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The Journal of Immunology, 2005, 175: 4433-4440.
Copyright © 2005 by The American Association of Immunologists

IFN-{gamma} Controls the Generation/Activation of CD4+CD25+ Regulatory T Cells in Antitumor Immune Response1

Hiroyoshi Nishikawa2,*, Takuma Kato{dagger}, Isao Tawara*, Hiroaki Ikeda3,*,{ddagger}, Kagemasa Kuribayashi{dagger}, Paul M. Allen{ddagger}, Robert D. Schreiber{ddagger}, Lloyd J. Old§ and Hiroshi Shiku4,*

* Second Department of Internal Medicine and {dagger} Department of Bioregulation, Mie University School of Medicine, Mie, Japan; {ddagger} Department of Pathology and Immunology, Center for Immunology, Washington University School of Medicine, St. Louis, MO 63110; and § Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunization with serological identification of Ags by recombinant expression cloning (SEREX)-defined self-Ags leads to generation/activation of CD4+CD25+ regulatory T cells with suppressive activities and enhanced expression of Foxp3. This is associated with increased susceptibility to pulmonary metastasis following challenge with syngeneic tumor cells and enhanced development of 3-methylcholanthrene-induced primary tumors. In contrast, coimmunization with the same SEREX-defined self-Ags mixed with a CTL epitope results in augmented CTL activity and heightened resistance to pulmonary metastasis, both of which depend on CD4+ Th cells. These active regulatory T cells and Th cells were derived from two distinct CD4+ T cell subsets, CD4+CD25+ T cells and CD4+CD25– T cells, respectively. In the present study, IFN-{gamma} was found to abrogate the generation/activation of CD4+CD25+ regulatory T cells by immunization with SEREX-defined self-Ag. CD4+CD25+ T cells from these IFN-{gamma}-treated mice failed to exhibit immunosuppressive activity as measured by 1) increased number of pulmonary metastasis, 2) enhanced development of 3-methylcholanthrene-induced primary tumors, 3) suppression of peptide-specific T cell proliferation, and 4) enhanced expression of Foxp3. The important role of IFN-{gamma} produced by CD8+ T cells was shown in experiments demonstrating that CD4+CD25+ T cells cotransferred with CD8+ T cells from IFN-{gamma}–/– mice, but not from wild-type BALB/c mice, became immunosuppressive and enhanced pulmonary metastasis when recipient animals were subsequently immunized with a SEREX-defined self-Ag and a CTL epitope. These findings support the idea that IFN-{gamma} regulates the generation/activation of CD4+CD25+ regulatory T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Naturally occurring CD4+CD25+ regulatory T cells play an important role in maintaining immunological balance in hosts by suppressing a wide variety of immune responses (1, 2, 3, 4). Although generation of these regulatory T cells has been demonstrated to be thymus dependent, their selection pathway through interaction with self-immunogenic peptides in the context of MHC class II molecules is not clearly understood (2, 3, 4). It has been suggested that CD4+CD25+ regulatory T cells experience a distinct intrathymic selection pathway as compared with other CD4+ T cells (5, 6, 7, 8).

We have recently proposed that a category of serologically defined self-Ags constitutes Ags recognized by both CD4+CD25+ naturally occurring regulatory T cells and CD4+ Th cells (9, 10, 11, 12). These self-Ags were identified by serological identification of Ags by recombinant expression cloning (SEREX),5 a widely used serological cloning method to detect tumor Ags (9, 13, 14). Immunogenic tumor-derived molecules are detected by IgG Abs in the sera of hosts bearing cognate tumors by screening {lambda} phage libraries prepared from cDNA of tumor cells. Although a small fraction of these Ags shows tumor specificity, due to mutations or restricted expression patterns, the great majority are wild-type self-Ags that are broadly expressed (9, 13, 14). In the previous study, we identified a number of immunogenic self-Ags by screening cDNA expression libraries from transplantable 3-methylcholanthrene (MCA)-induced sarcomas of BALB/c origin with sera of animals bearing cognate tumors. Among the array of genes detected, four of the most frequently detected and broadly expressed gene products were used for analysis after verifying the lack of any gene mutations based on the registered sequences in GenBank (9). For control purpose, clones encoding nonimmunogenic molecules were randomly selected from the SEREX screening library (9).

Immunization of BALB/c mice with plasmids encoding any one of the four immunogenic self-Ags resulted in enhancement of pulmonary metastasis following i.v. challenge with syngeneic tumor cells (10) and acceleration of tumor development induced by MCA (12). Detailed analysis showed that metastasis enhancement and acceleration of tumor development were due to the immunosuppressive effects of CD4+CD25+ T cells in the immunized hosts. These CD4+CD25+ T cells suppressed the activity of invariant NKT (iNKT) cells and NK cells (10, 12). In addition, the CD4+CD25+ T cells strongly suppressed in vitro peptide-specific proliferation of CD4+CD25– T cells and CD8+ T cells, indicating their suppressive activity in a wide range of immune responses (10, 11, 12). In contrast, coimmunization with a mixture of plasmids encoding a tumor-specific CTL epitope and any one of these SEREX-defined self-Ags led to a marked increase in the number of peptide-specific CD8+ CTL and to heightened resistance to challenge with syngeneic tumors expressing the CTL epitope. This heightened helper response was dependent on CD4+ T cells and on copresentation of the CTL epitope with the SEREX-defined self-Ag (9).

These results indicate that immunization with SEREX-defined self-Ags leads to generation/activation of either CD4+CD25+ regulatory T cells with potent suppressive activity or CD4+CD25– Th cells essential for amplification of CD8+ CTL. In relation to antitumor immunity, development of CD4+CD25+ regulatory T cells would result in tumor enhancement, whereas CD4+CD25– Th cells would lead to tumor rejection. The present study was designed to elucidate the cellular and molecular mechanisms underlying the generation/activation of CD4+CD25+ regulatory T cells. We report that IFN-{gamma} produced by peptide-specific CD8+ T cells controls the generation/activation of CD4+CD25+ regulatory T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female BALB/c mice and C.B-17 scid mice were purchased from CLEA Japan and used at 7–10 wk of age. IFN-{gamma}–/– mice with BALB/c background were purchased from The Jackson Laboratory. DUC18 mice, transgenic for {alpha}{beta}-TCR reactive with the Kd-restricted 136–144 peptide of mutated MAPK, ERK2 (mERK2) were established as described previously (15, 16). DO11.10 mice, transgenic for {alpha}{beta}-TCR reactive with the I-Ad-restricted 323–339 peptide of OVA, were kindly provided by Dr. K. M. Murphy (Washington University, St. Louis, MO) (17). Mice were maintained at the Animal Center of Mie University School of Medicine (Mie, Japan). The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Mie University School of Medicine.

Tumors

CMS5 is a MCA-induced sarcoma cell line of BALB/c origin (18). CMS5m is a subcloned cell line obtained from CMS5, a tumor expressing mERK2 (9, 15). P1.HTR is a subline of mastocytoma P815 of DBA/2 origin (19).

Immunization by gene gun

Gold particles coated with plasmid DNA (1 µg/injection) were prepared and delivered into shaved skin of the abdominal wall of BALB/c mice by a Helios Gene Gun System (Bio-Rad) at a helium discharge pressure of 350–400 pounds per square inch, as described (9).

Abs and reagents

Anti-CD3 mAb (145-2C11, hamster IgG) and anti-IFN-{gamma} mAb (H22, hamster IgG) were purified from ascetic fluid on a protein A column. FITC-conjugated anti-CD25 mAb (PC61, rat IgG1) was purchased from eBioscience. Synthetic mERK2136–144-9m peptide QYIHSANVL (15), HER2p63–71 (T) peptide TYLPTNASL (20), and chicken OVA323–339 peptide ISQAVHAAHAEINEAGR (17) were obtained from Takara Shuzo. SEREX-defined molecules and control SEREX-unrelated molecules were cloned into pBK-CMV (Stratagene) and purified using the EndoFree Plasmid Mega kit (Qiagen). A cDNA encoding the region of mERK2 containing CTL epitope 9m was cloned into pCAGGS-New (21). pCAGGS-New was kindly provided by Dr. J. Miyazaki (Osaka University, Osaka, Japan). IL-2, IL-4, IL-10, IL-12, IL-18, and IFN-{gamma} cloned into pCAGGS-New were donated by Dr. J. Miyazaki (Osaka University). {alpha}-Galactosylceramide ({alpha}-GalCer) was kindly provided by Pharmaceutical Research Laboratories (Kirin Brewery) (22). {alpha}-GalCer/CD1d tetramer was prepared as described previously (10).

Purification of cells

CD4+CD25+ and CD4+CD25– T cells were prepared as described previously (10, 11, 12) and were confirmed to contain >96% and >93% of respective cell population.

Spleen cell suspensions were mixed with anti-CD8-microbeads (Miltenyi Biotec) and subsequently separated into CD8+ T cells by positive selection on a MACS column. CD8+ T cell populations were confirmed to contain >95% CD8+ T cells.

Splenocytes from BALB/c mice were enriched for {alpha}-GalCer/CD1d tetramer+ cells using anti-PE microbeads (Miltenyi Biotec), as described previously (10). Purified cells were inoculated into mice through the lateral tail vein.

Proliferation assay

A total of 5 x 104 CD4+CD25– T cells obtained from DO11.10 mice or 5 x 104 CD8+ T cells obtained from DUC18 mice were cultured with 5 x 104 mitomycin C (MMC)-treated splenic Thy-1– APCs prepared from wild-type BALB/c mice in the presence of 10 µg/ml OVA323–339 peptide or mERK2136–144-9m peptide in 96-well-flat-bottom plates. To these cultures, CD4+CD25+ or CD4+CD25– T cells were added. Proliferation was evaluated by pulsing with 0.5 µCi/well [3H]thymidine for the last 6 h of 72 h culture. [3H]Thymidine incorporation was measured by a scintillation counter.

Real-time quantitative RT-PCR

Oligo-dT-primed first strand cDNA of Foxp3 or hypoxanthine-guanine phosphoribosyltransferase was synthesized for real-time RT-PCR. Real-time RT-PCR was performed as described previously (11, 23).

ELISPOT assay

The number of IFN-{gamma}-secreting peptide-specific CD8+ T cells was assessed by ELISPOT assays as described previously (9). Briefly, purified 1 x 105 CD8+ T cells were cultured for 24 h with 1 x 105 MMC-treated P1.HTR pulsed with mERK2136–144-9m or HER2p63–71 (T) peptides in 96-well nitrocellulose-coated microtiter plates (Millipore) coated with rat anti-mouse IFN-{gamma} (R4-6A2; BD Pharmingen). Spots were developed using biotinylated anti-mouse IFN-{gamma} (XMG1.2; BD Pharmingen), alkaline phosphatase-conjugated streptavidin (MabTech) and the Alkaline Phosphatase Substrate kit (Bio-Rad), and subsequently counted using a dissecting microscope and Axioplan 2 imaging system (Carl Zeiss).

Induction of tumors by MCA

Groups of 18–36 mice were inoculated s.c. in the right hind flank with 0.2 ml of peanut oil (Sigma-Aldrich) containing 50 µg of MCA and monitored weekly for the development of tumors. Tumors >5 mm in diameter and demonstrating progressive growth over 3 wk were counted as primary tumors.

Statistical analysis

Statistical significance was evaluated by a Mann-Whitney U test using SPSS 12.01 for Windows.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Active CD4+CD25+ regulatory T cells are elicited by immunization with Dna J-like 2 alone but not by coimmunization with Dna J-like 2 and CTL epitope 9m

CD4+CD25+ T cells or CD4+CD25– T cells were obtained 1 wk after two biweekly immunizations with one of the SEREX-defined self-Ags, Mus heat shock protein Dna J-like 2 (Dna J-like 2) (AF055664) (9, 10, 11, 12) or Dna J-like 2 and tumor-specific CTL epitope 9m derived from mERK2. Naive BALB/c mice were injected with 1 x 105 CD4+CD25+ T cells or 1 x 105 CD4+CD25– T cells i.v. and were challenged with 1 x 106 CMS5m i.v. 1 wk later. As shown in Fig. 1a, mice receiving CD4+CD25+ T cells from mice immunized with Dna J-like 2, but not from mice immunized with Dna J-like 2 and CTL epitope 9m, showed enhancement of pulmonary metastasis. Under similar conditions, no increase in the number of pulmonary nodules was observed in mice receiving CD4+CD25– T cells from either naive or immunized mice or CD4+CD25+ T cells from naive mice.



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  		FIGURE 1. Active CD4+CD25+ regulatory T cells are elicited by immunization with Dna J-like 2 alone but not by coimmunization with Dna J-like 2 and CTL epitope 9m. a, Mice were immunized with plasmids encoding Dna J-like 2 or plasmids encoding Dna J-like 2 and CTL epitope 9m. CD4+CD25+ T cells or CD4+CD25– T cells were obtained from spleens 1 wk after the second immunization. Naive BALB/c mice were injected i.v. with 1 x 105 CD4+CD25+ T cells or 1 x 105 CD4+CD25– T cells followed by i.v. injection of 1 x 106 CMS5m. Pulmonary metastatic nodules were counted with a dissecting microscope 28 days later. b, Graded numbers of CD4+CD25+ T cells or CD4+CD25– T cells derived from nonimmunized mice or mice immunized with Dna J-like 2 or Dna J-like 2 and CTL epitope 9m were added to cultures of 5 x 104 CD4+CD25– T cells from DO11.10 or 5 x 104 CD8+ T cells from DUC18 with 5 x 104 MMC-treated BALB/c splenic Thy-1– APCs and cognate peptides. Proliferation was assessed as described in Materials and Methods. c, Expression of Foxp3 was analyzed by real-time RT-PCR in 1 x 106 CD4+CD25+ T cells from nonimmunized mice or mice immunized with Dna J-like 2 or Dna J-like 2 and CTL epitope 9m. Normalized Foxp3 expression values were calculated as the ratio of Foxp3 expression to hypoxanthine-guanine phosphoribosyltransferase expression. These experiments were repeated three times with similar results. Data in a and c are mean + SD.

 
Next we examined the suppressive activity of these CD4+CD25+ T cells or CD4+CD25– T cells on peptide-specific proliferation of CD4+CD25– T cells and CD8+ T cells. For this purpose, we added graded numbers of CD4+CD25+ T cells or CD4+CD25– T cells to cultures of CD4+CD25– T cells from DO11.10 (transgenic mice with TCR specific for OVA323–339) or to cultures of CD8+ T cells from DUC18 (transgenic mice with TCR specific for mERK2136–144-9m) with splenic Thy-1– APCs from wild-type BALB/c mice and cognate peptides as described in Materials and Methods. CD4+CD25+ T cells from mice immunized with Dna J-like 2 alone, but not from mice immunized with Dna J-like 2 and CTL epitope 9m, had strong suppressive effect on peptide-specific proliferation of CD4+CD25– T cells and CD8+ T cells (Fig. 1b and Ref.11). CD4+CD25+ T cells from naive mice did not suppress peptide-specific proliferation of CD4+CD25– T cells or CD8+ T cells under these conditions (Fig. 1b and Ref.11).

Expression of Foxp3 in these CD4+CD25+ T cell populations was analyzed by real-time RT-PCR. CD4+CD25+ T cells from mice immunized with Dna J-like 2 alone showed several times higher expression of Foxp3 compared with naive CD4+CD25+ T cells as reported (Fig. 1c and Ref.11). In contrast, CD4+CD25+ T cells from mice immunized with Dna J-like 2 and CTL epitope 9m showed no increased expression of Foxp3. Considered together, the results indicate that immunization with Dna J-like 2 alone, but not with Dna J-like 2 and CTL epitope 9m, elicits active CD4+CD25+ regulatory T cells.

Dna J-like 2 responding Th cells and regulatory T cells are derived from distinct CD4+T cell subsets

As we previously reported, immunization of wild-type BALB/c mice with Dna J-like 2 and CTL epitope 9m leads to generation of a greatly increased number of 9m-specific CD8+ T cells that secrete IFN-{gamma} in a CD4+ T cell-dependent manner, as compared with immunization with CTL epitope 9m alone (9). The derivation of these Th cells was analyzed by reconstituting C.B-17 scid mice with 1 x 106 CD4+CD25+ T cells or 1 x 106 CD4+CD25– T cells, in addition to 1 x 106 CD8+ T cells, all of which were obtained from naive wild-type BALB/c mice. These mice were immunized with the CTL epitope 9m alone or Dna J-like 2 and CTL epitope 9m, and generation of 9m-specific CD8+ T cells was examined by ELISPOT assay. In mice reconstituted with CD4+CD25– T cells and CD8+ T cells, immunization with Dna J-like 2 and CTL epitope 9m resulted in an increased number of 9m-specific CD8+ T cells as compared with mice immunized with CTL epitope 9m alone (Fig. 2a). In contrast, a decreased number of 9m-specific CD8+ T cells was observed in mice reconstituted with CD4+CD25+ T cells and CD8+ T cells and immunized with CTL epitope 9m alone or Dna J-like 2 and CTL epitope 9m.



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  		FIGURE 2. Th cells and regulatory T cells responding to Dna J-like 2 are derived from distinct CD4+ T cell subsets. a, C.B-17 scid mice were reconstituted with 1 x 106 CD4+CD25+ T cells or 1 x 106 CD4+CD25– T cells, in addition to 1 x 106 CD8+ T cells, all of which were obtained from naive wild-type BALB/c mice. Mice were then immunized with CTL epitope 9m alone or Dna J-like 2 and CTL epitope 9m, and elicitation of 9m-specific IFN-{gamma}-secreting CD8+ T cells was examined by ELISPOT assay. Target cells were mERK2136–144-9m-pulsed P1.HTR (black bars) or HER2 p63–71 (T)-pulsed P1.HTR (open bars) as control. b, C.B-17 scid mice were reconstituted with 5 x 105 {alpha}-GalCer/CD1d tetramer+ cells, 1 x 106 CD8+ T cells and with or without 1 x 106 CD4+CD25+ T cells or 1 x 106 CD4+CD25– T cells. These mice were then injected i.v. with 1 x 106 CMS5m, followed by no further immunization (light gray bars), immunization with Dna J-like 2 (black bars), CTL epitope 9m (dark gray bars), or Dna J-like 2 and CTL epitope 9m (open bars). The number of pulmonary metastatic nodules was counted 28 days later. These experiments were repeated three times with similar results. Data are mean + SD.

 
We previously reported that the metastasis enhancement induced by immunization with SEREX-defined self-Ags was due to the inhibition of iNKT cell activity mediated by CD4+CD25+ regulatory T cells (10). To investigate this further, C.B-17 scid mice were reconstituted with 5 x 105 {alpha}-GalCer/CD1d tetramer+ cells as a source of iNKT cells, along with 1 x 106 CD8+ T cells, with or without 1 x 106 CD4+CD25+ T cells or 1 x 106 CD4+CD25– T cells. These mice were then challenged i.v. with 1 x 106 CMS5m tumor cells, followed by immunization with Dna J-like 2, CTL epitope 9m, or Dna J-like 2 and CTL epitope 9m, and the number of pulmonary metastatic nodules was counted 28 days later. As shown in Fig. 2b, the enhancement of pulmonary metastasis by immunization with Dna J-like 2 alone was observed when C.B-17 scid mice were reconstituted with {alpha}-GalCer/CD1d tetramer+ cells, CD8+ T cells, and CD4+CD25+ T cells but not with {alpha}-GalCer/CD1d tetramer+ cells, CD8+ T cells, and CD4+CD25– T cells. In contrast, the suppression of pulmonary metastasis by immunization with Dna J-like 2 and CTL epitope 9m was observed when C.B-17 scid mice were reconstituted with {alpha}-GalCer/CD1d tetramer+ cells, CD8+ T cells, and CD4+CD25– T cells but not with {alpha}-GalCer/CD1d tetramer+ cells, CD8+ T cells, and CD4+CD25+ T cells. Notably, the number of pulmonary nodules in C.B-17 scid mice reconstituted with {alpha}-GalCer/CD1d tetramer+ cells, CD8+ T cells, and CD4+CD25+ T cells without immunization was essentially the same as those mice immunized with Dna J-like 2 and CTL epitope 9m.

These results indicate that regulatory T cells responsible for the enhancement of pulmonary metastasis are generated/activated from CD4+CD25+ T cell population by immunization with Dna J-like 2 alone. The generation/activation of such CD4+CD25+ regulatory T cells is suppressed by coimmunization with CTL epitope 9m. Furthermore, Th cells for increased CTL induction are derived from CD4+CD25– T cells, whose activation requires coimmunization with Dna J-like 2 and CTL epitope 9m.

IFN-{gamma} abrogates enhanced pulmonary metastasis by immunization with Dna J-like 2 and prevents accelerated development of MCA-induced tumors

We next examined the possible involvement of cytokines that modulate generation/activation of CD4+CD25+ regulatory T cells responsible for the enhancement of the pulmonary metastasis. Wild-type BALB/c mice were immunized with Dna J-like 2 plasmids mixed with plasmids encoding IFN-{gamma}, IL-2, IL-4, IL-10, IL-12, or IL-12 and IL-18. Mice were then challenged with CMS5m and the number of pulmonary nodules was counted 28 days later. Mice immunized with plasmids encoding Dna J-like 2 and IFN-{gamma} showed no enhancement of pulmonary metastasis (Fig. 3). A similar effect was observed in mice coimmunized with plasmids encoding IL-12 and IL-18, and to a lesser degree in mice coimmunized with plasmids encoding IL-12. These effects were abrogated by treatment with anti-IFN-{gamma} mAb. Plasmids encoding IL-2, IL-4, and IL-10 showed no effect on the development of pulmonary metastasis (Fig. 3). To confirm this effect of IFN-{gamma}, BALB/c mice were injected with 1 x 105 CD4+CD25+ T cells i.v. derived from naive mice or mice immunized with Dna J-like 2 or Dna J-like 2 and IFN-{gamma} and were challenged with 1 x 106 CMS5m i.v. 1 wk later. Pulmonary metastasis was enhanced in mice receiving CD4+CD25+ T cells from Dna J-like 2 immunized mice, but not from mice immunized with Dna J-like 2 and IFN-{gamma} (Fig. 4a).



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  		FIGURE 3. IFN-{gamma} abrogates enhancement of pulmonary metastasis by immunization with Dna J-like 2. Wild-type BALB/c mice were challenged with CMS5m and then immunized 5 days later with plasmids coding for Dna J-like 2 mixed with plasmids encoding the following cytokines: IFN-{gamma}, IL-2, IL-4, IL-10, IL-12, or IL-12 and IL-18. Additional groups of mice were immunized with Dna J-like 2 mixed with plasmids encoding IL-12 or IL-12 and IL-18 and then treated with anti-IFN-{gamma} mAb. The number of pulmonary nodules was counted 28 days later. These experiments were repeated three times with similar results. Data are mean + SD.

 


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  		FIGURE 4. IFN-{gamma} abrogates the generation/activation of CD4+CD25+ regulatory T cells. a, Wild-type BALB/c mice received 1 x 105 CD4+CD25+ T cells from nonimmunized mice or mice immunized with plasmids encoding Dna J-like 2 alone, or Dna J-like 2 and IFN-{gamma}. The mice were then challenged with 1 x 106 CMS5m and the number of pulmonary metastasis was counted 28 days later. b, Mice were injected with 50 µg of MCA and 2 wk later, immunization with Dna J-like 2 with or without IFN-{gamma} was commenced and repeated at every 4 wk. Mice were monitored weekly for the development of tumors. The number of mice in each group is shown in parentheses. Statistical significance of immunization was determined by the Mann-Whitney U test. The percentage of tumor frequency of each group at 16 wk was compared with BALB/c mice receiving 50 µg of MCA alone (*, p < 0.001). c, Graded numbers of CD4+CD25+ T cells or CD4+CD25– T cells derived from nonimmunized mice or mice immunized with plasmids encoding Dna J-like 2 alone, or Dna J-like 2 and IFN-{gamma} were added to cultures of 5 x 104 CD4+CD25– T cells from DO11.10 or 5 x 104 CD8+ T cells from DUC18 with 5 x 104 MMC-treated BALB/c splenic Thy-1– APCs and cognate peptides. Proliferation was assessed as described in Materials and Methods. d, Expression of Foxp3 in CD4+CD25+ T cells from nonimmunized mice or from mice immunized with Dna J-like 2 or Dna J-like 2 and IFN-{gamma} was analyzed as described in legend of Fig. 1c. These experiments were repeated three times with similar results. Data in a and d are mean + SD.

 
In the previous work, immunization with Dna J-like 2 was found to accelerate tumor development induced by MCA in a CD4+CD25+ T cell-dependent manner (12). To investigate the effect of IFN-{gamma} on this effect, mice were injected with 50 µg of MCA and 2 wk later, immunization with Dna J-like 2 with or without IFN-{gamma} was commenced and repeated at a 4-wk interval. As shown in Fig. 4b, no accelerated tumor development was observed in mice immunized with Dna J-like 2 and IFN-{gamma}, in comparison with nonimmunized mice, whereas obvious acceleration of tumor development was observed in mice immunized with Dna J-like 2 alone.

IFN-{gamma} abrogates the generation/activation of CD4+CD25+ regulatory T cells

Based on the above results, we then examined whether coimmunization with IFN-{gamma} abrogated the generation/activation of CD4+CD25+ regulatory T cells elicited by immunization with Dna J-like 2. The effect of these CD4+CD25+ T cells was tested on peptide-specific proliferation of CD4+CD25– T cells and CD8+ T cells. CD4+CD25+ T cells from Dna J-like 2 and IFN-{gamma}-immunized mice did not suppress the proliferation of these cells (Fig. 4c). Furthermore, no increase of Foxp3 expression was observed in CD4+CD25+ T cells from Dna J-like 2 and IFN-{gamma}-immunized mice (Fig. 4d).

IFN-{gamma} produced by CD8+ T cells is critical for abrogating the generation/activation of CD4+CD25+ regulatory T cells

A critical role for IFN-{gamma} in abrogating the generation/activation of CD4+CD25+ regulatory T cells prompted us to examine the significance of IFN-{gamma} produced by CD8+ T cells specific for CTL epitope 9m. C.B-17 scid mice were reconstituted with whole CD4+ T cells or CD4+CD25+ T cells from wild-type BALB/c mice, and CD8+ T cells from either wild-type BALB/c mice or IFN-{gamma}–/– mice. The mice were subsequently immunized with Dna J-like 2 alone or Dna J-like 2 and CTL epitope 9m. CD4+CD25+ T cells from the C.B-17 scid mice receiving whole CD4+ T cells or CD4+CD25+ T cells and CD8+ T cells from IFN-{gamma}–/– mice suppressed the proliferation of CD4+CD25– T cells from DO11.10 mice and CD8+ T cells from DUC18 mice (Fig. 5, a and c). Similarly obtained CD4+CD25+ T cells from C.B-17 scid mice reconstituted with CD4+CD25+ T cells and CD8+ T cells from wild-type BALB/c mice had suppressive activity when the cells were derived from mice immunized with Dna J-like 2 alone, but not from mice immunized with Dna J-like 2 and CTL epitope 9m (Fig. 5b). Enhanced expression of Foxp3 was observed in the CD4+CD25+ T cells showing suppressive activity on proliferation of CD4+CD25– T cells and CD8+ T cells (Fig. 5d). To investigate this influence of IFN-{gamma} on enhanced pulmonary metastasis mediated by CD4+CD25+ regulatory T cells, C.B-17 scid mice were reconstituted with 5 x 105 {alpha}-GalCer/CD1d tetramer+ cells, 1 x 106 CD4+ T cells, and 1 x 106 CD8+ T cells derived from either wild-type BALB/c mice or from IFN-{gamma}–/– mice. These mice were then challenged i.v. with 1 x 106 CMS5m tumor cells, followed by no further immunization, or by immunization with Dna J-like 2, CTL epitope 9m, or Dna J-like 2 and CTL epitope 9m, and the number of pulmonary metastatic nodules was counted 28 days later. As shown in Fig. 5e, enhancement of pulmonary metastasis was abrogated when C.B-17 scid mice were reconstituted with {alpha}-GalCer/CD1d tetramer+ cells, CD4+ T cells, and CD8+ T cells derived from wild-type BALB/c mice and subsequently immunized with Dna J-like 2 and CTL epitope 9m. In contrast, such abrogation was not observed when CD8+ T cells were derived from IFN-{gamma}–/– mice. These results indicate that IFN-{gamma} produced by CD8+ T cells recognizing CTL epitope 9m abrogates the generation/activation of CD4+CD25+ regulatory T cells.



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  		FIGURE 5. IFN-{gamma} produced by CD8+ T cells is critical for abrogating the generation/activation of CD4+CD25+ regulatory T cells. C.B-17 scid mice received (a) whole CD4+ T cells or (b) CD4+CD25+ T cells and CD8+ T cells derived from either wild-type BALB/c mice or (c) from IFN-{gamma}–/– mice. Mice were then not immunized ({blacktriangleup}), or immunized with Dna J-like 2 alone ({blacksquare}), or with Dna J-like 2 and CTL epitope 9m (•). Bold and dotted lines represent control wild-type mice or reconstituted C.B-17 scid mice, respectively. Graded numbers of these CD4+CD25+ T cells derived from those mice were added to cultures of 5 x 104 CD4+CD25– T cells from DO11.10 mice or 5 x 104 CD8+ T cells from DUC18 mice with 5 x 104 MMC-treated BALB/c splenic Thy-1– APCs and cognate peptides. Proliferation was assessed as described in Materials and Methods. d, Expression of Foxp3 was examined in CD4+CD25+ T cells from C.B-17 scid mice reconstituted as indicated, followed by no further immunization, or immunization with Dna J-like 2 or with Dna J-like 2 and CTL epitope 9m as described in legend of Fig. 1c. e, C.B-17 scid mice were reconstituted with 5 x 105 {alpha}-GalCer/CD1d tetramer+ cells, 1 x 106 CD4+ T cells and 1 x 106 CD8+ T cells derived from either wild-type BALB/c mice or from IFN-{gamma}–/– mice. These mice were then injected i.v. with 1 x 106 CMS5m, followed by no further immunization (light gray bars), immunization with Dna J-like 2 (black bars), CTL epitope 9m (dark gray bars), or Dna J-like 2 and CTL epitope 9m (open bars). The number of pulmonary metastatic nodules was counted 28 days later. These experiments were repeated three times (a–d) or twice (e) with similar results. Data in d and e are mean + SD.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
SEREX-defined self-Ags have the capacity to generate two functionally distinct CD4+ autoreactive T cell populations–Th cells and regulatory T cells (9, 10, 11, 12). Whereas immunization with these self-Ags alone leads to generation/activation of CD4+CD25+ regulatory T cells with suppressive activities, coimmunization with the self-Ags together with CTL epitope 9m results in generation/activation of CD4+ Th cells amplifying tumor-specific CD8+ CTL (9, 10, 11, 12). Under the latter condition, CD4+CD25+ regulatory T cells are not generated/activated despite immunization with the same self-Ag. Thus, these two CD4+ subsets coexist in the periphery as functionally distinct cell populations. Although functionally distinct regulatory T cells and Th cells are present in distinct CD4+ T cell subsets, it is not clear whether CD4+CD25+ regulatory T cells and CD4+CD25– Th cells recognize identical Ag peptides in the context of self-MHC class II molecules.

The presumption is that constant stimulation by these SEREX-defined self-Ags under physiological conditions leads to the generation of CD4+CD25+ regulatory T cells that maintain immunological homeostasis by regulating a wide variety of immune responses, including those involving CD4+CD25– T cells. As shown in the present study, these suppressive effects of CD4+CD25+ regulatory T cells were able to be modulated by IFN-{gamma}. Generation/activation of CD4+CD25+ regulatory T cells following immunization with Dna J-like 2 was abrogated by IFN-{gamma}, but not by several other cytokines. This ability of IFN-{gamma} to nullify generation/activation of CD4+CD25+ regulatory T cells provides an explanation for the paradoxical effect of immunization with self-Ags and CTL epitopes, where strong helper activity is generated, rather than the suppression seen when mice are immunized with self-Ags alone. Under these conditions, IFN-{gamma} produced by CD8+ T cells responding to the CTL epitope inhibits generation/activation of CD4+CD25+ regulatory T cells, permitting CD4+CD25– Th cells to become dominant. Although IFN-{gamma} was shown to be sufficient to abrogate the enhancement of pulmonary metastasis by inhibiting generation/activation of CD4+CD25+ regulatory T cells, CD8+ T cells responding to the CTL epitope might be required to achieve complete blocking of pulmonary metastasis. It is likely that this action of CD8+ T cells uses not only IFN-{gamma} but also perforin and other factors. In this regard, it is important to note that the four SEREX-defined self-Ags, including Dna J-like 2 (9, 10, 11, 12), that generate/activate regulatory T cells lack dominant CTL epitope sequences binding to Kd or Dd. In fact, we have been unable to induce CD8+ CTL against P1.HTR target cells transfected with expression plasmids for any of these self-Ags, indicating the absence of potent CTL epitopes for BALB/c mice (data not shown).

The exact mechanism(s) underlying IFN-{gamma} interference with the elicitation of regulatory T cells remains elusive. There is circumstantial evidence to indicate that regulatory T cells favor Th2 immunological status (24, 25); the inclusion of IFN-{gamma} plasmids would promote a Th1 rather than a Th2 shift. It is also possible that IFN-{gamma} may modulate the function of APCs, such as dendritic cells, which are essential for activation of regulatory T cells (26, 27). Recently, it has been reported that stimulation with IFN-{gamma} up-regulates TLR9 expression in dendritic cells and enhances the response to CpG-oligodeoxynucleotides (28). Because it has also been reported that TLR signaling is able to inhibit the suppressive activity of regulatory T cells (29, 30), IFN-{gamma} may confer a favorable environment for the induction of Th cells through up-regulation of TLR signals. Another possibility is that IFN-{gamma} may induce the expression of Smad7, an antagonistic Smad, and prevent TGF-{beta} signaling via Smad3/4, which has been shown to be essential for maintenance of regulatory T cells (31, 32, 33, 34, 35). Alternatively, IFN-{gamma} may have a direct inhibitory effect through the IFN-{gamma}R on CD4+CD25+ regulatory T cells. It has been shown that IFN-{gamma} is critical for the generation and function of alloantigen-reactive regulatory T cells (36). These results appear to be contradictory to our finding that IFN-{gamma} inhibits generation/activation of CD4+CD25+ regulatory T cells. However, kinetic and quantitative differences in IFN-{gamma} production may explain these apparent contradictions and this issue needs further investigation. It is also possible that different levels of IFN-{gamma} are required for generation/activation of CD4+CD25+ regulatory T cells by self-Ags as compared with alloantigen-reactive regulatory T cells in their study. Alternatively, two experimental systems may examine the role of IFN-{gamma} in regulatory T cell generation/activation and function in different phases. Although IFN-{gamma} interfered with the generation/activation of CD4+CD25+ regulatory T cells by SEREX-defined self-Ags, it did not abrogate the suppressive activity of naturally occurring CD4+CD25+ regulatory T cells induced by anti-CD3 mAb (data not shown). The expression level of Foxp3 in CD4+CD25+ T cells from mice immunized with Dna J-like 2 and IFN-{gamma} or with Dna J-like 2 and CTL epitope 9m was comparable to that of naive CD4+CD25+ T cells, indicating persistent suppressor potential in these cellular populations (23, 37, 38).

IFN-{gamma} is considered one of the major molecules involved in the preventive and defensive responses against infectious pathogens, having multiple biological functions including regulation of Th cells (39, 40, 41). Our results suggest that in addition to its well-established role in balancing Th1 vs Th2 type immune responses, IFN-{gamma} is involved in regulating the generation/activation of CD4+CD25+ regulatory T cells. Although the present study emphasizes the role of CD8+ CTL-produced IFN-{gamma}, similar control of regulatory T cell generation/activation would result from IFN-{gamma} produced by other cell types, e.g., NK cells and NKT cells (42). As virtually all innate-type immunity involves the production of IFN-{gamma}, it may well be that the control of regulatory T cells represents an important early link between innate and adaptive immunity. IFN-{gamma} production during immune responses would also explain why suppression is not the predominant and permanent state of the immune system, given the constant stimulation of CD4+CD25+ regulatory T cells by self-Ags. Endogenous tumor-derived self-Ags with CTL epitopes, e.g., Melan A (43, 44) and NY-ESO-1 (45, 46), as well as self-Ags with CTL epitopes associated with autoimmune disease (47, 48) and exogenous Ags, e.g., viral or bacterial Ags (49), all have the capacity to elicit IFN-{gamma}-producing CD8+ T cells, and the ultimate outcome of recognition of these Ags would be immunological help rather than suppression.

Efforts to achieve immunologic control of cancer are currently receiving much attention. A number of promising tumor Ags have been identified and are being incorporated into cancer vaccines (50, 51, 52, 53). Self-Ags that are overexpressed in tumor cells represent attractive candidates for tumor vaccines because of their high frequency of expression in a range of tumor types. However, these Ags may cause the unexpected activation of regulatory T cells, similar to the response elicited by the four SEREX-defined self-Ags. Incorporating IFN-{gamma} in the construction of cancer vaccine may be a critical element in avoiding the potential danger of self-Ag-related generation/activation of CD4+CD25+ regulatory T cells.


   Acknowledgments
 
We thank Drs. S. Gnjatic and E. Sato for helpful discussion.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Back

2 Current address: Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY 10021. Back

3 Current address: Division of Immunoregulation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan. Back

4 Address correspondence and reprint requests to Prof. Hiroshi Shiku, Second Department of Internal Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan. E-mail address: shiku{at}clin.medic.mie-u.ac.jp Back

5 Abbreviations used in this paper: SEREX, serological identification of Ags by recombinant expression cloning; MCA, 3-methylcholanthrene; iNKT, invariant NKT; mERK2, mutated MAPK, ERK2; {alpha}-GalCer, {alpha}-galactosylceramide; MMC, mitomycin C; Dna J-like 2, Mus heat shock protein Dna J-like 2. Back

Received for publication May 31, 2005. Accepted for publication July 19, 2005.


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 Materials and Methods
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