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The Journal of Immunology, 2001, 166: 42-50.
Copyright © 2001 by The American Association of Immunologists

NK T Cell-Derived IL-10 Is Essential for the Differentiation of Antigen-Specific T Regulatory Cells in Systemic Tolerance1

Koh-Hei Sonoda*, Douglas E. Faunce*, Masaru Taniguchi{dagger}, Mark Exley{ddagger}, Steven Balk{ddagger} and Joan Stein-Streilein2,*,§

* Schepens Eye Research Institute, Harvard Medical School, Boston MA 02114; {dagger} Core Research and Evolutional Science and Technology Project (CREST), and Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan; {ddagger} Cancer Biology Program, Hematology/Oncology Division, Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and § Pulmonary and Critical Care Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In a model of systemic tolerance called Anterior Chamber-Associated Immune Deviation (ACAID), the differentiation of the T regulatory (Tr) cells depends on NK T cells and occurs in the spleen. We now show that the CD1d-reactive NK T cell subpopulation, required for development of systemic tolerance, expresses the invariant V{alpha}14J{alpha}281 TCR because J{alpha}281 knockout (KO) mice were unable to generate Ag-specific Tr cells and ACAID. The mechanism for NK T cell-dependent differentiation of Ag-specific Tr cells mediating systemic tolerance was studied by defining the cytokine profiles in heterogeneous and enriched NK T spleen cells. In contrast to there being no differences in most regulatory cytokine mRNAs, both mRNA and protein for IL-10 were increased in splenic NK T cells of anterior chamber (a.c.)-inoculated mice. However, IL-10 mRNA was not increased in spleens after i.v. inoculation. Finally, NK T cells from wild-type (WT) mice, but not from IL-10 KO mice, reconstituted the ACAID inducing ability in J{alpha}281 KO mice. Thus, NK T cell-derived IL-10 is critical for the generation of the Ag-specific Tr cells and systemic tolerance induced to eye-inoculated Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tolerance to self or foreign Ags is an active process that is mediated by multiple mechanisms (1). Central tolerance in the thymus is promoted by negative selection or central deletion of a vast array of lymphocytes capable of reacting to self molecules. However, not all Ag-reactive cells are eliminated in this fashion, and some self-reactive lymphocytes remain to be regulated by development of anergy (2), apoptosis by Fas ligand (3), or active suppression involving regulatory cells that prevent either development or expression of effector mechanisms (4, 5). T regulatory (Tr)3 cells that suppress effector mechanisms are the outcome of Ag presented in a variety of organs and tissues but are studied here in a model of immune privilege in the eye, known as Anterior Chamber-Associated Immune Deviation (ACAID) (6, 7).

Immune privilege in the eye is attributed to various local factors including the lack of lymphatic drainage (8), Fas ligand expression (9), and multiple immunosuppressive factors in aqueous humor (10, 11, 12, 13). ACAID is characterized by a selective deficiency in delayed-type hypersensitivity (DTH) and Ig isotypes that fix complement (14, 15). Central to the ACAID process are intraocular bone marrow-derived F4/80+ APCs that capture Ag within the anterior chamber (a.c.) and carry an Ag-specific ACAID-inducing signal via the blood directly to the spleen (14, 16). The effector phase of the DTH response is negatively regulated by spleen-generated CD8+ T cells within 7 days of a.c. inoculation (17, 18). CD1d-reactive NK T cells are central to the development of the Ag-specific Tr cell (19).

NK T cells belong to a specialized population of lymphocytes that coexpress the TCR {alpha}{beta}-chain and NK markers (20). A major subpopulation of NK T cells express a unique invariant V{alpha}14J{alpha}281 Ag receptor not expressed by conventional T cells (20, 21, 22, 23, 24). Similarly, NK T cells exists in the human and express the invariant V{alpha}24J{alpha}Q TCR{alpha}-chain (25, 26). NK T cells are restricted by MHC class I-like CD1d molecules (27, 28, 29), and because the CD1d molecule also is required for the development of NK T cells, CD1d knockout (KO) mice selectively lack NK T cells (30, 31, 32). Moreover, the NK T cell must interact with the CD1d molecule because blocking the CD1d interaction with a CD1d-specific Ab, either in vivo (19) or in vitro (K.-H.S., unpublished observations) blocks the development of Ag-specific Tr cells.

Following the NK T cell/CD1d interaction, the precise mechanism used by NK T cells to influence the development of Tr cells in the ACAID model is largely unknown but could involve soluble factors or cell-to-cell contact. It is well known that CD1d-restricted activated NK T cells produce large amounts of a variety of cytokines within minutes of signals (33). Because it was reported that IL-4 KO mice developed ACAID after Ag inoculation (a.c.) but IL-10 KO mice did not (34, 35, 36), we predicted that IL-10 and not IL-4 would be important in the development of the Tr cells. In this report, we demonstrate that IL-10 derived from NK T cells is absolutely required for the induction of Ag-specific Tr cells following the inoculation of Ag into the eye.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female, 8- to 10-wk-old mice were used in all experiments. C57BL/6 (B6) mice were obtained from Taconic Farms (Germantown, NY). CD1 KO mice were generated in the Transgenic Facility, Harvard Medical School (Boston, MA). In brief, the CD1d (both CD1.1 and CD1.2) deletion was created in strain 129/Sv-derived embryonic stem cells. Mutant embryonic stem cell clones were injected into C57BL/6 blastocysts to obtain chimeric mice. Heterozygous mutant animals were intracrossed in brother sister mating to obtain (C57BL/6 x 129/Sv) F2 homozygous mutants. The CD1d mutation was backcrossed to the B6 parent for six generations (N6). Progeny that lacked the CD1 gene, as determined by DNA analyses, were chosen as breeders. NK T KO mice (J{alpha}281 KO mice) were generated at Chiba University (Chiba, Japan) (24) and backcrossed nine times to B6 mice (N9). IL-10 KO mice (C57BL/6-IL-10tm1Cgn) were purchased from The Jackson Laboratory (Bar Harbor, ME). The animals were maintained on food and water ad libitum until they reached the desired weight (20–24 g). All animals were treated humanely and in accordance with the Schepens Animal Care and Use Committee and National Institutes of Health guidelines.

Induction of immune deviation and assay for DTH

ACAID was induced in mice by inoculating OVA (50 µg/2 µl in HBSS; Sigma, St. Louis, MO) into the a.c. (14) 7 days before sensitizing s.c. for DTH. Immune deviation was i.v. induced by inoculation of the Ag (OVA; 50 µg/100 µl in HBSS) into the tail vein with a 30-gauge needle 7 days before immunizing for DTH. To induce DTH, mice received an s.c. inoculation with OVA (100 µg/ml in HBSS, 50 µl) emulsified in CFA (50 µl), and 1 week later were tested for the development of DTH by an intradermal inoculation of OVA-pulsed peritoneal exudate cells (PECs) (prepared as described below, 2 x 105/10 µl HBSS) into the right ear pinnae. Ear swelling was measured 24 and 48 h later with an engineer’s micrometer (Mitutoyo, Paramus, NJ).

Local adoptive transfer (LAT)

To test for the efferent regulatory cell of ACAID, a modified LAT assay was performed as described elsewhere (18). In brief, T (effector) cells were generated in B6 mice by immunizing (s.c.) with OVA in HBSS and CFA, and 7 days later the primed T cells were enriched from dissociated spleen cells by removing B cells and macrophages using IMMULAN columns (Biotecx Laboratories, Houston, TX). Regulator cells were similarly enriched on IMMUNLAN columns from spleen cells of ACAID mice 7 days post a.c. inoculation of OVA. Stimulator cells were OVA-pulsed PECs as described below. Effector (5 x 105), stimulator (5 x 105), and regulator (5 x 105) cells were mixed and resuspended in 10 µl HBSS for inoculation into the right ear pinnae of naive mice. Ear swelling was measured with an engineer’s micrometer at 24 and 48 h. As a negative control, naive T cells from unmanipulated mice were used as effector cells and regulator cells. Primed T cells were used as effector cells, and naive T cells from unmanipulated mice were used as regulator cells for positive control.

Preparation of OVA-pulsed PECs

PECs were obtained from peritoneal washes of B6 mice 3 days after they received an i.p. inoculation of 2.5 ml of 3% aged thioglycolate solution (Sigma). After counting, PECs were cultured with OVA (5 mg/ml) in a 24-well culture plate in serum-free medium (RPMI 1640 medium, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin (BioWhittaker, Walkersville, MD), and supplemented with 0.1% BSA (Sigma) and ITD+ culture supplement (1 µg/ml iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml Na2Se, and 0.2 µg/ml Fe(NO3)3) (Collaborative Biomedical Products, Bedford, MA). Nonadherent cells were removed from the cultures after 18 h by three washes, and the remaining adherent cells were collected by vigorous pipetting with cold medium (4°C) before washing (three times with HBSS) to remove free OVA.

IFN-{gamma} assay

T (effector) cells were generated in B6 mice by immunizing (s.c.) with OVA in HBSS and CFA and 7 days later the primed T cells were enriched from spleens on IMMUNLAN columns. Regulatory cells were similarly enriched from spleen cells of ACAID mice 7 days post a.c. inoculation of OVA. Stimulator cells were OVA-pulsed PECs. Effector (2 x 105), stimulator (4 x 104), and regulator (2 x 105) cells were mixed and cultured in 200 µl serum-free medium. Supernatant were collected after 72 h, and IFN-{gamma} concentration was measured by quantitative capture ELISA, according to the manufacturer’s instructions (PharMingen, San Diego, CA). In brief, ELISA plates (Nunc-Immuno plate; VWR Scientific Products, Bridgeport, NJ) were coated with rat anti-mouse IFN-{gamma} mAb (R4-6A2; PharMingen). Recombinant mouse IFN-{gamma} (PharMingen) was used to construct a standard curve, and biotinylated rat anti-mouse IFN-{gamma} mAb (XMG1.2; PharMingen) was used as the detecting Ab. Plates were treated with alkaline phosphatase-conjugated ExtrAvidin (Sigma), substrated color by p-nitrophenyl phosphate (Sigma), and OD was measured at 405 nm by a MRX Microplate reader (Synateck Laboratories, Chantilly, VA).

Antibodies

The Abs used for flow cytometry analysis were: Fc block (anti-mouse FcR{gamma} II/III mAb, 2.4G2), biotin, or FITC-conjugated anti-NK1.1 mAb (PK136) and Cy-Chrome 5-conjugated anti-TCR{beta} mAb (H57–597), all purchased from PharMingen. Streptavidin-PE was purchased from Jackson ImmunoResearch (West Grove, PA).

Flow cytometry

Splenic NK and NK T cells were analyzed by flow cytometry. RBC were lysed by adding Tris-buffered ammonium chloride to a cell pellet of spleen cells. Staining was performed in the presence of a saturated concentration of Fc block (blocks FcR{gamma} II/IIIs). Cells were stained with the following reagents and colors (using concentrations recommended by the manufacturer): biotin-conjugated anti-NK1.1 mAb counterstained with streptavidin-PE; and Cy-Chrome 5-conjugated anti-TCR{beta}-chain mAb. Stained cells were analyzed on an EPICS XL flow cytometer (Beckman Coulter, Miami, FL). The absolute number of splenic NK T cells detected in flow cytometry was calculated from the percentage of NK T cells in the number of viable cells. The total number of viable cells harvested from the spleens before staining was determined by the trypan blue exclusion method.

Intracellular staining of IL-10

To prevent the intracellular cytokine protein from being secreted, freshly isolated splenocytes were harvested from naive or a.c.-inoculated (7 days post) B6 mice and immediately placed in HBSS with 5 µg/ml brefeldin A (Sigma) (37). Cells were incubated with Cy-Chrome 5-conjugated anti-TCR{beta}-chain and biotin-conjugated anti-NK1.1 mAb counterstained with streptavidin-PE in HBSS supplemented with 5 µg/ml brefeldin A. Three hours after harvesting the cells, they were fixed and permeabilized with PermeaFix (Ortho Diagnostics, Raritan, NJ) and stained by FITC-conjugated anti-IL-10 mAb or FITC-conjugated isotype control Ab (IgG2b, clone name: R35-38) and then analyzed by flow cytometry.

NK T/NK cell enrichment

For NK T/NK cell enrichment, IMMULAN column-enriched splenic T cells were harvested from wild-type (WT) or IL-10 KO mice. Cells were treated with FITC-conjugated anti-NK1.1 mAb before magnetic bead selection. Ab-labeled cells were treated with anti-FITC MicroBeads (Miltenyi Biotec, Auburn, CA) for 15 min, and washed twice. To harvest NK/NK T cell-enriched cells, cells were applied to type MS+ positive selection column with MiniMACS (Miltenyi Biotec). Positively selected cells were stained with Cy-Chrome 5-conjugated anti-TCR{beta}-chain mAb, and enrichment was confirmed by flow cytometry. The cell numbers of enriched populations were adjusted to approximate the number used in the control studies. NK T cells were further enriched by sorting for cells expressing intermediate density of the TCR{beta}-chain and NK1.1 molecules by a fluorescence-activated cell sorter (EPICS Cell Sorter; Beckman Coulter).

RNase protection assay (RPA)

Total RNA was extracted from whole splenocytes and in vitro Con A-stimulated splenocytes using Trizol (Life Technologies, Grand Island, NY) according to the manufacturer’s guidelines. Five micrograms of total RNA was then subjected to RiboQuant Multiprobe RNase Protection Analysis (PharMingen) using a riboprobe encoding multiple cytokine genes (mck-1, mck-3b). The hybridization products were resolved on a polyacrylamide sequencing gel that was dried and subjected to phosphorimaging with the FX Molecular Imaging System (Bio-Rad, Richmond, CA).

RT-PCR

Total RNA was extracted from whole splenocytes and enriched NK T cells 7 days after a.c. inoculation using Trizol. To identify {beta}-actin, IL-4, and IL-10 mRNAs total RNA was reverse transcribed and amplified by the Access RT-PCR System (Promega, Madison, WI) according to the manufacturer’s guidelines. PCR products were electrophoresed on 1.8% agarose gel in the presence of 100,000x GelStar nucleic acid gel stain (FMC BioProducts, Rockland, ME). Bands were photographed and quantified by FX Molecular Imaging System (Bio-Rad). The amount of RNA in each sample was standardized by preliminary amplification for {beta}-actin, and readjusting the sample concentration according to densitometry reading of {beta}-actin bands, as described above. The adjusting systems were repeated until {beta}-actin bands were equalized in serially diluted samples.

Semiquantitative analysis of IL-10 mRNA was preformed by a competitive RT-PCR as described before (38, 39). In brief, the IL-10 competitor cDNA was synthesized from Con A-stimulated splenocytes by RT-PCR using IL-10 competitor sense and IL-10 antisense. The initial sample mRNA level was normalized by {beta}-actin bands. An equivalent amount of samples were coamplified with an added constant concentration of the competitor. The competitor and target shared the same IL-10 sense and antisense primers used for target amplification. Both target and competitor were coamplified with equal amplification efficiencies in the same PCR tube. After PCR products were visualized, the target/competitor ratio was calculated for semiquantification.

Primers used in these experiments are listed below. For amplification: {beta}-actin; sense 5'-GTG GGC CGC TCT AGG CAC CAA-3' and antisense 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3' (product size: 539), IL-4; sense 5'-ATG GGT CTC AAC CCC CAG CTA GT-3' and antisense 5'-GCT CTT TAG GCT TTC CAG GAA GTC-3' (product size: 398), IL-10; sense 5'-ACC TGG TAG AAG TGA TGC CCC AGG CA-3' and antisense 5'-CTA TGC AGT TGA TGA AGA TGT CAA A-3' (product size: 237 bp). For IL-10 competitor construct: IL-10 competitor sense; 5'-ACC TGG TAG AAG TGA TGC CCC AGG CAT GGG TGA GAA GCT GAA GA-3' and antisense 5'-CTA TGC AGT TGA TGA AGA TGT CAA A-3' (product size: 197 bp).

Reconstitution of J{alpha}281 KO mice

J{alpha}281 KO mice were {gamma}-irradiated (cesium, 200 rad, Mark 1 irradiator, J.L. Shepherd and Associates, Glendale, CA) 1 day before receiving 106 mouse NK T/NK-enriched cells from either WT or IL-10 KO mice by i.v. route. NK T/NK cells were enriched as described above. Twenty-four hours after reconstitution, reconstituted NK T KO mice were inoculated (a.c.) with OVA (50 µg/2 µl in HBSS). Spleens were removed 1 wk after the a.c. inoculation, dissociated cells were pooled, and splenic T cells were enriched as described above. Enriched splenic T cells were transferred to naive B6 mice as regulator cells with effector (derived from B6 mice) and stimulator cells (derived from B6 mice) and tested in a LAT assay.

Statistics

Data were analyzed for significant differences among experimental groups by ANOVA and Scheffe’s test. A value of p <= 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Correlation of IL-10 mRNA production with development of Ag-specific Tr cells in ACAID

After Ag is inoculated into the eye, the development of systemic tolerance, in general, and Ag-specific Tr cells, in particular, are dependent on NK T cells interacting with CD1d molecules (19). Thus, we proposed that the CD1d interaction stimulated the NK T cell to produce cytokines that were influential in the differentiation of T cells into regulatory cells. Knowing that we were able to detect increased numbers of NK T cells in the spleen following a.c. inoculations, we reasoned that we would be able to detect increases in cytokines related to the induction of systemic tolerance, as well. Using various kinds of KO mice deficient in NK T cells, we tested whether the unique cytokines were dependent on the presence of the NK T cells. Initially, total RNA from spleens from WT (B6) and CD1d KO mice was screened for differences in cytokine mRNA.

Seven days after OVA was inoculated (a.c.) into WT or CD1d KO mice, the mRNA level of various cytokines in total splenic RNA was compared by RPA using RiboQuant MultiProbe. There were no remarkable differences in TFN{alpha}, IFN-{gamma}, or TGF{beta} from WT and CD1d KO mice, and we were unable to detect any bands of IL-4 or IL-10 mRNA in either sample by this RPA (Fig. 1GoA). However, the RT-PCR product from splenic mRNA from a.c.-inoculated WT mice produced more IL-10 mRNA than a.c.-inoculated CD1d KO mice (Fig. 1GoB).



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  		FIGURE 1. Measurement of cytokine mRNA in spleens of a.c.-inoculated mice. A, RPA. CD1d KO or WT (B6) mice (n = 3) were inoculated (a.c.) with OVA, and 7 days later total RNA was extracted from their spleen cells. The levels of various cytokine mRNAs were compared by RPA using RiboQuant MultiProbe. Left, Representative data using two different probes (left: mck-3b probe, right: mck-1 probe). Right, Bar graph of each cytokine mRNA density (IFN-{gamma}, TNF-{alpha}, and TGF{beta}) divided by the density of the housekeeping gene (GAPDH). The experiment was repeated twice. B, RT-PCR. mRNA levels for IL-10 and IL-4 in the whole spleen cells harvested from a.c.-inoculated CD1d KO mice or WT (B6) mice were compared by RT-PCR. Left, Bands on the agarose gel for IL-10, IL-4, and {beta}-actin (indicated with arrows). Right, Bar graph of the ratio of densitometer readings for IL-10 mRNA divided by density of the housekeeping gene ({beta}-actin). C, Competitive RT-PCR comparing IL-10 mRNA levels in various experimental groups. One microgram of total RNA from each pooled sample (n = 3) was added to each tube containing a standardized amount of competitor for the same IL-10 sense and antisense primers and coamplified (35 cycles). The loading technique was verified by RT-PCR {beta}-actin in a duplicate sample. The real IL-10 (237 bp) bands and the competitor (197 bp) bands are indicated by arrows. Both real target (IL-10) and competitor band volumes were measured in each sample by densitometry, and the IL-10/competitor product ratio was calculated (bar graph). The experiment was repeated three times.

 
Both semiquantitative and competitive RT-PCR were used to determine relative quantities of the IL-10 mRNA (Fig. 1GoC). The IL-10 mRNA level in spleen cells from naive (nontreated) mice was compared with that of a.c.-inoculated WT and CD1d KO mice, respectively. Analysis of RNA from a.c.-inoculated WT mice, but not from a.c.-inoculated CD1d KO mice, clearly showed an increase in IL-10 mRNA compared with RNA from naive mice (Fig. 1GoC). These observations confirmed the importance of IL-10 in ACAID (35, 36).

Deficit in IL-10 production correlates with failure of J{alpha}281 KO mice to develop Ag-specific Tr cells post a.c. inoculation

The majority of CD1d-reactive NK T cells in the spleen are V{alpha}14+ NK T cells that express a single invariant TCR{alpha}-chain encoded by the V{alpha}14J{alpha}281 segment (21, 22, 23). J{alpha}281 KO mice show marked reduction in total NK T cells in multiple organs (24) and, while lacking V{alpha}14 NK T cells, express CD1d molecules. WT (B6) mice and J{alpha}281 KO mice were inoculated a.c. or i.v. with OVA 7 days before s.c. sensitization with OVA and CFA, and tested 14 days later for a DTH response by inoculating OVA-pulsed PECs into the ear pinnae. In contrast to WT mice that developed decreased DTH expression regardless of the route of inoculation, J{alpha}281 KO mice inoculated with OVA a.c. (but not i.v.) failed to show suppression of the DTH response (Fig. 2GoA).



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  		FIGURE 2. ACAID in J{alpha}281 KO mice. A, In vivo analyses of OVA DTH. Control WT (B6) mice and J{alpha}281 KO mice (n = 5) were inoculated a.c. or i.v. with OVA 7 days before sensitization (s.c.) with OVA and CFA. OVA-inoculated mice were challenged in their ear pinnae with OVA-pulsed PECs from WT mice 7 days after sensitization. Changes ({Delta}) in ear swelling measurements (24 h post ear challenge) are shown on the ordinate, and treatment of mice is indicated below the abscissa for each bar. Significant differences (p <= 0.05) are indicated by an asterisk (*). The experiment was repeated twice. B, In vivo analyses of the regulatory cell in LAT assay. A LAT assay was performed to directly assess the development of efferent Tr cells. Regulatory cells were column-enriched splenic T cells that were harvested from a.c.-inoculated WT (B6) or J{alpha}281 KO mice (n = 5) that received OVA (a.c.) 7 days previously. Regulatory cells were cotransferred into naive B6 mice (five per group) with effector and stimulator cells from B6 mice. Changes ({Delta}) in ear swelling measurements (24 h post ear challenge) are shown on the ordinate, and the mixture of cells inoculated into the ear pinnae is indicated below the abscissa. Significant differences (p <= 0.05) are indicated by an asterisk (*). The experiment was repeated three times. C, In vitro analyses of regulatory cells in suppression of IFN-{gamma} production. IFN-{gamma} concentration in experimental culture supernatant was measured by ELISA. T cells from a.c.-inoculated mice were cocultured with OVA-primed T cells and OVA-pulsed PECs (mimics cell mixture in LAT assay). Culture supernatants were collected 72 h later, and the IFN-{gamma} concentration was measured by ELISA. A significant difference (p <= 0.05) is indicated by an asterisk (*). The experiment was repeated twice.

 
A LAT assay was used to test whether the lack of DTH suppression correlated with a defect in the Ag-specific Tr cells (18). J{alpha}281 KO mice were inoculated (a.c.) with OVA 7 days before harvesting, dissociating, and enriching T cells from the spleens for use as regulator cells. Tr cells were then cotransferred with OVA-primed T cells (effector cells from B6 mice) and OVA-pulsed PECs (stimulator cells from B6 mice) into the ear of naive B6 mice. In contrast to T cells from WT mice, T cells from J{alpha}281 KO mice who received OVA (a.c.) were unable to suppress the DTH response (Fig. 2GoB).

To confirm that Tr cells were generated we developed an in vitro assay. Because IFN-{gamma} is a Th1 cytokine that correlates in part with an in vivo DTH response, we tested whether 1) the Tr cells in ACAID interfered with in vitro production of IFN-{gamma}; and 2) whether suppression of IFN-{gamma} of Tr cells was dependent on V{alpha}14 NK T cells. T cells from a.c.-inoculated mice were cocultured with OVA-primed T cells and OVA-pulsed PECs (mimics CPU mixture in LAT assay). Culture supernatants were collected 72 h later, and the concentration of IFN-{gamma} was measured by ELISA. In contrast to T cells from WT mice, T cells from OVA a.c.-inoculated J{alpha}281 KO mice failed to suppress IFN-{gamma} production (Fig. 2GoC). Thus Tr cells generated in ACAID suppress IFN-{gamma} production in vitro. These results confirmed expectations that V{alpha}14 NK T cells were needed for the generation of the Ag-specific efferent Tr cells in ACAID.

We also examined total RNA from spleen cells from naive, a.c.-inoculated WT, or J{alpha}281 KO mice for levels of IL-10 mRNA. We observed that WT mice inoculated (a.c.) with OVA had increased levels of splenic IL-10 mRNA compared with naive B6 mice or a.c. inoculated J{alpha}281 KO mice (Fig. 3Go). Thus, induction of systemic tolerance through the eye correlated with increased production of IL-10 mRNA in the spleen.



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  		FIGURE 3. Competitive RT-PCR measurement of IL-10 mRNA in a.c.-inoculated J{alpha}281 KO mice. IL-10 mRNA was measured in total RNA harvested from spleens of naive and a.c.-inoculated WT and J{alpha}281 KO mice (n = 3) by competitive PCR (see Fig. 1GoC). The real IL-10 band (237 bp) bands and the competitor (197 bp) bands are indicated by arrows in the upper panel. The IL-10 and competitor bands were measured by densitometer, and the IL-10/competitor product ratio is shown in the bar graph. The experiment was repeated three times.

 
Splenic IL-10 mRNA does not increase post i.v. OVA inoculation

Unlike a.c.-induced tolerance, i.v.-induced tolerance is not dependent on NK T cells or the generation of an efferent negative regulatory cell (19). However, like a.c.-induced tolerance, i.v. tolerance is dependent on the spleen for the generation of immune deviation and suppression of DTH. Thus, the possibility was raised that tolerance mechanisms in the spleen might be shared by these two routes of inoculation. Similar to CD1d KO mice, J{alpha}281 KO mice developed tolerance after i.v., but not a.c., inoculation (Fig. 2GoA) (19). When the quantity of splenic IL-10 mRNA was compared, a.c.-, and not i.v.-, inoculated mice showed an obvious increase in IL-10 mRNA (Fig. 4Go) compared with naive mice at day 7 after inoculation. Thus, the IL-10 mRNA increase in the spleen correlated with the a.c. route of inoculation of the Ag and the need for NK T cells.



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  		FIGURE 4. Competitive RT-PCR measurement of IL-10 mRNA in i.v. inoculated mice. The splenic IL-10 mRNA was measured in total RNA harvested from spleens of naive, a.c.-inoculated, and i.v. inoculated B6 mice (n = 3) by competitive RT-PCR (see Fig. 1GoC). The real IL-10 bands (237 bp) and the competitor (197 bp) bands are indicated by arrows in the upper panel. The IL-10 and competitor bands were measured by densitometry, and the IL-10/competitor product ratio is shown in the bar graph. The experiment was repeated a minimum of three times.

 
The role of NK T cells in the production of IL-10

To examine whether NK T cells themselves produced IL-10, highly enriched FACS-sorted NK T cells were prepared from spleen cells harvested from the various experimental groups (Fig. 5GoA). When the total RNA was harvested from each NK T cell sample and the level of IL-10 mRNA was determined and compared, we observed that IL-10 mRNA was markedly increased in FACS-sorted NK T cells from a.c.-inoculated mice (Fig. 5GoB) compared with naive mice, thus indicating that splenic NK T cells produced more IL-10 following a.c. inoculation.



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  		FIGURE 5. Competitive RT-PCR measurement of IL-10 mRNA in enriched splenic NK T cells. A, Flow cytometry confirmation of NK T cell enrichment. NK T cells were enriched (see Materials and Methods). In brief, spleen cells were harvested from naive or a.c.-inoculated WT (B6) mice (n = 5) 7 days after OVA (a.c.) inoculation. T, NK T, and NK cells were enriched by depletion of B cells and macrophages using IMMULAN columns. Cells were stained with FITC-conjugated anti-NK1.1 mAb and enriched with anti-FITC magnetic microbeads before staining for Cy-Chrome 5-conjugated anti-TCR{beta}-chain. NK T cells were sorted for their dual positive (intermediate density) or TCR{beta}-chain and NK1.1 by EPICS Cell Sorter. The percentage of NK T cells is indicated in the square within dot plots before and after sorting for purity. B, Competitive RT-PCR measurement of IL-10 mRNA in FACS sorted NK T cell population. Total RNA was coamplified with IL-10 competitor as described in Fig. 1GoC. Both real and competitor IL-10 products are indicated in the left panel. The ratio of densitometer reading of IL-10/competitor products is shown in bar graph in the right panel. The experiment was repeated twice.

 
To determine whether the IL-10 mRNA was translated into protein, the number of splenic NK T cells expressing intracellular IL-10 from naive and a.c.-inoculated B6 mice was determined by flow cytometry. Spleen cell populations were gated according to their cell surface expression of intermediate levels of the TCR{beta}-chain and NK1.1 molecules (Fig. 6Go, left) and were examined for their intracellular expression of IL-10. We observed that the numbers of NK T cells with intracellular IL-10 were increased in a.c.-inoculated mice compared with i.v.-inoculated or naive mice (Fig. 6Go, right).



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  		FIGURE 6. Intracellular staining of IL-10 in NK T cells. Seven days after treatment, freshly isolated splenocytes from naive, a.c.-, or i.v. inoculated B6 mice (n = 4) were stained by Cy-Chrome 5-conjugated anti-TCR{beta}-chain and biotin-conjugated anti-NK1.1 mAb counterstained with streptavidin-PE. The flow cytometry gate was selected for dually positive NK T cells. Dot plots for flow cytometry analyses of intracellular IL-10 are shown in the top left panel. The dot plots to the right show a representative experiment for intracellular staining of IL-10 NK T cells (top right panel). The bar graph shows a comparison of IL-10+ NK T cells in the spleen in respect to the experimental condition indicated on the abscissa. The ordinate indicates the percentage of IL-10-positive cells. A significant difference (p <= 0.05) is indicated by an asterisk (*). The experiment was repeated three times.

 
Adoptive transfer of NK T cells from IL-10 KO mice could not reconstitute ACAID in J{alpha}281 KO mice

Previously we showed that NK T cells (and CD1d+ splenic APCs) reconstituted the ability to induce systemic tolerance via the eye in CD1d-deficient mice (19). To determine whether NK T cell-derived IL-10 was needed for the generation of the Tr cells that mediate the systemic tolerance, we enriched NK/NK T cells (106) from spleens of IL-10 KO (or WT) mice to reconstitute the ACAID-inducing ability of the J{alpha}281-deficient animals (Fig. 7GoA). Enriched mixed populations of NK/NK T cells were used as a source of the NK T cell because NK cells are unable to restore ACAID (19). Moreover, there was no deficiency in the numbers of NK T cells harvested from IL-10 KO mice compared with WT mice (data not shown).



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  		FIGURE 7. Ability of NK T cells from IL-10 KO mice to reconstitute ACAID in J{alpha}281 KO mice. A, Flow cytometry analyses of NK/NK T cell enrichment. Column-enriched splenic T cells were harvested from naive WT (B6) mice or IL-10 KO mice (n = 5) and NK/NK T cells were enriched by magnetic microbeads (see Fig. 5GoA). NK/NK T-enriched cells were stained as before and analyzed by flow cytometry. The percentage of NK T cells (upper rectangle) and NK cells (lower rectangle) is shown in the dot plot to the right. B, LAT assessment of Tr cell generation in NK T cell (WT)-reconstituted J{alpha}281 KO mice. A LAT assay was performed as described in Materials and Methods. Regulatory cells were cotransferred with responder and stimulator cells into naive recipient mice (n = 5). Mixtures of cells injected into the ear pinnae are indicated along the abscissa below each bar. Changes ({Delta}) in ear swelling measurements (24h post ear challenge) are shown on the ordinate. Significant differences (p <= 0.05) are indicated by an asterisk (*). The experiment was repeated three times. C, LAT assessment of Tr cell generation in NK T cell (IL-10 KO)-reconstituted J{alpha}281 KO mice. Regulatory cells were cotransferred with responder and stimulator cells into the ear pinnae of naive recipient mice (n = 5). The composition of cells inoculated is shown along the abscissa below each bar. Changes ({Delta}) in ear swelling measurements (24 h post ear challenge) are shown on the ordinate. Significant differences (p <= 0.05) are indicated by an asterisk (*). The experiment was repeated twice.

 
J{alpha}281 KO mice were reconstituted (i.v.) with NK/NK T-enriched cells (106) from WT or IL-10 KO mice 7 days before being inoculated (a.c.) with OVA. Eight days later, T cells were enriched from the spleens. The enriched T cell populations from a.c.-inoculated mice were then used as regulatory cells in the LAT assay to assess their ability to down-regulate a DTH response in vivo. Control regulatory cells were enriched T cells from spleens of a.c.-inoculated nonreconstituted (irradiated) J{alpha}281 KO mice. Tr cells from a.c.-inoculated WT mice suppressed the DTH response in the ear (Fig. 7GoB). However, NK/NK T-enriched spleen cells from IL-10 KO mice did not (Fig. 7GoC). Thus NK T cell-derived IL-10 is essential for the differentiation of Tr cells during systemic tolerance induction subsequent to a.c. inoculation of OVA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-10 is associated with immunosuppression (40), graft survival (41), tumor growth (42), and down-modulation of both Th1 and Th2 responses (43, 44). This report shows that IL-10 also plays a pivotal role in the development of systemic tolerance induced after Ag is inoculated into the eye, but not i.v., and that the V{alpha}14+ NK T cells are a required source of the IL-10 needed for the generation of the Ag-specific Tr cells in the ACAID spleen.

The observation that NK T cell-derived IL-10 is required for tolerance induction is different from previous data showing that APC-derived IL-10 is essential for ACAID induction (35, 36). In fact, although recent reports show that a variety of cells (45, 46), including NK T cells (47, 48), produce IL-10 in response to a variety of stimuli, the cellular source of IL-10 in most published studies on tolerance is the dendritic cell (DC) (49, 50, 51). It is thought that the induction of immune response or tolerance is mediated by corresponding subsets of DCs. It is well known that IL-10-treated DCs induce systemic tolerance (52). DC-derived IL-10 within the skin site of UV irradiation is important in UV-induced tolerance (53). Although the properties of the tolerogenic DC are not entirely clear, they share attributes with the ACAID inducing eye-derived F4/80 cells, in that they lack CD40 expression and IL-12 production, but produce IL-10 in association with induction of Tr cells and systemic tolerance (50).

The exact role of IL-10 in development of tolerance is not known, although it is often thought that regulatory cell-derived IL-10 directly down-regulates immune effector cells (54, 55, 56). Several reports suggested that IL-10 is important for the induction of Tr cells (57, 58, 59). Zeller et al. showed that IL-10 and TGF{beta} induced Tr cells during alloantigen-specific tolerance (58). Rizzo and colleagues showed that feeding mice interphotoreceptor retinoid binding protein (IRBP) before uveitogenic challenge (with the same Ag) provided protection against the development of uveitis. In their studies, both IL-10 and IL-4 were required for the development of TGF{beta}-producing Ag-specific Tr cells that, in turn, prevented the development of autoimmune uveitis after a low dose regimen of Ag (59). These observations support reports that show that feeding small amounts of protein produces Ag-specific tolerance by active suppression (60).

This is not the first report of a role for IL-10 in the development of ACAID. D’Orazio and colleagues reported that TGF{beta}-treated APCs (ACAID-inducing APCs) from IL-10 KO mice did not prevent a subsequent DTH response to immunizing Ag (35). Furthermore, Gao et al. suggested that in response to Ag inoculated into the a.c., Fas/Fas ligand-induced apoptotic cells in the eye produced IL-10 that was critical for the development of systemic immune deviation (36). Data in this report support a role for NK T cell-derived-IL-10 within the splenic microenvironment of Ag presentation for tolerance induction. Moreover, we propose that once the eye-derived APCs reach the spleen, recruited NK T cells produce IL-10 that is necessary for the generation of the Tr cell involved in mediating active suppression of DTH responses in the periphery. The data show that 5% of splenic NK T cells make IL-10 and support the notion that a subpopulation within the spleen, perhaps within the tolerance-inducing microenvironment, make the IL-10.

The target of the NK T cell-derived IL-10 is currently unknown but could be the Ag-specific T lymphocyte, destined to differentiate into a regulatory cell or the eye-derived APC that then interacts with the T lymphocytes. IL-10 has the capacity to down-regulate costimulatory molecules (including B7 and CD40) on APCs (61, 62). However, previous reports showed that the ACAID-inducing eye-derived APCs lacked expression of CD40 and were already making TGF{beta} (63, 64). Thus, in the ACAID paradigm for systemic tolerance there is no need for the NK T cell-derived IL-10 influence on the APCs in the spleen. Currently, our bias is that the target of the NK T cell-derived IL-10 is the T lymphocyte, and the purpose of the IL-10 is to influence the differentiation of the T cell into the Ag-specific regulatory cell.

Previous reports showed that IL-4 KO mice developed ACAID (34, 35), but IL-10 KO mice did not (35, 36). NK T cells produce a variety of cytokines (including IL-4, IFN-{gamma}, and IL-10) in response to signals (47, 48, 65). Here, we show that during the induction of systemic tolerance after Ag inoculation into the eye, IL-10 mRNA and protein is selectively up-regulated and is absolutely required for the development of active suppression. Thus, although IL-4 is the cytokine usually associated with down-regulation of inflammation (66) and induction of Th3 Tr cells in oral tolerance (67), during the development of eye-induced active suppression, NK T-derived IL-10 is the major player.

It is possible that other immunosuppressive cytokines also contribute to the development of active suppression mediated through the eye. Another potentially important cytokine for the generation of Tr cells is TGF{beta} (68). Although NK T cells are capable of producing TGF{beta} (69), we did not observe any effect of a.c. Ag inoculation on TGF{beta}1 mRNA levels in the spleens of WT or CD1d KO mice using either RPA (Fig. 1GoA) or RT-PCR (data not shown). Because TGF{beta} regulation occurs at the protein level by enzymatic conversion of latent TGF{beta} to active TGF{beta} (70, 71), future studies will use a biological assay to distinguish latent from active TGF{beta}.

Induction and maintenance of Ag-specific tolerance are required for immune homeostasis, prevention of autoimmune disorders, and are the goals of allotransplantation. A relationship among ACAID, self-tolerance, and autoimmunity is suggested by reports that induction of ACAID in mice both prevented the onset and the expression of existing experimental autoimmune uveitis (72). Moreover, several published reports imply a role for NK T cells in preventing certain autoimmune disease in humans (73, 74) and in mice (75, 76, 77). Together, these reports support the notion that autoimmunity associated with NK T cell defects may be mediated by disruption of organ-specific tolerance mechanisms that include NK T cell CD1d interactions and NK T cell-derived IL-10.


   Acknowledgments
 
We thank Dr. J. Wayne Streilein for his many helpful discussions on ACAID and his critical reading of the manuscript, Marie Ortega for management of the Schepens Eye Research Institute vivarium and breeding of the transgenic mice used in the experiments, Vladimir Russakovsky for his dependable and outstanding technical support, and Gayle Barry for her preparation of the manuscript.


   Footnotes
 
1 This work was supported in part by grants from the National Institutes of Health (R01 EY11989-01 to J.S.-S., R01 AI42955 to S.B., and R01 AI33911 to S.B.), and the Schepens Eye Research Institute. Back

2 Address correspondence and reprint requests to Dr. Joan Stein-Streilein, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. Back

3 Abbreviations used in this paper: Tr, T regulatory; a.c., anterior chamber; ACAID, Anterior Chamber-Associated Immune Deviation; DTH, delayed-type hypersensitivity; DC, dendritic cell; KO, knockout; LAT, local adoptive transfer; PEC, peritoneal exudate cell; RPA, RNase protection assay; WT, wild type. Back

Received for publication May 25, 2000. Accepted for publication September 27, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Van Parijs, L., A. K. Abbas. 1998. Homeostasis and self-tolerance in the immune system: turning lymphocytes off. Science 280:243.[Abstract/Free Full Text]
  2. Mueller, D. L., M. K. Jenkins, R. H. Schwartz. 1989. Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu. Rev. Immunol. 7:445.[Medline]
  3. Nagata, S., T. Suda. 1995. Fas and fas ligand: lpr and gld mutations. Immunol. Today 16:39.[Medline]
  4. Powrie, F.. 1995. T cells in inflammatory bowel disease: protective and pathogenic roles. Immunity 3:171.[Medline]
  5. Bonomo, A., P. Kehn, E. M. Shevach. 1995. Post-thymectomy autoimmunity: abnormal T-cell homeostasis. Immunol. Today 16:61.[Medline]
  6. Streilein, J. W.. 1987. Immune regulation and the eye: a dangerous compromise. FASEB J. 1:199.[Abstract]
  7. Streilein, J. W., B. R. Ksander, A. W. Taylor. 1997. Immune deviation in relation to ocular immune privilege. J. Immunol. 158:3557.[Abstract]
  8. Streilein, J. W.. 1993. Immune privilege as the result of local tissue barriers and immunosuppressive microenvironments. Curr .Opin. Immunol. 5:428.[Medline]
  9. Griffith, T. S., T. Brunner, S. M. Fletcher, D. R. Green, T. A. Ferguson. 1995. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 270:1189.[Abstract/Free Full Text]
  10. Taylor, A. W., J. W. Streilein, S. W. Cousins. 1992. Identification of {alpha}-melanocyte stimulating hormone as a potential immunosuppressive factor in aqueous humor. Curr. Eye Res. 11:1199.[Medline]
  11. Taylor, A. W., J. W. Streilein, S. W. Cousins. 1994. Immunoreactive vasoactive intestinal peptide contributes to the immunosuppressive activity of normal aqueous humor. J. Immunol. 153:1080.[Abstract]
  12. Streilein, J. W., D. Bradley. 1991. Analysis of immunosuppressive properties of iris and ciliary body cells and their secretory products. Invest. Ophthalmol. Vis. Sci. 32:2700.[Abstract/Free Full Text]
  13. Cousins, S. W., M. M. McCabe, D. Danielpour, J. W. Streilein. 1991. Identification of transforming growth factor-{beta} as an immunosuppressive factor in aqueous humor. Invest. Ophthalmol. Vis. Sci. 32:2201.[Abstract/Free Full Text]
  14. Streilein, J. W., J. Y. Niederkorn. 1981. Induction of anterior chamber-associated immune deviation requires an intact, functional spleen. J. Exp. Med. 153:1058.[Abstract/Free Full Text]
  15. Wilbanks, G. A., J. W. Streilein. 1990. Distinctive humoral immune responses following anterior chamber and intravenous administration of soluble antigen: evidence for active suppression of IgG2-secreting B lymphocytes. Immunology 71:566.[Medline]
  16. Wilbanks, G. A., J. W. Streilein. 1992. Macrophages capable of inducing anterior chamber associated immune deviation demonstrate spleen-seeking migratory properties. Reg. Immunol. 4:130.[Medline]
  17. Streilein, J. W., J. Y. Niederkorn. 1985. Characterization of the suppressor cell(s) responsible for anterior chamber-associated immune deviation (ACAID) induced in BALB/c mice by P815 cells. J. Immunol. 134:1381.[Abstract]
  18. Wilbanks, G. A., J. W. Streilein. 1990. Characterization of suppressor cells in anterior chamber-associated immune deviation (ACAID) induced by soluble antigen: evidence of two functionally and phenotypically distinct T-suppressor cell populations. Immunology 71:383.[Medline]
  19. Sonoda, K.-H., M. Exley, S. Snapper, S. Balk, J. Stein-Streilein. 1999. CD1 reactive NKT cells are required for development of systemic tolerance through an immune privileged site. J. Exp. Med. 190:1215.[Abstract/Free Full Text]
  20. Bendelac, A., M. N. Rivera, S. H. Park, J. H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15:535.[Medline]
  21. Lantz, O., A. Bendelac. 1994. An invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8- T cells in mice and humans. J. Exp. Med. 180:1097.[Abstract/Free Full Text]
  22. Makino, Y., R. Kanno, T. Ito, K. Higashino, M. Taniguchi. 1995. Predominant expression of invariant V{alpha}14+ TCR {alpha} chain in NK1.1+ T cell populations. Int. Immunol. 7:1157.[Abstract/Free Full Text]
  23. Shimamura, M., T. Ohteki, U. Beutner, H. R. MacDonald. 1997. Lack of directed V{alpha}14J{alpha}281 rearrangements in NK1+ T cells. Eur. J. Immunol. 27:1576.[Medline]
  24. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, M. Taniguchi. 1997. Requirement for V{alpha}14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623.[Abstract/Free Full Text]
  25. Porcelli, S., C. E. Yockey, M. B. Brenner, S. P. Balk. 1993. Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- {alpha}/{beta} T cells demonstrates preferential use of several V {beta} genes and an invariant TCR {alpha} chain. J. Exp. Med. 178:1.[Abstract/Free Full Text]
  26. Dellabona, P., E. Padovan, G. Casorati, M. Brockhaus, A. Lanzavecchia. 1994. An invariant V{alpha}24J{alpha}Q/V{beta}11 T cell receptor is expressed in all individuals by clonally expanded CD4-8-T cells. J. Exp. Med. 180:1171.[Abstract/Free Full Text]
  27. Exley, M., J. Garcia, S. P. Balk, S. Porcelli. 1997. Requirements for CD1d recognition by human invariant V{alpha}24+ CD4-CD8- T cells. J. Exp. Med. 186:109.[Abstract/Free Full Text]
  28. Bendelac, A.. 1995. Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. J. Exp. Med. 182:2091.[Abstract/Free Full Text]
  29. Bendelac, A., O. Lantz, M. E. Quimby, J. W. Yewdell, J. R. Bennink, R. R. Brutkiewicz. 1995. CD1 recognition by mouse NK1+ T lymphocytes. Science 268:863.[Abstract/Free Full Text]
  30. Chen, Y. H., N. M. Chiu, M. Mandal, N. Wang, C. R. Wang. 1997. Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice. Immunity 6:459.[Medline]
  31. Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, L. Van Kaer. 1997. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity 6:469.[Medline]
  32. Smiley, S. T., M. H. Kaplan, M. J. Grusby. 1997. Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells. Science 275:977.[Abstract/Free Full Text]
  33. Chen, H., W. E. Paul. 1997. Cultured NK1.1+ CD4+ T cells produce large amounts of IL-4 and IFN-{gamma} upon activation by anti-CD3 or CD1. J. Immunol. 159:2240.[Abstract/Free Full Text]
  34. Kosiewicz, M. M., P. Alard, J. W. Streilein. 1998. Alterations in cytokine production following intraocular injection of soluble protein antigen: impairment in IFN-{gamma} and induction of TGF-{beta} and IL-4 production. J. Immunol. 161:5382.[Abstract/Free Full Text]
  35. D’Orazio, T. J., J. Y. Niederkorn. 1996. A novel role for TGF-{beta} and IL-10 in the induction of immune privilege. J. Immunol. 160:2089.[Abstract/Free Full Text]
  36. Gao, Y., J. M. Herndon, H. Zhang, T. S. Griffith, T. A. Ferguson. 1998. Antiinflammatory effects of CD95 ligand (FasL)-induced apoptosis. J. Exp. Med. 188:887.[Abstract/Free Full Text]
  37. Asea, A., J. Stein-Streilein. 1998. Signalling through NK1.1 triggers NK cells to die but induces NKT cells to produce interleukin-4. Immunology 93:296.[Medline]
  38. Sun, B., J. Wells, E. Goldmuntz, P. Silver, E. F. Remmers, R. L. Wilder, R. R. Caspi. 1996. A simplified, competitive RT-PCR method for measuring rat IFN-{gamma} mRNA expression. J. Immunol. Methods 195:139.[Medline]
  39. Sun, B., L. V. Rizzo, S. H. Sun, C. C. Chan, B. Wiggert, R. L. Wilder, R. R. Caspi. 1997. Genetic susceptibility to experimental autoimmune uveitis involves more than a predisposition to generate a T helper-1-like or a T helper-2-like response. J. Immunol. 159:1004.[Abstract]
  40. Moore, K. W., A. O’Garra, R. de Waal Malefyt, P. Vieira, T. R. Mosmann. 1993. Interleukin-10. Annu. Rev. Immunol. 11:165.[Medline]
  41. Bromberg, J. S.. 1995. IL-10 immunosuppression in transplantation. Curr. Opin. Immunol. 7:639.[Medline]
  42. Maeda, H., A. Shiraishi. 1996. TGF-{beta} contributes to the shift toward Th2-type responses through direct and IL-10 mediated pathways in tumor-bearing mice. J. Immunol. 156:73.[Abstract]
  43. Mosmann, T. R., K. W. Moore. 1991. The role of IL-10 in crossregulation of TH1 and TH2 responses. Immunol. Today 12:A49.[Medline]
  44. Pretolani, M., M. Goldman. 1997. IL-10: a potential therapy for allergic inflammation?. Immunol. Today 18:277.[Medline]
  45. Woiciechowsky, C., K. Asadullah, D. Nestler, B. Eberhardt, C. Platzer, B. Schoning, F. Glockner, W. R. Lanksch, H. D. Volk, W. D. Docke. 1998. Sympathetic activation triggers systemic interleukin-10 release in immunodepression induced by brain injury. Nat. Med. 4:808.[Medline]
  46. Hennessy, A., H. L. Pilmore, L. A. Simmons, D. M. Painter. 1999. A deficiency of placental IL-10 in preeclampsia. J. Immunol. 163:3491.[Abstract/Free Full Text]
  47. Burdin, N., L. Brossay, M. Kronenberg. 1999. Immunization with {alpha}-galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis. Eur. J. Immunol. 29:2014.[Medline]
  48. Hammond, K. J. L., L. D. Poulton, L. J. Palmisano, P. A. Silveira, D. I. Godfrey, A. G. Baxter. 1998. {alpha}/{beta}-T cell receptor (TCR)+CD4-CD8- (NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10. J. Exp. Med. 187:1047.[Abstract/Free Full Text]
  49. Iwasaki, A., B. L. Kelsall. 1999. Freshly isolated Peyer’s patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells. J. Exp. Med. 190:229.[Abstract/Free Full Text]
  50. Gao, J. X., J. Madrenas, W. Zeng, M. J. Cameron, Z. Zhang, J. J. Wang, R. Zhong, D. Grant. 1999. CD40-deficient dendritic cells producing interleukin-10, but not interleukin-12, induce T-cell hyporesponsiveness in vitro and prevent acute allograft rejection. Immunology 98:159.[Medline]
  51. Enk, A. H., H. Jonuleit, J. Salonga, J. Knop. 1997. Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma. Int. J. Cancer 73:309.[Medline]
  52. Steinbrink, K., M. Wolfl, H. Jonuleit, J. Knop, A. H. Enk. 1997. Induction of tolerance by IL-10-treated dendritic cells. J. Immunol. 159:4772.[Abstract]
  53. Kurimoto, I., T. Kitazawa, J. W. Streilein. 2000. Studies of delayed systemic effects of ultraviolet B radiation (UVR) on the induction of contact hypersensitivity. 2. Evidence that interleukin-10 from UVR-treated epidermis is the critical mediator. Immunology 99:134.[Medline]
  54. Stohlman, S. A., L. Pei, D. J. Cua, Z. Li, D. R. Hinton. 1999. Activation of regulatory cells suppresses experimental allergic encephalomyelitis via secretion of IL-10. J. Immunol. 163:6338.[Abstract/Free Full Text]
  55. Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, F. Powrie. 1999. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190:995.[Abstract/Free Full Text]
  56. Sundstedt, A., I. Hoiden, A. Rosendal, T. Kalland, N. vanRooijen, M. Dohlsten. 1997. Immunoregulatory role of IL-10 during superantigen-induced hyporesponsiveness in vivo. J. Immunol. 158:180.[Abstract]
  57. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. DeVries, M. G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[Medline]
  58. Zeller, J. C., A. Panoskaltsis-Mortari, W. J. Murphy, F. W. Ruscetti, S. Narula, M. G. Roncarolo, B. R. Blazar. 1999. Induction of CD4+ T cell alloantigen-specific hyporesponsiveness by IL-10 and TGF-{beta}. J. Immunol. 163:3684.[Abstract/Free Full Text]
  59. Rizzo, L. V., R. A. Morawetz, N. E. Miller-Rivero, R. Choi, B. Wiggert, C. C. Chan, H. C. Morse 3rd, R. B. Nussenblatt, R. R. Caspi. 1999. IL-4 and IL-10 are both required for the induction of oral tolerance. J. Immunol. 162:2613.[Abstract/Free Full Text]
  60. Weiner, H. L.. 1997. Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol. Today 18:335.[Medline]
  61. Mottonen, M., P. Isomaki, R. Saario, P. Toivanen, J. Punnonen, O. Lassila. 1998. Interleukin-10 inhibits the capacity of synovial macrophages to function as antigen-presenting cells. Br. J. Rheumatol. 37:1207.[Abstract/Free Full Text]
  62. Ding, L., P. S. Linsley, L. Y. Huang, R. N. Germain, E. M. Shevach. 1993. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression. J. Immunol. 151:1224.[Abstract]
  63. Takeuchi, M., M. M. Kosiewicz, P. Alard, J. W. Streilein. 1997. On the mechanisms by which transforming growth factor-{beta}2 alters antigen-presenting abilities of macrophages on T cell activation. Eur. J. Immunol. 27:1648.[Medline]
  64. Takeuchi, M., P. Alard, J. W. Streilein. 1998. TGF-{beta} promotes immune deviation by altering accessory signals of antigen-presenting cells. J. Immunol. 160:1589.[Abstract/Free Full Text]
  65. Holmgren, J., L. A. Hanson, B. Carlson, B. S. Lindblad, J. Rahimtoola. 1976. Neutralizing antibodies against Escherichia coli and Vibrio cholerae enterotoxins in human mild from a developing country. Scand. J. Immunol. 5:867.[Medline]
  66. Falcone, M., N. Sarvetnick. 1999. Cytokines that regulate autoimmune responses. Curr. Opin. Immunol. 11:670.[Medline]
  67. Inobe, J., A. J. Slavin, Y. Komagata, Y. Chen, L. Liu, H. L. Weiner. 1998. IL-4 is a differentiation factor for transforming growth factor-{beta} secreting Th3 cells and oral administration of IL-4 enhances oral tolerance in experimental allergic encephalomyelitis. Eur. J. Immunol. 28:2780.[Medline]
  68. Wilbanks, G. A., M. Mammolenti, J. W. Streilein. 1992. Studies on the induction of anterior chamber-associated immune deviation (ACAID). III. Induction of ACAID depends upon intraocular transforming growth factor-{beta}. Eur. J. Immunol. 22:165.[Medline]
  69. Tamada, K., M. Harada, K. Abe, T. Li, H. Tada, Y. Onoe, K. Nomoto. 1997. Immunosuppressive activity of cloned natural killer (NK1.1+) T cells established from murine tumor-infiltrating lymphocytes. J. Immunol. 158:4846.[Abstract]
  70. Lyons, R. M., J. Keski-Oja, H. L. Moses. 1988. Proteolytic activation of latent transforming growth factor-{beta} from fibroblast-conditioned medium. J. Cell Biol. 106:1659.[Abstract/Free Full Text]
  71. Orlidge, A., P. A. D’Amore. 1987. Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells. J. Cell Biol. 105:1455.[Abstract/Free Full Text]
  72. Hara, Y., R. R. Caspi, B. Wiggert, C. C. Chan, G. A. Wilbanks, J. W. Streilein. 1992. Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber-associated immune deviation with interphotoreceptor retinoid-binding protein. J. Immunol. 148:1685.[Abstract]
  73. Sumida, T., A. Sakamoto, H. Murata, Y. Makino, H. Takahashi, S. Yoshida, K. Nishioka, I. Iwamoto, M. Taniguchi. 1995. Selective reduction of T cells bearing invariant V{alpha}24J{alpha}Q antigen receptor in patients with systemic sclerosis. J. Exp. Med. 182:1163.[Abstract/Free Full Text]
  74. Wilson, S. B., S. C. Kent, K. T. Patton, T. Orban, R. A. Jackson, M. Exley, S. Porcelli, D. A. Schatz, M. A. Atkinson, S. P. Balk, et al 1998. Extreme Th1 bias of invariant V{alpha}24J{alpha}Q T cells in type 1 diabetes: [Published erratum appears in 1999 Nature 399:84.]. Nature 391:177.[Medline]
  75. Mieza, M. A., T. Itoh, J. Q. Cui, Y. Makino, T. Kawano, K. Tsuchida, T. Koike, T. Shirai, H. Yagita, A. Matsuzawa, et al 1996. Selective reduction of V{alpha}14+ NK T cells associated with disease development in autoimmune-prone mice. J. Immunol. 156:4035.[Abstract]
  76. Gombert, J. M., A. Herbelin, E. Tancrede-Bohin, M. Dy, M. Carnaud, J. F. Bach. 1998. Early quantitative and functional deficiency of NK1+-like thymocytes in the NOD mouse. Eur. J. Immunol. 26:2989.
  77. Baxter, A. G., S. J. Kinder, K. J. Hammond, R. Scollay, D. I. Godfrey. 1997. Association between {alpha}{beta} TCR+CD4-CD8- T-cell deficiency and IDDM in NOD/Lt mice. Diabetes 46:572.[Abstract]



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A. Yamaura, C. Hotta, M. Nakazawa, L. Van Kaer, and M. Minami
Human invariant V{alpha}24+ natural killer T cells acquire regulatory functions by interacting with IL-10-treated dendritic cells
Blood, April 15, 2008; 111(8): 4254 - 4263.
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