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In Reply

Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD

Ferretti et al raise the still unresolved issue of whether the immunomodulatory effects associated with CTLA4 blockade in patients (autoimmunity and tumor regression¹) are mediated by alterations in T-regulatory cells (Treg). There is compelling evidence for a physiological role for Treg in mice where definitive depletion and adoptive T-cell transfer experiments can be done. Administration of CD4+ T-cell populations stripped of Treg cells to severe combined immunodeficiency (SCID) mice does induce autoimmune colitis.² In addition, administration of CD4+ CD25+ T cells to T-cell deficient mice (Rag-1–/–) undergoing CD8+ T-cell therapy for melanoma reduces the efficacy of that therapy while the CD4+ CD25- population improves the efficacy and spares the need for systemic interleukin (IL) -2 support.³ Yet even in mice, demonstrating these effects usually requires the use of immunodeficient recipients. When T-cell transfers are done in SCID or Rag-1–/– hosts, there is no background T-cell population and absolute control can be exerted over the T-cells populating those mice. Depletion of Treg from the peripheral repertoire of a normal, mature mouse (as opposed to congenital or germline depletion) to mediate autoimmunity or to augment antitumor responses does not work in most murine models.

In patients, no firm experimental data exists to verify the in vivo physiological role of Treg in modulating immune responses. Certainly cells with the appropriate phenotype exist and they can inhibit T-cell responses in vitro.⁴ In addition, germline loss of the gene for the Treg marker/transcriptional regulator forkhead box protein (FOXP) 3 results in an autoimmune syndrome, IPEX (immunodysregulation, polyendocrinopathy and enteropathy, X-linked), characterized by diabetes, diarrhea and enteropathy, autoimmune thyroid disease, dermatitis, and abnormal immune responses.⁵ Although this has hallmarks of immune dysregulation and autoimmunity, this disease could result from loss of a developmental role for FOXP3 rather than from simple release of autoimmune restraint on mature autoreactive T cells. These two hypotheses would have very different implications for the effects of Treg manipulation in cancer immunotherapy and autoimmunity. Perhaps questioning whether Treg depletion has ever had any significant demonstrated effects in immunoreplete patients should supercede the question of whether anti-CTLA4 antibody effects are mediated through Treg depletion.

If one grants the unsubstantiated conclusion that reductions in Treg in humans could have effects similar to complete depletions in immunosuppressed mice, then the issue becomes whether meaningful depletions occur with anti-CTLA4 antibody. In mice, Treg are the only naive T-cell population that constitutively expresses CTLA4, although it is induced on other T cells on activation.⁶ Studying the peripheral blood of patients with melanoma receiving ipilimumab, Maker et al⁷ found that there was no consistent depletion of Treg cells as determined by phenotype, FOXP3 expression by quantitative reverse transcriptase polymerase chain reaction or by in vitro assays of functional suppression. All such studies are hampered by the fact that there is no ideal lineage marker for Treg. It is increasingly clear that in humans, CD4+CD25+, glucocorticoid-induced tumor necrosis factor receptor family-related gene, and even FOXP3 can be induced on non-Treg populations by activation or IL-2. In fact, the lymphokine activated killer cell populations given to patients along with IL-2 with a 20% to 25% response rate in patients with melanoma and renal cancer (including 5% to 7% long-term complete responses), have extremely high levels of FOXP3 and show in vitro suppressive activity in Treg assays.⁸ Therefore, one must doubt either the role of Treg or the validity of FOXP3 as a human Treg marker. A study by Reuben et al⁹ of patients receiving ticilimumab found a small but significant reduction in the median percentage and number of peripheral blood Treg (ascertained by phenotype only) in patients with autoimmunity and tumor regression versus those with no tumor regression. The difference, 5.8% compared with 3.6%, was only seen at one of three time points tested and was of a magnitude that marginally impacts even in vitro Treg suppression assays. Treg modulation could also occur by changes in function after CTLA4 blockade rather than depletion. Reuben et al also found differences in IL-10 secretion by Treg and IL-2 secretion by stimulated peripheral blood mononuclear cells were also seen, but again only at one of three time points examined and no actual functional suppression assays were done. IL-2 is known to enhance the survival and function of Treg in mice and CD4+ FOXP3+ T cells are dramatically increased immediately after IL-2 therapy in patients.⁸ Yet when IL-2 was added to ipilimumab in patients with metastatic melanoma the response rate was 22% compared with historical response rates of 15% and 12%, respectively, for IL-2 and ipilimumab alone.⁷ No study has examined the impact of anti-CTLA4 antibodies on Treg at the site of tumor but lesion-to-lesion variability and the effects of multiple biopsies on infiltrating T cells makes this difficult to accomplish and more difficult to interpret. The current information on the effect of anti-CTLA4 antibodies on Treg in the peripheral T-cell compartment of patients ranges from frankly contradictory to somewhat suggestive. The larger issue of the role of Treg in the immunoreplete human also remains unanswered. It seems premature to cobble together a mechanism for the dramatic tumor regressions after anti-CTLA4 antibody treatment in some patients with renal cancer or melanoma from the bits of incomplete information we currently have available. Clinically, the treatment works in some patients, so broader immunological characterization of larger numbers of patients is warranted and should be pursued vigorously. Then perhaps we can fashion a mechanistic hypothesis that better explains all of the varied data and observations emanating from this new and exciting immunotherapeutic approach.

Author's Disclosures of Potential Conflicts of Interest

The author indicated no potential conflicts of interest.

REFERENCES

1. Beck KE, Blansfield JA, Tran KQ, et al: Enterocolitis in patients with cancer after antibody blockade of cytotoxic t-lymphocyte-associated antigen 4. J Clin Oncol 24:2283-2289, 2006[Abstract/Free Full Text]

2. Singh B, Read S, Asseman C, et al: Control of intestinal inflammation by regulatory T cells. Immunol Rev 182:190-200, 2001[CrossRef][Medline]

3. Antony PA, Piccirillo CA, Akpinarli A, et al: CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J Immunol 174:2591-2601, 2005[Abstract/Free Full Text]

4. Baecher-Allan C, Brown JA, Freeman GJ, et al: CD4+CD25 high regulatory cells in human peripheral blood. J Immunol 167:1245-1253, 2001[Abstract/Free Full Text]

5. Bennett CL, Christie J, Ramsdell F, et al: The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 27:20-21, 2001[CrossRef][Medline]

6. Takahashi T, Tagami T, Yamazaki S, et al: Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med 192:303-310, 2000[Abstract/Free Full Text]

7. Maker AV, Phan GQ, Attia P, et al: Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2: A phase I/II study. Ann Surg Oncol 12:1005-1016, 2005[Abstract/Free Full Text]

8. Ahmadzadeh M, Rosenberg SA: IL-2 administration increases CD4+ CD25(hi) FOXP3+ regulatory T cells in cancer patients. Blood 107:2409-2414, 2006[Abstract/Free Full Text]

9. Reuben JM, Lee BN, Li C, et al: Biologic and immunomodulatory events after CTLA-4 blockade with ticilimumab in patients with advanced malignant melanoma. Cancer 106:2437-2444, 2006[CrossRef][Medline]

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