View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Immunity Article Agonist-Selected T Cell Development Requires Strong T Cell Receptor Signaling and Store-Operated Calcium Entry Masatsugu Oh-hora,1,2,3,4,* Noriko Komatsu,1,3 Mojgan Pishyareh,2 Stefan Feske,5 Shohei Hori,6 Masaru Taniguchi,7 Anjana Rao,2,9,* and Hiroshi Takayanagi1,3,8,10 1Department of Cell Signaling, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan 2Department of Pathology, Immune Disease Institute and Program in Cellular and Molecular Medicine, Children’s Hospital, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA 3Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan 4Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO) program, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan 5Department of Pathology, New York University, School of Medicine, New York, NY 10016, USA 6Research Unit for Immune Homeostasis 7Laboratory for Immune Regulation RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan 8Japan Science and Technology Agency (JST), Explorative Research for Advanced Technology (ERATO) program, Takayanagi Osteonetwork Project, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan 9Present address: Division of Signaling and Gene Expression, La Jolla Institute for Allergy & Immunology, La Jolla, CA 92037, USA 10Present address: Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan *Correspondence: [email protected] (M.O.), [email protected] (A.R.) http://dx.doi.org/10.1016/j.immuni.2013.02.008 SUMMARY pMHC) interactions. In conventional TCRab+ T cell development, weak or absent TCR-pMHC interactions cannot support thymo- T cell receptor (TCR) signaling driven by interaction cyte survival, leading to death by neglect. Moderate-affinity of the TCR with specific complexes of self-peptide TCR-pMHC interactions lead to the development of functional and the major histocompatibility complex deter- T cells through positive selection. High-affinity TCR-pMHC inter- mines T cell fate in thymic development. However, actions normally trigger apoptosis of self-reactive thymocytes the signaling pathway through which TCR signal through negative selection. However, some self-reactive thymo- strength regulates distinct T cell lineages remains cytes mature into unconventional T-lineage cells through an alternative selection process defined as agonist selection (Bald- unknown. Here we have used mice lacking the endo- 2+ win et al., 2004; Stritesky et al., 2012). Agonist-selected uncon- plasmic reticulum Ca sensors stromal interaction ventional T cell subsets are thought to have a regulatory role in molecule 1 (STIM1) and STIM2 to show that STIM- immune system and are classified into three main cell types: 2+ induced store-operated Ca entry is not essential forkhead box P3 (Foxp3)+ regulatory T (Treg) cells, invariant + for thymic development of conventional TCRab natural killer T (iNKT) cells, and TCRab+ CD8aa+ intestinal intra- T cells but is specifically required for the develop- epithelial lymphocytes (IELs) (Hsieh et al., 2012; Kronenberg and ment of agonist-selected T cells (regulatory T cells, Gapin, 2002; Lambolez et al., 2007). It has been proposed that invariant natural killer T cells, and TCRab+ CD8aa+ these T cells require relatively strong and sustained TCR signals intestinal intraepithelial lymphocytes). The severe for their development (Baldwin et al., 2004). Although this affinity impairment of agonist-selected T cell development model is acknowledged, there remains a longstanding question is mainly due to a defect in interleukin-2 (IL-2) or concerning how the TCR signal strength and duration regulates the development of these distinct T cell subsets. IL-15 signaling. Thus, STIM1 and STIM2-mediated 2+ Engagement of TCR-pMHC activates several protein tyrosine store-operated Ca influx, leading to efficient kinases and ultimately phospholipase C (PLC)-g1. Activated activation of NFAT (nuclear factor of activated PLC-g1 hydrolyzes phosphatidylinositol 4,5-bisphosphate into T cells), is critical for the postselection maturation diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3), of agonist-selected T cells. which induces the release of Ca2+ from the endoplasmic retic- ulum (ER). In turn, ER store depletion triggers store-operated INTRODUCTION Ca2+ entry, the predominant mechanism for sustained increase 2+ 2+ of intracellular free Ca ([Ca ]i) downstream of the TCR. Thymic selection depends on the affinity of T cell receptor- Store-operated Ca2+ entry leads to activation of the phospha- peptide major histocompatibility complex glycoprotein (TCR- tase calcineurin, which in turn activates the transcription factor Immunity 38, 881–895, May 23, 2013 ª2013 Elsevier Inc. 881 Immunity T Cell Development and Calcium Signaling A C 220 Ca2+ WT 1KO 4 DKO 4 4 10 10 10 10.7 84.5 10.3 83.6 13.3 80.3 3 3 μ 1200 3 10 TG (1 M) 10 10 1200 2 2 10 2 10 (nM) WT 10 (nM) i i 800 ] 800 ] 2+ 1 2+ 1 1 10 CD4 10 10 400 400 [Ca [Ca 0 3.7 10 0 4.9 10 0 3.5 10 0 1 2 3 4 DKO * 0 1 2 3 4 10 10 10 10 10 10 0 10 1 102 103 104 10 10 10 10 10 CD8 0 0 0 200 400 600 WT DKO B 0Ca2 2+ WT 1KO DKO WT 1KO DKO 800 Bio- S. Avidin WT 1200 STIM1 STIM2 600 αCD3 (nM) i ] (nM) 800 i ] 2+ 400 2+ [Ca 200 400 DKO [Ca *** Actin Actin 0 0 0 200 400 600 WT DKO Time (s) D Male HY TCR tg E PBS SEB 30 Lymph nodes 25 4 Thymus 4 10 10 20 20 3 3 15 10 10 Control 58.8 10 34.6 10 10 2 1.25 10 2 13.4 Control 5 0 Gated on 10 1 10 1 0 10 0 10 1 102 103 104 10 0 10 1 102 103 104 50 + + 25 β 10 0 10 0 CD4 TCRV 8 10 0 10 1 102 103 104 10 0 10 1 102 103 104 4 4 40 10 10 20 30 15 3 3 10 10 DKO 20 71.7 59.4 DKO 10 2 2 10 2.86 10 15.2 10 5 0 0 1 1 10 10 10 0 10 1 102 103 104 10 0 10 1 102 103 104 CD24 10 0 10 0 CD4 10 0 10 1 102 103 104 10 0 10 1 102 103 104 CD8 Vβ8Vβ6 80 NS 80 NS Thymus LNs 3 20 NS ** NS 60 60 15 ** 2 40 40 10 T cells (%) immature cells (%) immature cells (%) T cells (%) 20 20 1 high high high high 5 CD8 CD8 0 0 CD24 00 CD24 PBS SEB PBS SEB PBS SEB PBS SEB DKO DKO Control Control Control DKO Control DKO F Female HY TCR tg 4 Thymus 4Lymph nodes 10 10 600 Thymus Lymph nodes 3 3 30 80 10 10 400 Control 10 2 11.5 10 2 37.1 60 200 20 10 1 10 1 *** 40 0 0 0 ** 10 10 T cells (%) NS 10 0 10 1 102 103 104 T cells (%) 10 0 10 1 102 103 104 10 0 10 1 102 103 104 4 4 ** 10 10 high high 10 20 300 3 3 10 10 CD8 CD8 DKO 200 0 0 10 2 8.0 10 2 17.6 100 10 1 10 1 DKO DKO DKO DKO Control Control Control Control 0 10 0 10 0 Cell number 0 1 2 3 4 CD4 10 10 10 10 10 10 0 10 1 102 103 104 10 0 10 1 102 103 104 6w 8w 6w 8w CD8 T3.70 (legend on next page) 882 Immunity 38, 881–895, May 23, 2013 ª2013 Elsevier Inc. Immunity T Cell Development and Calcium Signaling NFAT (Feske, 2007; Hogan et al., 2010). The induction of store- tion of IL-15 partially rescued differentiation into TCRab+ operated Ca2+ entry is controlled by two major molecules, the CD8aa+ IELs. These results suggest that postselection matura- ER Ca2+ sensor stromal interaction molecule 1 (STIM1) (Liou tion of all agonist-selected precursors requires continuous high et al., 2005; Roos et al., 2005) and calcium release-activated activity of NFAT by TCR-mediated store-operated Ca2+ entry, calcium (CRAC) channels ORAI1 (Feske et al., 2006; Vig et al., to induce efficient expression of downstream genes of NFAT 2006; Zhang et al., 2006). STIM1 is an acknowledged positive and NFAT-target transcription factors. regulator of store-operated CRAC channels. Loss of STIM1 abrogates TCR-induced store-operated Ca2+ entry and NFAT RESULTS activation, resulting in impaired proliferation and cytokine production by peripheral human and mouse T cells (McCarl Store-Operated Ca2+ Entry Is Dispensable for the et al., 2010; Oh-Hora et al., 2008; Picard et al., 2009). The related Development of Conventional TCRab+ T Cells protein STIM2 regulates sustenance of calcium entry and NFAT In both humans and mice, deficiency of STIM1 alone does not activation in mouse CD4+ T cells (Oh-Hora et al., 2008), but also influence the number of conventional TCRab+ T cells in the 2+ regulates basal concentration of [Ca ]i in HeLa cells (Brandman periphery (Beyersdorf et al., 2009; Picard et al., 2009) (data not et al., 2007). shown), suggesting that STIM2 may compensate for Ca2+ influx In thymocytes, TCR signal strength well correlates with magni- in thymocytes.