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Polarization of V24⁺ Vß11⁺ Natural Killer T Cells of Healthy Volunteers and Cancer Patients Using -Galactosylceramide-loaded and Environmentally Instructed Dendritic Cells¹

Departments of Medical Oncology [H. J. J. v. d. V., N. N., A. J. M., W. K., A. J. M. v. d. E., H. M. P., G. G., R. J. S.] and Pathology [H. J. J. v. d. V., A. J. M., W. K., B. M. E. v. B., R. J. S.], Vrije Universiteit Medisch Centrum, Amsterdam, the Netherlands; Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Gunma, 370-1295, Japan [N. N.]; and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 [S. A. P.]

	ABSTRACT

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CD1d-restricted natural killer T (NKT) cells play important regulatory roles in various immune responses. NKT cell-derived T helper (Th) 1 cytokines are important in the induction of antitumor immune responses in mice. Because the CD1d-restricted V24⁺ Vß11⁺ NKT cell population in cancer patients is decreased both in size and in its capacity to secrete IFN-, therapeutic strategies based on reconstitution of type 1 polarized V24⁺ Vß11⁺ NKT cells merit additional investigation. Here, we report the simultaneous strong expansion and type 1 polarization of human invariant V24⁺ Vß11⁺ NKT cells using -galactosylceramide-loaded type 1 dendritic cells and interleukin 15. Type 1 polarized V24⁺ Vß11⁺ NKT cells produced high levels of IFN-, tumor necrosis factor , and granulocyte macrophage colony-stimulating factor, and induced strong cytotoxicity in Jurkat cells in an -galactosylceramide-dependent manner. Importantly, the cytokine profile of V24⁺ Vß11⁺ NKT cells that were initially expanded under Th2 polarizing conditions could be reversed to a Th1 cytokine profile, indicating the plasticity of the cytokine profile of the human adult V24⁺ Vß11⁺ NKT cell population.

	INTRODUCTION

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NKT³ cells constitute a lymphocyte lineage sharing characteristics of both T cells and NK cells. Invariant NKT cells display an extremely restricted TCR repertoire, in humans consisting of a V24 chain preferentially paired with a Vß11 chain, and recognize antigen in the context of the monomorphic CD1d antigen-presenting molecule (1) . Invariant NKT cells play crucial roles in various immune responses, including antitumor, autoimmune, and antimicrobial immune responses (1) . Their regulatory role in immune responses that require opposite regulatory pathways has been attributed to an apparent flexibility of invariant NKT cells with regards to their predominant cytokine profile. NKT cell-derived Th1 cytokines (e.g., IFN-) are important in the initiation of antitumor immune responses although, NKT cell-derived Th2 cytokines (e.g., IL-4 and IL-10) are involved in down-regulation of autoimmune and antitumor immune responses (2, 3, 4) .

Murine invariant NKT cells play a role in tumor immune surveillance through production of IFN- and perforin, and are important in IL-12-induced antitumor effects (5, 6, 7) . The marine sponge derived glycosphingolipid -GalCer specifically activates invariant NKT cells (8) and induces strong, IFN- dependent, antitumor immune responses in mice (2 , 8 , 9) . Significant reductions in the size of the V24⁺ Vß11⁺ NKT cell population have been observed in cancer (10 , 11) . Because effects of -GalCer depend on the presence of invariant NKT cells (12) , -GalCer-induced immune responses can be expected to be compromised in patients with a reduced size of the V24⁺ Vß11⁺ NKT cell pool. Indeed, in a clinical Phase I trial of -GalCer in patients with solid tumors, we found immune activation only in patients with normal pretreatment V24⁺ Vß11⁺ NKT cell numbers (13) . Therefore, treatments based on reconstitution of invariant NKT cells potentially induce stronger antitumor immune responses compared with treatments based on in vivo -GalCer-induced NKT cell modulation. Importantly, murine studies have shown beneficial effects of adoptive transfer of invariant NKT cells in the inhibition of tumor metastasis (14) .

DCs are capable of inducing both Th1 and Th2 type immune responses in naive T cells (15 , 16) . Because V24⁺ Vß11⁺ NKT cells acquire a memory phenotype before birth, their cytokine profile can be expected to be more difficult to polarize compared with naive T cells (17) . Indeed, it was reported recently that adult V24⁺ Vß11⁺ NKT cells showed poor plasticity for Th1 or Th2 polarization (18) . However, the protocol used for Th1 polarization by these investigators could have been counterproductive, because it consisted of IL-7, known to induce Th2 cytokine production in murine NKT cells (19) , and moDC that, because of their 2-day maturation, produce only small amounts of the Th1 inducing cytokine IL-12 and have actually been reported to induce Th2 polarization in naive T cells (15 , 20) . Here, using IL-12-producing -GalCer-loaded type 1 moDC and IL-15, we report the simultaneous strong expansion and type 1 polarization of invariant V24⁺ Vß11⁺ NKT cells of healthy volunteers and cancer patients. These findings are important for the additional development of adoptive transfer strategies of V24⁺ Vß11⁺ NKT cells in cancer patients.

	MATERIALS AND METHODS

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Donors.
Expansion and polarization of V24⁺ Vß11⁺ NKT cells, defined by coexpression of the TCR V24 and Vß11 chain, was analyzed in 7 healthy adult volunteers and 9 cancer patients [age range: 49–68 years; tumor types: ovarian cancer, non-small cell lung cancer, metastatic colon cancer, laryngeal cancer, metastatic melanoma, metastatic breast cancer (n = 2), and metastatic renal cell cancer (n = 2)].

moDC.
Immature moDC, prepared from PBMCs as described previously (20) , were cultured for 4 or 48 h with LPS (100 ng/ml; Sigma-Aldrich, Zwijndrecht, the Netherlands) in the presence of 100 ng/ml -GalCer [KRN7000; (2S, 3S, 4R)-1-O-(-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1, 3, 4-octadecanetriol; Pharmaceutical Research Laboratory, Kirin Brewery], and rhIFN- (1000 units/ml; R&D Systems, Minneapolis, MN) or 1 x 10^-7 M PGE₂ (Sigma-Aldrich) as indicated. Mature -GalCer-loaded moDC were washed and used for flow cytometry or for cultures. Production of IL-12p70 was analyzed by ELISA after a 48-h stimulation of 4 x 10⁴ moDC with 4 x 10⁴ CD40L-transfected J558 cells (gift of Dr. Peter Lane, University of Birmingham, Birmingham, United Kingdom).

Expansion and Culture of V24⁺ Vß11⁺ NKT Cells.
For evaluation of V24⁺ Vß11⁺ NKT cell expansion, total PBMCs (3 x 10⁵) were cocultured with autologous -GalCer-loaded mature moDCs (3 x 10⁴) for 7 days in RPMI 1640 (BioWhittaker, Verviers, Belgium) supplemented with 8% human pooled serum (CLB Sanquin Blood Supply Foundation, Amsterdam, the Netherlands), 0.01 mM 2-ME, 1.6 mM L-glutamine, 25 mM HEPES, and 50 units/ml penicillin-streptomycin in the presence or absence of 1 x 10^-7 M dexamethasone (Sigma-Aldrich). In other experiments V24⁺ Vß11⁺ NKT cells were first enriched from PBMCs by magnetic isolation of V24⁺ T cells (autoMACS; Miltenyi Biotec, Bergisch-Gladbach, Germany) and subsequently expanded using -GalCer-loaded mature moDCs in the presence or absence of 10 ng/ml rhIL-7 (R&D Systems), 10 ng/ml rhIL-15 (R&D Systems), and 1 x 10^-7 M dexamethasone as indicated.

Characterization of Expanded V24⁺ Vß11⁺ NKT Cells.
For intracellular cytokine detection, expanded V24⁺ Vß11⁺ NKT cells were washed and stimulated with HeLa-CD1d in the presence of 100 ng/ml -GalCer and 3 µM monensin (Sigma-Aldrich) for 4 h. Intracellular stainings were performed as described previously (21) . For detection of cytokine secretion, expanded V24⁺ Vß11⁺ NKT cells were washed, coated with Ab-Ab conjugates directed against CD45 and IFN- or IL-4 (cytokine catch reagents; Miltenyi Biotec), and stimulated with HeLa-CD1d in the presence of 100 ng/ml -GalCer for 4 h. After washing, cells were incubated with PE-labeled anti-IL4 or anti-IFN- Ab (cytokine detection Ab; Miltenyi Biotec), anti-V24, and anti-Vß11 mAb, and analyzed by flow cytometry. For analysis of the kinetics of cytokine secretion upon restimulation, expanded V24⁺ Vß11⁺ NKT cells were washed, coated with IFN- or IL-4 catch reagents, and allowed to secrete cytokines for 45 min. Stainings were then performed as described above. ELISA was used to analyze the cytokine concentration in supernatants of 24 h cocultures of 1 x 10⁵ HeLa-CD1d cells and 2 x 10⁵ V24⁺ Vß11⁺ NKT cells in the presence of 100 ng/ml -GalCer. The following kits were used: IL-5 (PharMingen), IL-4, IL-6, IL-10, IL-13, IFN-, TNF- (CLB), TGF-ß1 (R&D Systems), and GM-CSF (Biosource, Camarillo, CA).

Flow Cytometry.
The following reagents were used: FITC-labeled IgG1, PE-labeled IgG_2a (BD, San Jose, CA); FITC and PE-labeled antihuman V24 and PE- and biotin-labeled antihuman Vß11 (Immunotech, Marseille, France); streptavidin-RPE-Cy5 (DAKO, Glostrup, Denmark); PE-labeled anti-IL-4 and anti-IFN- (PharMingen, San Diego, CA); PE-labeled CD40 and CD83 (Immunotech); PE-labeled CD80 (BD); and PE-labeled CD86 (PharMingen). CD1d expression was assessed using the antihuman CD1d27 mAb (22) , followed by a FITC-labeled antimurine-IgG1 mAb. The isotype control mouse IgG1 was obtained from Organon Technika-Cappel (Malvern, PA). Flow cytometry was performed on a FACStar plus (BD).

Cell-mediated Cytotoxicity.
Cytotoxicity was assessed using a standard 4-h ⁵¹Cr release assay at the indicated E:T ratios. V24⁺ Vß11⁺ NKT cells expanded using either -GalCer-loaded moDC1 and IL-15 or -GalCer-loaded moDC2, IL-7, and dexamethasone were used as effector cells 5 days after restimulation. U937 histiocytic lymphoma and Jurkat J32 T cell leukemia cells were used as target cells.

Statistical Analysis.
Statistical analyses were performed using Student t tests. P < 0.05 was considered significant.

	RESULTS

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Characterization of Polarized moDCs.
MoDCs were matured for 4 h using LPS (100 ng/ml) and IFN- (1000 units/ml), or for 48 h using LPS (100 ng/ml) and PGE₂ (1 x 10^-7 M) to generate type 1 and type 2 polarized moDCs, respectively (15 , 16) . Mature moDCs were characterized with respect to IL-12p70 production, and the expression of CD1d, CD40, CD80, CD83, and CD86 (Fig. 1A) . Levels of CD1d were similar on LPS/IFN- and LPS/PGE₂ matured moDCs, whereas levels of the costimulatory molecules CD40, CD80, and CD86 were higher on LPS/PGE₂ matured moDCs. LPS/IFN- matured moDCs produced significantly higher amounts of IL-12p70 on CD40 ligation (>2000 pg/ml; n = 3) compared with LPS/PGE₂ matured moDCs (20 ± 16 pg/ml; n = 3; P < 0.0001; paired Student’s t test).

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Characterization of polarized moDC. Immature moDC were matured with LPS/IFN- for 4 h or with LPS/PGE2 for 48 h. Expression of CD1d, CD40, CD80, CD83, and CD86 was assessed using flow cytometry (closed histograms). Open histograms indicate isotype controls. Representative histograms of 1 donor (of 4) are shown (A). Fold V24+ Vß11+ NKT cell expansion over 7 days induced by -GalCer-loaded moDC matured for 4 h using LPS/IFN- (n = 4) or for 48 h using LPS/PGE2 (n = 6). and indicate fold expansion in the absence or presence of dexamethasone during cocultures. Mean are shown (B); bars, ±SD.

Because dexamethasone was reported to enhance anti-CD3-mediated proliferation of V24⁺ Vß11⁺ NKT cells (23) , we investigated whether V24⁺ Vß11⁺ NKT cell expansion induced by polarized moDCs could be enhanced by adding dexamethasone. Dexamethasone enhanced V24⁺ Vß11⁺ NKT cell expansion induced by LPS/PGE₂-matured moDCs in the majority of tested individuals, but it uniformly abrogated V24⁺ Vß11⁺ NKT cell expansion induced by LPS/IFN--matured moDCs (Fig. 1B , closed bars).

Expansion and Polarization of Human Adult V24⁺ Vß11⁺ NKT Cells.
We have shown previously that although IL-7 and IL-15 can both potentiate V24⁺ Vß11⁺ NKT cell expansion induced by -GalCer-loaded moDCs, they exert different effects on V24⁺ Vß11⁺ NKT cell characteristics. IL-15 enhances GrB expression in V24⁺ Vß11⁺ NKT cells, whereas IL-7 decreases GrB expression in V24⁺ Vß11⁺ NKT cells (21) and reverses NK1⁺ T cell-defective IL-4 production in the nonobese diabetic mouse (19) . To evaluate whether V24⁺ Vß11⁺ NKT cells could be simultaneously expanded and polarized, the following culture protocols were used: for type 1 polarization, V24⁺ T cells were isolated from PBMCs and cocultured with 4-h LPS/IFN- matured- and -GalCer-loaded moDCs (moDC1) in the presence of 10 ng/ml rhIL-15. For type 2 polarization, V24⁺ T cells were cocultured with 48 h LPS/PGE₂ matured- and -GalCer-loaded moDC (moDC2), in the presence of 10 ng/ml rhIL-7, and 1 x 10^-7 M of the Th2 promoting glucocorticoid dexamethasone (24) . During a 12-day culture (primary stimulation and 1 restimulation) both protocols resulted in strong expansion of V24⁺ Vß11⁺ NKT cells [moDC1/IL-15: 356 ± 292 (mean ± SD); moDC2/IL-7/dexamethasone: 284 ± 156; n = 7; P = 0.63, paired Student’s t test]. The purity of V24⁺ Vß11⁺ NKT cells (preculture: 31.6 ± 24.7%) increased to 75.5 ± 32.8% using the type 1 protocol (n = 7; P = 0.004) and to 90.5 ± 8.6% using the type 2 protocol (n = 7; P = 0.0003), indicating that both protocols induced the preferential expansion of V24⁺ Vß11⁺ NKT cells.

On day 5 after restimulation, the intracellular cytokine profile of expanded V24⁺ Vß11⁺ NKT cells was determined after a 4-h stimulation with HeLa-CD1d in the presence of 100 ng/ml -GalCer and 3 µM monensin. Type 1 polarized V24⁺ Vß11⁺ NKT cells had a significantly higher IFN-:IL-4 ratio compared with type 2 polarized V24⁺ Vß11⁺ NKT cells (Table 1 ; n = 5; P = 0.007, paired Student’s t test). As can be observed in Table 1 , the lower IFN-:IL-4 ratio in type 2 polarized V24⁺ Vß11⁺ NKT cells mainly resulted from the decreased expression of IFN-. The percentage of V24⁺ Vß11⁺ NKT cells coexpressing IFN- and IL-4 showed substantial interdonor variability (5–40%) in type 1 polarized V24⁺ Vß11⁺ NKT cells (data not shown), whereas in type 2 polarized V24⁺ Vß11⁺ NKT cells the proportion of IFN-:IL-4 double-positive V24⁺ Vß11⁺ NKT cells as well as the proportion of IFN- single positive was strongly reduced. In addition to intracellular cytokine accumulation, secretion of IL-4 and IFN- was analyzed before restimulation, and on days 1, 3, and 5 after restimulation. Samples were washed, coated with Ab-Ab conjugates directed against CD45 and IFN- or IL-4, cultured in medium for 45 min, and analyzed by flow cytometry. This analysis confirmed the polarized secretion pattern of expanded V24⁺ Vß11⁺ NKT cells (Fig. 2A) . One day after restimulation, the percentage of IL-4- and IFN--producing V24⁺ Vß11⁺ NKT cells reached a maximum. The production of several cytokines was analyzed after a 24-h coculture of 2 x 10⁵ polarized V24⁺ Vß11⁺ NKT cells and 1 x 10⁵ HeLa-CD1d cells in the presence of 100 ng/ml -GalCer (Table 2) . The supernatant of 1 x 10⁵ HeLa-CD1d cells was used as a negative control, and contained 18 pg/ml IL-4, 98 pg/ml IL-6, 7 pg/ml IFN-, 912 pg/ml TGF-ß1, 8 pg/ml GM-CSF, and no detectable levels of IL-5, IL-10, IL-13, and TNF-.

View this table: [in this window] [in a new window]   Table 1 Cytokine profile of V24+ Vß11+ NKT cells expanded using -GalCer-loaded moDC1 and IL-15 or -GalCer-loaded moDC2, IL-7, and dexamethasone Intracellular expression of IL-4 and IFN- was determined by flow cytometry after a 4 h stimulation with HeLa-CD1d in the presence of -GalCer and monensin. Analyses were performed 5 days after restimulation. Data of 5 donors (of 5) are shown.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Kinetics of cytokine secretion and repolarization of polarized V24+ Vß11+ NKT cells. At indicated time points after restimulation, expanded V24+ Vß11+ NKT cells were washed, and the percentage V24+ Vß11+ NKT cells secreting IFN- and IL-4 during an additional 45-min culture in plain medium was determined using CD45/IFN- and CD45/IL-4 Ab-Ab conjugates. Data of secretion of V24+ Vß11+ NKT cells expanded using -GalCer-loaded moDC1 and IL-15 (open symbols) and -GalCer-loaded moDC2, IL-7 and dexamethasone (closed symbols) of 2 donors (of 2) are shown (A). The percentage of V24+ Vß11+ NKT cells secreting IL-4 and IFN- during a 4-h stimulation with HeLa-CD1d in the presence of -GalCer was determined before () and after () reversal of polarizing conditions. Analyses were performed 5 days after restimulation. Data of 4 donors (of 4) are shown (B).

View this table: [in this window] [in a new window]   Table 2 Cytokine production by V24+ Vß11+ NKT cells expanded using -GalCer-loaded moDC1 and IL-15 or -GalCer-loaded moDC2, IL-7, and dexamethasone Five days after restimulation, purified polarized V24+ Vß11+ NKT cells (2 x 105) were washed and cocultured with HeLa-CD1d (1 x 105) in the presence of -GalCer for 24 h (total volume 500 µl). Supernatants were harvested and used for ELISA [values (in pg/ml) represent specific V24+ Vß11+ NKT cell production]. Data of 3 donors (of 3) are shown.

Reversibility of V24⁺ Vß11⁺ NKT Cell Polarization.

Cytotoxicity of Polarized V24⁺ Vß11⁺ NKT Cells.
The cytotoxic potential of polarized V24⁺ Vß11⁺ NKT cells was assessed using a 4-h ⁵¹Cr release assay. Jurkat J32 T-cell leukemia cells and U937 histiocytic lymphoma cells were selected as target cells because they were reported previously to be susceptible to V24⁺ Vß11⁺ NKT cell-induced cytotoxicity (25 , 26) . Type 2 polarized V24⁺ Vß11⁺ NKT cells induced no detectable cytotoxicity against U937. Type 1 polarized V24⁺ Vß11⁺ NKT cells induced low levels of cytotoxicity, regardless of whether U937 cells were pulsed with -GalCer or not (Fig. 3 , top). Similarly, J32 cells were not lysed by type 2 polarized V24⁺ Vß11⁺ NKT cells, but they were clearly lysed by type 1 polarized V24⁺ Vß11⁺ NKT cells (Fig. 3 , bottom). Cytotoxicity of type 1 and type 2 V24⁺ Vß11⁺ NKT cells was strongly enhanced when J32 target cells were pulsed with -GalCer. Again, type 1 polarized V24⁺ Vß11⁺ NKT cells induced more cytotoxicity.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Cytotoxicity of polarized V24+ Vß11+ NKT cells. Cytotoxicity of V24+ Vß11+ NKT cells expanded using -GalCer-loaded moDC1 and IL-15, and -GalCer-loaded moDC2, IL-7, and dexamethasone against U937 cells (top) and J32 Jurkat cells (bottom) was assessed 5 days after restimulation. Target cells were pulsed with -GalCer (open symbols) or vehicle control (closed symbols) Representative data of 1 experiment (of 3) are shown.

Expansion and Type 1 Polarization of V24⁺ Vß11⁺ NKT Cells of Cancer Patients.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Expansion and cytokine profile of V24+ Vß11+ NKT cells of cancer patients. V24+ Vß11+ NKT cells were expanded using -GalCer-loaded moDC1 and IL-15. V24+ Vß11+ NKT cell purity (expressed as percentage of V24+ T cells) was assessed before culture, and at days 12 and 19. and indicate patients showing immediate (n = 4) and delayed (n = 5) proliferative responses, respectively. Data represent mean (A); bars, ±SD. Five days after restimulation, the percentage of V24+ Vß11+ NKT cells secreting IL-4 and IFN- during a 4-h stimulation with HeLa-CD1d in the presence of -GalCer was determined using CD45/IL-4 and CD45/IFN- Ab-Ab conjugates. Data of 4 healthy controls, 4 cancer patients with a delayed proliferative response, and 4 cancer patients with a normal proliferative response are shown (B).

	DISCUSSION

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The combination of -GalCer-loaded moDC1 and IL-15 or -GalCer-loaded moDC2, IL-7 and dexamethasone resulted in strong and selective expansion of V24⁺ Vß11⁺ NKT cells. Polarized V24⁺ Vß11⁺ NKT cells expressed the activation/memory markers CD25, CD45RO, and CD95, but did not express NK cell markers CD16, CD56, and CD161 (data not shown). Antigen-specific stimulation was used to determine the cytokine profile of expanded V24⁺ Vß11⁺ NKT cells. Using three techniques, we demonstrated that V24⁺ Vß11⁺ NKT cells expanded using -GalCer-loaded moDC1 and IL-15 had higher IFN-:IL-4 ratios, consistent with a Th1 cytokine profile, compared with V24⁺ Vß11⁺ NKT cells expanded using -GalCer-loaded moDC2, IL-7, and dexamethasone. Differences in the capacity of type 1 and type 2 polarized V24⁺ Vß11⁺ NKT cells to produce IFN-, TNF-, and GM-CSF were most striking. The high levels of IFN- produced by type 1 polarized V24⁺ Vß11⁺ NKT cells indicates that this protocol effectively generated Th1-type V24⁺ Vß11⁺ NKT cells, which have been shown to be important for -GalCer induced antitumor immune responses in mice by inducing cross-talk among invariant NKT cells, NK cells, and CTL (2 , 27 , 28) . The large amounts of TNF- and GM-CSF produced by type 1 polarized V24⁺ Vß11⁺ NKT cells strengthen the idea that these cells play an important role in the differentiation and maturation of myeloid DCs (29) . Still, total amounts of IL-4, IL-5, IL-6, IL-10, IL-13, and TGF-ß1 produced by both types of V24⁺ Vß11⁺ NKT cells were in the same range, and production of IL-6, IL-10, and IL-13 was somewhat higher in type 1 polarized V24⁺ Vß11⁺ NKT cells. It should be stressed that although IL-4 and IL-10 are well known for their roles in Th2-type immune responses, IL-4 is also involved in the generation of Th1-associated CTL-mediated tumor immunity (30) and, similarly, IL-10 promotes the maintenance of antitumor CTL effector function in situ (31) . Therefore, the production of these cytokines by type 1 polarized V24⁺ Vß11⁺ NKT cells does not need to impair, but could actually enhance antitumor effects mediated by type 1 polarized V24⁺ Vß11⁺ NKT cells. Of note, the type 1 polarized V24⁺ Vß11⁺ NKT cells described herein produce at least 2-fold higher amounts of IFN- and 5-fold lower amounts of IL-4 compared with those reported by others (18) , and consequently have a more pronounced Th1 profile.

Nicol et al. (26) demonstrated strong cytotoxicity of V24⁺ Vß11⁺ NKT cells against U937 cells, regardless of whether these were pulsed with -GalCer or not. In contrast, Metelitsa et al. (25) showed strong cytotoxicity of V24⁺ Vß11⁺ NKT cells only when target cells expressed CD1d (U937 and J32) and were pulsed with -GalCer. We found strong, -GalCer-dependent, cytotoxicity of V24⁺ Vß11⁺ NKT cells against J32 cells, but not against U937 cells. Differences in CD1d expression between our U937 cells (negative; data not shown) and those used by Metelitsa et al. (Ref. 25 ; positive) are likely to be responsible for this discrepancy. Importantly, type 1 polarized V24⁺ Vß11⁺ NKT cells induced more cytotoxicity than type 2 polarized V24⁺ Vß11⁺ NKT cells. This difference could be observed regardless of whether target cells were pulsed with -GalCer or not. As expected from our previous studies (21) , and in line with current results, GrB expression was higher in type 1 polarized V24⁺ Vß11⁺ NKT cells (data not shown).

Committed Th1 and Th2 cells develop from a common naive T-cell pool (32) . Stabilization of the cytokine profile of differentiated Th subsets is obtained after several rounds of cell division under Th1 or Th2 polarizing conditions (33) . V24⁺ Vß11⁺ NKT cells of prostate cancer patients were found to have a Th2-like cytokine profile, producing normal amounts of IL-4, but little IFN- (11) . Our demonstration that expanded and polarized V24⁺ Vß11⁺ NKT cells, which had undergone at least eight cell divisions under polarizing conditions, could be repolarized by reversing culture conditions, illustrates the plasticity of the cytokine profile of the V24⁺ Vß11⁺ NKT cell population, and suggests the potential use of expanded and type 1 (re)polarized autologous V24⁺ Vß11⁺ NKT cells for adoptive transfer in cancer patients.

-GalCer-loaded moDC1 and IL-15 also induced expansion and type 1 polarization of V24⁺ Vß11⁺ NKT cells of advanced cancer patients. Expansion was comparable with controls in 4 of 9 patients. In 5 of 9, V24⁺ Vß11⁺ NKT cells were activated but proliferated poorly. Of note, this defective proliferative response was transient. Such a transient defect in proliferation could be because of TCR signaling defects, e.g., as a result of in vivo down-regulation of the TCR chain (34) . Although an analysis of the cytokine profile of type 1 polarized V24⁺ Vß11⁺ NKT cells showed a Th1 cytokine profile in 6 of 8 patients tested, IFN- production remained low in 2 patients. This suggests that the type 1 polarization of V24⁺ Vß11⁺ NKT cells described here can be used in the majority of, but not in all, cancer patients.

In conclusion, we show the simultaneous expansion and polarization of adult human V24⁺ Vß11⁺ NKT cells. The combination of the reduced size of the V24⁺ Vß11⁺ NKT cell population in cancer patients, and the reported antitumor effects of adoptive transfer of type 1 polarized invariant NKT cells in mice, suggest that the type 1 polarization of human adult V24⁺ Vß11⁺ NKT cells described here could be of clinical benefit in cancer patients.

	ACKNOWLEDGMENTS

 
We thank Dr. Leonid S. Metelitsa (Children’s Hospital Los Angeles and Keck School of Medicine, University of Southern California, Los Angeles, CA) for providing the Jurkat J32 cell line and Dr. Mitchell Kronenberg (LIAI, San Diego, CA) for providing the CD1d-transfected HeLa cell line.

	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 Supported by a Spinoza Grant and grant nr 920-03-142 from the Netherlands Organization for Scientific Research (NWO).

² To whom requests for reprints should be addressed, at Department of Pathology, Vrije Universiteit Medisch Centrum, De Boelelaan 1117, 1081 HV, Amsterdam, the Netherlands. Phone: 31-20-4444031; Fax: 31-20-4442964; E-mail: rj.scheper{at}vumc.nl

³ The abbreviations used are: NKT, natural killer T; -GalCer, -galactosylceramide; DC, dendritic cell; moDC, monocyte-derived DC; rh, recombinant human; GrB, granzyme B; PBMC, peripheral blood mononuclear cell; NK, natural killer; TCR, T-cell receptor; Th, T helper; IL, interleukin; LPS, lipopolysaccharide; PGE₂, prostaglandin E₂; HeLa-CD1d, CD1d-transfected HeLa cell; PE, phycoerythrin; Ab, antibody; mAb, monoclonal antibody; TNF, tumor necrosis factor; GM-CSF, granulocyte macrophage colony-stimulating factor; BD, Becton Dickinson.

Received 7/31/02. Accepted 5/14/03.

	REFERENCES

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