Role of SLAM in NKT Cell Development Revealed by Transgenic Complementation in NOD Mice This information is current as Margaret A. Jordan, Julie M. Fletcher, Roby Jose, Shahead of September 26, 2021. Chowdhury, Nicole Gerlach, Janette Allison and Alan G. Baxter J Immunol 2011; 186:3953-3965; Prepublished online 25 February 2011; doi: 10.4049/jimmunol.1003305 Downloaded from http://www.jimmunol.org/content/186/7/3953 References This article cites 82 articles, 44 of which you can access for free at: http://www.jimmunol.org/content/186/7/3953.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 26, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Role of SLAM in NKT Cell Development Revealed by Transgenic Complementation in NOD Mice Margaret A. Jordan,* Julie M. Fletcher,* Roby Jose,* Shahead Chowdhury,* Nicole Gerlach,* Janette Allison,† and Alan G. Baxter* Allelic variation of SLAM expression on CD4+CD8+ thymocytes has been proposed to play a major role in NKT cell development. In this article, this hypothesis is tested by the production of subcongenic mouse strains and Slamf1 transgenic lines. The long isoform of the C57BL/6 allele of Slamf1 was transgenically expressed on CD4+CD8+ thymocytes under control of an hCD2 minigene. NOD.Nkrp1b.Tg(Slamf1)1 mice, which had a 2-fold increase in SLAM protein expression on CD4+CD8+ thymocytes, had a 2-fold increase in numbers of thymic NKT cells. The additional thymic NKT cells in NOD.Nkrp1b.Tg(Slamf1)1 mice were relatively immature, with a similar subset distribution to those of congenic NOD.Nkrp1b.Nkt1 and NOD.Nkrp1b.Slamf1 mice, which also express increased levels of SLAM on CD4+CD8+ thymocytes and produce larger numbers of NKT cells. Transgenic Downloaded from enhancement of SLAM expression also increased IL-4 and IL-17 production in response to TCR-mediated stimulation. Paradox- ically, NOD.Nkrp1b.Tg(Slamf1)2 mice, which had a 7-fold increase in SLAM expression, showed no significant increase in NKT cells numbers; on the contrary, at high transgene copy number, SLAM expression levels correlated inversely with NKT cell numbers, consistent with a contribution to negative selection. These data confirm a role for SLAM in controlling NKT cell development and are consistent with a role in both positive and negative thymic selection of NKT cells. The Journal of Immunology, 2011, 186: 3953–3965. http://www.jimmunol.org/ ype 1 NKT cells are a population of immunoregulatory to the marine sponge-derived glycolipid a-galactosylceramide T cells that can control the strength and character of (a-GalCer) when presented in the context of CD1d (12). This T responses against viruses, bacteria, fungi, parasites, tu- latter characteristic has been used to good advantage within the mors, allografts, and autologous tissues (reviewed in Refs. 1, 2). development of highly specific fluorescent CD1d/a-GalCer tet- Unlike conventional T cells, NKT cells exhibit various NK cell ramers, which have become the gold standard for the flow cyto- characteristics, including CD161c (NK1.1 in mice), and express metric identification of type 1 NKT cells (13, 14). a semi-invariant TCR consisting of an invariant Va24-Ja18 Upon activation, NKT cells rapidly respond with vigorous by guest on September 26, 2021 (Va14-Ja18 in mice) chain coupled to Vb11 (Vb8.2, Vb7, or production of a diverse range of cytokines, including IFN-g, TNF, Vb2 in mice) (3–5). The NKT-associated TCR binds CD1d, a b2- IL-4, IL-13, IL-10, and IL-17 (15). The contribution of NKT cells microglobulin–dependent integral membrane glycoprotein with to IL-4 production cannot be overstated: they are responsible for homology to MHC classes I and II (6, 7). The Ag-binding cleft of the majority of the IL-4 produced after anti-CD3 i.v. injection, CD1d is highly hydrophobic and presents glycolipids, such as they can produce IL-4 in the absence of IL-4 priming, and they glycosylceramides (8), GPI (8–10), and the lysosomal glyco- can do so within 30 min of TCR ligation (1). NKT cells can para- sphingolipid, iGb3 (11). Type 1 NKT cells are uniformly reactive doxically both suppress and promote cell-mediated immunity; although it is unclear how this dual functionality is regulated, the dominant hypothesis is that functionally different subsets are re- *Comparative Genomics Centre, James Cook University, Townsville, Queensland 4811, Australia; and †St. Vincent’s Institute, Melbourne, Victoria 2480, Australia sponsible for different activities (16). Received for publication October 6, 2010. Accepted for publication January 26, In ontogeny, NKT cells branch from the developmental pathway 2011. of conventional T cells at the CD4+CD8+ double-positive (DP) This work was supported by the Australian National Health and Medical Research stage after generation of the canonical Va14Ja18 TCR. These Council and intramural funding from James Cook University and James Cook Uni- versity Faculty of Medicine, Health and Molecular Sciences. A.G.B. is supported by cells are positively selected by CD1d-expressing DP thymocytes, a Senior Research Fellowship from the Australian National Health and Medical Re- which is in contrast with the selection of conventional T cells search Council. on thymic epithelial cells (17–20). This positive selection event M.A.J. and J.A. designed and produced the transgenic construct and performed requires ligation of both the TCR and a costimulatory molecule, genetic analyses; M.A.J., J.M.F., R.J., and S.C. performed flow cytometry and cel- lular assays; N.G. and J.M.F. performed tissue culture and cytokine assays and such as SLAM, that signals via SAP and the downstream Src generated figures; M.A.J., J.M.F., and N.G. analyzed results, contributed text, and kinase FYN; mice deficient in either SAP or FYN display severe produced figures; and A.G.B. contributed experimental design, analysis, and figures, and wrote the final draft. NKT cell defects (21–25, reviewed in Ref. 16). Address correspondence and reprint requests to Prof. Alan G. Baxter, Comparative The earliest NKT cells identified by binding of CD1d/a-GalCer + 2 2 2 Genomics Centre, Molecular Sciences Building 21, James Cook University, Towns- tetramers are CD4 CD8 NK1.1 and are precursors of the CD4 ville, Queensland 4811, Australia. E-mail address: [email protected] CD82NK1.12 and CD4+NK1.1+ subsets. Expression of NK1.1 is Abbreviations used in this article: a-DP, double-positive; GalCer, glycolipid a-gal- not required for migration to the periphery because the majority of actosylceramide; Klrb, killer cell lectin-like receptor subfamily B; LCR, locus con- 2 2 + trol region; SP, single-positive; SSLP, simple sequence length polymorphism; T1D, recent thymic emigrants are NK1.1 . Both NK1.1 and NK1.1 type 1 diabetes; TESS, Transcription Element Search Software; UTR, untranslated NKT cells can rapidly produce IL-4 and IFN-g on stimulation; region. NK1.1+ NKT cells produce high levels of both cytokines, whereas 2 Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 the NK1.1 subset produces high levels of IL-4 and relatively www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003305 3954 ROLE OF SLAM IN NKT CELLS lower levels of IFN-g (17). More recently, a third major subset has versity under specific pathogen-free conditions. The NOD.Nkrp1b strain been identified in lymph nodes. It is CD42NK1.12 and on acti- carries B6-derived alleles at the NK complex on chromosome 6 (from vation secretes large amounts of IL-17 (15). D6mit105 to D6mit135), permitting the use of the NK1.1 marker (32, 33). NOD.Nkrp1b.Nkt1 mice were originally produced by intercrossing NOD. NOD mice are susceptible to multiple autoimmune diseases, Nkrp1b and C57BL/6 mice, and performing serial backcrosses to NOD. including type 1 diabetes (T1D) (26), Sjo¨gren’s syndrome (27), Nkrp1b to N10, before intercrossing and selection of homozygous con- hemolytic anemia (28), systemic lupus erythematosus (29, 30), genic founders, as previously described (39). These studies have been and experimental autoimmune encephalomyelitis (31). The leu- reviewed and approved by the James Cook University institutional Animal Care and Ethics Committee. kocytes of the widely distributed NOD/Lt strain do not react with the NK1.1 Ab PK136, which binds two members of the killer cell Cell suspension preparation lectin-like receptor subfamily B (Klrb) type II transmembrane C- Thymocyte and splenocyte cell suspensions were prepared by gently type lectin receptors, Klrb1c/NKR-P1C and Klrb1b/NKR-P1B. A grinding the organ between frosted microscope slides in FACS buffer (PBS congenic NOD.Nkrp1b line was produced, which expresses NK1.1 containing 2 mM EDTA [Amresco, Solon, OH], 10% [v/v] bovine serum by virtue of carrying the C57BL/6 allele of Klrb1c, to characterize [Invitrogen, Melbourne, Victoria, Australia], and 0.02% [w/v] sodium NK and NKT cell deficiencies in NOD mice (32, 33). azide). Spleen cell suspensions were treated with RBC lysing buffer (Sigma Aldrich, Castle Hill, New South Wales, Australia).