Original Article Genetic and Functional Analysis of the Nkt1 Locus Using Congenic NOD Mice Improved V14-NKT Cell Performance but Failure to Protect Against Type 1 Diabetes Ana-Claudia Rocha-Campos, 1 Rahma Melki, 2 Ren Zhu, 1 Nathalie Deruytter, 2 Diane Damotte, 2,3 Michel Dy, 1 Andre ´ Herbelin, 1 and Henri-Jean Garchon 2 Defective invariant natural killer T-cells (iNKT cells) have been implicated in the etiology of type 1 diabetes in nonobese diabetic (NOD) mice. In a genome scan of a cross between NOD and C57BL/6 mice, the most significant locus controlling the number of iNKT cells, referred to as Nkt1, was recently mapped to distal chromosome 1. Here, using congenic mice for this chromosomal segment, we defini- tively demonstrate the existence of Nkt1 and show that introgression of the C57BL/6 allele onto the NOD back- ground improves both the number of iNKT cells and their rapid production of cytokines elicited by -galactosylcer- amide treatment, explaining at least half of the difference between the NOD and C57BL/6 strains. Using new subcon- genic lines, we circumscribed the Nkt1 locus to a 8.7-cM segment, between the NR1i3 and D1Mit458 markers, that notably includes the SLAM (signaling lymphocytic activa- tion molecule) gene cluster, recently involved in murine lupus susceptibility. However, despite a significant correc- tion of the iNKT cell defect, the Nkt1 locus did not alter the course of spontaneous diabetes in congenic mice. Our findings indicate a complex relationship between iNKT cells and autoimmune susceptibility. Congenic lines none- theless provide powerful models to dissect the biology of iNKT cells. Diabetes 55:1163–1170, 2006 T he development of type 1 diabetes results from T-cell–mediated destruction of insulin-producing -cells in the pancreas (1). Expression of effec- tor lymphocytes seems to be caused in large part by a defect of immunoregulatory cells (2), among which invariant natural killer T-cells (iNKT cells) are currently the focus of considerable attention. This unique popula- tion of -T-cells shows an invariant T-cell antigen recep- tor (TCR) -chain, including V14J18 in the mouse and V24JQ in humans, and also expresses natural killer cell receptors (3). Most remarkably, these cells are capable of rapidly producing large amounts of cytokines on stimula- tion by glycolipid-type ligands that must be presented by the major histocompatibility complex class I–like CD1d molecule (4). Although -galatosylceramide (-GalCer), a glycolipid isolated from marine sponge, has long been used as a surrogate antigen for iNKT cells, their endoge- nous ligand has been only recently characterized (5). Remarkably, mice of the nonobese diabetic (NOD) strain, the well-established murine model of spontaneous type 1 diabetes (6), exhibit both a numerical and a functional defect of their iNKT cells as early as 3 weeks of age compared with nondiabetic strains (7–9). The protec- tive role of iNKT cells is strongly suggested by adoptive transfer experiments (10) and by the study of NOD mice transgenic for a V14-J18 TCR (11) or for CD1d overex- pressed in pancreatic islets (12). Conversely, diabetes is exacerbated in CD1d knockout NOD mice, which are devoid of iNKT cells (13,14). Finally, diabetes can be prevented by in vivo treatment with -GalCer (15,16), opening a path for the therapeutic manipulation of this cell subset. Interstrain differences suggest that the number and function of iNKT cells might be under genetic control (8). Recently, a genome-wide screen of a cross between NOD and C57BL/6 mice localized two main loci controlling iNKT cell number: Nkt1 on distal chromosome 1 and Nkt2 on chromosome 2 (17). Importantly, the Nkt2 locus over- lapped with the Idd13 locus for insulin-dependent diabe- tes (Idd) susceptibility, suggesting that the iNKT cell defect of NOD mice might be a genetically determined component of their diabetes susceptibility. Consistent with this finding, mice congenic for certain Idd loci, including Idd6, Idd9, and Idd13, showed a significant From the 1 Centre National de Recherche (CNRS) Unite ´ Mixte de Recherche (UMR) 8147, Universite ´ Paris 5, Paris, France; the 2 Institut National de la Sante ´ et de la Recherche Me ´ dicale (INSERM) U580, Universite ´ Paris 5, Paris, France; and 3 Ho ˆ pital Europe ´ en George Pompidou, Paris, France. Address correspondence and reprint requests to Henri-Jean Garchon, INSERM U580, Ho ˆ pital Necker, 161 rue de Se ` vres, 75743 Paris Cedex 15, France. E-mail: garchon@necker.fr. Or Andre ´ Herbelin, CNRS INSERM 8147, Ho ˆ pital Necker, 161 rue de Se ` vres, 75743 Paris Cedex 15, France. E-mail: herbelin@necker.fr. Received for publication 15 July 2005 and accepted in revised form 3 January 2006. A.-C.R.-C. and R.M. contributed equally to this work. APC, allophycocyanin; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; -GalCer, -galatosylceramide; IFN-, -interfer- on; IL, interleukin; iNKT cell, invariant natural killer T-cell; mAb, monoclonal antibody; SLAM, signaling lymphocytic activation molecule; TCR, T-cell antigen receptor. © 2006 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. 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