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Type I IFN-Producing CD4 V14i NKT Cells Facilitate Priming of IL-10-Producing CD8 T Cells by Hepatocytes¹

Department of Internal Medicine I, University of Ulm, Ulm, Germany

	Abstract

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Upon entering the liver CD8 T cells encounter large numbers of NKT cells patrolling the hepatocyte (HC) surface facing the perisinusoidal space. We asked whether hepatic NKT cells modulate the priming of CD8 T cells by HC. Hepatic (-galactosyl-ceramide-loaded CD1d dimer binding) NKT cells produce predominantly IL-4 when stimulated with glycolipid-presenting HC but predominantly IFN- when stimulated with glycolipid-presenting dendritic cells. These NKT cells prime naive CD8 T cells to a (K^b-presented) peptide ligand if they simultaneously recognize a CD1d-binding glycolipid presented to them on the surface of the responding CD8 T cells that they prime. No IL-10-producing CD8 T cells are detected if these T cells are primed by either HC or NKT cells. In contrast, IL-10 is produced by HC-primed CD8 T cells if IFN--producing NKT cells are coactivated by the same HC presenting a glycolipid (in the context of CD1d) and an antigenic peptide (in the context of K^b). Hence, IL-10-producing CD8 T cells are generated in a type I IFN-dependent manner if the three cell types (CD8 T cells, NKT cells, and ligand-presenting HC) specifically and closely interact. IL-10-producing CD8 T cells generated under these conditions down-modulate IL-2 (and proliferative) responses of naive CD4 or CD8 T cells primed by DC. If in close proximity, NKT cells can thus locally modulate the phenotype of CD8 T cells during their priming by HC thereby limiting the local activation of proinflammatory immune effector cells and protecting the liver against immune injury.

	Introduction

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Natural killer T cells represent 1% of the murine T cell population (1). The TCR of V14i NKT cells (also known as type I or invariant (i) NKT cells) is composed of an invariant V14(V14i)³/J18-chain paired with a restricted set of V8.2, V7, or V2 chains (1). V14i NKT cells recognize glycolipids presented by CD1d molecules. Specific recognition of the CD1d-restricted superagonist glycosphingolipid -galactosyl-ceramide (GalCer) by V14i NKT cells is highly conserved through mammalian evolution (2, 3). Murine NKT cells express CD4 and NK1.1 (CD161c) on the surface and intermediate (int) levels of the CD3/TCR complex (CD3^intTCR^int), but these markers are not considered reliable (1). In contrast, CD1d tetramers or dimers loaded with glycolipids are valuable and unambiguous reagents for identifying in vitro or in vivo NKT cells (4, 5, 6), but the striking down-modulation of the TCR by NKT cells immediately after activation may underestimate the size of the NKT cell population under some experimental conditions. Unlike conventional T cells, NKT cells activate IL-4 and IFN- transcription during thymic development, populate the periphery as long-lived effector cells with both cytokine loci constitutively active (7), and rapidly secrete abundant amounts of cytokines for only a few hours after activation. Although intense research efforts focused on the immunobiology of NKT cells, many aspects of their immunoregulatory role are unresolved.

The murine liver harbors a large NKT cell population (8). The hepatic NKT cell population is expanded during liver regeneration (9), with age (10), or in mice constitutively expressing G-CSF in the liver (11). The appearance of NKT cells in the liver (but not in the spleen) depends on LFA-1 (12). Liver NKT cells can be locally activated either specifically through the TCR (e.g., by glycolipid-presenting cells) or nonspecifically through cytokines such as IL-12 (13, 14) or IL-18 (15). Activated intrahepatic NKT cells rapidly release cytokines (IFN- and IL-4) and are cytotoxic (7, 16, 17). NKT cells trigger liver damage in the experimental acute hepatitis model induced by Con A injection (18, 19, 20). NKT cell-mediated injury of hepatocytes (HC) after GalCer injection has been shown (21). NKT cells are sensitive and early sentinels that convey regulatory signals to other cells of the local immune system. They patrol within hepatic (peri)sinusoids at 10–20 µm/min and stop upon TCR-mediated activation, thus providing a local, intravascular immune surveillance (22). Cells in the (peri)sinusoidal compartment of the liver that can interact with NKT cells or are responsive to NKT cell-derived signals in situ include dendritic cells (DC), HC, NK cells, and T cells (23).

We reported that glycolipid-presenting HC stimulate mainly an IL-4 response of hepatic NKT cells while glycolipid-presenting DC stimulate mainly an IFN- response of hepatic NKT cells (24). Hepatic DC themselves are activated in the process of specifically stimulating CD1d-restricted NKT cells and recruit liver NK cells into the response (25). Murine type I IFN attenuate the DC-driven Th1 NKT/NK cell response in the liver and its associated immunopathology (25). We continued these studies by analyzing the specific interaction between NKT cells and CD8 T cells that recognizes their cognate ligand presented by HC.

NKT cells can enhance CD8 T cell priming and effector cell generation in vivo through a direct effect on DC function (26, 27). CD1d-dependent NKT cells play a role in the expansion of CD8 T cells and the amplification of their antiviral response (28). Naive and memory T cells express CD1d, but it is unknown whether they directly and specifically interact with NKT cells during priming or specific restimulation. NKT cells could either directly act as APC for MHC class I-restricted CD8 T cells (as they express MHC class I molecules on the surface) or modulate the response of CD8 T cells by an indirect effect on third party APC. The latter could be either a professional APC (e.g., a DC as shown by us previously (27)) or a nonprofessional APC (e.g., a HC) that acquires presenting functions through NKT cell activation. The functional outcome of such interactions merits interest especially in view of the close proximity between CD8 T cells entering the liver through sinusoids, numerous NKT cells constantly patrolling the entry site and a readily accessible and enlarged surface of HC (that can present class I-restricted antigenic determinants and CD1d-restricted glycolipid determinants).

We analyzed the cytokine expression profile in the specific interactions between NKT cells and naive CD8 T cells with HC. The novel finding emerging from this study is the IFN--dependent, rapid, and specific priming of IL-10-producing immunoregulatory CD8 T cells by Ag-presenting HC in the presence of NKT cell coactivation.

	Materials and Methods

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Mice

H-2^b C57BL/6 (B6) mice, CD1d^–/– B6 mice (29), MHC class II-deficient A^–/– B6 mice (30), IFN- receptor 1 (IFNAR1)^–/– B6 mice (31), IFN-^–/– B6 mice (32), and TCR transgenic OT-I and OT-II B6 mice (33) were bred and kept under standard pathogen-free conditions in the animal colony of Ulm University (Ulm, Germany). MHC class II-deficient A^–/– B6 mice were generated in the B6 strain. IFNAR1^–/– mice and IFN-^–/– mice were backcrossed for 16 generations to B6. Female and male mice were used at 8–12 wks of age. All animal experiments were approved by the respective institutional animal care and use committees in accordance with the applicable federal, provincial and local regulations.

Isolation of mouse liver cell subsets

The abdomen of an anesthetized mouse was opened and a needle was inserted into the portal vein. The vena cava was ligated and punctured. The liver was perfused with liver perfusion medium (catalog no. 17701-038; Invitrogen Life Technologies) and the perfusate was collected. To obtain nonparenchymal cell (NPC) populations, the liver was perfused with liver digestion medium (catalog no. 17703-034; Invitrogen Life Technologies), removed, and gently pressed through a mesh. NPC were separated from parenchymal HC by centrifugation at 50 x g for 5 min. NPC were collected, washed in PBS, resuspended in 40% Percoll (catalog no. L6145; Biochrom), diluted in RPMI 1640 medium, gently overlaid onto 70% Percoll, and centrifuged at 750 x g for 20 min. NPC collected from the interface were washed twice in PBS and resuspended in medium. HC washed twice in PBS plus 0.1% FCS were purged from lymphomyeloid cells by cell sorting (FACSAria; BD Biosciences). The purity of the isolated HC population was >98% as verified cytologically. Flow cytometric analyses showed that the HC populations were free of contaminating DC (B220, CD11c), stellate cells (CD44), macrophages (CD11b), T cells (CD3), or NK cells (DX5). HC were washed twice and resuspended in RPMI 1640 medium supplemented with 5% FCS (catalog no. 10270-106; Invitrogen Life Technologies). NKT cells from the livers of A^–/– B6 mice were sorted by MACS using anti-mouse CD4 Ab-coated microbeads (catalog no. 130-049-01; Miltenyi Biotec). NKT cells from normal B6, IFNAR1^–/– B6, or IFN-^–/– B6 mice were isolated by FACS (FACSAria; BD Biosciences). Liver NPC were stained with PE-conjugated anti-CD4 mAb and allophycocyanin-conjugated anti-CD3 mAb. CD4^intCD3^int lymphocytes were obtained by cell-sorting (purity >99%). Because the numbers of NKT cells isolated from the livers of normal B6 mice were limited, we used NKT cells from A^–/– B6 mice in many experiments.

Isolation of DC and T cells

Aseptically prepared spleen cell suspensions were washed, resuspended in NycoPrep (catalog no. 1002380; Axis-Shield), overlaid with RPMI 1640 medium, and centrifuged at 4°C and 9.500 x g for 20 min. Cells in the interface were collected and washed twice. Contaminating T cells, B cells, and NK cells were depleted from this cell population by MACS using PE-conjugated anti-CD3 mAb 145-2C11 (catalog no. 553064; BD Biosciences), PE-conjugated anti-B220 mAb RA3-6B2 (catalog no. 553089; BD Biosciences), PE-conjugated anti-NK mAb DX5 (catalog no. 130-052-501; BD Biosciences), and anti-PE microbeads (catalog no. 130-048-801; Miltenyi Biotec). CD11c⁺ DC were positively selected by MACS (catalog no. 130-090-485; Miltenyi Biotec) from the CD3^–DX5^– B220^– cell population as described (34). Flow cytometry analyses verified that these cell populations contained >98% CD11c⁺ DC. CD8 or CD4 T cells were purified from the spleen of OT-I or OT-II mice by MACS (catalog nos. 130-090-859 and 130-090-860; Miltenyi Biotec). The purity of the isolated CD8 or CD4 T cells was >98% as verified by flow cytometry.

Flow cytometry analyses

Cells were washed twice in PBS plus 0.3% (w/v) BSA supplemented with 0.1% w/v sodium azide and preincubated with the mAb 2.4G2 (catalog no. 01241D; BD Biosciences) to block nonspecific binding of Abs to Fc receptors. Cells were washed, incubated for 30 min at 4°C with 0.5 µg of the relevant mAb per 10⁶ cells and washed again. In most experiments, cells were subsequently incubated for 10 min at 4°C with a second-step reagent (streptavidin-PerCP; BD Biosciences; catalog no. 554064). Four-color flow cytometry analyses were performed using a FACSCalibur cytometer (BD Biosciences). Forward narrow angle light scatter was used as an additional parameter to facilitate the exclusion of dead cells and aggregated cell clumps. Data were analyzed using the WinMDI software.

Antibodies

The following mAbs from BD Biosciences were used: allophycocyanin-conjugated anti-CD8 mAb clone 53-6.7 (catalog no. 553035), PE-conjugated anti-CD25 mAb clone PC61 (catalog no. 553866), biotinylated anti-CD28 mAb clone 37.51 (catalog no. 553296), FITC-conjugated anti-CD122 mAb clone TM-1 (catalog no. 553361), FITC-conjugated anti-CD18 mAb clone C71/16 (catalog no. 553292), FITC-conjugated anti-CD102 mAb clone 3C4 (mIC2/4) (catalog no. 557444), PE-conjugated anti-CD69 mAb clone H1.2F3 (catalog no. 553237), PE-conjugated anti-PD-1 mAb clone J43 (catalog no. 551892), PE-conjugated anti-PD-L1 mAb clone MIH5 (catalog no. 558091), PE-conjugated anti-CTLA-4 mAb clone UC10-4F10-11 (catalog no. 553720), PE-conjugated anti-CD1d mAb clone 1B1 (catalog no. 553846), PE-conjugated anti-H-2K^b mAb clone AF6-88.5 (catalog no. 553570), PE-conjugated anti-CD54 mAb clone 3E2 (catalog no. 553253), PE-conjugated anti-CD278 (ICOS) mAb clone 7E.17G9 (catalog no. 552146), FITC-conjugated anti-CD4 mAb clone GK1.5 (catalog no. 553729), allophycocyanin-conjugated anti-CD3 mAb clone 145-2C11 (catalog no. 553066) and biotinylated anti-CD44 mAb clone IM7 (catalog no. 553132). PE-conjugated anti-B7-H3 mAb clone M3.2D7 (catalog no. 12-5973-82), anti-B7-H4 mAb clone 9 (catalog no. 12-5970-82), and anti-B and T lymphocyte attenuator (BTLA) mAb clone 6F7 (catalog no. 12-5950-82) were from eBioscience.

CD1d-dimer assay

We incubated 4 µg of the soluble, divalent mouse CD1d-IgG1 fusion protein DimerX (catalog no. 557599, BD Biosciences) overnight with 100 ng of GalCer at 37°C and neutral pH following the manufacturer’s instructions. The GalCer was a gift from Dr. Y. Koezuka (Pharmaceutical Research Laboratory, Kirin Brewery, Takasaki, Gunma, Japan). The GalCer-loaded CD1d-IgG1 dimers were incubated with PE-coupled (catalog no. 550083; BD Biosciences) or FITC-coupled anti-mouse IgG1 (catalog no. 553443; BD Biosciences) for 60 min at room temperature. Mouse NKT cells were labeled at 4°C for 15 min with GalCer-loaded CD1d-IgG1 dimers (1 µg of GalCer-loaded CD1d-IgG1 dimers per 10⁶ cells).

CFSE labeling

OT-I CD8 T cells or OT-II CD4 T cells were labeled with 2 µM CFSE (catalog no. C34554; Invitrogen Life Technologies) for 15 min at 37°C. The reaction was stopped with cold FCS at 4°C. The labeled cells were washed three times before culture.

Cocultures

Cells suspended in RPMI 1640 medium (supplemented with 5% FCS, 2 mM L-glutamine, and antibiotics) at a density of 1 x 10⁶/ml were pulsed for 2 h with the indicated dose the A^b-binding OVA_323–339 peptide ISQAVHAAHAEINEAGR (recognized by the transgene-encoded TCR of OT-II mice) and/or the K^b-binding OVA_257–264 peptide SIINFEKL (recognized by the transgene-encoded TCR of OT-I mice) and/or by GalCer. Cells were washed three times with medium and transferred into culture. Cells (1 x 10⁴ HC, 4 x 10⁴ NKT cells, and/or 1 x 10⁵ CD8 T cells) were cultured in 200-µl flat-bottom microwells in RPMI 1640 medium supplemented with 5% FCS, 2 mM L-glutamine, and antibiotics. Murine IFN- was obtained from a producer of transfectants as described (24, 25).

IL-10 detection

Cells were harvested from cocultures after 72 h of incubation and stimulated for 4 h with ionomycin (750 ng/ml) and PMA (50 ng/ml). Dead cells were removed by a kit (catalog no. 130-090-101; Miltenyi Biotec) to avoid nonspecific staining. IL-10-producing CD8 T cells were identified by a cytokine capture assay (catalog no. 130-090-489; Miltenyi Biotec) combined with surface staining with the allophycocyanin-conjugated anti-CD8 mAb 53-6.7 (catalog no. 553035; BD Biosciences).

Cytokine detection by ELISA

Cytokines in supernatants were detected by a conventional double-sandwich ELISA. The following mAbs (from BD Biosciences) were used for detection and capture: mAb R4-6A2 (catalog no. 551216) and biotinylated mAb XMG1.2 (catalog no. 554410) for IFN-, mAb JES6-1A12 (catalog no. 554424) and biotinylated mAb JES6-5H4 (catalog no. 554426) for IL-2, mAb 11B11 (catalog no. 554434), and biotinylated mAb BVD6-24G2 (catalog no. 554390) for IL-4. IL-10 was detected by the OptEIA mouse IL-10 ELISA set (catalog no. 555252; BD Biosciences). Extinction was measured at 405/490 nm on a TECAN microplate-ELISA reader (Tecan) using EasyWin software (Tecan).

Immunization of mice with ligand-presenting HC

Splenic CD8 T cells were purified from OT-I mice by MACS, labeled with CFSE, and injected i.p. into B6 mice (8 x 10⁶ cells/mouse). HC (2 x 10⁵ cells/mouse) were injected s.c. into the left lateral flank of these animals 24 h later. HC were either nonpulsed or pulsed with GalCer, the peptide SIINFEKL, or both GalCer and SIINFEKL. Cells from the regional inguinal lymph nodes (LN) were isolated 5 days later, incubated for 4 h with ionomycin (750 ng/ml) and PMA (50 ng/ml) in the presence of BFA, washed, surface stained with anti-CD8 mAb and anti-CD69 mAb, washed again and subjected to an intracellular stain with an anti-IL-10 mAb. The fraction of IL-10-producing CFSE⁺CD69⁺ CD8 T cells was determined by flow cytometry.

	Results

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NKT cells from different compartments of the mouse liver

CD1d:Ig dimers loaded with GalCer detect murine CD1d-restricted NKT cells with an invariant TCR (5). We used this reagent to enumerate NKT cells in the (peri)sinusoidal and periportal compartments as well as the (celiac) LN of the liver from normal B6 mice. Cells in the liver perfusate originate from the (peri)sinusoidal compartment but also contain peripheral blood cells. The number of NKT cells in the liver perfusate was high (20% of all CD3 T cells) and was 4-fold higher than the number of NKT cells in peripheral blood (Fig. 1 and Table I). Cells from the periportal compartment of the liver were prepared by in vitro collagenase digestion followed by isolation of the NPC population. Almost 50% of the T cells in the periportal compartment of the liver were NKT cells (Fig. 1). Only a few NKT cells seem to leave the liver through lymphatics, as <1% of the T cells of the celiac LN (the exclusive site of lymphatic drainage of this organ) were NKT cells (Fig. 1). Though low, NKT cell numbers in celiac LN were always higher than those in inguinal or popliteal LN (Table I). NKT cells are thus frequent in (peri)sinusoidal and periportal T cell populations of the liver but not in the spleen or LN.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. NKT cells in the (peri)sinusoidal and periportal compartments and celiac LN of the liver and in peripheral blood, spleen, and inguinal LNs from wild-type B6 mice identified by GalCer-loaded CD1d:IgG dimers (DimerX). Cells were gated on CD3 expression. Data from a representative mouse of three individual mice were analyzed.

View this table: [in this window] [in a new window]   Table I. NKT cells in the (peri)sinusoidal and periportal compartments and celiac LN of the liver and in the peripheral blood, spleen, and inguinal LN from wild-type B6 mice identified by GalCer-loaded CD1d:IgG dimers (DimerX)a

The surface phenotype of hepatic NKT cells

Most hepatic CD1d:Ig dimer⁺ NKT cells from the (peri)sinusoidal and periportal compartments were activated (CD69⁺CD44⁺) and expressed the 95-kDa -chain (CD18) of LFA-1, an integrin receptor that binds ICAM-1 (CD54) and ICAM-2 (CD102) (Fig. 2A and data not shown). Of the CD28 receptor family, NKT cells express CD28, ICOS, and BTLA but not CTLA-4 or programmed death 1 (PD-1). The expression of members of the B7 family of costimulatory/coinhibitory molecules includes that of PD-L1 and CD86 but not that of ICOSL, PD-L2 or B7-H4 (Fig. 2A and data not shown). No (or only low) expression of the IL-2R-chain (CD25) but readily detectable expression of the IL-2R-chain (CD122) supports the notion that NKT cell homeostasis depends on IL-15 but not IL-2 (35). Hepatic NKT cells thus express an activated surface phenotype (CD69⁺CD44⁺CD122⁺) and a distinct subset of costimulatory and coinhibitory molecules.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. A, Surface phenotype of liver NKT cells. Liver NPC were isolated from wild-type B6 mice and surface stained with DimerX and FITC-, PE-, or biotin-conjugated mAbs to the indicated markers and appropriate isotype control mAbs. Data from a representative mouse of three individual mice were analyzed. B, CD1d and MHC-I (Kb) expression was tested on purified NKT cells and OT-I CD8 T cells. Liver NKT cells were isolated from wild-type B6 mice. CD8 T cells were isolated from the spleen of OT-I mice by MACS and OT-I CD8 T blasts were activated for 72 h with the SIINFEKL peptide. Purified (DimerX+CD4+) NKT cells and OT-I CD8 T cells (CD3+CD8+) were surface stained with anti-CD1d mAb or anti-Kb mAb. Data from a representative of three independent experiments are shown.

Activation of NKT cells or naive CD8 T cells by HC

NKT cells were sorted from the liver NPC population of normal or A^–/– B6 mice. Naive CD8 T cells were isolated from the spleen of OT-I B6 mice (that express a transgene-encoded TCR specific for the OVA epitope SIINFEKL in the context of K^b). Flow cytometry analyses of this T cell population confirmed that its members contained either 88–92% CD1d:Ig dimer⁺ NKT cells or >98% CD3⁺CD8⁺ (CD69^–CD44^lowCD25^–ICOS^–) T cells. NKT cells cocultured for 48–96 h with (1, 10, or 100 ng/ml) GalCer-pulsed HC produced mainly IL-4, some IFN-, but no IL-10 (Fig. 3 and data not shown) confirming our previous report (24). CFSE-labeled NKT cells did not proliferate in these cocultures (data not shown). Only few NKT cells (<5% of the input numbers) were recovered from these cocultures after a 24- to 48-h incubation period. In contrast, CFSE-labeled naive OT-I CD8 T cells cocultured with peptide-pulsed HC proliferated and showed a dose-dependent release of IFN-, TNF, and IL-2, but no production of IL-4 or IL-10 (Fig. 3 and data not shown). Thus, HC prime class I-restricted (proliferative and cytokine), Th1-biased responses of CD8 T cells and CD1d-restricted, balanced (IFN-/IL-4) cytokine responses of NKT cells.

View larger version (5K): [in this window] [in a new window]   FIGURE 3. Specific NKT cell and CD8 T cell activation by pulsed HC. NKT cells were isolated from livers of A–/– mice by the CD4 T cell MACS isolation technique. CD8 T cells were isolated from the spleen of OT-I mice by MACS. Purified NKT cells (4 x 104/well) or OT-I CD8 T cells (105/well) were cocultured with HC (104/well) pulsed with either the SIINFEKL peptide or GalCer. IFN- and IL-4 in supernatants collected at the indicated time points were determined by ELISA. Mean values of triplicates (±SEM) of a representative of three independent experiments are shown.

Purified hepatic NKT cells were cocultured with purified, naive (OT-I) CD8 T cells. NKT cells were pulsed with antigenic peptide or CD8 T cells were pulsed with GalCer (Fig. 4A). Pulsed NKT were cocultured with nonpulsed CD8 T cells (group 1), nonpulsed NKT cells were cocultured with pulsed CD8 T cells (group 2), pulsed NKT cells were cocultured with pulsed CD8 T cells (group 3), and nonpulsed NKT cells were cocultured with nonpulsed CD8 T cells (control group 4). Peptide-pulsed NKT cells primed a response of naive OT-I CD8 T cells in vitro (group 1). NKT cell-primed CD8 T cells proliferated and produced low amounts of IFN- and some IL-2 but no IL-4 or IL-10 (Fig. 4B). GalCer-pulsed naive CD8 T cells triggered neither a proliferative nor a cytokine response of cocultured NKT cells or T cells (group 2). Glycolipid-presenting naive CD8 T cells thus cannot activate an NKT cell response. A coculture of GalCer-pulsed naive CD8 T cells with peptide-pulsed NKT cells induced a 5-fold higher IFN- and a 2- to 3-fold higher IL-2 response, no IL-4 or IL-10 response, but CD8 T cell proliferation (group 3). Hence, the response of naive CD8 T cells triggered by peptide-presenting NKT cells was enhanced when the peptide-presenting NKT cells simultaneously recognized their cogent glycolipid Ag presented by the responding CD8 T cell. We do not know what fraction of the IFN- and IL-2 detected in the supernatant by ELISA is derived from CD8 T cells or NKT cells. Activated OT-I CD8 T blasts were poor stimulators or responders in specific interactions with NKT cells because they produced only low amounts of IFN- and no IL-2, IL-4, or IL-10 when specifically restimulated by peptide-pulsed NKT cells (data not shown). NKT cells can thus prime naive CD8 T cells by presenting its class I-restricted peptide. This priming is strikingly enhanced when the peptide-presenting NKT cell recognizes its CD1d-restricted glycolipid ligand on the surface of the responding CD8 T cells.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. Specific interaction between NKT cells and naive OT-I CD8 T cells. A, Specific NKT cell/OT-I CD8 T cell interactions. The arrows point from the recognizing cells to the ligand-presenting cells. In group 1, the NKT cells were pulsed for 2 h with the OVA-derived peptide SIINFEKL (p) (1 µg/ml/106 cells) and CD8 T cells were not pulsed (–). In group 2, the T cells were pulsed for 2 h with GalCer (GC) (100 ng/ml/106cells) and the NKT cells were not pulsed (–). In group 3, the NKT cells were pulsed for 2 h with the SIINFEKL peptide (p) and CD8 T cells were pulsed for 2 h with GalCer (GC). B, Pulsed (with SIINFEKL peptide (p) or GalCer (GC)) or nonpulsed NKT cells (4 x 104/well) and naive OT-I CD8 T cells (105/well) were cocultured for 48 h, and IFN-, IL-2, IL-10, and IL-4 were determined in the supernatants by ELISA. Mean values of triplicates (±SEM) of one representative experiment of three are shown. C, Specific proliferative responses of CD8 T cells primed by NKT cells. CFSE-labeled OT-I CD8 T cells (105/well) (pulsed or not pulsed with GalCer) were cocultured for 72 h with SINFEKL peptide-pulsed or nonpulsed NKT cells (4 x 104/well). Cells were harvested and stained with anti-CD8 mAb. Open columns represent the CFSE profile from precultured OT-I CD8 T cells; filled columns represent the CFSE profile from cultured OT-I CD8 T cells. Representative CFSE profiles are shown.

We showed that first, NKT cells and naive CD8 T cells specifically interact with HC (Fig. 3), and second, that NKT cells and naive CD8 T cells specifically interact with each other (Fig. 4). NKT cells and CD8 T cells are in intimate contact with HC in the perisinusoidal space. In the next set of experiments, we analyzed in vitro the specific interactions of NKT cells, naive CD8 T cells, and HC. We asked whether NKT cells modulate the priming of naive CD8 T cells by HC (Fig. 5A). We cocultured naive CD8 T cells and NKT cells with HC that were pulsed with peptide, GalCer, or peptide plus GalCer (groups 1–3, respectively). Alternatively, we cocultured nonpulsed, naive CD8 T cells with peptide-pulsed NKT cells and GalCer-pulsed HC (group 4).

View larger version (21K): [in this window] [in a new window]   FIGURE 5. NKT cell help for specific CD8 T cell priming in the liver. A, Specific interactions between NKT cells, HC, and OT-I CD8 T cells. The arrows point from the recognizing cells to the ligand-presenting cell. In group 1 HC were pulsed for 2 h with GalCer (GC) (100 ng/ml in 106 cells). In group 2 HC were pulsed for 2 h with the SIINFEKL peptide (p) (1 µg/ml/106 cells). In group 3 HC were pulsed for 2 h with GalCer (GC) and with the SIINFEKL peptide (p). In group 4 HC pulsed for 2 h with GalCer (GC) and NKT cells were pulsed with the SIINFEKL peptide (p). OT-I CD8 T cells were not pulsed. B, Pulsed (SIINFEKL peptide (p) or GalCer (GC)) or nonpulsed (–) NKT cells (4 x 104/well) and HC (104/well) were cocultured with naive OT-I CD8 T cells (105/well). IFN-, IL-2, IL-10, and IL-4 were determined in 48-h supernatants by ELISA. Mean values of triplicates (±SEM) of a representative of four independent experiments are shown. C, Nonpulsed (–) OT-I CD8 T cells and nonpulsed (–) NKT cells were cocultured with HC that were pulsed with GalCer (GC) (group 1), SIINFEKL peptide (p) (group 2), or GalCer plus SIINFEKL peptide (GC/p) (group 3). In groups 1–3, pulsed HC (HC1) were mixed with nonpulsed HC (HC2) at a ratio of 1:1. In group 4, GalCer-pulsed HC (HC1) and SIINFEKL peptide-pulsed HC (HC2) were mixed at a ratio of 1:1. IFN-, IL-2, IL-10, and IL-4 were determined in 48-h supernatants by ELISA. Mean values of triplicates (±SEM) of a representative of five independent experiments are shown. D, HC were isolated from either normal or CD1d–/– knockout B6 mice. Nonpulsed OT-I CD8 T cells (105/well) were cocultured with nonpulsed NKT cells (–) and GalCer/SIINFEKL peptide-pulsed (GC/pep) CD1d–/– (group 1) or CD1d+/+ (group 3) HC or nonpulsed (–) or SIINFEKL peptide-pulsed NKT cells (p) and GalCer-pulsed (GC) CD1d–/– (group 2) or CD1d+/+ (group 4) HC. IL-10 and IL-4 in 48-h supernatants were determined by ELISA. Mean values of triplicates (±SEM) of one representative of two independent experiments are shown. OT-I CD8 T cells purified from either the liver NPC population (group 1) or the spleen NPC population (group 2) were cocultured with nonpulsed (–) NKT cells and GalCer-/SIINFEKL peptide-pulsed HC. The IL-10 in 48-h supernatants was determined by ELISA. Mean values of triplicates (±SEM) of a representative of two independent experiments are shown. E, NKT cells (4 x 104/well), OT-I CD8 T cells (105/well), and HC (104/well) were cocultured for 48 h with or without Con A (2 µg/ml). IFN-, IL-2, IL-10, and IL-4 in 48-h supernatants were determined by ELISA. Mean values of triplicates (±SEM) of one representative of four independent experiments are shown.

The IL-10 and IL-4 response was triggered only when the same HC presented simultaneously the antigenic peptide and the glycolipid ligand to naive CD8 T cells and NKT cells, because mixing peptide-pulsed and glycolipid-pulsed HC in the coculture (instead of pulsing HC with both ligands) did not elicit this cytokine response (Fig. 5C). The GalCer-pulsed HC had to express CD1d to prime the immunomodulating effect of NKT cells on cocultured naive CD8 T cells (Fig. 5D). This finding stresses the importance of cell contact between CD8 T and NKT responder cells interacting with the same APC. This was further supported by the observation that the effect of NKT cell coactivation on CD8 T cell priming by HC could not be replaced by adding medium conditioned by NKT cells specifically stimulated by glycolipid-pulsed HC to the CD8 T cell/pulsed HC priming cocultures (data not shown).

Splenic and hepatic OT-I CD8 T cells showed a similar IL-10 response when coactivated with NKT cells by ligand-presenting HC (Fig. 5D). The coculture of HC pulsed with the antigenic peptide SIINFEKL and/or the glycolipid GalCer with liver NKT cells and OT-I RAG1^–/– CD8 T cells purified from either the spleen or the liver NPC population induced a comparable IL-10 response. We thus found no evidence for a tissue-specific phenotype of CD8 T cells.

The IL-10/IL-4 response was also triggered by coculturing naive CD8 T cells, NKT cells, and HC with mitogenic concentrations of Con A (Fig. 5E). The polyclonal activation of both T cell subsets by a lectin can thus bypass the requirement for the specific coactivation of interacting NKT cells, CD8 T cells with HC. In the next set of experiments we elucidated the source of IL-10 in the cocultures.

HC-primed CD8 T cells are a major source for IL-10

Labeled and stimulated NKT cells are almost completely lost from the cocultures after a 24-h incubation. These cells may contribute to an early IL-10 response, although we did not detect IL-10 when purified NKT cells were stimulated by glycolipid-pulsed HC. In contrast, 10–15% of the CD8 T cells primed in the CD8 T cell/NKT cell/HC cocultures (and restimulated with PMA/ionomycin) produced IL-10 as shown at the single cell level by either intracellular cytokine staining or an IL-10 capture assay (Fig. 6A and data not shown). Furthermore, CD8 T cells primed in CD8 T cell/NKT cell/HC cocultures were purified from these cultures at day 3 of incubation. When these activated CD8 T blasts were restimulated in vitro with peptide-pulsed DC for 2 days they produced IL-10 only when primed by peptide-pulsed HC in the presence of NKT cells (Fig. 6B). Hence, the described type of NKT cell "help" facilitates the specific priming of IL-10-producing CD8 T cells by HC.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. The cytokine response of OT-I CD8 T cells primed by HC with or without NKT cell help. A, OT-I CD8 T cells (105/well) were cocultured with GalCer- and SIINFEKL peptide-pulsed HC (104/well) with (+) or without (–) NKT cells (4 x 104/well). Cells were harvested after a 72-h culture, incubated for 4 h with ionomycin (750 ng/ml) and PMA (50 ng/ml), washed, and tested for IL-10 secretion in a 3-h capture assay. Cells were labeled with a PE-conjugated anti-IL-10 mAb and an anti-CD8 mAb. Data from a representative of five independent experiments are shown. B, OT-I CD8 T cells (105/well) were cocultured for 72 h with GalCer-/SIINFEKL peptide-pulsed HC (104/well) with (+) or without (–) NKT cells (4 x 104/well). CD8 T blasts were harvested, purified, and restimulated in vitro for 48 h with peptide-pulsed DC. IFN-, IL-2, and IL-10 in 48-h supernatants of these secondary cultures were determined by ELISA. Mean values of triplicates (±SEM) of a representative of four independent experiments are shown.

NKT cell-derived IFN- facilitates priming of IL-10-producing CD8 T cells by HC

We tested whether type I IFNs play a role in generating IL-10-producing CD8 T cells in the liver. CD8 T cells were cocultured with peptide/GalCer-pulsed (normal or IFNAR^–/–) HC and (normal, IFN-^–/– or IFNAR^–/–) NKT cells (Fig. 7). As shown, NKT cell coactivation was required to prime IL-10-producing CD8 T cells (group 1 vs group 2). IFN- expression (group 4) but not IFN type I responsiveness (IFNAR expression) by the coactivated NKT cells promoted the IL-10 response (group 3). IFNAR expression by presenting HC was not required to prime IL-10-producing CD8 T cells (group 5). Recombinant IFN- added to CD8 T cell/peptide-pulsed HC cocultures (without NKT cells) partially reconstituted the IL-10 response (group 1 vs group 6). Hence, NKT cell-derived type I IFN facilitates IL-10 expression by HC-primed CD8 T cells. This has to be assumed to operate with very low concentrations of IFN- because: 1) we did not detect IFN- in supernatants of NKT cell/GalCer-pulsed HC cocultures; 2) we could not replace NKT cell help with supernatants from such cocultures; and 3) we demonstrated above the requirement for intimate cell contact to elicit this response.

View larger version (3K): [in this window] [in a new window]   FIGURE 7. NKT cell help that facilitates IL-10 expression by HC-primed CD8 T cells depends on IFN-. OT-I CD8 T cells (105/well) were cocultured with or without NKT cells (4 x 104/well) with GalCer- and SIINFEKL peptide-pulsed HC (104/well). NKT cells were isolated from either wild-type (wt) B6 mice (groups 2 and 5) mice, IFNAR–/– B6 mice (group 3), or IFN-–/– B6 mice (group: 4). These NKT cells were cocultured with HC from either wild-type B6 mice (groups 2–4 and 6) or IFNAR–/– B6 mice (group 5). In group 6, OT-I CD8 T cells (105/well) were cocultured without NKT cells with SIINFEKL peptide-pulsed HC (104/well) in the presence of 103 IU/ml murine IFN-. IL-10 was determined in 48-h supernatants by ELISA. Mean values of triplicates (±SEM) of a representative of three independent experiments are shown.

CD8 T cells primed by HC in the presence of NKT cell coactivation produce IL-10 but do not express Foxp3 (data not shown). Using in vitro coculture experiments we tested whether these CD8 T cells suppress the specific priming of naive CD4 or CD8 T cells by peptide-pulsed DC (Fig. 8). Naive CD4 (OT-II) or CD8 (OT-I) responder T cells were mixed (at a 1:1 ratio) with CD8 T blasts primed by peptide-pulsed HC either with or without activated NKT cells. These T cell mixtures were specifically restimulated by peptide-pulsed DC. In contrast to CD8 T blasts primed by HC without NKT cell help, specifically activated CD8 T blasts primed by HC with NKT cell help induced an IL-10 response and down-regulated the IL-2 response in cultures in which either naive CD4 or naive CD8 T cells were primed (Fig. 8, A and B). The proliferative response of naive (CFSE-labeled) T cells primed in the presence of IL-10-producing CD8 T blasts was reduced (Fig. 8B). IL-10-producing CD8 T cells primed by HC with NKT cell help (but not CD8 T blasts primed by HC without NKT cell help) thus down-modulate the specific priming of naive T cells.

View larger version (6K): [in this window] [in a new window]   FIGURE 8. Priming of naive CD4 or CD8 T cells is suppressed by CD8 T cells primed by HC with NKT cell help. A and B, OT-I CD8 T cells (105/well) and GalCer-/SIINFEKL-pulsed HC (104/well) were cocultured for 72 h with (filled symbols) or without (open symbols) NKT cells (4 x 104/well). CD8 T blasts (3 x 104/well) purified from these cultures by MACS were cocultured with (Kb-binding) SIINFEKL or (Ab-binding) ISQAVHAAHAEINEAGR peptide-pulsed DC (3 x 103/well) and either naive OT-I CD8 T cells (squares) (3 x 104/well) or naive OT-II CD4 T cells (circles) (3 x 104/well). IL-10 and IL-2 in supernatants collected from the cocultures at the indicated time points were determined by ELISA. Mean values of triplicates (±SEM) of one representative of four independent experiments are shown. Furthermore, naive OT-I CD8 T cells were stained with CFSE, cocultured with DC and CD8 T blasts (activated by HC in the presence or absence of activated NKT cells), surface stained with anti-CD8 mAb and anti-CD3 mAb, and their CFSE dilution profile was assessed. The CFSE profiles of DC-primed OT-I CD8 T cells cocultured with CD8 T blasts stimulated by HC with (filled column) or without (open column) activated NKT cells are shown. Data from a representative of two independent experiments are shown.

We tested whether HC pulsed with the antigenic peptide SIINFEKL can prime a specific response of IL-10-producing CD8 T cells in vivo if NKT cells are coactivated. CFSE-labeled OT-I CD8 T cells were adoptively transferred into normal B6 mice. These animals were challenged 24 h later by a s.c. injection of syngeneic HC pulsed with the antigenic peptide and/or the glycolipid GalCer. Five days postimmunization, the specific response of the OT-I CD8 T cells was read out in the regional LN. Cells from these LN were restimulated in vitro for 4 h with PMA and ionomycin followed by (CD69 and CD8) surface and (IL-10) intracellular staining (Fig. 9). HC that were not pulsed (group 2), pulsed only with GalCer-pulsed (group 3), or pulsed only with the antigenic peptide (group 4) induced only a low response of IL-10-producing CD8 T cells. In contrast, HC pulsed with both the SIINFEKL peptide and the GalCer primed a response in which 15% of all specifically activated CFSE⁺CD69⁺ OT-I CD8 T cells produced IL-10 (group 5). These in vivo data are similar to the in vitro data (compare Figs. 6A and 9). They support the notion that HC-activated NKT cells facilitate the generation of IL-10-producing CD8 T cells.

View larger version (3K): [in this window] [in a new window]   FIGURE 9. Priming of IL-10-expressing CD8 T cells by peptide-presenting HC in vivo. Splenic OT-I CD8 T cells (purified by MACS) were labeled with CFSE and injected i.p. into syngeneic B6 mice (8 x 106 cells/mouse). Twenty-four hours later, HC were injected s.c. into the left flank of these mice (2 x 105 cells/mouse). HC were nonpulsed (group 2), pulsed with GalCer (group 3), pulsed with the SIINFEKL peptide (group 4), or pulsed with both SIINFEKL peptide and GalCer (group 5) (p, SIINFEKL peptide; GC, GalCer). A control group that was transplanted with T cells but not injected with HC was also included (group 1). Five days postimmunization, the CD8 T cell response was assessed in the regional (inguinal) LN. LN cells were incubated for 4 h with ionomycin (750 ng/ml) and PMA (50 ng/ml) in the presence of brefeldin A, washed, surface stained with an anti-CD8 and an anti-CD69 Ab, washed, and submitted to intracellular staining for IL-10. The fraction (%) of the activated CFSE+CD69+ OT-I CD8 T cells that express IL-10 after short-term in vitro stimulation is shown. Data from a representative of two independent experiments are shown.

	Discussion

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The identification of NKT cells has been greatly facilitated by the availability of glycolipid-loaded, soluble CD1d:Ig dimers or CD1d tetramers (4, 5, 36). This detection system identifies NKT cells that recognize GalCer in the context of CD1d through the invariant V14/J18 TCR -chain preferentially paired with a V8.2-containing TCR -chain. It misses NKT cells that recognize glycolipids other than GalCer and/or are not restricted by CD1d. Using the DimerX readout we may therefore underestimate the number of NKT cells.

In contrast to other tissues, NKT cells represent a major component of the liver T cell population. NKT cells were found in the (peri)sinusoidal and periportal compartments but not in the celiac LN of the liver. This suggests that this T cell subset is a resident cell population with limited recirculation potential. Once exported from the thymus, NKT cells express an activated/memory surface phenotype and undergo continuous (IL-15-dependent but TCR/CD1d-independent) turnover (35, 37). Freshly isolated liver NKT cells expressed the costimulator molecules CD28 and ICOS but not the coinhibitor molecules PD-1 and CTLA-4 on the surface. The only coinhibitory molecule we identified on the NKT cell surface was the high constitutive expression of BTLA. This is of interest, because HC express the BTLA ligand for the herpes virus entry mediator (38). Almost all NKT cell express on the surface the B7 family members CD86 and PD-L1 but not other molecules of this family (PD-L2, ICOSL, and B7-H4). The surface phenotype of NKT cells from the (peri)sinusoidal and periportal compartments was similar. Costimulatory signals modulate the cytokine profile expressed by NKT cells in response to specific activation. Blocking CD28-CD80/CD86 costimulation during activation of NKT cells by GalCer-pulsed DC suppresses their IFN- and IL-4 production (17). In contrast, blocking CD40-CD154 interactions inhibits GalCer-induced IFN- production but not IL4 production by NKT cells (17). CD28-deficient mice show impaired IFN- and IL-4 production in response to GalCer stimulation in vitro and in vivo, whereas production of IFN- but not IL-4 is impaired in CD40-deficient mice (17). Both (CD28-CD80/CD86 and CD40-CD154) costimulatory pathways are not expected to operate in the stimulation of NKT cells by HC. NKT cell-derived IL-10 has been reported to stimulate the development of regulatory cells that mediate systemic tolerance (39, 40), but we could not detect IL-10 production by HC-stimulated NKT cells.

NKT cells are positively selected in the thymus where CD1d-expressing, double-positive (CD4 CD8) T cells present high avidity, self-agonistic glycolipid ligands to NKT cell precursors through a T-T cell interaction (41). We show that NKT cells interact with CD1d-expressing naive T cells in the periphery and influence their activation. Similar to MHC class I molecules, the MHC class-I-like molecule CD1d shows a broad tissue distribution (42). CD1d is expressed by professional APC (e.g., DC and macrophages) but also a wide range of nonprofessional APC (e.g., epithelia, HC, NK cells, and T cells). This allows NKT cells to specifically interact with a broad range of cells that can capture or produce a glycolipid ligand for CD1d. NKT cells could thereby influence the behavior of cell types of the specific and innate immune system, the parenchymal organs, or the epithelial barriers. We asked whether the specific interaction of NKT cells with either HC or naive CD8 T cells can modulate a CD8 T cell response. NKT cells primed naive CD8 T cells but were poorly activated by naive CD8 T cells presenting glycolipids. A bidirectional specific interaction between NKT and CD8 T cells was a more efficient way to prime a CD8 T cell response. But these interactions did not elicit the immuno modulatory (IL-10-producing) phenotype of CD8 T cells. The induction of this phenotype required NKT cell-derived type I IFN, close contact between NKT cells, CD8 T cells, and HC, and specific coactivation of CD8 T cells and NKT cells. We reported that type I IFN negatively regulates CD8 T cell responses through IL-10-producing CD4 T regulatory 1 cells (43). In the present study we demonstrate that NKT cell-derived IFN- directly acts on CD8 T cells to induce IL-10 expression during their priming by peptide-presenting HC. These data are of interest for the widespread use of type I IFNs in the treatment of chronic infection with hepatotropic viruses that may impair the reconstitution of local, antiviral CD8 T cell immunity.

The CD8 T cells that we describe may contribute to the tolerogenic milieu in the liver and/or may be hepatoprotective. IL-10 is an immunosuppressive cytokine that down-modulates the proinflammatory responses of the innate and specific immune system. IL-10 down-modulates the APC function of professional or nonprofessional APCs, including the sinusoidal endothelial cells of the liver (44). But IL-10 is also a growth cofactor for mature and immature T cells (45, 46, 47), drives CD8 T cell differentiation (48), induces antitumor reactivity of CD4 T cells (49) and chemoattracts CD8 T cells (50). It enhances the IL-18-stimulated IFN- production, proliferation, and cytotoxicity of NK cells (51, 52). IL-10-expressing CD8 T cells have been described. Natural CD8 regulatory T cells have been identified in rats that are CD45RC^low, produce IL-4, IL-10, and IL-13 after stimulation, and express Foxp3 and CTLA-4 but are not cytotoxic. These CD8 T_R cells suppress in vitro and in vivo Th1 CD4 T cell responses (53). The phenotype of these CD8 T_R cells differs from the Foxp3^–IL-10⁺ CD8 T cells that we describe in this study. IL-10 treatment enhances CD8 T cell priming but decreases clonal expansion during the secondary responses to a challenge (54). IL-10⁺ CD8 T cells appear late in AIDS and may play a role in HIV-associated immune dysfunction (55). The limited published evidence available thus indicates that CD8 T cells can express IL-10 under certain conditions and that IL-10 has many different effects on T cell responses.

IL-10 has been shown to play a hepatoprotective role in different preclinical liver injury models including galactosamine/endotoxin- (56, 57), Con A- (58), CD8 T cell- (59), or tetrachloride/thioacetamide-induced (59, 60, 61) injury. The antifibrogenic effect of IL-10 seems to be key to this protective effect (62). The hepatoprotective role of IL-10 in Con A-induced hepatitis (58) is interesting in view of: 1) the obligatory dependence of this hepatitis on NKT cells (18); 2) the protective role of IL-10 in this model (58); and 3) our finding that Con A can generate IL-10-producing CD8 T cells in the NKT cell/CD8 T cell/HC cocultures. Further work is required to identify the costimulator/coinhibitor molecules that mediate the complex and specific interactions of the three different cell types (of which two are resident in the liver and one is at its earliest point of entry into this organ) and to shed light on its role in organ-specific immunopathology.

	Acknowledgment

 
We greatly appreciate the expert technical assistance of Ellen Allmendinger.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported by Deutsche Forschungsgemeinschaft (DFG) Grant Rei 549/10-2 (to J.R.).

² Address correspondence and reprint requests to Dr. Jörg Reimann, Department of Internal Medicine I, University of Ulm, Albert Einstein Allee 11, Ulm, Germany. E-mail address: joerg.reimann{at}uni-ulm.de

³ Abbreviations used in this paper: V14i, invariant V14; GalCer, -galactosyl-ceramide; BTLA, B and T lymphocyte attenuator; DC, dendritic cell; HC, hepatocyte; IFNAR1, IFN- receptor 1; int, intermediate; NPC, nonparenchymal cell; PD-1, programmed death 1; LN, lymph node(s).

Received for publication July 11, 2006. Accepted for publication December 1, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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