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 Vα24+ Vβ11+ NKT cell population in cancer patients is decreased both in size and in its capacity to secrete IFN-γ, therapeutic strategies based on reconstitution of type 1 polarized Vα24+ Vβ11+ NKT cells merit additional investigation. Here, we report the simultaneous strong expansion and type 1 polarization of human invariant Vα24+ Vβ11+ NKT cells using α-galactosylceramide-loaded type 1 dendritic cells and interleukin 15. Type 1 polarized Vα24+ Vβ11+ NKT cells produced high levels of IFN-γ, tumor necrosis factor α, and granulocyte macrophage colony-stimulating factor, and induced strong cytotoxicity in Jurkat cells in an α-galactosylceramide-dependent manner. Importantly, the cytokine profile of Vα24+ Vβ11+ 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 Vα24+ Vβ11+ NKT cell population.

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 Vα24 chain preferentially paired with a Vβ11 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-γ) 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-γ and perforin, and are important in IL-12-induced antitumor effects (5, 6, 7). The marine sponge derived glycosphingolipid α-GalCer specifically activates invariant NKT cells (8) and induces strong, IFN-γ dependent, antitumor immune responses in mice (2, 8, 9). Significant reductions in the size of the Vα24+ Vβ11+ NKT cell population have been observed in cancer (10, 11). Because effects of α-GalCer depend on the presence of invariant NKT cells (12), α-GalCer-induced immune responses can be expected to be compromised in patients with a reduced size of the Vα24+ Vβ11+ NKT cell pool. Indeed, in a clinical Phase I trial of α-GalCer in patients with solid tumors, we found immune activation only in patients with normal pretreatment Vα24+ Vβ11+ 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 α-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 Vα24+ Vβ11+ 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 Vα24+ Vβ11+ 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 α-GalCer-loaded type 1 moDC and IL-15, we report the simultaneous strong expansion and type 1 polarization of invariant Vα24+ Vβ11+ NKT cells of healthy volunteers and cancer patients. These findings are important for the additional development of adoptive transfer strategies of Vα24+ Vβ11+ NKT cells in cancer patients.

Donors.

Expansion and polarization of Vα24+ Vβ11+ NKT cells, defined by coexpression of the TCR Vα24 and Vβ11 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 α-GalCer [KRN7000; (2S, 3S, 4R)-1-O-(α-d-galactopyranosyl)-2-(N-hexacosanoylamino)-1, 3, 4-octadecanetriol; Pharmaceutical Research Laboratory, Kirin Brewery], and rhIFN-γ (1000 units/ml; R&D Systems, Minneapolis, MN) or 1 × 10−7m PGE2 (Sigma-Aldrich) as indicated. Mature α-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 × 104 moDC with 4 × 104 CD40L-transfected J558 cells (gift of Dr. Peter Lane, University of Birmingham, Birmingham, United Kingdom).

Expansion and Culture of Vα24+ Vβ11+ NKT Cells.

For evaluation of Vα24+ Vβ11+ NKT cell expansion, total PBMCs (3 × 105) were cocultured with autologous α-GalCer-loaded mature moDCs (3 × 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 mml-glutamine, 25 mm HEPES, and 50 units/ml penicillin-streptomycin in the presence or absence of 1 × 10−7m dexamethasone (Sigma-Aldrich). In other experiments Vα24+ Vβ11+ NKT cells were first enriched from PBMCs by magnetic isolation of Vα24+ T cells (autoMACS; Miltenyi Biotec, Bergisch-Gladbach, Germany) and subsequently expanded using α-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 × 10−7m dexamethasone as indicated.

Characterization of Expanded Vα24+ Vβ11+ NKT Cells.

For intracellular cytokine detection, expanded Vα24+ Vβ11+ NKT cells were washed and stimulated with HeLa-CD1d in the presence of 100 ng/ml α-GalCer and 3 μm monensin (Sigma-Aldrich) for 4 h. Intracellular stainings were performed as described previously (21). For detection of cytokine secretion, expanded Vα24+ Vβ11+ NKT cells were washed, coated with Ab-Ab conjugates directed against CD45 and IFN-γ or IL-4 (cytokine catch reagents; Miltenyi Biotec), and stimulated with HeLa-CD1d in the presence of 100 ng/ml α-GalCer for 4 h. After washing, cells were incubated with PE-labeled anti-IL4 or anti-IFN-γ Ab (cytokine detection Ab; Miltenyi Biotec), anti-Vα24, and anti-Vβ11 mAb, and analyzed by flow cytometry. For analysis of the kinetics of cytokine secretion upon restimulation, expanded Vα24+ Vβ11+ NKT cells were washed, coated with IFN-γ 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 × 105 HeLa-CD1d cells and 2 × 105 Vα24+ Vβ11+ NKT cells in the presence of 100 ng/ml α-GalCer. The following kits were used: IL-5 (PharMingen), IL-4, IL-6, IL-10, IL-13, IFN-γ, TNF-α (CLB), TGF-β1 (R&D Systems), and GM-CSF (Biosource, Camarillo, CA).

Flow Cytometry.

The following reagents were used: FITC-labeled IgG1, PE-labeled IgG2a (BD, San Jose, CA); FITC and PE-labeled antihuman Vα24 and PE- and biotin-labeled antihuman Vβ11 (Immunotech, Marseille, France); streptavidin-RPE-Cy5 (DAKO, Glostrup, Denmark); PE-labeled anti-IL-4 and anti-IFN-γ (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. Vα24+ Vβ11+ NKT cells expanded using either α-GalCer-loaded moDC1 and IL-15 or α-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.

Characterization of Polarized moDCs.

MoDCs were matured for 4 h using LPS (100 ng/ml) and IFN-γ (1000 units/ml), or for 48 h using LPS (100 ng/ml) and PGE2 (1 × 10−7m) 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. 1 A). Levels of CD1d were similar on LPS/IFN-γ and LPS/PGE2 matured moDCs, whereas levels of the costimulatory molecules CD40, CD80, and CD86 were higher on LPS/PGE2 matured moDCs. LPS/IFN-γ 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).

The capacity of α-GalCer-loaded moDCs to induce Vα24+ Vβ11+ NKT cell expansion during a 7-day coculture of PBMCs and moDCs was studied. In all of the experiments, LPS/PGE2-matured moDCs induced stronger expansion of Vα24+ Vβ11+ NKT cells compared with LPS/IFN-γ matured moDCs (P = 0.11; unpaired Student’s t test; Fig. 1,B, open bars). The lower levels of Vα24+ Vβ11+ NKT cell expansion induced by LPS/IFN-γ-matured moDCs did not result from differences in the density of antigen-presenting molecules, because CD1d expression levels were comparable, but could be the result of the lower expression of CD40, CD80, and CD86 on LPS/IFN-γ-matured moDCs (Fig. 1 A).

Because dexamethasone was reported to enhance anti-CD3-mediated proliferation of Vα24+ Vβ11+ NKT cells (23), we investigated whether Vα24+ Vβ11+ NKT cell expansion induced by polarized moDCs could be enhanced by adding dexamethasone. Dexamethasone enhanced Vα24+ Vβ11+ NKT cell expansion induced by LPS/PGE2-matured moDCs in the majority of tested individuals, but it uniformly abrogated Vα24+ Vβ11+ NKT cell expansion induced by LPS/IFN-γ-matured moDCs (Fig. 1 B, closed bars).

Expansion and Polarization of Human Adult Vα24+ Vβ11+ NKT Cells.

We have shown previously that although IL-7 and IL-15 can both potentiate Vα24+ Vβ11+ NKT cell expansion induced by α-GalCer-loaded moDCs, they exert different effects on Vα24+ Vβ11+ NKT cell characteristics. IL-15 enhances GrB expression in Vα24+ Vβ11+ NKT cells, whereas IL-7 decreases GrB expression in Vα24+ Vβ11+ NKT cells (21) and reverses NK1+ T cell-defective IL-4 production in the nonobese diabetic mouse (19). To evaluate whether Vα24+ Vβ11+ NKT cells could be simultaneously expanded and polarized, the following culture protocols were used: for type 1 polarization, Vα24+ T cells were isolated from PBMCs and cocultured with 4-h LPS/IFN-γ matured- and α-GalCer-loaded moDCs (moDC1) in the presence of 10 ng/ml rhIL-15. For type 2 polarization, Vα24+ T cells were cocultured with 48 h LPS/PGE2 matured- and α-GalCer-loaded moDC (moDC2), in the presence of 10 ng/ml rhIL-7, and 1 × 10−7m of the Th2 promoting glucocorticoid dexamethasone (24). During a 12-day culture (primary stimulation and 1 restimulation) both protocols resulted in strong expansion of Vα24+ Vβ11+ 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 Vα24+ Vβ11+ 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 Vα24+ Vβ11+ NKT cells.

On day 5 after restimulation, the intracellular cytokine profile of expanded Vα24+ Vβ11+ NKT cells was determined after a 4-h stimulation with HeLa-CD1d in the presence of 100 ng/ml α-GalCer and 3 μm monensin. Type 1 polarized Vα24+ Vβ11+ NKT cells had a significantly higher IFN-γ:IL-4 ratio compared with type 2 polarized Vα24+ Vβ11+ NKT cells (Table 1; n = 5; P = 0.007, paired Student’s t test). As can be observed in Table 1, the lower IFN-γ:IL-4 ratio in type 2 polarized Vα24+ Vβ11+ NKT cells mainly resulted from the decreased expression of IFN-γ. The percentage of Vα24+ Vβ11+ NKT cells coexpressing IFN-γ and IL-4 showed substantial interdonor variability (5–40%) in type 1 polarized Vα24+ Vβ11+ NKT cells (data not shown), whereas in type 2 polarized Vα24+ Vβ11+ NKT cells the proportion of IFN-γ:IL-4 double-positive Vα24+ Vβ11+ NKT cells as well as the proportion of IFN-γ single positive was strongly reduced. In addition to intracellular cytokine accumulation, secretion of IL-4 and IFN-γ 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-γ or IL-4, cultured in medium for 45 min, and analyzed by flow cytometry. This analysis confirmed the polarized secretion pattern of expanded Vα24+ Vβ11+ NKT cells (Fig. 2,A). One day after restimulation, the percentage of IL-4- and IFN-γ-producing Vα24+ Vβ11+ NKT cells reached a maximum. The production of several cytokines was analyzed after a 24-h coculture of 2 × 105 polarized Vα24+ Vβ11+ NKT cells and 1 × 105 HeLa-CD1d cells in the presence of 100 ng/ml α-GalCer (Table 2). The supernatant of 1 × 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-γ, 912 pg/ml TGF-β1, 8 pg/ml GM-CSF, and no detectable levels of IL-5, IL-10, IL-13, and TNF-α.

Reversibility of Vα24+ Vβ11+ NKT Cell Polarization.

Vα24+ Vβ11+ NKT cells of prostate cancer patients were found recently to have a Th2-like cytokine profile, producing normal amounts of IL-4, but only little IFN-γ (11). To study whether the cytokine profile of polarized Vα24+ Vβ11+ NKT cells could be reversed, type 1 polarized Vα24+ Vβ11+ NKT cells were restimulated with moDC2/IL-7/dexamethasone, whereas type 2 polarized Vα24+ Vβ11+ NKT cells were restimulated with moDC1/IL-15. Five days after restimulation, IL-4 and IFN-γ secretion was assessed after a 4-h stimulation with HeLa-CD1d in the presence of α-GalCer. Fig. 2,B indicates that the cytokine profile of both types of Vα24+ Vβ11+ NKT cells could be altered by reversing culture conditions. Although Vα24+ Vβ11+ NKT cells restimulated with moDC2/IL-7/dexamethasone showed a strong decrease in IFN-γ secretion, it should be noted that IL-4 secretion was also reduced. Importantly, type 2 polarized Vα24+ Vβ11+ NKT cells that were restimulated with moDC1/IL-15 readily acquired a type 1 cytokine profile, characterized by increased secretion of IFN-γ (Fig. 2 B). Clearly, the degree of polarization of Vα24+ Vβ11+ NKT cells was mainly determined by the level of IFN-γ expression. The alterations in the proportion of IFN-γ-positive Vα24+ Vβ11+ NKT cells, which were induced by the reversal of culture conditions, were observed both in the IFN-γ/IL-4 double-positive Vα24+ Vβ11+ NKT cell population as well as in the IFN-γ single-positive Vα24+ Vβ11+ NKT cell population. These alterations in the proportion of Vα24+ Vβ11+ NKT cells that expressed IFN-γ were accompanied by alterations in the mean level of IFN-γ expression (as determined by mean fluorescence intensity; data not shown).

Cytotoxicity of Polarized Vα24+ Vβ11+ NKT Cells.

The cytotoxic potential of polarized Vα24+ Vβ11+ 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 Vα24+ Vβ11+ NKT cell-induced cytotoxicity (25, 26). Type 2 polarized Vα24+ Vβ11+ NKT cells induced no detectable cytotoxicity against U937. Type 1 polarized Vα24+ Vβ11+ NKT cells induced low levels of cytotoxicity, regardless of whether U937 cells were pulsed with α-GalCer or not (Fig. 3, top). Similarly, J32 cells were not lysed by type 2 polarized Vα24+ Vβ11+ NKT cells, but they were clearly lysed by type 1 polarized Vα24+ Vβ11+ NKT cells (Fig. 3, bottom). Cytotoxicity of type 1 and type 2 Vα24+ Vβ11+ NKT cells was strongly enhanced when J32 target cells were pulsed with α-GalCer. Again, type 1 polarized Vα24+ Vβ11+ NKT cells induced more cytotoxicity.

Expansion and Type 1 Polarization of Vα24+ Vβ11+ NKT Cells of Cancer Patients.

We evaluated whether Vα24+ Vβ11+ NKT cells of cancer patients could be expanded and polarized using α-GalCer-loaded moDC1 and IL-15. Of 9 advanced cancer patients tested, 4 showed Vα24+ Vβ11+ NKT cell expansions that were comparable with healthy controls. In the other 5 patients, Vα24+ Vβ11+ NKT cell expansion during the first 12 days of culture (primary stimulation and 1 restimulation) was significantly lower compared with healthy controls [2.9 ± 2.6-fold (mean ± SD) versus 356 ± 292-fold in healthy controls; P = 0.02, unpaired Student’s t test]. Importantly, although the initial proliferation of Vα24+ Vβ11+ NKT cells in these patients was poor, 98.6 ± 2.5% of the resulting Vα24+ Vβ11+ NKT cells expressed the activation marker CD25 (data not shown), and these cells expanded 294.4 ± 372.3-fold upon secondary restimulation. Fig. 4,A shows the increase in Vα24+ Vβ11+ NKT cell purity in cancer patients with an immediate or a delayed proliferative response to stimulation with α-GalCer-loaded moDC1 and IL-15. In 8 patients, the cytokine profile of Vα24+ Vβ11+ NKT cells was determined after a 4-h stimulation with HeLa-CD1d in the presence of α-GalCer. In 6 of 8 patients tested, the expanded Vα24+ Vβ11+ NKT cells showed a type 1 cytokine profile. IFN-γ was produced by only a low percentage of expanded Vα24+ Vβ11+ NKT cells in 2 patients. This defect in Vα24+ Vβ11+ NKT cell IFN-γ production was observed in 1 patient with a delayed proliferative response, and in 1 patient with a normal Vα24+ Vβ11+ NKT cell proliferative response (Fig. 4 B).

Here we describe the simultaneous expansion and polarization of human Vα24+ Vβ11+ NKT cells of healthy volunteers and cancer patients. Vα24+ Vβ11+ NKT cells expanded using α-GalCer-loaded moDC1 and IL-15 produced high levels of IFN-γ, TNF-α, and GM-CSF on triggering, and efficiently killed tumor cells in an α-GalCer-restricted manner.

The combination of α-GalCer-loaded moDC1 and IL-15 or α-GalCer-loaded moDC2, IL-7 and dexamethasone resulted in strong and selective expansion of Vα24+ Vβ11+ NKT cells. Polarized Vα24+ Vβ11+ 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 Vα24+ Vβ11+ NKT cells. Using three techniques, we demonstrated that Vα24+ Vβ11+ NKT cells expanded using α-GalCer-loaded moDC1 and IL-15 had higher IFN-γ:IL-4 ratios, consistent with a Th1 cytokine profile, compared with Vα24+ Vβ11+ NKT cells expanded using α-GalCer-loaded moDC2, IL-7, and dexamethasone. Differences in the capacity of type 1 and type 2 polarized Vα24+ Vβ11+ NKT cells to produce IFN-γ, TNF-α, and GM-CSF were most striking. The high levels of IFN-γ produced by type 1 polarized Vα24+ Vβ11+ NKT cells indicates that this protocol effectively generated Th1-type Vα24+ Vβ11+ NKT cells, which have been shown to be important for α-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-α and GM-CSF produced by type 1 polarized Vα24+ Vβ11+ 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-β1 produced by both types of Vα24+ Vβ11+ NKT cells were in the same range, and production of IL-6, IL-10, and IL-13 was somewhat higher in type 1 polarized Vα24+ Vβ11+ 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 Vα24+ Vβ11+ NKT cells does not need to impair, but could actually enhance antitumor effects mediated by type 1 polarized Vα24+ Vβ11+ NKT cells. Of note, the type 1 polarized Vα24+ Vβ11+ NKT cells described herein produce at least 2-fold higher amounts of IFN-γ 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 Vα24+ Vβ11+ NKT cells against U937 cells, regardless of whether these were pulsed with α-GalCer or not. In contrast, Metelitsa et al.(25) showed strong cytotoxicity of Vα24+ Vβ11+ NKT cells only when target cells expressed CD1d (U937 and J32) and were pulsed with α-GalCer. We found strong, α-GalCer-dependent, cytotoxicity of Vα24+ Vβ11+ 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 Vα24+ Vβ11+ NKT cells induced more cytotoxicity than type 2 polarized Vα24+ Vβ11+ NKT cells. This difference could be observed regardless of whether target cells were pulsed with α-GalCer or not. As expected from our previous studies (21), and in line with current results, GrB expression was higher in type 1 polarized Vα24+ Vβ11+ 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). Vα24+ Vβ11+ NKT cells of prostate cancer patients were found to have a Th2-like cytokine profile, producing normal amounts of IL-4, but little IFN-γ (11). Our demonstration that expanded and polarized Vα24+ Vβ11+ 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 Vα24+ Vβ11+ NKT cell population, and suggests the potential use of expanded and type 1 (re)polarized autologous Vα24+ Vβ11+ NKT cells for adoptive transfer in cancer patients.

α-GalCer-loaded moDC1 and IL-15 also induced expansion and type 1 polarization of Vα24+ Vβ11+ NKT cells of advanced cancer patients. Expansion was comparable with controls in 4 of 9 patients. In 5 of 9, Vα24+ Vβ11+ 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 ζ chain (34). Although an analysis of the cytokine profile of type 1 polarized Vα24+ Vβ11+ NKT cells showed a Th1 cytokine profile in 6 of 8 patients tested, IFN-γ production remained low in 2 patients. This suggests that the type 1 polarization of Vα24+ Vβ11+ 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 Vα24+ Vβ11+ NKT cells. The combination of the reduced size of the Vα24+ Vβ11+ 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 Vα24+ Vβ11+ NKT cells described here could be of clinical benefit in cancer patients.

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).

3

The abbreviations used are: NKT, natural killer T; α-GalCer, α-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.

Fig. 1.

Characterization of polarized moDC. Immature moDC were matured with LPS/IFN-γ 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 Vα24+ Vβ11+ NKT cell expansion over 7 days induced by α-GalCer-loaded moDC matured for 4 h using LPS/IFN-γ (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.

Fig. 1.

Characterization of polarized moDC. Immature moDC were matured with LPS/IFN-γ 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 Vα24+ Vβ11+ NKT cell expansion over 7 days induced by α-GalCer-loaded moDC matured for 4 h using LPS/IFN-γ (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.

Close modal
Fig. 2.

Kinetics of cytokine secretion and repolarization of polarized Vα24+ Vβ11+ NKT cells. At indicated time points after restimulation, expanded Vα24+ Vβ11+ NKT cells were washed, and the percentage Vα24+ Vβ11+ NKT cells secreting IFN-γ and IL-4 during an additional 45-min culture in plain medium was determined using CD45/IFN-γ and CD45/IL-4 Ab-Ab conjugates. Data of secretion of Vα24+ Vβ11+ NKT cells expanded using α-GalCer-loaded moDC1 and IL-15 (open symbols) and α-GalCer-loaded moDC2, IL-7 and dexamethasone (closed symbols) of 2 donors (of 2) are shown (A). The percentage of Vα24+ Vβ11+ NKT cells secreting IL-4 and IFN-γ during a 4-h stimulation with HeLa-CD1d in the presence of α-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).

Fig. 2.

Kinetics of cytokine secretion and repolarization of polarized Vα24+ Vβ11+ NKT cells. At indicated time points after restimulation, expanded Vα24+ Vβ11+ NKT cells were washed, and the percentage Vα24+ Vβ11+ NKT cells secreting IFN-γ and IL-4 during an additional 45-min culture in plain medium was determined using CD45/IFN-γ and CD45/IL-4 Ab-Ab conjugates. Data of secretion of Vα24+ Vβ11+ NKT cells expanded using α-GalCer-loaded moDC1 and IL-15 (open symbols) and α-GalCer-loaded moDC2, IL-7 and dexamethasone (closed symbols) of 2 donors (of 2) are shown (A). The percentage of Vα24+ Vβ11+ NKT cells secreting IL-4 and IFN-γ during a 4-h stimulation with HeLa-CD1d in the presence of α-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).

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Fig. 3.

Cytotoxicity of polarized Vα24+ Vβ11+ NKT cells. Cytotoxicity of Vα24+ Vβ11+ NKT cells expanded using α-GalCer-loaded moDC1 and IL-15, and α-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 α-GalCer (open symbols) or vehicle control (closed symbols) Representative data of 1 experiment (of 3) are shown.

Fig. 3.

Cytotoxicity of polarized Vα24+ Vβ11+ NKT cells. Cytotoxicity of Vα24+ Vβ11+ NKT cells expanded using α-GalCer-loaded moDC1 and IL-15, and α-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 α-GalCer (open symbols) or vehicle control (closed symbols) Representative data of 1 experiment (of 3) are shown.

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Fig. 4.

Expansion and cytokine profile of Vα24+ Vβ11+ NKT cells of cancer patients. Vα24+ Vβ11+ NKT cells were expanded using α-GalCer-loaded moDC1 and IL-15. Vα24+ Vβ11+ NKT cell purity (expressed as percentage of Vα24+ 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 Vα24+ Vβ11+ NKT cells secreting IL-4 and IFN-γ during a 4-h stimulation with HeLa-CD1d in the presence of α-GalCer was determined using CD45/IL-4 and CD45/IFN-γ 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).

Fig. 4.

Expansion and cytokine profile of Vα24+ Vβ11+ NKT cells of cancer patients. Vα24+ Vβ11+ NKT cells were expanded using α-GalCer-loaded moDC1 and IL-15. Vα24+ Vβ11+ NKT cell purity (expressed as percentage of Vα24+ 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 Vα24+ Vβ11+ NKT cells secreting IL-4 and IFN-γ during a 4-h stimulation with HeLa-CD1d in the presence of α-GalCer was determined using CD45/IL-4 and CD45/IFN-γ 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).

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Table 1

Cytokine profile of Vα24+ Vβ11+ NKT cells expanded using α-GalCer-loaded moDC1 and IL-15 or α-GalCer-loaded moDC2, IL-7, and dexamethasone

Intracellular expression of IL-4 and IFN-γ was determined by flow cytometry after a 4 h stimulation with HeLa-CD1d in the presence of α-GalCer and monensin. Analyses were performed 5 days after restimulation. Data of 5 donors (of 5) are shown.

DonorDC1 + IL-15DC2 + IL-7 + DEX
% IL-4 pos% IFN-γ posIFN-γ:IL-4 ratio% IL-4 pos% IFN-γ posIFN-γ:IL-4 ratio
AM 10.2 48.9 4.8 2.7 0.1 0.037 
HV 7.5 12.5 1.7 12.7 0.3 0.024 
B17.1 6.4 12 1.9 19 0.8 0.042 
B1.2 5.8 17 2.9 5.7 0.1 0.018 
B5.4 5.3 14.8 2.8 13.7 0.8 0.058 
Mean ± SD 7.0 ± 1.9 21.0 ± 15.7 2.8 ± 1.2 10.8 ± 6.5 0.4 ± 0.4 0.04 ± 0.02 
DonorDC1 + IL-15DC2 + IL-7 + DEX
% IL-4 pos% IFN-γ posIFN-γ:IL-4 ratio% IL-4 pos% IFN-γ posIFN-γ:IL-4 ratio
AM 10.2 48.9 4.8 2.7 0.1 0.037 
HV 7.5 12.5 1.7 12.7 0.3 0.024 
B17.1 6.4 12 1.9 19 0.8 0.042 
B1.2 5.8 17 2.9 5.7 0.1 0.018 
B5.4 5.3 14.8 2.8 13.7 0.8 0.058 
Mean ± SD 7.0 ± 1.9 21.0 ± 15.7 2.8 ± 1.2 10.8 ± 6.5 0.4 ± 0.4 0.04 ± 0.02 
Table 2

Cytokine production by Vα24+ Vβ11+ NKT cells expanded using α-GalCer-loaded moDC1 and IL-15 or α-GalCer-loaded moDC2, IL-7, and dexamethasone

Five days after restimulation, purified polarized Vα24+ Vβ11+ NKT cells (2 × 105) were washed and cocultured with HeLa-CD1d (1 × 105) in the presence of α-GalCer for 24 h (total volume 500 μl). Supernatants were harvested and used for ELISA [values (in pg/ml) represent specific Vα24+ Vβ11+ NKT cell production]. Data of 3 donors (of 3) are shown.

IL-4IL-5IL-6IL-10IL-13TGF-β1IFN-γTNF-αGM-CSF
AK          
 DC1/IL-15 368 33 6689 2495 524 443 68239 26317 6110 
 DC2/IL-7/DEX 531 46 5677 <1.2 187 558 304 241 116 
AM          
 DC1/IL-15 391 2450 5780 1274 168 780 29694 21597 25712 
 DC2/IL-7/DEX 108 5572 3406 <1.2 47 1419 484 205 9554 
HV          
 DC1/IL-15 631 2780 7518 1562 4861 <6.6 154762 3216 24302 
 DC2/IL-7/DEX 769 1298 4864 492 1355 <6.6 6568 116 2673 
IL-4IL-5IL-6IL-10IL-13TGF-β1IFN-γTNF-αGM-CSF
AK          
 DC1/IL-15 368 33 6689 2495 524 443 68239 26317 6110 
 DC2/IL-7/DEX 531 46 5677 <1.2 187 558 304 241 116 
AM          
 DC1/IL-15 391 2450 5780 1274 168 780 29694 21597 25712 
 DC2/IL-7/DEX 108 5572 3406 <1.2 47 1419 484 205 9554 
HV          
 DC1/IL-15 631 2780 7518 1562 4861 <6.6 154762 3216 24302 
 DC2/IL-7/DEX 769 1298 4864 492 1355 <6.6 6568 116 2673 

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.

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