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The Journal of Immunology, 2006, 177: 6747-6757.
Copyright © 2006 by The American Association of Immunologists, Inc.

CD4+ T Cells in the Absence of the CD8+ Cytotoxic T Cells Are Critical and Sufficient for NKT Cell-Dependent Tumor Rejection1

Changwan Hong*, Hyunji Lee*, Mihwa Oh*, Chang-Yuil Kang{dagger}, Seokmann Hong{ddagger} and Se-Ho Park2,*

* School of Life Sciences and Biotechnology, Korea University, Seoul, Korea; {dagger} Laboratory of Immunology, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea; and {ddagger} Department of Bioscience and Biotechnology, Sejong University, Seoul, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NKT cells perform crucial roles in tumor surveillance, functioning as regulators of early host response. In this study, we have assessed the effects of NKT activation at the time of tumor Ag immunization, and have evaluated the contributions of CD4+ and CD8+ T cells in tumor rejection during adaptive immune response against live tumor cells. Our data indicate that CD4+ T cells play critical roles, not only in assisting CTL, but also in the orchestration of host response against the tumor. The CD4+ T cells were found to reject the transplanted tumor cells very efficiently under conditions in which the CTLs were removed either genetically, or via the action of anti-CD8 Ab in mice that had been immunized with tumor extracts and {alpha}-galactosylceramide. Immunization resulted in an NKT cell-dependent antitumor adaptive immune response, which was associated with both CD4+ T cells and cytokine IFN-{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer T cells are a specialized subset of the T cells which are important regulators of several immune responses (1, 2, 3). NKT cells are special in that they express T cell (such as {alpha}β TCR) and NK-lineage (e.g., CD161 (NK1.1), CD16, Ly49A, and Ly49C) cell surface markers and key cytokines (e.g., IL-4 and IFN-{gamma}) which regulate the course of the immune response. NKT cells secrete large quantities of IL-4 after activation, both in vitro and in vivo (4, 5), and it has been theorized that they also direct the immune response toward a Th2 phenotype (6). NKT cells have also been demonstrated to generate the Th1 cytokine, IFN-{gamma}, after activation via NK1.1 (7) or TCR engagement (8), and have been determined to display Th0 cytokine profiles with both IFN-{gamma} and IL-4 (9). The majority of these T cells have also been shown to express highly restricted TCRs, comprised of an invariant V{alpha}14-J{alpha}18 TCR {alpha}-chain in association with a repertoire of Vβ8, Vβ7, or Vβ2 gene products (10). NKT cells exhibit the characteristics of activated memory cells, and also evidence autoreactivity in the absence of prior exposure to Ags. Thymic selection and development in NKT cells is restricted by CD1d, which is well-conserved throughout mammalian species. CD1d is nonpolymorphic as compared with conventional MHC I molecules and presents lipid/glycolipid Ags to NKT cells (10). The nonpolymorphic nature of the Ag-presenting CD1d, coupled with their restricted TCR repertoire, their prompt secretion of a variety of key cytokines, and their intrinsic memory phenotype, renders NKT cells excellent candidates for innate immune-regulatory cells.

The synthetic glycolipid, {alpha}-galactosylceramide ({alpha}-GalCer),3 a CD1d ligand, is a profound stimulator which is able to activate NKT cells, rendering it a powerful candidate for adjuvant therapy (11). It has been recently reported that {alpha}-GalCer induces dendritic cell (DC) maturation, effecting an integration of the innate immune response with the appropriate adaptive response. This conclusion was predicated on the observation that the treatment of soluble Ag in the presence {alpha}-GalCer-mediated NKT cell stimulation enhances CD4+ and CD8+ T cell responses via DC maturation (12, 13, 14). Gonzalez-Aseguinolaza et al. (15) previously reported that the coadministration of {alpha}-GalCer with a malaria Ag augments protective antimalaria immunity in mice. Although they used OVA or malaria Ag, both of which exhibit comparatively high antigenicity, the results of that study suggested that {alpha}-GalCer functions as a powerful, immune response-enhancing adjuvant. We reported in a previous study that {alpha}-GalCer can also function as an intranasal vaccine adjuvant (16). As this glycolipid appears to lack toxicity in humans, it may also prove to be useful in vaccine and pharmaceutical therapy design (17).

Although the antitumor effects of NKT cells, and their ligand {alpha}-GalCer, have been relatively well-documented, the relative contributions and importance of CD4+ and CD8+ T cells in the immunization and adaptive phases have yet to be clearly defined. On the basis of the distinctive features of NKT cells and {alpha}-GalCer, we elected to evaluate the {alpha}-GalCer-mediated mechanism that augments protective immune response during the adaptive phase, using whole-tumor Ags. NKT cells, MHC class II-restricted CD4+ T cells, and the IFN-{gamma} generated by the activated CD4+ T cells were all found to be critical with regard to the enhancement of the adaptive immune response against low-antigenicity tumor Ags. Surprisingly, MHC class I-restricted CD8+ T cells, which are commonly believed to function as the dominant effector cells that mediate tumor killing (18), were found to be dispensable in the mediation of antitumor adaptive immunity. Moreover, CD8+ T cell deficiency augmented both antitumor immune response and the survival of tumor-challenged animals in an {alpha}-GalCer-immunized model.

Collectively, our data demonstrate that IFN-{gamma}, NK cells, and Th1-type CD4+ T cells are crucial to tumor rejection. Our findings imply that activated NKT cells may induce a powerful adaptive immune response, operating via CD4+ T cells, against Ags with very low antigenicity, including tumor extracts.


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

C57BL/6 (B6) wild-type (WT), IFN-{gamma}-deficient (IFN-{gamma}–/–), IL-4-deficient (IL-4–/–), IL-12β-deficient (IL-12β–/–), and TNFR-deficient mice (TNFR–/–), all in the B6 background, were purchased from The Jackson Laboratory. The BALB/c mice were purchased from Charles River Laboratories. Mice deficient of KbDb (referred to as MHC class I deficient), I-Ab (MHC class II deficient), and CD1d (CD1d1/CD1d2 deficient) were used after >12 backcrosses to B6 (19, 20). All results reported in this study were derived from comparative analyses of littermates expressing –/– vs +/– or +/+ genotypes. All mice were raised in a specific pathogen-free environment at Korea University, and used at 7–10 wk of age, unless otherwise specified. The experimental protocols adopted in this study were consistent with the rules established by the Korean Animal Protection Law, and all protocols were approved by the Laboratory Animal Care and Use Committee of the College of Life Sciences and Biotechnology (Korea University).

B16 and EL-4 (all H-2b) cells were acquired from the American Type Culture Collection (ATCC). The 26-M3.1 colon cells (H-2d) used in the study was a gift from Dr. T. J. Yoon (Kyonggi University, Suwon, Korea). MB19 (H-2b), a methylcholanthrene (purchased from Sigma-Aldrich)-induced fibrosarcoma, was derived from mice injected with 100 µg of methylcholanthrene. All cell lines were maintained in RPMI 1640 complete medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS (HyClone), 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 10 µg/ml gentamicin sulfate, and 5 µM 2-ME (all from Invitrogen Life Technologies).

{alpha}-GalCer, CFA, and tumor Ag

The {alpha}-GalCer [(2S,3S,4R)-1-O-(-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol] used in this study was a gift from Dr. A. Bendelac (Chicago University, Chicago, IL). It was dissolved in 0.5% polysorbate-20 (Nikko Chemical) in 0.9% NaCl solution, and diluted with PBS before use. CFA (Mycobacterium tuberculosis) was purchased from Sigma-Aldrich. The tumor Ags were prepared via more than three freeze/thaw cycles or by the gamma-irradiation (30 Gy) of tumor cells. The tumor Ags were diluted in PBS equivalent to 1 x 107 cells/ml, and were cryopreserved at a temperature of –70°C.

Vaccinations and tumor models

Mice were immunized i.p. with 100 µl of tumor Ag, at a level equivalent to 5 x 105 tumor cells. Vehicle (Veh) or 1 µg of {alpha}-GalCer was coadministered with the tumor Ag when required. The immunization schedules are described in detail in the figure legends. Two weeks after immunization, 1 x 105 live tumor cells were injected i.p., s.c., or i.v.

In the i.p. tumor model mice, survival was monitored for up to 9 wk. In the s.c. tumor model mice, tumor weight at the site of inoculation was measured 3 wk after tumor injection. In the i.v. tumor model mice, each group was sacrificed 3 wk after tumor injection, and the lung foci (visible as black spots) were counted.

Collection and preparation of lymphocytes

The peritoneal cells were collected via two peritoneal cavity washes (PBS) of the tumor-bearing vaccinated mice. The cells were washed two additional times with PBS before their use in the in vitro assays.

The spleen and liver were removed from the vaccinated mice 1, 4, or 7 days after tumor challenge, minced, passed through a nylon mesh, and suspended in PBS. The erythrocytes were lysed in 150 mM ammonium chloride lysis buffer (ACK). The remaining cells were then suspended in PBS, centrifuged over a layer of Lympholyte-M (Cedarlane Laboratories) at 2000 rpm at room temperature for 20 min, and the lymphocytes were collected at the interface between the Lympholyte and PBS.

Cytotoxic assay ([3H]thymidine assay)

For the assay against tumor Ag, the mice were immunized with tumor Ag, coupled with either {alpha}-GalCer or Veh. Spleen cells were restimulated in vitro with 20 U/ml recombinant human IL-2 (PeproTech) plus tumor Ag for 5 days, after they were obtained at 14 days after immunization. The effector cells were harvested, washed, adjusted to 106 cells/ml, and then added to [3H]thymidine-labeled ([3H]TdR; 0.25 µCi/well; PerkinElmer Life Science) target cells (1 x 104 cells/well in 96-well round-bottom plates), to yield the desired E:T cell ratio (21, 22). After 6 h of incubation at 37°C in a humidified, 5% CO2 atmosphere, the plates were freeze-thawed three times, harvested, then measured using a micro-beta counter (Wallac). The percentage of specific lysis was calculated in triplicate, as follows: percent-specific lysis = ((cpm spontaneous value – cpm experimental value)/cpm spontaneous value) x 100.

Flow cytometric (FACS) analysis and mAbs

Cells were stained in FACS staining buffer (PBS containing 0.1% BSA and 0.01% sodium azide), incubated for 20 min at 4°C with anti-FcR-{gamma} mAb 2.4G2, then labeled for 30 min with the appropriate mAb conjugated to biotin (bio), FITC, R-PE, CyChrome, or allophycocyanin. When the mAbs were biotinylated, streptavidin (StAv)-PE or StAv-allophycocyanin was used as a second-step reagent. {alpha}-GalCer/CD1d-tetramers were produced and used for staining as described previously (23, 24). The following conjugates were used: FITC against CD4 (clone RM4-5), CD161 (clone PK136), TCRβ (clone H57), CD11c (clone HL3), CD11b (clone M1/70) (all BD Pharmingen), and I-Ab (clone Y3P) (provided by Dr. A. Bendelac, Chicago University); PE against CD44 (clone IM-7), CD28 (clone 37.51), CD49d (clone R1-2), CD86 (clone GL-1), CD161 (clone PK136), and TCRβ (clone H57); CyChrome against Gr-1 (clone RB6-8C5), CD8{alpha} (clone 53-6.7), CD44 (clone IM-7), and CD45 (clone RA3-6B2); allophycocyanin against CD11b (clone M1/70), TCRβ (clone H57), IL-4 (clone 11B11), IL-12 (clone C15.6), and IFN-{gamma} (XMG1.2); bio against CD49d (clone R1-2), CD69 (clone H1.2F3), CD95L (clone NOK-1), and CD11c (clone HL3) (all BD Pharmingen). The stained cells were then analyzed on a FACSCalibur, using CellQuest software (both BD Biosciences). Only viable cells, adjudged on the basis of their forward and side scatter (SSC), were used in the analyses. Figures with panel sets depict analyses from the same experiment, to allow for direct comparisons of fluorescence intensity.

Intracellular cytokine production analyses

Tumor cells (1 x 105) were i.p. inoculated into the immunized mice. One, 4, or 7 days later, single-cell suspensions of peritoneal cells from the tumor-injected mice were prepared as described above, and cultured for 6 h with Golgi stop (BD Pharmingen) in RPMI 1640 culture medium. To determine the intracellular cytokine levels, the cells were initially stained with the appropriate mAb, fixed and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen), and finally stained with allophycocyanin-conjugated anti-IL-4, anti-IL-12, and anti-IFN-{gamma} mAbs for 45 min on ice. The percentages of cells expressing cytoplasmic IL-4, IL-12, and IFN-{gamma} were determined via flow cytometry (FACSCalibur). IFN-{gamma} and IL-4 concentrations were measured using an enzyme-linked immunoassay kit (OPTEIA Mouse IFN-{gamma} and IL-4 set; BD Pharmingen), on the basis of a standard curve for recombinant mouse IFN-{gamma} and IL-4.

In vivo depletion of CD8+ T cells and DC transfer

For the in vivo depletion of CD8+ T cells, mice were injected with 250 µg of cytotoxic anti-CD8 mAb 2.43 (rat IgG2b). The mice were administered two i.p. injections on 3 days and 1 day before immunization or to tumor challenge. The control mice received 250 µg of rat IgG. This depletion was previously determined to be sufficient for the depletion of all CD8+ cells in mice (as determined via flow cytometry). For the transfer of the DCs, the DCs were obtained from naive mice spleens via MACS using magnetic bead-conjugated anti-CD11c mAb, bio-conjugated anti-CD8 mAb, and StAv-conjugated magnetic beads (Miltenyi Biotec), in accordance with the manufacturer’s instructions. The DCs were injected i.p. into naive mice on the day of the tumor challenge.

Statistical analysis

Student’s t tests were used to determine statistically significant differences between the two groups. Log-rank tests were used to analyze the mouse survival curves. Throughout the text, figures, and legends, the following terminology was used to denote statistical significance: ***, p < 0.0001 or p < 0.001; **, p < 0.01; *, p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NKT cells promote an Ag-specific adaptive immune response

It has been demonstrated that the coadministration of {alpha}-GalCer with a malaria Ag augments the level of protective antimalarial adaptive immunity (15). Several previous reports have indicated that {alpha}-GalCer-mediated NKT cell activation induces an antitumor immune response (14, 25). To assess the adjuvant activity of {alpha}-GalCer for tumor Ags, we immunized mice with tumor extracts. B6 mice immunized with B16 whole tumor extract (103-107 cells/mouse) alone evidenced no protective immune response against the live B16 tumor cells 2 wk after immunization (data not shown). Tumor extract immunized with CFA was associated with a marginally increased survival time, but did not result in complete protection. However, mice treated with {alpha}-GalCer at the time of the immunization with tumor Ag evidenced substantially enhanced survival as compared with what was seen with CFA, whereas the Veh-treated control mice demonstrated no such enhanced survival effects (Fig. 1A). This effect was determined to have been mediated by NKT cells, as only the littermate control heterozygotic (He, CD1d+/–) mice, and not the NKT cell-deficient CD1d–/– mice, were protected by this immunization. Furthermore, mice immunized 150 days before tumor challenge retained their antitumor immune response characteristics. These findings demonstrated that the activation of NKT cells at the time of tumor vaccination successfully established long-term memory cells against B16 tumors (Fig. 1A).


Figure 1
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  		FIGURE 1. {alpha}-GalCer induces protective antitumor adaptive immunity in broad tumor models and tumor cell lines when coadministered with tumor Ags. A, CD1d+/– (He) and CD1d–/– (KO) littermate mice were i.p. immunized with B16 tumor extract (ext.) in the presence of {alpha}-GalCer, CFA, or Veh. Mice were challenged i.p. with live B16 tumor cells (1 x 105 cells/mouse), either 2 wk or 5 mo after immunization. The survival rates of each of the groups were monitored over the following 8 wk. The data shown are representative results of at least three independent experiments, in which each of the experimental groups contained five to eight mice. B, CD1d+/– and CD1d–/– BALB/c mice were immunized i.p. with 26-M3.1 tumor extract, plus {alpha}-GalCer or Veh, then challenged i.p. with live 1 x 105 26-M3.1 tumor cells. CD1d+/– and CD1d–/– B6 mice were immunized i.p. with EL-4 extract (C) or MB19 extract (D) plus {alpha}-GalCer or Veh, then challenged i.p. with live 1 x 105 EL-4 or MB19 tumor cells. The survival rates of each group were monitored over the following 8–13 wk. E, CD1d+/– and CD1d–/– B6 mice were immunized with tumor extract and {alpha}-GalCer or Veh and were challenged s.c. with live 1 x 105 B16 tumor cells after 2 wk. All mice were sacrificed and tumor weights were determined 3 wk after tumor injection. F, Mice immunized as in C were challenged i.v. with live 1 x 105 B16 tumor cells 2 wk after immunization. Three weeks later, the mice were sacrificed, and the black metastatic lung foci were counted under a dissection microscope. The data shown are representative results of two independent experiments, in which each experimental group contained five mice.

 
Vaccination with {alpha}-GalCer induces effective antitumor immunity regardless of the mouse strain, tumor type, or tumor site

We attempted to determine whether the adjuvant effects of {alpha}-GalCer were applicable to other tumor cell lines and tumor injection tissues in different mouse strains. First, we immunized BALB/c CD1d–/– mice and littermate CD1d+/– mice with extracts of the 26-M3.1 colon cancer cell line. We then coadministered either {alpha}-GalCer or Veh, and initiated a challenge with live tumor cells 2 wk later. Only the CD1d+/– mice who had been administered {alpha}-GalCer were protected (Fig. 1B). To evaluate the protective effects of {alpha}-GalCer against different tumor lines, we immunized B6 mice with the extract of the lymphoma cell line, EL-4 (Fig. 1C), or the sarcoma cell line, MB19 (Fig. 1D). Only CD1d+/– mice that had been immunized with the Ag in the presence of {alpha}-GalCer were found to be protected against the live tumor challenge. All other groups, including the group of CD1d–/– mice immunized with Ag coupled with {alpha}-GalCer, were not protected. These findings clearly show that the adjuvant effects of {alpha}-GalCer occur independently of the mouse strain and the tumor type.

We also attempted to determine whether enhanced immunization with {alpha}-GalCer would affect the rejection of tumor cells injected into different anatomical sites. Rather than administering i.p. injections, {alpha}-GalCer and tumor Ag were immunized into He and CD1d–/– B6 mice either s.c. or i.v., into the left flank (Fig. 1E) or the tail vein (Fig. 1F), respectively. Three weeks later, the tumor masses growing on the left flanks of the injected mice were excised and weighed. The tail vein-injected mice were sacrificed 3 wk after the challenge, and the metastatic lung tumor nodules were counted. In all cases, the CD1d+/– mice immunized with tumor extract coupled with {alpha}-GalCer evidenced a controlled growth of the recently implanted tumor cells. Collectively, these data support the notion that {alpha}-GalCer exerts a profound and general adjuvant effect on antitumor response in a variety of mice strains, tumor lines, and tumor sites.

NKT cell activation enhances cytotoxic activity

NKT cells influence the quality of the adaptive immune response and augment cytotoxic activity (25). Therefore, we attempted to determine whether vaccination with {alpha}-GalCer augmented cytotoxic capacity. Mice were immunized with B16 extract, in the presence of {alpha}-GalCer or Veh. Splenocytes and peritoneal cells were obtained 2 wk after immunization. The splenocytes of the CD1d+/– mice immunized with {alpha}-GalCer evidenced an increase in cytotoxic activity against B16 (25% lysis with an E:T ratio of 10:1), as compared with the Veh-treated controls (25% lysis with an E:T ratio of 80:1, Fig. 2A, left panel). The peritoneal cells of the CD1d+/– mice that had been immunized with {alpha}-GalCer evidenced an increase in cytotoxic activity against B16 (40% lysis with an E:T ratio of 4:1) as compared with the Veh-treated controls (40% lysis with an E:T ratio of 50:1, Fig. 2A, right panel). Whereas, enhanced-cytotoxic activity was not observed when splenocytes and peritoneal cells from MHCI–/– or MHCII–/– mice were used as effector cells (Fig. 2A). Notably, peritoneal effector cells from CD4+ T cell-deficient MHCII–/– mice showed minimal cytotoxic activity compare with Veh-control groups.


Figure 2
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  		FIGURE 2. NKT cell activation at the time of immunization greatly augments CTL-mediated cytotoxicity and DC, macrophage, and B cell activation upon live tumor challenge. A, CD1d+/–, CD1d–/–, MHCI–/–, and MHCII–/– littermate mice were immunized with B16 tumor extract plus {alpha}-GalCer or Veh. Spleens and peritoneal cells were extracted 2 wk later, and the pooled splenocytes and peritoneal cells were restimulated in vitro with IL-2 plus B16 tumor extract for 5 days before assessing 6-h cytotoxic activity against [3H]thymidine-labeled B16 target cells. Once labeled, the cells were incubated with restimulated splenocytes or peritoneal cells at the designated E:T ratio. The data shown are representative results of three independent experiments, in which each experimental group contained three mice. B–D, CD1d+/– and CD1d–/– B6 mice were immunized i.p. with tumor extract plus {alpha}-GalCer or Veh, and challenged i.p. with live 1 x 105 B16 tumor cells 2 wk after immunization. Lymphocytes freshly isolated from the peritoneal cavity were stained with anti-CD11c, anti-CD11b, anti-B220, anti-Gr-1, anti-I-Ab, and anti-B7.2 Abs, 1, 4, and 7 days after B16 tumor challenge, and the DCs (B) and granulocyte/macrophages (C) were analyzed via flow cytometry. The average percentage of the CD11c+ cells and the absolute numbers of the CD11bint cells are shown on the right side of B and C. The data represent one of three independent experiments with similar results, in which each of the experimental groups contained three mice. D, Splenic and peritoneal DCs and splenic and peritoneal B cells were analyzed via flow cytometry. The average mean fluorescence intensities (MFI) for the B7.2 molecule on the cells 1 day after B16 tumor challenge are shown. The data represent one of three independent experiments with similar results, in which each experimental group contained three mice.

 
APC activation following tumor challenge is greatly enhanced in {alpha}-GalCer-immunized mice

Previous studies have reported that macrophages are recruited to tumor sites, where they function as effector or killer cells, facilitating the rejection of the tumor (26, 27). Treatment with {alpha}-GalCer results in the full maturation of DCs, supporting the cytokines and presenting tumor Ags, thereby combining CD4+ and CD8+ T cell immunity against the relevant Ag (14, 28). To determine whether macrophages and DCs are activated and recruited to B16 tumor sites in an {alpha}-GalCer-immunized system, we isolated tumor-infiltrating immune cells from the peritoneal cavity at various time points (1, 4, and 7 days) after the injection of live tumor cells. The cells were then stained with Abs against macrophages (CD11b) and DCs (CD11c), and analyzed via flow cytometry. The CD11c+ cells reached >20% of all of the peritoneal cells in the {alpha}-GalCer-immunized He mice within 1 day, and this level was maintained until 4 days after challenge with B16 tumor cells. Significantly fewer DCs were observed in the B16 tumor sites of the Veh-immunized WT mice and {alpha}-GalCer-immunized CD1d–/– mice (Fig. 2B). The CD11c+ cells also evidenced an activated phenotype, expressing higher levels of the costimulatory molecule, B7.2, in the spleen and peritoneal cavity cells of the WT mice immunized with {alpha}-GalCer, as compared with the mice that had been immunized with Veh (Fig. 2D). Both the absolute number and percentage of macrophages/granulocytes (CD11bint-gated, Fig. 2C) were increased soon after the injection of B16 tumor cells into the {alpha}-GalCer-immunized He mice, reaching their maximum level within 1 day. High levels, in this case, were maintained until day 4 (Fig. 2C). Among the CD11bint cells, highly granulated cells were found to constitute ~21 and 40% of all of the CD11bint cells in the {alpha}-GalCer-immunized WT mice on days 1 and 4, respectively, but the same population in the Veh-immunized mice constituted only 5~10% overall (data not shown). Highly granulated CD11bint (CD11bintSSChigh) cells expressed Gr-1, but not MHC class II, thereby indicating that the CD11bintSSChigh cells were matured and differentiated granulocytes (data not shown). Activated CD11bintSSClow macrophages were also detected, which expressed higher levels of MHC II molecules and the costimulatory molecule B7.2 in the He mice immunized with {alpha}-GalCer than in the mice that had been immunized with Veh (data not shown). We additionally determined that the expression of the costimulatory molecule B7.2 was highly elevated in the peritoneal B cells of the {alpha}-GalCer-immunized WT mice, whereas the levels of B7.2 remained relatively unchanged in the splenic B cells (Fig. 2D).

NK and CD4+ T cells are the source of IFN-{gamma} for antitumor response

We assumed that Ag-specific CTLs would infiltrate and generate effector molecules, most notably IFN-{gamma}, into the tumor inoculation site upon challenge with live tumor cells. Lymphocytes freshly isolated from the tumor challenge site were analyzed for the expression of IFN-{gamma}, the prototypical Th1 cytokine, on days 1, 4, and 7 after tumor injection. We determined that CD4+CD8– T cells from the He mice immunized with {alpha}-GalCer generated substantial amounts of IFN-{gamma} on day 1, which peaked on day 4 (Fig. 3A). Unexpectedly, however, we were unable to detect a substantial population of IFN-{gamma}-generating CD4–CD8+ T cells at any time point after the administration of tumor challenge (Fig. 3C). By way of contrast, a significant portion of the NK cells were also determined to generate IFN-{gamma} on day 1, but with a slight decline, and an ultimate plateau occurring over the next 3 days (Fig. 3B). However, neither the CD4+CD8– T cells nor NK cells expressed any IFN-{gamma} in the vaccinated CD1d–/– mice or the Veh-treated He mice, thereby suggesting that the development of IFN-{gamma}-producing CD4+ T cells is dependent on CD1d molecules and NKT cell activation at the time of vaccination. To test the possibility that some IFN-{gamma}-expressing CD4+ T cells may be NKT cells, we measured IFN-{gamma} production of NKT cells after staining with {alpha}-GalCer/CD1d-tetramer. CD4+ NKT cells from {alpha}-GalCer-immunized mice significantly secreted IFN-{gamma} compared with the Veh control group only on day 1 (Fig. 3D), indicating that NKT cells were also activated in early antitumor effector phase but constitute only a small part of IFN-{gamma}-producing CD4+ T cells.


Figure 3
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  		FIGURE 3. NKT cell activation at the time of immunization augments the secretion of IFN-{gamma} from CD4+ T and NK cells upon live tumor challenge. CD1d+/– and CD1d–/– B6 mice were immunized with tumor extract plus {alpha}-GalCer or Veh 2 wk before live tumor challenge (i.p, 1 x 105 B16 tumor cells). Lymphocytes freshly isolated from the peritoneal cavities of tumor-challenged mice were stained with anti-TCRβ, anti-CD4, anti-CD8, anti-NK1.1, anti-IFN-{gamma} Abs and {alpha}-Galcer/CD1d-tetramer 1, 4, and 7 days after tumor challenge. A–C, Peritoneal lymphocyte suspensions of the indicated mice were four-color stained to determine the prevalence of IFN-{gamma}-secreting cells (indicated in the upper right quadrants of each dot plot) among the CD4+ T (CD4+CD8–TCRβ+) cells, NK (NK1.1+TCRβ–) cells, and CD8+ T (CD4–CD8+TCRβ+) cells. The number of IFN-{gamma}-secreting CD4+ T cells, NK cells, and CD8+ T cells per peritoneal cavity is shown in the lower panel. D, Prevalence of IFN-{gamma}-secreting CD4+ cells among NKT ({alpha}-GalCer/CD1d-tetramer+ TCR+) cells. The number of IFN-{gamma}-secreting CD4+ NKT cells per peritoneal cavity is shown in the lower panel. The data shown represent one of three independent experiments with similar results, in which each experimental group contained three mice.

 
MHC class II-dependent CD4+ T cells are essentially required for NKT cell-dependent antitumor immunization, but MHC class I-dependent CD8+ T cells are dispensable

To determine whether the activation of NKT cells at the time of immunization facilitates its effects via the promotion of the activation of a tumor Ag-specific conventional T cell population, we immunized both MHC class I-deficient (Kb–/–Db–/–, marked as MHCI–/–) and MHC class II-deficient (I-Ab–/–, marked as MHCII–/–) mice with a combination of {alpha}-GalCer and tumor extract, 2 wk before the administration of a B16 tumor challenge. Surprisingly, the conventional CD8+ T cell-deficient MHCI–/– mice evidenced potent tumor rejection and survival characteristics, comparable to those of WT mice. Similar results were also observed when different tumor cell lines (EL-4 and MB19) were challenged (Table I). These data are consistent with the findings of Hung et al. (29), who demonstrated that a significant fraction of CD8–/– mice vaccinated with B16-GM-CSF also successfully rejected tumor tissue. However, CD4+ T cell-deficient MHCII–/– mice were not protected by the same immunization protocol, and died 3 wk postinjection, in a manner similar to that observed with the CD1d–/– mice (Fig. 4A). As the administered tumor Ag was prepared via a repeated freeze/thaw method, it is possible that the nature of the Ag induced the activation of a specific cell subpopulation. To address this question, we included tumor Ags prepared via gamma-ray irradiation to preserve cell integrity (Fig. 4B). In both setups, the MHCI–/– mice evidenced profound resistance against B16 tumors in cases in which the NKT cells had been activated at the time of immunization. Neither of the Ag preparation protocols resulted in any notable protection in the MHCII–/– mice. Table I shows the adjuvant effects of {alpha}-GalCer on protective antitumor immunity.


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  		Table I. Summary of {alpha}-GalCer effect on protective antitumor immunitya

 

Figure 4
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  		FIGURE 4. Memory CD4+ T cells, but not CD8+ T cells, are essential for the induction of adaptive antitumor immunity. WT, CD1d–/–, MHCI–/–, and MHCII–/– B6 mice were immunized i.p. with B16 extract (A) or gamma-irradiated (30 Gy) B16 tumor cells (B) plus {alpha}-GalCer or Veh. Two weeks later, all mice were challenged i.p. with live B16 tumor cells (1 x 105 cells). The survival rates of each group were monitored over the following 8 wk. The data shown are representative results of at least three independent experiments, in which each experimental group contained five to eight mice, respectively.

 
IFN-{gamma} is a key cytokine in NKT cell-mediated adaptive antitumor immunity

Both IFN-{gamma} and the lymphocytes perform crucial roles in the prevention of primary tumor development (30) in that they are both required during the early effector phase (31). Therefore, we evaluated the effects of vaccination with {alpha}-GalCer on cytokine levels in serum (Fig. 5A) and peritoneal lavage fluids (Fig. 5B), 1–7 days after a B16 tumor challenge. Soon after the injection of the live tumor cells, the levels of IFN-{gamma} rose significantly in both the peritoneal lavage fluid and sera of the B6 WT mice and MHCI–/– mice, but no such increase was observed in the Veh-immunized WT mice or the {alpha}-GalCer-immunized CD1d–/– and MHCII–/– mice. IL-4 production levels remained unaffected by {alpha}-GalCer vaccination in all experimental groups (data not shown). To directly assess the antitumor activity associated with IFN-{gamma} and other cytokines, we monitored the survival of IFN-{gamma}–/–, IL-12β–/–, IL-4–/–, and TNFR–/– mice vaccinated with {alpha}-GalCer for 2 mo postinjection. The vaccinated WT and TNFR–/– mice maintained 80 and 100% of pretreatment survival rates. A lack of antitumor immunity similar to that of the unvaccinated WT mice was observed in the vaccinated IFN-{gamma}–/– mice, thereby demonstrating the importance of IFN-{gamma} with regard to the prevention of B16 tumor development during the early effector phase. The IL-12β–/– and IL-4–/– mice also evidenced decreased rates of survival as compared with the WT mice, although this decrease was less severe than was observed in the IFN-{gamma}–/– mice. This finding illustrates the contributions of both IL-4 and IL-12β to a tumor-free environment (Fig. 5C).


Figure 5
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  		FIGURE 5. Vaccination with {alpha}-GalCer induces IFN-{gamma} expression and the expressed IFN-{gamma} plays critical roles in tumor rejection. WT, CD1d–/–, MHCI–/–, and MHCII–/– B6 mice were immunized with tumor extract plus {alpha}-GalCer or Veh, and challenged i.p. with live 105 B16 tumor cells 2 wk thereafter. IFN-{gamma} levels were determined via ELISA in freshly isolated serum (A) and peritoneal lavage (B) from all mice groups 1, 4, and 7 days after tumor challenge. The data represent one of three independent experiments with similar results, in which each experimental group contained three mice. C, WT, IFN-{gamma}–/–, IL-4–/–, IL-12β–/–, and TNFR–/– mice were immunized with tumor extract plus {alpha}-GalCer or Veh, and challenged i.p. with live 105 B16 tumor cells 2 wk later. The survival rates of each group were monitored over the next 8 wk. The data represent one of two independent experiments with similar results, in which each experimental group contained five to eight mice.

 
The conventional CTL-independent antitumor immunity requires CD8+ DCs

As the observed resistance of the MHCI–/– mice against tumor challenge was an unexpected result, we also attempted to evaluate this effect in mice in which the CD8+ T cells had been depleted via the anti-CD8 Ab. We depleted the CD8+ cells before immunization in a group of WT mice, and in another group of immunized WT mice, the CD8+ cells were depleted immediately before the challenge with live tumor cells. Treatment with anti-CD8 Ab before immunization did not impair the survival of the immunized mice, as compared with the isotype-matched control Ab-treated mice. However, the immunized mice to which anti-CD8 Ab had been administered before tumor challenge evidenced dramatically decreased survival rates (Fig. 6A). The former case shows that protective antitumor immune response can be generated in mice lacking CD8+ T cells at the time of immunization. However, as the results in the latter case suggested two possibilities–that either CD8+ T cells are one of the primary effector cells for tumor rejection, or that CD8+ DC is required for tumor rejection during the adaptive phase of the immune response–we transferred purified CD8+ DC to the anti-CD8 Ab-treated immune WT and MHCI–/– mice immediately before the tumor challenge. In the WT mice, the transferred DC were not able to restore protective immunity, thus demonstrating that the CD8+ T cells become the principal antitumor effector cells when they are present at the time of immunization. MHCI–/– mice, however, evidenced superior survival against anti-CD8 Ab treatment, as compared with the WT mice. Moreover, survival in these mice was restored completely by virtue of the transfer of the CD8+ DCs (Fig. 6B). These results indicate that the CD4+ T cells become the primary antitumor effector cells when CD8+ T cells are absent at the time of immunization, and that CD8+ DCs substantially augment the protective activity of tumor-specific CD4+ T cells.


Figure 6
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  		FIGURE 6. CD8+ DCs are required for the induction of conventional CTL-independent antitumor immunity. A, WT B6 mice were immunized i.p. with tumor extract plus {alpha}-GalCer or Veh. The mice were injected i.p. with anti-CD8 Ab ({alpha}CD8; 250 µg) or an isotype-control Ab, 1 day and 3 days before immunization, or the i.p. injection of B16 cells. Survival was evaluated at the indicated times after tumor injection. B, WT and MHCI–/– B6 mice immunized i.p. with tumor extract coupled with {alpha}-GalCer were treated with anti-CD8 Ab 1 day and 3 days before the i.p. injection of B16 cells. The mice were injected i.p. with spleen-derived CD8+ DCs at the time of tumor injection. The purity of the CD8+ DCs in this experiment was ≥90%. The data represent one of two independent experiments with similar results, in which each of the experimental groups contained five to eight mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NKT cells appear to initiate a rapid protective immune response against infection via the modulation of adaptive immune responses against pathogens (2, 10, 32). Although the results of a recent study showed clearly that a minor subpopulation of NKT cells can down-regulate tumor immunosurveillance (33), {alpha}-GalCer, an NKT cell ligand, is a promising immunomodulatory agent, which is currently being evaluated for its possible benefits in antitumor therapy (2, 17, 25, 34). The in vivo stimulation of NKT cells with {alpha}-GalCer has been shown to modulate acquired protective immunity. Gonzalez-Aseguinolaza et al. (15) reported that {alpha}-GalCer administered to mice immunized with malaria Ag augmented protective antimalaria immunity. Thus far, however, it remains unclear as to the manner in which {alpha}-GalCer, as an adjuvant, modulates acquired antitumor immunity. We determined that the coadministration of {alpha}-GalCer and tumor Ag successfully induced protective antitumor immune responses during the adaptive phase. Furthermore, the acquired antitumor immunity was maintained for as long as 150 days, thereby indicating that long-term memory was elicited by vaccination with {alpha}-GalCer. The increase of the CD44high memory type marker in CD8+ and CD4+ T cells supports the notion of this long-term memory antitumor effect (data not shown). During the time in which our manuscript was being prepared, Liu et al. (28) also reported the establishment of NKT cell-dependent adaptive immune responses against tumor Ags. In this report, they demonstrated that CD8+ T cells are direct antitumor effect cells, although both CD4+ and CD8+ T cells appear to be required for tumor rejection. Nishimura et al. (35) also showed the induction of CTL against MBL-2 tumors upon immunization in the context of {alpha}-GalCer.

In our study, the {alpha}-GalCer-based vaccine model clearly illustrates the pivotal role of tumor-specific CD4+ T cells and IFN-{gamma}-secreting NK cells during tumor eradication. The coadministration of {alpha}-GalCer and tumor Ags resulted in an increase in the number of IFN-{gamma}-secreting CD4+ T cells and NK cells after a live-tumor cell challenge, by ~10- and 4-fold on day 4, respectively. Although CD8+ T cells may play crucial roles in antitumor immunity (Fig. 2A), we determined that CD8+ T cells were dispensable. CD4+ T cells and NK cells, however, were found to elicit the antitumor response, and were essential for tumor rejection in cases in which {alpha}-GalCer was used as an adjuvant. This may be the consequence of FasL expression by B16, which has been determined to protect the tumor from direct CTL-mediated killing (36). However, our data appear consistent with the findings of previous adoptive transfer experiments, in which it was shown that the transfer of CD4+ T cell clones inhibited the growth of tumor cells which do not express MHC class II themselves without completely eliminating the tumor cells, and that the transfer of positively selected CD4+ T cells or CD8-depleted lymph node cells obtained from immunized mice resulted in tumor rejection in SCID mice (37).

This observation mandates the existence of a CD4+ T cell-dependent effector mechanism aside from the MHC class I-restricted CD8+ CTL. The weak involvement of CD8+ CTL in tumor rejection and the observed augmented protection of CTL-deficient mice (MHCI–/–) in the {alpha}-GalCer/tumor extract immunized model raised the possibility that the activation of NKT cells may, in turn, elicit the activation of both antitumor CD8+ CTLs and suppressive CD8+ T cells, which can down-regulate the activity of antitumor CD4+ T cells. Thus, CD8+ T cell-deficient animals establish strong antitumor CD4+ T cell responses with the assistance of NKT cells. However, in WT mice, antitumor CD4+ and CD8+ T cell responses may be accompanied by the activation of suppressive CD8+ T cell responses. We verified this possibility via the elimination of CD8+ T cells either before or after immunization using anti-CD8 mAb. When anti-CD8 mAb was injected, before immunization, the mice retained their tumor-rejection capacity. However, the administration of anti-CD8 mAb after immunization resulted in the abrogation of tumor-rejection capacity in the immunized mice. These results show that the subpopulation of suppressor CD8+ T cells generated by the activated NKT cells can down-regulate, to a certain degree, tumor-specific CD4+ T cell response (S. Hong and S.-H. Park, manuscript in preparation). Another possibility is that the MHC class I molecules may function as inhibitory molecules for NKT cells, as has been reported by Ikarashi et al. (38). Thus, in MHCI–/– mice, NKT cells are more profoundly activated by {alpha}-GalCer than is observed in WT mice. Lastly, activated CD8+ T cells may kill off APCs with cross-presenting tumor Ag, thus inhibiting the optimal priming or boosting of CD4+ T cells which require serial contacts with APC for expansion.

Treatment with {alpha}-GalCer appears to give rise to the activation of NK cells, B cells, and DCs, ultimately resulting in a selective proliferation of memory CD4+ and CD8+ T cells via NKT activation (12, 14, 39), and in the secretion of IFN-{gamma} by NK cells (40, 41). IFN-{gamma} is the principal cytokine mediating the effects of {alpha}-GalCer during the innate phase and tumor rejection in most tumor models (42, 43). IFN-{gamma} has also been shown to compel monocytes, macrophages, fibroblasts, and certain tumor cells to generate monokine induced by IFN-{gamma}, IFN-inducible protein 10, and CXC chemokines (44, 45, 46). These substances exert powerful antiangiogenic effects by damaging the tumor vasculature, resulting in both the inhibition of growth and tumor necrosis (47). Our finding that protective antitumor immune response is abrogated in mice lacking IFN-{gamma} shows that IFN-{gamma} is of central importance in the mediation of the adjuvant effects of {alpha}-GalCer. This is also consistent with the possibility that NK and CD4+ T cell-derived IFN-{gamma} causes macrophages to secrete antitumor factors. Indeed, we determined that the {alpha}-GalCer-vaccinated mice all exhibited elevated IFN-{gamma} levels, coupled with an increased quantity of CD11c+ cells and CD11bint granulocytes. A recent study asserted that granulocytes appear to play a role in tumor regression (48), whereas macrophages appear to participate in the antitumor activity of Ag-specific CD4+ T cells (29, 49). Thus, it seems conceivable that tumor stromal monocytes and macrophages are key effector cells that mediate IFN-{gamma} antitumor activity. Although the results of our study allowed us to conclude that IFN-{gamma} is required by CD4+ T cells for complete tumor cell elimination, the possibility cannot be excluded that other cytokines secreted by Ag-stimulated immune cells may have also participated and synergized with IFN-{gamma}, affecting the release of IFN-inducible protein 10 and monokine induced by IFN-{gamma}, and/or the activation of other tumoricidal cells. Our observation that mice lacking IL-4 or IL-12β did not reject the tumor transplants, yet evidenced a modest increase in survival rates, is consistent with this notion. These data are, again, concordant with previous studies, in which NKT activation and CD4+ T cells were required for the promotion of IFN-{gamma} production by CD4+ T cells and NK cells (29, 37, 40). We propose that the IFN-{gamma} generated by memory CD4+ T cells generates an early cytokine environment, allowing for the activation of NK cells and the recruitment of APCs.

Another interesting observation of this study was that the adjuvant potency of {alpha}-GalCer was independent of the tumor model (solid and metastasis) or tumor cell line used. We determined that vaccination with {alpha}-GalCer inhibited tumor growth in both the solid tumor model (after s.c. injection) and the lung metastasis model (after i.v. injection), thereby indicating that {alpha}-GalCer-induced protective immunity is not restricted to certain tumor injection sites, but is systemically effective. This further illustrates that tumor rejection is mediated not by the NKT cells themselves, but rather by immunized adaptive immune cells. Broad protection effects were observed not only with melanoma cells (B16, B6 originated), but also with different tumor cell lines, including lymphoma (EL-4 originated on B6), sarcoma (MB19 originated on B6), and carcinoma (26-M3.1 originated on BALB/c) cell lines. The adjuvant effects of {alpha}-GalCer were much more potent than those of CFA, a representative adjuvant. In animal studies, tumors modified to secrete IFN-{gamma} (50) or to express B7.1 (51) are often rejected, and these systems tend to generate systemic immunity against WT tumor challenges. In other studies, the cotransfection of the B7 molecule with MHC class II molecules is required for the induction of potent systemic immunity (52). Our vaccination system, however, achieved equal or superior antitumor effects, without the need for artificial genetic modifications such as the transfection of the B7 molecule or IFN-{gamma}. In this regard, the results of this study suggest that the activation of NKT cells, using the {alpha}-GalCer ligand, constitutes a simple and powerful method for the induction of a potent antitumor adaptive immune response against a variety of tumor models and tumor cell lines.

This study is, to our knowledge, the first report regarding the pivotal role of NKT cells in the establishment of an Ag-specific CD4+ T cell-mediated adaptive immune response and in the assistance of the T cell-mediated regulation of the adaptive immune response. It appears likely that an identical antitumor mechanism is operational in the human immune system, in which the CD1d/NKT pathways are quite well-conserved (53). This, if it is the case, may facilitate the design of more effective vaccines against other intracellular pathogens, in addition to tumors.


   Acknowledgments
 
We thank Drs. A. Bendelac and T. J. Yoon for providing the material and cell lines; H. S. Shin for the animal care and maintenance; Jiyeon Kim for assistance with flow cytometry; and Sundo Jung, M.S., and Eunjeong Cho, M.S., for helpful discussions.


   Disclosures
Top
 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 a Rheumatism Research Center Grant (R11-2002-098-05005-0) from the Korea Science and Engineering Foundation. Back

2 Address correspondence and reprint requests to Dr. Se-Ho Park, School of Life Sciences and Biotechnology, Korea University, Anam-Dong, Sungbuk-Gu, Seoul 136-701, Korea. E-mail address: sehopark{at}korea.ac.kr Back

3 Abbreviations used in this paper: {alpha}-GalCer, {alpha}-galactosylceramide; DC, dendritic cell; WT, wild type; Veh, vehicle; StAv, streptavidin; SSC, side scatter; int, intermediate. Back

Received for publication June 1, 2006. Accepted for publication August 30, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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