Abstract
CD8+ T cells in the circulation of patients with head and neck cancer (HNC) were previously shown to be significantly more sensitive to, and preferentially targeted for, apoptosis than CD4+ T cells (Hoffmann et al., Clin Cancer Res, 8:2553–2562, 2002). To distinguish global from CD8+ subset-specific apoptosis, we studied Annexin-binding to naïve, memory, and effector subsets of CD8+ cells by multicolor flow cytometry. Age-related changes in naïve and effector CD8+ cell subsets were observed in patients and normal controls (NC). The frequencies of naïve (CD28+CD45RO-) CD8+ T cells were lower and those of memory (CD28+CD45RO+) and effector (CD28-) CD8+ T cells significantly higher in the circulation of HNC patients relative to age-matched NC. Among CD8+ T cells, the CD28- effector cell subset contained the highest proportion of Annexin-binding cells, while the naïve CD28+CD45RO- subset contained the lowest. This suggested a high turnover rate of the CD8+CD28- effector cell subset in patients with HNC, which was being compensated by a rapid transition of naïve CD8+ T cells to the effector cell pool. Following tumor resection, the frequency of CD8+CD28- T cells normalized in the patients, an indication that the presence of tumor had an influence on the size of CD8+CD28- T-cell pool. Ex vivo, in mixed lymphocyte-tumor cultures (MLTC) with semiallogeneic T cells as responders, CD8+CD28- T cells could be generated from CD8+CD28+ cells by repeated stimulations with tumor cells. These CD8+CD28- effector cells lysed the tumor, produced IFN-γ in response to the tumor, and strongly expressed granzyme B. Thus, the high rate of their apoptosis in the circulation of patients with HNC might be expected to contribute to tumor progression. However, the ex vivo generation of this cell subset was suppressed by strong CD28/B7 ligation or by overexpresson of MHC molecules on tumor cells, suggesting that adequate costimulation is necessary for protection from apoptosis. It appears that interactions of immune and tumor cells might determine the fate of this terminally differentiated effector cell subset.
Keywords: Apoptosis, CD8+ T cells, CD8+CD28- subset, Head and neck cancer
Introduction
T-cell–mediated immune responses play a key role in the control of tumor progression. Nevertheless, recent data indicate that tumors can escape from immune surveillance by a number of different mechanisms, including the down-regulation of tumor antigens and/or HLA molecules [28], production of a variety of immunosuppressive factors [37, 38], or induction of apoptosis in T lymphocytes [8, 10, 12, 23, 27, 31]. Histologic and flow cytometry results have shown that lymphocyte apoptosis, as manifested by DNA fragmentation, is frequently observed at the tumor site as well as in the peripheral blood of cancer patients [10, 25, 31], including those with head and neck cancer (HNC). Results from our laboratory indicate that CD8+ T cells are preferentially targeted for spontaneous apoptosis in patients with cancer [26]. However, it remains to be determined whether this represents a global demise of CD8+ T cells or a selective removal of distinct subsets of effector T cells.
Circulating human T cells are not homogenous but rather comprise many phenotypically and functionally distinct subpopulations. Broadly, they can be divided into naïve and memory subsets based on the expression of isoforms of the leukocyte common antigen, CD45RA or CD45RO, respectively [1, 13, 22]. Moreover, expression of CD28, a costimulatory molecule, divides T lymphocytes into CD28+ and CD28- subsets. CD28 is primarily expressed on nonprimed, naïve T lymphocytes and is required for their activation and generation of antigen-specific cytolytic effector cells through ligation of B7.1 (CD80) or B7.2 (CD86) on antigen-presenting cells (APC) [24]. CD28- T lymphocytes are considered to be antigen-experienced effector cells, which have down-regulated expression of this surface molecule as a consequence of a previous engagement with APC [32]. The frequency of CD28- T cells has been reported to be significantly increased in the circulation of patients with cancer [21, 29, 32], infectious diseases [4, 30], as well as in old healthy individuals [5].
In this study, we have evaluated CD8+CD28+ and CD8+CD28- subsets of T cells for sensitivity to apoptosis in the circulation of patients with head and neck cancer (HNC). We report that CD8+CD28- T cells are the apoptosis-sensitive subset. We also characterize the CD8+CD28- subset as comprising terminally differentiated effector T cells, based on a series of ex vivo experiments with semiallogenic T-cell lines generated by coincubation of normal PBMC with a squamous cell carcinoma of the head and neck (SCCHN) cell line.
Materials and methods
Patients and controls
Thirty-two patients (aged 36 to 79 years; mean 61 years) with histologically proven squamous cell carcinoma of the head and neck (SCCHN) participated in the study after signing the IRB-approved informed consent. All patients asked to participate in the study were seen at the outpatient clinic of the Department of Otolaryngology. The patients were divided into two groups based on clinical evaluation: (1) 18 patients with active disease (AD), aged 36 to 76 years, mean 61 years; and (2) 14 patients with no evidence of disease (NED), aged 44 to 79 years, mean 61 years. The patients were not treated with chemotherapy or radiotherapy at the time of blood draws. Also, 30 healthy adult volunteers (aged 36 to 79 years; mean 59 years) participated in this study as normal controls (NC).
Lymphocyte separation
Venous blood was obtained from patients and controls. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque gradient centrifugation, washed and counted in the presence of the trypan blue dye to determine the cell number and viability. The cells were immediately evaluated for Annexin binding or cryopreserved for other studies.
Antibodies
The fluorochrome-labeled monoclonal antibodies used for staining of PBMC were FITC- or PE-conjugated anti-CD28 (CD28.2), ECD-conjugated anti-CD3 (UCHT1), anti-CD45RO (UCHL1), and PC5-conjugated anti-CD4 (13B8.2). All were purchased from Immunotec, France. In addition, PC5-conjugated anti-CD8 (SCCI21Thy2D3) purchased from Beckman Coulter (Miami, Florida); PE-conjugated anti-CD4 (Leu-3a) purchased from Becton Dickinson (San Jose, California); and PE-conjugated anti-CD95 (DX2) purchased from Pharmingen (San Diego, California). As negative controls, the respective Ig isotypes were purchased from Immunotec.
Staining of PBMC
In preparation for flow cytometry, staining of PBMC was performed as follows: cells (1×106) were placed in wells of a 96-well round bottom plate. An aliquot (1 μl) per well of an antibody was added, and the plate was incubated at 4°C for 30 min. After incubation, the cells were washed twice in FACS buffer and transferred to a flow tube. All antibodies were pretitered on normal PBMC to determine their optimal dilutions. Ig isotype controls were included in all experiments.
Flow cytometry
Four-color flow cytometry was performed on a Coulter Epics XL cytometer with a single 488-nm argon ion laser. At least 1×105 events were acquired for each sample. The amplification and compensation were set according to the standard procedure, using negative controls and tested cells stained in a single color or combination of colors (FL1, FITC-CD28; FL2, PE-CD4; FL3, ECD-CD3; and FL4, PC5-CD8). For evaluations of CD8+ T-cell subsets the gate was set on CD8bright T lymphocytes.
Annexin V binding assay
Annexin V binding to T lymphocytes was evaluated by multicolor flow cytometry. After surface staining with anti-CD28, anti-CD45RO, and anti-CD4 or anti-CD8 Abs, PBMC were washed once with FACS buffer and resuspended in Annexin-binding buffer (PharMingen), followed by incubation with FITC-conjugated Annexin V (PharMingen) for 15 min at room temperature. The events were acquired by flow cytometer within 60 min of staining. The percentages of apoptotic cells were calculated by scoring Annexin V–binding cells after backgating on CD4+ cells or CD8+ cells. All gated mononuclear cell populations were visualized on forward angle scatter/side angle scatter (FSC/SSC) dot plots, and the gate was set to eliminate cellular debris, which binds Annexin V and still may express CD3. Next, a cutoff was set using unstained control cells. To set the second (higher) cutoff, Jurkat cells pretreated with CH11 Ab to induce apoptosis were utilized. This allowed for the elimination of "dim" Annexin-staining cells, which were not apoptotic based on simultaneous staining of the positive control cells with Propidium Iodide (PI). This strategy of four-color staining combined with stringent gating provided the means for eliminating a majority of debris and for clear-cut discrimination between live and apoptotic cells among subpopulations of T lymphocytes in the gate. The debris, which was excluded using this gating, generally contained from 23±8% to 22±13% of Annexin+ events in patients and controls alike.
Intracytoplasmic staining
Effector cells were stimulated with PCI-13 or 1 ng/mL of phorbol 12-myristidate 13-acetate (PMA; Stigma, St. Louis, Missouri) and 1 μM of ionomycin (Sigma) for 24 hr at 37°C, in an atmosphere of 5% CO2 in air. At the end of the incubation period, cells were suspended in FACS buffer and stained with labeled antibodies selected to surface markers as described above. Next, cells were fixed for 10 min with 1% (w/vol) paraformaldehyde in PBS and washed with the permeabilization buffer (0.1% [w/vol] saponin in PBS). The cells were stained with FITC-conjugated anti-IFN-γ Ab (Pharmingen) or anti-granzyme B Ab (Alexis, San Diego, California) for 30 min at 4°C. As a negative control, cells were stained with mouse IgG1 or IgG2a isotype control (Becton Dickinson). Cells were washed once with permeabilization buffer and once with FACS buffer, and then analyzed by flow cytometry.
Generation of semiallogeneic CTL
The HLA-A2+ SCCHN cell line, PCI-13 was previously established from a moderately differentiated tumor and retrovirally transfected with the B7.1 gene (17). Tumor cells were maintained in DMEM supplemented with 10% fetal bovine serum, 1-mM l-glutamine, 100-μl/ml penicillin and 100-μg/ml streptomycin. Tumor cells were pretreated with 1000 IU/ml of IFN-γ (Roussel UCLAF, France) for 48 hr to enhance expression of MHC class I molecules prior to irradiation and were used as stimulator cells. PBMC from three HLA-A2+ healthy donors were coincubated with 1×105 irradiated (100 Gy) PCI-13 [14], or B7-transfected PCI-13 [17] at an effector/stimulator cell ratio of 10:1, and maintained in culture in the complete medium composed of AIM-V supplemented with 10% human AB serum. This stimulation was repeated weekly. IL-2 (20 U/ml) obtained from Chiron Cetus, Emeryville, California, was added to the medium beginning with the second cycle of stimulation.
Cytotoxicity assay
A 4-h 51Cr-release method was used for analyzing cytotoxicity of effector T cells at the effector/target ratio ranging from 2.5 to 50 as previously described [36]. To remove NK activity, 5×104 of K562 cells were incubated with effector cells for 30 min prior to the cytotoxicity assay. Aliquots of 1×103 of PCI-13 target cells were then added to each tube. The assay was performed in triplicate, and the percent specific lysis was calculated by the following formula: % specific lysis = [(maximal cpm − spontaneous cpm) / (experimental cpm − spontaneous cpm)] x 100.
Statistical analysis
The statistical significance of differences in expression of apoptotic markers between groups was determined by nonparametric Mann-Whitney U test for the unpaired analysis and Wilcoxon matched pairs test for the paired ansalysis. Linear regression analysis was performed to evaluate the influence of age, using Spearman's test. P values less than 0.05 were regarded as significant.
Results
Distribution of naïve, memory, and effector subsets of CD8± T cells in patients and NC
It has been previously suggested [2] that CD8+ T cells are heterogenous, comprising at least three distinct subsets: CD28+CD45RO- (naïve), CD28+CD45RO+ (memory) and CD28- (effector cells). By multicolor flow cytometry, backgating on CD8bright PBMC, it is possible to discriminate between these subsets (Fig. 1). In NC, CD28- T cells are few, and the naïve CD28+CD45RO- T cells are a major CD8+ subpopulation. However, in patients with HNC, the proportions of CD28+ subsets are decreased, while a significant increase in CD28- effector T cell is apparent (Fig. 1).
Fig. 1.
Distribution of naïve, memory and effector CD8+ T cells in the peripheral circulation of a normal control and a HNC patient. Representative cytofluorographic profiles are presented. Note diminished expression of CD28 on CD8+CD45RO+ T cells in the patient
Age-dependent changes in naïve, memory, and effector subsets of CD8± T cells
Patients with HNC are generally older than 50 years of age. Therefore, before comparing patients to NC, it was necessary to consider their age, since immune parameters could be altered in aging individuals. Indeed, as shown in Fig. 2, significant age-related changes were observed in the naïve and effector subsets of CD8+ T cells both in patients with HNC and NC. Thus, the proportions of naïve CD8+ T cells decreased, while those of CD28- effector T cells increased with age in both HNC and NC. In contrast, the memory T cells did not seem to significantly change with age (Fig. 2).
Fig. 2.
Proportions of naïve, memory, and effector CD8+ T-cell subsets relative to age in NC and patients with HNC
The frequency of CD8 T-cell subsets in HNC patients vs NC
Our data indicated that comparisons in the frequency of the naïve and effector subsets of CD8+ T cells between HNC patients and NC, would require that age-matched cohorts be used. We therefore matched the cohorts of HNC patients and NC in age within a 4-year window in order to compare the relative mean percentages of CD8+ T-cell subsets. The mean proportion of naïve CD8+ T cells was found to be decreased in HNC patients relative to controls (p<0.01), while that of memory (p<0.05) and effector (p<0.05) CD8+ T cells increased, as illustrated in Fig. 3.
Fig. 3.
Mean frequencies of naïve, memory, and effector subsets of CD8+ T cells in the circulation of age-matched NC (n=30) and patients with HNC (n=29). The data are means ± SD
Annexin V binding to CD8± T cells
We previously reported that CD8+ T cells preferentially bound Annexin V relative to CD4+ T cells and that proportions of such Annexin+CD8+ T cells were found to be elevated in patients with HNC [15]. We therefore examined binding of Annexin V to CD8bright cells and their subsets in our cohorts of patients and NC. As shown in Fig. 4A, HNC patients had a significantly higher total percentage of Annexin-binding CD8bright cells in the peripheral circulation than did NC (p<0.01), confirming our previous results. Because the frequency of CD8+CD28- T cells was significantly increased in HNC patients relative to NC, we did not expect to observe much apoptosis in this subset of effector cells. Surprisingly, CD28- T cells contained the highest proportion of Annexin+ cells among the three CD8+ T-cell subsets (CD8+CD28-Annexin V+), while the naïve CD28+CD45RO- subset contained the lowest (Fig. 4B). This counterintuitive result could be explained either by proliferation of CD8+CD28- cells or by cell influx from the naïve T-cell pool, which compensated for the loss of CD8+CD28-Annexin+ effector cells. This, in turn, implies that CD28- effector cells had a rapid turnover rate in patients with HNC. Both memory and effector CD8+ T-cell subsets contained a substantially higher proportion of CD95+ cells, as compared with naïve CD8+ T cells: 80–90% vs 15–30%, with patients consistently in the upper range.
Fig. 4.
A Percentages of Annexin V–binding CD8bright cells in the circulation of NC and patients with HNC (means ± SD). B Percentages of Annexin V–binding cells among the three subsets of CD8+ T cells (means ± SD)
In vivo changes in CD8± T-cell subsets after surgery
To determine whether the observed increased frequency of CD8+CD28- effector cells in HNC patients relative to NC was related to the presence of tumor, CD8+ T-cell subset analysis was performed before and after surgery. In five cases, we were able to obtain paired peripheral blood specimens. In three of five cases, normalization of the subset frequencies was observed after surgery, with a decrease in the percent of CD28- effector cells and an increase in the proportion of naïve CD28+CD45RO- T cells (Fig. 5). The proportion of CD95+ cells decreased slightly in the CD28- effector cell subset after surgery (data not shown). Although limited, these data suggest that the presence of tumor has an effect on the homeostasis of the CD8+ T-cell subsets, including that of CD8+CD28- effector cells.
Fig. 5.
Changes in the frequency of the subsets of naïve, memory, and effector CD8+ T cells in HNC patients after surgery. Pairs of PBMC obtained from five patients were monitored before and 1 month after tumor resection
Down-regulation of CD28 on tumor-reactive effector cells
A series of ex vivo studies was next performed to investigate whether the observed in vivo loss of CD28 from the surface of CD8+ T cells could be related to antigenic stimulation in the tumor microenvironment. Semiallogeneic T-cell lines were generated from PBMC of three normal HLA-A2+ donors in mixed lymphocyte tumor cultures (MLTC), using the PCI-13 cell line (HNC-derived, HLA-A2+) as a stimulator. The T-cell lines were tested for PCI-13–specific cytotoxicity in 4-h 51Cr-release assays performed after NK activity was removed by preincubation of T cells with K562 targets. Expression of CD28 molecules on CD8+ T cells and their cytotoxicity were repeatedly measured over a culture period of 12 weeks. Stimulations with the tumor were performed weekly. Early in MLTC, most cells had the CD8+CD28+CD45RO+ phenotype and mediated moderate but detectable antitumor activity (Fig. 6A). In the course of culture with repeated antigenic stimulations, gradual down-regulation of CD28 was observed in CD8+ cells, until more than 60% were CD28-. This gradual shift from the CD28+ to CD28- phenotype was accompanied by increasing antitumor cytotoxicity (Fig. 6A) and increased Annexin V binding, indicating that CD28- T cells were sensitive to apoptosis. When about 50–70% of the cells became CD28- (e.g., 9 weeks in culture), the down-regulation of CD28 leveled off and cytotoxicity of the population began to decline, while Annexin V binding was observed in 25–35% of the cells. These ex vivo experiments confirm that CD28+CD8+ T cells lose CD28, retain cytolytic function upon chronic (repeated) exposure to tumor antigens, and upon differentiating to CD28- cells become sensitive to apoptosis.
Fig. 6A, B.
Ex vivo model for generation of CD8+CD28- effector cells in MLTC. A Phenotypic changes in CTL generated from donor #3 were monitored at various time periods in culture after repeated stimulations with PCI-13 cells. Cytotoxic activities against PCI-13 were expressed as the % specific lysis at the E:T ratio of 10:1 for each time point (see the upper left quadrant). Circled areas indicate changes over time in the proportion CD8+CD28- T cells. B Comparisons of CD28 expression on CTL generated from PBMC of donor #1 repeatedly stimulated with untreated PCI-13, IFN-γ–treated PCI-13 or IFN-γ–treated B7-transfected PCI-13 and tested on week 6. Numbers next to the circled areas are percentages of CD28- cells among CD8+CD45RO+ T cells
On week 6 of these cultures, when antitumor cytotoxicity was high and CD28- cells began to reappear and constituted a substantial proportion of CD8+ T cells (Fig. 6A and B), we restimulated the culture with PCI-13 cells and 24 h later, performed intracytoplasmic staining for IFN-γ and granzyme B. By flow cytometry, it was possible to discriminate between CD28+ and CD28- cells and to define their functions (Fig. 7). We observed that both CD8+CD28+ and CD8+CD28- cells had strong granzyme-B and IFN-γ expression in response to the tumor (Fig. 7). In these cultures, the expanding CD28- cell population retained substantial antitumor functions.
Fig. 7A, B.
Intracellular expression of IFN-γ (A) and granzyme B (B) on the CTL generated in MLTC as described in the legend to Fig. 6. The CTL were generated from PBMC of donor #2 and were tested on week 6, at the time when cytotoxic activity against PCI-13 was around 70% as measured at the E:T ratio of 10:1. Numbers are percentages of cells within the areas defined by each quadrant. Controls were performed as described in "Materials and Methods"
Finally, when PBMC were cultured in the presence of IFN-γ treated PCI-13, which had significantly enhanced MHC class I expression (data not shown), down-regulation of CD28 was diminished (Fig. 6B). When PCI-13 transduced with the human B7.1 gene and overexpressing B7.1 was used for weekly stimulations, the shift from CD28+ to CD28- effector cells was suppressed (Fig. 6B). These data indicate that strong ligation of TCR by MHC molecules or of B7.1 by CD28 prevented down-regulation of CD28 in tumor-stimulated CD8+ T cells. Furthermore, no or little apoptosis was detected in these cultures.
Discussion
We have previously reported that preferential apoptosis of CD8+ T cells as compared with CD4+ T cells occurs in the peripheral circulation of healthy individuals and patients with cancer [15]. In HNC patients, apoptosis of CD8+ T cells was significantly greater than that in normal controls [15], as also confirmed in the present study. The CD8+ T-cell population contains at least three phenotypically and functionally distinguishable cell subsets: CD28+CD45RO- (naïve), CD28+CD45RO+ (memory), and CD28- (effector) [13, 21]. These populations undergo age-dependent changes in the course of a lifespan [5, 19]. We have determined that in NC and HNC patients alike, naïve CD8+CD28+CD45RO- cells decrease with age, effector CD8+CD28- cells increase with age, while the pool of memory CD8+CD28+CD45RO+ cells remains relatively constant during adulthood.
Of special interest was the observation that in patients with HNC, the frequency of the effector (CD8+CD28-) T-cell population was markedly increased while that of naïve CD8+CD28+CD45RO- cells was decreased relative to normal age-matched controls. This shift from a normal profile suggested a more rapid transition of naïve CD8+ T cells to the memory or effector pool. Since these pools were expanded in HNC patients (Fig. 3), it appeared that the naïve subset contributed to their repopulation. In general, the mechanism(s) involved in cellular differentiation and emergence of CD8+CD28- effector T cells have not yet been clarified, but it has been suggested that chronic antigenic stimulation or exposure to TNF-α might regulate these events in vivo [2, 6, 7, 20]. It was thus plausible that the observed increase in the frequency of CD8+CD28- effector cells in the circulation of patients with HNC might reflect persistent antigenic stimulation related to the presence of disease. Normalization in the CD8+ T-cell profile observed after tumor removal suggested that this might be the case.
Although the frequency of CD8+CD28- cells was increased in HNC patients relative to NC, most of these cells were observed to bind Annexin V. Binding of Annexin V to cell surface reflects phosphatidyl serine "flip" and is a measure of early apoptosis [33]. Such early apoptotic T cells in the peripheral circulation are propidium iodide (PI) negative (personal observations), although they are poised for apoptosis [15, 27]. In the circulation of HNC patients, we consistently detected a significantly expanded pool of CD8+CD28- effector T cells, although 15–30% of these cells bound Annexin V. In patients, the early apoptotic cells represented a significantly higher proportion of the effector subset than of the other two subsets of CD8+ T cells. It appears that expansion of effector cells in the CD8+CD28- pool was able to counterbalance their death, largely due to rapid transition of naïve cells from the naïve to memory and effector cell compartments. This implies a rapid turnover of these cells in patients with HNC, which might be influenced by the presence of the tumor, because in a few HNC patients, who were evaluated prior to and after surgical resection, the proportions of CD8+CD28- effector cells and naïve CD28+CD45RO- cells normalized in the peripheral circulation.
The nature and origin of CD8+CD28- effector cells in humans remains controversial [13]. Their increased proportions as well as increased apoptosis rate in cancer patients might suggest that chronic antigenic stimulation drives the recruitment, differentiation, and ultimately death of these cells. To gain a better understanding of the events leading to the loss of CD28 molecules on CD8+ T cells relative to their functional status, a series of ex vivo experiments was performed. CD28+ T cells responding to the tumor were subjected to persistent antigenic stimulation in MLTC and simultaneously tested for expression of CD28 and the ability to kill the stimulating tumor targets. These experiments clearly showed that CD28- cells are derived from CD28+ precursors in the peripheral blood, and that they retain cytolytic function even after the 12th cycle of stimulation with the tumor (Fig. 6A). By multicolor flow cytometry, it was possible to show that IFN-γ and granzyme-B expression were associated with both the CD28+ and CD28- cell fractions in these cultures. Thus, CD8+CD28- T cells are terminally-differentiated, antigen-experienced effector cells that emerge and expand in response to chronic antigenic stimulation. A substantial proportion of these effector cells bind Annexin V, an indication that they are sensitive to apoptosis. The generation of CD8+CD28- cells was arrested when costimulatory (B7.1) signals or effective TcR/MHC ligation was delivered (i.e., in the presence of tumor pretreated with IFN-α or transduced with the B7 gene). These ex vivo experiments suggest that in the presence of strong (i.e., two-signal) costimulation, such as that normally delivered by APC [3, 11], the differentiation of CD8+CD28- effector cells is down-regulated. In contrast, when tumor is the antigen source, as in MLTC, weak ligation of TCR and weak costimulatory signals promote rapid expansion of CD8+CD28- effector cells and their apoptosis. Therefore, it appears that in the tumor microenvironment, terminal differentiation of CD28+ to CD28- T cells and apoptosis of the latter subset might reflect the lack of, or weak, costimulation in the presence of chronic or persistent antigen delivery, preferentially leading to apoptosis of CD28- effector cells [18, 34]. Under the circumstances of strong TCR/MHC and B7/CD28 interaction (i.e., two-signal activation) expansion of CD8+CD28+ subset of T cells is favored and is accompanied by down-regulation of their terminal differentiation and the CD28 antigen loss [9, 16, 35].
At this point, it is only possible to speculate that expansion of CD28- effector T cells and their concomitant increased apoptosis in the circulation of cancer patients are a consequence of disturbed homeostasis within the immune system. It is highly likely that the presence of tumor influences lymphocyte homeostasis, which after surgery might normalize, as observed in our patients. It is also possible that the presence of tumor shifts the immunologic balance toward rapid expansion of effector T cells and their concomitant demise. The result might be that terminally differentiated CD8+CD28- effector cells are rapidly driven to apoptosis and are less able to exercise their antitumor functions. The observations we report contribute to a better understanding of the balance in immune-mediated interactions between the tumor and the host and show how the disturbance of this balance might contribute to tumor progression.
Footnotes
Supported in part by NIH grants: PO-1 DE 12321 and RO-1 CA 82016 to Theresa L. Whiteside.
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