1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

We investigated the relationship between transforming growth factor-β (TGF-β)-secreting T-regulatory (Tr) cells and anti-B16 melanoma immunity, and studied the association of early cytokines expressed at tumour sites with the generation of Tr cells. A large number of CD4+ Tr cells producing interleukin (IL)-4, IL-10 and TGF-β accumulated with functionally depressed CD8+ cytotoxic T lymphocytes (CTLs) at tumour sites on day 20 after subcutaneous (s.c.) inoculation of B16 tumour cells. Tr cells consisted of two populations, which were termed T helper 3 (Th3) and Tr1 cells. B16-infiltrating Tr cells strongly inhibited the generation of B16-specific T helper 1 (Th1) cells in a TGF-β-dependent manner and were assumed to suppress effective generation of CTLs. In addition, B16 cells markedly progressed in mice transferred adoptively by the cultured B16-infiltrating Tr cells compared with untreated mice. The capacity of these Tr cells to produce TGF-β was hampered by neutralizing anti-IL-10 and partly anti-IL-4 monoclonal antibodies (mAbs) injected intralesionally during the early development of B16 tumours, and this treatment markedly attenuated B16 growth. Furthermore, a lesional injection of recombinant mouse IL-10 at an early tumour site resulted in the vigorous progression of B16 tumours. These results provide evidence that Tr cells, belonging to the T helper 3/T-regulatory 1 (Th3/Tr1) type, are activated in B16-bearing hosts under the influence of T helper 2 (Th2) cytokines, mainly IL-10 (produced at early tumour lesions), and that this regulatory T-cell population functions as a suppressor of anti-B16 immunity.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

It is generally known that cytotoxic lymphocytes, including natural killer (NK) cells and tumour-specific cytotoxic T lymphocytes (CTLs) eliminate tumour cells in a major histocompatibility complex (MHC)-dependent manner.1 In addition, tumour-specific T helper 1 (Th1) cells and their cytokines, i.e. interleukin (IL)-2 and interferon-γ (IFN-γ), play a crucial role in the accomplishment of CTL-mediated antitumour immune responses and subsequent regression of tumours.1,2 Prior to the action of Th1 cells and CTLs, NK cells also serve as a source of IFN-γ at an early stage in tumour-bearers, further enhancing tumour immunity by assisting effective priming of Th1 and CTLs.3 In humans and rodents, however, tumour and immune cells implement diverse strategies against the generation/action of cytotoxic cells and Th1 cells. First, tumour cells down-regulate their expression of MHC molecules to avoid specific recognition by CTLs.4 Second, certain populations of co-infiltrating lymphocytes, as well as tumour cells, produce cytokines immunomodulatory to cytotoxic lymphocytes.5,6 In the latter case, IL-4, IL-10 and transforming growth factor-β (TGF-β) have been well defined to take a leading role as suppressive factors.5–8 IL-4 and IL-10, classified into T helper 2 (Th2) cytokines, function as down-modulators in Th1-cell generation.9 IL-10 is also known to decrease the expression of MHC molecules on tumour cells and to reduce the capacity of dendritic cells to generate antigen-specific Th1 cells.10 Finally, excess TGF-β derived from tumour cells vigorously down-regulates the proliferation of tumoricidal lymphocytes, especially Th1-type cells.11

Recently, a considerable number of studies on tissue-specific autoimmune diseases and oral tolerance have shown that TGF-β and/or IL-4 are the cytokines critical for suppression of inflammatory responses evoked by autoreactive Th1 cells12,13 and for induction of tolerance to orally administered exogenous antigens.14,15 In these systems, CD4+ cells secreting these cytokines and responsible for down-modulation are termed T helper 3 (Th3) or T-regulatory (Tr) 1 cells. Both types of T cells are differentiated intrathymically, like conventional T-cell populations. Th3 cells produce Th2 cytokines (IL-4 and IL-10) and TGF-β, while Tr1 cells secrete IL-10, TGF-β and a trace amount of IFN-γ.13,15 IL-10 is thought to be required for the generation of both Th3 and Tr1 cell populations.13,16 In addition, IL-4 seems to be concerned with skewing the generation of Th3 cells in co-operation with IL-10.17 There are similarities between tumour immunity and autoimmunity. For example, most melanoma antigens have been shown to be identical to a fragment of melanosomal protein derived from melanocytes,18 which are also targets of autoimmune vitiligo. This concept raises a possibility that Th3/Tr1-type cells also serve as down-regulator in antitumour immunity. In fact, there have been observations that in vivo neutralization of TGF-β activity in tumour-bearing mice results in the promotion of tumour growth,19,20 and that tumours are regressed by depleting a certain population of CD4+ cells from tumour-bearing mice.21

Our previous studies have demonstrated that the activity of NK cells serving as a source of IFN-γ and an eliminator of tumour cells at early B16 melanoma sites is depressed by IL-4, IL-10 and TGF-β, which are locally secreted by bystander γδ T and T-cell receptor (TCR)-αβ-intermediate (αβint) T cells.22–24 The percentages of these B16-infiltrating lymphocytes peak on day 5–7 after B16 cell inoculation and gradually decrease thereafter. Following this temporal accumulation of γδ T and αβint T cells, a significant number of other lymphocyte populations, expressing CD4 or CD8 molecules, gradually infiltrate into B16 lesions, as the total number of tumour-infiltrating lymphocytes (TILs) around day 20 surpasses that of the number found in lesions on days 5–7. CD4+ T cells are responsible for this numerical increment. This study was conducted to characterize these TILs propagating at the progressive stage of B16 tumour development. Results suggest that tumour-specific Th3/Tr1-type αβ T cells, instead of γδ T cells, accumulate on day 20 after B16 tumour-cell inoculation. IL-10 expressed early in the lesion was critical for the generation of these Tr cells. This unique population inhibits Th1-mediated antitumour responses and requires IL-10 and partly IL-4 to effectively produce TGF-β.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Mice and tumour cells

Eight to 12-week-old female C57BL/6 (B6) mice were obtained from Japan SLC (Hamamatsu, Japan). B16 (4A5), MH134 and YAC-1 tumour cells were used in this study. B16 (4A5) cells and MH134 cells are a melanoma cell line of C57BL/6 mice and a hepatoma cell line of C3H/He mice, respectively. B16 (4A5) cells are an in vitro cell line derived from subcutaneously (s.c.) inoculated B16F0 tumours and were purchased from the RIKEN cell bank (Wako, Japan). B16, MH134 and YAC-1 cells were maintained by culture in Dulbecco's modified Eagle's minimal essential medium (DMEM) (Nissui Pharm. Co., Tokyo, Japan) supplemented with 10% fetal calf serum (FCS). In in vivo experiments, B16 cells (1 × 105 cells/mouse) were inoculated s.c. into B6 mice. YAC-1 cells are an NK-sensitive cell line and were used as a control in the B16-specific cytotoxicity assay.

Preparation of lymphocytes

T-cell-enriched fractions of splenocytes from normal B6 and B16-bearing B6 mice were prepared according to an established method.24 Spleen cells were haemolysed with 0·17 m ammonium chloride. After washing three times, the cells were incubated at 37° for 1 hr on a plastic dish in RPMI-1640 (Nissui Pharm. Co.), supplemented with 10% FCS. Dish non-adherent cells were collected by gentle shaking and subjected to nylon wool column to enrich T cells. The T-cell-enriched fraction was used for cytokine expression and flow cytometry.

TILs were separated from B16 lesions. On day 20 after s.c. inoculation of B16 cells (1 × 105/mouse), tumour cell suspensions were prepared in phosphate-buffered saline (PBS) containing 0·1 m EDTA by scrubbing with a pair of tweezers. Ten millilitres of B16 cell suspensions (1 × 105 cells/ml of PBS supplemented with 10% FCS) applied on 5 ml of Histopaque 1083 (Sigma, St. Louis, MO) were subjected to centrifugation at 1000 g for 30 min at 20°. The cells at the interface were collected, washed three times with DMEM and used as B16 TILs.

Fractionation of T cells

RPMI-1640 supplemented with 25 mm HEPES, 2 mm l-glutamine, 1 mm non-essential amino acids, 5 × 10−2 mm 2-mercaptoethanol (2-ME), 1 mm sodium pyruvate, 100 µg/ml of gentamicin (all from Life Technologies, Grand Island, NY) and 10% FCS were used in this study.

Immunomagnetic beads were used for the preparation of CD4+ and CD8+ cells. Normal B6 splenocytes, B16-bearing B6 mice splenocytes and B16 TILs were preincubated with blocking monoclonal antibody (mAb) for mouse FcγII/III receptors (R) (2.4G2; PharMingen, San Diego, CA) at 4° for 30 min, and then washed three times with DMEM. The pretreated cells were mixed with anti-CD4 mAb- and anti-CD8 mAb-conjugated magnetic beads (Dynal Inc., Oslo, Norway), at a ratio of three beads per cell, for 1 hr at 4° on a rocking shaker, according to the manufacturer's instructions. Cells bound to CD4 beads were collected with a magnet and cultured in RPMI-1640 containing detaching beads (DETACHaBEAD mouse CD4; Dynal Inc.) in a CO2 incubator for 2 hr to separate cells from beads. To purify CD8+ cells, positive cells were collected with a magnet and cultured overnight. The cells separated from free beads were used as purified CD4+ and CD8+ cells, which were positive for CD3 and TCR-αβ and confirmed as being > 96% pure by flow cytometric analysis using fluorescein isothiocyanate (FITC)-conjugated mAbs to CD3 (145-2C11; PharMingen) and TCR-β (H57-597; PharMingen), and phycoerythrin (PE)-conjugated mAbs to CD4 (RM-4-5; PharMingen) and CD8 (53-6.7; PharMingen). CD25+ populations of CD4+ splenocytes were also selected with magnetic beads. The purified CD4+ cells were incubated with anti-CD25 mAb (1 µg/ml, 7D4; PharMingen) at 4° for 30 min, and washed three times with PBS. The mAb-treated CD4+ cells were mixed with anti-rat immunoglobulin G (IgG)-conjugated beads (Dynal Inc.) at a ratio of three beads per cell, and incubated at 4° for 30 min. The cells bound to beads were collected with a magnet, washed three times with PBS, cultured overnight to separate cells from beads and used as CD4+ CD25+ splenocytes.

To prepare CD4+ CD25 cell populations, CD4+ splenocytes (5 × 105 cells) from B16-bearing mice were treated with mAb specific to CD25, as described above, in FCS-free medium and centrifuged without dilution. The Ab-treated CD4+ cell pellets were gently suspended in FCS-free medium containing rabbit complement diluted 1 : 20 (Low toxic-M rabbit complement; Cedarlane Lab., Ontario, Canada) and incubated at 37° for 30 min. After washing three times with PBS, the CD4+ cell populations deprived of CD25+ cells were used for induction of B16-specific Th1 cells.

T-cell-enriched splenocytes (2 × 105 cells/ml) of normal B6 mice were cultured for 10 days in medium supplemented with recombinant (r)IL-10 (20 ng/ml; Genzyme, Cambridge, MA) and maintained with 10 U/ml of rIL-2 (Genzyme) at 37°. CD4+ cell populations separated from splenocytes or TILs of B16-bearing mice were cultured in the absence of cytokines for 10 days at 37°. A CD4+ TIL clone was obtained with 50 U/ml of rIL-2 by limiting dilution (0·5 cell/well in 96-well plates) of CD4+ TILs cultured for 7 days. Mitomycin C (MMC) (50 µg/ml, 30 min, 37°; Sigma)-treated T-cell-enriched splenocytes of B16-bearing mice were used as feeder cells (1 × 104 cells/well).

In vitro induction of tumour-specific Th cells with cultured dendritic cells (DCs)

DCs were differentiated from bone marrow cells of B6 mice by a previously described method.25,26 After removing muscle tissues, femurs and tibiae were rinsed in 70% ethanol for 5 min. Bone marrow cells were obtained from the bones by flashing out with medium in 25-gauge needle-connecting syringes. The cells were suspended in medium, passed through nylon mesh and treated with 0·17 m ammonium chloride to remove red blood cells. After washing three times with medium, lymphocytes and MHC class II+ cells were depleted with a cocktail of mAbs specific to CD4 (GK1.5), CD8 (53-6.7), Ia (2G9), Gr-1 (RB6-8C5) and B220 (RA3-6B2) (all from PharMingen), and complement at 37° for 60 min. DCs were prepared by culturing the mAb- and complement-treated bone marrow cells (1 × 105 cells/ml) in RPMI-1640 supplemented with 10% FCS, 5 × 10−5 mol/ml of 2-ME and antibiotics (penicillin G and streptomycin, 100 µg/ml) in the presence of the following recombinant mouse cytokines: 5 ng/ml of recombinant granulocyte–macrophage colony-stimulating factor (rGM-CSF), 20 ng/ml of stem cell factor (rSCF) and 25 ng/ml of recombinant tumour necrosis factor-α (rTNF-α) (all from Genzyme) for 20 days. In this culture, half of the medium was replaced with fresh medium at 4-day intervals. Weakly adhesive cells, used as antigen-presenting cells, had typical dendritic morphology, strongly expressed MHC class I (Db, Kb), II (Iab), CD80 (B7-1), CD86 (B7-2) and CD11b molecules, and weakly expressed CD11a, CD11c and CD54 (intracellular adhesion molecule-1 [ICAM-1]) molecules.

B16, MH134 and YAC-1 cells were treated with 50 µg/ml of MMC at 37° for 1 hr, and washed three times with medium. Immediately, the MMC-treated tumour cells and the cultured DCs were fused with PEG-1500 (Boehringer Mannheim GmbH, Mannheim, Germany) by gentle mixing at 37°, and washed twice with medium. The mixture contained DCs fused with and without tumour cells and tumour cells themselves. The fused DCs (B16-DCs, MH134-DCs or YAC-1-DCs) were used as stimulators for the specific induction of Th1 cells and CTLs.

To induce conventional Th1 cells, CD4+ CD25 cells (1 × 105 cells/well in 24-well plates) from splenocytes of B16-bearing mice were cultured with B16-DCs or MH134-DCs at a ratio of 10 CD4+ CD25 cells to one tumour DC in medium supplemented with 100 ng/ml of rIL-12 (Genzyme) and 10 µg/ml of anti-IL-4 mAb (PharMingen) at 37° for 7 days. The expanded cells were assayed for proliferation and cytokine production. In some experiments, cultured CD4+ TILs were employed for Th1 induction with neutralizing mAb to IL-4 (11B11; PharMingen), IL-10 (JES5-16E3; Pharmingen) or TGF-β1, -β2 or -β3 (Genzyme), or control rat IgG, at a concentration of 1 µg/ml.

Cytotoxicity assay

B16 cells treated for 24 hr with rIFN-γ (50 ng/ml; PharMingen) were used as target cells in the CTL assay. B16, MH134 or YAC-1 cells (5 × 105 cells/ml) were radiolabelled with medium containing 200 µCi of Na[51Cr] (Dupont NEN, Boston, MA) for 1 hr at 37°. Different numbers of CD8+ cells purified from B16 TILs and CD8+ cells induced by DC stimulation were mixed with 51Cr-labelled B16, MH134 or YAC-1 target cells (1 × 104 cells) at a final volume of 200 µl and incubated for 10 hr at 37°, as described previously.23,24 In cytotoxicity tests, the radioactivities of medium and the cells were counted by γ-counter, with the results expressed in counts per minute (c.p.m.), and the percentage specific lysis was calculated as follows:

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Proliferation assay

CD4+ and CD8+ cells (2 × 105 cells/ml) separated from TILs or splenocytes obtained under several different culture conditions, were incubated in triplicate for 24 hr in 96-well plates (Corning Glass Works, Corning, NY) in 100 µl of complete medium. Methyl tritiated thymidine ([3H]TdR) (1 µCi/well; Amersham Int, Arlington, IL) was added to the culture 8 hr before harvest. The cells were harvested on glass-fibre filters using a cell harvester (Cambridge Technologies, Watertown, MA), and their radiouptake was measured in a scintillation counter.

Enzyme-linked immunospot (ELISPOT) assay

Cytokine profiles of CD4+ cells from splenocytes, TILs and TIL clones were examined by ELISPOT assay, as described previously.23 mAbs (1 µg/ml in 100 µl of 0·1 m carbonate buffer, pH 9·0) to IL-4 (BVD4-1D11), IL-10 (JES5-2A5), IFN-γ (R4-6A2) or TGF-β1 (A75-2·1) (all from PharMingen), were added to each well of 96-well ELISPOT plates (MultiScreen-HA, Millipore, Bedford, MA) and incubated overnight at 4°. After incubation, the plates were washed twice with PBS, the wells were filled up with PBS containing 10% FCS and incubated at 37° for 1 hr to avoid non-specific reactions, and then washed twice with PBS. The cells (5 × 103 cells) suspended in medium were cultured for 1 day in each well (100 µl) of mAb-coated ELISPOPT plates at 37°. Each well was then washed vigorously 10 times with PBS and incubated with 0·5 µg/ml of biotin-conjugated mAb (prepared in 100 µl of PBS containing 10% FCS) to IL-4 (BVD6-24G2), IL-10 (SXC-1), IFN-γ (XMG1.3) or TGF-β1 (A75-3.1) (all from PharMingen) at 37° for 2 hr. After washing five times with PBS, the wells were incubated with 100 µl of PBS containing 10% FCS and streptavidin-peroxidase (1 : 1000 dilution; Boehringer Mannheim) at 37° for 30 min, and washed five times with PBS. The spots were visualized by incubation with 100 µl of 1 mg/ml substrate (3,3′-diaminobenzidine tetrahydrochloride plus 0·003% H2O2; both from Sigma) at 37° for 15 min.

In vivo injections of Abs and recombinant cytokines, and administration of lymphocytes

B16 cells (1 × 105) were inoculated s.c. into the hair-depilated lateral abdomen of B6 mice. On three consecutive days (either days 5–7 or days 12–14) after B16 cell inoculation, 50 µg of neutralizing mAb for IL-4 (11B11), IL-10 (JES5-16E3) or rat IgG, or 100 ng of rIL-4 or rIL-10 (Genzyme), was injected at a tumour site and subsequent tumour progression was observed by measurement of tumour diameters.

CD4+ or CD8+ cells (1 × 105 cells/well in 24-well plates), separated from B16 TILs, were cultured primarily in the presence or absence of B16-DCs (1 × 104 cells), respectively, at 37° for 7 days, and further maintained with rIL-2 (50 U/ml) for 7 days. The expanded CD4+ or CD8+ cells (1 × 106) were injected intravenously (i.v.) on day 7 after s.c. inoculation of B16 cells.

Reverse transcription–polymerase chain reaction (RT–PCR)

Cytokine mRNA profiles of CD4+ cells obtained from splenocytes and B16 TILs, CD4+ clones from B16 tumour-bearing mice, and IL-12- and DC-induced CD4+ CD25 splenocytes, were analysed by RT–PCR. Total RNAs of these cells were extracted using an RNA extraction kit (RNeasy; Qiagen GmbH, Hilden, Germany). First-strand cDNA was reverse transcribed using each RNA sample and amplified by the PCR using an RNA PCR kit (GeneAmp RNA PCR Kit; Takara Biomedicals, Osaka, Japan) according to the manufacturer's instructions. All pairs of primers for β-actin, IL-4, IFN-γ,27 IL-1028 and TGF-β29 were used. The PCR was run for 30 cycles on a thermal cycler (DNA amplifier; Sanyo Co., Osaka, Japan) as follows: 1·2 min at 95°, 2·5 min at 56° and 30 seconds at 72°. The PCR products and DNA molecular-weight marker VI (Boehringer Mannheim GmbH) were run on 2% agarose gels and visualized by UV light after staining with 1 µg/ml of ethidium bromide.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

CD4+ T cells as a major population in progressive B16 tumour lesions

We examined TIL populations on day 20 after s.c. inoculation of B16 melanoma cells in a comparison with those from 7-day (early) lesions, whose detail was reported previously.23,24 As shown in Fig. 1(a), 20-day TILs from B16-bearing mice contained 46% CD4+ cells and 21% CD8+ cells. These CD4+ or CD8+ cells bore TCR-αβ and CD3 (data not shown), and γδ T cells infiltrated at levels as low as 3%. Eleven per cent of NK1.1+ cells were present as another population. This is in contrast to 7-day lesions, in which TILs typically contain 52% of NK1.1+ cells and 11% of TCR-γδ+ cells.23 The CD4+ and CD8+ TIL populations were absent in 20-day B16 lesions of athymic nude B6 mice (data not shown), indicating that both T-cell populations are within the category of T cells that develop intrathymically.


Figure 1. Predominant accumulation of CD4+ cells and functionally depressed cytotoxic CD8+ cells in 20-day B16 lesions. (a) Freshly purified 20-day B16 TILs were dually incubated with phycoerythrin (PE)-conjugated anti-T-cell receptor (TCR)-γδ and fluorescein isothiocyanate (FITC)-conjugated anti-natural killer (NK)1.1 monoclonal antibodies (mAbs), or with PE-conjugated anti-CD8 and FITC-conjugated anti-CD4 mAbs. The stained cells were subjected to flow cytometric analysis. (b) The cytotoxicity of short-term cultured CD4+ or CD8+ B16 TILs, or freshly isolated B16 TILs (effector), was assayed using 51Cr-labelled MH134 or YAC-1 cells, or 51Cr-labelled B16 cells pretreated with recombinant interferon-γ (rIFN-γ) (target) at the indicated ratios. Data are expressed as the average of means from duplicate experiments.

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After separation with immunomagnetic beads, CD4+ and CD8+ TILs were cultured for a short time-period and subjected to cytotoxicity assay against tumour cells. The CD4+ T-cell population did not exhibit any cytotoxicity against B16, MH134 or YAC-1 cells (Fig. 1b). In contrast, CD8+ T cells lysed IFN-γ-pretreated B16 cells vigorously, depending on the cell number, whereas they had no cytotoxicity against MH134 or YAC-1 target cells, showing that the majority of CD8+ TILs, but not CD4+ cells, were CTLs specific for B16 cells. As freshly purified 20-day B16 TILs exhibited weak or no cytotoxicity against any tumour target, they were functionally down-regulated in this milieu.

Down-regulation of antitumour immunity by 20-day tumour-infiltrating CD4+ T cells with the Th3/Tr1 cytokine profile

As the CD4+ TIL population did not exert any cytotoxic effect on tumour cells, the possibility was considered that they are immunoregulatory for tumoricidal effector cells. To address this possibility, we first investigated the cytokine profile of the CD4+ TIL population and its clones. Figure 2 shows the results of an ELISPOT assay and representative bands of RT–PCR analysis. Freshly isolated CD4+ TILs showed high expression of IL-10 and TGF-β, moderate expression of IL-4 and weak expression of IFN-γ, in both analyses. Furthermore, ≈ 40% and 60% of CD4+ cell clones established from CD4+ TILs secreted Th3 cytokines (IL-4, IL-10 and TGF-β) and Tr1 cytokines (IL-10, trace amounts of IFN-γ, and TGF-β), respectively, indicating that 20-day CD4+ TILs were composed of Th3- or Tr1-type T cells. Conventional Th1 cells expressing only IFN-γ, but not IL-4, IL-10 or TGF-β, were absent. Therefore, it was speculated that the CD4+ T cell with a Th3/Tr1 cytokine profile inhibits propagation of Th1 cells, leading to down-regulation on Th1-mediated antitumour immunity, as has been reported in Th1-mediated autoimmune inflammation, the formation of which is inhibited by Tr cells in a TGF-β-dependent manner.13


Figure 2. Infiltration of T helper 3/T-regulatory 1 (Th3/Tr1)-type CD4+ T cells in 20-day B16 lesions. Freshly isolated B16 TILs and B16 TIL clones were subjected to enzyme-linked immunospot (ELISPOT) and reverse transcription–polymerase chain reaction (RT–PCR) analyses to examine cytokine profiles. Data are expressed as the mean ±SE of results from experiments performed in duplicate. In the right panel, data from a representative clone with a Th3- or Tr1-cytokine profile are shown.

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It has recently been shown that in tumour-bearing mice, CD4+ splenic T-cell populations co-expressing CD25 seem to play a pivotal role for attenuation of antitumour immunity.30 We examined the expression of CD25 on the surface of CD4+ TILs and splenocytes of 20-day B16-bearing mice. CD25 was detected on the vast majority of CD4+ TILs and ≈ 30% of splenocytes (Fig. 3a). As a control, normal splenocytes from B6 mice expressed CD25 at 7%. Freshly isolated CD4+ CD25+ splenic cells from tumour-bearers produced IL-4, IL-10 and TGF-β (Fig. 3b), indicating that CD4+ CD25+ Th3/Tr1 cells were increased in number, not only locally but also systemically.


Figure 3. T helper 3/T-regulatory 1 (Th3/Tr1) phenotype of CD4+ CD25+ cells in B16-bearing mice. (a) TILs and splenocytes freshly isolated from 20-day B16-bearing mice were double-stained with phycoerythrin (PE)-conjugated anti-CD4 and fluorescein isothiocyanate (FITC)-conjugated anti-CD25 monoclonal antibodies (mAbs). The stained cells were subjected to flow cytometric analysis. (b) Cytokine profile of short-term cultured CD4+ CD25+ splenic population from 20-day B16-bearing mice was examined by enzyme-linked immunospot (ELISPOT) and reverse transcription–polymerase chain reaction (RT–PCR) analysis with specific mAb or with primers for interleukin (IL)-4, IL-10, interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β), respectively. Data are expressed as the average of means from duplicate experiments.

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To explore the function of CD4+ CD25+ Th3/Tr1 cells, we generated B16 tumour-cell-specific Th1 cells in vitro from CD4+ CD25 splenic cells and examined the effect of CD4+ CD25+ T cells on the induction of this specific Th1 population. CD4+ CD25 cells prepared by depletion of CD25+ cells from CD4+ splenocytes were stimulated with B16 DCs in the presence of rIL-12 and anti-IL-4 mAb. With the use of this system, we tested the additional effect of Th3/Tr1-type CD4+ TILs on Th1 induction. As shown in Fig. 4(a), the CD4+ CD25 splenic population was differentiated into IFN-γ-producing Th1 cells in the absence of Th3/Tr1-type CD4+ TILs. The induced Th1 cells were specific for B16 cells, as they proliferated in response to B16 DCs but not to MH134 DCs or YAC-1 DCs (Fig. 4b). When CD4+ TILs were added to the culture, the induction of B16-specific Th1 cells, as estimated by [H3]TdR incorporation, was dramatically reduced in a cell number-dependent manner (Fig. 4c). This inhibitory effect of CD4+ TILs was abrogated by adding anti-TGF-β neutralizing mAb, but not anti-IL-4 or anti-IL-10 mAb, suggesting an essential role of TGF-β released from the CD4+ TIL population.


Figure 4. Inhibition of B16-specific T helper 1 (Th1) cell induction by T helper 3/T-regulatory 1 (Th3/Tr1)-type CD4+ B16 TILs. (a) CD4+ CD25 splenocytes of 20-day B16-bearing mice cultured in the presence of B16 dendritic cells (DCs) and interleukin (IL)-12 were subjected to reverse transcription–polymerase chain reaction (RT–PCR) analysis to examine the expression of the cytokines IL-4, IL-10, interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β). (b) Th1 cells induced from CD4+ CD25 splenocytes with B16-DCs and IL-12 were restimulated with B16-DCs, MH134-DCs or YAC-1-DCs, and their proliferation was assessed by [3H]thymidine ([3H]TdR) incorporation. The proliferative level before restimulation was used as a control. Data are expressed as the mean ±SE from experiments performed in duplicate. (c) In Th1 cell induction with B16-DCs and IL-12, short-term cultured Th3/Tr1-type CD4+ TILs were added to cultures with or without anti-IL-4, anti-IL-10, or anti-TGF-β neutralizing monoclonal antibody (mAb) at the indicated ratios.

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Cultured Th3/Tr1-type CD4+ cells or CD8+ cells from 20-day B16 TILs were administered i.v. on day 7 after inoculation of B16 cells, to confirm their in vivo suppressive or tumoricidal activity, respectively. As shown in Fig. 5, B16 tumours progressed more vigorously in the CD4+ T-cell-transferred group than those of the control group. In contrast, CD8+ cell administration markedly attenuated tumour growth. Furthermore, the tumour development was also inhibited, in mice reconstituted by cultured B16-specific Th1 cells, at a level comparable to that of the CD8+ cell-injected group, implying Th1 cell-dependent generation of anti-B16 CTLs.


Figure 5. In vivo effects of CD4+ TILs, CD8+ TILs and T helper 1 (Th1) cells on B16 cell growth. Cultured CD4+ and CD8+ B16 TILs, and Th1 cells expanded with B16-dendritic cells (DCs) and interleukin (IL)-12, were given intravenously (i.v.) on day 7 after tumour inoculation in B16-bearing mice, and the tumour diameter was measured. Untreated mice were used as a control. Vertical bars represent the SEM for five mice in each group. Data are representative results from two independent experiments.

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These results demonstrated that TGF-β produced by CD4+ B16 TILs down-regulated tumour-specific Th1 cell induction and subsequent Th1 cell-mediated CTL generation, leading to progression of B16 cells.

Inhibition of Th3/Tr1-type CD4+ T-cell generation by neutralization of IL-10 during early development of B16 tumours

Our recent study of early developing stage (7 days) of B16 tumours has shown that the cytotoxicity and IFN-γ production of NK-lineage cells are down-regulated by co-infiltrating CD4 CD8γδ T cells that produce IL-4, IL-10 and TGF-β.23,24 Although the down-regulatory action of TGF-β on NK activities was studied in detail, it was not elucidated whether IL-4 and IL-10 secreted by these γδ T cells played an important role in antitumour immunity. In addition, the reported finding of the involvement of IL-4 and IL-10 in the generation of Th3/Tr1 cells13–17 also urged us to examine the role of IL-4 and IL-10 released from γδ T cells in the generation of Th3/Tr1-type CD4+ cells, the appearance of which was preceded by that of γδ T cells.

B16-bearing mice were treated lesionally with neutralizing mAb for IL-4, IL-10 or rat IgG as a control consecutively on days 5–7 after tumour inoculation. The tumour growth level of anti-IL-4 and anti-IL-10 mAb-treated mice was significantly reduced compared to that of control mice (Fig. 6a). The growth inhibition was more marked with anti-IL-10 than with anti-IL-4 mAb. Furthermore, B16 tumours were completely regressed in 30% of mice treated with anti-IL-10 mAb, whereas no regression was observed with anti-IL-4 mAb. These findings were further confirmed by the study using rIL-4 and rIL-10. B16 tumours grew more rapidly in mice treated lesionally with rIL-10 on consecutive days 5–7 after B16-cell inoculation than in rIL-4-treated or untreated mice (Fig. 6b). T-cell-enriched splenocytes of B16 regressor mice, obtained by treatment with anti-IL-10 mAb, were tested in their cytotoxicity, phenotype and cytokine profile together with those from control mAb-treated B16-bearing mice. T cells from the regressor mice exhibited powerful cytotoxicity against IFN-γ-pretreated B16 cells, but showed no effect on MH134 cells (Fig. 7a). A decreased number of CD4+ CD25+ cells was found in these mice (Fig. 7b) as compared to 20-day B16-bearing mice (see splenocytes in Fig. 3a). Finally, the expression of TGF-β was decreased and that of IFN-γ was increased in these regressors (Fig. 7c). Therefore, IL-10 neutralization resulted in acquisition of B16-specific antitumour CTL and Th1 immunity, and impaired generation of Th3/Tr1 cells. It is suggested that IL-10, and partly IL-4, secreted during early development of B16 tumours play a crucial role for the effective maturation of Th3/Tr1-type CD4+ cells that allow tumour cells to evade from tumoricidal immunity.


Figure 6. Effects of lesional interleukin (IL)-4 and IL-10 at an early stage of B16 development on subsequent growth of B16 tumours. (a) Anti-IL-4, anti-IL-10 or control rat immunoglobulin G (IgG) monoclonal antibody (mAb), or (b) recombinant (r)IL-4, rIL-10 or phosphate-buffered saline (PBS), was injected on three consecutive days, days 5–7, after tumour inoculation into B16 tumour lesions, and the tumour diameter was measured. Vertical bars represent the SEM for five mice in each group. Data are representative results from two independent experiments.

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Figure 7. Insufficient generation of T helper 3/T-regulatory 1 (Th3/Tr1)-type cells and augmentation of anti-B16 activities in mice with regressed B16 tumours by early neutralization with interleukin (IL)-10. (a) T-cell-enriched splenocytes from mice with regression of B16 tumours following treatment with anti-IL-10 neutralizing monoclonal antibody (mAb) were subjected to cytotoxicity assay against 51Cr-labelled B16 (●) or MH134 (○) cells. (b) Flow cytometric analysis using phycoerythrin (PE)-conjugated anti-CD4 and fluorescein isothiocyanate (FITC)-conjugated anti-CD25 mAbs. (c) Reverse transcription–polymerase chain reaction (RT–PCR) analysis using cytokine-specific primers for IL-4, IL-10, interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β). In cytotoxicity assays and RT–PCR analysis, T-cell-enriched splenocytes from B16-bearing mice treated with control rat immunoglobulin G (IgG) were used as a control. All data are representative results of three independent experiments.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

It has recently been documented that Th3/Tr1-type T cells that secrete high amounts of IL-4, IL-10 and TGF-β function as suppressors in inflammatory responses of autoreactive Th1 cells and promote immunological tolerance to orally administered exogenous antigens.12–16 In this study using B16 melanoma cells, we demonstrated that CD4+ CD25+ T cells with the same cytokine profile serve as a down-regulator in antitumour immunity involving NK cells and CTLs. In addition, their inhibitory action on the Th1-cell induction and Th1-cell-mediated CTL generation was mediated mainly via release of TGF-β. IL-10 and partly IL-4, which are produced possibly by certain population(s) of T cells, such as γδ T cells, preexisting in early B16 lesions,24 seem to be required for the generation and TGF-β production of the Th3/Tr1-type CD4+ T-cell population. In fact, blocking of IL-10 with a specific mAb during early development of tumours led to insufficient induction of Th3/Tr1-type cells and consequent elevation of antitumour immunity. This was also proved by in vivo regression of tumours. Our findings are consistent with in vivo studies of other groups, which have documented that tumours are regressed by depletion of lesional CD4+ cells21 or of systemic CD25+ cells.30 In addition, attenuation of Th function by tumour-derived TGF-β19,20 also supports the importance of TGF-β function as a down-regulator in tumour rejection.

It has been found in infectious diseases, caused by micro-organisms, that innate immunity occurs through the actions of non-specific immune cells, including NK cells and certain populations of TCR-αβ- or TCR-γδ-bearing lymphocytes differentiated extrathymically, while acquired immunity is performed by antigen-presenting cells such as DCs and antigen-specific T cells, including conventional CTLs and Th cells differentiated through the thymus.31,32 Although acquired immunity follows early innate responses, antigen-specific priming via DCs in the process of development of intrathymically differentiated T cells is established during the innate period.32 IFN-γ primarily produced by innate immune cells seems to support effective priming of Th1 cells and CTLs.1–3 According to these viewpoints, our findings can be interpreted as an indication that infiltration of intrathymic Th3/Tr1 cells in 20-day tumour lesions duly follows temporal accumulation of extrathymic lymphocyte population(s), i.e. IL-4- and IL-10-producing inhibitory TCR-γδ+ cells present in early (7-day) tumour lesions.22–24 The significance of early IL-10 production by these γδ T cells in the effective generation of Th3/Tr1 cells provides the following insights:

  • • 
    both innate and acquired immune responses operate in tumour-bearing mice;
  • • 
    IL-10 produced during the innate period is a potent mediator for effective Th3/Tr1 cell generation; and
  • • 
    extrathymic TCRγδ+ cells may serve as an initiator in the formation of the immunosuppressive network.

Similarly to elimination of microbes, the observed elevation of antitumour CTL activity and IFN-γ secretion following neutralization of IL-10 also suggests the existence of a connecting network between innate and acquired immunity and the importance of Th1 cell mediation of CTL development.

The Th3/Tr1-type cells accumulating in 20-day B16 tumour lesions had unique properties, i.e. an IL-4-, IL-10- and TGF-β-producing cytokine profile, surface expression of CD25 and remarkable in vitro expansion, even without exogenous cytokines. The last property is in accordance with lack of enhanced proliferation by anti-CD3 stimulation (N. Seo et al., unpublished). Although the inhibitory action of TGF-β was investigated in this study, the immunomodulatory roles of IL-4 and IL-10 remains to be further elucidated. However, knowledge has been accumulated from autoimmunity and oral tolerance experimental systems. Inflammatory responses by autoreactive Th1 cells and immune reactions to orally administered antigens are partially blocked by TGF-β derived from CD4+ cells,13,17,33 and these CD4+ cells are expanded in vitro in the presence of IL-4 and IL-10·17,34 IL-4 and IL-10 produced by a Th3/Tr1-type TIL population may also promote their autocrine proliferation and maintenance of survival in tumour-bearing mice. In addition, the possibility of autocrine proliferation might explain their in vitro expansion in cytokine-free medium. Similarly to our cells, CD4+ CD25+ T-cell populations concerned with suppression of autoimmune formation were found not to expand in anti-CD3 mAb-immobilized plates.35 Thus, the Th3/Tr1-type TILs in our system resembled suppressive Th3/Tr1 cells found in autoimmune diseases.

Investigators have identified many kinds of tumour-associated antigen peptides that are presented in the context of MHC class I molecules and recognized by CTLs in a class I-dependent manner.18,36 Almost all antigen peptides of melanoma cells are derived from melanosomal proteins expressed in normal melanocytes.18 It is tempting to speculate that antitumour immunity mediated by CTLs is within the same category as autoimmunity. Melanoma immunotherapy using antigen peptides originating from melanosome proteins, if therapeutically effective, has been reported to evoke vitiligo skin as well as melanocyte lysis,37 verifying participation of autoreactive CTLs in the antitumour reactions. Thus, autoimmune reactions may hold the key for understanding complex antitumour immune reactions and for providing a more effective therapeutic strategy for tumour regression.

On the basis of several previously published studies, together with the present work,22–24,31 it is suggested that depletion of immunosuppressive factors in tumour-bearing mice and patients is an effective strategy for tumour immunotherapy. Removal of CD4+ Th3/Tr1-type T cells may be one conceivable way to treat progressive tumours. In addition, utilization of epitope peptides for tumour-specific CTLs and Th cells in combination with IL-12 is expected to promote cytotoxicity of CTLs and help by Th1 cells. However, as surface molecules capable of distinguishing Th3/Tr1-type T cells from other T cells have not been fully identified, it still remains difficult to delete this specific type of T cell in vivo. Alternatively, neutralization of IL-4, IL-10 or TGF-β with specific mAb may be useful in the treatment of small tumours or for the immunoprophylaxis of neoplasms.


  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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