NK T Cell-Derived IL-10 Is Essential for the Differentiation of Antigen-Specific T Regulatory Cells in Systemic Tolerance 1 Koh-Hei Sonoda,* Douglas E. Faunce,* Masaru Taniguchi, ² Mark Exley, ‡ Steven Balk, ‡ and Joan Stein-Streilein 2 * § 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 subpopu- lation, required for development of systemic tolerance, expresses the invariant Va14Ja281 TCR because Ja281 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 Ja281 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. The Journal of Immunology, 2001, 166: 42–50. T olerance 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 reg- ulated 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 reg- ulatory (Tr) 3 cells that suppress effector mechanisms are the out- come 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 –13). ACAID is characterized by a selective deficiency in de- layed-type hypersensitivity (DTH) and Ig isotypes that fix com- plement (14, 15). Central to the ACAID process are intraocular bone marrow-derived F4/80 1 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-gen- erated CD8 1 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 lympho- cytes that coexpress the TCR ab-chain and NK markers (20). A major subpopulation of NK T cells express a unique invariant Va14Ja281 Ag receptor not expressed by conventional T cells (20 –24). Similarly, NK T cells exists in the human and express the invariant Va24JaQ TCRa-chain (25, 26). NK T cells are restricted by MHC class I-like CD1d molecules (27–29), 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 –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 mecha- nism 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 cyto- kines 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 –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 ab- solutely required for the induction of Ag-specific Tr cells follow- ing the inoculation of Ag into the eye. Materials and Methods 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 *Schepens Eye Research Institute, Harvard Medical School, Boston MA 02114; ² Core Research and Evolutional Science and Technology Project (CREST), and De- partment of Molecular Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan; ‡ Cancer Biology Program, Hematology/Oncology Division, Beth Isra- el-Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and § Pul- monary and Critical Care Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115 Received for publication May 25, 2000. Accepted for publication September 27, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported 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. 2 Address correspondence and reprint requests to Dr. Joan Stein-Streilein, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. E-mail address: jstein@vision.eri.harvard.edu 3 Abbreviations used in this paper: Tr, T regulatory; a.c., anterior chamber; ACAID, Anterior Chamber-Associated Immune Deviation; DTH, delayed-type hypersensitiv- ity; DC, dendritic cell; KO, knockout; LAT, local adoptive transfer; PEC, peritoneal exudate cell; RPA, RNase protection assay; WT, wild type. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00