Originally published online as doi:10.1189/jlb.0607352 on April 11, 2008

Published online before print April 11, 2008

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(Journal of Leukocyte Biology. 2008;84:264-279.)
© 2008 by Society for Leukocyte Biology

Activation-induced NKT cell hyporesponsiveness protects from -galactosylceramide hepatitis and is independent of active transregulatory factors

Markus Biburger^*, and Gisa Tiegs^,,1

^* Laboratory for Experimental Immunology and Immunotherapy, Nikolaus-Fiebiger-Center for Molecular Medicine, Medical Department III, University Hospital Erlangen, Germany;
Center of Internal Medicine, Division of Experimental Immunology and Hepatology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany; and
Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuremberg, Erlangen, Germany

¹Correspondence: Center of Internal Medicine, Division of Experimental Immunology and Hepatology, University Medical Centre Hamburg-Eppendorf, Hamburg, Martinistr. 52, 20246 Hamburg, Germany. E-mail: g.tiegs{at}uke.de

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
NK T (NKT) cells, unique lymphocytes expressing features of NK and T lymphocytes, can specifically be activated with the glycolipid antigen -galactosylceramide (-GalCer). In humans and mice, this activation provokes pronounced cytokine responses. In C57BL/6 mice, -GalCer injection additionally induces NKT-mediated liver injury, representing a model for immune-mediated hepatitis in humans. However, a single -GalCer pretreatment of mice prevented NKT-mediated liver injury, cytokine responses (systemically and locally in the liver), and up-regulation of hepatocellular Fas upon -GalCer rechallenge. As -GalCer is used as a NKT cell-activating agent in clinical trials, an investigation of tolerance induction appears crucial. We demonstrate that -GalCer tolerance does not depend on Kupffer cells, IL-10, Caspase-3-mediated apoptosis, or CD4⁺CD25⁺ T regulatory cells (T_regs), which are crucial in other models of immunological tolerance. Amending relevant, earlier approaches of others, we cocultivated highly purified, nontolerized and tolerized liver NKT cells ex vivo and could convincingly exclude the relevance of transdominant NKT T_regs. These results strongly suggest -GalCer-induced tolerance to be exclusively caused by NKT cell intrinsic hyporesponsiveness. Tolerized mice showed specific diminishment of the intrahepatic CD4⁺ NKT cell subpopulation, with the CD4^– population largely unaffected, and revealed down-modulation of -GalCer-specific TCR and the NKT costimulator glucocorticoid-induced TNFR-related protein on liver NKT cells, whereas inhibitory Ly49I was increased. In conclusion, -GalCer tolerance could serve as a model for the frequently observed NKT cell hyporesponsiveness in tumor patients and might help to develop strategies for their reactivation. Conversely, approaches to render NKT cells hyporesponsive may constitute new therapeutic strategies for diseases, where aberrant NKT cell activation is causally involved.

Key Words: lymphocytes • natural killer T cells • tolerance • liver immunology • anergy • animal models

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
NK T (NKT) cells are a unique subpopulation of T cells, which in their majority, express NK surface markers such as NK1.1, IL-2Rß, and to some extent, Ly49 family members and reveal the Thy1^high, CD44^high, CD45RB^high phenotype of activated T cells (defined in C57BL/6 mice; see ref. [¹ ]). The predominant population of NKT cells expresses a TCR repertoire with an invariant V14-J18 TCR chain in mice or V24-J18 in humans and a restricted TCRß repertoire. This TCR mediates recognition of glycolipid antigens presented by the MHC class I-like molecule CD1d [¹ ], such as isoglobotrihexosylceramide, which has for some time been considered to be the physiological antigen [² ] (an assumption that is challenged by recent publications; refs. [³ , ⁴ ]), or synthetic -galactosylceramide. This surrogate antigen had been developed by Kirin Brewery Co., Ltd. (Tokyo, Japan) for cancer treatment [⁵ ], as NKT cells have been suggested to exert antitumor responses in several cancer types (reviewed in refs. [⁶ , ⁷ ]). In fact, NKT cell activation has already been analyzed in clinical trials as a therapeutic approach for cancer treatment [^{8 9 10 11 12} ]. Also, NKT cells are believed to be involved in the prevention of autoimmunity (see refs. [¹³ , ¹⁴ ]). However, besides such beneficial effects, there is in fact cumulating evidence that NKT cells play pivotal roles in the onset of pathological processes. They appear to be involved in several murine disease models such as atherosclerosis [¹⁵ , ¹⁶ ], allergen-induced airway inflammation and asthma [^{17 18 19} ], oxazolone-induced ulcerative colitis [²⁰ ], antibody-induced joint inflammation and arthritis [²¹ , ²² ], and depending on experimental design and mouse strain, also pristane-induced lupus [²³ ] or experimental autoimmune encephalomyelitis [²⁴ ]. They are also suggested to participate in the onset of several hepatic disorders in man such as immunopathogenesis of chronic hepatitis C virus-induced hepatitis [²⁵ ], intrahepatic bile duct lesions in primary biliary cirrhosis [²⁶ ], and cirrhosis progression in chronic viral hepatitis [²⁷ ]. Also, two well-established murine models of immune-mediated hepatitis induced by injection of Con A [²⁸ ] or -galactosylceramide (-GalCer) [²⁹ , ³⁰ ] are strictly NKT cell-dependent [³¹ , ³² ].

Several reports describe an impairment of NKT cell cytokine responses to -GalCer restimulation after a preceding -GalCer injection [^{33 34 35 36 37} ]. Also, other processes associated with NKT cell activation, such as, e.g., TCR down-modulation, have been reported to fail upon -GalCer reinjection [³⁶ ]. Considering the Janus-like capability of NKT cells to inhibit or augment pathologic processes [¹³ ] and our observation that a single -GalCer pretreatment ameliorated liver injury upon -GalCer rechallenge, we were interested in the mechanisms of -GalCer-induced immunosuppression and protection from liver injury. Because of our recent identification of T regulatory cells (T_regs), Kupffer cells (KCs), and IL-10 as important factors in a prima facie similar model of immunological tolerance induction by Con A [³⁸ ], we also wanted to characterize their potential role in -GalCer tolerance. Here, we demonstrate that neither these factors nor caspase-3-mediated apoptosis were relevant for -GalCer tolerance in terms of prevention of cytokine production and liver injury. In contrast, we could prove this tolerance to be based on a passive mechanism, i.e., activation-induced hyporesponsiveness, and disclose tolerization-induced changes among the intrahepatic NKT cell populations as well as phenotypic changes.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice
Male C57BL/6 wild-type or IL-10^–/– [³⁹ ] mice (8–12 weeks) were obtained from Harlan-Winkelmann (Borchen, Germany), Elevage Janvier (Le Genest-Saint-Isle, France), or animal facilities of the University of Erlangen-Nuremberg (Germany) and were maintained under controlled conditions (22°C, 55% humidity, 12 h day/night rhythm) and fed standard laboratory chow. All mice received human care according to the guidelines of the National Institutes of Health (Bethesda, MD, USA) and the legal requirements in Germany.

Animal treatments
-GalCer was kindly provided by Kirin Brewery Co., Ltd. Directly before i.v. injection, the stock solution of 200 µg/ml in vehicle (0.5% w/v polysorbate-20) was diluted in pyrogen-free saline to achieve (if not, different doses are mentioned for particular experiments) a dose of 1 µg per mouse in 200 µl. For hepatocyte-specific transcription inhibition with D-galactosamine (GalN), galactosamine-hydrochloride (Carl Roth GmbH, Karlsruhe, Germany) was administered i.p. in pyrogen-free saline (70 mg/ml) at 200 µl/20 g mouse 30 min prior to i.v. injection of 200 ng -GalCer. The effect of exogenous IL-10 on -GalCer hepatitis was analyzed by i.v. injection of 1 µg recombinant murine (rm)IL-10 (Peprotech, London, UK) 30 min prior to injection of 200 ng -GalCer. For KC depletion, 100 µl liposome-encapsulated dichloromethylene-bisphosphonate (Clodronate liposomes, derived from Dr. Nico van Rooijen, Vrije Universiteit, Amsterdam, The Netherlands) was injected i.v. 48 h before -GalCer rechallenge as described previously [⁴⁰ ]. Dichloromethylene-bisphosphonate, for their preparation, was a gift of Roche Diagnostics (Mannheim, Germany). Efficiency of KC depletion was verified by immunohistology (not shown).

For inhibition of caspase-3-like caspases, the irreversible inhibitors Z-Val-Ala-Asp(O-methyl)-fluoromethyl ketone [zVAD(OMe).fmk; R&D Systems, Wiesbaden, Germany] or Z-Val-Ala-fluoromethyl ketone (zVAD) fmk (Bachem, Weil am Rhein, Germany) were reconstituted in DMSO and diluted in pyrogen-free saline to the indicated doses directly before injection in a total volume of 200 µl per 20 g mouse with a final concentration of 4% DMSO. Application regimens were 5 mg/kg z-VAD(OMe).fmk i.p. 20 min prior to treatment with 1 µg -GalCer or alternatively, 10 mg/kg z-VAD.fmk i.v. 20 min prior to -GalCer treatment (200 ng) with an additional i.p. injection of 5 mg/kg 6 h thereafter. Activity of z-VAD.fmk had been verified before [³⁰ ].

In vivo depletion of CD4⁺CD25⁺ T_regs was achieved by i.v. injection of 300 µg rat anti-mouse CD25 mAb (clone PC-61.5) purified from hybridoma supernatant using Thiophilic-Superflow resin (BD-Clontech, Heidelberg, Germany) 1 day prior to -GalCer injection. Efficiency of T_reg depletion was verified by flow cytometry. Therefore, splenocytes were stained with anti-CD4 and anti-CD25 mAb. CD25 detection was carried out with clone 7D4, which recognizes another CD25 epitope than PC-61.5 to prevent false-negative staining of cells in PC-61.5-injected mice caused by epitope-masking.

For in vivo neutralization of TNF- or IFN-, mice were injected i.v. with 300 µg IgG, purified from sheep anti-mouse TNF- polyclonal antiserum [⁴¹ ] with 75 µl polyclonal rabbit anti-TNF- antibody IP-400 (Genzyme, Cambridge, MA, USA) or with 200 µl rabbit anti-mouse IFN- serum [⁴² ], one-half hour prior to treatment. Corresponding amounts of normal rabbit serum (Sigma Chemical Co., St. Louis, MO, USA) or purified, total IgG from normal sheep serum (Sigma Chemical Co.) were used as negative controls.

Sampling of material
Mice were lethally anesthetized (150 mg/kg i.v. methohexital+15 mg/kg heparin). Cardiac blood was withdrawn for analysis of plasma transaminases and cytokines. Livers were excised, frozen in liquid nitrogen, and stored at –20°C for preparation of RNA and subsequent real-time RT-PCR.

Analysis of liver injury
Hepatocyte damage was assessed 16–17 h after -GalCer treatment by measuring plasma enzyme activities of alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST) using the automated COBAS Mira system (Roche Diagnostics).

Cytokine quantification
Sandwich ELISAs for murine plasma cytokines were performed using Nunc-Immuno^TM 96-well flat-bottom Maxisorb^TM microtiter plates (Nunc GmbH, Wiesbaden, Germany). Antibodies were purchased from BD-PharMingen (San Diego, CA, USA) for IL-2, IL-4, IL-6, and IL-10. Streptavidin-peroxidase was purchased from Roche Diagnostics. IFN-, TNF- and TGF-ß were quantified using DuoSet^TM ELISA-Development systems from R&D Systems, together with tetramethyl benzidine substrate-reagent set (BD-PharMingen) as peroxidase chromogen according to the manufacturer’s instructions. For a single experiment, cytokine concentrations were measured in plasma of -GalCer-tolerized or control mice using the BD Cytometric Bead Array^TM (BD Biosciences, San Jose, CA, USA).

RNA isolation and real-time RT-PCR for cytokine mRNAs
Isolation of RNA from liver tissue was carried out using the Total-RNA isolation kit (Macherey-Nagel, Düren, Germany). mRNA was transcribed into cDNA using SuperScript^TM II RNase H^– RT, oligonucleotides, and oligo-(dT) primers from Invitrogen (Karlsruhe, Germany). Real-time RT-PCR was performed using a LightCycler rapid thermal cycler system (Roche Diagnostics) and LightCycler-FastStart DNA Master SYBR-Green I (Roche Diagnostics), according to the manufacturer’s instructions. Primer pairs were used as described previously [³⁰ ]. In addition, for Fas quantification, we used 5'-Fas: 5'-CGCTGTTTTCCCTTGCTGCA-3' and 3'-Fas: 5'-ACTGAGGTAGTTTTCACTCCA-3'. To confirm amplification specificity, melting curves of PCR products were analyzed. Relative mRNA levels were calculated by means of 2^CP (CP=difference of crossing-points of test and respective control samples, as extracted from amplification curves by the LightCycler^TM software) after normalization with respect to β-actin mRNA levels.

Isolation and flow cytometric analysis of liver mononuclear cells (MNCs)
To isolate hepatic MNCs, livers were passed through 100 µm nylon meshes, and hepatocytes were removed by centrifugation (800 g, 20 min) in isotonic 37% Percoll solution (Amersham-Biosciences, Freiburg, Germany) containing 100 U/ml heparin. Erythrocytes were lysed in 139 mM NH₄Cl, 19 mM Tris. For flow cytometry using a standard protocol, including preblocking FcRs, typically, 4 x 10⁵ liver leukocytes were stained using anti-mouse-CD16/32 mAb ("Fc-block"; clone 93, eBioscience, San Diego, CA, USA), FITC- or cychrome-labeled anti-mouse-CD3 mAb (clone 145-2C11), FITC- or PE-labeled anti-mouse-NK1.1 (clone PK136), PE-labeled anti-mouse-CD25 (clone PC-61.5) mAb, PE-labeled anti-mouse-Ly49I (all BD-PharMingen), biotinylated or PE-labeled anti-mouse glucocorticoid-induced TNFR-related protein (GITR; clone YGITR 765, BioLegend, San Diego, CA, USA), PE- or Tricolor-labeled anti-mouse-CD4 (clone RM4-5, Caltag, Hamburg, Germany), anti-mouse-CD25-PE mAb (clone 7D4, Miltenyi Biotec, Bergisch Gladbach, Germany), and FITC-, PE-, or CyChrome-conjugated streptavidin (Jackson Immunoresearch, West Grove, PA, USA). For preparation of -GalCer/CD1d tetramers to enable staining of -GalCer-specific TCR, biotinylated rmCD1d (kindly provided by Dirk Busch, Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Germany) was loaded with -GalCer for 20 h at room temperature. Subsequently, CD1d was incubated in molar excess at room temperature for 8 h with PE-conjugated streptavidin for labeling and tetramerization with stepwise addition of the streptavidin. Data were recorded and analyzed using a three-color FACScan^TM flow cytometer (BD Biosciences) with BD Biosciences CellQuest^TM software.

Purification of CD3⁺ NK1.1⁺ NKT cells and CD4⁺ CD25⁺ T_regs
For purification of CD3⁺NK1.1⁺ NKT cells, liver MNCs were stained with anti-CD3 and anti-NK1.1 mAb and sorted in the Cell Sorting Core Facility of the University of Erlangen-Nuremberg using a MoFlo^TM cell sorter (DakoCytomation GmbH, Hamburg, Germany; purity, 95%). For T_reg purification, a combined sorting procedure was carried out using magnetic bead separation (CD4⁺CD25⁺ T_reg isolation kit, mouse, Miltenyi Biotec) and subsequent FACS sorting as described previously [³⁸ ].

Ex vivo assays
Cells were isolated as described above, seeded in 96-well cell-culture plates, and stimulated with 15 ng/ml -GalCer or a corresponding volume of vehicle as negative control. iGB3 (Alexis Corp., Lausen, Switzerland) was solubilized in chloroform:methanol (2:1), dried, resuspended in methanol, and diluted in medium to reach a final concentration of 15 µg/ml with methanol remaining below 1%. In assays using liver MNCs (0.8–2x10⁵ cells/well), no additional APCs had to be added; in experiments with purified NKT cells (0.8x10⁵ cells/well), bone marrow-derived dendritic cells (DCs; 0.4x10⁵ cells/well; kind gift from Carsten Wiethe Department of Dermatology, University Hospital of Erlangen-Nuremberg, Erlangen) were used as -GalCer-presenting cells. In all assays analyzing potential suppressive effects of T_regs or cells from -GalCer-pretreated mice, close cell–cell contact was facilitated by using round-bottom plates. Cytokine concentrations in the supernatant were determined by ELISA.

Statistical analysis
All data are expressed as mean ± SEM (if n3). For calculation of statistical significance, data were analyzed using Student’s t-test if two groups were compared or the Bonferroni test if several groups were tested against one another. P 0.05 was considered significant.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Prevention of -GalCer-induced liver injury and cytokine production upon tolerance induction
To analyze the potential of NKT cell activation to induce a state of immunological tolerance, C57BL/6 mice were pretreated with 1 µg -GalCer and challenged 3 or 8 days thereafter. The -GalCer-induced cytokine response and/or the induction of hepatic injury were used in this study as readout for the ability of NKT cells to be activated efficiently in vivo. In comparison with control mice, mice pretreated with -GalCer at Days –3 or –8 revealed significantly reduced liver injury upon -GalCer challenge (Fig. 1A ). Reduced liver injury was also found in an experiment where only 50 ng -GalCer had been used for pretreatment (data not shown). -GalCer injection is well known to induce pronounced production of a broad range of Th0, Th1, and Th2 cytokines. To analyze the effect of -GalCer pretreatment, we measured cytokine responses in plasma and on the mRNA level in liver tissue of pretreated and control mice at 1.5 h or together with liver transaminases 16–17 h after rechallenge, i.e., at time-points where peak levels of intrahepatic mRNA and maximum plasma concentrations for several cytokines are detectable [³⁰ ]. Early plasma levels of IFN-, IL-2, IL-4, and TNF were decreased significantly by more than 80% (Fig. 1B) in -GalCer-pretreated mice. This corresponded well to significantly diminished mRNA levels in the liver at that time-point (Fig. 1C) . Also, 16–17 h after -GalCer rechallenge diminished IFN-, TNF-, and IL-6 responses were measured in liver and plasma (not shown). In contrast, pretreatment induced a tendency to increased IL-10 plasma levels at both time-points and significantly increased intrahepatic IL-10 mRNA 1.5 h after rechallenge (Fig. 1B and 1C) . Pretreatment had no relevant influence on TGF-ß production (not shown). It is worth mentioning that a BD Biosciences multiplex array analysis of a single experiment also revealed significantly reduced plasma levels of IL-1β, GM-CSF, and CXCL-1 in -GalCer-pretreated mice (data not shown). Moreover, we found significantly reduced intrahepatic Fas mRNA expression upon -GalCer rechallenge (measured 16 h after rechallenge) in tolerized mice in comparison with mock-pretreated mice (Fig. 1D) .

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Figure 1. A single pretreatment with -GalCer impaired susceptibility to -GalCer rechallenge in vivo. (A) Pretreatment with 1 µg -GalCer protected mice from liver injury upon rechallenge with 1 µg -GalCer 3 or 8 days thereafter in comparison with vehicle controls, as assessed by plasma ALT activity. Reduced susceptibility to rechallenge was reflected in reduced plasma cytokine concentrations in plasma of -GalCer-pretreated mice 1.5 h after rechallenge (B) and reduced relative intrahepatic cytokine mRNA expression at the same time-point as measured by real-time RT-PCR (C). Tolerance induction with respect to plasma ALT levels was detected in each of eight independent experiments (n3); reduced cytokine responses at 1.5 h were found in two independent experiments (n=4). (D) Tolerized mice revealed significantly reduced intrahepatic Fas mRNA expression upon -GalCer rechallenge 8 days after pretreatment. Relative mRNA expression was quantified by RT-PCR using liver samples that had been taken 16 h after rechallenge in parallel with cardiac blood withdrawal for ALT measurement. This impaired expression was detected in each of three independent experiments. The graph depicts the summary of these experiments (ntotal=9). (E) In -GalCer- and vehicle-treated mice, intrahepatic CD3+NK1.1+ NKT cell population sizes were quantified by FACS analysis. At both time-points where tolerance in pretreated mice had been demonstrated, i.e., 3 days or 8 days after -GalCer treatment, large NKT cell populations were detected in the livers, and their size was increased significantly after 3 days and moderately decreased after 8 days. The graphs represent the summary of two experiments per time-point (n=4). All results are expressed as mean ± SEM; *, P 0.05, versus vehicle control.

Modulation of NKT cell numbers/frequencies
Previous reports had documented NKT cells to become depleted or undetectable shortly after -GalCer injection into mice, followed by recurrence and modulation of the NKT population size within the following days. As disappearance of NKT cells would result in a tolerance-like phenotype, we characterized NKT cell numbers and frequencies in livers of -GalCer- and vehicle-treated animals. After 3 days, the number of CD3⁺NK1.1⁺ NKT cells in the liver of -GalCer-pretreated mice even exceeded that of vehicle-treated control mice. As shown in Figure 1E for two representative experiments, 8 days after pretreatment, the intrahepatic number of NKT cells was more than two-thirds of the NKT cells in the control group.

For the 8-day time-point, the modulation of the NKT population was analyzed in more detail by characterizing NKT cells also with regard to -GalCer/CD1d-tetramer staining. We found the frequency of NKT cells among intrahepatic lymphocytes on average to be reduced in tolerized mice by above two-fifths for CD3⁺NK1.1⁺-defined NKT cells (the average NKT cell frequency was 7.8% in tolerized compared with 14.3% in control mice in nine experiments) or above one-half for NKT cells being defined by -GalCer/CD1d-tetramer staining (5.6% compared with 10.1% in six experiments; for changes in NKT frequency, see also Fig. 7 ). However, as a result of increased total numbers of intrahepatic lymphocytes in tolerized mice, the total number of hepatic CD3⁺NK1.1⁺ NKT cells was measured to be reduced by only approximately one-fifth in tolerized mice (the average NKT cell number in tolerized mice calculated from mean values of nine experiments was 4.8x10⁵ cells compared with 6.2x10⁵ cells in control mice) and the total number of tetramer-positive cells by approximately one-third (3.4x10⁵ cells compared with 5.1x10⁵ cells in six experiments). This demonstrates that high numbers of NKT cells are still present in the liver of tolerized mice, and -GalCer pretreatment does not result in an extensive depletion of NKT cells.

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Figure 2. -GalCer-induced intrahepatic Fas expression is mainly restricted to hepatocytes and dependent on TNF-. To characterize the effect of -GalCer treatment on Fas expression in the liver, C57BL/6 mice were treated with vehicle as a negative control or with 200 ng -GalCer. To test for Fas expression by hepatocytes and its potential association with TNF-, some mice had been pretreated with GalN or anti-TNF- antibody 30 min prior to -GalCer injection. After -GalCer application (4.5 h), mice were killed, and the liver was excised for RT-PCR analysis (n=3; mean±SEM; *, P 0.05, vs. vehicle control; #, P0.05, vs. only -GalCer-treated group).

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Figure 3. -GalCer-induced tolerance is independent from IL-10 and KC and is not prevented by an inhibitor of caspase-3 mediated apoptosis. Injection of 1 µg IL-10 30 min prior to injection of 200 ng -GalCer failed to protect mice from -GalCer-induced liver injury as measured by ALT- and AST-activities as well as IFN concentrations in plasma 17 h after -GalCer rechallenge, indicating that -GalCer hepatitis is insensitive to IL-10. wt, Wild-type (A). Induction of -GalCer tolerance was also possible in C57BL/6 IL-10–/– mice as detected by reduced plasma transaminase activities in -GalCer-pretreated mice, indicating that the tolerogenic mechanism does not depend on IL-10 (B). Both graphs in panels A and B depict mean ± SEM; n = 4; *, P 0.05. (C) Eight days prior to -GalCer rechallenge, one group of mice had received vehicle as a control and two had received -GalCer pretreatment. In one of these two groups KCs had been depleted by injection of clodronate-liposomes 48 h prior to -GalCer pretreatment. Tolerance induction was achieved also in KC-depleted mice as revealed by plasma ALT activity 17 h after -GalCer rechallenge. The graph is representative of two experiments with corresponding results (n=3, mean±SEM; *, P0.05 vs. vehicle control). (D) Tolerance-induction under conditions of caspase-3 inhibition was analyzed by injecting control mice with vehicle and mice that were to be tolerized by injection of -GalCer either without additional treatment, or with zVAD.fmk treatment. Plasma ALT was measured after -GalCer rechallenge 8 days thereafter. For both C and D, dotted lines represent typical average ALT values of untreated mice (45 U/L). Both graphs are representative for two experiments with corresponding results, respectively. (n=3 or 4; mean±SEM; *, P0.05, vs. vehicle control).

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Figure 4. CD4+ CD25+ Tregs are not essential for -GalCer tolerance. (A) Treg depletion by i.p. injection of 300 µg anti-CD25 mAb 1 day prior to rechallenge did not inhibit tolerance, as reflected in the relative suppression of plasma ALT activities in tolerized mice (n=4; mean±SEM; *, P0.05, vs. nontolerized control). (B) i.p. injection of anti-CD25 mAb PC61.5 resulted in efficient down-regulation of CD25 on the cell surface of CD4+forkhead box P3 (FoxP3)+CD25+ Tregs (upper panels) without depletion of these cells (as judged by their FoxP3 expression, lower panels) within 1 day. The effects of anti-CD25 injection were analyzed by triple staining of peripheral blood leukocytes with anti-CD4, anti-FoxP3, and anti-CD25 mAb 24 h after PC-61.5 injection. CD25 detection was carried out with clone 7D4, which recognizes another CD25 epitope than PC-61.5 to prevent false-negative staining of cells in PC-61.5-injected mice caused by epitope masking. The cells depicted in these panels had been gated for being viable lymphocytes, according to light-scatter characteristics, and being CD4-positive. FSC, Forward-scatter. (C) Purified NKT cells and splenic Tregs were incubated alone (8x104 cells per well) or together at a 1:1 ratio in the presence of -GalCer (15 ng/ml) presented by DCs (4x104/well). Cytokine concentrations were measured in duplicate after 3 days of culture.

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Figure 5. -GalCer nonresponsiveness of purified liver NKT cells from -GalCer-pretreated mice is nontransferable to naïve cells. NKT cells were prepared separately from three tolerized and three nontolerized control mice 8 days after pretreatment and cultured together with DCs for antigen presentation (4x104 cells/well) in the presence or absence of 15 ng/ml -GalCer. (A) DCs cultured alone, with or without -GalCer, as well as NKT cells (8x104 cells/well) in the absence of -GalCer revealed no relevant cytokine production. Upon -GalCer stimulation for 3 days, NKT cells from nontolerized mice readily produced IFN-, IL-2, IL-4, and TNF-, whereas those from -GalCer-pretreated mice barely responded (n=3; mean±SEM; *, P0.05; one of two independent experiments is shown). (B) NKT cells were cultured separately at a density of 8 x 104 cells/well. In addition, they were cocultured at equal numbers (i.e., 8+8x104/well) in six different combinations (with respect to the mice from that had been isolated; mean±SEM; *, P0.05, vs. NKT cells from control mice; #, values below detection limit).

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Figure 6. -GalCer pretreatment devises phenotypic changes in the intrahepatic NKT cell population. Liver MNCs were isolated from -GalCer- or vehicle-treated mice 8 days after treatment. During FACS analysis, NKT cells were identified by gating on the lymphocyte population, according to light-scatter characteristics, and on cells being positively stained by PE-labeled -GalCer/CD1d tetramer (top left panel). Geometric mean fluorescence intensities of -GalCer/CD1d-tetramer staining of NKT cells from vehicle- or -GalCer-pretreated mice are depicted in the top right panel for one representative experiment, indicating a moderate down-regulation of -GalCer/CD1d-specific TCRs 8 days after -GalCer pretreatment. In addition, cells were stained with antibodies to CD4, Ly49I, or GITR (middle and bottom panels). Expression of CD4, Ly49I, or GITR on NKT cells from vehicle- and -GalCer-pretreated mice, respectively, is depicted in contour plots for one representative sample. Frequencies of relevant subpopulations among NKT cells from one representative experiment are shown. (n=3; mean±SEM; *, P0.05, vs. cells from control mice). Corresponding results were obtained in two additional independent experiments, and also, in experiments where NKT cells had been defined by being CD3+NK1.1+, qualitatively corresponding results were achieved.

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Figure 7. -GalCer pretreatment down-modulates the frequency of the intrahepatic CD4+ but not CD4– NKT subpopulations. Intrahepatic MNCs isolated from or vehicle- or -GalCer-treated mice 8 days after treatment were incubated with anti-CD4 mAb together in addition to -GalCer/CD1d tetramer (left panel) or CD3 and NK1.1 (right panel) for NKT cell identification. The graphs depict the frequency of the CD4+ and CD4– NKT cells among liver lymphocytes revealed. Whereas the significantly diminished frequency of NKT cells among liver lymphocytes of -GalCer-pretreated mice was largely based on a reduction of CD4+ NKT cells, the frequency of the CD4-negative NKT subpopulation was virtually unchanged in all experiments. This was true for NKT cells defined by being tetramer-positive or CD3/NK1.1 double-positive. The figure shows the results of one representative experiment of four independent tests for each of both NKT definitions (n=4; mean±SEM; *, P0.05).

Fas down-modulation in tolerance is coupled to reduced TNF- responses
As we had shown before that Fas ligand (FasL) expression on NKT cells was modulated by -GalCer-induced TNF- [³⁰ ], we were interested in whether Fas expression and correspondingly, its reduced expression in tolerized mice were also associated with respective TNF- responses. Injection of -GalCer to C57BL/6 mice induced significant up-regulation of Fas expression in the liver within a few hours, as detected by quantification of relative mRNA levels by real-time RT-PCR (Fig. 2 ). Hepatocyte-specific gene expression is studied frequently by application of GalN, which specifically inhibits transcription in hepatocytes by depletion of the hepatocellular pool of uracil nucleotides [⁴³ ]. Pretreatment of mice with GalN (700 mg/kg) 30 min prior to -GalCer injection (200 ng/mouse) abolished -GalCer-induced Fas up-regulation, suggesting that Fas was mainly expressed by hepatocytes. Pretreatment of mice with the TNF--neutralizing antibody (0.3 mg/mouse IgG purified from sheep anti-mouse TNF- polyclonal antiserum [⁴¹ ]) instead of GalN prior to -GalCer injection had virtually the same effect, clearly indicating that Fas up-regulation on hepatocytes is mediated by -GalCer-induced TNF-. Thus, down-modulated, intrahepatic Fas expression in tolerized mice upon rechallenge was probably not an independent effect but rather reflected diminished TNF- responses under these conditions.

-GalCer tolerance is not compromised by impairment of IL-10, T_regs, KC, or caspase-3-mediated apoptosis
Immunological tolerance can be mediated by passive mechanisms such as anergy and/or active tolerogenic/immunosuppressive factors. NKT cells have been suggested to become nonresponsive upon activation, which may account for or contribute to the observed reduction of immune-mediated liver injury induced by NKT cell-specific -GalCer [^{33 34 35} ]. As discussed below, IL-10, T_regs, and KCs are important factors in various models of immunological tolerance. Also recently, Sireci et al. [⁴⁴ ] demonstrated Th2 cytokine-mediated protection of mice from endotoxin shock by -GalCer treatment within 2 h before or after LPS injection. Moreover, we recently described an important role for IL-10, KCs, and CD4⁺CD25⁺ T_regs in a prima facie similar murine model of tolerance induction [³⁸ ], protecting from the NKT cell-dependent Con A-induced liver injury. In several models of immunological tolerance, apoptosis appears to play an important role in the onset of tolerance as well (reviewed, e.g., in refs. [^{45 46 47} ]). Also, a theoretically possible switch from an easily activatible to a less-responsive NKT subpopulation of extrahepatic origin populating the liver after -GalCer pretreatment might be accomplished by apoptosis within the NKT cell population, initially resident in the liver. Thus, we analyzed whether any of these factors also contribute to suppression of NKT-mediated hepatitis in -GalCer tolerance.

To analyze effects of IL-10 in -GalCer-hepatitis, mice were pretreated with 1 µg rIL-10 prior to -GalCer injection. However, neither liver injury nor IFN- production was affected by exogenous IL-10 (Fig. 3A ) at a dose that had been shown to prevent, e.g., fulminant galactosamine/LPS liver injury in mice [⁴⁸ ]. Concordantly, IL-10 deficiency in C57BL/6 IL-10^–/– mice did not inhibit the onset of -GalCer tolerance (Fig. 3B) .

To analyze their relevance in -GalCer tolerance, KCs were depleted by injection of clodronate liposomes prior to tolerogenic -GalCer pretreatment. This approach was experimentally feasible, as we have shown recently that KC depletion did not interfere with -GalCer-induced liver injury [³⁰ ]. However, KC depletion did not inhibit -GalCer tolerance induction (Fig. 3C) .

To investigate a potential role of apoptosis in the development of -GalCer tolerance, we used zVAD.fmk and zVAD(OMe).fmk, two irreversible inhibitors of caspase-3-like caspases (which according to manufacturers’ information, differ in their cellular permeability) to block apoptosis induction during the tolerogenic -GalCer pretreatment. However, measuring liver injury of mice upon -GalCer rechallenge, we found no hint for an interference of caspase-3-mediated apoptosis with the development of -GalCer tolerance (Fig. 3D) [zVAD(OMe).fmk; data not shown].

To test for a potential influence of CD4⁺CD25⁺ T_regs in -GalCer tolerance, mice were injected with anti-CD25 mAb, clone PC-61.5, 1 day prior to -GalCer rechallenge. We found that anti-CD25 treatment did not interfere with -GalCer tolerance, indicating that CD4⁺CD25⁺ T_regs were not relevant for tolerization-induced protection from liver injury upon -GalCer rechallenge (Fig. 4A ). In view of the ongoing discussion regarding a functional inactivation of CD4⁺CD25⁺ T_regs caused by anti-CD25 mAb-induced CD25 down-regulation or T_reg depletion (discussed below), we characterized the effects of anti-CD25 mAb treatment on CD25⁺ or FoxP3⁺ T cell populations. Efficient disappearance of CD4/CD25 double-positive cells within 1 day after PC-61.5 injection was verified by FACS analysis (Fig. 4B) . Also, functionality of PC-61.5 treatment in our hands had been proven earlier in a model of Con A tolerance [³⁸ ]. However, staining of transcription factor FoxP3 showed that the frequency of FoxP3-positive T_regs was virtually unchanged, indicating that PC-61.5 treatment did not cause T_reg depletion but rather down-modulation of CD25 surface expression. In an additional experiment, we analyzed the susceptibility of NKT cell activation by -GalCer to suppression by CD4⁺CD25⁺ T_regs. Ex vivo cocultivation of FACS-purified NKT cells together with CD4⁺CD25⁺ T_regs (purified by magnetic bead sorting plus FACS sorting; >98% purity), even at a 1:1 ratio, revealed no inhibitory effect of T_regs on -GalCer-induced cytokine secretion of NKT cells (Fig. 4C) . This insusceptibility of NKT cells to immunosuppression by T_regs further supports the notion that CD4⁺CD25⁺ T_regs are probably not involved in -GalCer tolerance.

Analysis of -GalCer tolerance in ex vivo assays
Immunological tolerance may be caused by a complex network of factors in an entire organism, be locally limited mainly to one organ, or occur on a single cell basis.

To investigate whether -GalCer tolerance might be detectable in the isolated population of hepatic leukocytes or would alternatively need factors deriving from other cells, liver MNCs from tolerized or control mice were isolated and restimulated in vitro. In fact, liver MNCs from vehicle-pretreated mice showed pronounced cytokine production upon -GalCer restimulation in vitro, whereas MNCs from -GalCer-pretreated mice revealed largely diminished production of these cytokines (IFN-, IL-2, IL-4, and TNF-; data not shown). Also, in initial experiments with restimulation by the ligand iGB3 that preferentially binds to the Vβ7 chain in contrast to -GalCer, which has higher affinity to Vβ8.2, we found a tendency to reduce IFN- production by MNCs from -GalCer-tolerized mice (258±148 pg/ml vs. 22±13 pg/ml for vehicle-pretreated vs. -GalCer-pretreated mice; mean±SEM; n=6; data were obtained from two independent experiments).

To exclude the possibility that reduced cytokine production might simply be caused by the observed differences in NKT cell frequencies within the liver MNC populations of -GalCer- and vehicle-pretreated mice, hepatic NKT cells from mice of both groups were purified by FACS sorting (purity, >95%) and stimulated with -GalCer using mouse DCs as APCs. Also, with these highly purified NKT cells, we found pronounced cytokine production by nontolerized NKT cells, whereas NKT cells from -GalCer-pretreated mice revealed significantly lower cytokine responses to -GalCer restimulation (Fig. 5A ).

To investigate the possibility that NKT cells from tolerized mice might have developed active tolerogenic features (such as, e.g., an increased fratricidal cytotoxicity), purified NKT cells from nontolerized and tolerized mice were cocultured at a 1:1 ratio. In fact, tolerized NKT cells did not suppress cytokine production by nontolerized NKT cells upon -GalCer stimulation (Fig. 5B) , thereby excluding -GalCer-induced development of transactive, tolergenic properties among intrahepatic NKT cells as a cause of impaired responses to restimulation.

To analyze if -GalCer tolerance might be mediated by any other -GalCer-induced, transactive mechanisms (such as regulatory cells, suppressive cytokines) in the liver, we tested whether hepatic MNCs from tolerized mice were able to impose immune suppression on MNCs from nontolerized mice. Whereas the cytokine response of tolerized MNCs to -GalCer restimulation in vitro was diminished by >95% (as measured by quantifying IFN- production during 3 days of culture), these tolerized MNCs could not suppress MNCs from nontolerized mice upon coculture in a 1:1 ratio (data not shown), similar to the results with purified NKT cells. This indicated that the observed disability to respond efficiently to -GalCer restimulation is also not caused by NKT cell-independent, transactive suppression factors among liver MNCs, which could have evolved upon -GalCer pretreatment but probably by intrinsic impairment of NKT cell responses.

Tolerance-associated phenotypic changes in the intrahepatic NKT cell population
To identify possible causes of -GalCer nonresponsiveness, we characterized changes of some surface markers of NKT cells, identified by being positive for CD3 and NK1.1 or for -GalCer/CD1d tetramers, 8 days after -GalCer tolerization by flow cytometry. One prominent effect of -GalCer pretreatment on the NKT population was a down-modulation of NK1.1 with the presence of NK1.1^low NKT cells still after 8 days (data not shown). However, as this NK1.1 down-regulation has been demonstrated in a large number of reports, we did not focus further on these results. NKT cells in the liver are known to be almost exclusively CD4⁺ or double-negative. In control mice, CD4⁺ NKT cells represented the largely predominant population with more than two-thirds of liver NKT cells. In contrast, only about one-half of hepatic tetramer-positive NKT cells from -GalCer-tolerized mice showed CD4 surface expression (Fig. 6 ). Measuring the geometric mean of -GalCer/CD1d-tetramer staining, we found that expression of the -GalCer-specific TCR was down-regulated significantly in the intrahepatic NKT population of tolerized mice (Fig. 6) . In addition, we analyzed the expression of GITR, which had been shown to act as an efficient costimulator for NKT cells [⁴⁹ ]. In nontolerized mice, virtually all intrahepatic NKT cells revealed strong staining for GITR (Fig. 6) , whereas only a minority of less than 10% of classical T cells, probably CD4⁺ CD25⁺ T_regs, which express high amounts of GITR (cited in ref. [⁴⁹ ]), was GITR⁺ (data not shown). In livers of tolerized mice, the fraction of NKT cells that lacked pronounced surface expression of this costimulator was increased significantly (Fig. 6) . This effect was found only for NKT cells but not for conventional T cell populations in livers of tolerized mice (data not shown). In contrast to CD4, TCR, and GITR down-regulation, -GalCer-pretreated mice showed a significantly higher fraction of NKT cells that expressed Ly49I, i.e., a representative inhibitory receptor (Fig. 6) .

It is important to mention that qualitatively identical results were received when expression of these markers was investigated using the classical CD3⁺NK1.1⁺ phenotype for NKT cell characterization. Quantitatively, the phenotypic changes described above were typically found to be equally or even more pronounced in analyses based on CD3⁺NK1.1⁺ (data not shown).

It is worth mentioning that in a single experiment, where we had analyzed TCR-Vß chain repertoires, we did not detect significant differences in the ratio of NKT cells expressing TCR β-chains Vß8.1/8.2 to those expressing Vß7 in livers of tolerized or control mice (data not shown).

Diverse effects of -GalCer pretreatment on CD4⁺ and CD4^– NKT subpopulations
As mentioned above, 8 days after -GalCer pretreatment, the frequency of NKT cells among liver lymphocytes was reduced. This, together with the observed reduction of the CD4⁺ NKT subpopulation, prompted us to analyze the modulation of CD4-positive or -negative NKT cells among liver lymphocytes after -GalCer pretreatment. Surprisingly, the observed reduction of the liver NKT cell frequency was virtually fully to the debit of CD4⁺ NKT cells. In contrast, the frequency of the CD4-negative NKT subpopulation was virtually unchanged in all experiments. This was true for NKT cells defined by being CD3⁺NK1.1⁺ or being tetramer-positive as shown in Figure 7 for one representative experiment using any of both modes of NKT definition, respectively. This result indicates a pronounced change in the distribution of phenotypically different intrahepatic NKT cell subpopulations upon -GalCer pretreatment.

Effects of IFN- or TNF- neutralization on tolerance induction
As a result of our observation that IFN- neutralization fulminantly increased -GalCer-induced liver injury [³⁰ ] and a report that high levels of IFN- inhibited cytotoxicity of NK cells in Con A-induced hepatitis [⁵⁰ ], we hypothesized that -GalCer-induced IFN- exerts suppressive effects on -GalCer-immune activation and might be a mediator of -GalCer tolerance. However, pretreatment with neutralizing anti-IFN- antiserum [⁴² ] prior to tolerogenic -GalCer injection did not impair tolerance. Rather, we found a tendency toward further declined cytokine responses to in vitro restimulation of -GalCer-tolerized liver MNCs from anti-IFN--pretreated mice [0.7±0.3 ng/ml vs. 15.4±7.2 ng/ml IFN by cells from -GalCer- vs. vehicle-pretreated, anti-IFN--injected mice (P<0.05) compared with 1.7±0.9 ng/ml vs. 15.0±6.3 ng/ml in -GalCer- vs. vehicle-pretreated control serum-injected mice (P>0.05); n=3]. However, these changes were associated with a further reduction of NKT cell frequencies among liver MNCs in these mice (58.6% -GalCer-induced reduction of NKT frequencies among liver MNCs in anti-IFN--injected mice compared with 23.2% reduction in control serum-injected mice, P<0.05), indicating that the onset of NKT cell nonresponsiveness, as reflected in cytokine responses to restimulation, was not crucially affected by IFN- neutralization.

As we and several others have described previously the important role of TNF- in NKT cell function, we wondered whether TNF- might also play a role in the induction of NKT cell hyporesponsiveness. In vivo analysis of -GalCer-induced tolerance in anti-TNF--pretreated mice was not feasible, as circulating anti-TNF- antibodies would probably also affect the outcome of -GalCer rechallenge several days after pretreatment. Thus, we analyzed the responsiveness of liver MNCs from mice of the different pretreatment groups in vitro. Mice were preinjected with rabbit control serum or polyclonal anti-TNF- serum (IP-400, Genzyme) one-half hour prior to injection of the vehicle or -GalCer. Similar to the anti-IFN- test, we found a tendency to an even more pronounced -GalCer-induced reduction of the NKT cell frequency upon anti-TNF- pretreatment (55.5% -GalCer-induced reduction of NKT frequencies among liver MNCs in anti-TNF--injected mice compared with 47.2% reduction in control serum-injected mice, P>0.05; n=6; summary of two experiments). In contrast to mice tolerized under conditions of IFN- neutralization, the in vitro IFN- response of MNCs from anti-TNF--pretreated, -GalCer-tolerized mice to -GalCer restimulation was increased in comparison with control serum-pretreated, tolerized mice [2.6±1.2 ng/ml vs. 9.4±2.8 ng/ml (P>0.05) by cells from -GalCer- vs. vehicle-pretreated, anti-TNF--injected mice compared with 1.1±1.0 ng/ml vs. 10.7±2.3 pg/ml in -GalCer- vs. vehicle-pretreated, control serum-injected mice (P<0.05)]. However, this anti-TNF--induced increase in -GalCer-induced hyporesponsiveness was not significant (P>0.05 between -GalCer-pretreated control and -GalCer-pretreated anti-TNF- mice), indicating that TNF- might be involved to some degree but is probably not a major factor in the development of -GalCer-induced hyporesponsiveness of NKT cells.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we demonstrated that already a single pretreatment with -GalCer rendered mice tolerant to -GalCer rechallenge with respect to cytokine production (except for IL-10) and liver injury. The finding that -GalCer treatment results in a "blocked" response to restimulation has gained particular importance, as -GalCer has already been used in clinical studies as an anti-tumor agent, and NKT cells appear to be involved in the onset of several diseases as mentioned above. If homologous effects should also apply to humans, it will be important to comprehend the mechanism of -GalCer-induced tolerance to find strategies for retaining or re-establishing the ability of NKT cells to be effectively activated to confer antitumoral activity. Inversely, intentional suppression of NKT cell activation per se or its physiological effects in vivo may enable treatment of NKT-mediated diseases.

Potential mechanisms of tolerance
The mechanism by which the first -GalCer injection might cause tolerance with respect to elementary NKT cell activation and/or NKT-mediated disease induction could be persisting depletion of NKT cells, induction of "active" immunologic tolerance conferred by regulatory cells or immunomodulatory cytokines, or induction of activation-induced nonresponsiveness.

In addition to mechanisms that directly modulate immune activation, reduced cytotoxic susceptibility may contribute to protection from liver injury.

In the context of -GalCer tolerance, reduced expression of intrahepatic Fas in -GalCer-challenged, tolerized mice compared with -GalCer-treated, control mice may be associated with reduced sensitivity of hepatocytes, consistent with studies reporting diminished -GalCer- or GalN/-GalCer-induced liver injury in Fas-deficient lpr^–/– mice [³² , ⁵¹ ]. As we could demonstrate that Fas up-regulation by hepatocytes upon -GalCer injection is presumably mediated by -GalCer-induced TNF-, the diminished intrahepatic Fas expression in tolerized mice probably reflects their diminished TNF- response to -GalCer rechallenge. This suggests that protection from liver injury, besides the more general aspect of immune suppression, includes an aspect of reduced susceptibility of hepatocytes to Fas/FasL cytotoxicity. Both aspects are interconnected by modulation of TNF- expression in -GalCer tolerance.

Recently, also Parekh et al. [³⁵ ] had investigated effects of -GalCer injection in mice, describing induction of nonresponsiveness as an outcome of this treatment. Whereas in their important manuscript, the authors mainly characterized NKT cell activation with respect to cytokine production and proliferation, in this work, we extended our analysis about physiological consequences by characterizing effects of tolerization on -GalCer-induced liver injury. Moreover, whereas Parekh et al. [³⁵ ] used splenic NKT cells in the majority of experiments, we focused on intrahepatic NKT populations. Also, in this regard, our work accounts for completing the comprehension of -GalCer tolerance, as presumably, there are profound differences between splenic and hepatic NKT cells (see, e.g., refs. [^{52 53 54} ]).

Intrahepatic NKT cell numbers
As NKT cells are the critical cell population that mediates -GalCer-induced liver injury and cytokine production, their absence could mimic a tolerogenic phenotype. It is well known that mouse NKT cells become transiently undetectable in the liver rapidly after activation by Con A [³¹ ], anti-CD3 [⁵⁵ ], or -GalCer [²⁹ , ^{56 57 58 59} ] treatment. Originally, this phenomenon was attributed to activation-induced death of NKT cells [⁵⁵ , ⁵⁶ ]. However, more recently, it has been shown that their activation is associated with transient down-modulation of the TCR complex and NK1.1, both of which are used for flow cytometric characterization [^{57 58 59} ], and that actually NKT cells are not persistently depleted. These results, indicating that -GalCer injection does not result in pronounced, persistent depletion of the intrahepatic overall NKT cell population could be reproduced in our experiments. Although the number of liver NKTs was somewhat decreased later on at Day 8 after pretreatment, at Day 3, when pretreated mice were also tolerized against -GalCer-induced liver injury, the intrahepatic NKT cell number of -GalCer-pretreated mice had even exceeded that of vehicle-treated control mice. This increase in intrahepatic NKT cell numbers at Day 3 after -GalCer injection corresponds to recently described kinetics of NKT cell expansion in spleen and/or liver, peaking 3 days after treatment, before returning to approximate normal levels [³⁶ , ³⁷ , ⁵⁷ , ⁵⁸ ]. In particular, Uldrich et al. [³⁷ ] have shown that the expansion of NKT populations in spleen and liver is dependent on costimulatory signaling by CD40 and CD28, whereas the subsequent contraction of these populations requires the proapoptotic Bcl-2 family member Bim. This expansion of the intrahepatic NKT population at Day 3, i.e., at a time-point where we had observed -GalCer-hepatitis protection, excluded pronounced, -GalCer-induced depletion of NKT cells as the main cause for this tolerance.

Active immune modulation in -GalCer tolerance?
Immunomodulatory leukocytes and immunosuppressive cytokines are important factors in a large number model of active immunological tolerance. Upon analysis of the cytokine responses to -GalCer in glycolipid- or vehicle-pretreated mice, we had found that in contrast to most other cytokines, there was a tendency to increase production of IL-10 in tolerized mice, one of the most prominent inhibitory cytokines. Moreover, IL-10 was recently found to be important for NKT cell activation-induced protection from experimental autoimmune encephalomyelitis [⁶⁰ ]. IL-10 also plays a role in the development of cellular tolerance, as it has been demonstrated to be important for the differentiation of Tr1 T_regs (reviewed, e.g., in ref [⁶¹ ]). However, the missing interference of exogenous IL-10 on -GalCer-induced liver injury as well as the possibility to induce tolerance in IL-10^–/– mice, demonstrated in this work, suggest that this cytokine is probably not an essential mediator of -GalCer tolerance. This was further supported by the observation that multiple vaccination of C57BL/6 mice with -GalCer-loaded DCs caused vast expression of IL-10 upon subsequent -GalCer injection but did not affect liver injury or proinflammatory cytokine production (Biburger, Tiegs, Manfred Lutz, and C. Wiethe, unpublished data).

KCs are not only responsible for phagocytic removal of dying cells but also for induction and maintenance of tolerance, as has been shown, e.g., in rat models of hepatic [⁶² ] or cardiac [⁶³ , ⁶⁴ ] allotransplantation. They have been described to induce apoptosis of liver-infiltrating, high-affinity CD8⁺ T cells [⁶⁵ ] and can produce immunoregulatory factors such as IL-10 [⁶⁶ ] as well as TNF-, IL-6, TGF- and -β, NO, and reactive oxygen species [⁶⁷ ]. However, we found that KC depletion by clodronate liposomes prior to tolerogenic -GalCer pretreatment did not affect -GalCer-induced tolerance to -GalCer-mediated hepatitis, thereby excluding an important role of intrahepatic macrophages in the development of -GalCer tolerance.

CD4⁺CD25⁺ T_regs are well known for their ability to confer immunological tolerance in humans and several mouse models. A common method for characterization of potential involvement of CD4⁺CD25⁺ T_regs in immunological processes in mice is the functional impairment of their regulatory function by injecting anti-CD25 mAb such as clone PC61.5. The prevention of CD4⁺CD25⁺ T_reg function upon this anti-CD25 treatment is attributed in the literature to depletion (e.g., ref. [⁶⁸ ]), down-regulation of CD25 associated with the functional inactivation of CD4⁺CD25⁺ T_regs (e.g., refs. [⁶⁹ , ⁷⁰ ]), or both [⁷¹ ]. Identification of CD25 down-regulation as a result of anti-CD25 treatment in the recent literature has been enabled as a consequence of the identification of FoxP3 as an additional, relevant, CD25-independent T_reg marker. In agreement with several reports, we also detected disappearance of CD25⁺ cells among CD4 lymphocytes without significant loss of CD4⁺FoxP3⁺ cells within the first day after PC61.5 treatment (without extending this analysis to longer periods). This supports the cumulating assessment in the literature of impairment of the regulatory capacities of CD4⁺CD25⁺ T_regs by anti-CD25 treatment (which we had also observed in a model of Con A tolerance [³⁸ ]) to be caused by functional inactivation associated with CD25 down-regulation rather than (or at least parallel to) T_reg depletion.

Recently, Ly et al. [⁷² ] described that protection from type 1 diabetes by -GalCer-activated, invariant NKT cells requires the activity of CD4⁺CD25⁺ T_regs and suggested that T_regs would regulate activation and anergy induction of NKT cells. The latter notion was based on data revealing significantly reduced IL-2 responses of splenocytes from -GalCer-pretreated mice versus vehicle-pretreated mice to in vitro -GalCer restimulation. These in vitro responses were claimed to be restored when CD4⁺CD25⁺ T_regs had been inactivated in the -GalCer-pretreated mice by anti-CD25 treatment. However, we do not fully agree with this interpretation, as the respective experiments reveal such an effect for IL-2 but for neither IFN- nor IL-4. In addition, the discussed in vitro IL-2 production of splenocytes from -GalCer-pretreated mice does not, in fact, appear to be caused by specific NKT cell activation at all, as the in vitro IL-2 production in response to -GalCer restimulation is virtually identical to that in response to vehicle in the anti-CD25- or control IgG-treated group, respectively. Thus, it rather appears that in splenocytes from -GalCer-pretreated mice, there is no specifically -GalCer-induced IL-2 production at all (the same appears to be true for IFN-), indicating that NKT cells are fully nonresponsive with regard to IL-2 production, independent from the presence or absence of T_reg activity.

This interpretation—together with our results that pretreatment with PC61.5 did not interfere with development of tolerance in vivo in our experiments, and purified T_regs did not suppress NKT cell activation, even under close cell–cell contact—prompts us to suppose that T_regs are not critically involved in -GalCer tolerance. Recently, in agreement with our results in mice, Jiang et al. [⁷³ ] provided evidence that in contrast to conventional T cells, which are susceptible to CD4⁺ CD25⁺ regulatory cell suppression, freshly isolated, human V24⁺ NKT cells were able to produce cytokines in the presence of CD4⁺CD25⁺ T_regs [⁷³ ]. It is not impossible, however, that as suggested by Ly et al. [⁷² ] (see above), T_regs might modulate the overall outcome of NKT cell activation in vivo (with regard to, e.g., cytokine production and activation of T, B, and NK cells), presumably by indirect effects such as modulation of third-party cells, like APCs.

It should be noted that our experiments, like those in all other reports based on techniques such as anti-CD25 treatment or T_reg purification using positive selection for CD25⁺ cells, do not account for a potential contribution of CD25-negative T_regs.

We had performed in vitro assays with liver MNCs to test whether any other active regulatory factors might have been evoked in this population by the tolerizing -GalCer pretreatment in vivo. However, whereas liver MNCs from tolerized mice revealed a pronounced suppression of cytokine responses to restimulation, cytokine production by nontolerized MNCs was not affected by coculture with respective cells from -GalCer-tolerized mice in a 1:1 ratio. This suggests that -GalCer-induced tolerance is not based on active, transferable factors but rather on a passive effect such as hyporesponsiveness. In principle, apparent overall hyporesponsiveness of the entirety of NKT cells might be caused by development of an actively suppressive subpopulation among activated NKT cells, by a phenotypic switch in the resident population, or repopulation with different NKT populations that could restrain activation of, in principle activatable, NKT cells. Parekh et al. [³⁵ ] had suggested nonresponsiveness of NKT cells as the basis of observed deficiencies in proliferation and cytokine production after -GalCer pretreatment, which would not enclose bystander suppression. This was assessed by coculture of splenocytes from -GalCer-pretreated and naïve mice, in principle, similar to coculture of liver MNCs, as accomplished in this work. However, the frequency of NKT cells among liver MNCs used in our study is below 20% and is even much lower among splenocytes used by Parekh et al. [³⁵ ] and in addition, differs between splenocytes from tolerized and nontolerized mice (3.4–3.9% among B220-negative splenocytes from naïve mice and 1.6–1.7% from mice pretreated 1 month earlier with -GalCer [³⁵ ]). In their physiological environment in vivo (e.g., as a result of local accumulation at relevant venues), immunoregulatory leukocytes may be able to exert efficient suppression, even if being present at rather low frequencies in the respective total organ/compartment. In vitro, however, such low frequencies might possibly be insufficient to reveal significant suppression. Thus, it cannot be excluded that a NKT cell subpopulation, which might have developed active, tolerogenic features upon -GalCer pretreatment, would have been ignored in these in vitro coculture assays as a result of the already low frequency of NKT cells and their even lower frequency in the tolerized group. This was the reason for us to cocultivate highly purified NKT cells from tolerized mice with those from nontolerized mice to assess a potential influence of conceivable regulatory NKT cells. These analyses represent an amendment to the important experiments of Uldrich et al. [³⁷ ] and Parekh et al. [³⁵ ]: Uldrich and coworkers [³⁷ ] used purified NKT cells from tolerized or nontolerized mice together with DCs but did not analyze potential transdominantly suppressive NKT cells by cocultivating NKT cells from both groups together. Inversely, Parekh et al. [³⁵ ], in addition, cocultivated NKT cells from both groups together but apparently only in the form of splenocytes with rather low and varying NKT frequencies as discussed above.

The results of our coculture experiments with highly purified NKT cells from nontolerized and tolerized mice convincingly demonstrated that -GalCer pretreatment does not induce a transactive, suppressive capacity among liver NKT cells.

Activation-induced nonresponsiveness
Taken together, the in vivo and ex vivo results clearly demonstrate that in contrast to Con A tolerance [³⁸ ], -GalCer-induced protection from liver injury and cytokine boost upon -GalCer restimulation is not mediated by active tolerogenic factors but independent from such factors by activation-induced nonresponsiveness of liver NKT cells. Thus, in summary, features of -GalCer-induced, long-term protection from -GalCer hepatitis appear to be fundamentally different from protection from Con A hepatitis [³⁸ ] (see comparison in Table 1 ) as well as those of Th2-dependent, -GalCer-induced protection from LPS shock [⁴⁴ ] and experimental autoimmune encephalomyelitis [⁶⁰ ] or T_reg-dependent protection from type 1 diabetes by -GalCer-activated, invariant NKT cells [⁷² ]. Our results with IL-10 knockout mice and KC depletion also show that these factors are not only dispensable as active factors but are also not relevant in the process of NKT anergization.

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Table 1. Comparison of Relevant Factors in -GalCer and Con A Hepatitis and Tolerance Models

Concomitants and potential elicitors of hyporesponsiveness induction
Upon analysis of phenotypic changes of the intrahepatic NKT population during tolerance, we found that 8 days after initial -GalCer injection, the frequency of CD4⁺ NKT among intrahepatic lymphocytes was reduced significantly, whereas the frequency of CD4^– NKT cells remained unchanged. This suggested that there may be a pronounced modulation in the allocation of different NKT subpopulations in the liver. This could be caused by differences in migration/retention as a result of divergent equipment with chemokine receptors or by distinct responses with respect to -GalCer-induced expansion/contraction. The observation that in the state of -GalCer-induced tolerance, the size of the CD4-negative population remains largely unaffected may be of pronounced importance for the attempts to use -GalCer as an antitumoral agent: In a s.c. sarcoma model, using sarcoma cell line MCA-1, Crowe et al. [⁵² ] demonstrated that among all NKT cell subsets tested, the CD4^– fraction of liver-derived NKT cells was mostly, if not completely, responsible for NKT-mediated tumor rejection. Also, in a lung metastases model using B16F10 melanoma cells, the authors found the hepatic CD4^– NKT subpopulation to be an efficient mediator of -GalCer-induced antitumor immunity, whereas other NKT subsets, including hepatic CD4⁺ NKT cells, were shown to be less potent [⁵² ].

In addition to the modulation of CD4+/– NKT cell populations in tolerized mice, we detected reduced expression of -GalCer-specific TCR on NKT cells. In a state of tolerance, 8 days after -GalCer injection, an increased proportion of NKT cells also failed to express high amounts GITR on their surface, which has been shown to be a potent costimulatory molecule for NKT cells and to be temporarily up-regulated for 3–4 days after an -GalCer encounter [⁴⁹ ].

As an interesting side-effect, the observation that GITR is substantially expressed by virtually all NKT cells (at least prior to tolerization), but only a small fraction of conventional T cells, may qualify this molecule as a novel marker for NKT cells, which in combination with additional markers, may aid and ameliorate NKT cell detection and identification. In comparison with the GITR staining using DTA-1 mAb, as shown in the work of Kim et al. [⁴⁹ ], staining with the anti-GITR clone YGITR 765 used in this work appeared to facilitate distinct discrimination between GITR-positive and -negative cells (as noticed for differentiation between conventional T cells and NKT cells in liver MNC preparations; not shown).

It is conceivable that in -GalCer-tolerized mice, GITR^–/low NKT cells and also, in particular, cells with reduced TCR may have an increased threshold for activation. In contrast to down-modulation of TCR and costimulatory GITR, expression of inhibitory Ly49I was increased on liver NKT cells in tolerized mice.

In summary, these data suggest that -GalCer-induced nonresponsiveness is accompanied and probably caused to some degree by variations of the intrahepatic repertoire of NKT subpopulations that may differentially respond to -GalCer and a shift of the balance between stimulatory and inhibitory receptors in favor of NKT cell suppression, as depicted in the model in Figure 8 . As none of the observed phenotypic changes appears to affect the NKT cells as a whole, it is tempting to speculate that to some degree, different subpopulations might be modulated by different mechanisms. However, this does not exclude the possibility that persistent changes of intracellular signaling pathways might contribute to -GalCer nonresponsiveness.

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Figure 8. Schematic model of tolerance induction by -GalCer pretreatment. An initial injection of -GalCer induces pronounced cytokine production by NKT cells including IFN-, IL-4, and TNF-, and the latter is an important mediator of liver injury [30 ]. This initial activation leads to phenotypical changes including down-regulation of activating receptors and up-regulation of inhibitory receptors on single cells or within the entire population with respect to changes in frequencies of the respective positive or negative NKT cell subsets. This, possibly together with additional modifications of signaling pathways, may render NKT cells nonresponsive to restimulation, resulting in reduced cytokine production and liver injury. NKT cells are represented as segments of cells to refer to the fact that variations in surface expression may take place on individual cells [such as for: TCR (glycolipid-specific TCR with invariant v chain) expression] and/or by changes in the ratios of positive/negative cells (such as for CD4), e.g., by alterations in their individual homing and/or proliferation.

In the literature, there are some discrepancies with respect to -GalCer-induced modulation of inhibitory receptors on NKT cells. Ota et al. [⁷⁴ ] reported an increased expression of inhibitory members of receptor families CD94/NKG2 (mainly NKG2A) and Ly49 (without distinguishing among Ly49-A, -C, -I, or -G2) on hepatic NKT cells 5–7 days after -GalCer injection. Moreover, the authors suggested an important role for interaction between NKG2A and its ligand Qa-1^b in negatively regulating NKT responses to rechallenge subsequent to -GalCer pretreatment [⁷⁴ ]. In contrast, Uldrich et al. [³⁷ ] found virtually no modulation of inhibitory Ly49 receptors (measuring expression of Ly49-A, -C/I, or -G2) on splenic NKT cells 1 or 2 weeks after -GalCer injection and even detected a persistent down-modulation of an inhibitory CD94/NKG2A (as measured by using antibodies against CD94, NKG2A, or NKG2A/C/E) receptor that persisted for at least 2 weeks on these cells. In the present work, we detected a significant increase in the frequency of Ly49-I-expressing liver NKT cells 8 days after -GalCer injection and could thus substantiate the results of Ota et al. [⁷⁴ ] on Ly49-A/C/I/G2. However, whereas Ota et al. [⁷⁴ ] reported an IFN--mediated up-regulation of NKG2A ligand Qa-1^b as a relevant factor for down-modulation of NKT cell responsiveness, in our hands, IFN- neutralization during -GalCer pretreatment did not interfere with its tolerogenic effect. Yet, these results are not contradictory, as in our experiments, IFN- activity was neutralized only at the time of tolerance induction, and tolerized cells were restimulated in vitro, whereas Ota et al. [⁷⁴ ] mainly characterized effects of persistent IFN- neutralization, e.g., by using IFN-^–/– mice or in vitro experiments in the presence of neutralizing antibodies. Thus, -GalCer-induced IFN- may actually transiently modulate the balance between stimulatory and inhibitory NK signals [⁵⁰ , ⁷⁵ ] and thereby, contribute to down-modulation of NKT cell responsiveness (e.g., by interaction of IFN--induced Qa-1^b with -GalCer-up-regulated NKG2A [⁷⁴ ]) but is probably not an important factor for the initiation of NKT cell anergy.

Fujii et al. [⁷⁶ ] had described that in contrast to injection of free -GalCer, an injection of -GalCer-loaded DCs does not induce hyporesponsiveness but rather, supports NKT cell activation. This had led to the conclusion that the outcome of an initial activation of NKT cells—anergy or pronounced capability of restimulation—may depend on the type of -GalCer-presenting cells; e.g., in the liver, the organ with the largest NKT population, the vast majority of cells that present previously injected -GalCer to liver-resident or circulating NKT cells may probably be hepatocytes. They are known to express CD1d [⁷⁷ ] but are clearly no highly specialized APCs. In comparison with DCs, hepatocytes have been shown to evoke only a limited repertoire of NKT cell responses to -GalCer [⁷⁸ ]. Thus, upon -GalCer injection, a large number of NKT cells might actually be stimulated under suboptimal conditions such as inappropriate costimulation. Activation by -GalCer without appropriate costimulation, which has been shown to modulate NKT cell responses [³⁷ ], might render NKT cells nonresponsive, analogous to the well-known anergy of conventional T cells.

As TNF- was pivotal in -GalCer-induced, NKT-mediated liver injury and appeared to play a contrary role to IFN- [³⁰ ], we wondered whether TNF- neutralization might affect not only hepatocyte damage [³⁰ ] but also the onset of hyporesponsiveness. In addition, we had found that -GalCer-induced FasL up-regulation on NKT cells was reduced under conditions of TNF- neutralization [³⁰ ], indicating a feedback signaling of -GalCer-induced TNF- on NKT cells. TNF- neutralization during -GalCer tolerization, to some extent but not significantly, abrogated hyporesponsiveness induction, as revealed by increased cytokine responses of respective liver MNCs upon -GalCer rechallenge. This indicates that TNF- might act auxiliary to a limited degree but is also not an essential factor for the development of NKT cell hyporesponsiveness upon -GalCer injection.

Implications for NKT cell investigation and their therapeutic use
There is compelling evidence in the literature that NKT cells can inhibit or augment pathologic processes by activation or suppression of immunologic processes in disease- and stage-specific manners (reviewed in ref. [¹³ ]). Our results about -GalCer-induced liver injury [³⁰ ] and -GalCer-induced protection from hepatitis induction (this work) now round off these findings by demonstrating that both outcomes are not excluding one another but can appear within the same disease model. Up- or down-modulation of NKT-based immune responses with their different effects on pathology can be two faces/phases of the same process, and primary activation is followed by a state of hyporesponsiveness.

The phenomenon of -GalCer-induced nonresponsiveness raises the necessity to thoroughly investigate NKT cell activation states in all experimental approaches comprising repeated cycles of NKT cell stimulation. It is necessary to show whether NKT cells are predominantly activated or rather rendered hyporesponsive by these procedures, as this will be crucial to correctly judge whether NKT cells act as promoters or suppressors in the respective model and at the relevant time-points.

As mentioned above, -GalCer has been used in clinical trials for cancer treatment [^{8 9 10 11 12} ]. If -GalCer-induced nonresponsiveness to subsequent -GalCer applications should also occur in humans, this would bear important implications for the use of -GalCer as a therapeutic agent for tumor treatment. In addition, -GalCer tolerance might serve as a model for the frequently found nonresponsiveness of NKT cells in tumor patients (reviewed in ref. [⁷⁹ ]), which might be caused by repeated contact with NKT cell-activating tumor antigens. Thus, improved knowledge of underlying mechanisms and identification of methods that overcome -GalCer tolerance might help to develop strategies for reactivation of hyporesponsive NKT cells against tumor entities.

If our observation that -GalCer treatment provokes differential modulation of total numbers of NKT subsets in mice also holds true for humans, this may also be highly relevant for NKT-based tumor therapy, as diverse NKT subpopulations have also been described repeatedly in humans to exert antithetic effects such as anti-tumor immunosurveillance or immune suppression.

NKT cells do not appear to have only beneficial effects such as suppression of autoimmunity or tumor repression. In fact, there is cumulating evidence that NKT cells may participate in the onset of a remarkable number of experimental pathological processes such as atherosclerosis, airway inflammation, asthma, ulcerative colitis, arthritis, lupus, experimental autoimmune encephalomyelitis, and the onset of several hepatic disorders as described above [^{15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30} ]. Injection of free -GalCer as a strategy for NKT cell decommissioning bears the risk of disadvantageous side-effects by NKT cell activation. However, as anergy induction is mainly unaffected by neutralization of the key cytokine TNF- (or IFN- as well), -GalCer treatment with simultaneous, anticytokine treatment might anergize NKT cells without respective cytokine-mediated side-effects.

Hence, we suggest the induction of persistent nonresponsiveness of NKT cells as a potential therapeutic approach for diseases or disease models, where inadequate NKT cell activation is involved in pathogenesis.

 
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants TI 169/6-3 and -4. The perfect technical assistance of Sonja Heinlein and Andrea Agli is gratefully acknowledged. We thank Kirin Brewery Co., Ltd. (Japan), for providing -GalCer and Prof. Dirk Busch (Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany) for -GalCer/CD1d tetramers.

Received June 6, 2007; revised February 29, 2008; accepted March 11, 2008.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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