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The Journal of Immunology, 2008, 181, 2446 -2454
Copyright © 2008 by The American Association of Immunologists, Inc.
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IFN-{gamma}-Producing Human Invariant NKT Cells Promote Tumor-Associated Antigen-Specific Cytotoxic T Cell Responses1

María Moreno*,{dagger}, Johan W. Molling2,*, Silvia von Mensdorff-Pouilly{dagger}, René H. M. Verheijen3,{dagger}, Erik Hooijberg*, Duco Kramer*, Anneke W. Reurs*, Alfons J. M. van den Eertwegh{ddagger}, B. Mary E. von Blomberg*, Rik J. Scheper* and Hetty J. Bontkes4,*

* Department of Pathology, {dagger} Department of Obstetrics and Gynecology, and {ddagger} Department of Medical Oncology of the VU University Medical Center, Cancer Center Amsterdam, Amsterdam, The Netherlands.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD1d-restricted invariant NKT (iNKT) cells can enhance immunity to cancer or prevent autoimmunity, depending on the cytokine profile secreted. Antitumor effects of the iNKT cell ligand {alpha}-galactosylceramide ({alpha}GC) and iNKT cell adoptive transfer have been demonstrated in various tumor models. Together with reduced numbers of iNKT cells in cancer patients, which have been linked to poor clinical outcome, these data suggest that cancer patients may benefit from therapy aiming at iNKT cell proliferation and activation. Herein we present results of investigations on the effects of human iNKT cells on Ag-specific CTL responses. iNKT cells were expanded using {alpha}GC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate type 1 cytokine production. Enhanced activation and increased IFN-{gamma} production was observed in iNKT cells, irrespective of CD4 expression, upon stimulation with IL-12-overexpressing dendritic cells. IL-12-stimulated iNKT cells strongly enhanced the MART-1 (melanoma Ag recognized by T cell 1)-specific CD8+ CTL response, which was dependent on iNKT cell-derived IFN-{gamma}. Furthermore, autologous IL-12-overexpressing dendritic cells, loaded with Ag as well as {alpha}GC, was superior in stimulating both iNKT cells and Ag-specific CTL. This study shows that IL-12-overexpressing allogeneic dendritic cells expand IFN-{gamma}-producing iNKT cells, which may be more effective against tumors in vivo. Furthermore, the efficacy of autologous Ag-loaded DC vaccines may well be enhanced by IL-12 overexpression and loading with {alpha}GC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Invariant NKT (iNKT)5 cells are T lymphocytes characterized by an invariant TCR{alpha}-chain gene rearrangement (V{alpha}24-J{alpha}18 paired with Vβ11 in humans) and coexpression of NK cell receptors (1). iNKT cells recognize glycolipid Ags in the context of the nonpolymorphic, MHC class I-like molecule CD1d via their highly restricted TCR repertoire. iNKT cells are considered to be primarily autoreactive-recognizing endogenous lipids (2), but they can also recognize bacterially derived glycolipids, suggestive for a role in antimicrobial defense (3). The synthetic glycolipid {alpha}-galactosylceramide ({alpha}GC) induces activation and proliferation of iNKT cells in vitro as well as in vivo. The main physiological function of iNKT cells remains to be elucidated, but based primarily on murine studies, modulation of innate and adaptive immune responses through activation or elimination of dendritic cells (DC) is thought to be the main function of iNKT cells (4, 5, 6, 7, 8, 9). Upon activation, iNKT cells rapidly secrete both type 1 (e.g., IFN-{gamma}, TNF-{alpha}) and type 2 (e.g., IL-4, IL-13) cytokines. Owing to this broad spectrum of cytokines, iNKT cells have the capacity to enhance host immunity to microorganisms and cancer, as well as to prevent autoimmunity. This has been experimentally demonstrated in various animal models and is also strongly suggested by low numbers of circulating iNKT cells in patients suffering from autoimmune diseases or cancer (10, 11, 12, 13, 14). We have recently demonstrated that a severe circulating iNKT cell deficiency predicts poor clinical outcome in head and neck squamous cell carcinoma patients (15). Additionally, increased iNKT cell infiltration at tumor sites is associated with prolonged survival in colon cancer and neuroblastoma patients (16, 17). These data suggest a critical contribution of iNKT cells to antitumor immune responses in humans. Subtypes of iNKT cells, based on CD4 expression, have been shown to express different cytokine profiles when analyzed directly ex vivo. Human CD4+ iNKT cells produce both type 1 and type 2 cytokines, whereas CD8+ and CD4–CD8– double-negative iNKT cells primarily produce type 1 cytokines (18, 19). Although iNKT cells possess the full lytic machinery, direct killing of tumor cells has predominantly been described for CD1d-positive leukemic cell lines (20, 21). Tumor cell lines of different origin were only susceptible to iNKT cell-mediated killing after CD1d transfection and pulsing with {alpha}GC (22). The antitumor effect of {alpha}GC in various tumor models has been shown to primarily depend on IL-12 (23, 24, 25). iNKT cells induce IL-12 production by DC through CD40 ligation and IFN-{gamma} production (26). IL-12 is a strong NK cell activator and is also considered to be a crucial third signal for induction of functional Ag-specific type 1 Th cell and CTL responses (27). In contrast to the vast number of studies in mice showing enhanced Ag-specific T cell activation by {alpha}GC and iNKT cells, the limited data on human iNKT cells demonstrate inhibition rather than enhancement of Ag-specific CTL responses in vitro. Isolated human CD4+ and CD8+ iNKT cells suppressed the expansion of Ag-specific CTL by type 2 cytokine production and lysis of APC and activated T cells, respectively (28, 29). Herein we expanded and stimulated human iNKT cells using {alpha}GC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate IFN-{gamma} production. These MUTZ-3 variants provided us with a standardized unlimited source of precursors to generate DC expressing CD1d and secreting IL-12. Effects of IL-12 on CD4– and CD4+ iNKT cell activation and cytokine production were analyzed, as was the effect of IL-12-stimulated iNKT cells on tumor-associated Ag (TAA)-specific CTL responses induced by autologous monocyte-derived dendritic cells (MoDC). In a more physiological and clinically relevant set-up, it was examined whether iNKT cells in total PBL can also enhance CTL expansion. To this end, PBL were cultured with {alpha}GC/MART-1 double-loaded, IL-12-overexpressing, autologous MoDC to simultaneously stimulate MART-1-specific CTL and iNKT cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Media, reagents, and cell lines

Recombinant human TNF-{alpha} (50 ng/ml), IL-7 (5 ng/ml), IL-15 (5 ng/ml), and IL-2 (10 or 50 IU/ml) were purchased from Strathmann Biotech. Recombinant human GM-CSF (Schering-Plough) was used at 100 ng/ml; IL-4 (R&D Systems) was used at 10 ng/ml; IFN-{gamma} (BioSource International) was used at 400 U/ml; blocking Abs against IL-4, IFN-{gamma}, and IL-10 (R&D Systems) were used at 4 µg/ml. IMDM (Cambrex) was supplemented with 10% FCS (Thermo Fisher Scientific) for culture of the melanoma cell lines Mel-JKO and Mel-AKR, the EBV-LCL JY and CD1d-transfected HeLa cells (a kind gift of Dr. M. Kronenberg, La Jolla Institute for Allergy & Immunology, San Diego, CA); with 8% human pooled serum (Sanquin) for culture of iNKT cells or Yssels supplement (30); and with 1% human AB serum (ICN Biomedicals) for CTL cultures. The CD34+ human acute myeloid leukemia cell line MUTZ-3 (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany) was cultured in MEM-{alpha} medium containing ribonucleosides and deoxyribonucleosides (Invitrogen) supplemented with 20% FCS (Thermo Fisher Scientific) and 10% 5637-conditioned medium (American Type Culture Collection). All media were supplemented with 100 IU/ml sodium penicillin (Yamanouchi Pharma), 100 µg/ml streptomycin sulfate (Radiumfarma-Fisiopharma), 2.0 mM L -glutamine (Invitrogen, Breda, Netherlands), and 0.01 mM 2-ME (Merck). FITC-, PE-PerCPCy5-, or APC-labeled isotype controls and mouse mAbs to CD1a, CD1d (clone CD1d42), CD40, CD80, CD86, iNKT TCR (6B11), CD8, CD4, CD3, CD69, CD161, CTLA4, CD25, IFN-{gamma}, IL-4, IL-10 (BD Pharmingen), CD83, V{alpha}24, Vβ11 (Immunotech), granzyme B (Sanquin), and CD56 (IQ Products) were used to determine the phenotype of iNKT cells and DCs. The CD1d-specific clone 51.1.3 was a kind gift from Dr. Mark Exley (Harvard Medical School, Boston, MA). Mean fluorescence index was calculated as mean fluorescence intensity marker/fluorescence intensity isotype.

Retroviral transduction

The human CD1d (Open Biosystems) and the IL-12elasti cassette containing the p35 and p40 subunits of IL-12 joined together by a flexible linker (InvivoGen) open reading frames were cloned into the Moloney murine leukemia virus-based retroviral vector LZRS (31). IL-12 was inserted into the multiple cloning site (mcs) of the bicistronic LZRS vector containing an internal ribosome entry site (IRES) followed by the truncated version of the nerve growth factor receptor ({Delta}NGFR) (LZRS-IL-12-IRES-{Delta}NGFR) (32). CD1d was cloned behind the IRES sequence (LZRS-mcs-IRES-CD1d). The constructs were transfected into the packaging cell line Phoenix A using Lipofectamine (Invitrogen), and retroviral supernatant was produced, followed by retroviral transduction of MUTZ-3 as described previously (33). Briefly, 5 x 105 MUTZ-3 cells were resuspended in retroviral supernatant supplemented with 10% 5367 conditioned medium and transferred to a fibronectin (RetroNectin; Takara Shuzo)-coated well of a non-tissue culture-treated 24-well plate (BD Biosciences). Plates were centrifuged, followed by a 5-h incubation at 37°C. The next day, retroviral transduction was repeated. NGFR-specific (Chromoprobe) and CD1d-specific (clone CD1d42, BD Biosciences) Abs were used to analyze transduction efficiency and isolate transduced cells by flow cytometry. IL-12-transduced cells were transduced with LZRS-mcs-IRES-CD1d after sorting of NGFR-positive cells to obtain double-transduced MUTZ-3 cells.

Expansion of iNKT cells and MART-126–35A27L peptide-specific CD8+ T cells from healthy donors

MoDC and DC derived from wild-type MUTZ-3 (M3) as well as CD1d (M3CD1d) and IL-12 and CD1d double-transduced (M312CD1d) cells were generated as described previously (34). Maturation was induced by 48 h culture in the presence of 30% (v/v) MCM and TNF-{alpha} as described (35). iNKT cells were enriched from 500 x 106 PBMC (isolated from buffy coats from healthy blood donors obtained after informed consent) by positive selection using the iNKT cell-specific Ab 6B11 (BD Pharmingen) and anti-mouse Ig-coated magnetic beads (Miltenyi Biotec) by MACS sorting. iNKT cells were expanded by weekly stimulation with irradiated {alpha}GC- (KRN7000, kindly provided by Dr. Shigeyuki Yamano, Kirin Brewery, Gunma, Japan) pulsed (100 µg/ml) mature M3CD1d-DC in the presence of IL-2 (50 U/ml), IL-15, and IL-7. If necessary, iNKT cells were further purified by MACS sorting (>90%). To enhance type 1 cytokine production, iNKT cells were stimulated for 5 days with {alpha}GC-pulsed mature M312CD1d-DC. To study proliferation of iNKT cell lines in response to various DC types, iNKT cell lines (>90% pure) were labeled with CFSE (1 µM; Molecular Probes) for 10 min at 37°C, washed twice in ice-cold PBS, and cultured in medium (supplemented with IL-2, IL-7, an IL-15) only or in the presence of {alpha}GC-pulsed MoDC or M3-, M3CD1d-, or M312CD1d-derived DC. Intensity of CFSE was measured or cell counts were performed after 1 wk culture. The relative CFSE intensity on 6B11-positive iNKT cells was calculated as follows: mean CFSE fluorescence intensity of iNKT cells cultured with DC/mean CFSE fluorescence intensity of iNKT cells cultured in medium.

CD8β-positive CTL precursors were isolated from buffy coats of HLA-A2.1-positive healthy donors using a CD8β-specific Ab (Beckman Coulter) by MACS sorting. Mature MoDC were pulsed with the HLA-A2.1-restricted epitope MART-126–35A27L or transfected with mRNA encoding a minigene of this epitope, ubi(MART)4 mRNA, or with ubi(MART)4–2A-IL12 mRNA, encoding both the MART minigene and IL-12. In vitro transcription and transfection methods were described in detail previously (35). After {alpha}GC and peptide pulsing or mRNA transfection, DC were washed and multiple bulk cultures containing 0.5–1 x 106 CD8β T cells or PBL and 0.5–1 x 105 {alpha}GC and peptide-pulsed or mRNA-transfected DC were set up. One thousand to 2000 (corrected for purity) autologous in vitro-expanded (iNKT) and IL-12-stimulated iNKT cells (iNKTIL-12) were added as indicated. Irradiated autologous CD8β T cell/iNKT cell/monocyte-depleted PBL (0.25–0.5 x 106) were added as helper cells to the CD8β cultures. The next day IL-7 was added. After 10 days, T cells were analyzed for specificity using PE- and/or APC-labeled HLA-A*0201 tetramers (Tm) presenting the MART-126–35A27L epitope (35). On day 10, the bulk cultures were restimulated with DCs and irradiated autologous CD8β T cell/iNKT cell/monocyte-depleted PBMC, fresh iNKT cells were added to the appropriate wells, and the next day IL-2 (10 U/ml) was added. Tm staining and functional analysis were performed after 7 days. To analyze the involvement of soluble factors, some experiments were done in transwells (0.4-µm pore size, Corning Life Sciences) or in the presence of cytokine-blocking Abs as indicated.

Expansion of iNKT cells and MART-126–35A27L peptide-specific CD8+ T cells from melanoma patients

Thirty to 40 ml of blood was drawn from four patients (two HLA-A2.1-positive and two HLA-A2.1-negative) with metastasized melanoma World Health Organization stage 0 or 1. Patients did not receive any immunosuppressive agents or chemotherapy in the 4 wk preceding blood sampling. iNKT cells were enriched from isolated PBMC and expanded and stimulated as described above. iNKT cells from HLA-A2 positive patients were added to cocultures of their autologous CD8β T cells and allogeneic MART peptide, {alpha}GC-pulsed, HLA-A2-matched MoDC. The iNKT cells derived from the HLA-A2-negative patients were added to cocultures of allogeneic HLA-A2-positive, healthy donor-derived CD8β T cells and MART peptide, {alpha}GC-pulsed MoDC. The medical ethics committee of the VU Medical Center (Amsterdam, The Netherlands) approved the study, and all patients gave informed consent.

Functional assays

Seven days after the second DC stimulation, the T cell bulk cultures were harvested and pooled per condition and incubated with target cells (JY, MART-126–35A27L-pulsed JY, Mel-JKO, or Mel-AKR). iNKT cells were harvested 5 days after stimulation with M3CD1d-DC or M312CD1d-DC and incubated with vehicle or {alpha}GC-pulsed target cells (Jurkat, Daudi, or HeLa-CD1d). Intracellular IFN-{gamma}/IL-4 staining was performed using the BD Cytofix/Cytoperm Plus kit (BD Biosciences) according to the manufacturer’s instructions. One hour after the start of the stimulation, GolgiPlug was added to each well (0.1% (v/v)). After 5 h, cells were washed and stained. CTL cultures were stained with APC-labeled tetramers and PE-labeled CD8 followed by intracellular staining with FITC-labeled anti-IFN-{gamma}. iNKT cells were stained with PE-labeled 6B11, PerCPCy5-labeled CD3, and APC-labeled CD4 followed by intracellular staining with FITC-labeled anti-IFN-{gamma} or anti-IL-4. Cytolytic activity of the iNKT cells to Daudi and Jurkat cells was determined using a standard chromium release assay as described (36).

Statistics

When n ≥ 5, parametric tests were used after confirmation of Gaussian distribution (normal distribution was confirmed in all cases); nonparametric tests were used when n < 5. Differences in iNKT cell characteristics after culture with or without IL-12 were analyzed with either the parametric two-sided paired Student’s t test or the nonparametric Wilcoxon rank sum test. Differences in MART-1-specific CTL frequencies were analyzed with either a two-sided Student’s t test or a two-sided Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
DC derived from CD1d-transduced MUTZ-3 support iNKT cell expansion

{alpha}GC-loaded autologous DC efficiently induce iNKT cell proliferation in vitro (37). Since iNKT cells are restricted by the nonpolymorphic CD1d molecule, allogeneic DC may be used to induce iNKT cell proliferation and activation, precluding the generation of autologous MoDC for each donor. Previously we have shown that the human acute myeloid leukemia cell line MUTZ-3 (M3) can be induced to differentiate into immature and mature DC upon cytokine stimulation. These DC display the full range of functional MHC-mediated Ag processing and presentation pathways and produce relatively low levels of IL-12 upon CD40 triggering (38). To further promote CD1d Ag presentation and type 1 cytokine production by iNKT cells, CD1d and IL-12 were introduced into M3 cells by retroviral transduction. CD1d expression was analyzed on wild-type M3, CD1d-transduced M3 (M3CD1d), IL-12/CD1d double-transduced M3 (M312CD1d), and monocytes before and after generation of mature DC using two Abs (Fig. 1, a and b). Expression levels were low to undetectable on wild-type M3 precursors and their derived mature DC. Monocytes and M3CD1d and M312CD1d precursors strongly expressed CD1d (particularly demonstrated with the 51.1.3 clone (the CD1d42 clone worked less well in our hands); Fig. 1b), which was reduced upon DC differentiation. Thus, both natural and ectopic CD1d expression was reduced upon DC differentiation. The 51.1.3 clone (more so than the CD1d42 antibody) has some cross-reactivity with CD1b, and it can therefore not be excluded that the remaining staining detected on mature DC is in fact CD1b. However, the {alpha}GC-dependent iNKT cell proliferation observed with {alpha}GC-pulsed mature DC (Fig. 1, c and d, and Table I) strongly suggests that the staining observed with 51.1 on mature DC is, at least in part, CD1d.


Figure 1
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  		FIGURE 1. Dendritic cells derived from CD1d-transduced MUTZ-3 support iNKT cell expansion. CD1d expression by MUTZ-3 (M3), CD1d-transduced M3 (M3CD1d), IL-12- and CD1d-transduced M3 (M312CD1d), and monocyte (Mo) precursors (prec) and their derived mature DC (DC) using the (a) CD1d42 and (b) 51.1.3 Abs. Gray histograms represent cells stained with the CD1d-specific Abs; open histograms (bold line) represent cells stained with the appropriate isotype control. Numbers in the upper right corner represent the mean fluorescence index (mean of three experiments). c, Example of CFSE dilution of an iNKT cell line after 7 days culture with the indicated {alpha}GC-pulsed mature DC. The vertical dotted line represents mean CFSE fluorescence of the medium control. d, Relative CFSE intensity (mean CFSE fluorescence intensity of iNKT cells cultured with DC/mean CFSE fluorescence intensity of iNKT cells cultured in medium) of iNKT cell lines after culture with the indicated {alpha}GC-pulsed DC (means ± SD of four independent experiments).

 

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  		Table I. DC derived from CD1d-transduced MUTZ-3 support iNKT cell expansion

 
Mature MoDC, M3-DC, M3CD1d-DC, and M312CD1d-DC expressed similar high levels of CD40, CD80, CD83, and CD86 (not shown and Refs. 38 , 39)). IL-12-transduced M3 precursors (M312CD1d) released 495 pg IL-12 (SD of 192) (2 x 105 cells/24 h) and M312CD1d-DC-produced 1600 pg IL-12 (SD of 586) (2 x 105 cells/24 h).

Next, we investigated whether ectopic CD1d and IL-12 expression had any effect on iNKT cell proliferation. Four iNKT cell lines were labeled with CFSE and cultured in medium supplemented with cytokines in the absence or presence of the different {alpha}GC-pulsed DC. CFSE dilution as a measure for proliferation was determined after 1 wk of culture (Fig. 1c). Proliferation rate was very low in response to M3DC, but high in response to M3CD1d-DC, M312CD1d-DC, and allogeneic MoDC, demonstrating that the increased ectopic CD1d expression in M3-DC lead to enhanced iNKT cell proliferation (Fig. 1, c and d). These results were confirmed with four other iNKT cell lines; 1.5 x 106 cells were stimulated with {alpha}GC-pulsed DC and counted after 1 wk (Table I). Proliferation induced by M3-DC was increased after CD1d transduction, and CD1d-transduced M3-DC were as effective as MoDC to induce iNKT cell proliferation. Based on these data, M3CD1d-DC and M312CD1d-DC were used to expand iNKT cells.

Stimulation with M312CD1d-DC induces iNKT cell activation and increased IFN-{gamma} production

Several investigators have demonstrated that CD4– and CD4+ iNKT cells represent different subsets with different cytokine secretion patterns when isolated directly ex vivo; CD4– iNKT cells produce predominantly type 1 cytokines, and CD4+ iNKT cells produce both type 1 and type 2 cytokines (18, 19). Herein, we investigated whether this also holds true after in vitro expansion using IL-12-producing DC. iNKT cells isolated from peripheral blood were expanded for 2–3 wk using {alpha}GC-pulsed M3CD1d-DC followed by a 5-day culture with {alpha}GC-pulsed M3CD1d-DC or M312CD1d-DC. The proportion of CD4+ iNKT cells was similar in cultures stimulated with or without IL-12 (67% (range 10–82%) and 68% (range 10–86%), respectively). Stimulation with M312CD1d-DC induced increased activation as measured by CD161, CD56, CD25, and intracellular CTLA4 and granzyme B expression compared with M3CD1d-DC (0.001 < p < 0.015 for all parameters, Fig. 2a). The increase was observed in both CD4– and CD4+ iNKT cells (data not shown). Rather than inducing a type 1-skewed cytokine secretion profile (i.e., increase in IFN-{gamma} and a decrease in IL-4 production), IL-12 stimulation resulted in increased numbers of IFN-{gamma}-producing iNKT cells (p = 0.0003), without significantly changing the number of IL-4-producing iNKT cells (Fig. 2b). The effect of IL-12 was the same on both CD4– and CD4+ iNKT cells (Fig. 2, c and d). The modulation of cytokine production by IL-12 resulted in a slight increase in the ratio of IFN-{gamma} and IL-4-producing iNKT cells, which was statistically significant for the whole population and for CD4+ iNKT cells and borderline significant for the CD4– iNKT cell population (p < 0.08) (Fig. 2e). Additionally, a very small but distinct population of IL-10-producing iNKT cells appeared after stimulation with M312CD1d-DC (Fig. 2f). Stimulation with M312CD1d-DC induced expression of the lymphoid homing receptor CD62L (known to be expressed on early effector CD8+ and central memory conventional T cells) on a subpopulation of both CD4+ and CD4– iNKT cells (Fig. 2g). The increased expression of granzyme B by M312CD1d-DC-stimulated iNKT cells (Fig. 2a) did not lead to enhanced killing of CD1d+ target cells: Jurkat (Fig. 2h), MOLT-4, and CD1d-transfected HeLa (not shown). Killing of CD1d– target cells (Daudi, HL60, HeLa) was always low (<5%) and could not be enhanced by IL-12 (not shown).


Figure 2
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  		FIGURE 2. Stimulation with M312CD1d-DC induces iNKT cell activation and increased IFN-{gamma} production. iNKT cells were expanded for 2–3 wk using M3CD1d-DC followed by 5 days culture with either M3CD1d-DC (open bars) or M312CD1d-DC (closed bars). Phenotype of the resulting iNKT cell lines was determined by FACS analysis after gating on CD3+6B11+ iNKT cells. All iNKT cell lines were >90% pure. a, Mean fluorescence index (means ± SD of six to eight individual experiments) of activation markers (CD161, CD56, CTLA4, granzyme B, and CD25). Percentage of IFN-{gamma}- and IL-4-producing cells (means ± SD of eight individual experiments) among (b) all iNKT cells and (c) CD4-negative and (d) CD4-positive iNKT cells. e, Ratio of IFN-{gamma}- and IL-4-producing iNKT cells (means ± SD of eight individual experiments). f, Stimulation with IL-12 induces a small increase in IL-10-producing iNKT cells (means ± SD of four individual experiments). g, Increase in number of iNKT cells expressing CD62L after stimulation with IL-12 (means ± SD of six individual experiments). p values are the result of a paired two-sided Student’s t test. h, No increase in {alpha}GC-dependent killing of CD1d-positive targets cells (Jurkat) by iNKT cells stimulated with IL-12-overexpressing DC.

 
IL-12-stimulated iNKT cells significantly enhance expansion of MART-1-specific CTL

To analyze the effects of iNKT cells on Ag-specific CTL responses, isolated CD8β+ T cells, depleted of iNKT cells, were cocultured with autologous MoDC. Autologous iNKT cell lines were generated using {alpha}GC-pulsed M3CD1d-DC (iNKT) and M312CD1d-DC (iNKTIL-12) as described above, and they were added to CD8β+ and MoDC cocultures. MoDC were pulsed with {alpha}GC to induce cytokine release by the added iNKT cells and/or MART-1 peptide to stimulate TAA-specific CTL. Specific CTL frequencies were measured using Tm staining after two in vitro stimulations as described previously (35). Addition of iNKT or iNKTIL-12 led to a significant increase in MART-1-specific CTL (p < 0.02 and p < 0.0005, respectively; Fig. 3a). The iNKT cell effect was {alpha}GC dependent; addition of iNKT cells had no effect when MoDC were pulsed with peptide alone without {alpha}GC. The MART-1-specific CTL were functionally active, as demonstrated by specific IFN-{gamma} production by Tm-positive CD8+ T cells in response to either peptide-pulsed targets or endogenously MART-1-expressing melanoma cells. However, addition of iNKTIL-12 did not increase the effector function of the induced MART-specific CTL (Fig. 3b). Peptide titration assays revealed no difference in functional avidity (not shown). To study the clinical relevance of this finding, iNKTIL-12 were generated from four melanoma patients as described above. Since the amount of blood that could be obtained was not sufficient to generate sufficient numbers of autologous MoDC, CD8β T cells from two HLA-A2+ patients were stimulated with allogeneic HLA-A2-matched MoDC in the absence or presence of iNKTIL-12 (Fig. 3, c and d). The two other melanoma patients were HLA-A2-negative and therefore their iNKTIL-12 cells were added to cocultures of CD8β and MoDC of HLA-A2+ healthy donors (Fig. 3, e and f). As was observed with the healthy donor-derived iNKT cells, addition of melanoma patient-derived iNKTIL-12 cells led to a significant increase in MART-specific CTL in three of the four donors tested (p < 0.03, Fig. 3, c, e, and f). In one of the HLA-A2-positive donors, a high proliferation of presumable alloresponsive CD8 T cells was observed, which resulted in overall relatively low percentages of MART-specific CTL. However, an increased, although not statistically significant, percentage of MART-specific CTL was observed when iNKTIL-12 cells were added (p = 0.11, Fig. 3d).


Figure 3
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  		FIGURE 3. IL-12-stimulated iNKT cells significantly enhance expansion of MART-1-specific CTL. a, iNKT cells and IL-12-stimulated iNKT cells (iNKTIL-12) were added (0.2%) to multiple bulk cocultures (n = 8) of isolated CD8β T cells and autologous MoDC pulsed with MART-1 peptide alone or with peptide and {alpha}GC. Percentage of Tm-positive CD8+ T cells is shown; horizontal lines represent the mean, p values are the result of a two-sided Student’s t test. b, Induced CTL were functional and produced IFN-{gamma} in response to HLA-A2.1-positive, MART-1 peptide-loaded JY cells, and the MART-1- and HLA-A2.1-positive melanoma cell line (Mel-AKR), MART-1-negative JY cells, and MART-1-positive, but HLA-A2.1-negative, Mel-JKO cells are not recognized. Percentage of IFN-{gamma}-positive Tm-positive T cells is shown. Results shown are from one representative donor out of four tested. c and d, Melanoma patient-derived, IL-12-stimulated iNKT cells (iNKTIL-12) were added (0.2%) to multiple bulk cocultures of autologous isolated CD8β T cells and allogeneic HLA-A2-matched MoDC pulsed with MART-1 peptide and {alpha}GC. The percentage of Tm-positive CD8+ T cells is shown; horizontal lines represent the median, p values are the result of a two-sided Mann-Whitney U test. e and f, Melanoma patient-derived, IL-12-stimulated iNKT cells (iNKTIL-12) were added (0.2%) to multiple bulk cocultures of healthy donor-derived CD8β T cells and MoDC pulsed with MART-1 peptide {alpha}GC. The percentage of Tm-positive CD8+ T cells is shown; horizontal lines represent the median (e) or mean (f), p values are the result of a two-sided Mann-Whitney U test (e) or a two-sided Student’s t test (f).

 
iNKT cell-mediated help is IFN-{gamma} dependent

To analyze whether the enhanced expansion of MART-1-specific CTL in the presence of iNKTIL-12 cells was dependent on cell-cell contact or soluble factors, cocultures were set up in transwells. Lower wells contained CD8β+ T cells and peptide and {alpha}GC-pulsed MoDC (–iNKT; Fig. 4a). iNKTIL-12 were added to the lower wells (+iNKTlow) or the upper wells alone (+iNKTup) or together with {alpha}GC-pulsed MoDC (+iNKTup+DCup). Similar CTL frequencies were obtained when iNKT cells were added to the lower wells and to the upper wells as long as {alpha}GC-presenting DC were also present to trigger the iNKT cells, suggesting that soluble factors were responsible for the observed enhanced effects (Fig. 4a). Since iNKTIL-12 cells provoked the strongest effect and contained high numbers of IFN-{gamma}, IL-10, and IL-4-producing cells, MART-1 CTL stimulation cultures were performed in the presence of iNKTIL-12 cells and IFN-{gamma}-, IL-10-, or IL-4-blocking Abs. Blocking of IFN-{gamma}, IL-4, and IL-10 had no effect on specific CTL frequencies in the absence of iNKT cells (Fig. 4b). However, the enhanced effect of iNKTIL-12 cells was completely blocked by anti-IFN-{gamma}, while anti-IL-4 Abs did not have any effect. Interestingly, in the presence of anti-IL-10 Abs the CTL frequencies increased even further (Fig. 4b). Finally, to confirm that IFN-{gamma} can indeed enhance the MART-1-specific CTL response, soluble recombinant IFN-{gamma} was added to cocultures and had similar enhancing effects as iNKTIL-12 (Fig. 4c).


Figure 4
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  		FIGURE 4. iNKT cell-mediated help is IFN-{gamma} dependent. a, Multiple bulk cocultures (n = 6/condition) of isolated CD8β T cells and autologous MoDC pulsed with MART-1 peptide and {alpha}GC were set up in a transwell system (–iNKT). IL-12-stimulated iNKT cells were added either to the lower well (+iNKTlow), to the upper well alone (+iNKTup), or in the presence of {alpha}GC-pulsed MoDC (+iNKTup +DCup). Percentage of Tm-positive CD8+ T cells is shown; horizontal lines represent the mean. Results shown are from one representative donor out of three tested; p values are the result of a two-sided Sudent’s t test. b, Multiple bulk cocultures (n = 4/condition) of isolated CD8β T cells and autologous MoDC pulsed with MART-1 peptide and {alpha}GC were set up in the presence or absence of IL-12-stimulated iNKT cells (iNKTIL-12) in the presence of blocking Abs against IFN-{gamma}, IL-4, IL-10, or isotype controls. Percentage of Tm-positive CD8+ T cells is shown; horizontal lines represent the median. Results are from one donor out of two tested; p values are from a two-sided Mann-Whitney U test. c, Multiple bulk cocultures (n = 6/condition) of isolated CD8β T cells and autologous MoDC pulsed with MART-1 peptide and {alpha}GC were set up (–); recombinant IFN-{gamma} or iNKTIL-12 cells were added. Percentage of Tm-positive CD8+ T cells is shown; horizontal lines represent the mean, results are from one donor out of three tested, p values are the result of a two-sided Student’s t test.

 
{alpha}GC and MART-1 double-loaded IL-12-overexpressing MoDC induce activation of iNKT cells and increase MART-1-specific CTL expansion

To examine whether IL-12-stimulated iNKT cells in PBMC physiologically enhance CTL expansion, cultures were started from PBL and {alpha}GC/MART-1 double-loaded MoDC. Multiple bulk cultures of total monocyte-depleted PBL were stimulated with autologous mature MoDC, transfected with MART-1 minigene ubi(MART)4 mRNA or ubi(MART)4–2A-IL-12 mRNA (35), either loaded with vehicle or {alpha}GC. Bulk cultures stimulated with {alpha}GC-loaded MoDC contained significantly higher numbers of iNKT cells, which were further increased when DCs were transfected with IL-12 mRNA (Fig. 5a). As expected and as previously described by us (35), stimulation with IL-12-overexpressing MoDC leads to an increased MART-1-specific CTL response (Fig. 5b). Pulsing with {alpha}GC without IL-12 overexpression had no significant effect; however, IL-12-overexpressing DC pulsed with {alpha}GC induced significantly more MART-specific CTL as compared with either IL-12-overexpressing DC or {alpha}GC-pulsed DC (Fig. 5b).


Figure 5
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  		FIGURE 5. {alpha}GC and MART-1 double-loaded IL-12-overexpressing MoDC induce activation of iNKT cells and increased MART-1-specific CTL expansion. Multiple bulk cultures (n = 8/condition) of monocyte-depleted PBL were stimulated with autologous MoDC transfected with MART-1 minigene mRNA or MART-1 minigene-2A-IL-12 mRNA, pulsed with either vehicle or {alpha}GC. The percentage of (a) iNKT cells and (b) MART-1 Tm-positive T cells was determined in each bulk culture 1 wk after the second stimulation. Horizontal lines represent the mean; results are from one donor out of two tested, p values are the result of a two-sided Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Herein we demonstrate that healthy donor-derived, expanded iNKT cells, and IL-12-stimulated iNKT cells in particular, enhance Ag-specific CTL responses in vitro. This effect was dependent on {alpha}GC-induced IFN-{gamma} production. We have previously demonstrated that despite impaired numbers of iNKT cells in cancer patients, IFN-{gamma} production by the remaining iNKT cells was not affected (14). Furthermore, iNKT cells from cancer patients could be expanded and skewed to produce IFN-{gamma} in vitro to similar levels as healthy donor-derived iNKT cells (40). In line with these findings, we demonstrate here that melanoma patient-derived, in vitro-expanded iNKT cells enhanced melanoma Ag-specific CTL as well, demonstrating their potential for the enhancement of CTL responses. Apart from IFN-{gamma}, IL-12 can also enhance IL-4 and IL-10 production by conventional T cells (41, 42). Indeed, after stimulation with IL-12-overexpressing DC, not only an increased number of IFN-{gamma}-producing iNKT cells was detected, but a small increase in IL-10-producing iNKT cells was observed as well. The positive effect of iNKT cell-derived IFN-{gamma} on CTL proliferation was not affected by concomitant IL-4 secretion; blocking of IL-10, however, further enhanced CTL expansion. Although only few iNKT cells produced IL-10 (see Fig. 2f), the amount of IL-10 may have been sufficient for the inhibiting effect. Alternatively, MoDC-derived IL-10 induced by iNKT cell-MoDC interactions may inhibit CTL expansion. In contrast to our findings here, Osada and colleagues previously demonstrated an inhibition of MART-1-specific CTL responses by expanded CD4+ iNKT cells (29). This inhibition was type 2 cytokine (among others, IL-4 and IL-10) mediated. In the present study most of the added iNKT cells were CD4+ (80–90%). However, there are several technical aspects that may explain the discrepancies. In healthy donors the frequency of circulating iNKT cells ranges from undetectable to 0.3% of circulating T cells. In the present study iNKT cells were added to the cocultures approximating physiological proportions, that is, 0.2% (1000 iNKT were added to 0.5 x 105 CD8β+ T cells). The much higher dose of iNKT cells (i.e., 10%) used by Osada and coworkers may have had different effects on CTL responses. IFN-{gamma} effects may become apparent at lower doses, while inhibition by IL-4 may become effective at higher concentrations. Both Osada et al. and Ho et al. observed a decline in Ag-specific CTL when free {alpha}GC and antigenic peptide were added to total PBMC and autologous DC cocultures (28, 29). It was concluded that this was due to type 2 cytokine release by CD4+ iNKT cells and lysis of APC and/or activated T cells by CD4–CD8{alpha}{alpha}+ iNKT cells, respectively. Addition of free MART peptide and {alpha}GC enables peptide presentation by (MART-specific) CTL as well as {alpha}GC presentation by activated T cells expressing CD1d (28) facilitating fratricide and iNKT cell-mediated killing of MART-specific CTL, respectively, which may lead to reduced MART-specific CTL rather than increased frequencies. Here, lysis of activated MART-1-specific CTL is less likely to occur because MART-1 was either introduced by mRNA transfection or the peptide was loaded, as was {alpha}GC, on DC before addition to total PBL or isolated CD8β+ T cells. In vivo, {alpha}GC loading specifically on DC also appears to be important, since in mice treatment with free {alpha}GC induced iNKT cell anergy in contrast to adoptive transfer with {alpha}GC-pulsed DC, which induces prolonged iNKT cell responsiveness (43). Similarly, in humans free {alpha}GC induces immune activation only after the first injection without iNKT expansion (44), while adoptive transfer with mature {alpha}GC-pulsed DC leads to a sustained expansion of iNKT cells (45). Addition of free {alpha}GC to total PBMC may lead to presentation by nonprofessional APC, leading to iNKT cell unresponsiveness. Furthermore, to provide iNKT cell-mediated help for Ag-specific CTL induction in vivo, the same DC had to present both the peptide and the glycolipid (7, 8). This is in contrast to our transwell results, where iNKT cells activated by {alpha}GC-pulsed DC in the upper well could enhance CTL responses induced by Ag-loaded MoDC in the lower well. This is probably due to the experimental conditions: a confined environment of a small well where cytokine concentrations remain high and can easily reach all the cells vs the in vivo situation, where cytokine levels rapidly diffuse and the differentially loaded DC have a low chance of ending up in close proximity to each other in the secondary lymphoid organs.

The cytokine neutralization experiments revealed that IFN-{gamma} is at least one soluble factor responsible for the enhanced proliferation of Ag-specific CTL, and that this effect was {alpha}GC dependent. It is not yet clear how the iNKT cell-derived IFN-{gamma} enhances CTL responses. It may act together with CD40 signaling provided by helper cells present in the cocultures to enhance IL-12 production by MoDC, which is an essential third signal for the development of CTL responses. However, as we have previously demonstrated for help provided by irradiated PBL (35) or isolated NK cells (46), CTL expansion is even further enhanced by iNKT cells (Fig. 5b) when DC overexpress IL-12. These results suggest that enhancing IL-12 secretion by DC is not the only mechanism at work here. Alternatively, IFN-{gamma} enhances CTL responses by positively affecting Ag presentation, at least in those experiments using MART-1 mRNA transfection for Ag loading of DC, through up-regulation of HLA class I expression and optimizing access of antigenic peptides to HLA class I (47) or by direct signaling through the IFN-{gamma}R on CD8+ T cells (48).

MART-1-presenting MoDC pulsed with {alpha}GC without IL-12 overexpression did not increase CTL expansion within PBL (Fig. 5b), in contrast to when expanded iNKT cells were added (Fig. 3a). This may be due to the type 1 skewing growth factor IL-15, which was added (next to IL-2 and IL-7) during the in vitro expansion of iNKT cells. This may generate iNKT cells more prone to produce IFN-{gamma} than the iNKT cells in the PBL experiments to which IL-2 and IL-7, but no IL-15, was added. This IL-15-mediated preactivation may be necessary in the absence of IL-12 for sufficient iNKT cell-derived IFN-{gamma}, while in the presence of IL-12, {alpha}GC does have an effect on CTL expansion.

The opposing effects of iNKT cells (i.e., enhancing antitumor immune responses on the one hand and inhibiting autoimmunity on the other hand) have been explained by the existence of iNKT cell subsets, based on CD4 and CD8{alpha}{alpha} expression, secreting distinct cytokine profiles ex vivo (18, 19). Herein we show that upon culture of human blood-derived iNKT cells the distinction based on CD4 expression becomes less clear. Both CD4– and CD4+ iNKT cells produced IFN-{gamma} as well as IL-4, and IFN {gamma} production was enhanced by IL-12 in CD4– as well as CD4+ iNKT cells, demonstrating the plasticity of iNKT cell subsets. The ratio of CD4–/CD4+ iNKT cells also changed during in vitro culture. Human CD4+ and CD4– iNKT cells have differential homeostatic requirements; that is, CD4– iNKT cells proliferate with a higher rate in response to IL-15, while CD4+ iNKT cells respond better to IL-7 in vitro (49). Nevertheless, the CD4–/CD4+ ratio is reduced upon culture in vitro irrespective of the cytokine used, due to a higher proliferation rate of CD4+ iNKT cells (50), which may be due to the fact that CD4– iNKT cells have been through more cell divisions in vivo, as illustrated by a decrease in TCR excision circles in CD4– iNKT cells (49). IL-12 stimulation induced an overall increase in activation markers, but despite the increase in tranzyme B expression, cytolytic activity was not enhanced. This is probably due to the fact that the expression of NKG2D, which is thought to play an important role in cytolytic activity by conventional NK and T cells (51), is not modulated by IL-12 (not shown).

In conclusion, IFN-{gamma} production by iNKT cells can be increased, irrespective of CD4 expression, by stimulating with IL-12-producing DC. These iNKT cells enhance Ag-specific CTL expansion in an IFN-{gamma}-dependent fashion. Phase I studies aiming at increasing iNKT cell numbers and activation by i.v. injection of {alpha}GC, {alpha}GC-pulsed DC, or in vitro-expanded iNKT cells were shown to be safe (44, 45, 52). Both vaccination with {alpha}GC-pulsed mature DC and iNKT cell adoptive transfer resulted in an increase in circulating iNKT cell numbers and IFN-{gamma}-producing PBMC, but no clinical responses were observed. Using the approach described herein, the efficacy of both treatment modalities may be enhanced. In vitro expansion of iNKT cells using IL-12-overexpressing (allogeneic) DC will lead to an iNKT cell population for adoptive T cell transfer that is superior in providing help for Ag-specific CTL. However, additional treatment with {alpha}GC, presented by DC, may be needed to reactivate the iNKT cells in vivo. An even more effective form of iNKT cell-mediated immunotherapy would be vaccination with Ag- and {alpha}GC-loaded, IL-12-overexpressing DC. This approach is expected to lead to an increased antitumor immune response mediated by TAA-specific CTL as well as IFN-{gamma}-producing iNKT cells.


   Acknowledgments
 
We thank Dr. M. Kronenberg (La Jolla Institute for Allergy & Immunology, San Diego, CA) for providing the CD1d-transfected HeLa cells (HeLa-CD1d), Dr. M Exley (Harvard Medical School, Boston, MA) for providing the CD1d-specific Ab (51.1.3), and Dr. S. Yamano (Kirin Brewery, Gunma, Japan) for providing KRN7000 ({alpha}GC). We thank Drs. J. Ruizendaal for preparation of Tm and FACS sorting. We thank Dr. T. D. de Gruijl for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts 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 the Dutch Cancer Society Grant VU2002-2607. Back

2 Current address: Nijmegen Center for Molecular Life Sciences (NCMLS), PO Box 9101, 6500 HB, Nijmegen, The Netherlands. Back

3 Current address: Department of Woman and Baby, Division of Surgical and Oncological Gynaecology, University Medical Center Utrecht, Utrecht, The Netherlands. Back

4 Address correspondence and reprint requests to Dr. H. J. Bontkes, Department of Hematology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands. E-mail address: hj.bontkes{at}vumc.nl Back

5 Abbreviations used in this paper: iNKT, invariant NKT cells; {alpha}GC, {alpha}-galactosylceramide; DC, dendritic cell; iNKTIL-12, IL-12-stimulated iNKT cells; IRES, internal ribosome entry site; MART-1, melanoma Ag recognized by T cell 1; MoDC, monocyte-derived dendritic cell; MUTZ-3 (M3), CD1d-transduced M3; M3CD1d, CD1d-transduced M3; M312CD1d, IL-12 and CD1d double-transduced M3; NGFR, nerve growth factor receptor; TAA, tumor-associated Ag; Tm, tetramer. Back

Received for publication January 3, 2008. Accepted for publication June 29, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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