Protective Roles of B and T Lymphocyte Attenuator in NKT Cell-Mediated Experimental Hepatitis

Arifumi Iwata, Norihiko Watanabe, Yoshihiro Oya, Takayoshi Owada, Kei Ikeda, Akira Suto, Shin-ichiro Kagami, Koichi Hirose, Hiroko Kanari, Saki Kawashima, Toshinori Nakayama, Masaru Taniguchi, Itsuo Iwamoto and Hiroshi Nakajima
J Immunol January 1, 2010, 184 (1) 127-133; DOI: https://doi.org/10.4049/jimmunol.0900389

Abstract

Although B and T lymphocyte attenuator (BTLA) was originally identified as an inhibitory coreceptor selectively expressed on Th1 cells and B cells, recent studies have revealed that BTLA is expressed on a variety of cells, including macrophages, dendritic cells, and NK cells, and modulates their functions. However, the role of BTLA in the regulation of NKT cell function remains unknown. In this study, we found that BTLA was expressed on NKT cells at the levels similar to those on T cells and that BTLA-deficient (BTLA−/−) NKT cells produced larger amounts of IL-4 and IFN-γ upon α-glactosylceramide stimulation as compared with wild-type (WT) NKT cells. In vivo, BTLA−/− mice produced larger amounts of IL-4 and IFN-γ upon Con A injection and were more susceptible to Con A-induced hepatitis than WT mice. In addition, the augmentation of Con A-induced hepatitis in BTLA−/− mice was not observed in BTLA/NKT-double deficient mice. Moreover, NKT−/− mice reconstituted with BTLA−/− NKT cells were significantly more susceptible to Con A-induced hepatitis as compared with NKT −/− mice reconstituted with WT NKT cells. These results suggest that BTLA functions as the inhibitory coreceptor of NKT cells and plays a critical role in the prevention of NKT cell-mediated liver injury.

Signals delivered through stimulatory and inhibitory coreceptors regulate lymphocyte activation in collaboration with primary AgR signals. Stimulatory coreceptors include CD28 and inducible T cell costimulator (ICOS), whereas inhibitory coreceptors include CTLA-4, programmed cell death 1 (PD-1), and B and T lymphocyte attenuator (BTLA) (1, 2). Accumulating evidence indicates that the balance between stimulatory and inhibitory cosignals is crucial for the effective immune responses to pathogens and the maintenance of self-tolerance (1, 2).

BTLA has originally been identified as an inhibitory coreceptor selectively expressed on Th1 cells and B cells (3). Thereafter, flow cytometric analyses using monoclonal Abs against BTLA have revealed that BTLA is expressed on certain lymphocyte subsets including γδ T cells and regulatory T cells as well as on some APCs such as macrophages and dendritic cells (DCs) (4, 5). BTLA has also been reported to be expressed at low levels on NK cells (4, 6). More recently, it has been shown that a TNFR family member herpesvirus entry mediator (HVEM) is a ligand for BTLA (5, 7, 8) and that the ligation of BTLA with HVEM transduces inhibitory cosignals (5).

In vivo function of BTLA has recently been addressed using BTLA-deficient (BTLA−/−) mice. Initially, we have shown that the sensitivity to experimental autoimmune encephalomyelitis as well as T cell-dependent Ab responses is increased in BTLA−/− mice (3). It has also been reported that BTLA−/− mice exhibit a rapid rejection of partially MHC-mismatched cardiac allograft (9), persistent allergic airway inflammation following Ag challenge (10, 11), and an acceleration of experimental colitis (12). These findings indicate that BTLA is crucial for dampening immune responses mediated by T cells. Moreover, we have recently shown that aged BTLA−/− mice spontaneously develop autoimmune hepatitis-like disease with an increase of NKT cells in the liver (13), suggesting that BTLA may prevent autoimmune hepatitis through the inhibition of NKT cell function.

NKT cells are characterized by coexpression of T cell markers such as TCR and NK cell markers such as NK1.1 (14). In mice, the majority of NKT cells express an invariant Vα14 TCR, which is essential for their development (14), and recognizes a specific ligand, α-galactosylceramide (α-GalCer), presented on CD1d molecules (14, 15). NKT cells rapidly produce both IL-4 and IFN-γ on activation (15, 16) and play a crucial role in various immune responses, including antitumor immunity, allergic reaction, and autoimmune diseases (14). Although the roles of stimulatory coreceptors in NKT cell function have been addressed (17, 18), the role of inhibitory coreceptors including BTLA in NKT cell function remains largely unknown.

In this study, we examined the role of BTLA in the regulation of NKT cell function. We found that BTLA was expressed on NKT cells at the levels similar to those on T cells. BTLA−/− NKT cells produced larger amounts of IL-4 and IFN-γ on α-GalCer stimulation as compared with wild-type (WT) NKT cells. Importantly, BTLA−/− mice were highly susceptible to Con A-induced hepatitis, in which NKT cells have been reported to play pathogenic roles (19, 20). Indeed, the augmentation of Con A-induced hepatitis in BTLA−/− mice was not observed in BTLA/NKT-double deficient mice. In addition, we found that NKT cell-deficient (NKT−/−) mice reconstituted with BTLA−/− NKT cells were significantly more susceptible to Con A-induced hepatitis as compared with NKT −/− mice reconstituted with WT NKT cells. Our results indicate that BTLA functions as the inhibitory coreceptor in NKT cells and thus prevents NKT cell-mediated tissue damage.

Materials and Methods

Mice

BTLA−/− mice (3) were backcrossed over eight generations onto C57BL/6 mice (Charles River Laboratories, Kanagawa, Japan). NKT−/− mice (Jα281−/− mice) on a C57/BL6 background were described previously (21). NKT −/− mice were crossed with BTLA−/− mice, and the offspring were intercrossed to obtain BTLA−/− NKT −/− mice. All mice were housed in microisolator cages under specific pathogen-free conditions, and the mice were used for the experiments at 6–12 wk of age. Animal procedures in this study were approved by the Chiba University Animal Care and Use Committee.

Flow cytometry

The following Abs were purchased from BD Biosciences (San Diego, CA): anti-CD3ε FITC, PE (145-2C11), anti-NK1.1 PE (PK136), anti-CD45R/B220 FITC, PE (RA3-6B2), anti-TCR β-chain FITC, PE (H57-597), anti-CD8α FITC (53-6.7), anti-CD11b FITC (M1/70), anti-CD11c FITC (HL3), anti-CD25 FITC (7D4), anti-CD69 FITC ([1H].2F3), anti-CD122 FITC (TM-β1), anti-Fas biotin (Jo2), anti-Fas ligand biotin (MFL3), streptavidin-PE, and streptavidin-allophycocyanin. Anti-BTLA PE (6F7) and anti-BTLA Alexa Fluor 647 (8F4) were purchased from eBioscience (San Diego, CA). After FcRs were blocked with anti-CD16/32 mAb (BD Biosciences), cells were stained with indicated Abs and analyzed on a FACSCalibur (BD Biosciences) using CellQuestPro software (BD Biosciences).

Preparation of allophycocyanin-conjugated α-GalCer/CD1d–dimer

Allophycocyanin-conjugated α-GalCer/CD1d–dimer was prepared as described previously (22). In brief, 2.75 ml α-GalCer (200 mg/ml; Kirin Pharma, Tokyo, Japan) and 6 ml mouse CD1d-Ig fusion protein (0.5 mg/ml; BD Biosciences) was conjugated at 37°C overnight. The α-GalCer/CD1d–Ig conjugates were then incubated with allophycocyanin-conjugated anti-mouse IgG1 (X56; BD Biosciences) for 60 min. Free allophycocyanin-conjugated anti-mouse IgG1 in the mixture was blocked by the addition of excess amounts of control mouse IgG1 mAb (A111-3; BD Biosciences) for 30 min at room temperature.

Preparation of mononuclear cells from the liver

The livers were removed from the mice after perfusion through the portal vein and inferior vena cava with PBS. Each liver was cut into small pieces, passed through a stainless steel mesh, and suspended in RPMI 1640 medium for 3 min. Mononuclear cells were then harvested from the supernatants and enriched by Percoll (GE Healthcare UK, Little Chalfont, United Kingdom) gradient centrifugation according to the manufacturer's instruction.

Preparation of intrahepatic NKT cells by magnetic-activated cell sorting

Liver mononuclear cells were incubated with anti-CD16/32 to block nonspecific binding and then stained with a mixture of FITC-conjugated Abs against B220, CD8α, CD11b, and CD11c, and allophycocyanin-conjugated α-GalCer/CD1d dimer. FITC-positive cells were depleted using anti-FITC MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. The remaining cells were then incubated with anti-allophycocyanin MicroBeads (Miltenyi Biotec), and allophycocyanin-positive cells were positively collected twice by magnet cell sorting. The purity of collected cells was determined by flow cytometry and was routinely >95% of TCR-β+ α-GalCer+ cells.

Coculture of NKT cells and α-GalCer–loaded cells

α-GalCer–loaded cells were prepared as described elsewhere (22) with a minor modification. Single cell suspension of splenocytes was irradiated (30 gray) and incubated with α-GalCer (10 or 100 ng/ml) in complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 5.5 μM β-mercaptoethanol, 2 mM l-glutamine, nonessential amino acids, and antibiotics) for 12 h. Hepatic NKT cells (5 × 104) were enriched by MACS as described above and were cocultured with α-GalCer loaded cells (5 × 104) in complete RPMI1640 medium in 96-well round bottom plates for 36 h.

Proliferation assay

The proliferation of NKT cells was measured using CellTiter-Glo reagent according to the manufacturer’s instruction (Promega, Madison, WI).

Con A-induced hepatitis

Con A (Sigma-Aldrich, St. Louis, MO) was dissolved in pyrogen-free PBS and injected into the mice (10 or 20 mg/kg i.v.). Sera were collected from the individual mice at the indicated time after Con A injection. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured by standard protocols (SRL, Tokyo, Japan).

Measurement of cytokine levels by enzyme-linked immunosorbent assay

The levels of TNF-α, IL-4, IFN-γ, and IL-10 in the sera and the supernatants were measured by ELISA kits according to the manufacturer's protocols (TNF-α, IL-4, and IL-10 kits from BD Biosciences; and IFN-γ kit from R&D Systems, Minneapolis, MN). The minimum significant value of the assay was 15 pg/ml TNF-α and IL-4, 30 pg/ml IFN-γ and IL-10.

Adoptive transfer of NKT cells

Purified intrahepatic NKT cells from WT mice or BTLA−/− mice were injected into the liver of NKT −/− mice (1× 106 cells/mouse) as described previously (19). One hour later, the mice were injected i.v. with Con A (10 mg/kg). Sera were obtained at 12 and 24 h after Con A injection.

Statistical analysis

Data are summarized as mean ± SD. The statistical analysis of the results was performed by the unpaired t test. p values < 0.05 were considered significant.

Results

BTLA is expressed on NKT cells but is dispensable for the development and maintenance of NKT cells

To determine whether BTLA is involved in the development and function of NKT cells, we first examined the expression of BTLA on NKT cells. Mononuclear cells from thymus, spleen, and liver in C57BL/6 mice were stained with anti-BTLA mAb (6F7), and the expression levels of BTLA on each cell type were evaluated by flow cytometry. As shown in Fig. 1, the expression of BTLA was detected on NKT cells, which were defined as CD3+ α-GalCer/CD1d-dimer+ cells, at the levels similar to those on CD3+ T cells. Consistent with previous reports (3, 6), BTLA was expressed at higher levels on B220+ B cells and at lower levels on NK cells (Fig. 1).

FIGURE 1.
FIGURE 1.

BTLA is expressed on NKT cells at the levels similar to those on CD3+ T cells. Mononuclear cells from thymus, spleen, and liver in C57BL/6 mice were stained with anti-BTLA mAb. Representative histograms of BTLA expression (solid line) on NKT cells (CD3+ α-GalCer/CD1d–dimer+), T cells (CD3high α-GalCer/CD1d–dimer−), B cells (CD3− B220+), and NK cells (NK1.1+ CD3−) are shown (n = 4 mice each). Shaded areas indicate control staining with isotype-matched Ab.

We next examined the development and activation state of NKT cells in BTLA−/− mice. As previously demonstrated (3), the numbers of thymocytes and splenocytes in BTLA−/− mice at 8 wk old were comparable to those in WT mice (data not shown). Flow cytometric analysis revealed that the frequencies of NKT cells (TCRβ+ α-GarCer/CD1d+cells) in the thymus and spleen of BTLA−/− mice were comparable to those in WT mice (Fig. 2A). The number of NKT cells in the livers of BTLA−/− mice was also similar to that in WT mice (Fig. 2A). In addition, the expression levels of activation markers such as CD25, CD69, and CD122 on NKT cells were similar between BTLA−/− mice and WT mice (Fig. 2B). Together, these results indicate that BTLA is dispensable for the development and the maintenance of steady state of NKT cells.

FIGURE 2.
FIGURE 2.

Normal development of NKT cells in BTLA−/− mice. Mononuclear cells from thymus, spleen, and liver in WT mice and BTLA−/− mice were stained with anti–TCR-β PE, allophycocyanin-conjugated α-GalCer/CD1d–dimer, and FITC-conjugated Abs against CD25, CD69, or CD122 and analyzed by flow cytometry. A, Representative FACS profiles of TCR-β versus α-GalCer/CD1d–dimer staining (left panels), and the percentages of TCR-β+ α-GalCer/CD1d–dimer+ cells (right panels) are shown. Data are means ± SD for 4 mice in each genotype. B, Representative histograms for CD25, CD69, and CD122 expression on TCR-β+ α-GalCer/CD1d–dimer+ cells are shown (n = 4 mice in each genotype). Dotted lines indicate control staining with isotype-matched Ab.

BTLA-deficient NKT cells are hyperreactive to Ag stimulation in vitro

We next examined whether BTLA regulated NKT cell function. Purified NKT cells from the livers of WT mice and BTLA−/− mice were stimulated with α-GalCer–loaded APCs for 36 h, and the levels of IFN-γ, IL-4, and TNF-α in the culture supernatants were measured by ELISA. In response to α-GalCer stimulation (100 ng/ml), BTLA−/− NKT cells secreted significantly larger amounts of IFN-γ and IL-4, compared with WT NKT cells (IFN-γ: BTLA−/− 14.9 ± 7.9 versus WT 3.7 ± 2.5 ng/ml; n = 6; p < 0.05; IL-4: BTLA−/− 1.8 ± 0.5 versus WT 1.0 ± 0.4 ng/ml; n = 6; p < 0.05; Fig. 3A). TNF-α was not detected in the culture supernatants of WT NKT cells and BTLA−/− NKT cells in the experimental condition (data not shown). In addition, BTLA−/− NKT cells showed enhanced proliferation in response to α-GalCer stimulation, compared with WT NKT cells (Fig. 3B). These results indicate that BTLA−/− NKT cells are hyperreactive to antigenic stimulation, suggesting that BTLA functions as an inhibitory coreceptor in NKT cell activation.

FIGURE 3.
FIGURE 3.

Increased activation of BTLA−/− NKT cells on AgR stimulation in vitro. NKT cells (5 × 104) were purified from the liver of WT and BTLA−/− mice and cocultured with α-GalCer–loaded irradiated splenocytes (5 × 104) as APCs for 36 h. A, The levels of IFN-γ and IL-4 in the culture supernatants were measured by ELISA. Data are means ± SD for 6 mice in each group. *p < 0.05. B, Proliferation of NKT cells was determined using CellTiter-Glo reagent. Data are means ± SD for 6 mice in each group. *p < 0.05.

BTLA−/− mice are highly susceptible to Con A-induced hepatitis

Con A-induced hepatitis is a widely used mouse model that resembles autoimmune hepatitis in humans in many aspects (23). The development of hepatitis after Con A injection has been shown to be attenuated in mice lacking NKT cells (19, 20), indicating that NKT cells are involved in causing Con A-induced hepatitis. We therefore chose this model to test the function of BTLA expressed on NKT cells in vivo. When WT mice and BTLA−/− mice were injected i.v. with Con A (20 mg/kg), all BTLA−/− mice died by 24 h after injection, whereas all WT mice survived over 48 h (Fig. 4A). These results indicate that BTLA−/− mice are highly susceptible to Con A-induced hepatitis.

FIGURE 4.
FIGURE 4.

Increased susceptibility to Con A-induced hepatitis in BTLA−/− mice. A, BTLA−/− mice (solid line) and WT mice (dotted line) were injected i.v. with Con A (20 mg/kg), and the survival of the mice was evaluated for 48 h (n = 5 mice in each group). BTLA−/− mice and WT mice were injected i.v. with a sublethal dose of Con A (10mg/kg). B, The liver was excised at 24 h after Con A injection. Representative photomicrographs (H&E staining) of the liver of BTLA−/− mice and WT mice are shown (n = 6 mice in each group). C, Sera were collected at 12 and 24 h after Con A injection, and the levels of ALT and AST were determined. Data are means ± SD (n = 6 mice in each group; *p < 0.05; * *p < 0.01). D, Sera were collected at 1, 3, 8, and 24 h after Con A injection, and the levels of TNF-α, IFN-γ, IL-4, and IL-10 were determined. Data are means ± SD (n = 4 mice in each group; *p < 0.05). E, One hour after Con A injection, liver mononuclear cells were isolated and the expression levels of Fas ligand (FasL) were analyzed by flow cytometry. Shown are representative histograms for FasL expression on T cells (CD3ε+ α-GalCer/CD1d–dimer−), NKT cells (CD3ε+ α-GalCer/CD1d–dimer+), and NK cells (CD3ε− NK1.1+) in the liver (n = 4 mice in each group).

To examine the immune responses to Con A in BTLA−/− mice in detail, we used a sublethal dose of Con A (10 mg/kg) for BTLA−/− mice in the following experiments. First, we performed histologic examination of the liver of Con A-injected BTLA−/− mice and WT mice. As shown in Fig. 4B, liver sections from Con A-injected BTLA−/− mice showed a massive necrosis and mononuclear cell infiltration in the portal area. In addition, the levels of ALT and AST in sera of BTLA−/− mice were significantly elevated, compared with those of WT mice at 12 and 24 h after Con A injection (n = 6; *p < 0.05; **p < 0.01; Fig. 4C), confirming the increased susceptibility to Con A-induced hepatitis in BTLA−/− mice.

A number of studies have suggested a proinflammatory role of TNF-α, IFN-γ, and IL-4 and a protective role of IL-10 in the Con A-induced hepatitis (24–26). It has also been demonstrated that IL-4 produced by NKT cells is implicated in liver damage in Con A-induced hepatitis (19, 27). Therefore, we measured cytokine levels in sera of Con A-injected BTLA−/− mice and WT mice. The levels of TNF-α and IFN-γ showed a sharp increase in Con A-injected BTLA−/− mice, and the peaks were higher than those in WT mice (Fig. 4D). The levels of TNF-α and IFN-γ reverted to the baseline at 24 h after Con A injection in WT mice, but remained elevated in BTLA−/− mice (Fig. 4D). Con A-injected BTLA−/− mice also exhibited prolonged IL-4 production, compared with WT mice (Fig. 4D). Alternatively, the levels of IL-10 were comparable between Con A-injected BTLA−/− mice and WT mice (Fig. 4D).

It has been reported that hepatic NKT cells rapidly upregulate FasL expression on the surface and induces apoptosis of hepatocytes upon Con A stimulation (19, 20). We therefore examined FasL expression on hepatic NKT cells in Con A-injected BTLA−/− mice and WT mice. As shown in Fig. 4E, FasL was upregulated on NKT cells but not on T cells or NK cells at 1 h after Con A injection in both BTLA−/− mice and WT mice. The levels of FasL on NKT cells were similar between BTLA−/− mice and WT mice, suggesting that FasL expressed on NKT cells could not account for the enhanced susceptibility to Con A-induced hepatitis in BTLA−/− mice.

NKT cells are required for the augmentation of Con A-induced hepatitis in BTLA−/− mice

To determine whether the augmentation of Con A-induced hepatitis in BTLA−/− mice depends on NKT cells, we examined the susceptibility to Con A-induced hepatitis in the mice lacking both BTLA and NKT cells (BTLA−/− NKT−/− mice). We first examined lymphocyte development in BTLA−/− NKT −/− mice and found that BTLA−/− NKT −/− mice lacked NKT cells, but had normal numbers of other lymphoid populations (data not shown). We then compared the levels of ALT and AST in BTLA−/− NKT −/− mice, BTLA−/− mice, NKT −/− mice, and WT mice upon Con A injection. Consistent with a previous study (19), Con A-induced hepatitis was significantly attenuated in NKT −/− mice compared with WT mice (Fig. 5A), indicating that NKT cells play an important role in causing Con A-induced hepatitis. Importantly, the augmentation of Con A-induced hepatitis in BTLA−/− mice was not observed in BTLA−/− NKT −/− mice (Fig. 5A). Histologic analysis confirmed the reduced liver damage in Con A-injected BTLA−/− NKT −/− mice compared with BTLA−/− mice (Fig. 5B). Moreover, serum levels of TNF-α, IFN-γ, and IL-4 were significantly decreased in BTLA−/− NKT−/− mice compared with BTLA−/− mice (Fig. 5C). These results indicate that NKT cells are critical for the augmentation of Con A-induced hepatitis in BTLA−/− mice.

FIGURE 5.
FIGURE 5.

NKT cells are required for the augmentation of Con A-induced hepatitis in BTLA−/− mice. WT mice, BTLA−/− mice, NKT−/− mice (Jα281−/− mice), and BTLA−/− NKT−/− mice were injected i.v. with a sublethal dose of Con A (10 mg/kg). A, Sera were collected at 12 h after Con A injection, and the levels of ALT and AST were determined. Data are means ± SD (n = 6; *p < 0.05; **p < 0.01). B, The liver was excised from WT mice, BTLA−/− mice, NKT−/− mice, and BTLA−/− NKT−/− mice at 24 h after Con A injection. Shown are representative photomicrographs (H&E staining) of the liver (n = 4 mice in each group). C, Sera were collected from WT mice, BTLA−/− mice, NKT−/− mice, and BTLA−/− NKT−/− mice at 3 and 8 h after Con A injection, and the levels of TNF-α, IFN-γ, and IL-4 were determined by ELISA. Data are means ± SD (n = 4; *p < 0.05).

BTLA expressed on NKT cells is involved in the inhibition of Con A-induced hepatitis

To determine whether BTLA expressed on NKT cells is crucial for the inhibition of Con A-induced hepatitis, we performed adoptive transfer experiments in which purified NKT cells from either WT or BTLA−/− mice were transferred to NKT −/− mice. As shown in Fig. 6, NKT−/− mice reconstituted with BTLA−/− NKT cells were significantly more susceptible to Con A-induced hepatitis, compared with NKT −/− mice reconstituted with WT NKT cells (n = 12 mice in each group; p < 0.05). Together, these results suggest that BTLA expressed on NKT cells is involved in the inhibition of Con A-induced hepatitis.

FIGURE 6.
FIGURE 6.

NKT−/− mice reconstituted with BTLA−/− NKT cells tended to be susceptible to Con A-induced hepatitis. Purified intrahepatic NKT cells from WT mice or BTLA−/− mice were injected into the liver of NKT−/− mice (1× 106 cells/mouse), and 1 h later the mice were injected with Con A i.v. Sera were obtained from the mice at 12 and 24 h after Con A injection, and serum ALT and AST levels were determined. Data are means ± SD (n = 12 mice in each group; *p < 0.05).

Discussion

In this study, we show that BTLA is expressed on NKT cells (Fig. 1) and that BTLA−/− NKT cells produce larger amounts of cytokines on Ag stimulation than WT NKT cells do (Fig. 3), suggesting that BTLA exerts inhibitory effects on NKT cells. In addition, it has recently been demonstrated that PD-1/PD-1 ligand 1 interaction is essential for the induction and maintenance of anergic state of NKT cells (28). It has also been demonstrated that stimulatory coreceptors such as CD28 (17) and ICOS (18) play a significant role in the induction of cytokine production from NKT cells. Together, these findings suggest that, analogous to T cells, the activation of NKT cells is regulated by the balance between stimulatory cosignals and inhibitory cosignals including BTLA.

We also show that BTLA−/− mice are highly susceptible to Con A-induced hepatitis (Fig. 4), in which NKT cells have been shown to play a significant role (19, 20). Indeed, although Con A-induced hepatitis was significantly attenuated in NKT−/− mice, compared with WT mice, the augmentation of Con A-induced hepatitis in BTLA−/− mice was not observed in BTLA−/− NKT −/− mice (Fig. 5A–C). Recently, Miller et al. (29) also reported that BTLA−/− mice were susceptible to Con A-induced hepatitis and that the increased susceptibility of BTLA−/− mice to Con A-induced hepatitis was diminished in BTLA−/− CD1d−/− mice, which lack most NKT cells (14). Importantly, we also show that NKT−/− mice reconstituted with BTLA−/− NKT cells are significantly more susceptible to Con A-induced hepatitis, compared with NKT−/− mice reconstituted with WT NKT cells (Fig. 6), indicating that BTLA expressed on NKT cells is involved in the regulation of the susceptibility to Con A-induced hepatitis.

Recently, it has been demonstrated that the mice lacking HVEM, a ligand for BTLA (5, 7, 8), are also highly susceptible to Con A injection to induce increased CD4+ T cell activation, but exhibit no significant liver damage (30). Because HVEM is a ligand for BTLA and a costimulatory receptor for lymphotoxins (inducible expression) that compete with HSV glycoprotein D for HVEM (a receptor expressed on T lymphocytes) (5), NKT cell activation might not be sufficient for inducing hepatitis in HVEM-deficient mice.

The effector mechanisms by which NKT cells cause Con A-induced hepatitis remain to be elucidated. Previous studies have shown that NKT cell-derived cytokines including IL-4 are crucial for NKT cell-mediated liver injury (19). Consistent with these findings, we found that IL-4 production from α-GalCer–activated NKT cells, and serum levels of IL-4 in Con A-injected mice were enhanced in BTLA−/− mice (Figs. 3A and 4D). Alternatively, FasL expression was similarly upregulated on BTLA−/− NKT cells and WT NKT cells on Con A injection (Fig. 4E). Together, it is suggested that the elevated levels of cytokines rather than FasL expression might be involved in the augmentation of Con A-induced hepatitis in BTLA−/− mice. In addition, it has recently been suggested that soluble lymphotoxins (inducible expression) that compete with HSV glycoprotein D for HVEM (a receptor expressed on T lymphocytes) released from NKT cells may also be an effector molecule for Con A-induced hepatitis (31).

We found that BTLA−/− NKT cells produced larger amounts of cytokines on α-GalCer stimulation in vitro (Fig. 3A). Because the expression levels of activation markers such as CD25, CD69, and CD122 on NKT cells were similar between BTLA−/− mice and WT mice (Fig. 2B), it is suggested that the enhanced cytokine production of BTLA−/− NKT cells is caused by a lack of BTLA-HVEM interaction during the in vitro Ag stimulation. However, in preliminary experiments, we found that the addition of anti-BTLA Ab (clone 6F7), which is reported to function as a neutralizing Ab (32), did not significantly enhance cytokine production from α-GalCer–stimulated WT NKT cells in vitro. Although this experiment is inconclusive, because the binding of 6F7 to BTLA may transduce some signals in NKT cells, this finding raises another possibility that the enhanced cytokine production of BTLA−/− NKT cells may be caused by developmental abnormality of BTLA−/− NKT cells rather than a lack of BTLA-HVEM interaction upon Ag stimulation. Further studies using inducible deletion of the BTLA gene in NKT cells are required to address the precise mechanism underlying the enhanced cytokine production from α-GalCer–stimulated BTLA−/− NKT cells.

More recently, it has been demonstrated that PD-1, another inhibitory coreceptor, is expressed on NKT cells (28) and that the engagement of PD-1 on NKT cells by PD-1 ligand 1 inhibits cytokine secretion by α-GalCer–activated NKT cells (33). CD94/NKG2A, an inhibitory receptor on NK cells, is also expressed on NKT cells, and the blockade of NKG2A-mediated inhibitory signal with an antagonistic Ab augments Con A- and α-GalCer–induced liver injury (34). Furthermore, CD4+ CD25+ regulatory T cells have been reported to be important for the suppression of Con A-induced liver injury via a TGF-β–dependent mechanism (35). Therefore, it is suggested that in addition to BTLA, multiple inhibitory pathways are involved in the regulation of NKT cell function in the liver.

In conclusion, we have shown that BTLA functions as an inhibitory coreceptor in NKT cells and prevents NKT cell-mediated experimental hepatitis. Although further studies are required to address the underlying mechanisms, our data suggest that the enhancement of BTLA signaling in NKT cells by agonistic ligands or by a stimulatory Ab might be applicable for the treatment of a number of diseases in which NKT cells play a pathogenic role.

Acknowledgments

We thank Dr. K. M. Murphy for BTLA−/− mice, Ms. Nagashima for animal care, and Ms. Sugaya for technical support on cell purification.

Disclosures The authors have no financial conflicts of interest.

Footnotes

  • This work was supported in part by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government, and by Global COE Program (Global Center for Education and Research in Immune System Regulation and Treatment), Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government, Japan.

  • Abbreviations used in this paper:

    ALT
    alanine aminotransferase
    AST
    aspartate aminotransferase
    BTLA
    B and T lymphocyte attenuator
    BTLA−/−
    BTLA-deficient
    DC
    dendritic cells
    α-GalCer
    α-galactosylceramide
    HVEM
    herpesvirus entry mediator
    ICOS
    inducible T cell costimulator
    PD-1
    programmed cell death 1
    NKT
    cell–deficient (NKT−/−)
    WT
    wild-type.

  • Received February 6, 2009.
  • Accepted October 21, 2009.

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