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Preferential Involvement of Tim-3 in the Regulation of Hepatic CD8⁺ T Cells in Murine Acute Graft-versus-Host Disease¹

Tsunekazu Oikawa^*,, Yosuke Kamimura^*, Hisaya Akiba, Hideo Yagita, Ko Okumura, Hiroki Takahashi, Mikio Zeniya, Hisao Tajiri and Miyuki Azuma^2,*

^* Department of Molecular Immunology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; Division of Gastroenterology and Hepatology, Department of Internal Medicine, Jikei University Medical School, Tokyo, Japan; and Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Tim-3, a member of the T cell Ig mucin (TIM) family regulates effector Th1 responses. We examined Tim-3 and its ligand expression as well as the effects of anti-Tim-3 mAb treatment in a murine model of acute graft-vs-host disease (aGVHD). In mice with aGVHD, Tim-3 expression was markedly up-regulated on splenic and hepatic CD4⁺ and CD8⁺ T cells, dendritic cells (DCs), and macrophages, and this was especially dramatic in hepatic CD8⁺ T cells. Both donor- and host-derived CD8⁺ T cells induced similar levels of Tim-3. Tim-3 ligand expression was also up-regulated in splenic T cells, DCs, and macrophages, but not in the hepatic lymphocytes. The administration of anti-Tim-3 mAbs accelerated aGVHD, as demonstrated by body weight loss, reduction in total splenocyte number, and infiltration of lymphocytes in the liver. IFN- expression by splenic and hepatic CD4⁺ and CD8⁺ T cells was significantly augmented by anti-Tim-3 mAb treatment. In addition, the cytotoxicity against host alloantigen by donor CD8⁺ T cells was enhanced. These results demonstrate that the anti-Tim-3 treatment in aGVHD augmented the activation of effector T cells expressing IFN- or exerting cytotoxicity. Our results suggest that Tim-3 may play a crucial role in the regulation of CD8⁺ T cells responsible for the maintenance of hepatic homeostasis and tolerance.

	Introduction

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The Tim gene family consists of eight members (Tims 1–8) on mouse chromosome 11B1.1 and three members (TIMs 1, 3, and 4) on human chromosome 5q33.2 (1, 2). Each of these genes is predicted to encode a type I membrane protein with a similar structure, consisting of a signal sequence followed by an Ig variable region (IgV)-like domain, a mucin-like domain, a transmembrane region, and an intracellular tail (3). Tim-1, Tim-2, and Tim-3 contain a predicted intracellular tyrosine phosphorylation motif and these surface molecules appear to have a role in the regulation of Th1 and Th2 effector T cell responses (1, 2, 3). The chromosomal region containing the TIM family has been linked to both autoimmune and allergic disease. Tim-3 is involved in negatively regulating Th1 T cell activation (4, 5), whereas Tim-2 and Tim-1 appear to positively regulate Th2 cells (6, 7, 8, 9, 10, 11).

Tim-3 is preferentially expressed on differentiated Th1 cells, but not on Th2 cells (4, 5). Treatment with anti-Tim-3 mAb enhanced the disease severity in a mouse model of Th1-mediated experimental autoimmune encephalomyelitis via the activation of macrophages (4). Administration of Tim-3Ig fusion protein to immunized mice with myelin proteolipid protein enhanced proliferation and cytokine production in Th1 cells (12). Tim-3Ig also abrogated the induction of Th1-mediated tolerance and the administration of either Tim-3Ig or anti-Tim-3 Ab in NOD-SCID mice clearly enhanced the onset of disease mediated by T cells transferred from diabetic NOD mice (5, 12). Tim-3-deficient mice were refractory to the induction of Th1-mediated high-dose tolerance (12). The absence of Tim-3 prevented the acquisition of transplantation tolerance typically induced by donor-specific transfusion in combination with CD40-CD154 costimulatory blockade by regulating the action of naturally arising CD4⁺CD25⁺ regulatory T cells (5, 12).

The ligand for Tim-3 (Tim-3L),³ as previously determined by Tim-3Ig fusion protein experiments, is expressed on resting CD4⁺ T cells and a minor population of CD11c⁺ dendritic cells (4, 5, 12). Recently, galectin-9, a member of the galectin family (also called S-type lectins), has been identified as a ligand for Tim-3 (13). Galectin-9 has been shown to induce calcium flux and aggregation and death of Th1 cells via Tim-3 in vitro. The administration of galectin-9 results in a selective reduction of IFN- producing cells and suppression of Th1-mediated autoimmune encephalomyelitis in vivo (13). Soluble form of Tim-3 inhibits Ag-specific T cell responses in vitro and reduced antitumor immunity in vivo by inhibiting effector Th1 responses (14). Overall, these data suggest that the Tim-3-Tim-3L pathway may contribute to the attenuation of Th1-mediated responses.

Acute graft-vs-host disease (aGVHD) is initiated by the activation of both donor CD4⁺ and CD8⁺ T cells against host alloantigens. In aGVHD, the activation of T cells and the ensuing production and release of inflammatory cytokine causes a systemic illness characterized by immunosuppression and tissue destruction of various organs, including liver, intestinal mucosa, and skin (15, 16, 17). Hepatic GVHD is characterized by donor T cell activation in the induction phase and by inflammatory reactions in the effector phase (18, 19, 20). Activation and infiltration of liver mononuclear cells, especially CD8⁺ T cells, induce liver injury and bile duct destruction in aGVHD. In contrast, hepatic immune responses are often associated with the induction of tolerance (21, 22). Tolerogenic properties of liver can be explained by unique intrahepatic CD8⁺ T cell responses, which result in the production of regulatory cytokines such as IL-10 and TGF- (23, 24). The precise molecular mechanisms that are responsible for such regulation have not been well elucidated.

To investigate roles of the Tim-3-Tim-3L pathway in a murine model of aGVHD, we examined expression of Tim-3 and its ligand on lymphocytes and APCs in the spleen and liver, and the effects of anti-Tim-3 mAb treatment.

	Materials and Methods

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Mice

Female 6-wk-old C57BL/6 (B6, H-2^b) and (C57BL/6 x DBA/2)F₁ (BDF₁, H-2^b/d) mice were purchased from Charles River Laboratories. Donors for B6 splenocytes were 6 to 10 wk old, and BDF₁ recipient mice were 8 to 10 wk old at the time of transfer. All mice were maintained in a specific pathogen-free animal facility at the Tokyo Medical and Dental University (Japan). All mice procedures were reviewed and approved by the animal care and use committee of the Tokyo Medical and Dental University.

Tim-3-Ig fusion proteins, transfectants, and anti-Tim-3 mAbs

The vectors for Tim-3/BALB-Ig and Tim-3/B6-Ig consisting of the extracellular domains (aa 1–191) of Tim-3 from BALB/c (Tim-3/BALB) (4) and C57BL/6 (Tim-3/B6) (25) mice linked to the Fc portion of mouse IgG2a in the pcDNA3.1(–) vector (Invitrogen) were prepared. Tim-3/BALB-Ig and Tim-3/B6-Ig were produced by transfection of each vector into Chinese hamster ovary (CHO) cells and were purified using protein G column (Amersham Biosciences) from the culture supernatant. Both full-length Tim-3/BALB and Tim-3/B6 cDNA were generated by RT-PCR from con A-activated splenocytes in each strain and were subsequently cloned into a pcDNA3.1/CT-GFP-TOPO vector (Invitrogen). Primers used to generate Tim-3/BALB and Tim-3/B6 cDNA were sense, 5'-ATGTTTTCAGGTCTTACCCTC-3', and antisense, 5'-GCTGCTGGCTGTTGACGT-3'. CHO cells were transfected with Tim-3/BALB- or Tim-3/B6-pcDNA3.1/CT-GFP-TOPO vector by LipofectAMINE (Invitrogen) and the cells expressing Tim-3 were selected by GFP expression. The anti-mouse Tim-3 mAbs (RMT3–23, rat IgG2a, and RMT3–8, rat IgG2b, ) were generated by immunizing SD rats with Tim-3/BALB-Ig or Tim-3/B6-Ig emulsified in CFA and fusing immunized lymph node cells with P3U1 myeloma cells; the fused cells were then subsequently screened for binding to Tim-3/BALB and/or Tim-3/B6 on CHO transfectants, but not parental CHO cells. RMT3–23 reacted with both Tim-3/BALB and Tim-3/B6 on CHO transfectants. RMT3–8 reacted with Tim-3/B6-CHO cells but not with Tim-3/BALB-CHO cells. These mAbs were purified from ascites by the caprylic acid and ammonium sulfate precipitation method, and the purity was verified by SDS-PAGE analysis.

Monoclonal Abs and immunofluorescence

Hybridomas against CD3 (145-2C11, hamster IgG), I-A^b,d,q (M5-114, rat IgG2b), CD24 (J11d, rat IgM), CD45R/B220 (RA3-3A1, rat IgM), CD4 (RL172.4, rat IgM), and F4/80 (rat IgG2b) were obtained from the American Type Culture Collection. mAbs against the following Ags were used for immunofluorescence analysis: CD3 (145-2C11), H-2K^d (SF1-1.1, mouse IgG2a), CD4 (RM4-5, rat IgG2a), CD8 (53-6.7, rat IgG2a), CD11c (N418, hamster IgG), CD25 (PC61.5, rat IgG1), CD69 (H1.2F3, hamster IgG), CD45R/B220 (RA3-6B2, rat IgG2a), IFN- (XMG1.2, rat IgG1), IL-4 (11B11, rat IgG1) and IL-10 (JES5-16E3, rat IgG2b), and mouse IgG2a (R19-15, rat IgG1). All FITC-, PE-, PerCP-, and allophycocyanin-conjugated or biotinylated mAbs were purchased from BD Pharmingen or e-Bioscience. FITC-conjugation and biotinylation for Tim-3 mAb (RMT3-23) were performed by a standard method in our laboratory. The cell surface expression of Tim-3L was confirmed by the staining with Tim-3/B6-Ig, followed by biotinylated anti-mouse IgG2a mAb. For biotinylated mAbs, PE-streptavidin (BD Pharmingen) was used for visualization. In select stainings, Fc blocking reagent (anti-CD16/32 mAbs) was used. Immunofluorescence, flow cytometry, and data analysis were performed using FACSCalibur and CellQuest software (BD Biosciences). Multicolor staining for intracellular cytokine and cell surface Ags was performed as previously described (26).

Induction of acute GVHD and mAb treatment

Single-cell suspensions of splenocytes from B6 mice were used as the source of GVHD-causing T cells. Unirradiated BDF₁ recipients received i.v. injection of 5 x 10⁷ splenocytes. Recipients receiving splenocytes were randomly divided into three groups of 5–10 mice and treated with either control rat IgG (Sigma-Aldrich), RMT3-23, or RMT3-8. A total of 300 µg of mAbs or control IgG was injected i.p. on 3 continuous days until 1 day before cell transfer and then every other day until day 13 for a total of nine times. After 14 days posttransfer, the spleens and livers were analyzed.

Histological assessment

Immunohistological staining was performed as described previously (27) with several modifications. Briefly, cryostat sections were fixed, pretreated with avidin-biotin blocking reagent (Vector Laboratories), and then incubated with anti-Tim-3 mAb (RMT3-23) or control rat IgG2a, followed by biotinylated rabbit-anti-rat IgG (Vector Laboratories). For detection, a Vectastain Elite ABC kit (Vector Laboratories) and diaminobenzidine (Merck) were used. All of the incubation steps were performed in a temperature-controlled microwave processor (MI-77; Azumaya) according to the manufacturer’s instructions. Digitalized images were captured using an inverted microscope and camera system (IX71 and Pro600ES-D; Olympus). For H&E staining, tissue samples were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained with H&E.

Isolation of hepatic mononuclear cells (HMC)

HMC were isolated from livers as described previously (28) with some modifications. Briefly, the livers were minced, pressed through a 200-gauge stainless steel mesh, and suspended in complete RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 5 x 10^–5 M 2-ME, and antibiotics. RBC were depleted with lysing solution and then hepatic parenchymal cells were removed by the gradient centrifugation using 40% and 80% Percoll. Cells in the interface were harvested as HMC and resuspended in complete RPMI 1640 medium.

CTL assay

Pooled splenocytes and HMC from each group of three mice were treated with hybridoma supernatants containing anti-I-A^b,d,q, anti-H-2D^d (34-2-12), anti-H-2K^d (31-3-4, provided by Dr. S Hirose, Juntendo University, Tokyo, Japan), anti-CD24, anti-CD45R, and anti-CD4 mAbs and rabbit complement as described previously (29). The donor CD8⁺ T cell fractions that contain over 90% CD8⁺CD3⁺ T cells were obtained and used as effector cells. Cytotoxicity against P815 (H-2^d), EL4 (H-2^b), and RDM4 (H-2^k) target cells was measured by a 6-h JAM test as described previously (30, 31).

Statistical analyses

Statistical analysis was performed using the Mann-Whitney U test. Values of p < 0.05 were considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Augmentation of Tim-3 expression in CD8⁺ T cells

To investigate the role of Tim-3 in the development of aGVHD, aGVHD was induced by transfer of B6 splenocytes into unirradiated BDF1 mice and Tim-3 expression was examined 14 days after transfer. In the liver from intact mice, a few Tim-3⁺ cells were found around the portal vein, whereas abundant Tim-3⁺ cells were observed at the portal areas and parenchymal sinusoids areas in aGVHD mice (Fig. 1A). Morphologically, these cells appeared to be infiltrating lymphocytes. The staining of serial sections using anti-CD8 or anti-CD4 mAb revealed that most of the Tim-3⁺ cells were CD8⁺ T cells (data not shown). In flow cytometric analysis of HMC, Tim-3 was slightly expressed on HMC from intact mice, whereas Tim-3 expression on CD3⁺ T cells was dramatically enhanced in aGVHD mice (Fig. 1B). The enhancement in Tim-3 expression was especially prominent in the CD8⁺ T cells. At this time point, both H-2^d– donor and H-2^d+ host T cells were present in the spleen and liver, but the proportion of donor T cells in the HMC was clearly higher than in the spleen (Fig. 1C). The donor to host ratio in both CD4⁺ and CD8⁺ T cells within Tim-3⁺ cells was 1:2 in the spleen, but the inverse ratio was seen in the liver. In the liver, over 50% of CD8⁺ T cells were of donor origin. There was no clear difference detected in the levels of Tim-3 expression between donor and host T cells in both spleen and liver. These results demonstrate that Tim-3 expression was observed on both donor and host T cells in the spleen and liver of the aGVHD mice, and this was especially notable in hepatic CD8⁺ T cells.

View larger version (55K): [in this window] [in a new window]   FIGURE 1. Induction of Tim-3 in aGVHD mice. A, The cryostat sections of the liver tissues from intact (a) and aGVHD (b) mice at 14 days after transfer were stained with anti-Tim-3 (RMT3–23) mAb. Scale bars = 25 µm. Control staining using isotype-matched rat Ig yielded negative results (data not shown). B and C, HMC and splenocytes from age-matched intact mice and the aGVHD mice 14 days after transfer were obtained. In B, cells were stained with FITC-conjugated anti-Tim-3 mAb, PerCP-conjugated anti-CD3 mAb and either allophycocyanin-conjugated anti-CD4 or anti-CD8 mAb, or with appropriate fluorochrome-conjugated control Igs. In C, cells were stained with FITC-conjugated anti-H-2Kd mAb, biotinylated-anti-Tim-3 mAb, followed by PE-streptavidin, PerCP-conjugated anti-CD3 mAb, and allophycocyanin-conjugated anti-CD4 or anti-CD8 mAb, or with appropriate fluorochrome-conjugated control Igs. Samples were analyzed by flow cytometry. If needed, an electronic gate was set on either the CD3+ (T), CD4+CD3+ (CD4 T), or CD8+CD3+ (CD8 T) lymphocytes; the expressions of the indicated Ags are shown as dot plots (four-decade log scale). Representative FACS profiles of ten mice in each group are shown. The quadrant markers have been positioned to include >98% of control Ig-stained cells (data not shown) in the lower left. The underlined values of the right corner in B show Tim-3 positive percentages within either CD3+, CD4+CD3+, or CD8+CD3+ T cells. The values of each quadrant in C are positive percentages within whole lymphocytes, CD4+ CD3+ or CD8+CD3+ T cells.

We next investigated the expression of Tim-3L using Tim-3/B6-Ig. In intact mice, Tim-3L was not detected on splenic and hepatic T cells. In aGVHD mice, Tim-3L was substantially induced on both CD4⁺ and CD8⁺ T cells in splenocytes but was not clearly observed on hepatic CD4⁺ T cells (Fig. 2). Similar to the expression of Tim-3, a higher percentage of splenic CD8⁺ T cells displayed increased expression of Tim-3L compared with the CD4⁺ T cells. However, despite the abundant Tim-3 expression, a much lower percentage of hepatic CD8⁺ T cells expressed Tim-3L. Tim-3L was not clearly observed on hepatic CD4⁺ T cells. Simultaneous staining for Tim-3 and Tim-3L in aGVHD mice revealed that most CD4⁺ and CD8⁺ T cells coexpressed Tim-3 and Tim-3L in the spleen, but a slight subpopulation of Tim-3⁺ T cells coexpressed Tim-3L in HMC. These results demonstrated that Tim-3L on splenic T cells was increased clearly, whereas the enhanced expression of Tim-3L on hepatic T cells was less.

View larger version (64K): [in this window] [in a new window]   FIGURE 2. Coexpression of Tim-3 and Tim-3L on T cells in aGVHD mice. Splenocytes and HMC from intact and aGVHD mice were obtained as described in Fig. 1. Cells were stained with FITC-conjugated anti-Tim-3 mAb, purified Tim-3/B6-Ig, followed by biotinylated anti-mouse IgG2a mAb and PE-streptavidin, PerCP-conjugated anti-CD3 mAb, and allophycocyanin-conjugated anti-CD4 or anti-CD8 mAb or with appropriate fluorochrome-conjugated control Igs. Samples were analyzed by flow cytometry. If needed, an electronic gate was set on either CD3+ (T), CD4+CD3+ (CD4 T) or CD8+CD3+ (CD8 T) lymphocytes; the expressions of the indicated Ags are shown as dot plots (four-decade log scale). Representative FACS profiles of ten mice in each group are shown. The quadrant markers have been positioned to include >98% of control Ig-stained cells (data not shown) in the lower left. The underlined values of the right corner show Tim-3L positive percentages within CD3+, CD4+CD3+, or CD8+CD3+ T cells. The values of each quadrant in the double staining with Tim-3 and Tim-3L are positive percentages within CD4+ CD3+ or CD8+CD3+ T cells.

We examined the expression of Tim-3 and Tim-3L on APCs. In both splenocyte and HMC from intact mice, a few CD11c⁺ DCs expressed either Tim-3 or Tim-3L (Fig. 3A). In contrast, a majority of CD11c⁺ splenic DCs from mice with aGVHD expressed Tim-3 and Tim-3L. In addition, CD11c⁺ hepatic DCs also increased Tim-3 expression. Note that no Tim-3L was induced on hepatic DCs in mice with aGVHD. Similar results were observed in macrophages (Fig. 3B). A substantial percentage of splenic and hepatic macrophages expressed Tim-3 in intact mice and their expression was dramatically enhanced in mice with aGVHD. In mice with aGVHD, Tim-3L was also induced on splenic macrophages, but not on hepatic macrophages. CD45R⁺ B cells in the both spleen and liver expressed neither Tim-3 nor Tim-3L even in mice with aGVHD (data not shown). These results demonstrated that similar to T cells, Tim-3 and Tim-3L were induced on DCs and macrophages in the spleen from aGVHD mice, but Tim-3L was not detected in the liver.

View larger version (39K): [in this window] [in a new window]   FIGURE 3. Expression of Tim-3 and Tim-3L on DCs and macrophages. Splenocytes and HMC from intact and aGVHD mice were stained with FITC-conjugated anti-CD11c or anti-F4/80 mAb, and either biotinylated anti-Tim-3 mAb, followed by PE-streptavidin, or unlabeled Tim-3Ig, followed by biotinylated anti-mouse IgG2a mAb and PE-streptavidin, or with appropriate fluorochrome-conjugated control Igs. Samples were analyzed by flow cytometry. Representative FACS profiles in each group of ten mice are displayed. The profiles of control Ig staining are shown in the left of each panel. The markers have been positioned to include >98% of control Ig-stained cells. A, the expressions of CD11c and either Tim-3 or Tim-3L within lymphocyte gate were analyzed. The underlined values of the right corner show Tim-3 or Tim-3L positive percentages within CD11c+ DCs. B, In the GVHD-induced mice, the F4/80+ cells did not contain a major population of forward scatter (FSC)high/side scatter (SSC)high characteristic macrophages. Therefore, an electronic gate was set on FSChigh/SSChigh cells, and then Tim-3 or Tim-3L expression was analyzed. The data are displayed as dot plots with Tim-3 or Tim-3L expression and FSC. Splenocytes and HMC within the macrophage gate contained 79% and 80% F4/80+ cells in an intact mouse and 58% and 30% F4/80+ cells in a GVHD mouse, respectively. The values are positive percentages in the FSChigh/SSChigh cells.

To investigate the involvement of the Tim-3 and Tim-3L pathway in the development of aGVHD, we examined the effects of administration of anti-Tim-3 mAbs. Recipient mice received RMT3–23, RMT3–8, or control rat IgG treatment nine times. The control IgG-treated mice showed a recovery of body weight after the temporal loss (Fig. 4A) and a significant reduction in the total splenocyte number (Fig. 4B). The mice treated with each clone of anti-Tim-3 mAb showed a continuous reduction in body weight and a significant reduction in the total splenocyte number compared with that of the control IgG-treated group. In histology, the liver from control IgG-treated mice showed a moderate infiltration of mononuclear cells around the portal area, whereas the livers from anti-Tim-3 mAb-treated mice showed a marked increase of liver infiltrating mononuclear cells and expansion of the portal tracts by cellular infiltration (Fig. 4C). The total cell count of HMC from control IgG-treated mice increased significantly compared with the intact mice, and this was increased dramatically by treatment with anti-Tim-3 mAb (Fig. 4B). These results indicated that the anti-Tim-3 mAb treatments accelerated the manifestation of aGVHD.

View larger version (56K): [in this window] [in a new window]   FIGURE 4. Effects of anti-Tim-3 mAb treatments on manifestation of acute GVHD. The BDF1 recipient mice transferred 5 x 107 B6 splenocytes were treated with either RMT3-23 (•), RMT3-8 (), or control rat IgG (). Body weight was measured on the indicated days. The change in mean body weight (A), total cell numbers of splenocytes and HMC at 14 days after transfer (B) are shown. The values are the mean ± SD from three experiments with each group of five mice. *, Statistically different from control IgG-treated group (p < 0.05) (A). *, Statistically different (p < 0.05) (B). C, H&E staining of the livers from aGVHD mice. Representative staining from each group of three mice are shown. Scale bars = 25 µm.

To investigate whether the anti-Tim-3 treatment affects T cell activation, we examined the proportion of CD4⁺ and CD8⁺ T cells, and activation Ags such as CD25 and CD69 (Table I). The induction of aGVHD increased the percentages of CD3⁺ and CD8⁺ T cells in the spleen. Both treatments with anti-Tim-3 mAbs did not clearly affect the ratio of CD3⁺, CD4⁺, or CD8⁺ T cells. In addition, although the percentages of CD25⁺ and CD69⁺ cells within CD4⁺ or CD8⁺ T cells were significantly enhanced in the control IgG-treated aGVHD mice, there was no clear differences with the anti-Tim-3 treated mice. In HMC from the control IgG-treated aGVHD mice, the ratios of CD3⁺ and CD8⁺ T cells increased and that of CD4⁺ T cells decreased, but these were not affected by the anti-Tim-3 treatments. Anti-Tim-3 mAb treatments did not significantly affect the increases seen in CD25⁺ or CD69⁺CD4⁺ and CD8⁺ T cells in aGVHD.

To evaluate changes in the functional properties of T cells, we examined cytokine expression and cytotoxicity against host alloantigens. In the control IgG-treated mice with aGVHD, the enhanced expression of IFN- in both splenic and hepatic CD4⁺ and CD8⁺ T cells was observed (Fig. 5A and Table II). Simultaneous staining with Tim-3 and IFN- in CD4⁺ and CD8⁺ T cells showed that most IFN- expressing cells also expressed Tim-3 (Fig. 5B). In hepatic T cells from control IgG-treated mice with aGVHD, the percentage of IFN--expressing CD4⁺ or CD8⁺ T cells was much higher than that in splenic T cells, especially in CD8⁺ T cells. The IFN- expression induced in both splenic and hepatic T cells was further enhanced by the anti-Tim-3 mAb treatments. IL-10 expression by CD4⁺ and CD8⁺ T cells in the spleen and liver was enhanced in the control IgG-treated mice with aGVHD, albeit modestly. The anti-Tim-3 mAb treatments enhanced IL-10 expression by CD4⁺ T cells. Expression of IL-4 was not detected in the control IgG- and anti-Tim-3-treated mice with aGVHD (data not shown).

View larger version (45K): [in this window] [in a new window]   FIGURE 5. Enhanced IFN- expression in T cells by the anti-Tim-3 mAb treatment. Splenocytes and HMC from aGVHD mice treated with either RMT3-23, RMT3-8, or control IgG were cultured with PMA and ionomycin in the presence of brefeldin A for 4 h. A, Cells were cell surface stained with FITC-conjugated CD8 mAb and PerCP-conjugated anti-CD3 mAb, or with appropriated fluorochrome-conjugated control Igs. After fixation and permeabilization, the cells were stained intracellularly with PE-conjugated anti-IFN- mAb or control Ig. B, Cells were cell surface stained with FITC-conjugated Tim-3 mAb and PerCP-conjugated anti-CD3 mAb, and either allophycocyanin-conjugated anti-CD4 or anti-CD8 mAb, or with appropriated fluorochrome-conjugated control Igs, and then stained intracellularly with PE-conjugated anti-IFN- mAb or control Ig. Samples were analyzed by flow cytometry. An electronic gate was set on either the CD3+ (A), CD3+CD4+ (CD4 T), or CD3+CD8+ (CD8 T) (B) cells; the expressions of the indicated Ags are shown as dot plots. Representative FACS profiles are shown. The quadrant markers have been positioned to include >98% of control Ig-stained cells (data not shown) in the lower left. Values are the mean ± SD for each group of ten mice.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. The anti-Tim-3 mAb treatment augmented cytotoxicity. Donor CD8+ T cells were isolated from pooled splenocytes and HMC from five aGVHD mice treated with either RMT3-23 (•), RMT3-8 (), or control IgG () and used as effector cells. Effector cells were cocultured with [3H]thymidine-labeled either P815 (H-2d), EL4 (H-2b), or RDM4 (H-2k) target cells for 6 h at the indicated E/T ratios. Cytotoxicity was measured as described in Materials and Methods. A, The values are the mean cytotoxicity ± SE from three independent experiments. B, Cytotoxicities against P815, EL4, and RDM4 by effector CD8+ T cells from control IgG-treated () or RMT3-23-treated () aGVHD mice are shown. The E/T ratio was 25. *, Statistically different from control IgG-treated aGVHD mice (p < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Tim-3 has been established as an immunoregulatory molecule on effector Th1 cells (4, 5, 12). In this study, we have highlighted for the first time the expression and functional role of Tim-3 in CD8⁺ T cells. Activation of CD8⁺ T cells plays a crucial role in the development of aGVHD, and CD8⁺ T cells are major effectors involved in the destruction of target organs in aGVHD (19, 32). A previous study demonstrated that the accumulation of liver-infiltrating mononuclear cells reached a maximum at 2 wk postallogeneic lymphocyte transfer. Therefore, we selected this time point for analysis in this study. We observed a clearly enhanced expression of Tim-3 on CD8⁺ T cells in the spleen and liver of the aGVHD mice. Consistent with previous reports (19, 33), infiltration of CD8⁺ T cells was especially dominant in the liver. Interestingly, Tim-3 was induced on both host- and donor-derived CD8⁺ T cells at similar levels. This suggests that generation of Tim-3 is not only induced on activated T cells in response to host alloantigen but is also induced on host T cells stimulated with soluble cytokines secreted from bystander cells. It appears that the Tim-3⁺ host T cells were already primed T cells and their Tim-3 expression was a temporal event in the early phase of aGVHD. It was shown that Tim-3 expression on CD4⁺ and CD8⁺ T cells was induced along with cell division, reached a plateau at the sixth to seventh cell cycle division, and that this time point was closely associated with the production of IFN- (5). Consistently, we also observed that most Tim-3-positive splenic and hepatic CD4⁺ and CD8⁺ T cells expressed IFN-, and the enhancement of IFN-⁺ cells seem to be closely correlated with Tim-3 expression, because highly activated hepatic CD8⁺ T cells induced higher levels of Tim-3 with IFN- (Fig. 5B). Although further studies are required to identify the molecular mechanisms that link Tim-3 and IFN- expression, we demonstrated that Tim-3 expression was closely associated with IFN- production in CD8⁺ T cells as well as Th1 cells. Induction of Tim-3 was also seen on splenic and hepatic DCs and macrophages but not on B cells in mice with aGVHD. The exact factors that play a role in the induction of Tim-3 on these cells remain unclear.

Tim-3L expression determined by Tim-3Ig fusion protein was also induced on CD4⁺ and CD8⁺ T cells, splenic DCs, and macrophages in mice with aGVHD, and most Tim-3L-expressing T cells and DCs coexpressed Tim-3. Therefore, it seems that Tim-3L expression on T cells coincides with Tim-3 expression. Interestingly, despite an efficient induction of Tim-3 in the liver, Tim-3L was not detected on liver-infiltrating CD4⁺ T cells, DCs, and macrophages, and hepatic CD8⁺ T cells induced only a limited level of Tim-3L. IFN- has been shown to be a potent inducer for galectin-9 (Tim-3L) expression in endothelial cells and fibroblasts (34, 35). IFN- might be abundant in the inflamed liver and, therefore, some regulatory factors for preventing Tim-3L expression may exist. The intrahepatic immune environment is often associated with tolerance rather than immunity (21, 22). An immunoregulatory cytokine, IL-10 is preferentially abundant in the liver (21, 36, 37). Such a microenvironmental factor may prevent the induction of Tim-3L on the hepatic T cells and APCs. Although we have not examined Tim-3L expression on hepatic parenchymal cells, galectin-9 is widely distributed in various tissues and is particularly abundant in the liver (38). In naive mice, galectin-9 was detected on hepatocytes, especially on the sinusoidal front (39). Therefore, it is possible that constitutive or up-regulated expression of Tim-3L on inflamed parenchymal cells of the liver directly interacts with the Tim-3 expressed on infiltrating CD8⁺ T cells, and their interactions down-regulate donor CD8⁺ T cell activation in the liver.

In conclusion, we demonstrated the involvement of the Tim-3 in regulating effector CD8⁺ T cells in a murine aGVHD model. Our results suggest that the Tim-3-Tim-3L interaction may play an important role in preventing the liver from T cell-mediated damage and may be responsible for maintaining hepatic homeostasis and tolerance.

	Acknowledgments

 
We thank Y. Shuda for technical assistance in H&E staining and Dr. S. Hirose for providing hybridomas.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

² Address correspondence and reprint requests to Dr. Miyuki Azuma, Department of Molecular Immunology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail address: miyuki.mim{at}tmd.ac.jp

³ Abbreviations used in this paper: Tim-3L, Tim-3 ligand; aGVHD, acute graft-versus-host disease; CHO, Chinese hamster ovary; HMC, hepatic mononuclear cell; DC, dendritic cell.

Received for publication February 17, 2006. Accepted for publication June 14, 2006.

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