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Originally published online as doi:10.1189/jlb.0807579 on January 11, 2008

Published online before print January 11, 2008
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(Journal of Leukocyte Biology. 2008;83:928-935.)
© 2008 by Society for Leukocyte Biology

Ly49 C/I-dependent NKT cell-derived IL-10 is required for corneal graft survival and peripheral tolerance

C. M. Watte*, T. Nakamura*,1, C. H. Lau*, J. R. Ortaldo{dagger} and J. Stein-Streilein*,2

* Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA; and
{dagger} Laboratory of Experimental Immunology, National Cancer Institute-Center for Cancer Research, Frederick, Maryland, USA

2 Correspondence: Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA. E-mail: joan.stein{at}schepens.harvard.edu


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ABSTRACT
 
Similar to their activity on NK cells, Ly49 molecules play a pivotal role in influencing how NKT cells respond. It is known that Ly49 C/I is an inhibitory receptor capable of down-modulating proliferation, IFN-{gamma} response, and cytotoxic activity in cells that express it. In a model of peripheral tolerance induced via the eye, we observed that Ly49 C/I-positive, invariant NKT cells were required. To test if the NK inhibitory receptor functionally contributed to tolerance development, we used blocking antibody, in vivo and in vitro, to interfere with the development of antigen-specific suppression. A result of blocking ligation of Ly49 C/I inhibitory receptor prevented NKT cell production of IL-10 and the subsequent development of tolerance. Ly49 C/I-blocking antibodies also prevented corneal graft survival, a phenomenon dependent on eye-induced tolerance. Furthermore, in the presence of TCR stimulation, cross-linking of Ly49 C/I on CD4+ NKT cells stimulated an increase in IL-10 mRNA and a decrease in IFN-{gamma}. The concept of Ly49 inhibitory receptors regulating immune reactivity to self by regulating immune activity of individual cells is thus expanded to include a role for the inhibitory receptors in the more global process of peripheral tolerance to foreign antigens.

Key Words: NK inhibitory receptors • cytokines • immunosuppression


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INTRODUCTION
 
Ly49 receptors were initially identified on NK cells, where they serve to recognize abnormal or foreign cells that do not express conventional levels of self-MHC class I molecules, a concept put forth as the "missing-self" hypothesis [1 ]. In addition to NK cells, NKT cells, {gamma}{delta} T cells, memory CD8+ T cells, B-1B cells, and a subset of dendritic cells [2 , 3 ] express Ly49 molecules. Although the family of Ly49 molecules contains inhibitory and activating receptors, there are no reports of activating Ly49 receptors being expressed by NKT cells [4 ]. Ly49 receptor expression on NKT cells is governed by several interacting mechanisms including NKT cell maturity, MHC context, and the nature of the TCR [5 ].

Similar to their role on NK cells, Ly49 molecules appear to influence the type of responsiveness of NKT cells during innate and adaptive immune responses. Engagement of Ly49 receptors inhibits inflammatory cytokine production by NKT cells [6 ], NKT cell proliferation [7 ], and cytotoxic activity [4 ]; hence, it is not unusual for Ly49 molecules to be associated with effector mechanisms of tolerance. However, the studies presented here extend our understanding of the role of Ly49 molecules in immune regulation to include the idea that NK inhibitory receptors provide positive signals for the efficient production of immunoregulatory cytokines. Therefore, Ly49 receptors not only influence individual cells toward tolerance to self-antigens but also contribute to the generation of antigen-specific T regulatory (Treg) cells and tolerance to foreign antigens.

Immune privilege is a transplantation term that describes sites that accept foreign grafts indefinitely. More detailed analyses of the immune privilege of the eye show that immune responses actually do occur but are not accompanied by inflammation [8 ]. In addition to the local regulation of immune responses, T cell regulation occurs in the periphery. Thus, studies about immune privilege are relevant to our understanding of peripheral tolerance induction in general.

During the induction of peripheral tolerance through the eye {using a classical model for the study of immune privilege, called anterior chamber (a.c.)-associated immune deviation (ACAID) [9 ]}, antigen is picked up by the indigenous F4/80+APC within the a.c. from the eye, carried through the trabecular meshwork into the blood. After a potential detour through the thymus [10 ], the F4/80+APCs eventually localize in the spleen. Additional F4/80+APCs from the periphery become educated by the few eye-derived APCs that migrate and aggregate in the marginal zone of the spleen with invariant (i)NKT cells [11 ], T cells, and B cells [12 , 13 ]. Within these aggregates, afferent CD4+ Treg cells and efferent CD8+ Treg cells are generated, and antigen-specific tolerance becomes available in the periphery [14 ]. Importantly, the NKT cells that were required for the differentiation of Treg cells expressed the NK inhibitory receptor, Ly49 C/I [15 ], and as we show here, the Ly49 C/I molecules not only identify the crucial iNKT cell subpopulation involved with induction of peripheral tolerance but also have a role in the production of NKT-derived IL-10.


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MATERIALS AND METHODS
 
Mice
Eight-week-old female C57BL/6Ntac (B6) and BALB/c mice were purchased from Taconic Farms (Germantown, NY, USA). B6.129-H2-Ab1tm1Gru N12 (MHC class II–/–) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). J{alpha}18–/– and V{alpha}14 transgenic (Tg; RAG–/–V{alpha}14TgVβ8.2Tg) mice (B6 background) were bred at the Schepens Eye Research Institute (Boston, MA, USA) from breeding pairs originally provided by Masanaru Taniguchi, Riken Research Center for Allergy and Immunology (Yokohama City, Kanagawa, Japan). All animals were treated humanely and in accordance with the National Institutes of Health (NIH) Guidelines and the approved protocols of the Schepens Animal Care and Use Committee.

Reagents and antibodies
Rat anti-mouse Fc{gamma}RII/III mAb (2.4G2 ascites), anti-Ly49 C/I (clone 5E6), anti-CD3 (2C11), anti-Ly49 C/I/H (1F8), and anti-Ly49 G (4D11) were produced in the laboratories of the authors. F(ab')2 fragments of clone 5E6 were made in the laboratory by standard techniques [16 ]. Biotinylated antibodies to Gr1, CD11b, CD8, biotin, and FITC-conjugated anti-Ly49 C/I (5E6), CD1d-dimer, and secondary PE-conjugated anti-mouse IgG1 were purchased from BD PharMingen (San Diego, CA, USA). FITC-conjugated anti-IL-10 mAb (JES5-16E3), isotype control antibody (rat IgG2b; LO-DNP-11), and PE-Cy5-conjugated anti-CD4 (GK1.5) were from eBioscience (San Diego, CA, USA); streptavidin and anti-CD19 Microbeads were from Miltenyi Biotec (Auburn, CA, USA).

Induction and assessment of ACAID
ACAID was induced as described previously [17 ]. In brief, antigen (OVA, 50 µg/2 µl in PBS) was injected (a.c.) using a glass needle. One week later, mice were immunized (s.c.) with OVA (100 µg/50 µl in PBS), emulsified 1:1 in CFA. Following the lapse of another week, mice were assessed for their delayed hypersensitivity (DH) responses by injecting OVA-pulsed, thioglycolate-induced peritoneal exudate cells (PEC; 5x105 cells/10 µl) intradermally into the ear pinnae. Ear thickness was measured 24 h later with an engineer’s micrometer (Mitutoyo, Paramus, NJ, USA) and compared with ear measurement before challenge.

Reconstitution of iNKT cell-deficient mice
J{alpha}18–/– mice were reconstituted with T cells harvested from spleens of class II–/– mice. T cells were enriched by density separation (Lympholyte M, Cedarlane Laboratories, Burlington, NC, USA) and negative selection using goat anti-mouse-coated Immulan beads (Biotecx, Houston, TX, USA). Nonspecific binding of the antibodies was blocked with 2.4G2 mAb (anti-Fc{gamma}RII/III) prior to specific antibody treatment. Ly49 C/I+ cells were further depleted with biotinylated 5E6 mAb and streptavidin-coated magnetic beads on an auto MACS (Miltenyi Biotec). There was less than 1% contamination of the resultant cell population with Ly49 C/I+ cells. Enriched T cells (2x106 cells in 100 µl HBSS/mouse) were injected i.v. into J{alpha}18–/– mice by retro-orbital injection 24 h before a.c. inoculation of antigen to induce peripheral tolerance.

Corneal transplantation
Female C57BL/6 mice were used as donors and female BALB/c as recipients. One day before corneal transplantation, recipient mice received an injection (i.p.) of 5E6 (100 µg/mouse), F(ab')2 of 5E6 (10 µg/mouse), or IgG2a (isotype control, 20 µg/mouse). Under general anesthesia (120 mg/kg ketamine/20 mg/kg xylazine), a 2-mm diameter, full-thickness donor corneal button from C57BL/6 was sutured to a 1.5-mm diameter recipient bed with at least eight interrupted 11-0 nylon sutures (Sharpoint Surgical Specialties Corp., Reading, PA, USA). Gentamicin antibiotic ointment was applied, and eyelids were closed with an 8-0 nylon mattress suture (Ethicon, Somerville, NJ, USA) for 3 days. Corneal sutures were removed on Day 7. Graft status was assessed weekly up to 8 weeks, and graft rejection was determined by a graft opacity score of greater than or equal to two [18 ].

Flow cytometry
Lymphocytes were enriched from dissociated spleen cells by density separation (Lympholyte M, Cedarlane Laboratories). Events were collected on an EPICS XL flow cytometer (Beckman Coulter, Miami, FL, USA) or FACSCalibur (BD Biosciences, Bedford, MA, USA) and analyzed with FlowJo software (Treestar, Ashland, OR, USA). The relative number of cells was calculated according to the following formula: number of enriched spleen cells x percent positive events. A Cytofix/Cytoperm kit (BD PharMingen) was used to detect intracellular IL-10 in cells after their incubation (3 h) in complete media containing GolgiStop (BD PharMingen).

In vitro ACAID
In vitro ACAID was performed by incubating freshly harvested spleen cells (25x106/well in a six-well plate) with PEC (3x106/well) that were previously treated with TGF-β (5 ng/ml) and pulsed with OVA (5 mg/ml). In some experiments, F(ab')2 of anti-Ly49 C/I mAb (5E6, 2 µg/ml) was added at the start of the culture. After a 5-day culture period, iNKT cells were enriched by depletion with Gr1+, CD11b+, CD8+ biotinylated antibody (BD PharMingen), streptavidin, and anti-CD19 Microbeads (Miltenyi Biotec) and then further purified on a MoFlo cell sorter (DakoCytomation, Carpinteria, CA, USA) with an {alpha}-galactosylceramide ({alpha}GalCer)-loaded CD1d dimer or CD4 mAb. Purified cells were processed immediately for isolation of RNA.

Cross-linking studies
Anti-CD3 mAb ranging from 0 to 1 µg/ml in 50 µl PBS were added to 96-well, flat-bottomed, tissue-culture plates and incubated overnight at 4°C. Following the incubation, the plates were coated with antibodies to Ly49 C/I (5E6 mAb, 10 µg/ml in 50 µl/each well of a 96-well plate) and incubated for another 4 h at room temperature. In other experiments, antibodies were coated on protein G agarose beads (Invitrogen, Carlsbad, CA, USA) instead of the plate and added to the cell culture. NKT cells were enriched from spleens of V{alpha}14Tg mice or class II-deficient mice by density separation (Lympholyte M) and T cell enrichment (Immulan column). CD4+ NKT cells from class II-deficient mice were further enriched by FACS. NKT cells were added to the prepared plates and incubated for 3 h (37°C, 5% CO2, and air). The resulting FACS-sorted population was ≥98% CD4+ NKT cells.

RT-PCR
Total RNA was extracted using Trizol, reverse-transcribed with SuperscriptTMII RT polymerase, and amplified with Platinum Taq DNA polymerase (all from Invitrogen). The following primers were used: IL-10 sense (5'-ACCTGGTAGAAGTGATGCCCCAGGCA-3') and antisense (5'-CTATGCAGTTGATGAAGATGTCAAA-3'), IFN-{gamma} sense (5'-TGAACGCTACACACTGCATCTTGG-3') and antisense (5'-CGACTCCTTTTCCGCTTCCTGAG-3'), β-actin sense (5'-GTGGGCCGCTCTAGGCACCAA-3') and antisense (5'-CTCTTTGATGTCACGCACGATTTC-3'). Semiquantitative analysis of cytokine mRNA expression was performed by measuring the ratio of cytokine band density compared with the housekeeping gene β-actin.

Statistics
Data were analyzed for significant differences among experimental groups using a one-way ANOVA with Bonferroni’s Multiple Comparison test. Statistical differences of flow cytometry data were calculated with a one-tailed Student’s t-test, P ≤ 0.05. Kaplan-Meier survival curves were constructed, and a Mantel-Cox test was used to compare the probability of corneal graft survival.


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RESULTS
 
Ly49 C/I inhibitory receptor identifies the CD4+ iNKT cell population that is required for ACAID
Previously, it was established that a splenic population of NKT cells that bound CD1d (and expressed the iTCR) was critical for the induction of peripheral tolerance [15 ]. This cell population is required for low-dose oral tolerance induction as well [19 ]. We also showed that the iNKT cells that accumulate within the spleen after tolerance induction express CD4 [20 ] and Ly49 C/I [15 ]. Ly49 C/I expression was first determined by indirect studies, where mAb 5E6 (anti-Ly49 C/I) treatment removed the NKT cell population required for tolerance induction. Here, we directly tested by flow cytometry if the increased population of NKT cells expressed the Ly49 C/I receptor.

Peripheral tolerance was induced via the eye by inoculating OVA into the a.c. of C57BL/6 mice. Five days later, the percentage of NKT cells in the enriched splenic T cells that expressed CD4, the iTCR, and Ly49 C/I was determined. As before, CD4+iNKT cells were increased in the spleens of a.c.-inoculated compared with uninoculated mice [20 ] (Fig. 1A ), but for the first time, we documented that the CD4+ population contained an increased number of Ly49 C/I+CD4+iNKT cells (Fig. 1B) . These data support the postulate that the subpopulation of CD4+iNKT cells that is increased in the spleens during the induction of ocular peripheral tolerance expresses Ly49 C/I.


Figure 1
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  		Figure 1. Flow cytometry analyses of Ly49 C/I+CD4+iNKT cells. Mice received an a.c. inoculation of OVA 5 days prior to enrichment of iNKT cells from their spleens. (A) Left column (dot-plot) shows the percentage of CD4+NKT cells with the ordinate representing binding to the {alpha}GalCer CD1d dimer and the abscissa representing anti-CD4 mAb binding; right column (histogram) is the percentage of Ly49 C/I+ cells (5E6 mAb anti-Ly49 C/I) within the gated CD4+iNKT cells (indicated in the left column). The ordinate also indicates whether the cells were from naïve or a.c.-inoculated mice. (B) Bar graph shows relative number of Ly49 C/I+ CD4+iNKT cells in total number of lymphocytes enriched from spleens of a.c.-inoculated or naïve animals (indicated in abscissa). n = Five animals per group; *, P ≤ 0.05. The experiment was performed three times with similar results.

Ly49 C/I+ iNKT cells are necessary to reconstitute ACAID in NKT cell-deficient mice
To verify that the CD4+ Ly49 C/I+ iNKT subpopulation was functionally required for induction of peripheral tolerance through the eye, we reconstituted NKT-deficient mice with CD4+ NKT cells containing, or not, Ly49 C/I+ cells (Fig. 2 ). Previously, we and others [19 , 21 ] reported that neither ACAID nor low-dose oral tolerance could be induced in iNKT cell-deficient mice. However, peripheral tolerance in the oral [19 ] and ocular tolerance models could be restored in iNKT cell-deficient mice if they were reconstituted with cells containing CD4+ iNKT cells [22 ]. CD4+NKT cells were enriched from spleens of class II-deficient mice and then further depleted, or not, of the Ly49 C/I-positive cell population. Class II-deficient mice were used, since in the absence of traditional CD4+ T cells, the T cells that express CD4 are exclusively CD4+ NKT cells [20 ]. The enriched T cells were injected (i.v.) into J{alpha}18–/– mice 24 h before a.c. injection of antigen to induce peripheral tolerance. As expected, peripheral tolerance was induced following a.c. inoculation of antigen when J{alpha}18–/– mice were reconstituted with cells that contained the Ly49 C/I+CD4+ but not if Ly49 C/I–CD4+ iNKT cells were used.


Figure 2
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  		Figure 2. DH response after ACAID induction in iNKT cell-deficient mice reconstituted with Ly49 C/I+ cells. The ordinate represents the change in ear thickness, as measured by an engineer’s micrometer 24 h postchallenge of experimental animals with the immunizing antigen. The experimental treatment of the mice in each group is indicated under the abscissa. *, P ≤ 0.05. Bar graph represents the sum of two individual experiments. A positive ACAID experiment is shown in Figure 3 .

Blocking Ly49 C/I in vivo interferes with the induction of ACAID
Having established that the critical subpopulation of iNKT cells for peripheral tolerance expressed Ly49 C/I, we reasoned that the Ly49 C/I protein might actually have a function in the generation of peripheral tolerance. To test this idea, we analyzed if ACAID-induced peripheral tolerance were modulated in mice when Ly49 C/I ligation was blocked. B6 mice received injections (i.p.) of anti-Ly49 C/I/H (IF8) or Ly49 G (4D11) twice (3 days and 24 h) prior to tolerance induction (Fig. 3A ). The anti Ly49G (4D11) mAb was an isotype control, and a control to show that another Ly49 molecule expressed by the NKT cells did not have the capacity to interfere with DH suppression. As whole antibodies might remove the cell population that expresses the molecule and/or block-binding, we also used F(ab')2 fragments of mAb 5E6 to block binding of the Ly49 C/I receptor (Fig. 3B) .


Figure 3
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  		Figure 3. DH response in mice treated with mAb to Ly49 C/I prior to induction of peripheral tolerance via the eye. The ordinate represents the change in ear thickness 24 h post intradermal inoculation of antigen. The experimental treatment of the mice is indicated below the abscissa. (A) The mice were treated with mAb specific for Ly49 C/I/H (mAb 1F8, rat IgG2a) or isotype-matched control antibody specific for Ly49 G (mAb 4D11, rat IgG2a). *, P ≤ 0.001; **, P < 0.05; NS, nonsignificant. Data were pooled from six experiments. (B) The experimental group was treated with the F(ab')2 preparation of mAb 5E6 (specific for Ly49 C/I). *, P ≤ 0.001; **, P ≤ 0.01. A repeat experiment produced similar results.

We observed that 1F8 mAb (Ly49 C/H/I) or the F(ab')2 fragments of 5E6 mAb (Ly49 C/I) abolished the ability to induce tolerance in the mice. In contrast, tolerance induction was uncompromised in mice treated with the isotype-matched control mAb (4D11) with specificity to Ly49 G (Fig. 3A) . The results support the notion that the Ly49 C/I molecule on the CD4+ iNKT cell must be available to interact with its ligand, MHC class I, for the establishment of peripheral tolerance induced through the eye.

Blocking Ly49 C/I interferes with corneal graft survival
As ACAID is a model for immune privilege, and immune privilege is essential for corneal graft survival, we next tested the role of Ly49 C/I in this ACAID-related transplantation model. It is reported that when B6 corneas are transplanted to BALB/c mice, 50% survive indefinitely, as mice develop peripheral tolerance to the alloantigens of the graft [23 ]. Previously, it was proposed [23 , 24 ] and later proven that acceptance of corneal grafts was dependent on ACAID mechanisms [25 ]. Therefore, we tested if Ly49 C/I ligation were necessary for corneal graft survival by blocking its binding with a specific antibody in vivo. Indeed, we observed that BALB/c mice given whole or F(ab')2 fragments of 5E6 mAb specific for Ly49 C/I rejected 100% of the grafts (Fig. 4 ). Thus, Ly49 C/I participates in tolerance to corneal allografts.


Figure 4
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  		Figure 4. Kaplan-Meier graft survival curves for allogeneic corneal transplants. The ordinate shows the percent of cornea grafts that survived. The time that the grafts were evaluated is indicated on the abscissa. BALB/c recipients were treated with IgG2a isotype control ({blacksquare}), 5E6 ({diamondsuit}), and F(ab')2 of 5E6 ({blacktriangleup}). The difference in graft survival rate in mice treated with 5E6 or F(ab')2 of 5E6 compared with IgG2a isotype control was significant (P≤0.05). Each group contained 10 mice.

Blocking Ly49 C/I receptor interactions interferes with the production of IL-10 by iNKT cells in vivo and in vitro
We analyzed the outcome of ligating Ly49 C/I on NKT cells and its role in the development of ACAID. Although we suspected the Ly49 C/I molecule would prevent NKT cell production of IFN-{gamma}, we wondered if it could also be involved in the production of immunosuppressive cytokines needed for the tolerance outcome. Since iNKT-derived IL-10 is required for the induction of ocular tolerance [22 ], we began with the postulate that the Ly49 C/I+ molecule might stimulate the production of IL-10 by iNKT cells. CD4+ Ly49 C/I+ NKT cells were enriched from dissociated spleen cells harvested from naïve and ACAID mice and further assessed for intracellular IL-10 by flow cytometry analyses. Clearly, there were more IL-10-producing Ly49 C/I+ iNKT cells in spleens of mice inoculated (a.c.) with OVA than in naïve mice (Fig. 5A ). Thus, Ly49 C/I+ iNKT cells participate in tolerance induction, in part, by producing IL-10. As stated above, NKT cell-derived IL-10 is essential for the development of the subsequent Treg cells that mediate peripheral tolerance [21 ], although other cell types are capable of producing IL-10 in this model [26 ]. The data that supported this hypothesis showed that NKT cells from B6 or class II-deficient, but not from IL-10-deficient, mice restored the ability of J{alpha}18–/– mice to develop ACAID post-a.c. inoculation of antigen [22 ].


Figure 5
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  		Figure 5. IL-10 expression by iNKT cells during ACAID. (A) Flow cytometry analysis of spleen cells from an individual ACAID and naïve mouse. Cells were gated on {alpha}GalCer ({alpha}GC)-CD1d dimer+ and Ly49 C/I+ cells and analyzed for intracellular expression of IL-10. Numbers represent the percentages of IL-10-producing cells among the Ly49 C/I+ cell population. The experiment is representative of three independent experiments with minor variations. (B and C) Semiquantitative RT-PCR analysis of IL-10 mRNA transcripts in NKT cells harvested from vitro ACAID cultures using (B) wild-type (WT) B6 and (C) class II–/– mice, in the presence or absence of blocking 5E6 F(ab')2 reagent. The treatment and origin of the cells are indicated below the abscissa.

It was reported previously that the necessity for injecting antigen into the eye can be bypassed by treating APC in vitro with TGF-β and OVA to produce tolerogenic or surrogate ACAID APC [9 ]. The in vitro-generated APC then can be injected (i.v.) into naive mice to induce ACAID in the recipients or further cultured with naive spleen cells to generate ACAID Treg cells [27 , 28 ]. The use of in vitro ACAID allows for an in-depth, cellular analysis of the ACAID system. With this in mind, iNKT cell production of IL-10 in spleen cells that had been cultured with TGF-β-treated, OVA-pulsed APCs in the presence or absence of anti-Ly49 C/I F(ab')2 (5E6) was assessed. IL–10 mRNA content from FACS-sorted iNKT cells from in vitro ACAID cultures (Fig. 5B and 5C) was analyzed by semiquantitative RT-PCR. As expected, NKT cells from ACAID cultures that contained blocking antibody produced less IL-10 mRNA than NKT cells from cultures without antibody (Fig. 5B and 5C) . These data support the postulate that engagement of Ly49 C/I molecules on the NKT cell is needed for their efficient production of IL-10.

TCR activation is required for Ly49 C/I-dependent production of IL-10
To address the role of the Ly49 C/I molecule in IL-10 production more directly, we tested if signaling through Ly49 C/I could stimulate the production of IL-10 directly or secondarily to an activating signal (TCR). Various concentrations of anti-CD3 and a constant concentration of anti-Ly49 C/I mAb were bound to the culture wells. iNKT cells enriched from spleens from V{alpha}14Tg mice were added to the wells and incubated for 3 h prior to isolation of their RNA and the analyses of IL-10 mRNA by semiquantitative RT-PCR. IL-10 mRNA was increased in the wells containing suboptimal concentrations of anti-CD3 mAb (10 ng/ml) and anti-Ly49 C/I (Fig. 6A ). IL-10 mRNA, however, was not increased in the cultures that contained either antibody alone bound to the plate. Similar results were obtained when CD4+NKT cells from class II–/– mice were used for cross-linking (Fig. 6B) . IL-10 mRNA was only weakly expressed when stimulated with suboptimal concentration of anti CD3 alone, compared with its expression when CD3 and Ly49 C/I were simultaneously stimulated. In accordance with previously published reports describing the inhibitory role of Ly49 C/I on inflammatory cytokine production, IFN-{gamma} mRNA was decreased with the simultaneous cross-linking of Ly49 C/I and CD3 (Fig. 6A and 6B) . Thus, the data support the postulate that ligation of Ly49 C/I in the presence of a TCR-activating signal not only down-regulates IFN-{gamma} production but also promotes the production of IL-10 by Ly49 C/I+, CD4+ iNKT cells.


Figure 6
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  		Figure 6. Semiquantitative RT-PCR analysis of IL-10 and IFN-{gamma} mRNA transcripts. Cells were cross-linked for 3 h with plate-bound anti-Ly49 C/I and anti-CD3. (A) Dose-response curve to cross-linking of iNKT cells (V{alpha}14Tg mice) with anti-CD3 (10–103 ng/ml) and anti-Ly49 C/I (10 µg/ml). Cross-linking of anti-CD3 alone (•). (B) Cross-linking of CD4+NKT cells (class II–/– mice) with anti-Ly49 C/I and anti-CD3 (10 µg and 10 ng/ml, respectively) or isotype control and anti-CD3. The experiment was performed twice with similar results.


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DISCUSSION
 
This report shows that Ly49 C/I ligation on NKT cells is required for the development of peripheral tolerance induced via the a.c. (ACAID) and is necessary for corneal graft survival. Blocking Ly49 C/I prevented the increase in production of NKT cell-derived IL-10. Cross-linking studies documented that in the presence of TCR activation, ligation of Ly49 C/I led to increases in IL-10 mRNA as well as decreases in IFN-{gamma} mRNA. Thus, in addition to suppressing the production of IFN-{gamma}, ligation of the NK inhibitory molecule, Ly49 C/I, is required for efficient production of IL-10 in iNKT cells. This novel finding broadens our understanding of mechanisms of tolerance through the eye as well as reveals a new role for the Ly49 inhibitory receptors in costimulating the production of an immunosuppressive cytokine. Thus, Ly49 inhibitory receptors may have a broader biological function than previously thought. The Ly49 C/I-dependent production of iNKT cell-derived IL-10 most likely contributes to the maintenance of the immunosuppressive milieu, in which the induction of peripheral tolerance occurs [29 ].

Ly49 molecules belong to a multigene family of homodimeric C-type, lectin-like glycoprotein receptors that recognize MHC class I molecules. Importantly, a functionally analogous yet structurally different set of receptors, called killer Ig-like receptors, is expressed on human cells. Different isoforms within the Ly49 family bind to different allelic forms of MHC class I molecules [2 ]. Inhibitory receptors mediate their effect through an ITIM in their cytoplasmic domains [30 ]. It is generally believed that ITIM motifs recruit Src homology 2-containing tyrosine phosphates that down-regulate signaling from ITAMs. Inhibition by Ly49s requires coclustering and coligation of an activating immune recognition receptor [31 ]. As stated previously, only inhibitory Ly49 isoforms are found on NKT cells and until now, were thought to play a major role in dampening the Ly49 C/I+ NKT cell inflammatory responsiveness [4 , 6 , 7 ]. Although the regulatory role of Ly49 inhibitory receptors has been studied in many models of immune activation, this report shows a new role for Ly49 inhibitory molecules in the development of peripheral tolerance and Treg cells.

NKT cells are a distinct subset of innate lymphocytes that possess features of T cells and NK cells. Most of the NKT cells in the mouse display an iTCR{alpha} chain using the conserved V{alpha}14-J{alpha}18 TCR rearrangement and are termed V{alpha}14+NKT or iNKT. Mouse iNKT cells are further subdivided into CD4+ or CD4–CD8– double-negative cells [32 ]. Studies of mouse NKT cells are important, as an analogous V{alpha}24-J{alpha}18 iNKT cell population is present in humans. In contrast with conventional T cells that recognize peptide antigens presented by MHC class I, the TCR on the iNKT cells recognize glycolipid antigens presented by CD1d, a nonpolymorphic MHC class I-like molecule. Their ability to rapidly produce large amounts of cytokines upon activation (Th1 and/or Th2) suggests that they may be a regulatory cell type important to the initiation and regulation of various immune responses and thus, could form a bridge between the innate and adaptive immune response [33 ]. NKT cells indeed play important roles in various and contrasting immune conditions such as anti-tumor immunity, host defense, autoimmunity, and tolerance [34 ]. The characteristic of promoting immunity or tolerance may not only reflect the activity of distinct iNKT cell subsets within a particular organ but also, the physiological context in which iNKT cells are activated [35 ]. To date, iNKT cell biology is incompletely understood. The studies presented here present a novel insight into NKT cell function/activation and how its contradicting abilities might be regulated.

In many models of peripheral tolerance, IL-10 is a critical factor. A variety of hematopoietic cells, other than the NKT cell, including the APC and Treg cells, produces IL-10 [26 ]. Fas/Fas ligand-initiated apoptotic cells produce IL-10 and induce the macrophages that phagocytize them to produce IL-10 as well [36 ]. IL-10 is widely regarded as an important immunosuppressive cytokine and has attracted much attention because of its anti-inflammatory properties. IL-10 appears to act as a pleiotropic immunomodulatory cytokine with activating and deactivating properties [37 ]. Although the cellular source of IL-10 is not always known, it is known that NKT cell-derived IL-10 is essential for eye-induced peripheral tolerance [22 ], and data in this report further show that NKT-derived IL-10 is essential for corneal graft survival, as Ly49 C/I-specific, blocking antibodies prevented long-term graft survival.

A question raised by this report but not studied is how signaling through inhibitory Ly49 receptors could allow for IL-10 production. Lucas and colleagues [38 ] showed that macrophages stimulated through their Fc{gamma}R by immune complexes produce high levels of IL-10 and that MAPKs were required for the IL-10 synthesis. Macrophage and NKT cell production of IL-10 requires signals through two receptors (FcR{gamma} and Ly49), which have ITIM motifs in their cytoplasmic domains [30 , 39 ]. Thus, a potential pathway exists through ITIM for the activation of IL-10 production and adds to the controversy that there might be ambiguity in immunoreceptor signaling [40 ].

Finally, the idea that Ly49 C/I cosignaling on NKT cells leads to its production of IL-10 does not necessarily contradict the notion that Ly49 C/I is an inhibitory receptor but expands the understanding of how NK inhibitory receptors mechanistically contribute to the down-regulation of an immune inflammatory response.


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ACKNOWLEDGEMENTS
 
This work was supported in part by NIH Public Health Service grants EY11983 and EY016476. We thank Drs. T. Yamamura and S. Miyake (National Institute of Neuroscience, Tokyo, Japan) for generously providing the {alpha}GalCer used in these studies and Dr. M. Taniguchi for the gift of the original breeding pairs for J{alpha}18–/– and V{alpha}14Tg mice. We are grateful to Dr. C. Benarafa for his help on the FACSCalibur flow cytometer at the CBR Institute for Biomedical Research (Boston, MA, USA). We thank Mr. Peter Mallen for his assistance with graphics. We are indebted to Frits Hogewind, a former student from Leiden Medical College (The Netherlands), for initiating the work with the Ly49 receptors and ACAID in our laboratory. We thank Ms. Amelia Margolis for her assistance with the preparation and submission of this manuscript.


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FOOTNOTES
 
1 Current address: Department of Ophthalmology, Kure Medical Center, National Hospital Organization, Aoyama 3-1, Kure, Japan. Back

Received August 28, 2007; revised November 19, 2007; accepted December 13, 2007.


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REFERENCES
 
    1
  1. Ljunggren, H. G., Karre, K. (1990) In search of the ‘missing self’: MHC molecules and NK cell recognition Immunol. Today 11,237-244[CrossRef][Medline]
    2. 2
  2. Anderson, S. K., Ortaldo, J. R., McVicar, D. W. (2001) The ever-expanding Ly49 gene family: repertoire and signaling Immunol. Rev. 181,79-89[CrossRef][Medline]
    3. 3
  3. Raulet, D. H., Held, W., Correa, I., Dorfman, J. R., Wu, M. F., Corral, L. (1997) Specificity, tolerance and developmental regulation of natural killer cells defined by expression of class I-specific Ly49 receptors Immunol. Rev. 155,41-52[CrossRef][Medline]
    4. 4
  4. Ortaldo, J. R., Winkler-Pickett, R., Mason, A. T., Mason, L. H. (1998) The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells J. Immunol. 160,1158-1165[Abstract/Free Full Text]
    5. 5
  5. Skold, M., Stenstrom, M., Sidobre, S., Hoglund, P., Kronenberg, M., Cardell, S. (2003) MHC-dependent and -independent modulation of endogenous Ly49 receptors on NK1.1+ T lymphocytes directed by T-cell receptor type Immunology 110,313-321[CrossRef][Medline]
    6. 6
  6. Ikarashi, Y., Mikami, R., Bendelac, A., Terme, M., Chaput, N., Terada, M., Tursz, T., Angevin, E., Lemonnier, F. A., Wakasugi, H., Zitvogel, L. (2001) Dendritic cell maturation overrules H-2D-mediated natural killer T (NKT) cell inhibition: critical role for B7 in CD1d-dependent NKT cell interferon {gamma} production J. Exp. Med. 194,1179-1186[Abstract/Free Full Text]
    7. 7
  7. Maeda, M., Lohwasser, S., Yamamura, T., Takei, F. (2001) Regulation of NKT cells by Ly49: analysis of primary NKT cells and generation of NKT cell line J. Immunol. 167,4180-4186[Abstract/Free Full Text]
    8. 8
  8. Niederkorn, J. Y. (1990) Immune privilege and immune regulation in the eye Adv. Immunol. 48,191-226[Medline]
    9. 9
  9. Streilein, J. W. (2003) Ocular immune privilege: therapeutic opportunities from an experiment of nature Nat. Rev. Immunol. 3,879-889[CrossRef][Medline]
    10. 10
  10. Wang, Y., Goldschneider, I., O'Rourke, J., Cone, R. E. (2001) Blood mononuclear cells induce regulatory NK T thymocytes in anterior chamber-associated immune deviation J. Leukoc. Biol. 69,741-746[Abstract/Free Full Text]
    11. 11
  11. Faunce, D. E., Sonoda, K-H., Stein-Streilein, J. (2001) MIP-2 recruits NKT cells to the spleen during tolerance induction J. Immunol. 166,313-321[Abstract/Free Full Text]
    12. 12
  12. D'Orazio, T. J., Niederkorn, J. Y. (1998) Splenic B cells are required for tolerogenic antigen presentation in the induction of anterior chamber-associated immune deviation (ACAID) Immunology 95,47-55[CrossRef][Medline]
    13. 13
  13. Sonoda, K-H., Stein-Streilein, J. (2002) CD1d on antigen-transporting APC and splenic marginal zone B cells promotes NKT cell-dependent tolerance Eur. J. Immunol. 32,848-857[CrossRef][Medline]
    14. 14
  14. Wilbanks, G. A., Streilein, J. W. (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 71,383-389[Medline]
    15. 15
  15. Sonoda, K-H., Exley, M., Snapper, S., Balk, S., Stein-Streilein, J. (1999) CD1 reactive NKT cells are required for development of systemic tolerance through an immune privileged site J. Exp. Med. 190,1215-1225[Abstract/Free Full Text]
    16. 16
  16. Andrew, S. M., Titus, J. A. (2001) Pilot fragmentation of IgG to F(ab')2 using pepsin Current Protocols in Immunology, Suppl. 21, Unit 2.8 ,2.8.4 John Wiley & Sons New York, NY, USA.
    17. 17
  17. Streilein, J. W., Niederkorn, J. Y. (1981) Induction of anterior chamber-associated immune deviation requires an intact, functional spleen J. Exp. Med. 153,1058-1067[Abstract/Free Full Text]
    18. 18
  18. Sonoda, Y., Streilein, J. W. (1993) Impaired cell-mediated immunity in mice bearing healthy orthotopic corneal allografts J. Immunol. 150,1727-1734[Abstract]
    19. 19
  19. Roelofs-Haarhuis, K., Wu, X., Gleichmann, E. (2004) Oral tolerance to nickel requires CD4(+) invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells J. Immunol. 173,1043-1050[Abstract/Free Full Text]
    20. 20
  20. Nakamura, T., Sonoda, K. H., Faunce, D. E., Gumperz, J., Yamamura, T., Miyake, S., Stein-Streilein, J. (2003) CD4+ NKT cells, but not conventional CD4+ T cells, are required to generate efferent CD8+ T regulatory cells following antigen inoculation in an immune privileged site J. Immunol. 171,1266-1271[Abstract/Free Full Text]
    21. 21
  21. Sonoda, K. H., Faunce, D. E., Taniguchi, M., Exley, M., Balk, S., Stein-Streilein, J. (2001) NK T cell-derived IL-10 is essential for the differentiation of antigen-specific T regulatory cells in systemic tolerance J. Immunol. 166,42-50[Abstract/Free Full Text]
    22. 22
  22. Sonoda, K-H., Faunce, D. E., Taniguchi, M., Exley, M., Balk, S., Stein-Streilein, J. (2001) NKT cell-derived IL-10 is essential for the differentiation of antigen-specific T regulatory cells in systemic tolerance J. Immunol. 166,42-50[Abstract/Free Full Text]
    23. 23
  23. Yamada, J., Streilein, J. W. (1997) Induction of anterior chamber-associated immune deviation by corneal allografts placed in the anterior chamber Invest. Ophthalmol. Vis. Sci. 38,2833-2843[Abstract/Free Full Text]
    24. 24
  24. Niederkorn, J. Y., Mellon, J. (1996) Anterior chamber-associated immune deviation promotes corneal allograft survival Invest. Ophthalmol. Vis. Sci. 37,2700-2707[Abstract/Free Full Text]
    25. 25
  25. Sonoda, A., Sonoda, Y., Muramatu, R., Streilein, J. W., Usui, M. (2000) ACAID induced by allogeneic corneal tissue promotes subsequent survival of orthotopic corneal grafts Invest. Ophthalmol. Vis. Sci. 41,790-798[Abstract/Free Full Text]
    26. 26
  26. D'Orazio, T. J., Niederkorn, J. Y. (1998) A novel role for TGF-β and IL-10 in the induction of immune privilege J. Immunol. 160,2089-2098[Abstract/Free Full Text]
    27. 27
  27. Keino, H., Masli, S., Sasaki, S., Streilein, J. W., Stein-Streilein, J. (2006) CD8* T regulatory cells use a novel genetic program that includes CD103 to suppress Th1 immunity in eye-derived tolerance Invest. Ophthalmol. Vis. Sci. 47,1533-1543[Abstract/Free Full Text]
    28. 28
  28. Keino, H., Takeuchi, M., Kezuka, T., Hattori, T., Usui, M., Taguchi, O., Streilein, J. W., Stein-Streilein, J. (2006) Induction of eye-derived tolerance does not depend on naturally occurring CD4+CD25+ T regulatory cells Invest. Ophthalmol. Vis. Sci. 47,1047-1055[Abstract/Free Full Text]
    29. 29
  29. Grutz, G. (2005) New insights into the molecular mechanism of interleukin-10-mediated immunosuppression J. Leukoc. Biol. 77,3-15[Abstract/Free Full Text]
    30. 30
  30. Long, E. O. (1999) Regulation of immune responses through inhibitory receptors Annu. Rev. Immunol. 17,875-904[CrossRef][Medline]
    31. 31
  31. Billadeau, D. D., Leibson, P. J. (2002) ITAMS versus ITIMs: striking a balance during cell regulation J. Clin. Invest. 109,161-168[CrossRef][Medline]
    32. 32
  32. Benlagha, K., Weiss, A., Beavis, A., Teyton, L., Bendelac, A. (2000) In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers J. Exp. Med. 191,1895-1903[CrossRef][Medline]
    33. 33
  33. Taniguchi, M., Seino, K., Nakayama, T. (2003) The NKT cell system: bridging innate and acquired immunity Nat. Immunol. 4,1164-1165[CrossRef][Medline]
    34. 34
  34. Kronenberg, M., Gapin, L. (2002) The unconventional lifestyle of NKT cells Nat. Rev. Immunol. 2,557-568[Medline]
    35. 35
  35. Seino, K., Taniguchi, M. (2005) Functionally distinct NKT cell subsets and subtypes J. Exp. Med. 202,1623-1626[Abstract/Free Full Text]
    36. 36
  36. Ferguson, T. A., Herndon, J., Elzey, B., Griffith, T. S., Schoenberger, S., Green, D. R. (2002) Uptake of apoptotic antigen-coupled cells by lymphoid dendritic cells and cross-priming of CD8+ T cells produce active immune unresponsiveness J. Immunol. 168,5589-5595[Abstract/Free Full Text]
    37. 37
  37. Taylor, A., Verhagen, J., Blaser, K., Akdis, M., Akdis, C. A. (2006) Mechanisms of immune suppression by interleukin-10 and transforming growth factor-β: the role of T regulatory cells Immunology 117,433-442[CrossRef][Medline]
    38. 38
  38. Lucas, M., Zhang, X., Prasanna, V., Mosser, D. M. (2005) ERK activation following macrophage Fc{gamma}R ligation leads to chromatin modifications at the IL-10 locus J. Immunol. 175,469-477[Abstract/Free Full Text]
    39. 39
  39. Won, W. J., Foote, J. B., Odom, M. R., Pan, J., Kearney, J. F., Davis, R. S. (2006) Fc receptor homolog 3 is a novel immunoregulatory marker of marginal zone and B1 B cells J. Immunol. 177,6815-6823[Abstract/Free Full Text]
    40. 40
  40. Barrow, A.D., Trowsdale, J. (2006) You say ITAM and I say ITIM, let’s call the whole thing off: the ambiguity of immunoreceptor signaling Eur. J. Immunol. 36,1646-1653[CrossRef][Medline]




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