Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag α-galactosylceramide. Numerous studies have investigated the mechanisms leading to Th1 and Th2 cytokine production by iNKT cells, as well as the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. In this study, we investigated the effect of Ag availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver, and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the Ag-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently used for tumor protection. Therefore, unlike NK cells, which rely mostly on perforin/granzyme-mediated mechanisms, the Ag-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway.
Invariant NKT (iNKT) cells are a unique subset of T lymphocytes characterized by the expression of an invariant Vα14-Jα18 TCR rearrangement (Vα14iNKT cells) and the recognition of CD1d, a nonpolymorphic MHC class I homolog. CD1d binds lipid structures, and one of the best studied Vα14iNKT cell Ags is α-galactosylceramide (αGalCer), a glycolipid originally isolated from a marine sponge, or perhaps more likely, from a Sphingomonas microorganism associated with the sponge (1, 2). Activation with this model Ag leads to rapid induction of effector functions by Vα14iNKT cells. Indeed, the identification of αGalCer was largely based on the discovery of its antitumor activity (1, 2), and numerous studies have described the strong iNKT cell- and CD1d-dependent antitumor properties of αGalCer.
Previous studies on iNKT cell effector functions have focused principally on their strong and rapid production of Th1 and Th2 cytokines (3–6). However, less attention has been given to their cytotoxic potential. Some reports demonstrate NK cell-like cytotoxicity of iNKT cells following activation with IL-12 (7–14) or αGalCer (1, 2, 15–27). However, few studies have addressed the Ag specificity of this cytotoxicity. We previously demonstrated Ag-specific cytotoxicity by transgenic, non–Vα14i-expressing CD8+ NKT (tgNKT) cells (28). However, little is known about the properties of and requirements for Ag-specific cytotoxicity by iNKT cells (24, 29, 30).
To achieve a better understanding of the cytotoxic potential of iNKT cells in vitro and in vivo, we used an Ag-specific in vivo cytotoxicity assay (28, 31). We investigated the effect of Ag availability and strength on iNKT cell cytotoxicity and found a positive correlation between these two parameters. Furthermore, we demonstrate that the Ag-specific iNKT cell cytotoxicity in vivo depends on the interaction of CD95 and its ligand CD178. Additionally, we demonstrate that the direct Ag-specific cytotoxicity by iNKT cells can be used for antitumor responses.
Materials and Methods
Mice and cell lines
All mice were housed under specific pathogen-free conditions at the animal facilities of the La Jolla Institute for Allergy and Immunology (La Jolla, CA) and The Scripps Research Institute (La Jolla, CA) in accordance with the Institutional Animal Care Committee guidelines. Experiments were performed according to animal experimental ethics committee guidelines. C57BL/6J mice (no. 000664), CD45.1 congenic B6.SJL mice (no. 002014), and mice deficient for perforin (no. 002407), CD95/Fas (no. 000482), and CD178/FasL (CD95L; no. 001021) on the C57BL/6 background were purchased from the The Jackson Laboratory (Bar Harbor, ME). B6.129-Tcra-Jtm1Tgi (Ja18−/−) mice and CD1d-deficient mice (CD1d−/−) on the C57BL/6 background were a gift from Dr. M. Taniguchi (RIKEN Institute, Yokohama, Japan) and Dr. Luc Van Kaer (Vanderbilt University, Nashville, TN), respectively. The B lymphoma A20 (BALB/cAnN; no. TIB-208) and the melanoma B16-F10 (C57BL/6; no. CRL-6475) were purchased from the American Type Culture Collection (Manassas, VA) and were virally transfected to stably express CD1d as previously described for A20 (32), resulting in the lines A20-CD1d and B16-CD1d.
Reagents and monoclonal Abs
Single-cell suspensions were prepared from the liver, spleen, and thymus. Prior to extraction, the liver was perfused with PBS via the portal vein until opaque and meshed through a 100-μm cell strainer (BD Biosciences, San Diego, CA) and washed. Total liver cells were then resuspended in a 40% isotonic Percoll solution (Amersham Biosciences, Piscataway NJ) and underlaid with a 70% isotonic Percoll solution. After centrifugation for 20 min at 900 × g, mononuclear cells were isolated from the 40%/70% interface. The cells were washed once with medium. For the spleen and the thymus, lymphocytes were filtered through a 70-μm cell strainer (BD Biosciences). With splenocytes indented for in vitro cultures, RBCs were lysed (red cell lysis buffer [RCLB] from Sigma-Aldrich, St. Louis, MO) and WBCs were washed once with medium (RPMI 1640; Invitrogen, Carlsbad, CA) supplemented with 5% (v/v) FCS (Mediatech, Manassas, VA), 1% (v/v) penicillin-streptomycin-glutamine (10,000 U/ml penicillin, 10,000 μg/ml streptomycin, 29.2 mg/ml l-glutamine; Invitrogen), and 50 μM β-mercaptoethanol (Sigma-Aldrich). For the in vivo cytotoxicity assay, spleen B cells were purified by positive selection with anti–CD19-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. For enrichment of Vα14i NKT cells from splenocytes and thymocytes, cells were incubated with PE-conjugated NK1.1 (PK136) followed by positive selection with anti-PE magnetic beads (Miltenyi Biotec). Liver-associated lymphocytes were used directly.
Lung metastases with B16 melanoma cells
B16 and B16-CD1d melanoma cells were either loaded with 250 ng/ml αGalCer (37°C, 90 min) or mock treated, washed twice with PBS, and 1 × 105 tumor cells were injected i.v. into C57BL/6 mice as indicated. Fourteen days after challenge the numbers of metastatic nodules on the lung surface were counted. Three hundred fifty tumor nodules were established as the upper limit for counting, as at higher densities discrete tumor nodules could not be separated accurately.
For staining of cell surface molecules, cells were suspended in staining buffer (PBS, 1% BSA, 0.01% NaN3) and stained with fluorochrome-conjugated Ab at 0.1–1 μg/106 cells for 15 min in a total volume of 50 μl. FcγR-blocking Ab anti-CD16/32 (2.4G2) and unconjugated rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) were added to prevent nonspecific binding. If biotin-conjugated Abs were used, cell-bound Abs were detected with streptavidin conjugates (1:200) in a second incubation step. Staining of T cells with αGalCer-loaded CD1d tetramers (34) was performed as described previously. In brief, cells were stained with the tetramer together with other surface mAbs in staining buffer at 4°C for 30 min. For analysis of intracellular cytokines, cells were fixed and permeabilized using the Cytofix/Cytoperm reagents (BD Biosciences, San Diego, CA) for 10 min at 37°C. Cells were washed twice and incubated for 30 min with fluorochrome-conjugated Ab and unconjugated rat and mouse IgG in Perm/Wash solution (BD Biosciences), which was followed by an additional 5 min incubation in Perm/Wash solution without mAb. For in vitro experiments intended for intracellular staining, GolgiPlug and GolgiStop (BD Biosciences) were added for the last 4 h of incubation. Cells were analyzed with FACSCalibur, FACSCanto, or LSR II (BD Biosciences), and data were processed with CellQuest Pro (BD Bioscience) or Flow Jo (Tree Star, Ashland, OR) software. Graphs derived from digital data are displayed on a “logical scale” (35).
In vivo cytotoxicity assays were performed according to Coles et al. (31) with minor alterations. Splenic B cells were purified as described and either pulsed with the indicated glycolipids (250 ng/ml, 1 h at 37°C) and labeled with a high concentration of CFDA-SE (1 μM, 15 min at 37°C; CFSEhigh cells) or were mock treated and labeled with a low concentration of CFDA-SE (0.15 μM; CFSElow cells). Cells were washed three times with PBS and equal numbers of cells from each population were injected i.v. (total of 1 × 107 target cells). Animals were sacrificed after indicated times, and the presence of target cells in spleen and liver was determined by flow cytometry. To calculate specific lysis of the in vivo cytotoxicity assay, the following formula was used: percentage specific cytotoxicity = 100 − [100 × (CFSEhigh/CFSElow)C57BL/6/(CFSEhigh/CFSElow)iNKT cell-deficient]. For in vitro cytotoxicity assays the tumor cells were either pulsed with αGalCer (250 ng/ml, 1 h at 37°C) or were mock treated, mixed at an equal ratio, and 1 × 105 cells were incubated with iNKT cell-enriched lymphocytes at indicated ratio for 4 h. Tumor cells used were either A20 and A20-CD1d or RMA together with B16 or B16-CD1d. Later pairings were labeled with CFDA-SE (0.15 μM; 15 min at 37°C). A20/A20-CD1d targets cells were distinguished based on forward light scatter (FSC)/side light scatter (SSC) characteristics and CD1d expression. RMA/B16 and RMA/B16-CD1d target cells were distinguished based on FSC/SSC characteristics and TCRβ expression.
Results are expressed as mean ± SEM. Comparisons were drawn using a two-tailed Student t test (Excel; Microsoft, Redmond, WA) or ANOVA test (GraphPad Prism; GraphPad Software, San Diego, CA). Each experiment was repeated at least twice. Graphs were generated with GraphPad Prism (GraphPad Software).
In vivo cytotoxicity of iNKT cells correlates with CD1d expression
We applied an in vivo cytotoxicity assay that has been used to study conventional CD8+ T cells (31) and tgNKT cells (28) for the investigation of the Ag-specific cytotoxicity of iNKT cells. To generate targets, whole splenocytes were loaded in vitro with αGalCer or incubated with medium as a control, differentially labeled with CFSE, injected i.v., and 16 h later the cytotoxicity was analyzed. We detected Ag-specific cytotoxicity of 30.4 ± 1.7% against the αGalCer-loaded splenocytes when compared with control targets (Fig.1A, 1B). As reported previously (29), this in vivo cytotoxicity was dependent on iNKT cells, as it was not detected in mice deficient for Jα18 (Fig. 1A) or CD1d (data not shown). Using congenic and surface markers, we then dissected the different splenocyte subsets to determine whether there was preferential elimination of certain cell types by iNKT cells in vivo. As shown in Fig. 1B, the in vivo cytotoxicity of iNKT cells varied significantly for different cell populations, ranging from no cytotoxicity against NK cells (0.4 ± 2.8%) to almost complete elimination of marginal zone B cells (87.3 ± 0.3%). Based on the hypothesis that cytotoxicity is likely influenced by the amount of Ag presented on the cell surface of the target cell, and because it is known that marginal zone B cells have very high expression levels of surface CD1d (36, 37), we analyzed CD1d expression by different splenic cell populations. The CD1d expression levels indeed varied between the different lymphoid and myeloid populations (Fig. 1C), and they correlated directly with the observed in vivo cytotoxicity by iNKT cells (Fig. 1D, R2 = 0.912). Purified CD19+ B cells were thereafter used as target cells to maximize cytotoxicity and consequently the sensitivity of the in vivo assay. This optimization based on target cell type allowed us to detect in vivo cytotoxicity of 40–60% within 4 h of incubation in vivo.
Ag-loaded target cells accumulate in the liver
The highest proportion of iNKT cells within the lymphocyte compartment is found in the liver (4, 38, 39), and liver iNKT cells have been reported to be more effective in the in vivo response to a methylcholanthrene-induced sarcoma (40). Therefore, we were interested to compare the in vivo cytotoxicity of splenic versus hepatic iNKT cells. Interestingly, when we analyzed the Ag-specific iNKT cell cytotoxicity against αGalCer-loaded B cells in the liver we obtained values that were either very low or in most cases negative (Fig. 2A and data not shown). The local immune system of the liver has previously been shown to actively promote tolerance rather than immunity (41, 42). Therefore, it could be argued that the tolerogenic environment of the liver was blocking the cytotoxicity of the liver iNKT cells in situ. However, due to the measured negative values of the in vivo cytotoxicity assay, we hypothesized that the αGalCer-loaded target cells might be trapped in the liver, thus distorting the ratio of αGalCer-loaded to unloaded B cell targets. Therefore, we analyzed the ratio of both target populations in liver and spleen over time. As expected, the ratio of Ag-loaded target cells to control target cells decreased continuously in the spleen (Fig. 2B), in line with the increased cytotoxic removal of Ag-loaded target cells over time. In contrast, this ratio increased rapidly in the liver and was followed by an Ag-specific reduction (Fig. 2B). This observation is in agreement with the notion that αGalCer-loaded B cell targets accumulate in the liver in an Ag- and iNKT cell-dependent manner during at least the first hour in vivo after injection. This accumulation in the liver cannot account for the decrease in the spleen, as the liver has many fewer targets cells. We estimate that if all of the ∼50% increase in αGalCer-loaded target B cells in the liver were due to cells from spleen, this would contribute <5–10% of the total estimated cytotoxicity. Therefore, the decrease in the spleen likely reflects mostly cytotoxicity there. Consequently, we concluded that the accumulation of αGalCer-loaded B cell targets in the liver exceeds the capacity of the normal cytotoxic function of the liver resident iNKT cells at early time points. Nevertheless, the cytotoxic capacity of spleen and liver resident iNKT cells appears similar.
Mutual activation of αGalCer-loaded targets and iNKT cells in spleen and liver
We next analyzed the activation of the iNKT cells by αGalCer-loaded B cell targets. As shown in Fig. 3, the αGalCer-loaded B cell targets stimulated iNKT cells in spleen and liver to upregulate the activation markers CD69 and CD25 after 4 h. This upregulation was seen as early as 2 h after the injection of the B cell targets (data not shown). Furthermore, the iNKT cells produced the cytokines IFN-γ, TNF-α, and IL-4 within 4 h after the injection of the target cells (Fig. 3B). These data demonstrate that αGalCer-loaded B cell targets activate iNKT cells in the spleen and liver. As with the iNKT effector cells, the B cell targets also were activated, as measured by the upregulation of CD69 and CD25, both in spleen and liver (Fig. 3C). Taken together, these data indicate that liver iNKT cells do not have an activation defect during the in vivo cytotoxicity assay.
In vitro cytotoxicity by iNKT cells
To compare the intrinsic cytotoxicity of iNKT cells derived from different organs side by side, we used a flow cytometric assay similar in principle to the in vivo assay. A20 B lymphoma cells and A20 cells transfected with a construct driving the expression of mouse CD1d (A20-CD1d) were used as targets. Although A20 cells are H2d haplotype, we could not detect in vitro NK type killing during the 4 h incubation time with H2b iNKT cells (data not shown). The A20 cell line does not express detectable levels of CD1d and is not killed by iNKT cells when loaded with αGalCer (data not shown). As A20 and A20-CD1d displayed identical growth within 48 h, both cell lines could be used as targets without the need of prior CFSE labeling. Target cells were identified by size (FSC versus SSC) and CD19 expression and differentiated based on their CD1d expression levels. Using this protocol we assessed the Ag-specific cytotoxicity of iNKT cells from thymus, spleen, and liver. In all experiments the cytotoxicity of iNKT cells from all three organs was comparable (Fig. 4), demonstrating that iNKT cells have similar cytotoxic capability irrespective of the organ of their origin.
In vivo cytotoxicity by iNKT cells correlates with Ag potency
We next analyzed the Ag-specific cytotoxicity of iNKT cell for CD1d-presented glycosphingolipid Ags with different antigenic potencies. We compared iNKT cell cytotoxicity against αGalCer-loaded targets with two of its synthetic derivatives, OCH and C-Gly (43). OCH is a weaker iNKT cell Ag than αGalCer and induces a systemic Th2 response (43–46). C-Gly is the weakest of these three Ags and induces a systemic Th1 response (43). As shown in Fig. 5 the cytotoxicity of iNKT cells in vivo directly correlated with the potency of the iNKT cell Ag (αGalCer > OCH > C-Gly). Although we could not detect any cytotoxicity against B cell targets loaded with C-Gly, the cytotoxicity against OCH-loaded targets reached 22–35% of the cytotoxicity observed with αGalCer (Fig. 5A). Furthermore, the in vivo cytotoxicity correlated with the intensity of CD25 upregulation on the Ag-loaded B cell targets (Fig. 5B) and the intensity of the observed iNKT cell stimulation, as measured by upregulation of activation markers and cytokine production (data not shown). We also tested the in vivo iNKT cell cytotoxicity against B cells loaded with a synthetic version of the Sphingobium yanoikuyae glycolipid GalA-GSL (called GSL-1′ in earlier publications) (47, 48). The iNKT cell cytotoxicity against the GalA-GSL–loaded targets was low, reaching only 3–9% of the cytotoxicity observed with αGalCer-loaded targets (Fig. 5C). Furthermore, we did not observe upregulation of CD25 on the GalA-GSL–loaded B cell targets (data not shown).
iNKT cell cytotoxicity depends on CD95/CD178 interaction
To gain insight into the Ag-specific cytotoxic mechanisms employed by iNKT cells in vivo, we analyzed perforin- and CD95-deficient hosts. As shown in Fig. 6, the lack of perforin had no significant effect on the observed in vivo iNKT cell cytotoxicity, either alone or in conjunction with the lack of CD95 on the targets cells (Fig. 6A, 6B). In contrast, deficiency for either CD178 in the host (Fig. 6A) or of CD95 on the B cell targets (Fig. 6B) resulted in a significant decrease in the observed Ag-specific in vivo iNKT cell cytotoxicity. The remaining cytotoxicity in the absence of the CD95/CD178 pathway was between 17.6 and 28.5%, with a mean 23.4% of the C57BL/6 value, when normalized to 100%. In accordance with the results of the in vivo cytotoxicity assay, we observed upregulation of CD178 on iNKT cells stimulated with αGalCer-loaded B cells (Fig. 6C) and the upregulation of CD95 expression by the B cell targets from the same mice (Fig. 6D). Furthermore, we could not detect granzyme B or perforin by either stimulated or control iNKT cells (data not shown). Collectively, these data demonstrate that the in vivo cytotoxicity of iNKT cells depends mainly on the CD95/CD178 interaction.
CD1d expression by B16 melanomas augments αGalCer-mediated protection
Given the strong cytotoxicity we observed against CD1d-expressing targets, we determined if this direct Ag-specific cytotoxicity could be used for antitumor responses. The B16 melanoma aggressively grows in vivo, but protection can be conferred by concurrent stimulation of iNKT cells via IL-12 (10, 12, 49) or αGalCer (17, 19, 26, 27). However, the B16 melanoma does not express CD1d (50, 51 and data not shown), excluding a direct presentation of αGalCer. We stably transfected B16 melanoma cells with mouse CD1d (B16-CD1d) and used them as targets in an in vivo cytotoxicity assay. B16 or B16-CD1d cells were loaded with αGalCer, mixed 1:1 with RMA cells as internal control, labeled with CFSE, and incubated for 4 h with iNKT cell-enriched splenocytes in vitro. Whereas no cytotoxicity was detected against the control B16 melanoma (data not shown), the αGalCer-loaded B16-CD1d cells were eliminated (Fig. 7A). We also observed an upregulation of CD95 on the B16-CD1d melanomas (Fig. 7B), but not on the B16 (data not shown), during the 4-h incubation with the splenocytes. To determine the CD95/CD178 dependency of the observed cytotoxicity, we inhibited this death pathway using a blocking anti-CD178 Ab. This resulted in a decrease of the cytotoxicity against the B16-CD1d cells of 41–59% (Fig. 7A), indicating that the CD95/CD178 pathway is important for the observed cytotoxicity. We next determined whether expression of CD1d on the B16 melanoma could also confer protection in vivo. B16 and B16-CD1d melanomas were pulsed shortly (90 min) with αGalCer before i.v. injection, and the numbers of formed tumor metastases in the lung were recorded 14 d later. It has previously been shown that loading of B16 cells in vitro with αGalCer for 2 d protects mice from tumor metastases (51, 52). In contrast, αGalCer loading for only 1.5 h resulted in a minor protective effect compared with unloaded B16 tumor cells (Fig. 7C). In contrast, αGalCer loading of B16-CD1d melanomas protected the animals from metastases (Fig. 7C). These data indicate that direct Ag presentation by the B16-CD1d melanoma leads to an efficient eradication of the tumor in vivo.
Although it is well known that iNKT cells can augment the cytotoxic activity of NK cells, herein we have analyzed the cellular and molecular parameters involved in the in vivo and in vitro Ag-dependent cytotoxicity of target cells by iNKT cells. In particular, we provide evidence that the in vivo cytotoxicity of iNKT cells correlates directly with CD1d expression levels on target cells and with the potency of the iNKT cell Ag. The interaction of iNKT cells with their targets led to a mutual activation of the both cell types, and the iNKT cells from spleen and liver showed a similar degree of cytotoxicity. Most surprising, we show that the Ag-specific cytotoxicity of iNKT cells in vivo relies almost entirely on the interaction between CD95 (Fas) and CD178 (FasL).
It has been reported for αGalCer and its derivatives OCH and C-Gly that the intensity of cytokine produced by the iNKT cells following i.v. injection correlated directly with the antigenic potency (i.e., αGalCer > OCH > C-Gly) (43). Using αGalCer, OCH, C-Gly, and a synthetic version of the S. yanoikuyae–derived Ag GalA-GSL, we demonstrate that the antigenic strength directly correlated in a similar fashion with the iNKT cell in vivo cytotoxicity (Fig. 5). Surprisingly, limited cytotoxicity was observed with the weaker Ags, including C-Gly and the S. yanoikuyae Ag GalA-GSL. These data suggest that the described in vivo potency of C-Gly in tumor rejection (53) or the antimalarial response (54) may not be due principally to antigenic-specific killing by iNKT cells. Similarly, although mice that have iNKT cells clear S. yanoikuyae more rapidly when compared with iNKT cell-deficient animals, this also may occur independently of an Ag-specific cytotoxic function (48, 55). Our experiments focused on testing the immediate or short-term cytotoxic function of iNKT cells, and therefore we cannot rule out the possibility that iNKT cell-mediated Ag-specific killing is important in immune responses that take place over several days, such as during tumor or pathogen challenges.
Furthermore, under different circumstances, some of the less potent compounds may stimulate cytotoxic activity more efficiently. For example, dendritic cells may be more effective at taking up C-Gly and loading it into CD1d than in B lymphocytes; similarly, uptake of whole Sphingomonas bacteria or membrane fragments may promote more effective loading of GalA-GSL into the groove of mouse CD1d. Additionally, the overall avidity of an Ag depends not only on its strength of interaction with the TCR when bound to CD1d (i.e., the TCR affinity), but also on its density, that is, on the amount of available epitopes presented and recognized by the T cell. In line with this notion, we show that the iNKT cell cytotoxicity in vivo also correlated with the amount of CD1d expressed on the target cells (Fig. 1). Although NK cells expressed lower levels of CD1d than did any other cell type analyzed (Fig. 1C), it was still surprising that we did not observe αGalCer-induced cytotoxicity against splenic NK cell targets (Fig. 1B). The expression of CD95 induced on the NK cells was lower than on B cells (data not shown), which together with the low amount of CD1d may explain the reduced NK cell susceptibility to killing. Furthermore, several mechanisms have been reported that protect cytotoxic lymphocytes against cytotoxicity (56–58), which could be involved in our experimental system as well.
The two major mechanisms to induce cell death by cytotoxic lymphocytes involve, on the one hand, different surface receptors, most prominently CD95/CD178, but also TNF-α/TNFR and TRAIL/TRAILR, or alternatively, soluble mediators, mainly perforin and granzymes (59, 60). iNKT cell express CD178 following activation with Con A (61) and αGalCer (Fig. 6) (17, 62). Furthermore, human iNKT cell lines have been reported to express TRAIL (CD253) following restimulation with αGalCer-loaded DCs, and they exerted TRAIL-dependent cytotoxic activity against some leukemia cells in vitro (62, 63). Despite this diversity of mechanisms, most studies have implicated the perforin/granzyme B pathway in the cytotoxic activity of iNKT cells (19, 64–66). However, these studies relied on chemical inhibitors of granule release, and most likely addressed NK-type cytotoxicity rather than Ag-specific CD1d-dependent cytotoxicity. A typical experimental set-up for these studies involved the injection of αGalCer i.v., and the determination of the cytotoxicity of purified splenocytes 1 d later against NK cell-sensitive targets in vitro. Such an approach does not directly address the mechanisms or even the role of iNKT cells in the observed cytotoxicity. Indeed, in several studies it was shown that the perforin required for the observed cytotoxicity against tumors resided not within the iNKT cells, but rather in NK cells that had been activated downstream of the iNKT cell activation (25, 67, 68). In fact, this trans-activation of NK cells appears to be the general mechanism for the αGalCer-induced antitumor activity. Following αGalCer exposure, NK cells are activated by several mechanisms, including iNKT cell-derived IFN-γ [in mice (27, 67, 69–71)] or IL-2 [in humans (24)], leading to NK cell cytotoxicity and cytokine production. The importance of this trans-activation of NK cells by iNKT cell-derived IFN-γ has been demonstrated for pathogen infections (39, 72) and in tumor models (17, 27, 67, 73). Apparently, NK cells act generally in amplifying the iNKT cell signal in a feed-forward loop (14, 49, 74).
In contrast with these earlier studies, our experiments were designed to investigate the direct, short-term, CD1d-dependent, Ag-specific cytotoxicity of iNKT cells. CD1d-dependent cytotoxicity of iNKT cells has been reported previously (2, 24, 29, 30, 65, 75–77), but the underlying mechanism was not addressed. In this study, we demonstrate that the CD1d and Ag-dependent cytotoxicity of iNKT cells requires the CD95/CD178 pathway and is completely independent of the perforin/granzyme pathway (Fig. 6). iNKT cells expressed CD178 following interaction with αGalCer-loaded B cell targets and induced CD95 expression on these target B cells (Fig. 6). The remaining, relatively minor cytotoxicity observed in the absence of CD95 and perforin expression could be due to TRAIL (CD253) and/or TNF-α–mediated apoptotic pathways. However, we could not detect any TRAIL staining (clone N2B2) on iNKT cells by flow cytometry in our experiments (data not shown). Our data are in agreement with earlier studies suggesting an iNKT cell-mediated elimination of B cells during nickel tolerance that was dependent upon the CD95/CD178 pathway (78) and an elimination of hepatotcytes following ConA-induced hepatitis that depended on both the CD95/CD178 and the perforin/granzyme pathway (61). However, the requirements for expression of CD95 by iNKT cells, as well as the role of CD1d and TCR recognition, were not addressed in these studies.
Numerous studies have established the strong antitumor activity following the activation of iNKT cells with IL-12 (7–14) or αGalCer (1, 2, 15–27). Importantly, NKT cells that were not intentionally Ag-stimulated were also shown to be involved in tumor surveillance (25, 68, 79, 80). It has been demonstrated previously that loading of αGalCer onto B16 and other tumor lines can induce a strong immune response and protection against tumor growth in vivo (51, 52). However, in these studies the loading of αGalCer was carried out for 2 d, which may allow the tumor cells to take up high amounts of this glycolipid Ag (51). Consequently, in this experimental format, the responses of iNKT cells (51) and dendritic cells (52), after injection of either αGalCer-loaded B16 or αGalCer-loaded B16-CD1d cells, were indistinguishable. This indicates that direct presentation of αGalCer by the tumor cells was not required, suggesting that potent cross-presentation of αGalCer can occur in vivo. As mentioned above, the antitumor response following αGalCer treatment in vivo depends on IFN-γ–mediated trans-activation of NK cells (17, 67, 81). Furthermore, it has been shown that IFN-γ induces upregulation of CD95 on B16 tumor cells (82, 83). Therefore, the αGalCer-induced antitumor response for most of these studies can be explained by the trans-activation of NK cells, which then attack the tumor. In line with this interpretation is the observation that protocols that augment the IFN-γ response by iNKT cells, thereby intensifying the NK cell trans-activation, augment the antitumor response. This could be achieved either by using dendritic cells loaded with αGalCer instead of free αGalCer (22, 84), or by utilizing the Th1 cytokine–inducing iNKT cell Ag C-Gly (85).
In contrast, we show herein that expression of CD1d by B16 melanoma cells makes them susceptible to cognate Ag-dependent cytotoxicity by iNKT cells, mainly via the CD95/CD178 pathway. Furthermore, we demonstrate that following a short pulsing of B16 tumor cells with αGalCer, the efficiency of the antitumor response depends on the expression of CD1d and most probably on direct presentation of the Ag by the tumor cells (Fig. 7). Several studies have shown that tumor cells that express CD1d can be lysed by iNKT cells in vitro when the tumor cells were loaded with αGalCer (24, 30, 65, 76, 77). In this study, we extended these reports by demonstrating the CD1d dependency of the Ag-specific iNKT cell cytotoxicity against tumors in vivo (Fig. 7). Therefore, our data suggest that activation of iNKT cells for an antitumor therapy, a strategy currently applied in clinical trials (86), might be most effective against those tumors that express CD1d. In such cases, the activation of iNKT cells might facilitate cognate-Ag killing as well as trans-activation of other cell types.
We thank Archana Khurana for excellent technical assistance. We are grateful for the scientific contributions of Barbara Sullivan, Bo Pei, Aaron Tyznik, and Jennifer Matsuda.
Disclosures The authors have no financial conflicts of interest.
This work was supported in part by National Institutes of Health Grants RO1 AI45053 and R37 AI71922 (to M.K.), an Outgoing International Fellowship by the Marie Curie Actions (to G.W.), and by a long-term European Molecular Biology Organization fellowship and a fellowship from the Swiss National Science Foundation (to P.K.).
Abbreviations used in this paper:
- carboxyfluorescein diacetate–succinimidyl ester
- follicular B cell
- forward light scatter
- galacturonosyl glycosphingolipid
- marginal zone B cell
- side light scatter
- transgenic NKT
- invariant Vα14 to Jα18 TCR rearrangement.
- Received March 29, 2010.
- Accepted June 20, 2010.
- Copyright © 2010 by The American Association of Immunologists, Inc.