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Clinical Cancer Research Vol. 12, 2394-2403, April 15, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Alterations in the T-Cell Receptor Variable ß Gene–Restricted Profile of CD8+ T Lymphocytes in the Peripheral Circulation of Patients with Squamous Cell Carcinoma of the Head and Neck

Andreas E. Albers1,2, Carmen Visus1,2, Takashi Tsukishiro1, Robert L. Ferris1,3,4, William Gooding1, Theresa L. Whiteside1,2,3,4 and Albert B. De Leo1,2

Authors' Affiliations: 1 Divisions of Basic Research and Biostatistics, University of Pittsburgh Cancer Institute and Departments of 2 Pathology, 3 Immunology, and 4 Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Requests for reprints: Albert B. De Leo, University of Pittsburgh Cancer Institute, Research Pavilion, Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-3228; Fax: 412-1415; E-mail: deleo{at}imap.pitt.edu.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 References
 
Purpose: Apoptosis of activated CD8+ T cells is often seen in tumor-infiltrating lymphocytes and circulating peripheral blood mononuclear cells (PBMC) in patients with squamous cell carcinoma of the head and neck (SCCHN). We investigated whether T-cell receptor (TCR) variable ß chain (Vß)–restricted T cells were more sensitive to apoptosis than non–TCR Vß-restricted T cells.

Experimental Design: Flow cytometry analysis with anti-TCR Vß antibodies was used to define expansions and contractions of Vß-restricted T cells in patients with SCCHN relative to normal donors. This staining was combined with Annexin V binding to indicate early T-cell apoptosis.

Results: The TCR Vß profiles of CD3+ T cells in tumor-infiltrating lymphocytes and PBMCs of patients with SCCHN were altered relative to controls, with one to five expansions and numerous contractions of TCR Vß-restricted T cells detected. These types of alterations were significantly greater in CD8+ than CD4+ T cells. Enhanced Annexin V binding to CD8+ T cells was evident in PBMCs obtained from all patients, with 3 of 13 showing preferential targeting for apoptosis of TCR Vß-restricted T cells.

Conclusions: TCR Vß profiles of CD8+ T cells were altered in patients with SCCHN relative to normal controls. This may reflect increased apoptosis of expanded or contracted CD8+ T cells, which define the TCR Vß profile of antigen-responsive T-cell populations in patients with cancer.

The T-cell receptor (TCR) repertoire of T cells in any individual is determined by their HLA haplotype and exposure to determinants derived from self and non-self molecules. Although studies of cloned T cells indicate that variable {alpha} chain (V{alpha}) segment restrictions define TCR specificity for an epitope more precisely than Vß segments, current knowledge of the molecular genetics of TCR Vß family genes is more extensive (1, 2). Consequently, analysis of TCR Vß rather than V{alpha} usage has been more frequently done. The analysis of TCR Vß repertoire of T cells in the peripheral circulation and at tumor sites has been useful in characterizing tumor-specific immune responses in patients with cancer (3). Several studies have identified TCR Vß restrictions in tumor-infiltrating lymphocytes (TIL) relative to peripheral blood mononuclear cells (PBMC) obtained from these patients (4–6). Our past PCR-based studies of Vß usage by T cells in patients with melanoma, hepatoma, or squamous cell carcinoma of the head and neck (SCCHN) also showed that Vß restrictions are present in circulating T cells in cancer patients, and that some of Vß restrictions were common to or shared by T cells of these patients (7, 8). These observations of shared T-cell Vß specificities were tentatively interpreted as expansions of tumor antigen (TA)–specific T cells. However, although detectable by sensitive methods, such as tetramer analysis, TA-specific T cells are, in general, present at low frequencies in the peripheral circulation of patients with cancer and dysfunctional (9–11). This could contribute to tumor progression and facilitate tumor escape from the host immune system.

A variety of mechanisms may be responsible for tumor escape, one of which involves apoptosis of effector CD8+ T cells at the tumor site and in the peripheral circulation of patients with cancer (12–15). Based on the paucity of TA-specific T cells and evidence for their impaired functions, we hypothesized that TA-specific T cells preferentially undergo apoptosis in patients with cancer. To begin investigating this possibility, with the caveat that TCR Vß-restricted CD8+ T cells are TA specific, we did a flow cytometry–based analysis of the TCR Vß repertoire of CD3+ T cells in PBMCs and TILs of patients with SCCHN. Subsequently, the objective was to quantify TCR Vß usage in CD8+ and CD4+ T-cell subsets in PBMCs obtained from patients and to determine the sensitivity of their circulating TCR Vß-restricted CD8+ T cells to spontaneous apoptosis.

Approximately 70% of the human TCR Vß repertoire can presently be readily identified using a panel of 24 commercially available chromophore-conjugated monoclonal antibodies (mAb) that target specific TCR Vß segments. This methodology readily lends itself to additional flow cytometry–based analyses that combine phenotypic and functional characterization of Vß-restricted T cells. Using this strategy in multivariable flow cytometry, we found that the binding of Annexin V, an indicator of early apoptosis, to TCR Vß-restricted populations of CD8+ T cells is enhanced in peripheral circulation of some patients with SCCHN.


   Patients and Methods
Top
 Abstract
 Patients and Methods
Results
 Discussion
 References
 
Patients and normal controls. Peripheral blood samples (10-30 mL) were obtained by venipuncture from patients with SCCHN seen in the Outpatient Otolargyngology Clinic at the University of Pittsburgh Cancer Institute and from normal healthy donors. Subjects involved in this study were screened for HLA-A2 expression and included 45 HLA-A2+ patients with SCCHN and 20 HLA-A2+ healthy normal donors. The patients were not on any therapy at the time of blood draws and represent the usual mix of those with active disease tested before treatment or no evident disease tested after curative therapy. The Institutional Review Board at the University of Pittsburgh approved the study, and written informed consents were obtained from all participating individuals.

PBMCs and TILs. PBMCs were isolated from peripheral blood samples obtained from HLA-A2+ SCCHN patients and normal donors by centrifugation over Ficoll-Hypaque gradients (Amersham Pharmacia Biotech, Piscataway, NJ). TILs were isolated from tumor samples at the University of Pittsburgh Cancer Institute Immunologic Monitoring and Cellular Products Laboratory using a standard operating procedure, as previously described (7, 8). The TIL samples were cryopreserved in freezing medium that consisted of human AB serum (Nabi, Miami, FL) plus 10% (v/v) DMSO (Fisher Scientific, Pittsburgh, PA) using Cryomed. Before flow cytometry, TILs were thawed, washed in medium, and tested by trypan blue exclusion for viability, which was generally >95%.

TCR Vß family usage. The TCR Vß family usage in PBMCs and TILs was determined by four-color flow cytometry analysis (16), using the TCR Vß repertoire kit, IO Test Beta Mark (Beckman Coulter, San Diego, CA), which consists of mAbs designed to identify 24 distinct TCR Vß families (Table 1 ). Each set consisted of three distinct anti-Vß family–specific mAb labeled with FITC, phycoerythrin, or doubly labeled with FITC/phycoerythrin. Fresh or thawed PBMCs or TILs (5 x 105) were stained simultaneously with allophycocynanin-conjugated anti-CD3 (clone UCHT1) and cychrome-conjugated anti-CD8 mAb (clone HIT8a), both obtained from BD Biosciences (San Jose, CA) and one of the eight sets of mAb directed against three distinct TCR Vß families. In this manner, T-cell usage of a total of 24 distinct TCR Vß families could be detected, using eight individual tubes and a total of <3 x 106 cells. The gate was set on the CD3+ population and then either on the CD8+ or CD8-CD4+ T-cell population. Individual TCR Vß families were evaluated based on T cells showing exclusive staining for FITC, phycoerythrin, or FITC/phycoerythrin. The total percentages of CD3+ TCR Vß+ cells detected using the IO Test Beta Mark kit in PBMC (fresh or cryopreserved and thawed), and TIL samples were generally 70%, consistent with the manufacturer's literature. Data acquisition was done using a FACSCalibur flow cytometer and Cellquest software (BD Biosciences). Expo32 software (Beckman Coulter) was used for data analysis.


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  		Table 1. Box and whisker analysis of TCR Vß usage by circulating CD3+ T cells in PBMCs obtained from 10 normal donors

 
Flow cytometry analysis for Annexin V binding. Annexin V staining was done on freshly isolated PBMCs obtained from patients or normal donors and combined with staining for TCR Vß families. Freshly harvested PBMCs were maintained at room temperature and analyzed within 1 to 2 hours of phlebotomy. PBMCs were stained with anti-CD8 mAb and the panel of anti-TCR Vß mAb for 30 minutes at room temperature, washed twice with flow buffer consisting of PBS supplemented with 1% fetal bovine serum and 0.1% NaN3 followed by wash with Annexin V binding buffer, and stained with allophycocynanin-conjugated Annexin V (BD PharMingen, San Diego, CA) for 15 minutes at room temperature immediately before analysis (17).

Statistical analysis. The samples analyzed were divided into subsets consisting of samples from up to 14 patients, which were then compared with those obtained from cohorts of 7 or 10 normal controls. The distributions of percentages of a TCR Vß family in CD3+, CD4+, or CD8+ T cells were characterized by box and whisker statistics. Box and whisker statistics are the values used to draw box-and-whisker diagrams (Table 1). Results of box and whisker statistics consist of the median, the interquartile range (box), and 1.5 times the interquartile upper and lower range (whiskers). The latter can then be used to identify outliers or observations falling above or below the whiskers. To define TCR Vß restrictions in CD3+, CD4+, and CD8+ T cells, first, the whiskers were calculated for the normal controls for each of the 24 families of the TCR Vß repertoire being analyzed. Next, the percentages of patient's TCR Vß components were compared with the whisker values for normal controls. Any patient's TCR Vß family value that was greater than the high whisker for that TCR Vß family was defined as a TCR Vß expansion. Similarly, any patient's TCR Vß value lower than the low whisker was defined as a TCR Vß contraction. A patient with either a TCR Vß expansion or contraction was considered to have a TCR Vß restriction. Hierarchical clustering, a multivariate test, was used to compare the TCR Vß repertoire of patients to normal controls. First, the Euclidean distance between all subjects (cases and controls) was computed. Then, observations were clustered based on proximity in multidimensional space and distances between clusters was then computed based on the average distance (the average linkage method). With respect to Annexin V binding to TCR Vß+ CD8+ T cells, these cells were classified into one of three groups according to whether they fit the definition of expansion, contraction, or the lack of restriction. The percentage of CD8+Vß+Annexin V+ T cells was compared for each of the 24 TCR Vß components, and the difference was tested with the Kruskal-Wallis test. If a difference was found among the three groups, the specific paired differences were probed with the Wilcoxon test.


   Results
Top
 Abstract
 Patients and Methods
 Results
Discussion
 References
 
TCR Vß restrictions in circulating CD3+ T cells of patients with SCCHN. A panel of commercially available anti-TCR Vß segment mAb was first used to determine TCR Vß usage in circulating CD3+ T cells of 10 normal donors. PBMCs obtained from these subjects were analyzed by flow cytometry, and the results indicated that the mAb in the panel stained 72 ± 6% (mean ± SD) of CD3+ T cells present in the PBMC samples, a level comparable with the 70% value supplied by the manufacturer of the IO Test Beta Mark kit. A similar analysis of PBMCs obtained from 10 patients with SCCHN stained a mean of 68 ± 7% CD3+ T cells. Descriptive statistics based on the quartile estimates for a box and whisker plot of the data were calculated for the individual TCR Vß family usage by CD3+ T cells obtained from the normal donors is shown in Table 1. To define expansions and contractions of individual TCR Vß+ CD3+ T-cell populations in PBMCs obtained from patients with SCCHN, the TCR Vß family usage values obtained from the PBMCs that were in excess of the upper whisker values or below the lower whisker values for each of the 24 TCR Vß specificities defined for CD3+ T cells obtained from normal donors were considered to be significant. A diverse repertoire of TCR Vß expansions and/or contractions involving all 24 TCR Vß families was evident in the PBMC samples obtained from all the 10 patients with SCCHN who were initially tested (Table 2 ). Importantly, contractions of CD3+ T cells were more common than expansion (Table 2). Contractions of CD3+ T cells expressing TCR Vß 2, 8, 12, and 13.6, each seen in at least 5 of 10 patients, were the most frequently detected shared contractions, whereas TCR Vß 4 and 11 were the most common shared T-cell expansions detected, but each involved only 3 of 10 patients. These results showed that relative to the normal TCR Vß T-cell profile, expanded as well as contracted T cells were present in PBMCs of patients with SCCHN and could be clearly identified and quantified by this flow cytometry–based method. There was no relationship between the frequency of TCR Vß restrictions in T lymphocytes and the disease activity (active disease versus no evident disease).


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  		Table 2. Analysis of TCR Vß restrictions in CD3+ T cells in PBMCs obtained from 10 patients with SCCHN

 
TCR Vß usage in TILs and PBMCs. Based on the hypothesis that TCR Vß T-cell restrictions in PBMCs of patients with SCCHN might represent tumor-related responses, we expected to detect the same restrictions in TIL/PBMC pairs. We compared matched sets of archived TIL and PBMC samples obtained from eight patients for TCR Vß usage by CD3+ T cells using flow cytometry. Relative to the TCR Vß values obtained for normal donor PBMCs detailed in Table 1, all eight sets of matched TIL/PBMC samples from patients 11 to 18 showed at least one shared expansion or contraction of a TCR Vß-specific CD3+ T-cell population (Table 3 ). For five of eight patients (patients 11-15), the same TCR Vß expansions were present in the analyzed TIL and PBMC samples. Expansions involving TCR Vß 13.6, detected in paired samples of three of eight patients, were most frequently encountered (Table 3). In all eight sets of paired samples, the TIL samples had an equal or greater number of TCR Vß clonal expansions than the PBMCs. A representative example is shown in Fig. 1 , in which an expansion of CD3+ T cells expressing TCR Vß 23 in the TIL sample but not the PBMC sample obtained from patient 17 was detected.


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  		Table 3. Analysis of TCR Vß usage in CD3+ T cells present in paired TIL and PBMC samples obtained from patients with SCCHN

 

Figure 1
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  		Fig. 1. Flow cytometry analysis of TCR Vß usage by CD3+ T cells present in paired TIL/PBMC samples using flow cytometry. The gate was set on CD3+ T cells using allophycocynanin-conjugated anti-CD3 antibody. Analysis of paired PBMC/TIL samples obtained from a patient with SCCHN (patient 17; Table 3), shows an expansion of TCR Vß23+CD3+ T cells in TILs relative to PBMCs.

 
Relative to TCR Vß contractions, all matched sets of patients' samples, except those from patient 12, had two or more contractions of TCR Vß usage. Two TIL samples, those from patients 15 and 16, had an equal or greater number of contractions than the matched PBMCs. Compared with expansions of TCR Vß-restricted CD3+ T cells, a greater number of shared contractions of the same TCR Vß-restricted cells were present in the TIL and PBMC samples. The most frequently detected contractions, present in five or more patients' samples, involved TCR Vß8, 9, 11, and 18. Of these four TCR Vß families, only contractions involving TCR Vß8 were also noted in the previous analysis of CD3+ T cells present in PBMCs of the first 10 patients with SCCHN we studied (Table 2).

The expansions and/or contractions of certain TCR Vß specificities consistently observed in TILs and PBMCs of patients with SCCHN might reflect expansions of antitumor effectors at the tumor sites or the periphery and/or their enhanced apoptosis, respectively. This latter possibility is consistent with previously reported apoptosis of T cells that is often seen at the tumor site (14).

TCR Vß by circulating CD8+ and CD4+ T cells in patients with SCCHN. To determine whether the TCR Vß restrictions observed in CD3+ T cells included CD8+ as well as CD4+ T cells, we analyzed the TCR Vß usage in these T-cell subsets using PBMCs of additionally recruited cohorts of 14 patients with SCCHN and seven normal donors. The percentages and ratios of CD4+ to CD8+ T cells in PBMCs of the SCCHN patients were comparable with those detected in normal donor PBMCs as previously reported (17). The box and whisker analysis of TCR Vß usage by CD4+ and CD8+ T cells in PBMCs obtained from seven normal donors is shown in Table 4 . Based on these values and our definitions of expansion and contraction, numerous expansions and contractions of TCR Vß-restricted specificities could be identified in the CD4+ and CD8+ T cells present in the patients' PBMCs. The changes in TCR Vß usage by CD4+ T cells (Fig. 2 ), however, were less frequent and relatively modest compared with those observed in CD8+ T cells present in the same samples (Fig. 3 ). The total count of individual TCR Vß restrictions was greater in CD8+ T cells compared with CD4+ T cells. These data are consistent with a selective expansion or contraction of CD8+ T cells expressing certain TCR Vß specificities in the peripheral circulation of patients with SCCHN. Comparing the profiles of the TCR Vß restrictions identified in CD4+ and CD8+ T cells of patients with those of normal donors by cluster analysis reveals that the profiles of TCR Vß restrictions in CD8+ T cells of patients are more distinct from those of normal donors than the profiles of the CD4+ T cells are (Fig. 4 ). These results are also consistent with our hypothesis that homeostasis of the CD8+ T-cell subset is modulated in patients with SCCHN, and that changes in the TCR Vß repertoire in PBMCs reflect expansions and contractions of antigen-responsive T effector cells.


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  		Table 4. Box and whisker analysis of TCR Vß usage by circulating CD4+ and CD8+ T cells in PBMCs obtained from seven normal donors

 

Figure 2
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  		Fig. 2. Expansions and contractions of TCR Vß usage by CD4+ T cells in PBMCs obtained from patients with SCCHN. Percentage changes in TCR Vß usage in CD4+ T cells obtained from 14 patients relative to either upper or lower whisker values for TCR Vß family usage by CD4+ T cells in PBMCs of seven normal donors, as listed in Table 4.

 

Figure 3
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  		Fig. 3. Expansions and contractions of TCR Vß usage by CD8+ T cells in PBMCs obtained from patients with SCCHN. Percentage changes in TCR Vß usage in CD8+ T cells in PBMCs obtained from 14 patients relative to either upper or lower whisker values for TCR Vß family usage by CD8+ T cells in PBMCs of seven normal donors, as listed in Table 4.

 

Figure 4
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  		Fig. 4. Hierarchical cluster analysis of the profiles of TCR Vß usage by CD4+ and CD8+ T cells in PBMCs of patients with SCCHN and normal donors. A hierarchical cluster analysis for TCR Vß expression in CD4+ (A) and CD8+ (B) T cells present in PBMCs of 14 patients with SCCHN (P#19-32) and seven normal donors (HD#11-17). For CD8+ but not CD4+ T cells, patients (red) clustered separately from controls (blue).

 
Annexin V binding to TCR Vß-restricted CD8+ T cells in PBMCs obtained from SCCHN patients. We previously showed that CD8+ T cells have significantly higher rates of apoptosis than CD4+ T cells in the circulation of patients with SCCHN and age-matched normal donors (14, 16). When PBMCs obtained from additional 13 patients with SCCHN were analyzed in this study for Annexin V binding to CD8+ T cells, the mean % ± SD of Annexin V+CD8+ T cells detected was 17.7 ± 11.7%, with a range from 0% to 67% (data not shown). In normal donors, it was ≤3% and conformed to our previously reported results (16). Given the distinctive TCR Vß profiles of the CD8+ T-cell subset in PBMCs of patients with SCCHN relative to those in PBMCs of normal controls (Fig. 4), it was reasonable to ask whether spontaneous apoptosis of these cells contributed to changes in TCR Vß profiles (i.e., whether Annexin V binding was preferentially targeted to TCR Vß-expanded or Vß-contracted CD8+ T cells). We reasoned that if TCR Vß restrictions or contractions observed in CD8+ T cells reflect TCR Vß repertoire changes due to rapid expansion and/or demise of T cells, then enhanced apoptosis of TCR Vß-restricted CD8+ T-cell populations would be expected. Although considerable variation in Annexin V binding to TCR Vß-expanded or Vß-contracted versus non–TCR Vß-restricted CD8+ T cells within PBMCs in each patient was detected (Fig. 5 ), statistically significant differences in Annexin V binding between TCR Vß expansions, contractions, or non–TCR Vß-restricted CD8+ T cells were only detected in samples of 3 of 13 patients (patients 39, 40, and 43). In PBMCs of patients 39 and 40, the percentages of Annexin V binding TCR Vß expansions were higher relative to contractions or non–TCR Vß-restricted T cells. For example, in patient 39, only 4.7 ± 2.2% of CD8+ T cells showing TCR Vß contractions bound Annexin V, a value significantly lower than the 16.3 ± 9.7% of non–TCR Vß-restricted CD8+ T cells and 23.2 ± 11.8% of CD8+ T cells showing TCR Vß expansions (P = 0.0015; Fig. 5). An example of enhanced Annexin V binding to an expansion of TCR Vß2+CD3+CD8+ T cells compared with non–TCR Vß-restricted T cells in PBMCs of patient 39 is shown in Fig. 6 . In contrast, the apoptotic rate of TCR Vß-constricted CD8+ T cells in PBMCs of patient 43 was significantly greater than that of the TCR Vß expansion or non–TCR Vß-restricted CD8+ T-cell populations (P = 0.0062). Consistent with previous findings (18), no significant differences were observed in the percentage of Annexin V binding CD8+ TCR Vß-restricted or Annexin V binding CD8+ TCR Vß nonrestricted T cells in patients with active disease versus those with no evident disease.


Figure 5
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  		Fig. 5. Annexin V (Anx) binding to TCR Vß-restricted CD8+ T cells in PBMCs obtained from patients with SCCHN. TCR Vß expression was classified as expanded (+), restricted (–), or nonrestricted (=). Box-and-whisker plots for percentages of CD8+ TCR Vß+ANX+ T cells for all TCR Vß specificities in each of the three groups were then plotted for each patient. P values for the Kruskal-Wallis test, comparing the percentages of CD8+Vß+ANX+ T cells among the three groups are below the box plots.

 

Figure 6
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  		Fig. 6. Restricted Vß2 subset and Annexin V (ANX) binding to CD8+ T cells in the circulation of a patient with SCCHN. Cells were stained with anti-TCR Vß12/FITC, anti-Vß5.2/PE, and anti-Vß2/FITC-PE (doubly labeled) antibodies. To evaluate Annexin V binding to TCR Vß2+ T cells, the gate was set as shown by the upper arrow. To evaluate Annexin V binding to TCR Vß2–T cells, the gate was reset as shown by the lower arrow. Note that cell numbers were much greater in the lower than the upper frame.

 

   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
References
 
The analysis of PBMCs and TILs obtained from patients with SCCHN was designed to show that the TCR Vß repertoire distinguished these patients from normal donors and to test the hypothesis that TCR Vß-restricted T cells are especially sensitive to spontaneous apoptosis. The data suggest that TCR Vß restrictions, including expansions and contractions of the Vß profiles, and increased levels of apoptosis of CD8+ T cells present in these samples may be related to altered lymphocyte homeostasis in patients with cancer. It is well documented that lymphocyte homeostasis in the peripheral circulation is regulated by proliferation and survival of mature T cells (19, 20). Expansion of antigen-reactive T cells determines the immunologic profile of circulating T cells. At the same time, proliferation of T cells must be regulated, and those T cells that have completed their function are destined for apoptosis. Hence, a balance between antigen-driven expansion and apoptosis is maintained under physiologic conditions. This balance is reflected in TCR Vß repertoire of T cells in the peripheral circulation of normal donors, where all TCR Vß families are represented, and Vß restrictions are uncommon. In disease, including chronic infections and malignancies, this balance is lost, leading to a distorted or altered TCR Vß profile, which is manifested by expansion of certain T cells and a reduction of others (21).

To date, the vast majority of studies of TCR Vß restrictions in disease have focused on analyses of T cells that expanded, presumably in response to disease-relevant antigens (22, 23). In cancer, including SCCHN, TCR Vß restrictions are frequently seen among TILs and/or PBMCs (1, 5–9). Because certain TCR Vß restrictions are often shared in patients with the same type of cancer, as determined by a sensitive spectratyping for the TCR Vß variable complementarity-determining region 3, it has been suggested that they represent expansions of TA-specific T cells (4–7). In some cases, TA specificity of such TCR Vß-restricted T cells has been confirmed (24, 25). A more recent analysis suggests that TCR Vß profiles in patients with cancer reflect expansions of clonal as well as non–clonal-reactive T cells (26), and that contractions of TCR Vß-restricted T cells are common.

It has been observed that the presence of TA-specific CD8+ T cells (as defined by various in vitro assays) at tumor sites or in the circulation of patients with cancer does not correlate with improved prognosis or better survival (reviewed in ref. 27). This has been attributed to the state of T-cell dysfunction in patients with cancer, which primarily affects T-cell subsets responsible for antitumor activity (28). Several distinct mechanisms, including rapid terminal differentiation and apoptosis of effector CD8+ T cells, seem to be responsible for this dysfunction and to contribute to tumor escape, disease progression, and its recurrence (reviewed in ref. 29). A demise of antitumor effector cells and decreased absolute numbers of T-cell subsets in patients with SCCHN are a consequence of tumor-associated immune suppression (16, 17). The current report shows that profound and widespread alterations in the TCR Vß repertoire of CD3+ T cells exist in TILs and PBMCs of patients with SCCHN. The alterations consist of numerous expansions and contractions of various TCR Vß specificities. In general, TCR Vß contractions outnumber expansions. These types of changes in TCR Vß repertoire of T cells in SCCHN were shown to be highly significant by cluster analysis in the CD8+ T-cell subset but not in the CD4+ T-cell subset.

We have previously reported that turnover of lymphocytes is enhanced in the peripheral circulation of patients with SCCHN, based on TREC analyses, changes in the phenotype of naive versus memory pools, and decreased absolute counts of T-cell subsets (17, 30, 31). This rapid turnover is orchestrated by enhanced apoptosis of activated CD8+ T cells (16, 30). Because CD8+ and not CD4+ T cells are sensitive to spontaneous apoptosis in the peripheral circulation or at the tumor site in cancer patients, our current observations suggest that the T-cell repertoire might well be shaped by enhanced apoptosis of antigen-driven, TCR Vß-restricted populations of CD8+ T cells. As most tumor antigens are "self" in nature (32), enhanced levels of apoptosis in circulating CD8+ T cells in cancer patients could be responsible for persistent "editing" of the TCR Vß repertoire and for maintaining peripheral tolerance.

Our earlier finding that CD8+ T cells bound Annexin V at significantly higher levels in patients with cancer than in normal donors (18) was confirmed in this study and suggested that survival of Vß-restricted, antigen-responsive CD8+ T cells may be shorter in the circulation of patients than normal donors, whose T cells bind little, if any, Annexin V. We next considered a possibility that Annexin V binding targeted TCR Vß-restricted CD8+ T cells. Annexin V binding detects early changes (phosphatidylserine "flip") in the cell membrane of preapoptotic cells, which are marked for apoptosis and rapidly removed by the reticuloendothelial system (33). As actively expanding T cells must be regulated, activation-induced cell death could be the mechanism responsible for the onset of apoptosis. Annexin V binding to TCR Vß-restricted subsets of CD8+ T cells was, therefore, used to determine sensitivity of expanded as well as contracted subpopulations to spontaneous apoptosis. The expectation was that populations of TCR Vß-restricted CD8+ T cells representing antigen-responding cells might bind more Annexin V than all other (non-Vß restricted) CD8+ T cells. However, as shown in Fig. 5, we observed a broad variability in Annexin V binding to CD8+ T cells in different TCR Vß families within each patient. The greater tendency for Annexin V binding to TCR Vß-expanded than Vß-contracted CD8+ T cells can be seen (Fig. 5) but is not statistically significant, except in 2 of 13 patients (patients 39 and 40). In one patient (patient 43), Vß-constricted CD8+ T cells bound most Annexin V. Thus, showing enhanced apoptosis of TCR Vß-restricted CD8+ T-cell populations was apparently more difficult than anticipated, perhaps, due to the overall increased rate of Annexin V binding to patients' CD8+ T cells relative to those in normal donors, which might well mask the enhanced Annexin V binding to circulating TCR Vß-restricted antitumor CD8+ T cells in patients with SCCHN.

The profile of TCR Vß restrictions seen by fluorescence-activated cell sorting analysis of CD8+ T cells in the peripheral circulation of SCCHN patients is also consistent with the rapid turnover of lymphocytes. Although the fluorescence-activated cell sorting analysis we performed did not permit us to identify the antigenic specificity of CD8+ T cells that underwent apoptosis, our other results involving analysis of Annexin V binding to tetramer-positive, tumor peptide-specific, or viral-specific CD8+ T cells suggest that enhanced apoptosis of T cells in the circulation of cancer patients is antigen specific but not necessarily tumor antigen specific.5 Nevertheless, tumor antigen-specific, TCR Vß-restricted CD8+ effector T cells are undoubtedly caught in this rapid turnover of lymphocytes, and their survival is impaired. The pronounced alterations in the TRC Vß-restricted profile of CD8+ T cells in PBMCs of patients with SCCHN together with general enhanced apoptosis of CD8+ T cells suggest that a shortened survival of antigen-responsive T cells is a characteristic feature of this cancer. Similar changes in the T-cell repertoire have been reported in cutaneous T-cell lymphoma, HIV, and after myeloablative bone marrow transplantation (20, 34, 35). The overall impression is that alterations of the T-cell repertoire characterized by short-lived expansion of few antigen-reactive T cells and contraction or depression of others are the hallmark of diseases associated with rapidly increased turnover of circulating T cells.


   Footnotes
 
Grant support: NIH grants PO-1 DE-12321, RO-1 DE-13918, and RO-1 CA-82016 and The Stout Family Fund for Head and Neck Cancer Research at The Eye and Ear Foundation of Pittsburgh.

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.

Note: A. Albers and C. Visus equally contributed to this work.

5 A. Albers et al. Spontaneous apoptosis of epitope-specific tetramer+ CD8+ T lymphocytes in the peripheral circulation of patients with cancer. Submitted for publication. Back

Received 8/18/05; revised 1/31/06; accepted 2/ 9/06.


   References
Top
 Abstract
 Patients and Methods
 Results
 Discussion
 References
 

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