. The data sets were analyzed for the presence of correlations between local and systemic effects within each patient as well as for correlations between immune and tumor-related end points.
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-Chain Expression in TIL and PBL. chain, which is often depressed or absent in lymphocytes of patients with cancer, was compared in paired autologous TIL and PBL. Overall, reduced expression of in TIL was observed in 9 of 28 (32%) and in PBL in 12 of 28 (43%) of the patients (Table 3)
. Concomitant reduction of in paired TIL and PBL was seen in 8 of 28 (29%) of the patients with oral carcinoma (Table 3)
. Thus, expression was reduced only in a subset of these patients, but when present, the defect in signaling was usually detectable both in TIL and PBL, indicating a significant correlation for decreased expression in TIL and PBL (P = 0.0012). Among the 8 patients with reduced in both TIL and PBL, 6 (75%) had advanced (stage IV) disease. However, of 15 patients with normal expression in both TIL and PBL, 10 (67%) had stage IV disease. These results provide an indication that abnormalities in are not always associated with advanced disease. Furthermore, statistical analyses of expression in lymphocytes and of all other immune measures studied in this cohort of patients with oral carcinoma indicated no significant correlations with disease stage.
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, associated with TcR in lymphocytes, implies that their functions are depressed. When proliferative responses of the patients’ lymphocytes to mitogens and to anti-CD3 Ab were examined, a subgroup of 12 patients with low expression in PBL was found to have significantly depressed responses (Fig. 1
; P < 0.0012). In contrast, PBL obtained from 16 patients with normal chain expression in T cells responded to anti-CD3 Ab by robust proliferation, which was comparable with that of normal T lymphocytes (data not shown). Thus, lymphocyte proliferation was significantly reduced when expression in PBL was decreased.
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. Responses of these PBLs were found to be significantly depressed (Fig. 2
; P < 0.0112) as would be predicted from a significant correlation established between concomitant low expression in TIL and PBL. In contrast, if TIL expressed normally, PBL responses to anti-CD3 were equal to those of normal T cells (Fig. 2)
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expression is not a local but generalized phenomenon present in a subset of patients with oral carcinoma who may or may not have advanced disease.
Apoptosis in TIL and PBL.
Apoptosis of TIL in situ was studied using a TUNEL assay in combination with staining for CD3 to identify T lymphocytes. It was detected in 13 of 27 (48%) tumors. Fig. 3
illustrates high (in A) and low (in B) levels of TIL apoptosis in representative tumor specimens. In general, apoptosis was more pronounced in TIL than in circulating T lymphocytes in individual patients (Fig. 3C)
. An elevated percentage of circulating apoptotic T cells (5%) was seen in 11 of 28 (39%) patients. An association between the presence of apoptosis in paired TIL and PBL specimens was observed (Table 4)
and was statistically significant at P = 0.0322. Furthermore, 4 of 6 patients with high TIL apoptosis were noted to have >5% of apoptotic T lymphocytes in the peripheral circulation. Interestingly, these 4 patients belonged to the subgroup with reduced expression in both PBL and TIL. Therefore, we analyzed the data for associations of apoptosis with chain expression and proliferation in TIL and PBL of the patients (Table 5)
. In TIL as well as PBL, there was a significant correlation (P = 0.0015 and P < 0.0019, respectively) between apoptosis and low expression in CD3+ cells. The correlation of apoptosis in PBL with decreased proliferation was weaker at P < 0.0170, with the median values of 277% for the 11 patients with evident apoptosis and 425% for the 17 patients with none or <5% apoptosis (data not shown). These data suggest that the chain loss in both PBL and TIL might be related to the process of spontaneous apoptosis of T cells, which occurs both at the tumor site and in the circulation of patients with oral carcinoma.
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, although tumors could be distinguished as high (17 of 28) or low (11 of 28) FasL expressors. Even in tumors with relatively weak FasL expression, apoptotic CD3+ T cells were present at the tumor border (Fig. 5)
. Expression of FasL on the tumor was not related to the tumor stage or lymph node involvement. It was interesting to note that of 17 tumors that expressed high levels of FasL, 12 also contained apoptotic (TUNEL+) TIL. Of 11 tumors with low FasL expression, only one had some evidence (low) of TIL apoptosis (Table 6)
. As can be seen, P for the association of high FasL expression on a tumor with TIL apoptosis was 0.001. Also, there was a significant correlation between FasL expression on the tumor cells and reduced in TIL (P = 0.0002) as well as PBL (P = 0.0013). These results substantiate the relationship between FasL expression on tumor and functional changes in TIL as well as PBL-T of patients with oral cancer, including defects in TcR signaling and apoptosis of immune cells.
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Density and Phenotype of TIL.
In view of the evidence that TIL was undergoing apoptosis at the tumor site, we also evaluated the relative density of lymphocytic infiltrates in the tumor, with particular attention to the CD4 and CD8 phenotype of infiltrating T cells (Fig. 6)
. The tumors were variably infiltrated with CD3+ T cells, and the density of the infiltrate correlated weakly with FasL expression on the tumor as well as the extent of TIL apoptosis (data not shown). There was no correlation observed between the density of T-cell infiltrate and expression in TIL or apoptosis in PBL-T. In comparing the relative proportions of CD4+ and CD8+ T cells at the tumor site, it appeared that the number of CD4+ plus CD8+ cells did not add up to total CD3+TIL, especially in the tumors with high levels of TIL apoptosis as determined by TUNEL positivity. In these tumors, a substantial proportion of CD3+ cells were DN cells (Fig. 6)
. A loss of both CD4 and CD8 membrane markers in up to 50% of TIL was seen in 4 patients whose tumors were T3 or T4 and in 1 patient whose tumor was T1N1. All 5 patients had tumors expressing high levels FasL and containing elevated numbers of TUNEL+ TIL. Also, 4 of 5 of these patients showed reduced expression in TIL. These data suggest that in those patients with advanced disease whose tumors are strong FasL expressors, TIL is more likely to be undergoing apoptosis and thus contain more DN cells and more cells with low than TIL infiltrating less advanced tumor lesions.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our own work has long been directed toward providing evidence for the deleterious effects of the tumor microenvironment on immune cells (27) . Earlier, we demonstrated that signaling defects in T cells, which specifically impair TcR functions, are a hallmark of those immune cells that accumulate at the tumor site (7 , 9) . Although the mechanisms responsible for these defects may vary, depending on the nature of the tumor microenvironment, the final result is invariably linked to tumor progression. Human solid tumors progress and metastasize, whereas TIL, generally present in substantial numbers in or around these tumors, fails to restrict or arrest tumor progression. This failure of TIL to intervene is now thought to be associated with their functional impairments, which have been amply described in the existing literature (14) . The major unsolved question is the origin and the mechanism(s) responsible for immune dysfunction in tumor-associated lymphocytes. The newer data describing signaling dysfunctions and spontaneous apoptosis in circulating T cells of patients with cancer strongly suggest that immunosuppressive effects of the tumor extend beyond its microenvironment (13 , 28) . If this hypothesis is correct, then a direct correlation should exist between dysfunction seen in TIL and that in autologous PBL, and that dysfunction should be attributable to the presence of tumor.
In this study, we tested the above hypothesis by combining recently developed methods for in situ analysis of TIL and tumor cells with studies of paired PBL-T in 28 patients with oral carcinoma. The results clearly indicated that a high degree of correlation exists between local and systemic effects on functions of TIL and PBL-T, respectively. The systemic signaling defects (low ) appeared to be restricted to a subset of patients and were not related to the presence of advanced disease. On the other hand, all patients with concomitant low expression in both TIL and PBL had high levels of FasL expression in the tumors and nearly all had detectable TUNEL+ T cells in the peripheral circulation. Although these data suggest that the observed systemic effects are attributable to the tumor, this clearly is not a complete explanation. Other patients with advanced disease or poor prognostic factors such as node involvement did not show similar systemic effects. A microenvironment of progressing tumor may or may not be immunosuppressive depending on the factors produced by the tumor and/or immune cells and on their mutual interactions. Thus, local or systemic influence of the tumor on the host is likely to be broadly variable and, therefore, difficult to assess at the experimental or clinical level. This is an important consideration because of the possibility that the patients with concomitant local and systemic T-cell dysfunction have a poorer prognosis than those with local immune dysfunction only. The presence and degree of immune dysfunction in TIL and PBL might reflect tumor aggressiveness in respect to its ability to mediate immunosuppressive effects. If confirmed, this biological characteristic of solid tumors might be a useful guide to selection of future immunotherapy. It is likely that tumors able to induce and sustain defects in local as well as systemic immunity might require a particularly robust immune intervention targeted at overcoming these defects.
Our data set is unique because it has allowed for a simultaneous examination of the tumor, TIL, and PBL-T, and because we were able to study not only the phenotype but also functional characteristics of these immune cells. We evaluated responses to anti-CD3 Ab, which provided a model of TcR-driven activity and, in combination with expression, were considered to be an adequate surrogate end point for evaluation of immune competence of T cells. As expected, we observed a significant correlation between T-cell proliferation and expression. We initially suspected that depressed functions of T cells seen in patients with cancer might be linked to apoptosis, largely based on the evidence that chain degradation could be a manifestation of apoptosis (29)
. In support of this mechanism, we now demonstrate a significant association between decreased expression in TIL or PBL and the frequency of spontaneous apoptosis of these cell populations.
A robust correlation of FasL expression on the tumor with TIL apoptosis, as well as a convincing association between apoptosis in autologous TIL and PBL, suggest that the tumor could influence immune cell survival through the Fas/FasL pathway, as suggested previously (30) . The contribution of sFasL to this process remains unclear, although it apparently can cross-link Fas expressed on T cells and thus mediate apoptosis (31) . Our results indicate that nearly all circulating T cells are Fas+ in patients with cancer (13 , 32) and, thus, are susceptible to apoptosis upon interaction with FasL. Indeed, preliminary data from our laboratory indicate that these Fas+ T cells preferentially bind Annexin V, i.e., are in early stages of apoptosis (32) .
The presence of CD3+ T-cell infiltrates in oral carcinomas was highly variable and characterized in some tumors by the unusual CD3+CD4-CD8- or DN phenotype. These DN cells might represent T cells that are in the process of apoptosis, as our preliminary observations suggest that they are predominant infiltrating cells in tumors expressing high levels of FasL and containing many TUNEL+ T cells. In vitro, the DN phenotype is characteristic of CD3+ T cells induced to undergo apoptosis by CH11 Ab and binding Annexin V, as shown by us previously (13) . In aggregate, our present data as well as our recent reports on increased apoptosis of circulating T cells in patients with cancer (13 , 32) point to tumor-mediated cell death as a prominent, although certainly not the only, mechanism responsible for abnormalities in function and phenotype of immune cells in the tumor microenvironment.
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FOOTNOTES |
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1 This work was supported by from Deutsche Forschungsgemeinschaft Grant (to T. E. R.) and by NIH Grant P01-DE12321 (to T. L. W.). 
2 To whom requests for reprints should be addressed, at University of Pittsburgh Cancer Institute, W1041 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213-2582. Phone: (412) 624-0096; Fax: (412) 624-0264; E-mail: whitesidetl{at}msx.upmc.edu
3 The abbreviations used are: TIL, tumor-infiltrating lymphocyte; Fas L, Fas ligand; sFasL, soluble Fas ligand; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; PBL, peripheral blood lymphocyte; Ab, antibody; HPF, high-power field; TcR, T-cell receptor; DN, double negative.
Received 2/25/02; revised 6/20/02; accepted 6/21/02.
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