Proximal Lck Promoter−Driven Cre Function Is Limited in Neonatal and Ineffective in Adult δγ T Cell Development This information is current as Gina J. Fiala, Anna-Maria Schaffer, Katja Merches, Anna of September 27, 2021. Morath, Jeremy Swann, Laurenz A. Herr, Miriam Hils, Charlotte Esser, Susana Minguet and Wolfgang W. A. Schamel J Immunol 2019; 203:569-579; Prepublished online 5 June 2019; Downloaded from doi: 10.4049/jimmunol.1701521 http://www.jimmunol.org/content/203/2/569 Supplementary http://www.jimmunol.org/content/suppl/2019/06/04/jimmunol.170152 http://www.jimmunol.org/ Material 1.DCSupplemental References This article cites 76 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/203/2/569.full#ref-list-1 Why The JI? Submit online. by guest on September 27, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • 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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Proximal Lck Promoter–Driven Cre Function Is Limited in Neonatal and Ineffective in Adult gd T Cell Development Gina J. Fiala,*,†,‡,x Anna-Maria Schaffer,*,†,‡ Katja Merches,{ Anna Morath,*,†,‡,‖ Jeremy Swann,# Laurenz A. Herr,*,†,‡ Miriam Hils,†,‡ Charlotte Esser,{ Susana Minguet,*,†,‡,** and Wolfgang W. A. Schamel*,†,‡,** During T cell development, Lck gene expression is temporally controlled by its proximal and distal promoters. The pLckCre transgenic mouse available from The Jackson Laboratory, in which the proximal promoter of Lck drives Cre expression, is a commonly used Cre driver line to recombine genes flanked by loxP sites in T cells. pLckCre drives recombination early in thymocyte development and is frequently used to delete genes in ab and gd T cells. We found that pLckCre failed to efficiently delete floxed genes in gd T cells in contrast to a complete deletion in conventional as well as unconventional ab T cells. Mechanistically, gd T cells inefficiently transcribed the endogenous proximal Lck promoter compared with ab T cells during Downloaded from adult thymic development. A small population of gd T cells that had activated pLckCre was detected, many of which were located in nonlymphoid organs as well as precommitted IL-17– or IFN-g–producing gd T effector cells. In newborn thymi, both pLckCre and endogenous Lck proximal promoter expression were substantially enhanced, giving rise to an elevated fraction of gd T cells with recombined floxed genes that were increased in unique gd T subsets, such as the IL-17–producing gd T cells. Our data point out striking differences in Lck transcription between perinatal and adult gd T cell development. Taken together, the data presented in this study shed new light on gd T cell development and stimulate a reanalysis of data generated using the pLckCre http://www.jimmunol.org/ transgenic mice. The Journal of Immunology, 2019, 203: 569–579. he use of conditional knockout (ko) mice, in which the in vivo. The gene of interest is flanked by loxP sites, called a floxed gene of interest is deleted only in certain cell types, has gene, and its recombination is mediated by a Cre recombinase T substantially advanced the analysis of gene function (Cre) expressed under the control of a cell type–specific promoter (1). This strategy enables a detailed analysis of gene function in a given cell type while leaving the remaining organism unaffected *Centre for Biological Signalling Studies BIOSS, University of Freiburg, 79104 Freiburg, by guest on September 27, 2021 Germany; †Center for Chronic Immunodeficiency, Medical Center Freiburg and Faculty of (reviewed in Ref. 2). Medicine, University of Freiburg, 79106 Freiburg, Germany; ‡Institute Biology III, Faculty To generate T cell–specific ko mice, a commonly used deleter x of Biology, University of Freiburg, 79104 Freiburg, Germany; Instituto de Medicina strain is the proximal lymphocyte protein tyrosine kinase (Lck) Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisbon, Portugal; { Leibniz Research Institute for Environmental Medicine, 40225 Du¨sseldorf, Germany; promoter–driven Cre (pLckCre) transgenic line (1, 3). In these ‖Spemann Graduate School of Biology and Medicine, University of Freiburg, 79104 mice, Cre expression is controlled by the proximal promoter of the # Freiburg, Germany; Department of Developmental Immunology, Max Planck Institute Lck gene, which is expressed in T cells. Endogenous Lck ex- of Immunology and Epigenetics, 79108 Freiburg, Germany; and **Centre for Integrative Biological Signalling Studies CIBSS, University of Freiburg, 79104 Freiburg, Germany pression is tightly controlled during thymocyte development by ORCIDs: 0000-0002-8226-5584 (G.J.F.); 0000-0002-2561-0820 (A.-M.S.); 0000- two separate promoters, the proximal and the distal promoter, 0002-1035-2558 (L.A.H.); 0000-0002-2957-4636 (C.E.); 0000-0001-8211- giving rise to transcripts differing in their 59 untranslated regions 5538 (S.M.). (4–7). Immature murine thymocytes transcribe Lck from both Received for publication November 2, 2017. Accepted for publication May 8, 2019. promoters, whereas mature ab thymocytes and peripheral ab This work was supported by the Deutsche Forschungsgemeinschaft through EXC294 T cells dominantly use the distal promoter (5). Previous studies (Centre for Biological Signalling Studies BIOSS) and FOR 2799 to W.W.A.S., have demonstrated deletion of floxed genes by pLckCre in T cells MI1942/2-1 and SFB-1160-TP5 to S.M., ES103/7-1 to C.E., and GSC-4 (Spemann 2 2 Graduate School of Biology and Medicine) to A.M., the German Federal Ministry of of the ab lineage starting at the CD4 CD8 double-negative Education and Research (Grant BMBF 01EO1303) to W.W.A.S., and partially sup- (DN) stage of thymocyte development (8–10), as well as ported by a European Molecular Biology Organization long-term fellowship (ALTF 252-2017) and a Marie-Curie Intra-European Fellowship to G.J.F. pLckCre-mediated reporter gene expression starting at the DN2 + + Address correspondence and reprint requests to Dr. Gina J. Fiala at the current address: (CD44 CD25 ) stage (11, 12). Thus, pLckCre mice delete floxed Instituto de Medicina Molecular Joao Lobo Antunes, Faculdade de Medicina da Uni- genes very early in ab T cell development and are broadly used versidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisbon, Portugal. E-mail and commercially available (e.g., from The Jackson Laboratory addresses: gina.fi[email protected]/gina.fi[email protected] and Taconic Biosciences). The online version of this article contains supplemental material. Two lineages of T cells emerge from the thymus, the well-studied Abbreviations used in this article: AhR, arylhydrocarbon receptor; Cre, Cre recom- 2 2 ab T cells and the enigmatic gd T cells, whose function in the binase; DETC, dendritic epidermal T cell; DN, CD4 CD8 double-negative; DP, CD4+CD8+ double-positive; E, embryonic day; floxed, gene of interest flanked by immune system is just beginning to be understood (13–15). The loxP sites; icTCRb, intracellular TCRb; IEL, intraepithelial lymphocyte; iNKT, potential use of gd T cells in cancer immunotherapy has stimu- invariant NKT cell; ko, knockout; Lck, lymphocyte protein tyrosine kinase; MAIT, mucosal-associated invariant T cell; MFI, mean fluorescence intensity; p/d, proximal lated renewed interest in this lineage (16). gd T cell subsets are to distal Lck transcript ratio; pLckCre, proximal Lck promoter–driven Cre; qRT-PCR, diverse and reside in various tissues; they have subset-specific quantitative RT-PCR; ROSA26, Gt(ROSA)26Sor locus; tdTomato, tandem dimeric functions, and their generation is temporally controlled during dsRed. ontogeny, correlating with specific Vg-chain expression (17, Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 reviewed in Ref. 18). In contrast to conventional ab T cells, the gd www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701521 570 pLckCre INEFFICIENTLY DELETES FLOXED GENES IN gd T CELLS counterparts can acquire effector functions already during thymic Reagents development (19, 20). + The following Abs were used: anti-CD3-FITC (17A2), anti-CD4-FITC In mice, Vg5 dendritic epidermal T cells (DETCs) are the first (GK1.5), anti-CD19-FITC (1D3), anti-CD19-V450 (6D5), anti-CD24- gd T cells to be generated and leave the thymus between em- PE-Cy5 (M1/69), anti-IL-17A-FITC (17B7), anti-IFN-g-FITC bryonic day (E) 13 and E17. DETCs are committed IFN-g pro- (XMG1.2), anti-CD8a-allophycocyanin-eF660 (53-6.7), anti-CD8a-FITC ducers and home to the epidermis. During the second wave, gd (53-6-7), anti-CD8a-biotin (53-6.7), streptavidin–PECy7, anti-TCRd- allophycocyanin (GL3), anti-TCRd-eFluor 450 (GL3) and anti-TCRd- T cells preprogrammed to IL-17 production emerge from E14 to biotin (GL3), anti-TCRb-allophycocyanin-eFluor 780 (H57), polyclonal around birth.