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Effects of the Administration of High-Dose Interleukin-2 on Immunoregulatory Cell Subsets in Patients with Advanced Melanoma and Renal Cell Cancer

Authors' Affiliations: ¹ Cancer Biology Program, Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; ² Department of Internal Medicine, Vrije Universiteit Medisch Centrum, Amsterdam, the Netherlands; and ³ Department of Immunology, University of Washington, Seattle, Washington

Requests for reprints: Mark A. Exley, Harvard Institutes of Medicine, 330 Brookline Avenue, Boston, MA 02115. Phone: 617-667-0982; E-mail: mexley{at}bidmc.harvard.edu.

	Abstract

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Purpose: High-dose recombinant human interleukin-2 (IL-2) therapy is of clinical benefit in a subset of patients with advanced melanoma and renal cell cancer. Although IL-2 is well known as a T-cell growth factor, its potential in vivo effects on human immunoregulatory cell subsets are largely unexplored.

Experimental Design: Here, we studied the effects of high-dose IL-2 therapy on circulating dendritic cell subsets (DC), CD1d-reactive invariant natural killer T cells (iNKT), and CD4⁺CD25⁺ regulatory-type T cells.

Results: The frequency of both circulating myeloid DC1 and plasmacytoid DC decreased during high-dose IL-2 treatment. Of these, only a significant fraction of myeloid DC expressed CD1d. Although the proportion of Th1-type CD4^– iNKT increased, similarly to DC subsets, the total frequency of iNKT decreased during high-dose IL-2 treatment. In contrast, the frequency of CD4⁺CD25⁺ T cells, including CD4⁺Foxp3⁺ T cells, which have been reported to suppress antitumor immune responses, increased during high-dose IL-2 therapy. However, there was little, if any, change of expression of GITR, CD30, or CTLA-4 on CD4⁺CD25⁺ T cells in response to IL-2. Functionally, patient CD25⁺ T cells at their peak level (immediately after the first cycle of high-dose IL-2) were less suppressive than healthy donor CD25⁺ T cells and mostly failed to Th2 polarize iNKT.

Conclusions: Our data show that there are reciprocal quantitative and qualitative alterations of immunoregulatory cell subsets with opposing functions during treatment with high-dose IL-2, some of which may compromise the establishment of effective antitumor immune responses.

CD1d-restricted invariant NK T cells (iNKT) display a semi-invariant T-cell receptor (V24/J18 in human and V14/J18 in mouse) and are characterized by their capacity to rapidly produce large amounts of cytokines on triggering. iNKT cells have been shown to promote antitumor immune responses by rapidly producing copious amounts of IFN-, resulting in the activation of NK cells, dendritic cells (DC), and conventional CD4⁺ and CD8⁺ T cells as well as in the inhibition of tumor angiogenesis (reviewed in refs. 6, 7). Importantly, the circulating pool of iNKT cells shows quantitative and qualitative defects in cancer patients (8, 9), which seem to be clinically relevant, as increased numbers of intratumoral or circulating iNKT cells are associated with improved prognosis (10, 11).⁴

In contrast to iNKT cells, CD4⁺CD25⁺ regulatory T cells, a population of CD4⁺ T cells constitutively expressing the IL-2 receptor -chain, have been shown to suppress immune responses, including antitumor immune responses, via both contact-dependent (CTLA-4 and CD30) and contact-independent (IL-10 and transforming growth factor-ß) pathways (reviewed in refs. 6, 12, 13). As elevated numbers of CD4⁺CD25⁺ regulatory T cells have been observed in melanoma and various other types of cancer (14, 15) and are associated with reduced survival (16), a recent study evaluated whether depletion of CD4⁺CD25⁺ regulatory T cells using recombinant IL-2 diphtheria toxin (DAB₃₈₉IL-2) would enhance tumor-specific T-cell responses in patients with RCC, as indicated by earlier reports in mice (17), and found that this was indeed the case (18).

As discussed above, both iNKT cells and CD4⁺CD25⁺ regulatory T cells seem to play important roles in the regulation of antitumor immune responses. As CD4⁺CD25⁺ regulatory T cells constitutively express the IL-2 receptor -chain (CD25) and IL-2 is an important growth factor for both iNKT cells and CD4⁺CD25⁺ regulatory T cells, both these regulatory T-cell subsets could be important cellular targets during high-dose IL-2. Here, we evaluated the in vivo effects of high-dose IL-2 on circulating iNKT cells, CD4⁺CD25⁺ regulatory T cells, and the DC with which they interact.

	Materials and Methods

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Patients. Patients (mean age, 53 years; range, 30-68 years; 15 males; 10 females) in this study had been diagnosed with either advanced melanoma (n = 15) or RCC (n = 10). All were treated with high-dose IL-2 as described (3). Peripheral blood was obtained on four occasions: before treatment, after the first week of high-dose IL-2, before the second week of high-dose IL-2, and after the second week of high-dose IL-2. Informed consent was obtained from all patients, and the study was approved by the Committee for Clinical Investigation of Beth Israel Deaconess Medical Center (Boston, MA). Clinical responses were assessed using previously reported criteria (19).

Flow cytometry. Peripheral blood mononuclear cells were obtained by Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden) density gradient centrifugation of heparinized peripheral blood. Peripheral blood mononuclear cells were stained using combinations of the following reagents after an initial incubation with 10% human pooled serum to reduce nonspecific binding. FITC-labeled CD3, FITC-labeled CD25, phycoerythrin (PE)-labeled CD56, PE-labeled CD1d, PE-labeled CD8, PE-labeled HLA-DR, PE-labeled CD86, PE-labeled CD80, PE-Cy5-labeled CD19, PE-Cy5-labeled CD25, PE-Cy7-labeled CD4, PE-Cy7-labeled CD14, FITC-labeled anti-invariant V24J18 T-cell receptor monoclonal antibody (mAb) 6B11 (9), and the appropriate isotype controls were obtained from BD PharMingen (San Jose, CA). PE-labeled V24 and FITC-labeled Vß11 were obtained from Immuno tech (Marseille, France). FITC-labeled BDCA-1, BDCA-2, and BDCA-3 were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). FITC-labeled anti-CTLA-4, FITC-labeled CD30, and PE-labeled anti-GITR were obtained from R&D Systems (Minneapolis, MN). The anti-human Foxp3 polyclonal antibody was obtained by repeated immunization of rabbits (20). Flow cytometry was done on a Cytomics FC 500 (Beckman Coulter, Fullerton, CA).

Generation of iNKT cell lines. iNKT cells were enriched from peripheral blood mononuclear cells of healthy adult volunteers by positive selection using biotinylated anti-invariant T-cell receptor mAb (6B11) in combination with anti-biotin microbeads (Miltenyi Biotec) and subsequently expanded using -galactosylceramide (-GalCer)-loaded (KRN7000, kindly provided by the Pharmaceutical Research Laboratory, Kirin Brewery, Ltd., Gunma, Japan) and lipopolysaccharide-matured monocyte-derived DCs and 100 units/mL recombinant human IL-2 (National Biological Response Modifier Program, National Cancer Institute, Frederick, MD). In some cases, iNKT cell lines were further purified using high-speed sorting (Modular Flow FACS, Cytomation, Fort Collins, CO).

Functional evaluation of CD4⁺CD25⁺ regulatory T cells. For evaluation of the suppressive properties of CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁺ and CD4⁺CD25^– T cells were purified using high-speed fluorescence-activated cell sorting (MoFlo Cytomation, Boulder, CO) or by CD25 mAb magnetic microbeads at relatively low density to favor removal of CD25^hi cells and cocultured with purified iNKT cell lines (purity >95%) and -GalCer–pulsed monocyte-derived DCs for 48 h essentially as described elsewhere (21).

CD25⁺ cells were obtained by magnetic-activated cell sorting. Cells were rested overnight after sorting to release beads before use. Assays were carried out in 96-well plates. CD25^– cells (column "flow through") were used in all wells plus variable levels of autologous CD25⁺ cells. For the highest level of CD25⁺ cells ("high"), a 1:1 ratio to CD25^– cells was used. At lowest level ("low"), the relative ratio was 1:16 of CD25⁺ cells to CD25^– cells, respectively. Results of the middle dilution of CD25⁺ cells were intermediate and therefore not shown for clarity.

Supernatants were harvested for detection of IFN- and IL-4 by standard capture ELISA with matched antibody pairs in relation to cytokine standards (Endogen, Cambridge, MA).

Statistical analysis. Statistical analyses were done using paired Student's t tests, Wilcoxon matched pairs tests, or ANOVA as appropriate. P < 0.05 was considered significant.

	Results

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Effect of high-dose IL-2 therapy on T cells, NK cells, and B cells. First, we evaluated the effect of high-dose IL-2 therapy on the main peripheral blood lymphocyte subsets (Fig. 1 ). The frequency of both CD4⁺ and CD8⁺ T cells decreased during the first week of treatment with high-dose IL-2 (P = 0.0001 and 0.009, paired Student's t test), but levels recovered before the second week of treatment (P = 0.003 and 0.02). The second week of high-dose IL-2 induced no significant changes in the frequency of either CD4⁺ or CD8⁺ T cells. High-dose IL-2 induced a substantial increase in the frequency of NK cells during the first week of IL-2 (P = 0.0005), and their frequency remained at higher than pretreatment levels thereafter. In contrast to NK cells, B cells substantially decreased in frequency during both the first week (P = 0.006) and the second week (P = 0.02) of high-dose IL-2 treatment.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Frequency of CD4+ T cells, CD8+ T cells, NK cells, and B cells during high-dose IL-2 therapy. Columns, mean frequency of CD4+ T cells, CD8+ T cells, NK cells, and B cells as a percentage of total lymphocytes during high-dose IL-2 therapy; bars, SD. Frequencies were assessed before (pre) and after (post) the first (1) and second (2) week of high-dose IL-2.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Frequency and CD1d expression of circulating DC subsets during high-dose IL-2 therapy. Columns, mean frequency (top row) and CD1d expression (bottom row) of circulating mDC1, pDC, and mDC2 during high-dose IL-2 therapy; bars, SD. Analyses were done before (pre) and after (post) the first (1) and second (2) week of high-dose IL-2.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Response and phenotype of circulating iNKT cells during high-dose IL-2 therapy. Columns, circulating iNKT cell frequency (A, median ± IQR), absolute number of circulating iNKT cells (B, median ± IQR), and proportion of double-negative iNKT cells of the total circulating iNKT cell pool (C, mean and SD) during high-dose IL-2 therapy. iNKT cells were quantitated as double positive for V24 Vß11 T-cell receptor mAb. Similar results were obtained with anti-invariant V24J18 T-cell receptor mAb 6B11. Analyses were done before (pre) and after (post) the first (1) and second (2) week of high-dose IL-2.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Response of circulating CD4+CD25+ T cells during high-dose IL-2 therapy. Columns, circulating CD4+CD25+ T-cell frequency (A, median ± IQR), absolute number of circulating CD4+CD25+ T cells (B, median ± IQR) during high-dose IL-2 therapy, and mean fluorescence intensity (MFI) of CD25 expression on CD4+CD25+ T cells during high-dose IL-2 therapy (C, median ± IQR). Analyses were done before (pre) and after (post) the first (1) and second (2) week of high-dose IL-2.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Response of circulating CD4+CD25+ regulatory T cells during high-dose IL-2 therapy. Representative flow cytometric dot plots of one donor showing the association between changes in the frequency of circulating CD4+CD25+ T cells and CD4+Foxp3+ T cells during high-dose IL-2 therapy. Right of each dot plot, frequency of double-positive cells. Analyses were done before (pre) and after (post) the first (1) and second (2) week of high-dose IL-2.

View larger version (9K): [in this window] [in a new window]   Fig. 6. Surface expression of GITR, CD30, and CTLA-4 on circulating CD4+CD25+ T cells during high-dose IL-2 therapy. Columns, expression of GITR, CD30, and CTLA-4 (median ± IQR) on circulating CD4+CD25+ T cells during high-dose IL-2 therapy. Analyses were done before (pre) and after (post) the first (1) and second (2) week of high-dose IL-2.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Effects of CD4+CD25+ T cells and CD4+CD25– T cells on the IFN-/IL-4 responses of iNKT cells and CD25– T cells. A and B, CD25– T cells were incubated at increasing levels with CD25+ T cells in the presence or absence of CD3 mAb. Columns, mean; bars, SD. IL-4 (A) and IFN- (B) release were determined as above. "CD25 lo" indicates a ratio of 1:16, and "CD25 hi" represents a ratio of 1:1. Data from three individual healthy and three high-dose IL-2–treated patient donors after week 1 of high-dose IL-2 (peak of CD25+ cells). C, IFN-/IL-4 ratios were determined in supernatants collected after a 48-h coculture of CD1d-transfected HeLa and iNKT cells in the presence of 100 ng/mL -GalCer, 20 units/mL recombinant human IL-2, and either CD4+CD25+ T cells (white columns) or CD4+CD25– T cells (black columns). CD4+CD25+ and CD4+CD25– T cells were isolated from peripheral blood after 1 wk of high-dose IL-2 therapy and added at a ratio of 1:2 iNKT cells. Data of six individual high-dose IL-2–treated patient donors.

Relation between clinical response and iNKT cells and CD4⁺CD25⁺ T cells during high-dose IL-2 therapy. Finally, we evaluated whether the pretreatment frequency of iNKT cells or CD4⁺CD25⁺ T cells or whether changes in the frequency of CD4⁺CD25⁺ T cells could be related to clinical outcome. Clinical responses were assessed at 6 and 12 weeks after initiation of high-dose IL-2 therapy. The pretreatment CD4⁺CD25⁺ T-cell frequency was comparable in patients with a partial response [7.4 ± 6.1% (mean ± SD)], mixed response (3.6 ± 1.9%), stable disease (4.2 ± 2.6%), or progressive disease (12.8 ± 12.0%) at 6 weeks (P = 0.26, one-way ANOVA). Similarly, the pretreatment iNKT cell frequency was not predictive of the clinical outcome (P = 0.58 and 0.55 for response assessment at 6 and 12 weeks, respectively). As high-dose IL-2 resulted in an increase in the frequency of CD4⁺CD25⁺ T cells in most, but not all, patients, we also evaluated whether the fold change in CD4⁺CD25⁺ T-cell frequency (pretreatment versus 1 week after high-dose IL-2) could be used to predict disease outcome. We found that the fold change in the frequency of CD4⁺CD25⁺ T cells was similar in patients with partial response (3.7 ± 4.0–fold), mixed response (3.1 ± 0.6–fold), stable disease (2.8 ± 1.6–fold), or progressive disease (2.7 ± 2.1–fold) at 6 weeks (P = 0.91). Similarly, neither the pretreatment CD4⁺CD25⁺ T-cell frequency nor the fold change in CD4⁺CD25⁺ T cells during the first week of high-dose IL-2 was statistically significantly different in patients with a partial response, mixed response, stable disease, or progressive disease at 12 weeks (P = 0.49 and 0.40, respectively; data not shown).

	Discussion

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Treatment with high-dose bolus IL-2 can be used in selected patients with metastatic melanoma or RCC (3, 4). IL-2, best known as a T-cell growth factor, plays a dominant role in the biology of iNKT cells and CD4⁺CD25⁺ regulatory T cells, both of which have been shown to play important roles in the regulation of antitumor immune responses. Here, we evaluated the in vivo effects of high-dose IL-2 therapy on iNKT cells, CD4⁺CD25⁺ regulatory T cells, and the DC with which they interact.

As professional antigen-presenting cells, DCs play critical roles in the initiation of immune responses that, depending on the microenvironment and DC subtype (e.g., myeloid versus lymphoid), can support either Th1- or Th2-type immune responses (26). We analyzed the frequency and CD1d expression of three types of circulating DC that can be readily identified by expression of BDCA-1 (mDC1), BDCA-2 (pDC), and BDCA-3 (mDC2; ref. 22). We found that CD1d was predominantly expressed by mDC1, confirming previous data (27), and that this mDC1-specific expression did not fluctuate during high-dose IL-2 therapy. The frequency of mDC1 and pDC, but not mDC2, temporarily decreased during treatment with high-dose IL-2. Because of the very low frequency of circulating mDC1 and pDC during high-dose IL-2, we were unable to assess whether the decreases in DC frequencies were accompanied by functional changes.

As expected from previous reports (8, 9), we found low pretreatment circulating iNKT cell numbers in the evaluated patients. Interestingly, although both the frequency and the absolute number of iNKT cells decreased further during treatment with high-dose IL-2, the absolute number of iNKT cells thereafter recovered to higher than pretreatment levels, reflecting either redistribution of iNKT cells from tissue to peripheral blood or an actual expansion of the total iNKT cell pool. As the size of the circulating iNKT cell pool predicts disease outcome in patients with squamous cell head and neck cancer (11) and immunologic responses to the iNKT cell ligand -GalCer (28), this suggests potential value of evaluating the antitumor effects of -GalCer after pretreatment of patients with IL-2. As CD4⁺ iNKT cells and CD4^–CD8^– double-negative iNKT cells represent distinct functional lineages of iNKT cells (23, 24), we assessed whether the relative proportion of each subset would change during high-dose IL-2. Although not sustained, the contribution of proinflammatory double-negative iNKT cells to the total iNKT cell pool did increase significantly after the first week of high-dose IL-2, potentially resulting in a more Th1-biased iNKT cell response when high-dose IL-2 therapy would be followed by -GalCer therapy. In several patients, we found that purified iNKT cells from these patients could indeed produce IFN- when activated by -GalCer in the context of CD1d, but a clear increase in iNKT cell IFN- secretion was only detected anecdotally.

Strikingly, high-dose IL-2 therapy resulted in an increase in both the frequency and the absolute number of CD4⁺CD25⁺ T cells. These increases of CD4⁺CD25⁺ T cells were accompanied by an increase in the level of CD25 expression, indicating the predominant expansion of CD4⁺CD25^high T cells previously shown to exert the majority of the suppressive effects attributed to the population of CD4⁺CD25⁺ T cells (25). As CD4⁺CD25⁺ regulatory T cells are critically dependent on the X chromosome–encoded FOXP3 gene (6, 12), we similarly evaluated the levels of CD4⁺Foxp3⁺ T cells during high-dose IL-2 therapy. Importantly, the increase in the frequency of CD4⁺CD25⁺ T cells was accompanied by a marked increase in CD4⁺Foxp3⁺ T cells to levels comparable with total CD4⁺CD25⁺ T cells, strongly suggesting that high-dose IL-2 indeed results in an increase in CD4⁺CD25⁺ regulatory T cells. We found limited surface expression on CD4⁺CD25⁺ T cells of GITR, CD30, and CTLA-4, molecules implicated in the function of CD4⁺CD25⁺ regulatory T cells. Among these, the increase in surface expression during high-dose IL-2 was most prominent for CD30, although the expression of GITR and CTLA-4 also increased. As IL-2 is not only a vital cytokine for the thymic generation and peripheral maintenance and function of CD4⁺CD25⁺ regulatory T cells but also seems to reduce the CD4⁺CD25⁺ regulatory T-cell inhibitory effects at higher doses (29), we additionally tested whether the expanded pool of CD4⁺CD25⁺ T cells could still suppress -GalCer–induced iNKT cell activation as previously shown for healthy donor–derived CD4⁺CD25⁺ T cells (21). As we found no evidence for suppressive effects of the expanded pool of CD4⁺CD25⁺ T cells, one can conclude that, although high-dose IL-2 results in the expansion of CD4⁺CD25⁺ T cells, it may also impair their suppressive effects, at least temporarily, perhaps due to activated conventional CD25⁺ T cells overwhelming the truly functionally suppressive CD4⁺CD25⁺ T cells. Indeed, in patients with HIV or cancer who are treated with lower doses of IL-2, an increase in CD4⁺CD25⁺ T cells was also noted, and although these cells were Foxp3⁺, they were also poor functional suppressors (30–32). In line with this, we found no relation between either the pretreatment CD4⁺CD25⁺ T-cell frequency or the high-dose IL-2–induced increase in CD4⁺CD25⁺ T-cell frequency and clinical responses. As previous studies showed that patients with higher CD4⁺CD25⁺ T-cell frequencies had a worse prognosis compared with patients with lower CD4⁺CD25⁺ T-cell frequencies, one mecha nism through which high-dose IL-2 might induce effective antitumor immunity may be ironically through the (temporary) inhibition of the suppressive effects of CD4⁺CD25⁺ T cells. It is likely that the suppressive effects of CD4⁺CD25⁺ T cells are indeed only temporarily inhibited, as two recent articles showed that suppressive effects of CD4⁺CD25⁺ T cells were intact from a few days to 4 weeks after high-dose IL-2 therapy (33, 34). Interestingly, these authors confirmed the increase in the frequency of circulating CD4⁺CD25⁺ T cells in patients with metastatic melanoma and RCC during high-dose IL-2 therapy, but Cesana et al. (33) also noted a significant decrease in CD4⁺CD25⁺ regulatory T cells 4 weeks after the second cycle of high-dose IL-2 in patients achieving an objec tive clinical response, perhaps reflecting migration of nonsuppressing CD4⁺CD25⁺ regulatory T cells to sites of antigenic stimulation.

In conclusion, our data show reciprocal effects of high-dose IL-2 therapy on circulating immunoregulatory iNKT and CD4⁺CD25⁺ T cells. Although the frequency and absolute number of iNKT cells decrease during high-dose IL-2 therapy, this is accompanied by an increase in the proportion of potentially proinflammatory double-negative iNKT cells and is followed by an increase in the total number of circulating iNKT cells. In contrast, both the frequency and the absolute number of CD4⁺CD25⁺ regulatory T cells increase during high-dose IL-2 therapy, and although the frequency returns to baseline levels, the absolute number remains elevated thereafter. Significantly, the expanded population of CD4⁺CD25⁺ T cells did not consistently exert suppressive effects, suggesting surprisingly that high-dose IL-2 therapy may also reverse CD4⁺CD25⁺ T-cell suppressive effects.

	Acknowledgments

 
We thank the patients for providing serial samples, Kirin Brewery for -GalCer, and colleagues for helpful discussions.

	Footnotes

 
Grant support: Netherlands Organization for Scientific Research NWO-TALENT grant and grant 920-03-142, Dutch Cancer Society (Koningin Wilhelmina Fonds) academic grant, NIH grants R01 DK066917 and R01 AI42955, Dana-Farber/Harvard Cancer Center Skin Cancer Specialized Program of Research Excellence grant P50 CA93683, Harvard Medical School Center for Human Cell Therapy pilot grant, and Hershey Family Prostate Cancer Research Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

⁴ J.W. Molling, personal communication.

Received 7/ 7/06; revised 11/10/06; accepted 11/28/06.

	References

Top Abstract Materials and Methods Results Discussion References

 

1. Morgan DA, Ruscetti FW, Gallo R. Selective in vitro growth of T lymphocytes for normal human bone marrows. Science 1976;193:1007–8.[Abstract/Free Full Text]
2. Rosenberg SA, Mule JJ, Speiss PJ, et al. Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2. J Exp Med 1985;161:1169–88.[Abstract/Free Full Text]
3. Atkins MB, Lotze MT, Dutcher JP, et al. High-dose recombinant interleukin-2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin Oncol 1999;17:2105–16.[Abstract/Free Full Text]
4. McDermott DF, Regan MM, Clark JI, et al. Randomized phase III trial of high-dose interleukin-2 versus subcutaneous interleukin-2 and interferon in patients with metastatic renal cell carcinoma. J Clin Oncol 2005;23:133–41.[Abstract/Free Full Text]
5. Lotze MT, Matory YL, Ettinghausen SE, et al. In vivo administration of purified human interleukin 2. II. Half life, immunologic effects, and expansion of peripheral lymphoid cells in vivo with recombinant IL 2. J Immunol 1985;135:2865–75.[Abstract]
6. Kronenberg M, Rudensky A. Regulation of immunity by self-reactive T cells. Nature 2005;435:598–604.[CrossRef][Medline]
7. van der Vliet HJ, Molling JW, von Blomberg BM, et al. The immunoregulatory role of CD1d-restricted natural killer T cells in disease. Clin Immunol 2004;112:8–23.[CrossRef][Medline]
8. Molling JW, Kolgen W, van der Vliet HJ, et al. Peripheral blood IFN--secreting V24⁺Vß11⁺ NKT cell numbers are decreased in cancer patients independent of tumor type or tumor load. Int J Cancer 2005;116:87–93.[CrossRef][Medline]
9. Tahir SM, Cheng O, Shaulov A, et al. Loss of IFN- production by invariant NK T cells in advanced cancer. J Immunol 2001;167:4046–50.[Abstract/Free Full Text]
10. Tachibana T, Onodera H, Tsuruyama T, et al. Increased intratumor V24-positive natural killer T cells: a prognostic factor for primary colorectal carcinomas. Clin Cancer Res 2005;11:7322–7.[Abstract/Free Full Text]
11. Molling JW, Langius JA, Langendijk JA, et al. Low levels of circulating invariant natural killer T cells predict poor clinical outcome in patients with head and neck squamous cell carcinoma. J Clin Oncol 2007 (in press).
12. Sakaguchi S. Naturally arising Foxp3-expressing CD4⁺CD25⁺ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005;6:345–52.[CrossRef][Medline]
13. Shevach EM. Certified professionals: CD4⁺ CD25⁺ suppressor T cells. J Exp Med 2001;193:F41–6.[Free Full Text]
14. Chattopadhyay S, Chakraborty NG, Mukherji B. Regulatory T cells and tumor immunity. Cancer Immunol Immunother 2005;54:1153–61.[CrossRef][Medline]
15. Viguier M, Lemaitre F, Verola O, et al. Foxp3 expressing CD4⁺CD25^high regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells. J Immunol 2004;173:1444–53.[Abstract/Free Full Text]
16. Curiel TJ, Coukos G, Zou L, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004;10:942–9.[CrossRef][Medline]
17. Onizuka S, Tawara I, Shimizu J, Sakaguchi S, Fujita T, Nakayama E. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor ) monoclonal antibody. Cancer Res 1999;59:3128–33.[Abstract/Free Full Text]
18. Dannull J, Su Z, Rizzieri D, et al. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells. J Clin Invest 2005;115:3623–33.[CrossRef][Medline]
19. Oken MM, Creech RH, Tormey DC, et al. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol 1982;5:649–55.[Medline]
20. Gavin MA, Torgerson TR, Houston E, et al. Single-cell analysis of normal and FOXP3-mutant human cells: FOXP3 expression without regulatory T cell development. Proc Natl Acad Sci U S A 2006;103:6659–64.[Abstract/Free Full Text]
21. Azuma T, Takahashi T, Kunisato A, Kitamura T, Hirai H. Human CD4⁺CD25⁺ regulatory T cells suppress NKT cell functions. Cancer Res 2003;63:4516–20.[Abstract/Free Full Text]
22. Dzionek A, Fuchs A, Schmidt P, et al. BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood. J Immunol 2000;165:6037–46.[Abstract/Free Full Text]
23. Gumperz JE, Miyake S, Yamamura T, Brenner MB. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J Exp Med 2002;195:625–36.[Abstract/Free Full Text]
24. Lee PT, Benlagha K, Teyton L, Bendelac A. Distinct functional lineages of human V24 natural killer T cells. J Exp Med 2002;195:637–41.[Abstract/Free Full Text]
25. Baecher-Allan C, Wolf E, Hafler DA. Functional analysis of highly defined, FACS-isolated populations of human regulatory CD4⁺CD25⁺ T cells. Clin Immunol 2005;115:10–8.[CrossRef][Medline]
26. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245–52.[CrossRef][Medline]
27. van der Vliet HJ, Molling JW, von Blomberg BM, et al. Circulating V24⁺Vß11⁺ NKT cell numbers and dendritic cell CD1d expression in hepatitis C virus infected patients. Clin Immunol 2005;114:183–9.[CrossRef][Medline]
28. Giaccone G, Punt CJ, Ando Y, et al. A phase I study of the natural killer T-cell ligand -galactosylceramide (KRN7000) in patients with solid tumors. Clin Cancer Res 2002;8:3702–9.[Abstract/Free Full Text]
29. Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G. Ex vivo isolation and characterization of CD4⁺CD25⁺ T cells with regulatory properties from human blood. J Exp Med 2001;193:1303–10.[Abstract/Free Full Text]
30. Sereti I, Imamichi H, Natarajan V, et al. In vivo expansion of CD4CD45RO-CD25 T cells expressing foxP3 in IL-2-treated HIV-infected patients. J Clin Invest 2005;115:1839–47.[CrossRef][Medline]
31. Shah MH, Freud AG, Benson DM, et al. A phase I study of ultra low dose interleukin-2 and stem cell factor in patients with HIV infection or HIV and cancer. Clin Cancer Res 2006;12:3993–6.[Abstract/Free Full Text]
32. Zorn E, Nelson EA, Mohseni M, et al. IL-2 regulates FOXP3 expression in human CD4⁺CD25⁺ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood 2006;108:1571–9.[Abstract/Free Full Text]
33. 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 2006;24:1169–77.[Abstract/Free Full Text]
34. Ahmadzadeh M, Rosenberg SA. IL-2 administration increases CD4⁺CD25hi Foxp3⁺ regulatory T cells in cancer patients. Blood 2006;107:2409–14.[Abstract/Free Full Text]

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