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Journal of Clinical Oncology, Vol 24, No 7 (March 1), 2006: pp. 1169-1177 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.03.6830 Characterization of CD4+CD25+ Regulatory T Cells in Patients Treated With High-Dose Interleukin-2 for Metastatic Melanoma or Renal Cell CarcinomaFrom the Tumor Immunology Laboratory, Department of Surgery and Biostatistics, Columbia University Medical Center, New York, NY Address reprint requests to Howard L. Kaufman, MD, Columbia University Medical Center, 177 Fort Washington Ave, New York, NY 10032; e-mail: hlk2003{at}columbia.edu
PURPOSE: To characterize the number and functional status of CD4+CD25+ regulatory T cells (Tregs) in patients with metastatic melanoma (MM) and renal cell carcinoma (RCC) treated with high-dose bolus interleukin-2 (IL-2). PATIENTS AND METHODS: Patients with MM or RCC treated with high-dose bolus IL-2 (600,000 IU/kg every 8 hours) at a single center provided pre- and post-treatment whole blood specimens. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, separated into cellular subsets, and analyzed by flow cytometry or used for in vitro proliferation assays. RESULTS: Between September 2003 and July 2005 57 patients were enrolled in the study with 48 patients available for analysis (45 MM, 12 RCC). Tregs were defined as CD4+CD25hi T cells, and this subset was significantly elevated in the cancer patients compared with normal donors (7.75% v 2.24%). The CD4+CD25hi T-cell pool in the patients constitutively expressed intracellular FoxP3, CTLA-4, and produced high amounts of IL-10. The Tregs were CCR7+ with 50% representing naïve and 50% central-memory T cells. The cells were functionally suppressive in mixed in vitro proliferation assays. Following IL-2 administration, the number and frequency of Tregs increased in patients with progressive disease but returned to normal levels in patients with objective clinical responses. CONCLUSION: The number of Tregs, defined as CD4+CD25hi T cells is increased in patients with MM and RCC. High-dose IL-2 resulted in a significant decrease of Tregs in those patients achieving an objective clinical response to IL-2 therapy.
Interleukin-2 (IL-2) is a T-cell growth factor that is thought to play a critical role in T-cell–dependent immune responses. Human IL-2 is a 15 to 17 kD glycoprotein, belongs to the four-bundle  -helical cytokine family, and is produced primarily by activated T cells. The IL-2 receptor (IL-2R) consists of three subunits, an -chain (CD25), ß-chain (CD122), and a common cytokine-receptor  -chain (c or CD132).1 High-dose IL-2 has been extensively studied and results in a 16% to 20% objective response rate in patients with metastatic melanoma (MM) and renal cell carcinoma (RCC), with significant durability in selected patients.2-4 The mechanism of therapeutic activity in responding patients, however, is not known, and there are few pretreatment (Pre-Tx) predictive biomarkers to identify those patients more likely to respond to treatment. Recently, a subpopulation of thymus-derived CD4+ T cells, displaying high levels of CD25, have been described that function to suppress self-reactive T cells in the periphery.5-7 These so-called regulatory T cells (Tregs) express all three subunits of the IL-2R but do not secrete IL-2 and, therefore, depend on exogenous IL-2 for survival and function. This has been demonstrated through in vitro mixed culture assays in which CD4+CD25+ Tregs mediate suppression of CD4+CD25– T cells only in the presence of IL-2.8 Furthermore, in IL-2 knockout mice, characterized as having significant impairment in Tregs, animals develop severe autoimmunity that can be reversed by reconstitution of Tregs from normal mice. The possibility that Tregs may influence patients with cancer has been suggested by the increased numbers of Tregs demonstrated in a variety of human cancers.9-14 The increased levels of Tregs have been associated with a poor prognosis in women with advanced ovarian cancer and were implicated in preventing the induction of effective antitumor immunity.15 Thus, the emerging evidence suggests that IL-2 may be more appropriately thought of as a regulatory cytokine involved in mediating peripheral tolerance through its effects on Tregs. This role does not adequately explain why some patients with MM and RCC derive a therapeutic benefit from IL-2 administration. While in vitro studies have clearly supported the role of low-dose IL-2 in promoting the survival and differentiation of Tregs, the influence of high-dose IL-2 on the Treg population in vivo has not been previously examined. Therefore, we sought to assess the number, phenotypic characteristics, and functional status of CD4+CD25+ Tregs in patients undergoing standard high-dose IL-2 administration.
Patient Characteristics Patients with MM or RCC treated with high-dose bolus IL-2 between September 2003 and July 2005 were eligible for participation. The clinical protocol was approved by the institutional review board, and all patients were required to give written informed consent. Patients were also required to meet the institutional standards for high-dose IL-2 administration.16 Eligible patients underwent Pre-Tx imaging within 4 weeks of starting and completing each course of therapy.
Study Design
Peripheral Blood Mononuclear Cell (PBMC) Isolation
Quantification of Absolute Cell Numbers
Phenotypic Characterization of Tregs
Functional Characterization of Tregs
Statistical Analysis
Patient Characteristics and Clinical Results Fifty-seven patients were eligible and provided consent to participate in the study with 48 patients available for complete PBMC and clinical analysis. The summary of patient characteristics is presented in Table 1. Forty-five patients had MM (78.9%) and 12 patients had RCC (21.1%). The maximum number of doses given during one course of treatment was 14; the mean was seven. Objective clinical responses were documented in three patients with RCC and eight patients with MM. There were three complete responses (one RCC and two MM), all of whom remain free of disease at a median follow-up of 21 months, and eight partial responses.
CD4+CD25hi Is the Regulatory T-Cell Phenotype in Humans Because the definition of Tregs in the human population has been controversial, we first determined the functional status of CD4+CD25+ T cells in standard proliferation assays. CD4+CD25+ T cells were separated into high and low level CD25 expression on the basis of the level of CD25 expression in nonCD4+ T cells, as previously described17 (Fig 1A). Suppression of CD4+CD25– T-cell proliferation was observed when cocultured with CD4+CD25hi T cells, but not CD4+CD25lo T cells (Fig 1B). The CD4+CD25hi T cells were also nonresponsive to in vitro stimulation via their T-cell receptors, even in the presence of anti-CD28.18 Thus, we defined the CD4+CD25hi T-cell pool as Tregs in all assays.
CD4+CD25hi Tregs Are Increased in the Peripheral Blood of MM and RCC Patients To establish a baseline level of Tregs in the normal population, 16 normal donors had PBMC collected and the absolute number and frequency of CD4+CD25hi T cells was determined by FACS analysis. The mean number of CD4+CD25hi T cells was 22,280 ± 2,720 in these donors and the frequency was 2.24% with an SD of 0.59% (range, 1.01% to 2.86%; coefficient of variance = 0.19; Fig 2). The number and frequency of CD4+CD25hi T cells was significantly increased in patients with MM and RCC (Figs 2B and 2C). The mean frequency was 7.93% with an SD of 5.36% (range, 0.54% to 26.35%; n = 36) for MM patients and 7.21% with an SD of 5.36% (range, 1.13% to 11.61%; n = 12) for patients with RCC (Fig 2D). There was a corresponding increase in the mean absolute number of Tregs to 77,862 with an SD of 7,624 and 56,298 with an SD of 5,823 in patients with MM and RCC, respectively (Fig 2E). This increase was significant compared with normal donor CD4+CD25hi T-cell levels (P < .001 for MM and P < .05 for RCC). A comparison between healthy donors and the metastatic cancer patients revealed a 3.5-fold increased frequency of CD4+CD25hi T cells in MM and a 3.2-fold increased frequency in RCC (P < .01). Thus, the number and frequency of CD4+CD25+ Tregs were elevated in patients with MM and RCC.
CD4+CD25hi Tregs Are Phenotypically and Functionally Similar in Healthy Donors and Patients With Cancer Although the number of Tregs was clearly increased in the cancer patients, we also sought to assess the functional and phenotypic characteristics of these cells. The functional status was tested in a suppression assay using a mixed culture assay where CD4+CD25hi T cells obtained from patients before IL-2 treatment were added to cultures of CD4+CD25– T cells. The Tregs derived from the cancer patients were functional and inhibited proliferation in all patients tested. Figure 3A shows two representative patients where Tregs suppressed proliferation by 38.71% ± 12.12% (range, 22.5% to 54%). In agreement with previous reports, there was a significant increase in the expression of FoxP3, IL-10, CTLA-4, and GITR, common markers of the Treg phenotype, in patient derived CD4+CD25hi T cells compared with CD4+CD25lo T cells (P < .01). When we compared the level of expression of these markers between normal donor Tregs and patient-derived Tregs, there were similar levels of FoxP3, IL-10, and CTLA-4 expression, but a slightly decreased level of GITR expression was noted in Tregs collected from cancer patients (Fig 3B).
To further define the Treg population, we determined the expression of CD45 and CCR7 by flow cytometry in the CD4+CD25hi T cells in normal donors, MM, and RCC patients. The use of these markers identifies subsets of CD4+ T cells, including naive (CD45RA+CCR7+), central-memory (CD45RA–CCR7+), and effector-memory (CD45RA–CCR7–) cells.19 The Tregs in our study were largely CCR7+ (mean, 98.96%; SD, 1.08% in healthy donors; mean, 97.82%; SD, 3.14% in MM patients; and mean, 97.05%; SD, 3.51% in RCC patients; Fig 3). In healthy donors, Tregs were defined as naive in 45.06% (SD, 16.7%) and central-memory in 53.90% (SD, 16.42%) of the samples. Similarly, Tregs were characterized as naïve in 51.43% (SD, 21.61%) and central-memory in 46.39% (SD, 20.28%) of MM patients. RCC patients had 54.47% (SD, 17.35%) naive and 42.58% (SD, 14.84%) central-memory T cells (Fig 4). Only 0.91% ± 0.95% of the Tregs from healthy donors, 1.73% ± 2.59% in MM patients, and 2.51% ± 3.44% in RCC patients had an effector-memory T-cell phenotype. The frequency of effector-memory T cells was significantly higher (P < .01) in the CD4+CD25– and CD4+CD25lo T-cell subsets (data not shown). Collectively, this data suggests that the phenotypic and functional characteristics of Tregs derived from MM and RCC patients are similar to Tregs in normal donors.
Clinical Response to High-Dose IL-2 Therapy Is Correlated With Changes in the Frequency of Tregs The administration of high-dose IL-2 induces an initial lymphopenia followed by recovery.20 This lymphopenia was manifest in our patients after the fourth dose of IL-2, and so we collected PBMC Pre-Tx and after the fourth dose of IL-2 during the first and second cycle of therapy (cycle 1 and cycle 2, respectively). In addition, we collected a post-treatment sample 2 weeks after the first cycle (post 1) and 4 weeks after the second cycle (post 2). Figure 5 A demonstrates a typical histogram of PBMCs from a single patient Pre-Tx and after the fourth dose of IL-2 (cycle 1), demonstrating the appearance of cells with reduced size and increased granularity (cells gated in the triangle in Fig 5A), consistent with the onset of apoptosis at the time of maximum lymphopenia.
The total absolute lymphocyte count (ALC) was decreased after the fourth dose of IL-2 during cycle 1 (cycle 1) but rebounded within 2 weeks (post 1) of completing treatment (Fig 5B). A similar pattern was observed with the second cycle of IL-2 therapy. Before treatment the ALC was 21,789/mL (39.8%) and decreased to 1,164/mL (12.3%) after the first cycle of IL-2 and 3,287/mL (17.8%) after the second cycle of treatment (Fig 5B). The absolute number of CD4+ T cells and Tregs were also decreased during the IL-2–induced lymphopenia after both cycles of treatment, although the ratio of Tregs to CD4+ T cells remained constant (Fig 5B). Next, we compared those patients with progressive disease with those who obtained an objective clinical response. The mean number of lymphocytes before treatment was 22,296 (40.1%) in patients with disease progression, 21,578 (35.6%) in those with a partial response, and 16,461 (45.4%) in patients with a complete response (P = .84 using a linear model on the basis of the lymphocyte counts). In patients with progressive disease, the mean frequency of Tregs was 7.72% before treatment and increased to 16.7% after the first cycle of IL-2 and remained elevated after cycle 1 (772.3/mL, 9.06%). These increased levels were maintained through the second cycle of treatment (Fig 5C). In contrast, while patients achieving a complete response had slightly higher mean frequency of Tregs (9.0%) before treatment and maintained at a mean of 9.2% after cycle 1, the mean frequency dropped to 1.2% after cycle 2. The differences among the three response status groups are statistically significant with P = .004 (Table 2). Furthermore, this correlation remains significant after adjusting for the ALCs (Table 3).
Tregs Phenotype and Function Were Not Affected by High-Dose IL-2 Therapy To assess the effects of high-dose IL-2 on Treg function, we used Tregs derived from IL-2 patients in coculture suppression assays and determined the presence of known Treg markers. Tregs derived from patients Pre-Tx and after IL-2 administration significantly suppressed CD4+CD25– T-cell proliferation in all patients tested and a representative assay is shown in Figure 6A. All CD4+ T-cell subpopulations were indiscriminately activated after IL-2 therapy as evidenced by an increase in activation markers CD69 and CTLA-4 (P < .01; Fig 6B). IL-2 administration did not alter the expression of FoxP3 or IL-10 (Fig 6B). GITR, which is usually expressed by activated cells, was significantly upregulated after IL-2 in Tregs (P < .01; Fig 6C).
High-dose IL-2 has been approved for the treatment of metastatic RCC and MM since 1992 and 1998, respectively. While the overall response rates are relatively low, complete clinical responses appear to be durable in the majority of patients.21 The mechanism of IL-2 activity in vivo, however, is not well understood. This is particularly perplexing given recent evidence suggesting that IL-2 regulates the homeostasis and activation of Treg suppressor function.22 The effects of IL-2 on Tregs have largely been based on murine studies and in vitro assays utilizing low doses of IL-2.8 We sought, therefore, to determine the effects of high-dose IL-2 on Tregs in vivo. We observed an increased number of Tregs in our patients with metastatic MM and RCC before treatment, consistent with increases in Tregs observed in cancer patients with several other types of tumors.9-15 The number of Tregs increased after exposure to IL-2 and remained elevated in patients with disease progression, but fell to normal levels within 4 weeks in those patients with objective clinical responses. The phenotypic characteristics of the Tregs did not change, suggesting that the change in Treg frequency, but not the functional status of the Tregs, was associated with clinical responses to high-dose IL-2 therapy. Given the relatively low response rate and toxicity profile of high-dose IL-2 it would be useful to identify biomarkers predictive of clinical outcome. Previous reports have postulated a relationship between IL-2 response in melanoma patients and expression of HLA-A11 and induction of HLA-DR.23-24 In a review of 270 patients treated with high-dose IL-2, only prior systemic therapy and baseline performance status were predictive of response.2 Prior immunotherapy was also reported as a Pre-Tx factor associated with induction of complete response in a series of MM and RCC patients treated with high-dose IL-2.25 In this report, however, the total amount of IL-2 administered and rebound lymphocytosis after treatment cessation was also associated with complete tumor regression. The presence of soft-tissue–only metastases, increased number of doses during course one, development of abnormal thyroid function tests, and appearance of vitiligo were also associated with antitumor responses.26 Putative biomarkers with possible predictive value include lactate dehydrogenase, 5-S-cysteinyldopa, melanoma-inhibiting activity protein, S100ß, the presence of circulating melanoma cells, erythropoietin levels, and molecular profiling of metastatic lesions, but none have yet proven reliable in terms of patient selection.27-29 Recently, carbonic anhydrase IX, which plays a role in hypoxia-mediated cell proliferation, has been identified as a potential marker of IL-2 responsiveness in RCC, but prospective validation is not yet available.30 In our study, clinical responses were associated with the frequency of Tregs in the circulating PBMC 4 weeks after completing the second cycle of IL-2. Thus, Treg frequency does not appear to be useful as an initial predictor of treatment response but may warrant further investigation regarding its use as a method for monitoring patients after an initial positive response to immunotherapy. In our trial, we defined Tregs as CD4+ T cells expressing high levels of CD25 since this population consistently suppressed in vitro proliferation of CD4+CD25– T cells and also expressed characteristic markers of Tregs. These cells are a subset of the CD4+CD25+ T-cell pool and represent 2.5% of the CD4+ T-cell population in normal donors. While these cells were elevated in the cancer patients before treatment, the frequency is lower than reported in other series where the Treg population included CD25dim CD4+ T cells. The CD4+CD25hi T cells constitutively expressed FoxP3, the X-linked Forkhead/winged helix transcription factor, described as the most important marker of Tregs.31-33 These cells also constitutively expressed CTLA-4 and produced high levels of IL-10, suggesting that this population may contain Tr1 type cells.34,35 We also observed low level expression of the human analog of murine GITR (glucocorticoid-induced TNFR family-related gene).36-37 GITR was described as an important marker for Tregs in mice because GITR-specific monoclonal antibodies against CD25+ Tregs blocks their suppressive activity,38 and because the gene array analysis of CD4+CD25+ T cells revealed constitutive expression of high GITR levels.39 Our data suggest that GITR is not a critical marker for human Tregs as there was no difference in GITR expression between CD4+CD25hi and CD4+CD25lo T cells (data not shown). We also analyzed Tregs for CCR7 and CD45RA expression with the majority characterized as naive (CCR7+CD45RA+) or central-memory (CCR7+CD45RA–) T cells. While the implications of this are not entirely clear, central-memory Tregs are purported to be antigen-specific and traffic to secondary lymphoid tissues where they may make priming of effector T-cell responses more difficult in the cancer patient.38,39 Data from IL-2 and IL-2R knockout mice suggest that IL-2 is required for the generation and peripheral maintenance of Tregs. There is also significant evidence that low-dose IL-2 is essential for Treg activity in vitro.40 Thus, it is paradoxical that IL-2 induces clinical responses and decreases Tregs in vivo.41 While it is possible that high-dose IL-2 may induce qualitatively different responses from low-dose regimens, it is also possible that polymorphisms in immune response genes may mediate the diverse responses to IL-2 in individual patients.42 In support of dosing differences, it is clear that high-dose bolus IL-2 induces more durable objective clinical responses in MM and RCC.43-45 In support of the latter, it has been shown that polymorphisms in the chemokines receptor CCR5 is protective against non-Hodgkin's lymphoma.46 These mechanisms are not mutually exclusive and future studies of the molecular and genetic features of Tregs will be important to better define the mechanism of IL-2 therapy, to predict which patients respond to treatment, and to develop new strategies for the immunotherapy of cancer.
Although all authors completed the disclosure declaration, the following author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000-99,999 (C) > $100,000 (N/R) Not Required
Supported by Grant No. RO1 93696 from the National Institutes of Health and the Chiron Corporation. Presented in part at the Masir Congress on Measurement of Antigen-Specific Immune Responses, Courmayeur, Italy, January 26-29, 2005 and at the 58th Annual Cancer Symposium of the Society of Surgical Oncology, Atlanta, GA, March 3-5, 2005. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
