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Journal of Clinical Oncology, Vol 25, No 18 (June 20), 2007: pp. 2628-2630 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.08.0192
CD4+CD25highFOXP3+ Regulatory T Cells in Peripheral Blood Are Primarily of Effector Memory PhenotypeMolecular Tumor Biology and Tumor Immunology, Clinic I for Internal Medicine, University of Cologne, Cologne, Germany To the Editor: We noted with interest the article by Cesana et al1 in the March 1, 2006, issue of the Journal of Clinical Oncology, entitled "Characterization of CD4+CD25+ Regulatory T Cells in Patients Treated With High-Dose Interleukin-2 for Metastatic Melanoma or Renal Cell Carcinoma." Since the first description of CD4+CD25+ T cells as regulatory T cells (Tregs) by Sakaguchi et al,2 a number of studies have addressed the frequency of CD4+CD25high Tregs in cancer patients. The increase of Tregs in cancer-bearing patients either in the tumor microenvironment or peripheral blood is now well accepted in the literature.3,4 How interleukin (IL)-2 therapy influences the frequency of Tregs has been addressed in different settings with similar results.5-7 A further increase of Tregs after IL-2 administration seems to be associated with progressive disease while a response to IL-2 treatment seems to be associated with decreased Tregs numbers.1 However, we would like to draw your attention to the authors' statement that the Tregs were almost exclusively of naïve or central memory phenotype. In 1999, Sallusto et al8 were the first to describe the differentiation of total CD4+ and CD8+ T cells into CCR7+CD45RA+ naïve T cells, CCR7+CD45RA– central memory and CCR7–CD45RA– effector memory T cells. Since then, this T-cell classification has been applied by many groups confirming the initial observation. For Tregs, however, it was assumed for adults that these cells are exclusively of memory phenotype as defined by surface expression of CD45RO and absence of CD45RA. Only very recently has the existence of human naïve Tregs cells in healthy individuals been demonstrated in two elegant studies.9,10 Both studies revealed a subset of CD4+CD25+ T cells with full regulatory capacity coexpressing CD45RA. These cells were termed natural naïve Tregs. Unfortunately costainings for CD45RA and CCR7 were not reported in both studies; therefore, not allowing distinction between central and effector memory Tregs cells. However, both studies came to the conclusion that the cell population coexpressing CD4, CD25, and CD45RA constitute approximately one third of the total Tregs population. In addition to the demonstration of the regulatory capacity of the T-cell population under study, it is mandatory to measure the expression of the transcription factor Forkhead Box Protein 3 (FOXP3) to classify the cells as Tregs. In fact, both studies demonstrated a relative increase of FOXP3 mRNA expression in CD4+CD25+CD45RA+ T cells compared with CD4+CD25–CD45RA+ T cells.9,10 Unfortunately, FOXP3 protein expression was not determined in these studies, so it remained elusive whether few CD4+CD25+CD45RA+ T cells express large amounts of FOXP3 or many CD4+CD25+CD45RA+ T cells little FOXP3. Using the same gating strategy, we assessed 19 healthy donors and confirmed the existence of a CD4+CD25+CD45RA+ T-cell population presenting approximately 30% of the total CD4+CD25+ Tregs population (Fig 1B). However, when assessing FOXP3 protein expression in context of CD45RA expression (Fig 1C), only few of the CD4+CD45RA+ T cells were weakly stained for FOXP3 in contrast to a distinct subpopulation of CD4+CD45RA–FOXP3+ T cells. In fact, only 14.6% ± 15.5% of all CD4+CD25+FOXP3+ T cells coexpress the naïve T-cell marker CD45RA (Table 1). This is in sharp contrast to the data reported by Cesana et al1 who reported almost one half of the Tregs to be of naïve phenotype. To further study the distribution of central and effector memory Tregs, we assessed CCR7 expression in the CD4+ T-cell population in relation to FOXP3 protein expression. Again, this data revealed significantly different results (Fig 1D).11 While Cesana et al1 did not find any effector memory Tregs, our results indicate that the majority of CD4+FOXP3+ T cells are indeed of the effector memory phenotype.
To further control for these findings, we applied a second approach (Fig 2). In this case, we first separated CD4+ T cells based on their expression of CD25 into CD25–, CD25low, and CD25high T cells. Next CD25highFOXP3+ cells were determined and CD45RA and CCR7 expression was assessed in comparison with CD25highFOXP3– cells. This approach clearly demonstrated that not all CD4+CD25high T cells are also FOXP3+ as suggested by Cesena et al.1 Our findings are in line with previous data demonstrating an increase of both FOXP3+ as well as FOXP3–CD4+CD25high T cells after IL-2 therapy.6 As shown in Figure 2C, within the 19 healthy donors studied, the large majority of CD4+CD25highFOXP3+ T cells are of effector memory phenotype. Only very few Tregs are of a naïve phenotype. In CD4+CD25highFOXP3– T cells, the distribution differs as more cells are of a naïve respectively central memory phenotype (Fig 2D).
As an important control, we also studied CD4+CD25low (Fig 2E) and CD4+CD25– T cells (Fig 2F). Because these cells comprise the majority of human CD4+ T cells, it is not surprising that we can confirm the original observation by Sallusto et al7 (Fig 2E and F) demonstrating that 20% to 40% of human CD4+ T cells are of naïve phenotype, between 20% and 40% of central memory and the remaining cells of effector memory phenotype. Taken together, we come to the conclusion that human Tregs—in contrast to conventional T cells—are mainly of effector memory phenotype. The discrepancy to the data reported by Cesana et al1 might be simply due to different technical approaches. Because the distribution of naïve, central, and effector memory T cells within the CD4+CD25– T cell population was not shown by Cesana et al,1 it is not easy to determine whether the gating strategy applied by Cesana et al1 would result in data compatible with previous findings7 concerning the distribution of the different subpopulations within conventional T cells. Clarifying this issue will be of importance in context of malignant disease since the expansion of naïve versus memory Tregs might have significant impact of further therapeutic strategies. In the end, however, the best way to resolve such discrepancies will be the identification of truly specific markers for Tregs, which then will allow better subclassification of these cells and to determine their dynamics during disease or therapeutic intervention. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Although all authors completed the disclosure declaration, the following authors or their 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: Joachim Schultze, Funds, Becton Dickinson USA Testimony: N/A Other: N/A
ACKNOWLEDGMENTS Supported by the Sofja Kovalevskaja Award of the Alexander von Humboldt-Foundation (J.L.S.), the Wilhelm-Sander Stiftung (J.L.S., M.B.), and the Nationales Genomforschungsnetz (J.L.S.). REFERENCES
1. Cesana GC, DeRaffele G, Cohen S, et al: Characterization of CD4+CD25+ regulatory T cells in patients treated with high-dose interleukin-2 for metastatic melanoma or renal cell carcinoma. J Clin Oncol 24:1169-1177, 2006 2. Sakaguchi S, Sakaguchi N, Asano M, et al: Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25): Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155:1151-1164, 1995[Abstract] 3. Beyer M, Schultze JL: Regulatory T cells in cancer. Blood 108:804-811, 2006 4. Beyer M, Kochanek M, Darabi K, et al: Reduced frequencies and suppressive function of CD4+CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine. Blood 106:2018-2025, 2005 5. Zhang H, Chua KS, Guimond M, et al: Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells. Nat Med 11:1238-1243, 2005[CrossRef][Medline] 6. Ahmadzadeh M, Rosenberg SA: IL-2 administration increases CD4+ CD25(hi) Foxp3+ regulatory T cells in cancer patients. Blood 107:2409-2414, 2006 7. Zhou G, Drake CG, Levitsky HI: Amplification of tumor-specific regulatory T cells following therapeutic cancer vaccines. Blood 107:628-636, 2006 8. Sallusto F, Lenig D, Forster R, et al: Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708-712, 1999[CrossRef][Medline] 9. Seddiki N, Santner-Nanan B, Tangye SG, et al: Persistence of naive CD45RA+ regulatory T cells in adult life. Blood 107:2830-2838, 2006 10. Valmori D, Merlo A, Souleimanian NE, et al: A peripheral circulating compartment of natural naive CD4 Tregs. J Clin Invest 115:1953-1962, 2005[CrossRef][Medline] 11. Beyer M, Kochanek M, Giese T, et al: In vivo peripheral expansion of naive CD4+CD25high FoxP3+ regulatory T cells in patients with multiple myeloma. Blood 107:3940-3949, 2006
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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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