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Human CD4⁺ CD25⁺ Regulatory T Cells Suppress NKT Cell Functions

Departments of Hematology and Oncology [T. A., T. T., A. K., H. H.] and Urology [T. A., T. K.], Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan

	ABSTRACT

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CD4⁺CD25⁺ regulatory T cells play an important role in peripheral tolerance. These cells have been reported to be capable of suppressing the response of CD4⁺CD25^- T cells in vitro. The depletion of these cells evokes effective immune responses to tumor cells in vivo. In this study, we demonstrate that CD4⁺CD25⁺ T cells also suppress all subsets of V24⁺NKT cells (V24⁺CD4^-CD8^- double negative, V24⁺CD4⁺, and V24⁺CD8⁺) in both proliferation and cytokine production [IFN-, interleukin-4 (IL-4), IL-13, and IL-10]. This suppression is mediated by cell-to-cell contact but not by a humoral factor or the inhibition of antigen-presenting cells. Moreover, the cytotoxic activity of V24⁺NKT cells against some tumor cell lines is suppressed by CD4⁺CD25⁺ T cells. This finding is important in developing an effective immunotherapy for cancer.

	INTRODUCTION

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Immunotherapy is a promising treatment for cancer. However, vaccination with a tumor antigen or the adoptive transfer of ex vivo propagated lymphocytes may lead to the development of autoimmune diseases because there are antigenic cross-reactions between a tumor antigen and normal tissue antigens (1, 2, 3) . These findings suggest the possibility that the mechanisms maintaining immunological tolerance to self-constituents impede the generation of effective tumor immunity.

CD4⁺CD25⁺ T cells have been shown to represent a unique population of immunoregulatory cells. These cells constitute 5–10% of peripheral CD4⁺ T cells and resemble anergic cells in vitro (4, 5, 6, 7, 8) . After T-cell receptor-mediated stimulation, CD4⁺CD25⁺ T cells suppress the activation and proliferation of CD4⁺CD25^- T cells in an antigen-nonspecific manner (4, 5, 6, 7, 8) . Their inhibitory capacity is mediated by direct cell-to-cell contact but not by cytokines, such as IL-10² and TGF-ß (4 -8) . It has been reported that the depletion of CD4⁺CD25⁺ T cells can disrupt immunological unresponsiveness to autologous tumors in vitro and in vivo, leading to the spontaneous development of tumor-specific and tumor-nonspecific effector cells (9 , 10) .

V24⁺ NKT cells have been recently defined as a novel lymphocyte lineage, characterized by the expression of an invariant T-cell receptor encoded by V24-JQ and an NK receptor (11 , 12) . These cells recognize a glycolipid antigen, such as -galactosylceramide in a CD1d-dependent fashion (13, 14, 15, 16) . Recently, three subsets of V24⁺ NKT cells have been identified, which had different cytokine production patterns (17) . V24⁺ NKT cells had cytotoxicity against some tumor cells (13 , 18) . Studies using J281 knockout mice, which lack V14⁺ NKT cells, indicate that V24⁺ NKT cells contribute to antitumor immunity (13) . Moreover, the in vivo stimulation of invariant NKT cells with -GalCer stimulates IL-12 production and NK cell activation and augments antitumor responses (13 , 19, 20, 21) . Thus, V24⁺ NKT cells are promising for antitumor immunotherapy.

Although it has been evident for years that CD4⁺CD25⁺ T cells are a unique population of immunoregulatory cells, the potential inhibitory activity of CD4⁺CD25⁺ T cells on non-CD4⁺ T cells has not been studied in detail. In this study, we demonstrate that CD4⁺CD25⁺ T cells can suppress proliferation, cytokine secretion, and cytotoxic activity in each subset of V24⁺ NKT cells. This inhibitory effect is dependent on cell-to-cell contact. This finding is important in developing an effective immunotherapy for cancer.

	MATERIALS AND METHODS

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Abs and Reagents
The following Abs were purchased from Immunotech (Marseilles, France): IgG1 (679.1 Mc7), IgG2b (MOPC-195), anti-CD3 (UCHT-1), anti-CD4 [SFCI12T4D11(T4)], anti-CD8ß (2ST8.5H7), anti-CD161 (191B8), anti-V24 (C15), anti-Vß11 (C21), anti-CD25 (m-A251), anti-CD45RA (HI 100), anti-CD45RO (UCHL1), and anti-CD152 (BNI3.1). Anti-CD1d (55.3.1) Ab (22) was a gift from Dr. S. Porcelli (Albert Einstein College of Medicine, Bronx, NY). -GalCer was obtained from Kirin (Gunma, Japan). rhGM-colony-stimulating factor and rhIL-4 were purchased from CellGenix (Freiburg, Germany), and rhIL-2 was obtained from Shionogi (Osaka, Japan). Anti-TGF-ß (R&D Systems, Abingdon, United Kingdom) and anti-IL-10 (JES3–19F1) Abs (BD PharMingen, San Diego, CA) were used for neutralization experiments.

Cell Populations
CD4⁺CD25^- and CD4⁺CD25⁺ T Cells.
PBMCs were isolated from peripheral blood samples by density gradient centrifugation using Ficoll-Hypaque (Pharmacia, Upsala, Sweden). PBMCs were incubated in the AIM medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS at 37°C on tissue culture dishes (IWAKI, Tokyo, Japan) for 1 h to remove adherent cells. Nonadherent cells were collected and washed. CD4⁺ T cells were purified by negative selection with a cocktail of antibodies [anti-CD8 (OKT8), anti-CD14 (ME2), anti-CD56 (N901), anti-CD19 (BU12), and anti-class II (L243)] followed by magnetic bead sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated CD4⁺ T cells were incubated with FITC-conjugated anti-CD25 Ab and then with anti-FITC magnetic beads. CD4⁺CD25⁺ T cells were obtained by positive selection and CD4⁺CD25^- T cells by negative selection.

Establishment of V24⁺NKT Cells.
Monocytes from healthy human donors were cultured in the AIM medium supplemented with 10% FCS, 500 units/ml rhIL-4, and 500 units/ml rhGM-colony-stimulating factor for 5 days and used as Mo-DCs. Lymphocytes from the same donor were cultured with irradiated (50 Gy) Mo-DCs, which were pulsed for 12 h with -GalCer (100 ng/ml). After 7 days, V24⁺ cells were established by positive (V24) magnetic bead sorting and maintained in the presence of 40 units/ml rhIL-2. The cells were restimulated every 7 days. Allogeneic Mo-DCs pulsed with -GalCer were used in restimulation. V24⁺NKT cells recognized monomorphic CD1d expressed in allogeneic Mo-DCs; thus, they could be stimulated by this method. When the cells were expanded, V24⁺Vß11⁺CD4^-CD8^-, V24⁺Vß11⁺CD4⁺, and V24⁺Vß11⁺CD8⁺ subsets were sorted using the FACSVantage apparatus (Becton Dickinson, San Jose, CA) and maintained in the presence of 40 units/ml rhIL-2 and by restimulation every 7–10 days.

	Flowcytometry and Sorting

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Cells were analyzed by flowcytometry (FACScalibur; Becton Dickinson). Immunofluorescence staining using specific monoclonal antibody was performed according to a standard procedure. V24⁺Vß11⁺CD4^-CD8^-, V24⁺Vß11⁺CD4⁺, and V24⁺Vß11⁺CD8⁺ subsets were sorted using the FACSVantage apparatus (Becton Dickinson).

	Proliferation of NKT Cells in Response to -GalCer-pulsed Mo-DCs

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The bulk of 1 x 10⁵ NKT or CD4⁺CD25^- T cells as responders and 5 x 10⁴ irradiated allogeneic Mo-DCs (all allogeneic monocytes were derived from the same donor) as stimulators in 0.2 ml of the culture medium was seeded onto roundbottomed microtiter wells with or without 100 ng/ml -GalCer. For the assessment of their suppression properties, CD4⁺CD25⁺ T cells were added at different concentrations. The culture was incubated for 72 h. At the final 12 h of incubation, 1 µCi of [³H]dThd was added to each well, and the incorporation of [³H]dThd was determined by liquid scintillation counting.

	Cytokine Production Assay

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NKT cells (1 x 10⁵) and 5 x 10⁴ irradiated--GalCer-pulsed Mo-DCs (all allogeneic monocytes were derived from the same donor) were suspended in 200 µl of the culture medium in 96-well plates. After 18 h, the supernatant was collected from each well and subjected to ELISA of the concentrations of IFN-, IL-4, IL-10, and IL-13 by ELISA according to the manufacturer’s protocol (AN'ALYZA; Genzyme, Cambridge, MA).

	Transwell Experiments

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Transwell experiments were performed in 24-well plates. NKT cells (1 x 10⁵) were stimulated with 5 x 10⁴ irradiated allogeneic Mo-DCs pulsed with -GalCer. In addition, 1 x 10⁵ CD4⁺CD25^- T cells were either added directly to the culture or placed in transwell chambers (Millicell, 0.4µ; Millipore). After 72 h of coculture, NKT cells were transferred to 96-well plates in triplicates. Proliferation was measured by liquid scintillation counting after pulsing with [³H]dThd for 12 h.

	Cytotoxic Assay

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⁵¹Cr-labeled (Na₂⁵¹CrO₃; Amersham, Arlington Heights, IL) MOLT-4 or Jurkat cells (5 x 10³) as the target and effector cells of various numbers in 200 µl of the culture medium were seeded onto roundbottomed microtiter wells. The culture was incubated for 4 h, and 100 µl of the supernatant were collected from each well. The percentage of specific ⁵¹Cr release was calculated using [(cpm experimental release - cpm spontaneous release)/(cpm maximal release - cpm spontaneous release)] x 100.

	RESULTS

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Phenotypic Characterization of CD4⁺CD25⁺ Regulatory T and V24⁺NKT Cells.
CD4⁺CD25⁺ T cells represented 5–10% of CD4⁺ T cells. Both CD4⁺CD25^- and CD4⁺CD25⁺ cells were isolated with purities of >90%. The expressions of surface antigens on these populations were compared by FACS analysis (Fig. 1) . CD4⁺CD25⁺ T cells expressed CD45RO but not CTLA-4. No surface expression of CTLA-4 was observed in both populations. However, CD4⁺CD25⁺ T cells expressed CTLA-4 intracellularly. Furthermore, CTLA-4 expression was detectable after activation by allogeneic DCs (data not shown). Two subsets (CD45RA^- and CD45RA⁺) of CD4⁺CD25⁺ T cells were identified. CD45RA⁺ had a lower CD25 expression level than CD45RA^-. We considered that the CD45RA⁺ comprised CD4⁺CD25^+low T cells, which were isolated by Baecher-Allan et al. (7) .

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Isolation and cell surface phenotype of CD4+CD25+ T cells. CD4+ T cells were isolated from PBMCs and separated into CD25+ and CD25- fractions. The phenotypes of CD4+CD25+ and CD4+CD25- T cells were analyzed by two-color flow cytometry using the indicated monoclonal antibodies. In addition, intracellular CTLA-4 expression was analyzed. Representative results of three independent experiments are shown.

CD4⁺CD25⁺ Regulatory T Cells Suppress the Proliferation and Cytokine Secretion of V24⁺NKT Cells in Response to -GalCer-pulsed Mo-DCs.
We confirmed that CD4⁺CD25⁺ T cells had regulatory properties (4, 5, 6) . These cells suppressed the proliferative response of alloreactive CD4⁺CD25^- T cells (data not shown). Then, to investigate the ability of CD4⁺CD25⁺ T cells to suppress the proliferation of V24⁺NKT cells, each subset of V24⁺NKT cells was stimulated with allogeneic Mo-DCs using -GalCer, and variable numbers of autologous CD4⁺CD25⁺ T cells were added. CD4⁺CD25⁺ T cells inhibited the proliferation of all subsets of V24⁺NKT cells in a dose-dependent manner. CD4⁺CD25⁺ T cells themselves failed to proliferate in response to alloantigens (Fig. 2A) .

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. In A, CD4+CD25+ T cells suppress the proliferation of V24+ DN, V24+CD4+, and V24+CD8+ NKT cells in a dose-dependent manner. The proliferative response of each NKT population (1 x 105 cells/well) was stimulated with irradiated allogeneic Mo-DCs (5 x 104 cells/well) in the presence of CD4+CD25+ T cells of various numbers from the same donor. Proliferation was determined after 72 h of culture by the addition of [3H]dThd at 12 h. All data represent the means ± SD of triplicate assays. Representative results of three independent experiments. In B, CD4+CD25+ T cells do not secrete cytokines when stimulated with allogeneic Mo-DCs but suppress the secretion of IFN-, IL-4, IL-10, and IL-13 by NKT cells in a dose-dependent manner. The supernatants were collected from the proliferation cultures incubated for 18 h. Concentrations of IFN-, IL-4, IL-10, and IL-13 were determined by ELISA. All data represent the means ± SD of triplicate assays. Representative results of three independent experiments are shown.

We investigated the cytotoxicity of CD4⁺CD25⁺ T cells to analyze mechanisms underlying the above suppression. The cytotoxicity of CD4⁺CD25⁺ T cells against V24⁺NKT cells was not observed (data not shown).

The Inhibitory Effect of CD4⁺CD25⁺ Regulatory T Cells on V24⁺NKT Cells Is Direct and Contact Dependent but Cytokine Independent.
We investigated whether cell-to-cell contact or soluble factors mediate the inhibitory effect of CD4⁺CD25⁺ T cells. As shown in Fig. 3 , the addition of neutralizing antibodies against IL-10 or TGF-ß could not restore the proliferation of all V24⁺NKT subsets. ICAM-1 is an adhesion molecule that plays important roles in cell-to-cell contact. The blockade of ICAM-1 using anti-ICAM-1 Abs could reverse the inhibitory effect. These observations suggest that direct cell contact rather than humoral factors is responsible for the inhibitory effect of CD4⁺CD25⁺ T cells.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. CD4+CD25+ T-cell inhibitory function is direct cell contact dependent but cytokine independent. NKT cells (1 x 105 cells/well) were stimulated with irradiated allogeneic Mo-DCs in the bottom chamber of the transwell plate; CD4+CD25+ T cells (1 x 105 cells/well) were then added and activated with irradiated allogeneic Mo-DCs separately in the top chamber. Alternatively, CD4+CD25+ T cells (1 x 105 cells/well) were cocultured with NKT cells at a ratio of 1:1 (1 x 105 cells/well) and stimulated with allogeneic Mo-DCs (5 x 104 cells/well) with or without 10 µg/ml anti-IL-10, 10 µg/ml anti-TGF-ß, and 10 µg/ml anti-ICAM-1 Abs. All data represent the means ± SD of triplicate assays. Representative results of three independent experiments are shown.

CD4⁺CD25⁺ Regulatory T Cells Inhibit the Cytotoxic Activity of V24⁺NKT Cells against Tumor Cell Lines.
V24⁺NKT cells have cytotoxic activity against some tumor cell lines. We investigated whether CD4⁺CD25⁺ T cells regulate the cytotoxic activity of V24⁺NKT cells. The cytotoxicity of V24⁺NKT cells against MOLT-4 and Jurkat cells was examined after coculture in vitro with or without CD4⁺CD25⁺ T cells for 8 h. These T-cell leukemia cell lines were chosen as the target cells because these cell lines were the most sensitive to human V24⁺NKT cells among the cell lines tested (data not shown). CD4⁺CD25⁺ T cells inhibited the cytotoxic activity of all subsets of V24⁺NKT cells against these tumor cell lines. At a ratio of 1:1, cytotoxicity was inhibited by 50%, and the separation of the two populations in transwell chambers abolished this inhibitory effect (Fig. 4) .

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. CD4+CD25+ T cells inhibit the cytotoxicity of NKT cells against MOLT-4 and Jurkat cells. 51Cr-labeled MOLT-4 (5 x 103) or Jurkat cells and effector cells were incubated with or without CD4+CD25+ T cells for 4 h. The percentage of specific 51Cr release was calculated. All data represent the means ± SD of triplicate assays. Representative results of three independent experiments are shown.

	DISCUSSION

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Many reports have clarified the phenotypic and functional characteristics of human CD4⁺CD25⁺ T cells (4, 5, 6 , 8) . Our isolated cells had a distinctive phenotype, expressing a mixture of markers of memory and naïve T cells, and inhibited the proliferation of CD4⁺CD25^- T cells (data not shown). Our CD4⁺CD25⁺ T cells were suggested to be of the same population as in previous reports (4, 5, 6 , 8) .

We showed that CD4⁺CD25⁺ T cells could suppress proliferation, cytokine secretion, and the cytotoxic activity of V24⁺NKT cells. Fifty percent suppression was observed in the proliferation assay at a cell ratio of 1:1. Previous studies on the inhibitory activity of human CD4⁺CD25⁺ T cells against CD4⁺CD25^- T cells showed 80–50% inhibition at a cell ratio of 1:1 (4, 5, 6, 7, 8) . It is important to note that this inhibition is in a dose-dependent manner.

V24⁺NKT cells can produce large amounts of IL-4 and IFN- (18) , which can potentially influence immune regulation, such as the determination of the Th1/Th2 profile. In several mouse models, it has been indicated that V24⁺NKT cells augment protective cellular Th1-like immune responses (28, 29, 30, 31, 32) . Moreover, previous studies showed the decreased numbers of V24⁺NKT cells in patients with melanoma and prostate cancer (33 , 34) . These data suggest that the loss of the V24⁺NKT cell function is a general finding in advanced cancers. Our data may support the idea that CD4⁺CD25⁺ T cells are associated with the prevention of the increase in the number of V24⁺NKT cells and can suppress the secretion of both Th1 and Th2 cytokines. Recently, it has been reported that there are three subsets of V24⁺NKT cells (V24⁺DN, V24⁺CD4⁺, and V24⁺CD8⁺ NKT cells), which show different cytokine production patterns (17) . V24⁺CD4⁺ NKT cells can produce a larger amount of Th2 cytokines (17) . However, there was no difference in the degree of suppression among the three subsets.

V24⁺NKT cells have also been considered to be promising for cancer immunotherapy for several reasons (13 , 18 , 33 , 35) . These cells exhibit a high cytotoxic activity against some tumor cells, and normal tissue cells are not susceptible to them. -GalCer can be applied to all patients, irrespective of MHC haplotype, because the activation of V24⁺NKT cells is restricted to CD1d molecules, which are monomorphic among individuals. The initial number of V24⁺NKT cells in cancer patients is significantly lower than that in healthy volunteers. Recently, it has been reported that -GalCer can effectively induce antitumor cytotoxic activity through the specific activation of NKT cells in mouse models (19 , 33 , 35) . It is unclear whether CD4⁺CD25⁺ T cells directly suppress the cytotoxicity of effector cells. We showed that CD4⁺CD25⁺ T cells suppress the cytotoxicity of V24⁺NKT cells against tumor cells. It has also not been reported that CD4⁺CD25⁺ T cells suppress the cytotoxicity of CD4⁺CD25^- T cells. This is the first report on the suppression of cytotoxicity of CD4⁺CD25⁺ T cells. Our results suggest that the combination of the depletion of CD4⁺CD25⁺ T cells and immunotherapy using the NKT cell system might result in the maximal efficacy.

The mechanism underlying CD4⁺CD25⁺ T-cell suppression remains to be fully elucidated. Our results demonstrate that the addition of anti-IL-10 or anti-TGF-ß Ab has no effect on the activity of CD4⁺CD25⁺ T cells, whereas the addition of anti-ICAM-1 Ab or transwell analysis abolishes the inhibitory effect of CD4⁺CD25⁺ T cells. Thus, it is not dependent on inhibitory cytokines, IL-10 or TGF-ß, but requires cell-to-cell contact initially. There remains a possibility that the suppression mechanism might be associated with a soluble factor after initial cell-to-cell contact. Next, we evaluated the possibility that the inhibitory function of CD4⁺CD25⁺ T cells is mediated by adenomatous polyposis colis. Recently, it has been reported that mouse CD4⁺CD25⁺ T cells down-regulate the expressions of CD80 and CD86 in Mo-DCs (36) . However, our results showed that they have no effect on the development of Mo-DCs. We also confirmed that there is no significant down-regulation of CD1d molecule expression, which is necessary for V24⁺NKT cell activation (13, 14, 15, 16) . Moreover, we confirmed that CD4⁺CD25⁺ T cells do not inhibit Mo-DC function by the mixed leukocyte reaction. These results suggest that CD4⁺CD25⁺ T cells may directly regulate the function of V24⁺NKT cells in a cell-to-cell contact manner.

In conclusion, we demonstrated that CD4⁺CD25⁺ T cells suppress the proliferation and function of V24⁺NKT cells. Most of the effects of CD4⁺CD25⁺ T cells on V24⁺NKT cells were similar to those observed in CD4⁺ T cells (4, 5, 6 , 8 , 26 , 27) . Two important findings should be noted: (a) in addition to cell proliferation, secretion of various cytokines by all subsets of V24⁺NKT cells is inhibited by CD4⁺CD25⁺ T cells; and (b) the cytotoxicity of V24⁺NKT cells against tumor cell lines is suppressed by CD4⁺CD25⁺ T cells. These data are important in developing an effective immunotherapy for human cancers.

	ACKNOWLEDGMENTS

 
We thank Eri Nagata for technical assistance.

	FOOTNOTES

 
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.

¹ To whom requests for reprints should be addressed, at Department of Urology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-8655, Japan. Fax: 81-3-3812-7838; E-mail: t-azuma{at}tc4.so-net.ne.jp

² The abbreviations used are: IL, interleukin; TGF, transforming growth factor; DN, double-negative; rh, recombinant human; Mo-DC, monocyte-derived dendritic cell; NK, natural killer; Ab, antibody; CTLA-4, CTL-associated antigen-4; ICAM, intercellular adhesion molecule; FACS, fluorescence-activated cell sorter; dThd, thymidine; PBMC, peripheral blood mononuclear cell.

Received 11/14/02. Accepted 5/27/03.

	REFERENCES

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