Tryptophan metabolite 3-hydroxyanthranilic acid selectively induces activated T cell death via intracellular GSH depletion

Immunology Letters 132 (2010) 53–60

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Tryptophan metabolite 3-hydroxyanthranilic acid selectively induces activated T cell death via intracellular GSH depletion Sun-Mi Lee a,b , Young-Suk Lee a,b , Jae-Hyeog Choi a , Sae-Gwang Park a , Il-Whan Choi a , Young-Don Joo c , Won-Sik Lee c , Jeong-Nyeo Lee d , Inhak Choi a,b , Su-Kil Seo a,b,∗ a

Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735, Republic of Korea Department of Hemato/Oncology, Busan Paik hospital, Inje University College of Medicine, Busan 614-735, Republic of Korea d Department of Laboratory Medicine, Inje University College of Medicine, Busan 614-735, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 18 February 2010 Received in revised form 19 May 2010 Accepted 30 May 2010 Available online 4 June 2010 Keywords: Indoleamine 2,3-dioxygenase 3-Hydroxyanthranilic acid T cells Glutathione

a b s t r a c t Tryptophan-derived metabolites, initiated by indoleamine 2,3-dioxygenase (IDO), preferentially induce activated T cell death, which is an important mechanism in IDO-mediated T cell suppression. However, the mechanism of this phenomenon remains unclear. We found that 3-hydroxyanthranilic acid (3-HAA), the most potent metabolite, selectively eliminated activated T cells, which were stimulated with the bacterial superantigen staphylococcal enterotoxin A (SEA), but not resting T cells, by inducing apoptosis. We observed 3-HAA-induced depletion of intracellular glutathione (GSH) in activated T cells. When GSH levels were maintained by addition of N-acetylcysteine (NAC) and GSH, 3-HAA-mediated T cell death was completely inhibited. This was associated with extrusion of GSH from activated T cells without increased reactive oxygen species (ROS) formation. Finally, we showed that administration of 3-HAA in mice after allogeneic bone marrow transplantation reduced acute graft-versus-host disease (GVHD) lethality by inhibition of alloreactive T cell expansion through intracellular GSH depletion. Our data suggest that direct depletion of intracellular GSH is the major mechanism of 3-HAA-mediated activated T cell death. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Indoleamine 2,3-dioxygenase (IDO) is a cytoplasmic enzyme that converts tryptophan to catabolic products, collectively known as kynurenines [1,2]. The functional activity of IDO contributes to T cell tolerance in mammalian pregnancy [3], tumor resistance [4,5], allograft acceptance [6,7], autoimmunity [8–10], and allergy [11]. It has been suggested that the effector mechanism of IDO may involve tryptophan Trp metabolites or Trp starvation [12]. Tryptophan starvation by IDO induces an increase in the level of uncharged tRNA in T cells, which follows activation of the amino acid-sensitive general control non-depressible 2 (GCN2) stress kinase pathway. This causes cell cycle arrest of T cells and induces these cells to become more sensitive to apoptosis [13]. Several studies have demonstrated that exogenous treatment with Trp metabolites, such as l-kynurenine, 3-hydroxykynurenine (3-HK), and 3-hydroxyanthranilic acid (3-HAA), preferentially promotes apoptosis in activated T cells [14,15]. Fallarino et al. showed that

∗ Corresponding author at: Department of Microbiology and Immunology, Inje University College of Medicine, 633-165 Gaegum-Dong, Busan, Republic of Korea. Tel.: +82 51 890 6434; fax: +82 51 891 6004. E-mail address: [email protected] (S.-K. Seo). 0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2010.05.008

both 3-HAA and quinolinic acid induce apoptosis in Th1 cells, but not Th2 cells, by Fas-independent mechanisms involving activation of caspase8 and release of cytochrome c from mitochondria [16]. It has also been suggested that 3-HAA suppresses T cell antigen receptor-triggered NF-␬B activation by directly inhibiting PDK1 phosphorylation [17]. However, it remains unclear how Trp metabolites induce activated T cell death without affecting resting T cells although both cell types are exposed to it. Intracellular glutathione (GSH) is a tripeptide thiol that is required for cellular processes during primary T cell activation, such as cell cycle progression [18,19] and cytokine production [20,21]. Depletion of intracellular GSH with l-buthionine sulfoximine (BSO) inhibits T cell proliferation in response to mitogenic stimulation by cell cycle arrest [22]. This response could be reverted by reconstituting GSH levels with N-acetylcysteine (NAC) [23] and GSH [24]. These previous reports demonstrated that GSH depletion increases the susceptibility of activated T cells to apoptosis much more than resting T cells, potentially via target selectivity. In this study, we investigated the intracellular molecular mechanism of 3-HAA-mediated activated T cell death. We found that 3-HAA selectively induced apoptosis by depleting GSH in activated T cells, independent of increased reactive oxygen species (ROS) generation. Moreover, we found that this mechanism of 3-HAAmediated GSH depletion is physiologically relevant to a mouse allogeneic bone marrow transplantation (BMT) model.

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2. Materials and methods 2.1. Antibodies and reagents The following mAbs and reagents were purchased from BD Biosciences (San Jose, CA): PE- and PE-Cy5-anti-human CD3 (UCHT1), FITC- and PE-anti-human CD25 (BC96), PE-Cy5-antimouse Ly5.1 (A20), PE-anti-mouse CD3 (145-2C11), PE-anti-mouse CD4 (L3T4), PE-anti-mouse CD8 (Ly-2), purified mouse IgG, PEAnnexin V, BrdU Flow Kit, and 7-AAD. Purified anti-mouse cleaved caspase3 (Asp175) was obtained from Cell Signaling (Beverly, MA). The 3-hydroxyanthranilic acid (3-HAA), monobromobimane (MBB), N-acetylcysteine (NAC), glutathione reduced ethyl ester (GSH), ␣-tocopherol, and allopurinol (AP) were obtained from Sigma–Aldrich (St. Louis, MO). Manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) was obtained from Calbiochem (La

Jolla, CA). SEA was purchased from Toxin Technology (Sarasota, FL). Carboxyfluorescein succinimidyl ester (CFSE), dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (DCFDA), and 2-mercaptoethanol (2-ME) were obtained from Invitrogen (Carlsbad, CA). 2.2. Cell culture and treatments Peripheral blood samples were collected from healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were obtained by density centrifugation using Ficoll-Histopaque (Sigma). Cells (1 × 107 ) were suspended in PBS and incubated with 5 ␮M CFSE for 8 min at room temperature, and the reaction was stopped with cold 5% FBS/PBS. Cells (1 × 106 cell/ml) were washed with HBSS and suspended in RPMI 1640 tissue culture medium, which was supplemented with 10% FBS (Invitrogen), 100 U/ml penicillin (Cambrex),

Fig. 1. 3-HAA selectively induces activated T cell death. (A) Human PBMCs were labeled with CFSE and stimulated with SEA in the presence of the indicated concentrations of 3-HAA for 72 h. The cells were then stained with PE-anti-CD3. Data represent the cell proliferation of gated 7-AAD− CD3+ T cells, as determined by flow cytometry. The numbers indicate the percentages of gated T cells among total live cells. (B) CFSE-labeled PBMCs were pre-stimulated with SEA for 48 h prior to treatment with 100 ␮M 3-HAA for the indicated incubation periods (B) and for 24 h (C). (B) The cells were then stained and analyzed as in (A). (C) Cells were stained with PE-Cy5-anti-CD3 and PE-Annexin V and analyzed by flow cytometry (left). Quantification of the percentage of Annexin V+ cells at each T cell division (right). Similar results were obtained with PBT cells from five individuals.

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and 100 ␮g/ml streptomycin (Cambrex), and 0.2 ml of the suspension was then plated in 96-well plates (Corning Costar, Cambridge, MA) and stimulated with 0.15 ␮g/ml of SEA, either in the presence or absence of the indicated concentration of 3-HAA for 3 days. To determine the effects of 3-HAA on pre-stimulated cells, CFSElabeled or unlabeled PBMCs were first stimulated with SEA for 48 h and then treated with 100 ␮M 3-HAA. The cells were harvested for analysis at the indicated times. 2.3. Flow cytometry To immunostain CFSE-labeled cells, cultured cells were harvested, washed with FACS buffer (PBS containing 2% FCS and 0.01% NaN3), and pre-incubated with purified human IgG for 10 min at 4 ◦ C to block non-specific binding to Fc receptors. Cells were incubated with PE- or PE-Cy5-anti-CD3 for 25 min at 4 ◦ C. Cells were washed and resuspended in 0.3 ml of FACS buffer and treated with 7-AAD before analysis with a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software. To discriminate between activated and resting T cells, cells were stained with PE- or PE-Cy5anti-CD3 and FITC- or PE-anti-CD25. For analysis of apoptosis, cells were stained with PE-Annexin V according to the manufacturer’s instructions (BD bioscience). Caspase3 activation was measured using anti-mouse cleaved caspase3 according to the manufacturer’s instructions (Cell Signaling).

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(H-2b/d ) recipient mice were given two separate doses of radiation (950 cGy) within 3 h to minimize the degree of gastrointestinal toxicity. T cells and T cell-depleted bone marrow cells (TCD BM) were purified from donor naïve C57BL/6 mice using a microbead separation system (Miltenyi Biotec). Recipient mice were transplanted with T cells (3 × 106 ) and TCD BM cells (5 × 106 ) via tail vein injection. 3-HAA was dissolved in 1 M NaOH and brought to a final concentration of 10 mg/ml in PBS after adjusting pH to 7.2–7.4. Recipient mice received 3-HAA or control vehicle at a dose of 50 or 100 mg/kg daily by intraperitoneal (i.p.) injection on days 0–13 following transplantation. For adoptive transfer experiments, T cells (H-2b , Ly5.1) were labeled with CFSE and transferred into lethally irradiated (1000 cGy) Balb/c (H-2d ) mice. Mice were injected i.p. with 3-HAA or vehicle daily on days 0–4. Spleens from recipient mice were harvested on day 4 after transfer, and T cell division was analyzed by dilution of CFSE in the donor T cells by flow cytometry. To measure GSH concentration in alloreactive donor T cells, donor T cells were isolated from spleens of recipient mice using a biotinanti-Ly5.1 and anti-biotin-microbead separation system (Miltenyi Biotec) and measured as described above.

2.4. Measurement of intracellular GSH Intracellular GSH levels in T cells were determined using previously described methods [25]. Briefly, cells were first stained with PE-Cy5-CD3 and PE-anti-CD25 and then washed with PBS. MBB (40 ␮M) was added, and the samples were incubated at room temperature for 10 min. MBB fluorescence was measured by flow cytometry. The concentration of intracellular GSH was measured using the Glutathione assay kit II (Calbiochem). Briefly, cultured cells were harvested at different time points, and T cells were isolated using anti-human CD3 magnetic beads (Miltenyi Biotech, Auburn, CA). Isolated T cells (1 × 105 ) were resuspended in 100 ␮l of 50 mM phosphate buffer, pH 7.0, containing 1 mM EDTA and homogenized. After centrifugation of the homogenate at 10,000 g for 15 min, 50 ␮l of the supernatant was transferred to 150 ␮l of assay cocktail solution according to the manufacturer’s instructions. The mixture was incubated at 25 ◦ C for 10 min in the dark on an orbital shaker. The absorbance at 405 nm was measured, and the GSH concentration was then determined based on the GSH standard curve. 2.5. Intracellular ROS generation Intracellular ROS levels in T cells were determined using previously described methods [26]. The cells were first stained with PE-Cy5-anti-CD3 and FITC- or PE-anti-CD25, then incubated with DHA or DCFDA anti-CD3 and CD25, and then washed with PBS. For DHE staining, cells were resuspended in pre-warmed PBS. The dye (2 ␮M) was added, and cells were incubated at room temperature for 1 h. For DCFDA staining, cells were incubated at 37 ◦ C for 30 min following addition of the dye (5 ␮M). After washing, the cells were placed on ice and analyzed by flow cytometry. 2.6. In vivo studies Female C57BL/6 (H-2b , Ly5.2), BALB/c (H-2d , Ly5.2), and B6D2F1 (H-2b/d ) mice were obtained from Charles River (Tokyo, Japan). B6.SJL-Ptprca Pep3b /BoyJ (H-2b , Ly5.1) mice were purchased from Jackson Laboratory (Bar Harbor, ME). For GVHD induction, B6D2F1

Fig. 2. 3-HAA depletes intracellular GSH in activated T cells. PBMCs were prestimulated with SEA for 48 h and then treated with 100 ␮M 3-HAA for 24 h (A) and for the indicated times (B). (A) Representative flow cytometry data. The cells were stained with PE-Cy5-nti-CD3 plus PE-anti-CD25 and then stained with 40 ␮M MBB for 15 min at RT. The data represent the intracellular GSH levels (MFI value) of gated CD25+ CD3+ T cells (activated) and CD25- CD3+ T cells (resting). Unstained cells were used as the negative control (filled curve). (B) Quantification of intracellular GSH levels. Cultured cells were harvested at the indicated time points, and T cells were isolated using anti-CD3 magnetic beads. The intracellular GSH concentration in isolated T cells (1 × 105 ) was measured as described in Section 2. * p < 0.05, compared with the control treatment. The data are means ± S.E. Similar results were obtained with PBT cells from three individuals.

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2.7. Statistics The Student’s t-test was used for statistical analysis of the in vitro data. P values less than 0.05 were defined as statistically significant. The Kaplan–Meier product-limit method was used to obtain survival curves. Survival data were analyzed by the log-rank test. 3. Results 3.1. Selective elimination of activated T cells by 3-HAA Previous reports showed that Trp metabolites preferentially inhibit activated T cells by promoting cell death [19,20]. To investigate the selective inhibitory effect of 3-HAA in human peripheral T cells, we first labeled human PBMCs with CFSE and stimulated them with bacterial superantigen SEA to activate a high percentage of T cells expressing a particular TCR V beta region [27], either in the presence or absence of various concentrations of 3HAA. In the control treatment, both CFSElow proliferating CD3+ cells, identified as activated T cells (CD25+ CD27+ CD45RO+ , data not

shown), and CFSEhi CD3+ cells, identified as resting T cells, were observed. However, treatment with 3-HAA decreased the activated T cell population in a dose-dependent manner. Treatment with 100 ␮M 3-HAA completely inhibited the activated T cell population (4.5 ± 1.2% of total cells) compared with the control treatment (35.1 ± 2.6% of total cells), while demonstrating no effects on resting T cells (41.1 ± 2.3% of total cells versus control, 37.2 ± 2.5%). We next evaluated the selective inhibition mediated by 3-HAA on the simultaneous presence of activated and resting T cells. To generate culture conditions in which both activated and resting T cells are present, we pre-stimulated PBMCs with SEA for 48 h prior to 3-HAA treatment. SEA-reactive activated T cells continuously expanded following the control treatment (27.5 ± 2.3% at 48 h, 33.3 ± 3.2% at 48 h + 24 h, 37.2 ± 4.6% at 48 h + 48 h, and 50.6 ± 4.2% at 48 h + 60 h), whereas treatment with 3-HAA selectively eliminated activated T cells over a period of time without any cytotoxic effects on resting T cells (Fig. 1B). We then assessed whether 3-HAA selectively eliminated activated T cells through induction of apoptosis. Treatment of pre-stimulated cells with 3-HAA induced significantly increased

Fig. 3. Maintenance of intracellular GSH protects against 3-HAA-mediated activated T cell death via inhibition of caspase3 activation. (A) GSH supplementation prevents 3-HAA-mediated activated T cell death. CFSE-labeled PBMCs were stimulated with SEA in the presence of 3-HAA (100 ␮M) alone, 3-HAA plus NAC (5 mM), or 3-HAA plus GSH (10 mM) for 72 h. The cells were then stained and analyzed as in Fig. 1A. (B and C) PBMCs were pre-stimulated with SEA for 48 h and then treated with 3-HAA (100 ␮M) or 3-HAA plus NAC (5 mM) for 24 h (B) and the indicated time periods (C). (B) Inhibition of caspase3 activation by NAC in 3-HAA-treated activated T cells. The cells were stained for intracellular activated caspase3 as described in Section 2. The data represent the activated caspase3 levels of gated CD25+ CD3+ T cells, as determined by flow cytometry. (C) Maintenance of GSH levels with NAC in 3-HAA-treated T cells. The intracellular GSH concentration was measured as in Fig. 2B. * p < 0.05, ** p < 0.001, compared with 3-HAA treatment. The data are means ± S.E. Similar results were obtained with PBT cells from three individuals.

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apoptosis (Annexin V+ ) among activated T cells compared with the control treatment (Fig. 1C). The apoptotic sensitivity of these cells following 3-HAA treatment was markedly increased following cell division. Taken together, these results show that 3-HAA selectively eliminates activated T cells by inducing apoptosis. 3.2. Depletion of intracellular GSH by 3-HAA GSH depletion is an early hallmark of apoptosis observed in primary activated T cells exposed to apoptotic stimuli, including death receptor-induced [28] and drug-induced cell death [29]. To determine whether 3-HAA depletes intracellular GSH in activated T cells, we measured intracellular GSH levels in T cells by MBB staining. Consistent with previous studies [30], we found that intracellular GSH levels increased in activated T cells compared with resting T cells (Fig. 2A). However, treatment of pre-stimulated cultures with

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3-HAA decreased GSH levels in activated T cells (Fig. 2A). To determine the kinetics of intracellular GSH depletion following 3-HAA treatment, we next measured intracellular GSH concentration in T cells at different time points. As shown in Fig. 2B, intracellular GSH was dramatically depleted in T cells after 24 h treatment with 3-HAA (9.1 ± 0.80 ␮M) compared with the control treatment (18.0 ± 0.95 ␮M). These results suggest that 3-HAA depletes intracellular GSH, which may be related to the induction of apoptosis in activated T cells. 3.3. Replenishment of intracellular GSH protects against 3-HAA-elicited selective elimination To determine whether GSH depletion by 3-HAA is responsible for the selective elimination of activated T cells, we analyzed the effect of 3-HAA on activated T cells after intracellular levels of GSH

Fig. 4. GSH depletion by 3-HAA is not associated with ROS generation in activated T cells. (A) Kinetics of ROS generation. PBMCs were pre-stimulated with SEA for 48 h and then treated with 100 ␮M 3-HAA for the indicated time periods. The cells were stained with PE-Cy5-anti-CD3 plus FITC- or PE-anti-CD25 and then incubated with DHA or DCFDA as described in Section 2. The data represent the DHA or DCFDA oxidation (MFI value) of gated CD25+ CD3+ activated T cells. * p < 0.05, compared with the control treatment. The data are means ± S.E. (B) Effect of non-thiol antioxidants. CFSE-labeled PBMCs were stimulated with SEA in the presence of 3-HAA (100 ␮M) alone, 3-HAA plus MnTBAP (50 ␮M), 3-HAA plus AP (10 ␮M), or 3-HAA plus ␣-toc (1 ␮M) for 72 h. The cells were then stained and analyzed as in Fig. 1A. Similar results were obtained with PBT cells from three individuals. (C) GSH levels in culture supernatants. PBMCs were pre-stimulated with SEA for 48 h and then treated with 100 ␮M 3-HAA for 24 h. The GSH concentration in the culture supernatants was measured as described in Fig. 2B.

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were replenished with NAC and GSH. As shown in Fig. 3A, activated T cells supplemented with NAC and GSH were completely protected from 3-HAA-elicited elimination. This was associated with inhibition of caspase3 activation (Fig. 3B). We confirmed that the inhibition of 3-HAA-induced apoptosis was related to maintenance of intracellular GSH levels with thiol treatment. Maintenance of GSH levels in T cells treated with NAC was observed during 3HAA treatment (Fig. 3C). Together, these data suggest that 3-HAA induces activated T cell death by depleting intracellular GSH via caspase3 activation. 3.4. 3-HAA induces activated T cell death by extrusion of GSH GSH depletion has been found to provoke a rise in ROS and to induce apoptotic cell death [31]. It has also been reported that 3-HAA exhibits pro- [32] and antioxidant activities [33]. To evaluate whether GSH depletion by 3-HAA is associated with elevated ROS levels in activated T cells, we used DHE and DCFDA staining to examine O2 − and H2 O2 levels, respectively, in activated T cells by flow cytometry. When pre-stimulated cultures were treated with 3-HAA, intracellular ROS levels were not increased in activated T cells compared with those that received the con-

Fig. 5. 3-HAA treatment limits expansion of alloreactive T cells through GSH depletion. Lethally irradiated (950 cGy) Balb/c mice were adoptive transferred with CFSE-labeled T cells from B6 (Ly5.1, H-2b ) mice. Recipients were injected i.p. with 3-HAA (50 or 100 mg/kg) or control vehicle daily from day 0 to 3. (A) Spleens from recipients were harvested on day 4 after transfer, and T cell division was analyzed by dilution of CFSE in the donor T cells (Ly5.1+ ) by flow cytometry. (B) Donor T cells (Ly5.1+ ) were isolated from recipient spleens 4 days after transplant as described in Section 2, and intracellular GSH concentration (1 × 105 ) was measured as described in Fig. 2B. Naïve T cells were used as the negative control. *p < 0.05, compared with the control treatment. The data are means ± S.E.

trol treatment, as determined by both DHE and DCFDA staining (Fig. 4A). In addition, non-thiol antioxidants, MnTBAP, AP, and ␣tocopherol, were unable to reverse the effect of 3-HAA on activated T cells (Fig. 4B). Recent studies have reported that increasing apoptosis sensitivity following GSH depletion is independent of ROS generation in activated T cells [28,37]. It has been mostly associated with GSH extrusion across the plasma membrane [38]. We found that GSH was increased in the culture supernatants following 3-HAA treatment (Fig. 4C), which indicates that 3-HAA induces releasing GSH from activated T cells. Taken together, these results demonstrate that 3-HAA-mediated activated T cell death by GSH depletion is associated with GSH extrusion rather than oxidative pathway.

3.5. Administration of 3-HAA limits alloreactive T cell expansion through GSH depletion To further assess the physiological relevance of 3-HAAmediated GSH depletion, we adoptively transferred CFSE-labeled donor T cells into lethally irradiated allogeneic recipient mice, which were treated with 3-HAA or the control vehicle. Control treatment resulted in extensive expansion of alloreactive donor T cells. However, 3-HAA treatment inhibited alloreactive T cell expansion, which primarily involved CD8 T cells, rather than CD4 T cells (Fig. 5A). To determine whether alloreactive T cell inhibition following 3-HAA treatment is associated with intracellular GSH depletion, we measured intracellular GSH levels in purified donor T cells isolated from spleens of recipients. As shown in Fig. 5B, 3HAA treatment resulted in significantly decreased intracellular GSH compared with control treatment (2.87 ± 0.47 ␮M versus control 6.1 ± 0.05 ␮M, p = 0.02). These data indicate that 3-HAA treatment inhibits alloreactive T cell expansion in vivo, which is mediated by GSH depletion.

Fig. 6. Administration of 3-HAA reduces acute GVHD lethality after allogeneic BMT. Lethally irradiated (950 cGy) B6D2F1 recipients were transplanted with 3 × 106 T cells and 5 × 106 TCD BM cells from C57BL/6 donors. Groups of 10 mice each were injected daily with control vehicle or 3-HAA at 50 or 100 mg/kg from day 0 to 13. Percent survival (A) and body weight change (B) of recipients after BMT.

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3.6. Administration of 3-HAA reduces acute GVHD lethality Because 3-HAA significantly suppressed alloreactivity in vivo, we next assessed its therapeutic effects on a murine model of acute lethal GVHD (C57BL/6 → B6D2F1). As shown Fig. 6A, recipients that were administered 3-HAA demonstrated significantly improved survival rates from acute GVHD in a dose-dependent manner. At a dose of 100 mg/kg, long-term survivor mice were free of clinical GVHD, as indicated by the pattern of body weight gain (Fig. 6B). Taken together, these data indicate that administration of 3-HAA reduces acute GVHD lethality.

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GVHD lethality through alloantigen-reactive T cell suppression, which is mediated by GSH depletion. Our results provide not only valuable information for the development of Trp metabolite-based drugs but also suggest an IDO-mediated downstream mechanism. Acknowledgements This research was supported by a grant (0920040 to SKS) from the National R&D Program for Cancer Control, Ministry of Health, Welfare and Family affairs, Republic of Korea, and by the 2006 Inje University research grant (WSL).

4. Discussion References In addition to Trp depletion, generation of Trp metabolites is known to be a key pathway of functional IDO activity in T cell suppression. Previous studies have demonstrated the beneficial effects of exogenous treatment with these metabolites on T cell suppression. L-Kyn, 3-HK, and 3-HAA preferentially induce death of activated T cells without causing functional defects in resting T cells [14,15]. Therefore, Trp metabolites have been identified as potential targets in the development of therapeutic drugs for the treatment of T cell-mediated immunological diseases, such as autoimmune disease and transplantation [34–36]. However, the intracellular molecule mechanism of these metabolites is still unclear. In the present study, we demonstrate one possible molecular mechanism for the selective death of activated T cells induced by 3-HAA. Our data show that 3-HAA selectively induces apoptosis in activated T cells (Fig. 1) by intracellular GSH depletion (Fig. 2). When GSH levels are replenished by the addition of exogenous NAC or GSH, the inhibition of T cell expansion induced by 3-HAA was completely reversed (Fig. 3). In fact, intracellular GSH plays a key role in promoting the proliferation, cytokine production, and survival of primary activated T cells [18–21]. Therefore, because GSH depletion increases the susceptibility of activated T cells to apoptosis much more than that of resting T cells, it may provide target selectivity. Recently, Platten et al. reported that 3-HAA treatment skews the cytokine profile of myelin-specific TH cells from TH 1 to TH 2, thereby ameliorating established experimental autoimmune encephalomyelitis (EAE) [35]. It has also been suggested that 3HAA suppresses T cell antigen receptor-triggered NF-␬B activation by directly inhibiting PDK1 phosphorylation [17]. We found that the effect of 3-HAA on GSH depletion is physiologically relevant in a murine allogeneic T cell transplant model (Fig. 5). Moreover, we also observed that 3-HAA improved the survival rate following acute GVHD (Fig. 6). As demonstrated by earlier studies and our data, 3-HAA might affect activated T cells by multiple pathways in vivo. GSH is the most abundant antioxidant in many animal cells. GSH depletion has been found to provoke a rise in ROS and to induce apoptotic cell death [31]. However, its function in T cells remains controversial. Recent studies have reported that increasing apoptosis sensitivity following GSH depletion is independent of ROS generation in activated T cells [28,37]. It has been mostly associated with GSH extrusion across the plasma membrane [38]. Although 3-HAA depletes GSH in activated T cells, our data showed that 3-HAA treatments did not increase ROS generation in activated T cells, and antioxidants did not reverse 3-HAA-induced activated T cell death (Fig. 4). In contrast, 3-HAA treatment increased GSH level in the culture supernatants. These results demonstrate that GSH extrusion by 3-HAA treatment increases the susceptibility to apoptosis of activated T cells without increasing ROS generation. In summary, the data presented here demonstrate that 3-HAA directly induces activated T cell death via depletion of intracellular GSH. Importantly, administration of 3-HAA is able to reduce acute

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