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Institute of Respiratory Diseases, First Affiliated Hospital, Guangxi Medical University, Nanning, Guangxi, People's Republic of China
ABSTRACT
Background: Active suppression by CD4+CD25+ regulatory T lymphocytes plays an important role in the downregulation of T-cell responses to foreign and self-antigens.
Objective: To analyze whether the CD4+CD25+ regulatory T lymphocytes exist and function normally in malignant pleural effusion.
Methods: The percentages of CD4+CD25+ T lymphocytes in pleural effusion and peripheral blood from patients with lung cancer with malignant effusion, pleural lavage and peripheral blood from patients with lung cancer without effusion, and peripheral blood from healthy control subjects were determined by flow cytometry. The expressions of forkhead transcription factor Foxp3 and cytotoxic lymphocyte-associated antigen-4 were also examined. CD4+CD25+ and CD4+CD25– T cells from pleural effusion and peripheral blood were isolated, and were cultured to observe the effects of CD4+CD25+ cells on proliferation response of CD4+CD25– T cells in vitro.
Main Results: There were increased numbers of CD4+CD25+ T cells in malignant pleural effusion from patients with lung cancer compared with pleural lavage from patients with lung cancer without pleural effusion, and that these cells have constitutive high-level expression of Foxp3 and cytotoxic lymphocyte-associated antigen-4. Furthermore, CD4+CD25+ T cells mediate potent inhibition of proliferation response of CD4+CD25– T cells, and anticytotoxic lymphocyte-associated antigen-4 monoclonal antibody could reduce the inhibitory activity of CD4+CD25+ T cells.
Conclusions: The increased CD4+CD25+ T cells found in malignant pleural effusion express high levels of Foxp3 transcription factor and potently suppress the proliferation of CD4+CD25– T cells, and cytotoxic lymphocyte-associated antigen-4 is involved in the suppressive activity of pleural CD4+CD25+ T cells.
Key Words: CD4+CD25+ T cells lung cancer pleural effusion regulatory T lymphocyte
Accumulation of lymphocytes in the pleural effusion (PE) frequently occurs in neoplastic effusions secondary to direct pleural involvement and/or metastases from malignancy (1, 2). However, these lymphocytes have very few natural killer cells and low antitumor cytolytic activity (3, 4). Malignant PE is frequently observed in lung cancer, and a diagnosis of malignant PE in lung cancer carries a poor prognosis (5, 6). In malignant PE, CD4+ T lymphocytes are dominant, and the proportion of CD8+ T cells is significantly lower than that of CD4+ T cells (7). In contrast, the proportion of CD4+ T cells in the pleural cavity of patients with lung cancer without malignant PE is significantly lower than that of CD8+ T cells (8). Invasion of cancer cells into the pleural cavity may be affected by both the nature of the cancer cells and host factors of patients with lung cancer.
Immunoregulatory T cells have long been believed to be involved in the control of the local immune response and in the growth of malignant tumors (9–12). Studies ongoing for more than a decade have provided firm evidence for the existence of a unique CD4+CD25+ T-cell population of "professional" regulatory/suppressor T cells that actively and dominantly prevent both the activation and the effector function of autoreactive T cells that have escaped other mechanisms of tolerance (13, 14). The involvement of such CD4+CD25+ T cells in patients with cancer was recently questioned by different groups. Some of them studied CD4+CD25+ T cells from peripheral blood (PB) mononuclear cells of patients with colorectal cancer or melanoma (15, 16), whereas others suggested the presence of these cells among tumor-infiltrating lymphocytes in breast, pancreas, lung cancer, ovarian, and, more recently, in Hodgkin lymphoma (17–19). However, the possible implication of CD4+CD25+ T cells in downregulating antitumor responses, which could explain the poor clinical response of patients with cancer under immunotherapeutic protocols, remains to be demonstrated. The present study investigated the idea that CD4+CD25+ T cells could be involved in the control of the local immune response in malignant PE. Our data provide the first evidence that CD4+CD25+ T cells infiltrating into human malignant PE behave as regulatory T cells. These regulatory T cells may play a role in inducing or maintaining tolerance to tumors in patients with lung cancer, and manipulation of this subpopulation could be an important component of cancer immunotherapy.
METHODS
Patients and Samples
The study protocol was approved by our institutional review board for human studies, and informed consent was obtained from all subjects.
PE samples were collected from 15 patients (8 men) with a median age of 54 yr (range, 35–70 yr) with newly diagnosed lung cancer with malignant effusion. Histologically, all samples were adenocarcinoma. A diagnosis of malignant PE was established by the demonstration of malignant cells in pleural fluid and/or on a closed pleural biopsy specimen. At the time of sample collection, none of the patients had received any anticancer treatment, corticosteroids, or other nonsteroidal antiinflammatory drugs. The PE was collected in heparin-treated tubes from each subject, using a standard thoracocentesis technique within 24 h after hospitalization. A pleural biopsy was performed after the collection of PE. The PE specimens were immersed in ice immediately.
After obtaining informed consent, pleural lavage (PL) was performed in 13 patients (7 men) with a median age of 51 yr (range, 32–73 yr) with resectable primary lung cancer not associated with PE. None of the patients had received any anticancer therapy before the study. Computed tomographic scanning was used to determine whether PE was present. Histologically, there were 10 adenocarcinomas and three squamous cell carcinomas. All of these patients were further confirmed to have no PE at the time of thoracotomy. PL was performed by the method described previously by Takahashi and colleagues (8, 20). The pleural cavity was lavaged with 1,000 ml of 0.9% NaCl solution at 37°C. The PL fluid was collected aseptically in heparin-treated tubes, which were immediately immersed in ice.
Both PE and PL samples were centrifuged at 1,200 rpm for 5 min; the cell pellets were resuspended in 15 ml Hanks' balanced salt solution for later use. Analyses of both PE and PL for total nucleated cell and differentials counts were performed.
Ten milliliters of PB from all studied patients and 14 healthy volunteers (6 men) with a median age of 45 yr (range, 28–63 yr) were drawn simultaneously for total and differential cell counts and isolating mononuclear cells.
Analysis of Lymphocyte Subsets
Mononuclear cells in PE, PL, and PB were separated by centrifugation on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient and were processed for flow cytometry to determine lymphocyte phenotype. Four-color flow cytometry was performed to determine phenotypes in T lymphocytes in PE, PL, and PB. The monoclonal antibodies (mAbs) used were anti–CD3-allophycocyanin (UCHT1), anti–CD4-fluorescein isothiocyanate (RPA-T4), anti–CD25-Cy-chrome (M-A251), and anti–CD152-phycoerythrin (BNI3; cytotoxic lymphocyte-associated antigen-4 [CTLA-4]). Appropriate isotype controls were performed for each experiment. All mAbs and controls were purchased from BD PharMingen (San Diego, CA). Briefly, cells were incubated in the dark at room temperature for 30 min with mAbs at the concentrations recommended by the manufacturer, washed once in fluorescence-activated cell sorter buffer (calcium/magnesium-free Hanks' balanced salt solution containing 1 mg/ml bovine serum albumin and 0.1 mg/ml sodium azide), and fixed with 2% formaldehyde. Flow cytometry was performed on a Coulter Epics XL-MCL flow cytometer using System II software (Beckman Coulter, Miami, FL).
Cell Isolation
In the preliminary experiments, we failed to obtain enough lymphocytes from PL for purifying CD4+CD25+ and CD4+CD25– T cells. In the present study, CD4+ T cells from PE and PB were purified by negative selection (by depletion of CD8+, CD11b+, CD16+, CD19+, CD36+, and CD56+ cells) with the Untouched CD4+ cell isolation kit (Miltenyi Biotec, Auburn, CA). After isolation of CD4+ T cells, CD25+ T cells were stained with phycoerythrin-coupled anti-CD25 mAbs and purified after the addition of anti–phycoerythrin-coupled magnetic beads (Miltenyi Biotec). Eventually, CD4+CD25+ T cells were obtained with a purity ranging from 90 to 95%. CD4+CD25– T cells were also collected, with a purity ranging from 80 to 90%.
Quantitative Real-Time Polymerase Chain Reaction
For quantitative real-time polymerase chain reaction (PCR) analysis of mRNA expression of the forkhead transcription factor Foxp3, total RNA was isolated from CD4+CD25+ or CD4+CD25– T cells by use of a total RNA extraction kit for mammalian RNA (Sigma Aldrich, St. Louis, MO) according to the manufacturer's instructions, and cDNA was prepared with 2.5-μM random hexamers (Applied Biosystems, Foster City, CA). Real-time PCR was performed with a LightCycler (Roche, Mannheim, Germany) according to the manufacturer's instructions, with the following cycling conditions: 10 min at 95°C, followed by 45 cycles of 15 s at 95°C, and 1 min at 60°C. All samples were run in triplicate, and data were expressed as normalized expression obtained by dividing the relative level for each sample by the relative level of GAPDH for the same sample, where GAPDH = 1. Primer sequences were as follows: GAPDH: 5'-CCACATCGCTCAGACACCAT-3' and 5'-GGCAACAATATCCACTTTACCAGAGT-3'; Foxp3: 5'-CAGCTGCCCACACTGCCCCTAG-3' and 5'-CATTTGCCAGCAGTGGGTAG-3'. The mean values from duplicates were used for calculations.
Proliferation Assay
To analyze proliferation in response to polyclonal stimulation, freshly isolated CD4+ T cells (5 x 104 cells), CD4+CD25– cells (5 x 104 cells) alone or with different numbers of CD4+CD25+ cells in a final volume of 200 μl of complete medium were incubated in 96-well round-bottomed plates in the presence of 10 μg/ml of platebound anti-CD3 plus 10 μg/ml soluble anti-CD28 mAbs. In some experiments, 10 μg/ml anti–CTLA-4 (BNI3; BD PharMingen) or 10 μg/ml IgG2 isotype mAbs were added to the cultures. After 4 d of culture at 37°C in a 5% CO2 humidified atmosphere in Roswell Park Memorial Institute 1640 medium supplemented with 10% normal human serum (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, [3H]thymidine (37 KBq/well) was added for an additional 16 h. [3H] Thymidine incorporation (cpm) was measured using a liquid scintillation counter.
Statistics
Values are presented as mean ± SEM. Nonparametric tests were used to analyze variables of PE, PL, and/or PB because these variables were not normally distributed. Comparisons of the data between different groups were performed using a Mann-Whitney U test or Kruskal-Wallis one-way analysis of variance on ranks. p values of less 0.05 were considered significant.
RESULTS
Increased Proportion of CD4+CD25+ T Cells in Malignant PE
Percentages of lymphocytes in total nucleated cells in PE, PL, and PB are illustrated in Figure 1A. Percentages of lymphocytes represented the highest values in malignant PE, showing a significant increase in comparison with those in PL and all PB samples from three groups of subjects (all p < 0.001). Lymphocytes from PE, PL, and PB from all subjects studied were analyzed by flow cytometry (Figure 1B). A total of 18.9 ± 1.7% of CD4+ T cells in PE were CD4+CD25+; in contrast, only 10.7 ± 0.6% of CD4+ T cells in PL had this phenotype (p < 0.001). CD4+CD25+ T-cell numbers in PB from patients with PE, patients with lung cancer without PE, and control subjects were 9.0 ± 0.6, 9.9 ± 0.8, and 11.5 ± 0.9%, respectively; they were not different statistically, and did not differ from those in PL (all p > 0.05), but all were lower than those in PE (all p < 0.001).
PE CD4+CD25+ T Cells Express the Foxp3 Transcription Factor
Foxp3 was recently described as an important transcription factor involved in the development and function of CD4+CD25+ T cells and appears to be the most specific molecular marker available to date (21). In the present study, Foxp3 mRNA expression was analyzed by real-time PCR in both purified CD4+CD25+ and CD4+CD25– T cells in PE and PB from six patients with PE, and PB from six healthy control subjects. As expected, PB CD4+CD25+ T cells from both healthy subjects and patients with PE expressed high levels of Foxp3, whereas their PB CD4+CD25– T cells expressed only very low levels (Figure 2). Similarly, we also noted that PE CD4+CD25+ T cells highly expressed Foxp3, whereas PE CD4+CD25– T cells expressed very low levels. Thus, these data revealed that CD4+CD25+ T cells infiltrating malignant PE express the Foxp3 transcription factor, strongly suggesting that this population includes regulatory T cells.
Surface Expression of CTLA-4 on PE CD4+CD25+ T Cells
Recent studies have shown that CTLA-4 is upregulated on mouse and human CD4+CD25+ T cells (22, 23). Therefore, we analyzed the lymphocytes from PE, PL, and PB for expression of CD4, CD25, and CTLA-4 by flow cytometry. As shown in Figure 3, in PE CD4+CD25+ T cells, 77.4 ± 3.3% were positive for CTLA-4, whereas only 26.6 ± 2.3% of PL CD4+CD25+ T cells were positive for CTLA-4 (p < 0.001). In contrast to the high expression of CTLA-4 on PE CD4+CD25+ T cells, only 24.2 ± 1.8% of PB CD4+CD25+ T cells from patients with lung cancer with PE and 24.1 ± 2.3% from those without PE stained positive for CTLA-4; both were similar to those in normal control PB (25.1 ± 2.0%, all p > 0.05).
Inhibition of Proliferation of CD4+CD25– T Cells by CD4+CD25+ T Cells
To assess the function of CD4+CD25+ T cells in PE, we separated CD4+CD25+ and CD4+CD25– T cells from PE and PB of six patients with PE, as well as PB from six healthy subjects, then determined their proliferative capacity and the effect of CD4+CD25+ T cells on CD4+CD25– T-cell proliferation. As shown in Figure 4A, proliferative responses to anti-CD3 and CD28 mAbs were observed in the total CD4+ T-cell populations from PE and their counterparts in PB from both patients with PE and healthy subjects. On depletion of the CD4+CD25+ T cells, the proliferation of the remaining CD4+CD25– T-cell population was increased significantly, whereas proliferation of CD4+CD25+ T cells from the three groups was low, indicating that PE CD4+CD25+ T cells, as those in normal PB, were anergic. When CD4+CD25– T cells were cocultured with CD4+CD25+ T cells, proliferative response of CD4+CD25– T cells decreased as numbers of CD4+CD25+ T cells increased. We also noted that no difference could be observed in suppressive capacity of PE CD4+CD25+ T cells and PB CD4+CD25+ T cells (Figure 4B). When the ratio of CD4+CD25– T cells and CD4+CD25+ T cells was 1:1, the mean percentage inhibition values of the T-cell response were 69.6 ± 2.1% in PE (n = 6), 72.0 ± 1.7% in autologous PB (n = 6), and 70.1 ± 2.6% in control PB (n = 6); these values were almost the same (all p > 0.05).
The nature of molecules present on the surface of CD4+CD25+ T cells that were responsible for their suppressive activity was also investigated in the present study. To do this, different numbers of purified CD4+CD25+ T cells were added to cultures of 5 x 104 CD4+CD25– T cells in the absence or presence of neutralizing anti–CTLA-4 or isotype control mAbs. The results of these experiments are summarized in Figure 5. The addition of isotype control mAbs did not exert any effect on the inhibitory activity of CD4+CD25+ T cells on the proliferative response of CD4+CD25– T cells. By contrast, the addition of anti–CTLA-4 mAbs partially, but consistently, reduced the inhibitory activity of CD4+CD25+ T cells.
DISCUSSION
Studies ongoing for more than a decade have provided firm evidence for the existence of a unique CD4+CD25+ population of "professional" regulatory/suppressor T cells that actively and dominantly prevent both the activation and the effector function of autoreactive T cells that have escaped other mechanisms of tolerance (13, 14). The elimination or inactivation of CD4+CD25+ T cells resulted in severe autoimmunue disease and was also found to enhance immune responses to alloantigens and even tumors (12, 24, 25). CD4+CD25+ T cells have unique immunologic characteristics compared with other regulatory or suppressor T cells induced by certain routes of exogenous immunization or tolerance induction. For example, they do not proliferate in response to antigenic stimulation in vitro (i.e., they are naturally anergic) and can potently suppress the activation and proliferation of CD4+CD25– T cells in an antigen-nonspecific manner through cell-to-cell interaction (26).
There was a significant elevation in the numbers of CD4+CD25+ T cells in patients with melanoma in comparison with the control subjects (16). Similarly, the prevalence of CD4+CD25+ T cells in both patients with breast and pancreatic cancer was also significantly higher than in normal individuals (17). However, PB of patients with non–small cell lung cancer did not demonstrate a significant increase in the CD4+CD25+ T-cell population (18). These previous studies have confirmed that CD4+CD25+ T cells are functional through their inhibition of nonspecific T-cell activation in vitro, providing indirect evidence that implicates CD4+CD25+ T cells in the immunopathogenesis of cancer. In a more recent study, Curiel and colleagues (27) have shown that CD4+CD25+ T cells in patients with ovarian cancer inhibit tumor-associated antigen-specific immunity in vitro and in vivo, and contribute to tumor growth, and that there is an inverse correlation between tumor CD4+CD25+ T cells and patient survival. These data therefore provide direct in vitro and in vivo evidence that CD4+CD25+ T cells have an important immunopathologic role in human ovarian cancer by suppressing endogenous antigen-associated, antigen-specific T-cell immunity. In the present study, our data showed that the numbers of CD4+CD25+ T cells in malignant PE were much higher than those in PL and PB. We noted that CD4+CD25+ T cell numbers in PL did not differ from those in PB from patients with PE, patients with lung cancer without PE, or healthy control subjects.
We did not study the mechanisms by which CD4+CD25+ T cells were recruited into the malignant PE in the present study. The primary aim of this study was to explore the presence of CD4+CD25+ T cells in malignant PE, as well as the immunosuppressive activity of these PE CD4+CD25+ T cells. It has been reported that the long-term effects of adoptively transferred CD4+CD25+ T cells induced ex vivo are due to their ability to generate new cytokine-producing CD4+CD25+ T cells in vivo (28). We speculated that an increased percentage of CD4+CD25+ T cells in malignant PE might be due to active recruitment or local differentiation. It has been demonstrated that human CD4+CD25+ T cells preferentially move to and accumulate in tumors and ascites, but rarely enter draining lymph nodes in later cancer stages, and that tumor cells and microenvironmental macrophages produce the chemokine CCL22, which mediates trafficking of CD4+CD25+ T cells to the tumors and ascites (27). In the previous study, we provided direct evidence that interleukin (IL)-16 is capable of inducing CD4+ T-cell infiltration into the pleural space (29). Therefore, as a subpopulation of CD4+ T cells, CD4+CD25+ T cells might also be recruited in to malignant PE by local production of IL-16, because the IL-16 level is significantly higher in PE than in serum (29).
The molecular basis for the development and function of CD4+CD25+ T cells is not clear. Molecules including CTLA-4, glucocorticoid-induced tumor necrosis factor receptor family–related gene (GITR), transforming growth factor , and IL-10 have been implicated in the function of these cells (14, 30). However, none of these are specific for CD4+CD25+ T cells, and in any case, the requirement for any individual factor in their suppressive activity may vary according to the effector response being regulated. The mechanism by which CD4+CD25+ T cells suppress proliferation of naive T cells in vitro seems to be independent of soluble factors and requires cell–cell contact, but the molecular basis for this effect is not known (13, 30). Both GITR and CTLA-4 are implicated in CD4+CD25+ T-cell activity (13, 31, 32) but are also expressed by other activated CD4+ T cells. Thus, in addition to possible effects via CD4+CD25+ T cells, it is possible that these surface molecules have a role in intrinsically controlling the threshold of T-cell stimulation. Recent studies (33–35) have shown that Foxp3 is specifically expressed in CD4+CD25+ T cells and is necessary for their development and function. Foxp3 is not simply a marker of activation because CD4+CD25– T cells do not express Foxp3 after activation (33–35). More recently, Voo and colleagues (36) have demonstrated that the suppressive activity of these GITR+Foxp3+ regulatory CD4+CD25+ T cells is dependent on antigen specificity. Our present data revealed that CD4+CD25+ T cells infiltrating malignant PE express the Foxp3, indicating that this population includes regulatory T cells. Our results suggested that an increase in these regulatory T cells may be relevant to the development of malignant PE. It is possible that an increase in CD4+CD25+ T cells at the site of malignant pleuritis may promote local tumor growth.
Our data also demonstrated that proliferative responses to anti-CD3 and CD28 mAbs were observed in the total CD4+ T-cell populations in PE. On depletion of the CD4+CD25+ T cells, the proliferation of the remaining CD4+CD25– T-cell population was increased significantly, providing indirect evidence that CD4+CD25+ T cells suppressed the proliferative response. Proliferation of the CD4+CD25+ T cells alone was low, indicating that these cells were anergic. To investigate the direct suppressive capacity of CD4+CD25+ T cells, we cocultured CD4+CD25– T cells with CD4+CD25+ T cells and stimulated them with anti-CD3 and CD28 mAbs. Of note, when CD4+CD25+ T cells were added to the coculture, suppression could be observed. We observed no difference in inhibitory effects of between CD4+CD25+ T cells from PE and their counterparts from PB. These findings indicate that CD4+CD25+ T cells in PE of patients with lung cancer are not impaired with regard to suppressive function. An increase in PE CD4+CD25+ T-cell numbers, as well as a possible functional impairment of CD4+CD25+ T cells, might thus lead to a disturbed balance between immunity and tolerance.
CTLA-4 may have multiple biological roles during different stages and conditions of T-cell activation. These concepts have important implications for the targeting of CTLA-4 in immunotherapy. Direct demonstration of this concept came from studies showing that induction of B7 expression by transfection was sufficient to induce T-cell–mediated rejection of many immunogenic tumors (37, 38). CTLA-4 blockade may allow the expansion of a critical number of tumor-reactive T cells needed to mediate tumor rejection from a limited number of responders (39). It has been known that CD4+CD25+ T cells constitutively express intracellular CTLA-4. Of note was the striking surface expression of CTLA-4 in PE CD4+CD25+ T cells that we observed in this study. The explanation for the striking surface CTLA-4 expression that we observed in PE CD4+CD25+ T cells is not yet clear, although it most likely reflects vigorous ongoing activation of the cells in the tumor microenvironment, perhaps by tumor antigens. We asked whether the neutralization of CTLA-4 molecules expressed by these CD4+CD25+ T cells could inhibit their suppressive activity. Although the addition in culture of isotype control mAbs had no effect, blocking of CTLA-4 partially, but consistently, inhibited the suppressive activity of CD4+CD25+ T cells. These findings suggested CTLA-4 molecules are involved in the suppressive activity of CD4+CD25+ T cells. Blockade of CTLA-4 abrogates CD4+CD25+ T-cell–mediated suppression in vitro, elicits autoimmune disease, and evokes tumor immunity in vivo.
In conclusion, our data showed that CD4+CD25+ T-cell numbers in malignant PE were much higher than those in PL from patients with lung cancer without malignant effusion, as well as those in PB. Our data also revealed that CD4+CD25+ T cells infiltrating PE were regulatory T cells because they express high levels of Foxp3 transcription factor. Moreover, pleural CD4+CD25+ T cells could potently suppress the proliferation of CD4+CD25– T cells, and CTLA-4 was involved in the suppressive activity of pleural CD4+CD25+ T cells. These data provide the basis for developing novel immune-boosting strategies based on eliminating this cell population in patients with cancer.
FOOTNOTES
Supported in part by research grant 30460051 from the National Natural Science Foundation of China, and in part by research grant 200260 from the Ministry of Education, P. R. China.
Originally Published in Press as DOI: 10.1164/rccm.200504-588OC on September 8, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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