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Journal of Clinical Oncology, Vol 25, No 18 (June 20), 2007: pp. 2630-2632 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.08.4954
In ReplyTumor Immunology Laboratory, Columbia University, New York, NY High-dose interleukin-2 (IL-2) is an approved treatment regimen for metastatic melanoma and renal cell carcinoma with objective tumor response rate of 16% to 20% and significant durability in selected patients.1-3 Despite these results, the mechanism of IL-2–mediated tumor rejection is not yet defined. In addition to promoting the proliferation and activation of effector T cells, IL-2 is necessary for the development and homeostasis of a regulatory T cell (Tregs) population, defined by coexpression of CD4 and high levels of the IL-2 receptor alpha subunit (CD25). Tregs comprise 1% to 4% of the CD4+ T cell population in healthy adult humans, but are elevated in cancer patients where they can exert inhibitory effects on primed T cells and thus prevent effective antitumor immunity.4-10 We recently reported that Tregs are elevated in melanoma and renal cell carcinoma patients and that high-dose IL-2 administration is associated with a decrease in the number of Tregs in patients exhibiting objective clinical response after IL-2 administration.11 To address this result, we established a validated assay for the identification of Tregs by confirming their functional characteristic through coculture proliferation assays and their phenotypes through flow cytometry of peripheral blood mononuclear cells (PBMCs) derived from healthy donors or cancer patients eligible for IL-2 therapy. The phenotype was characterized by immunofluorescence staining, using anti-CD45RA and anti-CCR7, intracellular staining was performed to analyze expression of Forkhead Box Protein P3 (FOXP3), CTLA-4, IL-10, and glucocorticoid-induced tumor necrosis factor receptor. We showed that in humans the phenotype of Tregs and their functional characteristic correspond to CD4+CD25high T cells, consistent with other reports.12,13 After staining cells with anti-CCR7 and anti-CD45RA, we obtained three different subpopulations of cells, as previously described14: naïve (CD45RA+CCR7+), central memory (CD45RA–CCR7+), and effector T cells (CD45RA–CCR7–). In our study, Tregs resulted largely CCR7+ both in donors and in cancer patients, almost equally subdivided in naïve and central memory populations. A very low percentage of Tregs resulted effector memory cells. In another study, Beyer et al analyzed 19 healthy donors and noticed that only 14.6% ± 15.5% of CD4+CD25+FOXP3+ T cells were naïve cells and that most of them were effector memory cells. They also observed that not all CD4+CD25high T cells were FOXP3+. We agree with Beyer et al that different technical approaches may create discrepancy among studies. Our gating strategy was decided on healthy donors total PBMCs and then applied to CD4+CD25–, CD4+CD25high, and CD4+CD25low T cells. To clarify the issue, in Figure 1 we show the initial gating on total PBMCs, established using APC and PeCy7 IgG controls. In the PBMCs of 10 healthy donors, naïve cells resulted to be 34.70% ± 9.65% of the total, central memory cells were 7.60% ± 3.31%, and effector memory cells were 11.40% ± 5.19% (Fig 1). Compared with total PBMC population, the number of naïve cells and central memory cells resulted increased in CD4+CD25– T cells (55.96% ± 15.54% and 14.05% ± 7.98%, respectively), in CD4+CD25low T cells (68.71% ± 18.06% and 25.13% ± 15.82%, respectively), and in CD4+CD25high T cells (45.06% ± 16.70% and 53.90% ± 16.42%, respectively). In contrast, the number of effector memory T cells remained similar to PBMCs in CD4+CD25– T cells (11.30% ± 8.30%), but decrease significantly in CD4+CD25low T cells (3.73% ± 3.65%) and in CD4+CD25high T cells (0.91% ± 0.95%).
To better understand the mechanism of action of Tregs in humans, it is necessary to identify them in a clear population. Many researchers have focused their studies on finding a Tregs' specific marker, but at present, except for CD25, no other molecules seem to exclusively identify the regulatory T population. Even FOXP3, which has been described as the most important marker for Tregs,15-17 is not so specific: it has been demonstrated that it can be induced by transforming growth factor beta in CD4+CD25– T cells, too.18 Another problem is that authors use different markers to define a specific pattern of cells. For example, to describe the memory population some authors use just CD45RA, some use CD45RO, and some others the combination between CD45RA and CCR7. These discrepancies make the comparison of data difficult. Exactly because the characterization of Tregs in the human population has been controversial, we decided to define them firstly determining their functional status. Only CD4+CD25high T cells were nonresponsive to in vitro stimulation via T-cell receptor, even in the presence of anti-CD28, and able to suppress CD4+CD25– cells. These two functional characteristics allowed us to define CD4+CD25high T cells as Tregs. At present, functional assay remains the best method to identify Tregs in humans. Once we identified CD4+CD25high T cells as Tregs, we tried to analyze the expression of certain molecules, which were described to be very present in Tregs, such as FOXP3, CTLA4, IL-10, and GITR. We noticed that, except for GITR, CD4+CD25high Tregs were expressing high levels of those molecules. Finally, we tried to describe the CCR7/CD45RA phenotype of Tregs, to evaluate if IL-2 activity consisted also in a change of naïve/memory T-cells pattern, which did not occur. It is very important to precisely identify the Tregs population to better understand its real role in the tumor microenvironment setting. Future studies on IL-2 therapy and Tregs will focus on better characterizing the molecular and genetic features of these cells. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST 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. Employment: N/A Leadership: N/A Consultant: N/A Stock: N/A Honoraria: N/A Research Funds: Howard L. Kaufman, Chiron Corp Testimony: N/A Other: N/A
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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