1 Scientific RepoRts | 6:27375 | DOI: 10.1038/srep27375 www.nature.com/scientificreports Mutation of the Traj18 gene segment using tALeNs to generate Natural Killer T cell defcient mice Jingjing Zhang 1 , Romain Bedel 1,† , s. Harsha Krovi 1 , Kathryn D. tuttle 1 , Bicheng Zhang 2 , James Gross 2 , Laurent Gapin 1,2 & Jennifer L. Matsuda 2 Invariant Natural Killer t (iNKt) cells are a unique subset of t lymphocytes that have been implicated in both promoting and suppressing a multitude of immune responses. In mice, iNKt cells express t cell antigen receptors (tCRs) comprising a unique tCRα rearrangement between the Trav11 and Traj18 gene segments. When paired with certain trbv tCRβ chains, these tCRs recognize lipid antigens presented by the major histocompatibility complex (MHC) class I-like molecule, CD1d. Until recently, the sole model of iNKT defciency targeted the Jα18, which is absolutely required to form the TCR with the appropriate antigenic specifcity. However, these mice were demonstrated to have a large reduction in TCR repertoire diversity, which could confound results arising from studies using these mice. Here, we have created a new NKT-defcient mouse strain using transcription activator-like efector nuclease (TALEN) technology to only disrupt the expression of Jα18, leaving the remaining Jα repertoire unperturbed. We confrm that these mice lack iNKT cells and do not respond to lipid antigen stimulation while the development of conventional t cells, regulatory t cells, and type Ib NKt cells is normal. this new mouse strain will serve as a new model of iNKT cell defciency to facilitate our understanding of iNKT biology. Although the majority of αβ TCR + T cells recognize peptide antigens presented by conventional polymorphic MHC I or MHC II molecules, a fraction of T cells deviates from this rule. Instead, these αβ T cells recognize lipid or glycolipid antigens presented by members of the monomorphic molecules of the CD1 family, or they recognize microbial ribofavin precursor derivatives presented by the monomorphic MR1 molecules 1 . Of these, the most extensively studied lipid-reactive T cells are the natural killer T (NKT) cells, which detect a number of glycolipid antigens in association with CD1d. Two broad classes of NKT cells have been defned on the basis of TCR expression and antigen reactivity 2 . Most studies of these cells focus on type I, or iNKT cells (for invariant NKT cells), which are the most prevalent NKT cells in mice 3 . iNKT cells express a TCR that is the product of a canonical rearrangement between the Trav11 (Vα14) gene segment (Trav10 or Vα24 in human) and the Traj18 (Jα18) gene segment, with a CDR3α region invariant at the amino acid level 4,5 . Tis Vα14 invariant chain is co-expressed with a limited set of Vβ chains, predominantly Trbv13-2 (Vβ8.2), Trbv29 (Vβ7) and Trbv1 (Vβ2) in mice and Trbv25-1 (Vβ11) in humans 4–7 . iNKT cells expressing these TCRs recognize several microbe-derived glycosphingolipid 8,9 and diacylglycerol anti- gens 10 , including the prototypical glycosphingolipid antigen α-galactosylceramide (αGC) 11,12 , and can be iden- tifed using CD1d tetramers loaded with this antigen 13,14 . Because of their unique ability to rapidly and potently secrete cytokines and infuence downstream responses 15 , iNKT serve as an important link between the innate and adaptive immune systems and are ofen regarded as potential therapeutic targets. Studies have linked iNKT cell defects with increased susceptibility to a wide range of disease processes including autoimmunity 16 , cancer 17 , and even obesity 18 . Additionally, the potential for therapies involving iNKT cells is especially attractive since the use of αGC to activate iNKT cells has proven safe in humans 19–21 . Ever emerging roles for this unconventional subset of lymphocytes make the study of their development and regulation both relevant and signifcant. In addition to iNKT cells, there exists other CD1d-reactive T cells, which do not express the invariant Vα 14-Jα18 TCR. Tese cells were frst described when Cardell and colleagues examined the TCR usage of T cell 1 Department of immunology and Microbiology, University of colorado Denver School of Medicine and national Jewish Health, Aurora, CO 80206, USA. 2 Department of Biomedical Research, national Jewish Health, Denver, cO 80206, USA. † Present address: Ludwig Center for Cancer Research, University of Lausanne, CH-1066 Epalinges, Switzerland. Correspondence and requests for materials should be addressed to L.G. (email: Laurent.gapin@ ucdenver.edu) Received: 11 March 2016 accepted: 13 May 2016 Published: 03 June 2016 opeN