Tuberous Sclerosis 1 Promotes Invariant NKT Cell Anergy and Inhibits Invariant NKT Cell−Mediated Antitumor Immunity This information is current as of March 6, 2015.
Jinhong Wu, Jinwook Shin, Danli Xie, Hongxia Wang, Jimin Gao and Xiao-Ping Zhong J Immunol 2014; 192:2643-2650; Prepublished online 14 February 2014; doi: 10.4049/jimmunol.1302076 http://www.jimmunol.org/content/192/6/2643
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References
The Journal of Immunology
Tuberous Sclerosis 1 Promotes Invariant NKT Cell Anergy and Inhibits Invariant NKT Cell–Mediated Antitumor Immunity Jinhong Wu,*,† Jinwook Shin,* Danli Xie,*,‡ Hongxia Wang,*,x Jimin Gao,‡ and Xiao-Ping Zhong*,{
T
he invariant NKT (iNKT) cells belong to a distinct subset of T lymphocytes characterized by the cell surface expression of an invariant Va14-Ja18 TCR (iVa14TCR) a-chain in mice. The activation of iNKT cells is initiated by the engagement of the iNKT receptor with lipid Ags such as a-GalCer presented by the MHC class I-like molecule CD1d, which leads to the rapid production of a variety of cytokines such as IFN-g, IL-4, and other cytokines. iNKT cells greatly impact innate immunity, shape adaptive immune responses, and contribute to or regulate the pathogenesis of diseases via these cytokines (1–5). Evidence has implicated iNKT cells in tumor surveillance. Deficiency of iNKT cells impairs antitumor immune responses, whereas activation of iNKT cells has been demonstrated to enhance tumor eradication (6–10). Considerable effort has been expended to employ the immunoregulatory functions of iNKT cells for immunotherapy of cancer via iNKT cell transfer and/or repetitive stimulation of iNKT cells using iVa14TCR ligands such as a-galactosylceramide (a-GalCer) (11, 12). However, iNKT cell *Division of Allergy and Immunology, Department of Pediatrics, Duke University Medical Center, Durham, NC 27710; †Division of Pediatric Pulmonology, Department of Internal Medicine, Shanghai Children’s Medical Center affiliated with Shanghai Jiaotong University School of Medicine, Shanghai 200127, China; ‡School of Laboratory Medicine, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China; xLaboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China; and {Department of Immunology, Duke University Medical Center, Durham, NC 27710 Received for publication August 6, 2013. Accepted for publication January 15, 2014. This work was supported by National Institutes of Health Grants AI076357, AI079088, and AI101206 and American Cancer Society Grant RSG-08-186-01-LIB. Address correspondence and reprint requests to Dr. Xiao-Ping Zhong, Division of Allergy and Immunology, Department of Pediatrics, Room 133 MSRB, Research Drive, Box 2644, Duke University Medical Center, Durham, NC 27710. E-mail address: [email protected] Abbreviations used in this article: BFA, brefeldin A; cabT, conventional ab T; a-GalCer, a-galactosylceramide; iNKT, invariant NKT; iVa14TCR, invariant Va14Ja18 TCR; KO, knockout; MNC, mononuclear cell; mTOR, mammalian target of rapamycin; PD-1, programmed death-1; qPCR, quantitative PCR; TSC, tuberous sclerosis; WT, wild-type. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302076
anergy is a significant challenge to the success of such immunotherapy. Following in vivo activation by a-GalCer, iNKT cells undergo dynamic changes characterized by robust cytokine secretion, clonal expansion, homeostatic contraction, and then acquisition of an anergic phenotype (13, 14). Similar to conventional ab T (cabT) cells, iNKT cell anergy is an unresponsive or hyporesponsive state to secondary Ag stimulation following initial TCR stimulation. iNKT cell anergy thwarts the effectiveness of repeated administration of a-GalCer for cancer immunotherapy (13). Although TCR signal strength (15, 16), programmed death-1 (PD-1) (17, 18), the E3-ubiquitin ligase Cbl-b (19), and Wnt/ b-catenin signaling (20) are implicated in iNKT cell anergy, the mechanisms that control iNKT cell anergy remain poorly understood. Tuberous sclerosis (TSC)1 associates with TSC2 to form a complex that functions as a critical regulator of the mechanistic or mammalian target of rapamycin (mTOR). mTOR signals through two complexes, mTORC1 and mTORC2, to integrate numerous environmental stimuli, including growth factors, nutrients, and stressactivated signals to regulate cell metabolism, survival, growth, and proliferation (21, 22). The GTP-binding protein Rheb (Ras homolog enriched in brain) is crucial for mTORC1 activation. TSC2 inactivates RheB through its GTPase-activating protein activity, and TSC1 is crucial for TSC2 stability in many cell types, including immune cells (23). The PI3K-Akt pathway activates mTORC1 signaling through phosphorylation of TSC2, leading to its degradation (24, 25). In T cells, RasGRP1 is critical for mTOR activation via activating the Ras-Mek1/2-Erk1/2 pathway and for early iNKT cell development (26, 27). Coinciding with this observation, Erk1/2 was found to be able to inactivate TSC2 via phosphorylation (28). The importance of TSC1 in the immune system has only recently gained recognition. TSC1 maintains hematopoietic stem cells in a normally quiescent state by preventing them from proliferation (29). It is critical for T cell quiescence, survival, and anergy, and for the maintenance of a normal peripheral T cell pool (30–35). Additionally, it plays important roles in B cell development and function (36), controls macrophage and dendritic cell response to
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Development of effective immune therapies for cancer patients requires better understanding of hurdles that prevent the generation of effective antitumor immune responses. Administration of a-galactosylceramide (a-GalCer) in animals enhances antitumor immunity via activation of the invariant NKT (iNKT) cells. However, repeated injections of a-GalCer result in long-term unresponsiveness or anergy of iNKT cells, severely limiting its efficacy in tumor eradication. The mechanisms leading to iNKT cell anergy remain poorly understood. We report in this study that the tuberous sclerosis 1 (TSC1), a negative regulator of mTOR signaling, plays a crucial role in iNKT cell anergy. Deficiency of TSC1 in iNKT cells results in resistance to a-GalCer– induced anergy, manifested by increased expansion of and cytokine production by iNKT cells in response to secondary Ag stimulation. It is correlated with impaired upregulation of programmed death-1, Egr2, and Grail. Moreover, TSC1-deficient iNKT cells display enhanced antitumor immunity in a melanoma lung metastasis model. Our data suggest targeting TSC1/2 as a strategy for boosting antitumor immune therapy. The Journal of Immunology, 2014, 192: 2643–2650.
2644 TLR ligands (37, 38), promotes MHC class II expression and Ag presentation of dendritic cells (38) and dendritic cell development (39), and regulates mast cell survival and function (40). In mice with T cell–specific TSC1 deletion, iNKT cell numbers are decreased due to increased iNKT death, and such decrease can be rescued with Bcl-2 overexpression (31). However, the importance of TSC1 in iNKT cell function remains unclear. We report in this study that acute ablation of TSC1 does not obviously impact iNKT cell activation but results in resistance of iNKT cells to anergy induced by Ag stimulation, correlated with impaired upregulation of anergy-promoting molecules PD-1, Erg2/3, and Grail. Moreover, TSC1-deficient iNKT cells display enhanced antitumor immunity in a lung metastasis tumor model. In addition, anergic iNKT cells express high levels of TSC1 and TSC2 with concomitant decrease of mTORC1 signaling. Our data suggest targeting TSC1/2 to boost iNKT cell function as a new strategy for cancer immunotherapy.
Mice and cells The TSC1f/f-ERCre+ and TSC1f/f-ERCre2 mice backcrossed to C57BL/6J background for eight to nine generations, as previously reported (37). These mice were i.p. injected with tamoxifen (100 mg/kg body weight) on days 1, 2, and 5 and then euthanized for experiments on day 8 or other indicated days. Ja182/2 mice (10) were provided by K. Nichols (Children’s Hospital of Philadelphia, Philadelphia, PA), L. Van Kaer (Vanderbilt University, Nashville, TN), and M. Taniguchi (RIKEN, Center for Integrative Medical Sciences, Yokohama, Japan). The Va14Ja18 iNKT TCR transgenic (iVa14TCRtg) mice (41) were purchased from The Jackson Laboratory and bred with the TSC1deficient mice. All mice were housed in a pathogen-free facility and used according to protocols approved by the Institutional Animal Care and Use Committee of Duke University. Splenocytes and liver mononuclear cells (MNCs) were made according to previously published protocols (42).
Reagents, Abs, and flow cytometry IMDM was supplemented with 10% (v/v) FBS, penicillin/streptomycin, and 50 mM 2-ME (IMDM-10). PE- or allophycocyanin-conjugated mouse CD1d tetramers loaded with PBS-57 (CD1dTet) were provided by the National Institutes of Health Tetramer Facility. The Live/Dead Fixable Violet Dead Cell Stain Kit was purchased from Invitrogen. Fluorescence-conjugated antimouse TCRb (H57-597), NK1.1 (PK136), CD44 (IM7), B220 (RA3-6B2), Gr-1 (RB6-8C5), CD11c (N418), CD11b (M1/70), CD4 (GK1.5), CD8 (536.7), IFN-g (XMG1.2), IL-4 (11B11), PD-1 (RMP1-30), and ICOS (D10. G4.1) Abs were purchased from BioLegend. Anti–phospho-p44/42 MAPK T202/Y204 (197G2), anti–phospho-S6 S240/244 (D68F8), anti–phospho-4EBP1 T37/46 (236B4), anti–phospho-Akt S473 (193H12), TSC1 (D43E2), and TSC2 (D93F12) were purchased from Cell Signaling Technology. Anti–bactin Ab was purchased from Sigma-Aldrich. Cell surface staining was performed with 2% FBS-PBS. Intracellular staining for Ki67 was performed using the eBioscience Foxp3 staining buffer set. Ki67 was detected with an anti-Ki67 Ab (B56; BD Biosciences), followed by detection with AlexaFluor488conjugated goat anti-mouse IgG (H+L; Invitrogen). Intracellular staining for IFN-g and IL-4 was performed using the BD Biosciences Cytofix/Cytoperm and perm/wash solutions. Dead cells were excluded from the analysis using Live/Dead Fixable Violet Dead Cell Stain Kit (Invitrogen). The iNKT cell population was identified as Lin2 (B2202Gr12CD11b2CD11c2CD82) TCRb+CD1dTet+ cells. Data were collected using FACS Canto-II (BD Biosciences) and analyzed using Flowjo software (Tree Star).
Purification of iNKT cells and real-time quantitative PCR Mice either untreated or treated with a-GalCer (2 mg, i.v., one injection) 7 days early were euthanized to isolate splenocytes and liver MNCs. iNKT cells from pooled splenocytes and liver MNCs were enriched with CD1dTet and MACS beads, according to a previously published protocol (27). Enriched iNKT cells were stained with anti-TCRb and 7-aminoactinomycin D. Live TCRb+CD1dTet+ iNKT cells were sorted using MoFlo with .98% purity and were immediately lysed in TRIzol for RNA preparation. cDNA was made using the iScript Select cDNA Synthesis Kit (Bio-Rad). Real-time quantitative PCR was performed and analyzed, as previously described (34). Expressed levels of target mRNAs were normalized with b-actin and calculated using the 22DDCT method. Primers were as follows: Egr2 (forward, 59-TTGACCAGATGAACGGAGTG-39;
reverse, 59-CAGAGATGGGAGCGAAGCTA-39), Egr3 (forward, 59-CGCGCTCAACCTCTTCTC-39; reverse, 59-GATTGGGCTTCTCGTTGGT-39), Itch (forward, 59-GGACCACAGCTTGATTCCAT-39; reverse, 59-GACTTGGTCCAAACCAATTCTT-39), Grail (forward, 59-CAACCGTGGGCTATTTCATC-39; reverse, 59-GCAGCTGAAGCTTTCCAATA-39), PD-1 (forward, 59-CTGGAAGCAAGGACGACACT-39; reverse, 59-TGGTGGCATATTCTGTGTGC-39), P27 (forward, 59-CCTGACTCGTCAGACAATCC-39; reverse, 59-TCTGTTCTGTTGGCCCTTTT-39), Cbl-b (forward, 59GGCAAGGAAAGGATGTACGA-39; reverse, 59-AAGATCGCCTTGATTTCTGC-39), TSC1 (forward, 59-ACATCTTTGGCCGTCTCTCGTCAT-39; reverse 59-ATGGGTACATCCCATAAAGGCGGT-39), TSC2 (forward, 59-ACTGCCAACCAGACAAGGTGTACT-39; reverse, 59-GTTTCTTGTCACAGCGGTGCTTGT-39), b-actin (forward, 59-TGTCCACCTTCCAGCAGATGT-39; reverse, 59-AGCTCAGTAACAGTCCGCCTAGA-39).
Stimulation of iNKT cells and induction of iNKT cell anergy For in vivo iNKT cell stimulation, mice were first i.p. injected with 150 mg brefeldin A (BFA; Sigma-Aldrich), followed by i.v. injection with 2 mg a-GalCer 90 min later. Two hours after a-GalCer injection, spleens and livers were harvested for iNKT cell staining and intracellular staining of IFN-g and IL-4. a-GalCer–induced iNKT cell anergy in mice was performed according to a previously published protocol (13). Briefly, mice were i.v. injected with 2 mg a-GalCer on days 1 and 8, respectively. The following experiments were performed for anergy evaluation. 1) Sera were collected 4 h after each injection for measurement of IL-4 and IFN-g concentrations. 2) Three days after a-GalCer injection, splenocytes and liver MNCs were stained for iNKT cells and for Ki67 expression. 3) One and one-half hours before a-GalCer injection, mice were injected with BFA and IFN-g, and IL-4 intracellular staining in splenic and liver iNKT cells was performed, as described above.
Western blot analysis Cell lysates were prepared from thymocytes of Raptorf/f and Raptorf/fERCre mice following tamoxifen treatment or from purified iNKT cells isolated from naive mice or a-GalCer–experienced mice. Cell lysates were subjected to Western blot analysis, as previously described (26, 30). Western blot films were scanned, and phosphorylation and protein intensities were quantified using the Adobe Photoshop CS4 software.
Determination of B16 melanoma lung metastases iNKT cells were isolated from pooled thymocytes, splenocytes, and liver MNCs from TSC1f/f-ERCre2-iVa14-TCRtg or TSC1f/f-ERCre+-iVa14-TCRtg mice on day 0 after i.p. injected with 200 ml 10 mg/ml tamoxifen on days 28, 27, and 24. After i.v. receiving 5 3 105 B16F10 melanoma cells on day 0, female Ja182/2 mice were injected with 1 3 106 wild-type (WT) or TSC1-deficient iNKT cells. Recipient mice were i.v. injected with 2 mg a-GalCer at 1, 4, and 7 d and euthanized on day 14. The number of metastatic nodules in the lung was counted.
Results Effects of acute TSC1 deletion on iNKT cell homeostasis and cytokine production Mice with T cell–specific deletion of TSC1, TSC1f/f-CD4Cre, contain reduced iNKT cells (31) and are not ideal for examining mature iNKT cell function. To delete TSC1 in mature iNKT cells, we generated TSC1f/f (WT) and TSC1f/f-ERCre+ (knockout [KO]) mice and injected tamoxifen into the mice on days 1, 2, and 5. Mice were euthanized on day 8 for evaluation of TSC1 deletion efficiency as well as iNKT cells. As shown in Fig. 1A, TSC1 protein was drastically decreased in TSC1KO thymocytes and splenocytes, indicating efficient deletion of the gene. TSC2 protein was also reduced, consistent with the importance of TSC1 for TSC2 stability. Moreover, S6 and 4E-BP1 phosphorylation, which is dependent on mTORC1 activation, was increased in TSC1KO thymocytes and splenocytes, suggesting enhanced mTORC1 signaling. AKT phosphorylation at serine 473, a mTORC2-mediated event, was slightly decreased in TSC1KO splenocytes but was not decreased in thymocytes. ERK1/2 phosphorylation was not obviously affected by TSC1 deficiency. Thus, acute deletion of TSC1
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Materials and Methods
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resulted in elevated mTORC1 signaling in both thymocytes and splenocytes with minimal impact on mTORC2 signaling. Staining of iNKT cells with CD1dTet and TCRb revealed similar percentages and numbers in the WT and TSC1KO thymus, spleen, and liver (Fig. 1B). Moreover, percentages and numbers of stage 1 (CD442NK1.12), stage 2 (CD44+NK1.1+), and stage 3 (CD44+NK1.1+) iNKT cells were also similar between these two groups of mice (Fig. 1C). Thus, in the time frame we tested, TSC1 deficiency did not obviously affect iNKT cell homeostasis. To determine whether acute deletion of TSC1 may affect iNKT cell activation and cytokine production, we injected BFA into WT and
TSC1KO mice to block protein secretion and subsequently injected with a-GalCer 90 min later. IFN-g and IL-4 expression in iNKT cells 2 h after a-GalCer injection was determined by intracellular staining. As shown in Fig. 2, both splenic and liver iNKT cells from TSC1KO mice expressed similar levels of IFN-g and IL-4 to their respective WT controls. Thus, acute TSC1 ablation does not obviously affect Ag-induced cytokine production by iNKT cells. Resistance of TSC1-deficient iNKT cells to anergy induction Following initial Ag stimulation, iNKT cells undergo expansion that peaks on day 3 and then contract to close to basal level on day 7.
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FIGURE 1. Effects of acute deletion of TSC1 on iNKT cell homeostasis. (A) Efficient ablation of TSC1 in TSC1f/f-ERCre+ mice following tamoxifen treatment. Total thymocyte and splenocyte lysates from TSC1f/f (WT) and TSC1f/f-ERCre+ (KO) mice following tamoxifen treatment were subjected to immunoblotting analysis with the indicated Abs. (B and C) iNKT cell staining in TSC1f/f and TSC1f/f-ERCre+ mice following tamoxifen treatment. Thymocytes, splenocytes, and liver MNCs were stained with TCRb, PBS570-loaded CD1dTet, CD44, NK1.1, and Live/Dead and subjected to flow cytometry analysis. (B) TCRb and CD1dTet staining of live-gated thymocytes, splenocytes, and liver MNCs. (C) CD44 and NK1.1 staining of gated TCRb+ CD1dTet+ cells. Bar graphs are mean 6 SEM presentation of percentages and cell numbers from multiple mice (n = 5). Data shown represent three experiments.
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FIGURE 2. Acute deletion of TSC1 does not obviously affect iNKT cell cytokine production. Tamoxifen-treated TSC1f/f and TSC1f/f-ERCre+ mice were i.p. injected with 150 mg BFA and then injected with 2 mg a-GalCer 90 min later. Two hours after a-GalCer injection, splenocytes and liver MNCs were cell surface stained with CD1d-Tet and anti-TCRb and intracellularly stained with anti–IFN-g and anti–IL-4. (A) Representative dot plots showing IFN-g and IL-4 staining in gated iNKT cells. (B) Bar graphs are mean 6 SEM of IFN-g– or IL-4–positive cells in CD1dTet+TCRb+ cells (n = 3). Data shown represent three experiments.
FIGURE 3. Elevated TSC1 and TSC2 expression in anergic iNKT cells. WT mice were i.v. injected with a-GalCer or PBS. Seven days later, mice were euthanized and iNKT cells were purified from the spleen and liver for isolation of total RNA and making cell lysates. (A) TSC1 and TSC2 mRNA levels determined by real-time qPCR; (B) TSC1/2 protein levels and phosphorylation of the indicated proteins determined by Western blot analysis. Data shown are representative of two experiments. **p , 0.01, ***p , 0.01, determined by Student t test.
the last injection for evaluation of iNKT cell expansion. In the 1˚ group, we observed similar numbers of iNKT cells in WT and TSC1KO spleens and livers (Fig. 4A). Moreover, expression of Ki67, a marker for cell proliferation, was only slightly increased in TSC1KO iNKT cells (Fig. 4B). Together, these data suggest that iNKT cell expansion during initial Ag encountering was not substantially affected by TSC1 deficiency. Different from the initial Ag-induced response, WT iNKT cell numbers were much less than TSC1KO iNKT cells following secondary a-GalCer injection (Fig. 4A). Moreover, Ki67 expression was much lower in 2˚ WT iNKT cells than in 1˚ WT iNKT cells, suggesting hypoproliferative response of Ag-experienced WT iNKT cells to secondary Ag stimulation (Fig. 4B). However, Ag-experienced TSC1KO iNKT cells (2˚) expressed much higher levels of Ki67 compared with their WT controls in response to a secondary a-GalCer stimulation. Thus, different from initial Ag encountering, Ag-experienced TSC1-deficient iNKT cells retained great proliferative responses to secondary Ag stimulation. In addition to a hypoproliferative response, another hallmark of anergic iNKT cells is defective cytokine production (13). Following primary a-GalCer injection, serum IL-4 and IFN-g levels were similarly induced in WT and TSC1KO mice, which was consistent with the observation shown in Fig. 2. After secondary a-GalCer injection, WT mice produced much less IFN-g and IL-4 compared with mice with only primary injection. However, Agexperienced TSC1KO mice still produced high levels of IFN-g and IL-4 following secondary a-GalCer injection (Fig. 5A). Consistent with elevated serum IL-4 and IFN-g levels, TSC1KO iNKT cells expressed higher levels of IFN-g and IL-4 2 h after a-GalCer injection determined by intracellular staining (Fig. 5B, 5C). Of note, the percentages of IL-4– and IFN-g–producing iNKT cells from Ag-experienced TSC1KO mice were still lower than those from naive mice, suggesting that TSC1 deficiency causes iNKT cells to partially resist anergy induction. The high serum IL-4 and IFN-g levels in Ag-experienced TSC1 KO mice following the second a-GalCer injection are most likely an additive effect of increased percentages of cytokine-producing iNKT cells and a ∼50% increase of iNKT cell numbers in these mice compared with WT control mice before the second a-GalCer injection (data not shown).
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These Ag-experienced iNKT cells become anergic and thus hyporesponsive to subsequent Ag stimulations (13). Interestingly, anergic iNKT cells expressed higher levels of TSC1 and TSC2 at the mRNA and protein levels (Fig. 3A). Moreover, phosphorylation of 4E-BP1 was obviously decreased in anergic iNKT cells, suggesting decreased mTORC1 signaling in anergic iNKT cells (Fig. 3B). To examine whether TSC1 is involved in iNKT cell anergy, we injected a-GalCer into WT and TSC1KO mice once on day 0 (1˚) or twice on days 0 and 8 (2˚), respectively. Mice were euthanized 3 d after
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FIGURE 4. Enhanced expansion of TSC1-deficient iNKT cells following repeated a-GalCer injection in vivo. TSC1f/f and TSC1f/f-ERCre+ mice were treated with tamoxifen on days 1, 2, and 5. Mice were either injected with a-GalCer on day 8 (1˚) and examined on day 11 or injected on days 8 and 15 (2˚) and examined on day 18. (A) Bar graphs are mean 6 SEM presentation of splenic and liver iNKT cell numbers (1˚, n = 4; 2˚, n = 5). (B) Intracellular staining of Ki67 in gated iNKT cells. Data shown are representative of three experiments. *p , 0.05, **p , 0.01, determined by Student t test.
At present, only a limited number of molecules is known to be involved in iNKT cells’ anergy. PD-1, a coinhibitory molecule expressed on T cells, is upregulated in iNKT cells following Ag encountering and is important for iNKT cell anergy (17, 18). Before a-GalCer injection, WT and TSC1KO iNKT cells expressed similar low levels of PD-1. Seven days after a-GalCer injection, PD1 was upregulated in WT iNKT cells. However, iNKT cells from similarly treated TSC1KO mice expressed lower levels of PD-1 than WT iNKT cells (Fig. 6A). To determine whether TSC1 controls PD1 expression at the mRNA level, we sorted iNKT cells from WT and TSC1KO splenocytes 7 d after primary a-GalCer injection to make total RNA. PD-1 mRNA level was measured by real-time quantitative PCR (qPCR). PD-1 mRNA level was induced in WT iNKT cells but not in TSC1KO iNKT cells following a-Galcer injection (Fig. 6B). These observations suggest that TSC1 may
promote PD-1 expression for anergy induction via increasing its transcription. The E3 ubiquitin ligases Cbl-b, Itch, and Grail; transcription factors Egr2/Egr3; and the cell cycle inhibitor p27kip are anergypromoting molecules in cab T cells (43–46). Recent research has demonstrated the importance of Cbl-b for iNKT cell anergy (19). We observed increased Egr2, Egr3, and Grail expression in Agexperienced WT iNKT cells compared with non-Ag–experienced iNKT cells. However, the induction of these molecules was abolished in the absence of TSC1. In addition, p27kip mRNA was reduced in Ag-experienced TSC1KO iNKT cells compared with WT controls (Fig. 6B). Together, these observations suggest that TSC1 positively controls the expression of PD-1, Egr2/3, Grail, and p27kip to promote iNKT cell anergy. Enhanced antitumor immunity by TSC1-deficient iNKT cells iNKT cells play important roles in antitumor immune responses (11, 12). To further understand whether TSC1-mediated iNKT cell
FIGURE 5. Resistance of TSC1-deficient iNKT cells to anergy induction. TSC1f/f and TSC1f/f-ERCre+ mice were treated with tamoxifen on days 1, 2, and 5. Mice either were injected with a-GalCer on day 8 (1˚) or injected on days 8 and 15 (2˚). (A) Serum IFN-g and IL-4 concentrations 4 h after the last injection measured by ELISA (first, n = 4; second, n = 5). Bar graphs are mean 6 SEM. (B and C) Intracellular staining of IFN-g and IL-4 in gated iNKT cells. Mice were injected with BFA 1.5 h before the last injection of a-GalCer. Splenocytes were harvested 2 h after the last a-GalCer injection for iNKT and cytokine staining. (B) Representative dot plots; (C) mean 6 SEM presentation of percentages of cytokine-producing iNKT cells (first, n = 4; second, n = 5). Data shown are representative of four experiments. ***p , 0.001.
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anergy induction regulates antitumor immune responses, we used the B16F10 melanoma lung metastasis model, a model for assessing iNKT cell antitumor immunity (10, 47, 48). Because TSC1 is systemically deleted in TSC1KO mice, we adoptively transferred iNKT cells from WT and TSC1KO mice carrying the iVa14TCR transgene (41) into the iNKT cell–deficient Ja182/2 mice. The recipient mice were injected with B16F10 melanoma on the same day (day 0) and received three a-GalCer injections on days 1, 4, and 7. On day 14, recipient mice were euthanized for evaluation of tumor metastasis in the lung. The numbers of tumor nodules in the lung were fewer in recipient mice reconstituted with TSC1KO iNKT cells than in those reconstituted with WT iNKT cells (Fig. 7A, 7B). Moreover, adoptively transferred TSC1KO iNKT cells expressed lower levels of PD-1 than WT iNKT cells in the a-GalCer–injected tumor-bearing recipient mice (Fig. 7C). Together, these data suggest that TSC1-deficient iNKT cells conferred stronger antitumor immunity against melanoma than WT iNKT cells.
Discussion iNKT cells play important roles in tumor immunity. Although initial injection of a-GalCel into mice activates iNKT cells, Ag-
FIGURE 7. Enhanced antitumor immunity by TSC1-deficient iNKT cells. A total of 2 3 105 B16F10 melanoma cells was injected into Ja182/2 mice on day 0. The recipient mice were subsequently i.v. injected with 1 3 106 enriched iNKT cells from tamoxifen-treated iVa14TCRtg-TSC1f/f and iVa14TCRtg-TSC1f/f-ERCre+ mice. Mice were also injected with a-GalCer on days 1, 4, and 7. Mice were euthanized on day 14 for evaluation of lung metastasis of melanoma. (A) Representative lung morphology. (B) Average of melanoma nodule numbers per lung. Each circle represents one mouse (n = 17). (C) Overlaid histograms show PD-1 expression in gated iNKT cells from the spleens. Bar graphs are mean 6 SEM of the geometric mean fluorescence intensity of PD-1 and percentages of iNKT cells in the indicated gate that expressed high levels of PD-1 (n = 7). Data shown are representative of four experiments. **p , 0.01, ***p , 0.001.
experienced iNKT cells render anergic, which hinders the effectiveness of repetitive application of iNKT agonists for enhancing antitumor immunity. Thus, overcoming the induction of anergy may overcome the hurdle in iNKT cell–mediated tumor immunotherapy. In this study, we demonstrate that TSC1 plays a critical role in iNKT cell anergy. We reveal that TSC1 differentially controls iNKT cell activation in agonist-unexperienced and -experienced iNKT cells. Acute deletion of TSC1 in mature iNKT cells does not obviously affect iNKT cell proliferation or cytokine production following initial encountering with a-GalCer in vitro and in vivo. In contrast to Ag-experienced and anergic WT iNKT cells, Ag-experienced TSC1-deficient iNKT cells retain
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FIGURE 6. Altered expression of anergy-related molecules in TSC1deficient iNKT cells. TSC1f/f and TSC1f/f-ERCre+ mice were treated with tamoxifen on days 1, 2, and 5. Mice were injected with either PBS or a-GalCer on day 8. On day 15, splenocytes were harvested for assessing iNKT cells. (A) Overlaid histograms show PD-1 expression on CD1dTet+ TCRb+ iNKT cells. Bar graph represents mean 6 SEM of the geometric mean florescence intensity (gMFI) of PD-1 (n = 5). (B) Altered mRNA levels of anergy-related genes. iNKT cells were enriched with CD1dTet and MACS beads, followed by FACS sorting. The levels of mRNA of the indicated molecules from the sorted iNKT cells were quantified by realtime qPCR. Bar graphs are mean 6 SEM (n = 3). Data shown represent three experiments. ***p , 0.001.
TSC1 FOR iNKT CELL ANERGY
The Journal of Immunology
Acknowledgments We thank Drs. Kim Nichols, Luc Van Kaer, and Masaru Taniguchi for providing the Ja182/2 mice, the National Institutes of Health Tetramer Core Facility for CD1d tetramers, and the flow cytometry core facility at Duke University for cell sorting.
Disclosures The authors have no financial conflicts of interest.
References 1. Bendelac, A., P. B. Savage, and L. Teyton. 2007. The biology of NKT cells. Annu. Rev. Immunol. 25: 297–336. 2. Godfrey, D. I., S. Stankovic, and A. G. Baxter. 2010. Raising the NKT cell family. Nat. Immunol. 11: 197–206. 3. Brutkiewicz, R. R., and V. Sriram. 2002. Natural killer T (NKT) cells and their role in antitumor immunity. Crit. Rev. Oncol. Hematol. 41: 287–298. 4. Van Kaer, L. 2007. NKT cells: T lymphocytes with innate effector functions. Curr. Opin. Immunol. 19: 354–364. 5. Cerundolo, V., and M. Kronenberg. 2010. The role of invariant NKT cells at the interface of innate and adaptive immunity. Semin. Immunol. 22: 59–60. 6. Yamasaki, K., S. Horiguchi, M. Kurosaki, N. Kunii, K. Nagato, H. Hanaoka, N. Shimizu, N. Ueno, S. Yamamoto, M. Taniguchi, et al. 2011. Induction of NKT cell-specific immune responses in cancer tissues after NKT cell-targeted adoptive immunotherapy. Clin. Immunol. 138: 255–265. 7. Renukaradhya, G. J., M. A. Khan, M. Vieira, W. Du, J. 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Harnessing invariant NKT cells in vaccination strategies. Nat. Rev. Immunol. 9: 28–38. 13. Parekh, V. V., M. T. Wilson, D. Olivares-Villago´mez, A. K. Singh, L. Wu, C. R. Wang, S. Joyce, and L. Van Kaer. 2005. Glycolipid antigen induces longterm natural killer T cell anergy in mice. J. Clin. Invest. 115: 2572–2583. 14. Uldrich, A. P., N. Y. Crowe, K. Kyparissoudis, D. G. Pellicci, Y. Zhan, A. M. Lew, P. Bouillet, A. Strasser, M. J. Smyth, and D. I. Godfrey. 2005. NKT cell stimulation with glycolipid antigen in vivo: costimulation-dependent expansion, Bim-dependent contraction, and hyporesponsiveness to further antigenic challenge. J. Immunol. 175: 3092–3101. 15. Iyoda, T., M. Ushida, Y. Kimura, K. Minamino, A. Hayuka, S. Yokohata, H. Ehara, and K. Inaba. 2010. Invariant NKT cell anergy is induced by a strong TCR-mediated signal plus co-stimulation. Int. Immunol. 22: 905–913. 16. Joshi, S. K., G. A. Lang, J. L. Larabee, T. S. Devera, L. M. Aye, H. B. Shah, J. D. Ballard, and M. L. Lang. 2009. Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy. PLoS Pathog. 5: e1000588. 17. Chang, W. S., J. Y. Kim, Y. J. Kim, Y. S. Kim, J. M. Lee, M. Azuma, H. Yagita, and C. Y. Kang. 2008. Cutting edge: programmed death-1/programmed death ligand 1 interaction regulates the induction and maintenance of invariant NKT cell anergy. J. Immunol. 181: 6707–6710. 18. Parekh, V. V., S. Lalani, S. Kim, R. Halder, M. Azuma, H. Yagita, V. Kumar, L. Wu, and L. V. Kaer. 2009. PD-1/PD-L blockade prevents anergy induction and enhances the anti-tumor activities of glycolipid-activated invariant NKT cells. J. Immunol. 182: 2816–2826. 19. Kojo, S., C. Elly, Y. Harada, W. Y. Langdon, M. Kronenberg, and Y. C. Liu. 2009. Mechanisms of NKT cell anergy induction involve Cbl-b-promoted monoubiquitination of CARMA1. Proc. Natl. Acad. Sci. USA 106: 17847–17851. 20. Deng, Z. B., X. Zhuang, S. Ju, X. Xiang, J. Mu, Q. Wang, H. Jiang, L. Zhang, M. Kronenberg, J. Yan, et al. 2013. Intestinal mucus-derived nanoparticlemediated activation of Wnt/b-catenin signaling plays a role in induction of liver natural killer T cell anergy in mice. Hepatology 57: 1250–1261. 21. Zoncu, R., A. Efeyan, and D. M. Sabatini. 2011. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 12: 21–35. 22. O’Brien, T. F., and X. P. Zhong. 2012. The role and regulation of mTOR in T-lymphocyte function. Arch. Immunol. Ther. Exp. 60: 173–181. 23. Huang, J., and B. D. Manning. 2009. A complex interplay between Akt, TSC2 and the two mTOR complexes. Biochem. Soc. Trans. 37: 217–222. 24. Inoki, K., Y. Li, T. Zhu, J. Wu, and K. L. Guan. 2002. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4: 648–657. 25. Cai, S. L., A. R. Tee, J. D. Short, J. M. Bergeron, J. Kim, J. Shen, R. Guo, C. L. Johnson, K. Kiguchi, and C. L. Walker. 2006. 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their responsiveness to subsequent Ag stimulation and display strong antitumor immunity. Conventional ab T cell activation is dependent on mTOR signaling. Inhibition of mTOR renders these cells anergic (34, 35, 49, 50). TSC1 negatively controls mTORC1 activation in cabT cells and is important for T cell anergy (30, 31, 34). The resistance of TSC1-deficient cabT cells to anergy induction is at least in part due to increased mTORC1 signaling and subsequent elevation of the expression of the costimulatory molecule ICOS (34). Similar to cabT cells, anergic iNKT cells also contain elevated TSC1 and TSC2 expression but diminished mTOCRC1 and mTORC2 signaling. The ability of TSC1-deficient iNKT cells to resist anergy induction is most likely due to enhanced mTORC1 signaling in these cells. However, different from cabT cells, acute deletion of TSC1 does not lead to increased ICOS expression in iNKT cells, suggesting a limited role of elevated ICOS expression in the resistance to anergy by TSC1-deficient iNKT cells. PD-1 is upregulated in Ag-experienced iNKT cells. Although it is believed that upregulated PD-1 plays an important role in iNKT cell anergy (17, 18), contradictory results have also been reported (15). PD-1 is able to recruit Src homology 2–containing tyrosine phosphatase 2 to inhibit signal transduction (51). The mechanism controlling the upregulation of PD-1 in iNKT cells has been unclear. Our data reveal that TSC1 is an important positive regulator for PD-1 upregulation in Agexperienced iNKT cells and suggest that TSC1 may promote iNKT cell anergy via upregulating PD-1. Of note, PD-1–deficient iNKT cells are fully resistant anergy (18). However, TSC1 deficiency does not lead to complete reversal of iNKT cell anergy, which is consistent with the partial inhibition of PD-1 induction in TSC1-deficient iNKT cells. In addition to PD-1, Ag-experienced TSC1-deficient iNKT cells do not upregulate Egr2/3 and Grail expression. In cabT cells, Egr2/3 promotes Grail expression, and expression of Egr2/3 themselves is controlled by NFAT downstream of the calcium–calcineurin pathway (45, 46). The decreased expression of Egr2/3 and Grail suggests the possibility that TSC1 may regulate the NFAT function in iNKT cells. In a cell line model, mTOR can phosphorylate NFAT to prevent its nuclear translocation (52). It is possible that elevated mTOR signaling may inhibit NFAT nuclear translocation in TSC1-deficient iNKT cells to prevent the induction of anergy-promoting molecules. Further studies are required to define whether such a mechanism exists in iNKT cells and to determine how TSC1/mTOR signaling controls PD-1 expression in Ag-experienced iNKT cells. Additionally, multiple approaches, such as intradermal administration of a-GalCer, administration of a-GalCer–loaded dendritic cells, and PD-1 blockade with Abs, have been used to inhibit iNKT cell anergy (17, 18, 53). Because PD-1 is still upregulated in a small portion of TSC1KO iNKT cells in B16-melanoma–bearing mice, future studies should determine whether combination of TSC1 inhibition and PD-1 blockade may synergistically enhance antitumor immunity. mTOR integrates diverse signals to promote cell growth and proliferation. Elevated mTOR signaling contributes to the development and expansion of many tumor cells (21). mTOR inhibitors have been developed and used for tumor therapies (54). However, the importance of mTOR signaling in T cell activation and the ability of iNKT cells with elevated mTOR signaling to be resistant to anergy induction for promoting antitumor immune responses suggest the requirement for designing proper therapeutic strategies that take into consideration both tumor inhibition and immune suppression.
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TSC1 FOR iNKT CELL ANERGY
Jinhong Wu, Jinwook Shin, Danli Xie, Hongxia Wang, Jimin Gao and Xiao-Ping Zhong J Immunol 2014; 192:2643-2650; Prepublished online 14 February 2014; doi: 10.4049/jimmunol.1302076 http://www.jimmunol.org/content/192/6/2643
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References
The Journal of Immunology
Tuberous Sclerosis 1 Promotes Invariant NKT Cell Anergy and Inhibits Invariant NKT Cell–Mediated Antitumor Immunity Jinhong Wu,*,† Jinwook Shin,* Danli Xie,*,‡ Hongxia Wang,*,x Jimin Gao,‡ and Xiao-Ping Zhong*,{
T
he invariant NKT (iNKT) cells belong to a distinct subset of T lymphocytes characterized by the cell surface expression of an invariant Va14-Ja18 TCR (iVa14TCR) a-chain in mice. The activation of iNKT cells is initiated by the engagement of the iNKT receptor with lipid Ags such as a-GalCer presented by the MHC class I-like molecule CD1d, which leads to the rapid production of a variety of cytokines such as IFN-g, IL-4, and other cytokines. iNKT cells greatly impact innate immunity, shape adaptive immune responses, and contribute to or regulate the pathogenesis of diseases via these cytokines (1–5). Evidence has implicated iNKT cells in tumor surveillance. Deficiency of iNKT cells impairs antitumor immune responses, whereas activation of iNKT cells has been demonstrated to enhance tumor eradication (6–10). Considerable effort has been expended to employ the immunoregulatory functions of iNKT cells for immunotherapy of cancer via iNKT cell transfer and/or repetitive stimulation of iNKT cells using iVa14TCR ligands such as a-galactosylceramide (a-GalCer) (11, 12). However, iNKT cell *Division of Allergy and Immunology, Department of Pediatrics, Duke University Medical Center, Durham, NC 27710; †Division of Pediatric Pulmonology, Department of Internal Medicine, Shanghai Children’s Medical Center affiliated with Shanghai Jiaotong University School of Medicine, Shanghai 200127, China; ‡School of Laboratory Medicine, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China; xLaboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China; and {Department of Immunology, Duke University Medical Center, Durham, NC 27710 Received for publication August 6, 2013. Accepted for publication January 15, 2014. This work was supported by National Institutes of Health Grants AI076357, AI079088, and AI101206 and American Cancer Society Grant RSG-08-186-01-LIB. Address correspondence and reprint requests to Dr. Xiao-Ping Zhong, Division of Allergy and Immunology, Department of Pediatrics, Room 133 MSRB, Research Drive, Box 2644, Duke University Medical Center, Durham, NC 27710. E-mail address: [email protected] Abbreviations used in this article: BFA, brefeldin A; cabT, conventional ab T; a-GalCer, a-galactosylceramide; iNKT, invariant NKT; iVa14TCR, invariant Va14Ja18 TCR; KO, knockout; MNC, mononuclear cell; mTOR, mammalian target of rapamycin; PD-1, programmed death-1; qPCR, quantitative PCR; TSC, tuberous sclerosis; WT, wild-type. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302076
anergy is a significant challenge to the success of such immunotherapy. Following in vivo activation by a-GalCer, iNKT cells undergo dynamic changes characterized by robust cytokine secretion, clonal expansion, homeostatic contraction, and then acquisition of an anergic phenotype (13, 14). Similar to conventional ab T (cabT) cells, iNKT cell anergy is an unresponsive or hyporesponsive state to secondary Ag stimulation following initial TCR stimulation. iNKT cell anergy thwarts the effectiveness of repeated administration of a-GalCer for cancer immunotherapy (13). Although TCR signal strength (15, 16), programmed death-1 (PD-1) (17, 18), the E3-ubiquitin ligase Cbl-b (19), and Wnt/ b-catenin signaling (20) are implicated in iNKT cell anergy, the mechanisms that control iNKT cell anergy remain poorly understood. Tuberous sclerosis (TSC)1 associates with TSC2 to form a complex that functions as a critical regulator of the mechanistic or mammalian target of rapamycin (mTOR). mTOR signals through two complexes, mTORC1 and mTORC2, to integrate numerous environmental stimuli, including growth factors, nutrients, and stressactivated signals to regulate cell metabolism, survival, growth, and proliferation (21, 22). The GTP-binding protein Rheb (Ras homolog enriched in brain) is crucial for mTORC1 activation. TSC2 inactivates RheB through its GTPase-activating protein activity, and TSC1 is crucial for TSC2 stability in many cell types, including immune cells (23). The PI3K-Akt pathway activates mTORC1 signaling through phosphorylation of TSC2, leading to its degradation (24, 25). In T cells, RasGRP1 is critical for mTOR activation via activating the Ras-Mek1/2-Erk1/2 pathway and for early iNKT cell development (26, 27). Coinciding with this observation, Erk1/2 was found to be able to inactivate TSC2 via phosphorylation (28). The importance of TSC1 in the immune system has only recently gained recognition. TSC1 maintains hematopoietic stem cells in a normally quiescent state by preventing them from proliferation (29). It is critical for T cell quiescence, survival, and anergy, and for the maintenance of a normal peripheral T cell pool (30–35). Additionally, it plays important roles in B cell development and function (36), controls macrophage and dendritic cell response to
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Development of effective immune therapies for cancer patients requires better understanding of hurdles that prevent the generation of effective antitumor immune responses. Administration of a-galactosylceramide (a-GalCer) in animals enhances antitumor immunity via activation of the invariant NKT (iNKT) cells. However, repeated injections of a-GalCer result in long-term unresponsiveness or anergy of iNKT cells, severely limiting its efficacy in tumor eradication. The mechanisms leading to iNKT cell anergy remain poorly understood. We report in this study that the tuberous sclerosis 1 (TSC1), a negative regulator of mTOR signaling, plays a crucial role in iNKT cell anergy. Deficiency of TSC1 in iNKT cells results in resistance to a-GalCer– induced anergy, manifested by increased expansion of and cytokine production by iNKT cells in response to secondary Ag stimulation. It is correlated with impaired upregulation of programmed death-1, Egr2, and Grail. Moreover, TSC1-deficient iNKT cells display enhanced antitumor immunity in a melanoma lung metastasis model. Our data suggest targeting TSC1/2 as a strategy for boosting antitumor immune therapy. The Journal of Immunology, 2014, 192: 2643–2650.
2644 TLR ligands (37, 38), promotes MHC class II expression and Ag presentation of dendritic cells (38) and dendritic cell development (39), and regulates mast cell survival and function (40). In mice with T cell–specific TSC1 deletion, iNKT cell numbers are decreased due to increased iNKT death, and such decrease can be rescued with Bcl-2 overexpression (31). However, the importance of TSC1 in iNKT cell function remains unclear. We report in this study that acute ablation of TSC1 does not obviously impact iNKT cell activation but results in resistance of iNKT cells to anergy induced by Ag stimulation, correlated with impaired upregulation of anergy-promoting molecules PD-1, Erg2/3, and Grail. Moreover, TSC1-deficient iNKT cells display enhanced antitumor immunity in a lung metastasis tumor model. In addition, anergic iNKT cells express high levels of TSC1 and TSC2 with concomitant decrease of mTORC1 signaling. Our data suggest targeting TSC1/2 to boost iNKT cell function as a new strategy for cancer immunotherapy.
Mice and cells The TSC1f/f-ERCre+ and TSC1f/f-ERCre2 mice backcrossed to C57BL/6J background for eight to nine generations, as previously reported (37). These mice were i.p. injected with tamoxifen (100 mg/kg body weight) on days 1, 2, and 5 and then euthanized for experiments on day 8 or other indicated days. Ja182/2 mice (10) were provided by K. Nichols (Children’s Hospital of Philadelphia, Philadelphia, PA), L. Van Kaer (Vanderbilt University, Nashville, TN), and M. Taniguchi (RIKEN, Center for Integrative Medical Sciences, Yokohama, Japan). The Va14Ja18 iNKT TCR transgenic (iVa14TCRtg) mice (41) were purchased from The Jackson Laboratory and bred with the TSC1deficient mice. All mice were housed in a pathogen-free facility and used according to protocols approved by the Institutional Animal Care and Use Committee of Duke University. Splenocytes and liver mononuclear cells (MNCs) were made according to previously published protocols (42).
Reagents, Abs, and flow cytometry IMDM was supplemented with 10% (v/v) FBS, penicillin/streptomycin, and 50 mM 2-ME (IMDM-10). PE- or allophycocyanin-conjugated mouse CD1d tetramers loaded with PBS-57 (CD1dTet) were provided by the National Institutes of Health Tetramer Facility. The Live/Dead Fixable Violet Dead Cell Stain Kit was purchased from Invitrogen. Fluorescence-conjugated antimouse TCRb (H57-597), NK1.1 (PK136), CD44 (IM7), B220 (RA3-6B2), Gr-1 (RB6-8C5), CD11c (N418), CD11b (M1/70), CD4 (GK1.5), CD8 (536.7), IFN-g (XMG1.2), IL-4 (11B11), PD-1 (RMP1-30), and ICOS (D10. G4.1) Abs were purchased from BioLegend. Anti–phospho-p44/42 MAPK T202/Y204 (197G2), anti–phospho-S6 S240/244 (D68F8), anti–phospho-4EBP1 T37/46 (236B4), anti–phospho-Akt S473 (193H12), TSC1 (D43E2), and TSC2 (D93F12) were purchased from Cell Signaling Technology. Anti–bactin Ab was purchased from Sigma-Aldrich. Cell surface staining was performed with 2% FBS-PBS. Intracellular staining for Ki67 was performed using the eBioscience Foxp3 staining buffer set. Ki67 was detected with an anti-Ki67 Ab (B56; BD Biosciences), followed by detection with AlexaFluor488conjugated goat anti-mouse IgG (H+L; Invitrogen). Intracellular staining for IFN-g and IL-4 was performed using the BD Biosciences Cytofix/Cytoperm and perm/wash solutions. Dead cells were excluded from the analysis using Live/Dead Fixable Violet Dead Cell Stain Kit (Invitrogen). The iNKT cell population was identified as Lin2 (B2202Gr12CD11b2CD11c2CD82) TCRb+CD1dTet+ cells. Data were collected using FACS Canto-II (BD Biosciences) and analyzed using Flowjo software (Tree Star).
Purification of iNKT cells and real-time quantitative PCR Mice either untreated or treated with a-GalCer (2 mg, i.v., one injection) 7 days early were euthanized to isolate splenocytes and liver MNCs. iNKT cells from pooled splenocytes and liver MNCs were enriched with CD1dTet and MACS beads, according to a previously published protocol (27). Enriched iNKT cells were stained with anti-TCRb and 7-aminoactinomycin D. Live TCRb+CD1dTet+ iNKT cells were sorted using MoFlo with .98% purity and were immediately lysed in TRIzol for RNA preparation. cDNA was made using the iScript Select cDNA Synthesis Kit (Bio-Rad). Real-time quantitative PCR was performed and analyzed, as previously described (34). Expressed levels of target mRNAs were normalized with b-actin and calculated using the 22DDCT method. Primers were as follows: Egr2 (forward, 59-TTGACCAGATGAACGGAGTG-39;
reverse, 59-CAGAGATGGGAGCGAAGCTA-39), Egr3 (forward, 59-CGCGCTCAACCTCTTCTC-39; reverse, 59-GATTGGGCTTCTCGTTGGT-39), Itch (forward, 59-GGACCACAGCTTGATTCCAT-39; reverse, 59-GACTTGGTCCAAACCAATTCTT-39), Grail (forward, 59-CAACCGTGGGCTATTTCATC-39; reverse, 59-GCAGCTGAAGCTTTCCAATA-39), PD-1 (forward, 59-CTGGAAGCAAGGACGACACT-39; reverse, 59-TGGTGGCATATTCTGTGTGC-39), P27 (forward, 59-CCTGACTCGTCAGACAATCC-39; reverse, 59-TCTGTTCTGTTGGCCCTTTT-39), Cbl-b (forward, 59GGCAAGGAAAGGATGTACGA-39; reverse, 59-AAGATCGCCTTGATTTCTGC-39), TSC1 (forward, 59-ACATCTTTGGCCGTCTCTCGTCAT-39; reverse 59-ATGGGTACATCCCATAAAGGCGGT-39), TSC2 (forward, 59-ACTGCCAACCAGACAAGGTGTACT-39; reverse, 59-GTTTCTTGTCACAGCGGTGCTTGT-39), b-actin (forward, 59-TGTCCACCTTCCAGCAGATGT-39; reverse, 59-AGCTCAGTAACAGTCCGCCTAGA-39).
Stimulation of iNKT cells and induction of iNKT cell anergy For in vivo iNKT cell stimulation, mice were first i.p. injected with 150 mg brefeldin A (BFA; Sigma-Aldrich), followed by i.v. injection with 2 mg a-GalCer 90 min later. Two hours after a-GalCer injection, spleens and livers were harvested for iNKT cell staining and intracellular staining of IFN-g and IL-4. a-GalCer–induced iNKT cell anergy in mice was performed according to a previously published protocol (13). Briefly, mice were i.v. injected with 2 mg a-GalCer on days 1 and 8, respectively. The following experiments were performed for anergy evaluation. 1) Sera were collected 4 h after each injection for measurement of IL-4 and IFN-g concentrations. 2) Three days after a-GalCer injection, splenocytes and liver MNCs were stained for iNKT cells and for Ki67 expression. 3) One and one-half hours before a-GalCer injection, mice were injected with BFA and IFN-g, and IL-4 intracellular staining in splenic and liver iNKT cells was performed, as described above.
Western blot analysis Cell lysates were prepared from thymocytes of Raptorf/f and Raptorf/fERCre mice following tamoxifen treatment or from purified iNKT cells isolated from naive mice or a-GalCer–experienced mice. Cell lysates were subjected to Western blot analysis, as previously described (26, 30). Western blot films were scanned, and phosphorylation and protein intensities were quantified using the Adobe Photoshop CS4 software.
Determination of B16 melanoma lung metastases iNKT cells were isolated from pooled thymocytes, splenocytes, and liver MNCs from TSC1f/f-ERCre2-iVa14-TCRtg or TSC1f/f-ERCre+-iVa14-TCRtg mice on day 0 after i.p. injected with 200 ml 10 mg/ml tamoxifen on days 28, 27, and 24. After i.v. receiving 5 3 105 B16F10 melanoma cells on day 0, female Ja182/2 mice were injected with 1 3 106 wild-type (WT) or TSC1-deficient iNKT cells. Recipient mice were i.v. injected with 2 mg a-GalCer at 1, 4, and 7 d and euthanized on day 14. The number of metastatic nodules in the lung was counted.
Results Effects of acute TSC1 deletion on iNKT cell homeostasis and cytokine production Mice with T cell–specific deletion of TSC1, TSC1f/f-CD4Cre, contain reduced iNKT cells (31) and are not ideal for examining mature iNKT cell function. To delete TSC1 in mature iNKT cells, we generated TSC1f/f (WT) and TSC1f/f-ERCre+ (knockout [KO]) mice and injected tamoxifen into the mice on days 1, 2, and 5. Mice were euthanized on day 8 for evaluation of TSC1 deletion efficiency as well as iNKT cells. As shown in Fig. 1A, TSC1 protein was drastically decreased in TSC1KO thymocytes and splenocytes, indicating efficient deletion of the gene. TSC2 protein was also reduced, consistent with the importance of TSC1 for TSC2 stability. Moreover, S6 and 4E-BP1 phosphorylation, which is dependent on mTORC1 activation, was increased in TSC1KO thymocytes and splenocytes, suggesting enhanced mTORC1 signaling. AKT phosphorylation at serine 473, a mTORC2-mediated event, was slightly decreased in TSC1KO splenocytes but was not decreased in thymocytes. ERK1/2 phosphorylation was not obviously affected by TSC1 deficiency. Thus, acute deletion of TSC1
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Materials and Methods
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resulted in elevated mTORC1 signaling in both thymocytes and splenocytes with minimal impact on mTORC2 signaling. Staining of iNKT cells with CD1dTet and TCRb revealed similar percentages and numbers in the WT and TSC1KO thymus, spleen, and liver (Fig. 1B). Moreover, percentages and numbers of stage 1 (CD442NK1.12), stage 2 (CD44+NK1.1+), and stage 3 (CD44+NK1.1+) iNKT cells were also similar between these two groups of mice (Fig. 1C). Thus, in the time frame we tested, TSC1 deficiency did not obviously affect iNKT cell homeostasis. To determine whether acute deletion of TSC1 may affect iNKT cell activation and cytokine production, we injected BFA into WT and
TSC1KO mice to block protein secretion and subsequently injected with a-GalCer 90 min later. IFN-g and IL-4 expression in iNKT cells 2 h after a-GalCer injection was determined by intracellular staining. As shown in Fig. 2, both splenic and liver iNKT cells from TSC1KO mice expressed similar levels of IFN-g and IL-4 to their respective WT controls. Thus, acute TSC1 ablation does not obviously affect Ag-induced cytokine production by iNKT cells. Resistance of TSC1-deficient iNKT cells to anergy induction Following initial Ag stimulation, iNKT cells undergo expansion that peaks on day 3 and then contract to close to basal level on day 7.
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FIGURE 1. Effects of acute deletion of TSC1 on iNKT cell homeostasis. (A) Efficient ablation of TSC1 in TSC1f/f-ERCre+ mice following tamoxifen treatment. Total thymocyte and splenocyte lysates from TSC1f/f (WT) and TSC1f/f-ERCre+ (KO) mice following tamoxifen treatment were subjected to immunoblotting analysis with the indicated Abs. (B and C) iNKT cell staining in TSC1f/f and TSC1f/f-ERCre+ mice following tamoxifen treatment. Thymocytes, splenocytes, and liver MNCs were stained with TCRb, PBS570-loaded CD1dTet, CD44, NK1.1, and Live/Dead and subjected to flow cytometry analysis. (B) TCRb and CD1dTet staining of live-gated thymocytes, splenocytes, and liver MNCs. (C) CD44 and NK1.1 staining of gated TCRb+ CD1dTet+ cells. Bar graphs are mean 6 SEM presentation of percentages and cell numbers from multiple mice (n = 5). Data shown represent three experiments.
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FIGURE 2. Acute deletion of TSC1 does not obviously affect iNKT cell cytokine production. Tamoxifen-treated TSC1f/f and TSC1f/f-ERCre+ mice were i.p. injected with 150 mg BFA and then injected with 2 mg a-GalCer 90 min later. Two hours after a-GalCer injection, splenocytes and liver MNCs were cell surface stained with CD1d-Tet and anti-TCRb and intracellularly stained with anti–IFN-g and anti–IL-4. (A) Representative dot plots showing IFN-g and IL-4 staining in gated iNKT cells. (B) Bar graphs are mean 6 SEM of IFN-g– or IL-4–positive cells in CD1dTet+TCRb+ cells (n = 3). Data shown represent three experiments.
FIGURE 3. Elevated TSC1 and TSC2 expression in anergic iNKT cells. WT mice were i.v. injected with a-GalCer or PBS. Seven days later, mice were euthanized and iNKT cells were purified from the spleen and liver for isolation of total RNA and making cell lysates. (A) TSC1 and TSC2 mRNA levels determined by real-time qPCR; (B) TSC1/2 protein levels and phosphorylation of the indicated proteins determined by Western blot analysis. Data shown are representative of two experiments. **p , 0.01, ***p , 0.01, determined by Student t test.
the last injection for evaluation of iNKT cell expansion. In the 1˚ group, we observed similar numbers of iNKT cells in WT and TSC1KO spleens and livers (Fig. 4A). Moreover, expression of Ki67, a marker for cell proliferation, was only slightly increased in TSC1KO iNKT cells (Fig. 4B). Together, these data suggest that iNKT cell expansion during initial Ag encountering was not substantially affected by TSC1 deficiency. Different from the initial Ag-induced response, WT iNKT cell numbers were much less than TSC1KO iNKT cells following secondary a-GalCer injection (Fig. 4A). Moreover, Ki67 expression was much lower in 2˚ WT iNKT cells than in 1˚ WT iNKT cells, suggesting hypoproliferative response of Ag-experienced WT iNKT cells to secondary Ag stimulation (Fig. 4B). However, Ag-experienced TSC1KO iNKT cells (2˚) expressed much higher levels of Ki67 compared with their WT controls in response to a secondary a-GalCer stimulation. Thus, different from initial Ag encountering, Ag-experienced TSC1-deficient iNKT cells retained great proliferative responses to secondary Ag stimulation. In addition to a hypoproliferative response, another hallmark of anergic iNKT cells is defective cytokine production (13). Following primary a-GalCer injection, serum IL-4 and IFN-g levels were similarly induced in WT and TSC1KO mice, which was consistent with the observation shown in Fig. 2. After secondary a-GalCer injection, WT mice produced much less IFN-g and IL-4 compared with mice with only primary injection. However, Agexperienced TSC1KO mice still produced high levels of IFN-g and IL-4 following secondary a-GalCer injection (Fig. 5A). Consistent with elevated serum IL-4 and IFN-g levels, TSC1KO iNKT cells expressed higher levels of IFN-g and IL-4 2 h after a-GalCer injection determined by intracellular staining (Fig. 5B, 5C). Of note, the percentages of IL-4– and IFN-g–producing iNKT cells from Ag-experienced TSC1KO mice were still lower than those from naive mice, suggesting that TSC1 deficiency causes iNKT cells to partially resist anergy induction. The high serum IL-4 and IFN-g levels in Ag-experienced TSC1 KO mice following the second a-GalCer injection are most likely an additive effect of increased percentages of cytokine-producing iNKT cells and a ∼50% increase of iNKT cell numbers in these mice compared with WT control mice before the second a-GalCer injection (data not shown).
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These Ag-experienced iNKT cells become anergic and thus hyporesponsive to subsequent Ag stimulations (13). Interestingly, anergic iNKT cells expressed higher levels of TSC1 and TSC2 at the mRNA and protein levels (Fig. 3A). Moreover, phosphorylation of 4E-BP1 was obviously decreased in anergic iNKT cells, suggesting decreased mTORC1 signaling in anergic iNKT cells (Fig. 3B). To examine whether TSC1 is involved in iNKT cell anergy, we injected a-GalCer into WT and TSC1KO mice once on day 0 (1˚) or twice on days 0 and 8 (2˚), respectively. Mice were euthanized 3 d after
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FIGURE 4. Enhanced expansion of TSC1-deficient iNKT cells following repeated a-GalCer injection in vivo. TSC1f/f and TSC1f/f-ERCre+ mice were treated with tamoxifen on days 1, 2, and 5. Mice were either injected with a-GalCer on day 8 (1˚) and examined on day 11 or injected on days 8 and 15 (2˚) and examined on day 18. (A) Bar graphs are mean 6 SEM presentation of splenic and liver iNKT cell numbers (1˚, n = 4; 2˚, n = 5). (B) Intracellular staining of Ki67 in gated iNKT cells. Data shown are representative of three experiments. *p , 0.05, **p , 0.01, determined by Student t test.
At present, only a limited number of molecules is known to be involved in iNKT cells’ anergy. PD-1, a coinhibitory molecule expressed on T cells, is upregulated in iNKT cells following Ag encountering and is important for iNKT cell anergy (17, 18). Before a-GalCer injection, WT and TSC1KO iNKT cells expressed similar low levels of PD-1. Seven days after a-GalCer injection, PD1 was upregulated in WT iNKT cells. However, iNKT cells from similarly treated TSC1KO mice expressed lower levels of PD-1 than WT iNKT cells (Fig. 6A). To determine whether TSC1 controls PD1 expression at the mRNA level, we sorted iNKT cells from WT and TSC1KO splenocytes 7 d after primary a-GalCer injection to make total RNA. PD-1 mRNA level was measured by real-time quantitative PCR (qPCR). PD-1 mRNA level was induced in WT iNKT cells but not in TSC1KO iNKT cells following a-Galcer injection (Fig. 6B). These observations suggest that TSC1 may
promote PD-1 expression for anergy induction via increasing its transcription. The E3 ubiquitin ligases Cbl-b, Itch, and Grail; transcription factors Egr2/Egr3; and the cell cycle inhibitor p27kip are anergypromoting molecules in cab T cells (43–46). Recent research has demonstrated the importance of Cbl-b for iNKT cell anergy (19). We observed increased Egr2, Egr3, and Grail expression in Agexperienced WT iNKT cells compared with non-Ag–experienced iNKT cells. However, the induction of these molecules was abolished in the absence of TSC1. In addition, p27kip mRNA was reduced in Ag-experienced TSC1KO iNKT cells compared with WT controls (Fig. 6B). Together, these observations suggest that TSC1 positively controls the expression of PD-1, Egr2/3, Grail, and p27kip to promote iNKT cell anergy. Enhanced antitumor immunity by TSC1-deficient iNKT cells iNKT cells play important roles in antitumor immune responses (11, 12). To further understand whether TSC1-mediated iNKT cell
FIGURE 5. Resistance of TSC1-deficient iNKT cells to anergy induction. TSC1f/f and TSC1f/f-ERCre+ mice were treated with tamoxifen on days 1, 2, and 5. Mice either were injected with a-GalCer on day 8 (1˚) or injected on days 8 and 15 (2˚). (A) Serum IFN-g and IL-4 concentrations 4 h after the last injection measured by ELISA (first, n = 4; second, n = 5). Bar graphs are mean 6 SEM. (B and C) Intracellular staining of IFN-g and IL-4 in gated iNKT cells. Mice were injected with BFA 1.5 h before the last injection of a-GalCer. Splenocytes were harvested 2 h after the last a-GalCer injection for iNKT and cytokine staining. (B) Representative dot plots; (C) mean 6 SEM presentation of percentages of cytokine-producing iNKT cells (first, n = 4; second, n = 5). Data shown are representative of four experiments. ***p , 0.001.
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anergy induction regulates antitumor immune responses, we used the B16F10 melanoma lung metastasis model, a model for assessing iNKT cell antitumor immunity (10, 47, 48). Because TSC1 is systemically deleted in TSC1KO mice, we adoptively transferred iNKT cells from WT and TSC1KO mice carrying the iVa14TCR transgene (41) into the iNKT cell–deficient Ja182/2 mice. The recipient mice were injected with B16F10 melanoma on the same day (day 0) and received three a-GalCer injections on days 1, 4, and 7. On day 14, recipient mice were euthanized for evaluation of tumor metastasis in the lung. The numbers of tumor nodules in the lung were fewer in recipient mice reconstituted with TSC1KO iNKT cells than in those reconstituted with WT iNKT cells (Fig. 7A, 7B). Moreover, adoptively transferred TSC1KO iNKT cells expressed lower levels of PD-1 than WT iNKT cells in the a-GalCer–injected tumor-bearing recipient mice (Fig. 7C). Together, these data suggest that TSC1-deficient iNKT cells conferred stronger antitumor immunity against melanoma than WT iNKT cells.
Discussion iNKT cells play important roles in tumor immunity. Although initial injection of a-GalCel into mice activates iNKT cells, Ag-
FIGURE 7. Enhanced antitumor immunity by TSC1-deficient iNKT cells. A total of 2 3 105 B16F10 melanoma cells was injected into Ja182/2 mice on day 0. The recipient mice were subsequently i.v. injected with 1 3 106 enriched iNKT cells from tamoxifen-treated iVa14TCRtg-TSC1f/f and iVa14TCRtg-TSC1f/f-ERCre+ mice. Mice were also injected with a-GalCer on days 1, 4, and 7. Mice were euthanized on day 14 for evaluation of lung metastasis of melanoma. (A) Representative lung morphology. (B) Average of melanoma nodule numbers per lung. Each circle represents one mouse (n = 17). (C) Overlaid histograms show PD-1 expression in gated iNKT cells from the spleens. Bar graphs are mean 6 SEM of the geometric mean fluorescence intensity of PD-1 and percentages of iNKT cells in the indicated gate that expressed high levels of PD-1 (n = 7). Data shown are representative of four experiments. **p , 0.01, ***p , 0.001.
experienced iNKT cells render anergic, which hinders the effectiveness of repetitive application of iNKT agonists for enhancing antitumor immunity. Thus, overcoming the induction of anergy may overcome the hurdle in iNKT cell–mediated tumor immunotherapy. In this study, we demonstrate that TSC1 plays a critical role in iNKT cell anergy. We reveal that TSC1 differentially controls iNKT cell activation in agonist-unexperienced and -experienced iNKT cells. Acute deletion of TSC1 in mature iNKT cells does not obviously affect iNKT cell proliferation or cytokine production following initial encountering with a-GalCer in vitro and in vivo. In contrast to Ag-experienced and anergic WT iNKT cells, Ag-experienced TSC1-deficient iNKT cells retain
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FIGURE 6. Altered expression of anergy-related molecules in TSC1deficient iNKT cells. TSC1f/f and TSC1f/f-ERCre+ mice were treated with tamoxifen on days 1, 2, and 5. Mice were injected with either PBS or a-GalCer on day 8. On day 15, splenocytes were harvested for assessing iNKT cells. (A) Overlaid histograms show PD-1 expression on CD1dTet+ TCRb+ iNKT cells. Bar graph represents mean 6 SEM of the geometric mean florescence intensity (gMFI) of PD-1 (n = 5). (B) Altered mRNA levels of anergy-related genes. iNKT cells were enriched with CD1dTet and MACS beads, followed by FACS sorting. The levels of mRNA of the indicated molecules from the sorted iNKT cells were quantified by realtime qPCR. Bar graphs are mean 6 SEM (n = 3). Data shown represent three experiments. ***p , 0.001.
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Acknowledgments We thank Drs. Kim Nichols, Luc Van Kaer, and Masaru Taniguchi for providing the Ja182/2 mice, the National Institutes of Health Tetramer Core Facility for CD1d tetramers, and the flow cytometry core facility at Duke University for cell sorting.
Disclosures The authors have no financial conflicts of interest.
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their responsiveness to subsequent Ag stimulation and display strong antitumor immunity. Conventional ab T cell activation is dependent on mTOR signaling. Inhibition of mTOR renders these cells anergic (34, 35, 49, 50). TSC1 negatively controls mTORC1 activation in cabT cells and is important for T cell anergy (30, 31, 34). The resistance of TSC1-deficient cabT cells to anergy induction is at least in part due to increased mTORC1 signaling and subsequent elevation of the expression of the costimulatory molecule ICOS (34). Similar to cabT cells, anergic iNKT cells also contain elevated TSC1 and TSC2 expression but diminished mTOCRC1 and mTORC2 signaling. The ability of TSC1-deficient iNKT cells to resist anergy induction is most likely due to enhanced mTORC1 signaling in these cells. However, different from cabT cells, acute deletion of TSC1 does not lead to increased ICOS expression in iNKT cells, suggesting a limited role of elevated ICOS expression in the resistance to anergy by TSC1-deficient iNKT cells. PD-1 is upregulated in Ag-experienced iNKT cells. Although it is believed that upregulated PD-1 plays an important role in iNKT cell anergy (17, 18), contradictory results have also been reported (15). PD-1 is able to recruit Src homology 2–containing tyrosine phosphatase 2 to inhibit signal transduction (51). The mechanism controlling the upregulation of PD-1 in iNKT cells has been unclear. Our data reveal that TSC1 is an important positive regulator for PD-1 upregulation in Agexperienced iNKT cells and suggest that TSC1 may promote iNKT cell anergy via upregulating PD-1. Of note, PD-1–deficient iNKT cells are fully resistant anergy (18). However, TSC1 deficiency does not lead to complete reversal of iNKT cell anergy, which is consistent with the partial inhibition of PD-1 induction in TSC1-deficient iNKT cells. In addition to PD-1, Ag-experienced TSC1-deficient iNKT cells do not upregulate Egr2/3 and Grail expression. In cabT cells, Egr2/3 promotes Grail expression, and expression of Egr2/3 themselves is controlled by NFAT downstream of the calcium–calcineurin pathway (45, 46). The decreased expression of Egr2/3 and Grail suggests the possibility that TSC1 may regulate the NFAT function in iNKT cells. In a cell line model, mTOR can phosphorylate NFAT to prevent its nuclear translocation (52). It is possible that elevated mTOR signaling may inhibit NFAT nuclear translocation in TSC1-deficient iNKT cells to prevent the induction of anergy-promoting molecules. Further studies are required to define whether such a mechanism exists in iNKT cells and to determine how TSC1/mTOR signaling controls PD-1 expression in Ag-experienced iNKT cells. Additionally, multiple approaches, such as intradermal administration of a-GalCer, administration of a-GalCer–loaded dendritic cells, and PD-1 blockade with Abs, have been used to inhibit iNKT cell anergy (17, 18, 53). Because PD-1 is still upregulated in a small portion of TSC1KO iNKT cells in B16-melanoma–bearing mice, future studies should determine whether combination of TSC1 inhibition and PD-1 blockade may synergistically enhance antitumor immunity. mTOR integrates diverse signals to promote cell growth and proliferation. Elevated mTOR signaling contributes to the development and expansion of many tumor cells (21). mTOR inhibitors have been developed and used for tumor therapies (54). However, the importance of mTOR signaling in T cell activation and the ability of iNKT cells with elevated mTOR signaling to be resistant to anergy induction for promoting antitumor immune responses suggest the requirement for designing proper therapeutic strategies that take into consideration both tumor inhibition and immune suppression.
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TSC1 FOR iNKT CELL ANERGY
