FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2057673313> ?p ?o ?g. }

• W2057673313 endingPage "2143" @default.
• W2057673313 startingPage "2132" @default.
• W2057673313 abstract "Background & Aims: Vα14 invariant natural killer T cells (iNKT) are localized in peripheral tissues such as the liver rather than lymphoid tissues. Therefore, their role in modulating the stimulation of conventional, major histocompatibility complex (MHC)-restricted T-cell responses has remained ambiguous. We here describe a role for Vα14 iNKT cells in modulating conventional T-cell responses to antigen expressed in liver, using transferrin-mOVA (Tf-mOVA) mice. Methods: Naïve ovalbumin-specific class I MHC-restricted T cells (OTI) were adoptively transferred into Tf-mOVA mice in the presence or absence of iNKT-cell agonist α-galactosylceramide, after which OTI T-cell priming, antigen-specific cytokine production, cytotoxic killing ability, and liver damage were analyzed. Results: Transfer of OTI cells resulted in robust intrahepatic, antigen-specific proliferation of T cells. OTI T cells were activated in liver, and antigen-specific effector function was stimulated by coactivation of Vα14 iNKT cells using α-galactosylceramide. This stimulation was absent in CD1d−/−Tf-mOVA mice, which lack Vα14 iNKT cells, and was prevented when interferon-γ and tumor necrosis factor-α production by Vα14 iNKT cells was blocked. Conclusions: CD1d-restricted Vα14 iNKT cells stimulate intrahepatic CD8 T-cell effector responses to antigen expressed in liver. Our findings elucidate a previously unknown intervention point for targeted immunotherapy to autoimmune and possibly infectious liver diseases. Background & Aims: Vα14 invariant natural killer T cells (iNKT) are localized in peripheral tissues such as the liver rather than lymphoid tissues. Therefore, their role in modulating the stimulation of conventional, major histocompatibility complex (MHC)-restricted T-cell responses has remained ambiguous. We here describe a role for Vα14 iNKT cells in modulating conventional T-cell responses to antigen expressed in liver, using transferrin-mOVA (Tf-mOVA) mice. Methods: Naïve ovalbumin-specific class I MHC-restricted T cells (OTI) were adoptively transferred into Tf-mOVA mice in the presence or absence of iNKT-cell agonist α-galactosylceramide, after which OTI T-cell priming, antigen-specific cytokine production, cytotoxic killing ability, and liver damage were analyzed. Results: Transfer of OTI cells resulted in robust intrahepatic, antigen-specific proliferation of T cells. OTI T cells were activated in liver, and antigen-specific effector function was stimulated by coactivation of Vα14 iNKT cells using α-galactosylceramide. This stimulation was absent in CD1d−/−Tf-mOVA mice, which lack Vα14 iNKT cells, and was prevented when interferon-γ and tumor necrosis factor-α production by Vα14 iNKT cells was blocked. Conclusions: CD1d-restricted Vα14 iNKT cells stimulate intrahepatic CD8 T-cell effector responses to antigen expressed in liver. Our findings elucidate a previously unknown intervention point for targeted immunotherapy to autoimmune and possibly infectious liver diseases. The initiation of adaptive immune responses generally entails the antigen-specific stimulation of naïve major histocompatibility complex (MHC)-restricted T cells by professional antigen-presenting cells (APCs), a process that requires prolonged contact between T cells and APCs. CD1d-restricted invariant natural killer T (iNKT) cells, in contrast, can secrete cytokines within only few hours of activation. In mice, the majority of CD1d-restricted iNKT cells are relatively invariant and are collectively called Vα14 iNKT cells.1Brossay L. Burdin N. Tangri S. et al.Antigen-presenting function of mouse CD1: one molecule with two different kinds of antigenic ligands.Immunol Rev. 1998; 163: 139-150Crossref PubMed Scopus (49) Google Scholar Human iNKT cells mostly express a Vα24-Jα18 rearranged T-cell receptor (TCR) α chain with a Vβ11-containing TCRβ chain.2Kronenberg M. Gapin L. The unconventional lifestyle of NKT cells.Nat Rev Immunol. 2002; 2: 557-568Crossref PubMed Scopus (662) Google Scholar Vα14 iNKT cells in mice (referred to as iNKT cells hereafter) are highly enriched in liver, in which they can represent up to 30% of lymphocytes,3Matsuda J.L. Naidenko O.V. Gapin L. et al.Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers.J Exp Med. 2000; 192: 741-754Crossref PubMed Scopus (755) Google Scholar, 4Bendelac A. Rivera M.N. Park S.H. et al.Mouse CD1-specific NK1 T cells: development, specificity, and function.Annu Rev Immunol. 1997; 15: 535-562Crossref PubMed Scopus (1198) Google Scholar and are likely to play an important role in local immune responses. Their activation, through stimulation with the glycolipid α-galactosylceramide (αGalCer), can enhance T-cell responses to soluble protein antigens by directly interacting with dendritic cells (DCs) in a CD40-dependent manner5Carnaud C. Lee D. Donnars O. et al.Cutting edge: cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells.J Immunol. 1999; 163: 4647-4650PubMed Google Scholar, 6Hermans I.F. Silk J.D. Gileadi U. et al.NKT cells enhance CD4+ and CD8+ T-cell responses to soluble antigen in vivo through direct interaction with dendritic cells.J Immunol. 2003; 171: 5140-5147Crossref PubMed Scopus (406) Google Scholar, 7Fujii S. Shimizu K. Smith C. et al.Activation of natural killer T cells by α-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T-cell immunity to a coadministered protein.J Exp Med. 2003; 198: 267-279Crossref PubMed Scopus (608) Google Scholar and can enhance antitumor cytotoxicity of NK cells and CD8+ T cells to inhibit metastasis to the liver.8Nakagawa R. Nagafune I. Tazunoki Y. et al.Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by α-galactosylceramide in mice.J Immunol. 2001; 166: 6578-6584Crossref PubMed Scopus (207) Google Scholar, 9Smyth M.J. Crowe N.Y. Pellicci D.G. et al.Sequential production of interferon-gamma by NK1.1(+) T cells and natural killer cells is essential for the antimetastatic effect of α-galactosylceramide.Blood. 2002; 99: 1259-1266Crossref PubMed Scopus (334) Google Scholar Intrahepatic iNKT cells moreover play a protective role in the clearance of multiple pathogens such as picornavirus,10Exley M.A. Bigley N.J. Cheng O. et al.CD1d-reactive T-cell activation leads to amelioration of disease caused by diabetogenic encephalomyocarditis virus.J Leukoc Biol. 2001; 69: 713-718PubMed Google Scholar herpes simplex virus,11Grubor-Bauk B. Simmons A. Mayrhofer G. et al.Impaired clearance of herpes simplex virus type 1 from mice lacking CD1d or NKT cells expressing the semivariant V α 14-J α 281 TCR.J Immunol. 2003; 170: 1430-1434Crossref PubMed Scopus (170) Google Scholar and Pseudomonas earuginosa.12Nieuwenhuis E.E. Neurath M.F. Corazza N. et al.Disruption of T helper 2-immune responses in Epstein-Barr virus-induced gene 3-deficient mice.Proc Natl Acad Sci U S A. 2002; 99: 16951-16956Crossref PubMed Scopus (151) Google Scholar In transgenic mouse models of hepatitis B virus (HBV) replication, Vα14 iNKT cells contribute to interferon (IFN)-α/β- and IFN-γ-dependent inhibition of viral replication,13Kakimi K. Lane T.E. Chisari F.V. et al.Cutting edge: inhibition of hepatitis B virus replication by activated NK T cells does not require inflammatory cell recruitment to the liver.J Immunol. 2001; 167: 6701-6705Crossref PubMed Scopus (102) Google Scholar and livers of hepatitis C virus (HCV) patients and patients with primary biliary cirrhosis contain large numbers of iNKT cells.14Kita H. Naidenko O.V. Kronenberg M. et al.Quantitation and phenotypic analysis of natural killer T cells in primary biliary cirrhosis using a human CD1d tetramer.Gastroenterology. 2002; 123: 1031-1043Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 15Lucas M. Gadola S. Meier U. et al.Frequency and phenotype of circulating Vα24/Vβ11 double-positive natural killer T cells during hepatitis C virus infection.J Virol. 2003; 77: 2251-2257Crossref PubMed Scopus (97) Google Scholar, 16Nuti S. Rosa D. Valiante N.M. et al.Dynamics of intra-hepatic lymphocytes in chronic hepatitis C: enrichment for Vα24+ T cells and rapid elimination of effector cells by apoptosis.Eur J Immunol. 1998; 28: 3448-3455Crossref PubMed Scopus (158) Google Scholar These findings support the hypothesis that iNKT cells contribute to the pathogenesis of these liver diseases. Still, the liver is an organ with paradoxical immunologic properties, functioning either as a site amendable to effective immune responses or to generation of tolerance, as appropriate.17Crispe I.N. Giannandrea M. Klein I. et al.Cellular and molecular mechanisms of liver tolerance.Immunol Rev. 2006; 213: 101-118Crossref PubMed Scopus (191) Google Scholar We asked how intrahepatic Vα14 iNKT cells are involved in antigen-specific hepatitis whereby antigen-specific conventional CD8 T cells instigate liver injury. We here describe the role of iNKT cells in stimulating CD8 T-cell responses to antigen restricted to the liver, using a recently developed mouse model, transferrin (Tf)-mOVA mice. Tf-mOVA,18Derkow K. Loddenkemper C. Mintern J. et al.Differential priming of CD8 and CD4 T cells in animal models of autoimmune hepatitis and cholangitis.Hepatology. 2007; 46: 1155-1165Crossref PubMed Scopus (58) Google Scholar CD1d-deficient,10Exley M.A. Bigley N.J. Cheng O. et al.CD1d-reactive T-cell activation leads to amelioration of disease caused by diabetogenic encephalomyocarditis virus.J Leukoc Biol. 2001; 69: 713-718PubMed Google Scholar, 19Cyster J.G. Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs.Annu Rev Immunol. 2005; 23: 127-159Crossref PubMed Scopus (715) Google Scholar and OTI20Hogquist K.A. Jameson S.C. Heath W.R. et al.T-cell receptor antagonist peptides induce positive selection.Cell. 1994; 76: 17-27Abstract Full Text PDF PubMed Scopus (2256) Google Scholar RAG1-deficient mice were maintained in a rodent barrier facility at Harvard Medical School. CD1d-deficient Tf-mOVA mice were generated by crossbreeding. All mice were on C57Bl/6J background and were used at 6–8 weeks of age. Studies were performed according to institutional guidelines for animal use and care. Wild-type control mice were obtained from The Jackson Laboratory (Bar Harbor, ME). OTI T cells were extracted from lymph nodes and spleen of RAG1−/− OTI mice (>95% purity). OTI T cells (4 × 106) were injected via tail vein, with or without 100 ng αGalCer. αGalCer was synthesized in the laboratory of Dr Gurdyal S. Besra. For proliferation measurements (at 36 hours post-transfer), OTI T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen-Molecular Probes, Carlsbad, CA). For blocking of lymph node homing, OTI T cells were preincubated with neutralizing antibody to CD62L (clone MEL14) or isotype control IgG2a (clone RTK2758, 50 μg at 4°C, 30 minutes; both Biolegend, San Diego, CA). At the time of transfer and 24 hours later, 50 μg/mouse of anti-CD62L or isotype control antibody were injected intraperitoneally (IP) into splenectomized Tf-mOVA mice. For blocking of lymph node exit, Tf-mOVA mice received FTY 720 at the time of T-cell adoptive transfer and 24 hours posttransfer (Cayman Chemical, Ann Arbor, MI; 1 mg/kg) intraperitoneally. Littermate controls received phosphate-buffered saline (PBS). Mice were anesthetized before surgery, and hair was removed at the surgical site. A small incision was made on the left side of the mouse and through the peritoneal cavity. The spleen was ligated with 5-0 Ethilon (Ethicon, Inc, Sommerville, NJ) and excised from the opening. The abdominal wall incision was closed with staples (Precise; 3M Healthcare, St Paul, MN). Experiments were performed at least 7 days postsurgery. After perfusion (2–5 mL PBS), the liver was removed and pressed through 70-μm mesh (BD Pharmingen, Frankin Lakes, NJ). After washing in PBS, mononuclear cells were resuspended in 33% Percoll (GE Health Care, Piscataway, NJ), overlayed onto 80% Percoll and centrifuged (20 minutes at 900g). Mononuclear cells were collected from the interface. After erythrocyte lysis using NH4Cl, cells were washed twice in PBS and resuspended in PBS supplemented with 10% fetal calf serum (FCS) for flow cytometry or in RPMI medium supplemented with 5% FCS and Pen/Strep (Invitrogen-Gibco, Carlsbad, CA) for culture. Mononuclear cells were also isolated from spleen and from inguinal, axillary, mesenteric, and portal lymph nodes. Cells were prepared for flow cytometry or culture as described below. DCs used in T-cell stimulation assays were purified from mesenteric and skin-draining lymph nodes (inguinal and axillary), from spleen and from liver based on CD11c expression using MACS (Miltenyi Biotec Inc, Auburn, CA). One × 105 DC were incubated with 1 × 105 naïve OTI T cells in round-bottom 96-wells plates (BD Pharmingen), in RPMI supplemented with 5% FCS and PenStrep, for 24 hours at 37°C. As a positive control, crystalline ovalbumin (OVA) was added (10 μg/mL; Sigma-Aldrich, St Louis, MO). As a negative control, 1 × 105 OTI T cells were incubated without DC. After 24 hours, CD25 and CD69 expression by OTI T cells was determined by flow cytometry. T cells, iNKT cells, and DC were preincubated 5 minutes on ice with Fc-block 1:200 (clone 2.4G2; BD Pharmingen). Immunostaining was performed for 15 minutes on ice with fluorophore-conjugated antibodies (Ab) against CD11c (clone HL3; BD Biosciences), CD8 (clone 53-6.7; Abcam Inc., Boston, MA), Thy1.1 (clone OX7; Antigenix America Inc., Huntington Station, NY), CD69 (clone H1.2F3; BD Pharmingen), CD25 (clone PC61; Biolegend), NK1.1 (clone PK136; eBiosciences, San Diego, CA), TCRβ (clone H57; eBiosciences), Vα2 (clone B20.1; BD Pharmingen), and PBS57-loaded CD1d-tetramers (NIH Tetramer Facility, Atlanta, GA). Flow cytometry analysis was performed on a FACS-Canto flow cytometer (BD Pharmingen). For intracellular cytokine staining, OTI T cells were restimulated with SIINFEKL peptide in 96- or 48-well plates (10 μmol/L, 5 hours, 37°C) in the presence of brefeldin A (10 μg/mL; Sigma-Aldrich). Whole OVA was used as negative control (10 μg/mL; Sigma-Aldrich). For intracellular cytokine staining of iNKT cells, mice were injected intravenously (IV) with brefeldin A (250 ng/mouse) 15 minutes prior to αGalCer IV injections (100 ng/mouse). Liver iNKT cells were isolated at indicated times. Cells were stained for surface markers, fixed, permeabilized, and stained intracellular using a Cytofix/Cytoperm kit (BD Pharmingen) and fluorophore-conjugated Ab to IFN-γ (clone XMG1.2; eBiosciences), interleukin (IL)-4 (clone 11B11; BD Pharmingen), or TNF-α (clone MP6 XT22; eBiosciences). To assay for apoptosis, OTI T cells were extracted at days 3 and 5 after adoptive transfer in Tf-mOVA mice, and nonparenchymal cells were isolated from the liver and stained for CD8 (clone 5H10; Invitrogen-Caltag Laboratories, Carlsbad, CA) and Vα2 (clone B20.1; BD Pharmingen). For detection of caspase 3, cells were resuspended at 1 × 106 cells/mL in RPMI containing 10% FCS, 1% Pen/Strep, and 50 μmol/L β-mercaptoethanol, and 5 × 105 cells were incubated with 1 μL RED-DEVD-FMK (Red Caspase-3 Staining Kit, PromoKine, Heidelberg, Germany) for 45 minutes at 37°C. Anti-IFN-γ (clone XMG1.2), anti-TNF-α (clone XT3.11), or rat IgG1 control antibody (200 μg/mL each) were IP injected at days −1, 0, 2, and 4 of IV αGalCer and OTI T-cell injection. At day 5, serum ALT levels were measured, liver pathology was scored, and OTI T cells were isolated from the liver. After restimulation with SIINFEKL, intracellular cytokines were measured. Blood was collected from the tail of mice, centrifuged at 5000g for 10 minutes, and the serum was extracted. Serum alanine aminotransferase (ALT) activity was determined in serum using ALT (SGPT) reagent set (colorimetreic method by Teco Diagnostics, Anaheim, CA) according to manufacturer's instructions. Liver tissue was fixed in 4% formalin (pH 7.0) for at least 24 hours and embedded in paraffin. Two-micrometer-thick paraffin sections were stained with H&E and were blindly scored by a pathologist (R. T. Bronson). IFN-γ production by OTI T cells, their absolute cell counts, serum ALT levels, and the frequencies of iNKT and NK cells were compared using the Mann-Whitney U test (Windows; Microsoft Corp, Redmond, WA; SPSS, Chicago, IL). The self-antigen Tf-mOVA is expressed by hepatocytes and may be presented as peptide/class I MHC complexes to CD8 T lymphocytes in antigen-draining lymphoid tissues.21Lanzavecchia A. Sallusto F. Regulation of T-cell immunity by dendritic cells.Cell. 2001; 106: 263-266Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar To determine in which anatomic location(s) OTI T-cell priming occurs in Tf-mOVA mice, we extracted DCs from different tissues and performed DC/T-cell cocultures (Figure 1A). DC from Tf-mOVA, but not wild-type mice, activated OTI T cells, as measured by CD69 and CD25 expression at 24 hours (Figure 1A). DC from Tf-mOVA mesenteric lymph nodes and spleen induced most OTI cell activation, whereas DC from liver and skin-draining lymph nodes (axillary and inguinal combined) induced more modest OTI T-cell activation. To establish whether OTI T cells are activated in naïve Tf-mOVA mice in vivo, we adoptively transferred CFSE-labeled OTI T cells and analyzed their anatomic location and proliferation status at 36 hours posttransfer (Figure 1B). In the Tf-mOVA mice by far, most proliferating OTI T cells were recovered from the liver, and some were detected in the spleen. L-selectin (CD62L) expression of lymphocytes is critical for their homing to peripheral lymph nodes, through binding to endothelial sulfated carbohydrate ligands in the high endothelial venules.22Gallatin W.M. Weissman I.L. Butcher E.C. A cell-surface molecule involved in organ-specific homing of lymphocytes.Nature. 1983; 304: 30-34Crossref PubMed Scopus (1234) Google Scholar Injection of neutralizing antibody to L-selectin, MEL14, blocks lymph node homing,22Gallatin W.M. Weissman I.L. Butcher E.C. A cell-surface molecule involved in organ-specific homing of lymphocytes.Nature. 1983; 304: 30-34Crossref PubMed Scopus (1234) Google Scholar whereas spleen-directed migration is L-selectin independent. To investigate where CD8 T-cell priming occurs, we transferred CFSE-labeled OTI T cells to splenectomized Tf-mOVA mice in which lymph node homing is blocked using MEL14. At 36 hours posttransfer, most OTI T cells (>96%) were recovered from livers regardless of antibody treatment (Figure 2A). Additionally, we made use of the immune-modulator FTY720, which induces the migration of lymphocytes into secondary lymphoid tissues and blocks their egress into efferent lymphatics.23Matloubian M. Lo C.G. Cinamon G. et al.Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1.Nature. 2004; 427: 355-360Crossref PubMed Scopus (2047) Google Scholar Again, the majority of OTI T cells was recovered from livers of Tf-mOVA mice (Figure 2B, data are shown for 6 mice/group). A fraction of the cells (30%) had proliferated in the spleen, but only very few OTI T cells were present in lymph nodes. Thus, even though in vitro DC derived from lymph nodes of Tf-mOVA mice are potent OTI T-cell activators, in vivo OTI T cells are primarily primed in the liver. We hypothesized that rapid secretion of cytokines by iNKT cells may stimulate intrahepatic MHC-restricted T-cell responses. We therefore injected naïve CFSE-labeled OTI T cells into Tf-mOVA mice in the presence or absence of 1 single, low dose of αGalCer (100 ng/mouse). Vα14 iNKT-cell activation by αGalCer treatment did not significantly affect the intrahepatic proliferation rate of OTI T cells nor did it affect OTI proliferation in the lymph nodes or spleen (Figure 3). Proliferation of T cells does not necessarily imply acquisition of effector cell capabilities. We therefore determined whether αGalCer injection potentiates the production of IFN-γ by liver-resident OTI T cells (Figure 4A). Coinjecting αGalCer with OTI T cells increased the frequency of liver-resident IFN-γ-producing OTI T cells from 5.1% (3.8%–13.5%) to 15.3% (6.5%–26.6%; P = .017). The potentiation of OTI T-cell function was mediated by iNKT cells because αGalCer did not promote IFN-γ production by OTI T cells in CD1d−/−Tf-mOVA mice that lack iNKT cells: 6.5% (2.1%–11.7%; P > .05) (Figure 4B). Restimulation of OTI T cells with whole OVA (10 μg) as a negative control induced background IFN-γ levels. To investigate further whether iNKT-cell activation facilitates intrahepatic effector function of OTI T cells in Tf-mOVA mice, in vivo cytolysis assays were performed. Coinjecting αGalCer with OTI T cells caused a profound increase in specific lysis of SIINFEKL-pulsed splenocytes in the liver (Figure 4C). The activation of iNKT cells using αGalCer induces leukocyte recruitment to the liver, including NK cells, T cells, and iNKT cells.8Nakagawa R. Nagafune I. Tazunoki Y. et al.Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by α-galactosylceramide in mice.J Immunol. 2001; 166: 6578-6584Crossref PubMed Scopus (207) Google Scholar, 24Osman Y. Kawamura T. Naito T. et al.Activation of hepatic NKT cells and subsequent liver injury following administration of α-galactosylceramide.Eur J Immunol. 2000; 30: 1919-1928Crossref PubMed Scopus (242) Google Scholar Treatment of Tf-mOVA mice with αGalCer indeed induced a profound increase of intrahepatic leukocytes, and a large fraction consisted of NK cells and iNKT cells (Figure 5A). However, αGalCer treatment did not result in a significant increase in intrahepatic OTI T cells: coinjection of αGalCer changed the median OTI T-cell number in Tf-mOVA livers from 1.8 × 106 to 1.6 × 106 (day 5 post-transfer). From αGalCer-coinjected CD1d−/−Tf-mOVA mice, we retrieved 1.4 × 106 intrahepatic OTI T cells (P > .05) (Figure 5B). Does αGalCer treatment induce apoptosis of recruited OTI cells? Only a small fraction of OTI T cells in livers of mice that did not receive αGalCer was undergoing apoptosis, which was only slightly increased after αGalCer treatment. Apoptotic OTI T cells were characterized by Vα2 costaining for active caspase 3 or the probe DEVD-FMK (Figure 5C). Taken together, in Tf-mOVA mice, iNKT-cell activation by αGalCer does not stimulate proliferation or liver-directed migration of antigen-specific CD8 T cells. Instead, iNKT-cell activation facilitates the intrahepatic effector function of OTI T cells. To determine whether iNKT-cell activation promotes liver damage in Tf-mOVA mice, we analyzed serum levels of ALT at 5 days post-OTI transfer in the presence or absence of αGalCer (Figure 6A). Transfer of OTI T cells induced mild hepatitis (median ALT level, 37.5 IU/mL). Injection of αGalCer alone caused a modest increase of serum ALT levels in Tf-mOVA and WT mice, which is most likely due to nonspecific liver damage after activation of iNKT cells.25Fujii H. Seki S. Kobayashi S. A murine model of NKT cell-mediated liver injury induced by α-galactosylceramide/d-galactosamine.Virchows Arch. 2005; 446: 663-673Crossref PubMed Scopus (21) Google Scholar Coinjection of both factors in Tf-mOVA mice increased the median serum ALT level (57 IU/L; P > .05). Furthermore, severe hepatitis (>5× upper limit of normal) only occurred in Tf-mOVA mice that received both OTI and αGalCer. Analysis of liver histology revealed that activation of OTI T cells by transfer into Tf-mOVA mice induced severe hepatitis by day 5, regardless of αGalCer presence (Figure 6B). Treatment with αGalCer in the absence of OTI T cells caused only mild inflammation. Treatment with αGalCer induces iNKT cells to secrete TNF-α and IFN-γ.26Inui T. Nakagawa R. Ohkura S. et al.Age-associated augmentation of the synthetic ligand- mediated function of mouse NK1.1 ag(+) T cells: their cytokine production and hepatotoxicity in vivo and in vitro.J Immunol. 2002; 169: 6127-6132Crossref PubMed Scopus (51) Google Scholar, 27Inui T. Nakashima H. Habu Y. et al.Neutralization of tumor necrosis factor abrogates hepatic failure induced by α-galactosylceramide without attenuating its antitumor effect in aged mice.J Hepatol. 2005; 43: 670-678Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Indeed, shortly after αGalCer injection, intrahepatic iNKT cells produced TNF-α and IFN-γ (Figure 7A). Is potentiation of intrahepatic OTI T-cell function by αGalCer injection mediated by TNF-α and IFN-γ? Antibody-mediated neutralization of both cytokines significantly reduced the frequency of intrahepatic IFN-γ-producing OTI T cells compared with levels observed using isotype control Ab (Figure 7B). Interestingly, only anti-TNF-α Ab and not anti-IFN-γ Ab pretreatment could prevent liver damage after transfer of OTI T cells + αGalCer as measured by increased serum ALT levels (Figure 7B) and liver damage (Figure 7C). We here describe the role of Vα14 iNKT cells in stimulating intrahepatic antigen-specific CD8 T-cell responses using a recently established mouse model that allows for study of intrahepatic immune responses in vivo.18Derkow K. Loddenkemper C. Mintern J. et al.Differential priming of CD8 and CD4 T cells in animal models of autoimmune hepatitis and cholangitis.Hepatology. 2007; 46: 1155-1165Crossref PubMed Scopus (58) Google Scholar The presence of the antigen on Tf-mOVA hepatocytes led to retention of antigen-specific CD8 T cells in the liver. These intrahepatic CD8 T cells were fully functional because they produced IFN-γ after brief restimulation with SIINFEKL, for induction of hepatitis in vivo. Using a different transgenic mouse model in which alloantigen was present within both liver and lymph nodes, it was recently suggested that the site of primary T-cell activation determines their functional faith: CD8 T cells activated in the periphery were able to cause liver injury, whereas intrahepatically activated T cells exhibited defective function.28Bowen D.G. Zen M. Holz L. et al.The site of primary T-cell activation is a determinant of the balance between intrahepatic tolerance and immunity.J Clin Invest. 2004; 114: 701-712Crossref PubMed Scopus (274) Google Scholar As recently suggested by Derkow et al in their study describing the Tf-mOVA model,18Derkow K. Loddenkemper C. Mintern J. et al.Differential priming of CD8 and CD4 T cells in animal models of autoimmune hepatitis and cholangitis.Hepatology. 2007; 46: 1155-1165Crossref PubMed Scopus (58) Google Scholar we here show that, whereas Tf-mOVA antigen is only expressed in hepatocytes,18Derkow K. Loddenkemper C. Mintern J. et al.Differential priming of CD8 and CD4 T cells in animal models of autoimmune hepatitis and cholangitis.Hepatology. 2007; 46: 1155-1165Crossref PubMed Scopus (58) Google Scholar DCs in lymphoid organs presented this antigen to OTI T cells (Figure 1A). However, when lymph node homing and exit were blocked, the large majority of transferred CD8 T cells was recovered from livers. Thus, even though the antigen is present in the periphery, in Tf-mOVA mice, OVA-specific CD8 T cells are most likely primed in the liver, and this is in accordance with previous studies.28Bowen D.G. Zen M. Holz L. et al.The site of primary T-cell activation is a determinant of the balance between intrahepatic tolerance and immunity.J Clin Invest. 2004; 114: 701-712Crossref PubMed Scopus (274) Google Scholar, 29Klein I. Gassel H.J. Crispe I.N. Cytotoxic T-cell response following mouse liver transplantation is independent of the initial site of T-cell priming.Transplant Proc. 2006; 38: 3241-3243Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 30Wuensch S.A. Pierce R.H. Crispe I.N. Local intrahepatic CD8+ T-cell activation by a non-self-antigen results in full functional differentiation.J Immunol. 2006; 177: 1689-1697Crossref PubMed Scopus (70) Google Scholar Several studies have investigated the role of iNKT cells in intrahepatic immunity. αGalCer-activated iNKT cells can cause extensive liver damage25Fujii H. Seki S. Kobayashi S. A murine model of NKT cell-mediated liver injury induced by α-galactosylceramide/d-galactosamine.Virchows Arch. 2005; 446: 663-673Crossref PubMed Scopus (21) Google Scholar and inhibited tumor metastasis to the liver,8Nakagawa R. Nagafune I. Tazunoki Y. et al.Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by α-galactosylceramide in mice.J Immunol. 2001; 166: 6578-6584Crossref PubMed Scopus (207) Google Scholar, 31Nakagawa R. Inui T. Nagafune I. et al.Essential role of bystander cytotoxic CD122+CD8+ T cells for the antitumor immunity induced in the liver of mice by α-galactosylceramide.J Immunol. 2004; 172: 6550-6557Crossref PubMed Scopus (33) Google Scholar which involved the activation of tumor nonspecific CD8 T cells.8Nakagawa R. Nagafune I. Tazunoki Y. et al.Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by α-galactosylceramide in mice.J Immunol. 2001; 166: 6578-6584Crossref PubMed Scopus (207) Google Scholar, 31Nakagawa R. Inui T. Nagafune I. et al.Essential role of bystander cytotoxic CD122+CD8+ T cells for the antitumor immunity induced in the liver of mice by α-galactosylceramide.J Immunol. 2004; 172: 6550-6557Crossref PubMed Scopus (33) Google Scholar Very little was known, however, about whether iNKT cells mediate antigen-specific hepatitis whereby antigen-specific conventional T cells instigate liver injury. Here, we demonstrate that activation of CD1d-restricted iNKT cells with αGalCer facilitates the effector function of intrahepatic antigen-specific CD8 T cells. Activation of iNKT cells tripled the frequency of intrahepatic IFN-γ-producing OTI T cells and increased their cytolytic capacity, contributing to liver damage. αGalCer-mediated iNKT-cell activation did not appear to increase intrahepatic effector function of OTI T cells by promoting their proliferation or influx from the periphery. Neither could we demonstrate a switch in polarization phenotype of OTI T cells as measured by IL-4 production: regardless of αGalCer coinjection, only very few (<1%) intrahepatic OTI T cells produced IL-4 (data not shown). Instead, iNKT-cell activation influences the cytolytic ability and IFN-γ production of intrahepatic mOVA-specific CD8 T cells primed in the liver. Because αGalCer did not promote mOVA-specific IFN-γ production in CD1d−/−mOVA mice, its enhancement of OTI T-cell effector function is CD1d dependent. Surface expression of CD1d in liver is detected on hepatocytes,32Trobonjaca Z. Leithauser F. Moller P. et al.Activating immunity in the liver I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-γ release by liver NKT cells.J Immunol. 2001; 167: 1413-1422Crossref PubMed Scopus (103) Google Scholar DCs,32Trobonjaca Z. Leithauser F. Moller P. et al.Activating immunity in the liver I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-γ release by liver NKT cells.J Immunol. 2001; 167: 1413-1422Crossref PubMed Scopus (103) Google Scholar Kupffer cells,33Schmieg J. Yang G. Franck R.W. et al.Glycolipid presentation to natural killer T cells differs in an organ-dependent fashion.Proc Natl Acad Sci U S A. 2005; 102: 1127-1132Crossref PubMed Scopus (104) Google Scholar and Ito cells.34Winau F. Hegasy G. Weiskirchen R. et al.Ito cells are liver-resident antigen-presenting cells for activating T cell responses.Immunity. 2007; 26: 117-129Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar Display by DCs of both CD1d/αGalCer and specific peptide-loaded class I MHC molecules can trigger iNKT cells to enhance T-cell responses to soluble antigen.6Hermans I.F. Silk J.D. Gileadi U. et al.NKT cells enhance CD4+ and CD8+ T-cell responses to soluble antigen in vivo through direct interaction with dendritic cells.J Immunol. 2003; 171: 5140-5147Crossref PubMed Scopus (406) Google Scholar Kupffer cells and hepatic stellate cells are additional candidate cells that may mediate the CD8 T-cell stimulation we report here because these cells are potent Vα14 iNKT-cell activators.33Schmieg J. Yang G. Franck R.W. et al.Glycolipid presentation to natural killer T cells differs in an organ-dependent fashion.Proc Natl Acad Sci U S A. 2005; 102: 1127-1132Crossref PubMed Scopus (104) Google Scholar, 34Winau F. Hegasy G. Weiskirchen R. et al.Ito cells are liver-resident antigen-presenting cells for activating T cell responses.Immunity. 2007; 26: 117-129Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar We show that αGalCer induces iNKT cells to produce TNF-α and IFN-γ. The production of these cytokines by iNKT cells can be crucial in mediating antimetastatic effects of αGalCer treatment.26Inui T. Nakagawa R. Ohkura S. et al.Age-associated augmentation of the synthetic ligand- mediated function of mouse NK1.1 ag(+) T cells: their cytokine production and hepatotoxicity in vivo and in vitro.J Immunol. 2002; 169: 6127-6132Crossref PubMed Scopus (51) Google Scholar, 27Inui T. Nakashima H. Habu Y. et al.Neutralization of tumor necrosis factor abrogates hepatic failure induced by α-galactosylceramide without attenuating its antitumor effect in aged mice.J Hepatol. 2005; 43: 670-678Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 35Hayakawa Y. Takeda K. Yagita H. et al.Critical contribution of IFN-γ and NK cells, but not perforin-mediated cytotoxicity, to anti-metastatic effect of α-galactosylceramide.Eur J Immunol. 2001; 31: 1720-1727Crossref PubMed Scopus (177) Google Scholar In our study, pretreatment with neutralizing antibodies to either anti-TNF-α or anti-IFN-γ reduced the frequency of IFN-γ-producing OTI T cells in liver to the levels observed in the absence of αGalCer. Therefore, both cytokines appear to be potent effectors of αGalCer-mediated stimulation of intrahepatic CD8 T cells, either directly or indirectly by stimulating surrounding lymphocytes such as NK cells.8Nakagawa R. Nagafune I. Tazunoki Y. et al.Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by α-galactosylceramide in mice.J Immunol. 2001; 166: 6578-6584Crossref PubMed Scopus (207) Google Scholar, 31Nakagawa R. Inui T. Nagafune I. et al.Essential role of bystander cytotoxic CD122+CD8+ T cells for the antitumor immunity induced in the liver of mice by α-galactosylceramide.J Immunol. 2004; 172: 6550-6557Crossref PubMed Scopus (33) Google Scholar It is yet unclear whether iNKT cell-derived cytokines stimulate intrahepatic CD8 T cells directly or through stimulation of antigen presentation by APCs such as Kupffer cells, Ito cells, and DCs. Importantly, anti-TNF-α treatment, but not anti-IFN-γ treatment, prevented serum ALT elevations and liver damage in all animals. This is in accordance with previous studies that suggest that activated iNKT cells use the production of IFN-γ and TNF-α as different effector tools, whereby especially the latter plays an essential role in the hepatic injury induced by αGalCer.27Inui T. Nakashima H. Habu Y. et al.Neutralization of tumor necrosis factor abrogates hepatic failure induced by α-galactosylceramide without attenuating its antitumor effect in aged mice.J Hepatol. 2005; 43: 670-678Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 36Biburger M. Tiegs G. α-Galactosylceramide-induced liver injury in mice is mediated by TNF-α but independent of Kupffer cells.J Immunol. 2005; 175: 1540-1550Crossref PubMed Scopus (145) Google Scholar The observed stimulatory effect of iNKT-cell activation on the intrahepatic antigen-specific immune response suggests the possibility to use αGalCer in immune-modulatory therapy for human conditions characterized by insufficient Th1 responses in the liver. αGalCer-mediated iNKT-cell activation may stimulate weak virus-specific immune responses generally observed in patients with chronic HBV and HCV infection (reviewed in Bertoletti and Ferrari37Bertoletti A. Ferrari C. Kinetics of the immune response during HBV and HCV infection.Hepatology. 2003; 38: 4-13Crossref PubMed Scopus (234) Google Scholar). However, as shown by others previously,8Nakagawa R. Nagafune I. Tazunoki Y. et al.Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by α-galactosylceramide in mice.J Immunol. 2001; 166: 6578-6584Crossref PubMed Scopus (207) Google Scholar, 24Osman Y. Kawamura T. Naito T. et al.Activation of hepatic NKT cells and subsequent liver injury following administration of α-galactosylceramide.Eur J Immunol. 2000; 30: 1919-1928Crossref PubMed Scopus (242) Google Scholar, 25Fujii H. Seki S. Kobayashi S. A murine model of NKT cell-mediated liver injury induced by α-galactosylceramide/d-galactosamine.Virchows Arch. 2005; 446: 663-673Crossref PubMed Scopus (21) Google Scholar and also shown here, αGalCer-mediated iNKT-cell activation can cause liver injury. Moreover, use of especially repeated dosages of αGalCer somehow causes redirection of the immune response toward T helper cell 2 rather than T helper cell 1 reactivity (reviewed in Van Kaer38Van Kaer L. α-Galactosylceramide therapy for autoimmune diseases: prospects and obstacles.Nat Rev Immunol. 2005; 5: 31-42Crossref PubMed Scopus (267) Google Scholar). Therefore, several obstacles need to be overcome before αGalCer can be used as treatment to stimulate inadequate T helper cell 1 immune responses in human liver. In conclusion, functional antigen-specific CD8 T cells can be activated in the liver. Stimulation of iNKT cells with 1 single low dose of αGalCer facilitates the intrahepatic effector function of these antigen-specific T cells. Activation of iNKT cells may stimulate effector function of antigen-specific CD8 T cells that are primed in the liver, leading to hepatitis and subsequent liver damage. Thus, cross talk between CD1d-restricted Vα14 iNKT cells and APCs in the liver may be critically involved in controlling intrahepatic MHC-restricted T-cell responses. The authors thank the Boes laboratory for helpful discussions and Teresa Bianchi also for tail vein injections; Dr Mark Exley for providing CD1d−/− mice; and The NIH Tetramer Facility for supplying the CD1d tetramers. Download .tif (.03 MB) Help with tif files Supplementary Figure S1FTY720 blocks T-cell exit from lymph nodes. To block lymph node exit of T cells, FTY720 was injected into mice. To make sure FTY720 treatment blocks the number of circulating CD8+ T cells, in every experiment using FTY720-treated and nontreated mice, the absolute number of total CD8+ cells/mL peripheral blood was determined (bar graph)." @default.
• W2057673313 created "2016-06-24" @default.
• W2057673313 creator A5009960850 @default.
• W2057673313 creator A5016293777 @default.
• W2057673313 creator A5018682708 @default.
• W2057673313 creator A5036407951 @default.
• W2057673313 creator A5041153123 @default.
• W2057673313 creator A5043626370 @default.
• W2057673313 creator A5073821908 @default.
• W2057673313 date "2008-06-01" @default.
• W2057673313 modified "2023-10-17" @default.
• W2057673313 title "Critical Role for CD1d-Restricted Invariant NKT Cells in Stimulating Intrahepatic CD8 T-Cell Responses to Liver Antigen" @default.
• W2057673313 cites W1481001557 @default.
• W2057673313 cites W1506174227 @default.
• W2057673313 cites W1523753658 @default.
• W2057673313 cites W1580852520 @default.
• W2057673313 cites W1599518596 @default.
• W2057673313 cites W1653749361 @default.
• W2057673313 cites W1748261727 @default.
• W2057673313 cites W1959677146 @default.
• W2057673313 cites W1966259152 @default.
• W2057673313 cites W1968141301 @default.
• W2057673313 cites W1985970818 @default.
• W2057673313 cites W1987904745 @default.
• W2057673313 cites W1989018151 @default.
• W2057673313 cites W2006985918 @default.
• W2057673313 cites W2008596165 @default.
• W2057673313 cites W2016548343 @default.
• W2057673313 cites W2016598959 @default.
• W2057673313 cites W2044828065 @default.
• W2057673313 cites W2053954731 @default.
• W2057673313 cites W2061140050 @default.
• W2057673313 cites W2062813847 @default.
• W2057673313 cites W2087287657 @default.
• W2057673313 cites W2091602751 @default.
• W2057673313 cites W2097302710 @default.
• W2057673313 cites W2099465341 @default.
• W2057673313 cites W2101722063 @default.
• W2057673313 cites W2103614304 @default.
• W2057673313 cites W2111828785 @default.
• W2057673313 cites W2114441652 @default.
• W2057673313 cites W2116813965 @default.
• W2057673313 cites W2121895986 @default.
• W2057673313 cites W2122582242 @default.
• W2057673313 cites W2127064328 @default.
• W2057673313 cites W2144821901 @default.
• W2057673313 cites W2150998778 @default.
• W2057673313 cites W2157797015 @default.
• W2057673313 cites W2161422743 @default.
• W2057673313 cites W2170944561 @default.
• W2057673313 cites W4244017533 @default.
• W2057673313 cites W4321429285 @default.
• W2057673313 doi "https://doi.org/10.1053/j.gastro.2008.02.037" @default.
• W2057673313 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18549881" @default.
• W2057673313 hasPublicationYear "2008" @default.
• W2057673313 type Work @default.
• W2057673313 sameAs 2057673313 @default.
• W2057673313 citedByCount "21" @default.
• W2057673313 countsByYear W20576733132012 @default.
• W2057673313 countsByYear W20576733132013 @default.
• W2057673313 countsByYear W20576733132015 @default.
• W2057673313 countsByYear W20576733132016 @default.
• W2057673313 countsByYear W20576733132018 @default.
• W2057673313 countsByYear W20576733132019 @default.
• W2057673313 countsByYear W20576733132020 @default.
• W2057673313 countsByYear W20576733132021 @default.
• W2057673313 crossrefType "journal-article" @default.
• W2057673313 hasAuthorship W2057673313A5009960850 @default.
• W2057673313 hasAuthorship W2057673313A5016293777 @default.
• W2057673313 hasAuthorship W2057673313A5018682708 @default.
• W2057673313 hasAuthorship W2057673313A5036407951 @default.
• W2057673313 hasAuthorship W2057673313A5041153123 @default.
• W2057673313 hasAuthorship W2057673313A5043626370 @default.
• W2057673313 hasAuthorship W2057673313A5073821908 @default.
• W2057673313 hasBestOaLocation W20576733131 @default.
• W2057673313 hasConcept C139563560 @default.
• W2057673313 hasConcept C147483822 @default.
• W2057673313 hasConcept C154317977 @default.
• W2057673313 hasConcept C167672396 @default.
• W2057673313 hasConcept C202751555 @default.
• W2057673313 hasConcept C203014093 @default.
• W2057673313 hasConcept C2778607024 @default.
• W2057673313 hasConcept C55493867 @default.
• W2057673313 hasConcept C71924100 @default.
• W2057673313 hasConcept C86803240 @default.
• W2057673313 hasConceptScore W2057673313C139563560 @default.
• W2057673313 hasConceptScore W2057673313C147483822 @default.
• W2057673313 hasConceptScore W2057673313C154317977 @default.
• W2057673313 hasConceptScore W2057673313C167672396 @default.
• W2057673313 hasConceptScore W2057673313C202751555 @default.
• W2057673313 hasConceptScore W2057673313C203014093 @default.
• W2057673313 hasConceptScore W2057673313C2778607024 @default.
• W2057673313 hasConceptScore W2057673313C55493867 @default.
• W2057673313 hasConceptScore W2057673313C71924100 @default.
• W2057673313 hasConceptScore W2057673313C86803240 @default.
• W2057673313 hasIssue "7" @default.
• W2057673313 hasLocation W20576733131 @default.
• W2057673313 hasLocation W20576733132 @default.

• next