Proximal Lck Promoter−Driven Cre Function Is Limited in Neonatal and Ineffective in Adult δγ T 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

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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 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 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 (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 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 ; 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. During fetal development, this subset consists goat anti-rabbit IgG–DyLight 633, and streptavidin–eFluor 450, all from mainly of Vg6+ cells, but, around birth, other Vg-chains are used, Thermo Fisher Scientific; anti-CD3-PE-Cy7 (17A2), anti-CD4-PE (RM4- especially Vg4 (21, 22). Importantly, the generation of pre- 5), anti-CD24-biotin (M1/69), anti-CD44-FITC (IM7), anti-CD73-FITC/ TY/11.8), anti-NK1.1-FITC (PK136), anti-TCRVg1.1/Cr4-FITC (2.11), committed IL-17 producers is restricted to the fetal and perinatal anti-TCRVg1.1/Cr4-allophycocyanin (2.11), anti-TCRVg2-FITC (UC3- period (23), and these gd T cells populate peripheral organs such 10A6), anti-TCRVg2-allophycocyanin (UC3-10A6), anti-TCRd-PE as the dermis, uterus, and peritoneal cavity [reviewed in Ref. (UC7-13D5), anti-TCRb-biotin (H57), FcR block anti-CD16/32 (93), and (24)]. The third functional wave of gd T cells generates gd anti-I-A/I-E (M5/114.15.2), all from BioLegend; anti-TCRb-FITC (H57) NKT cells, which mainly express Vg1 and are potent IFN-g from SouthernBiotech; rabbit anti-Cre Ab (D3U7F) from Cell Signaling Technology; anti-Vg5-FITC (536) from BD Biosciences, and goat anti-rat producers. Fetal progenitor-derived gd NKT cells undergo a IgG–TexasRed and anti-TCRb-PE-Cy7 (H57) from Invitrogen. Mouse substantial expansion in early neonatal life and predominantly CD1d-unloaded and PBS-57–loaded monomers as well as MR1-Ac-6- localize to the liver (25, 26). A role of TCR signaling in effector FP– and 5-OP-RU–loaded (51) were obtained from the National Institutes fate commitment has been suggested (19, 21, 23, 27–31). In ad- of Health Tetramer Core Facility and tetramers were produced according Downloaded from to the National Institutes of Health Tetramer Core Facility protocol using dition, uncommitted gd T cells are generated during the late streptavidin–allophycocyanin. embryonic phase, and their development is further sustained T cells were expanded ex vivo with 5 mg/ml Con A (Sigma-Aldrich) or throughout adult life (20, 32). 1 mg/ml anti-TCRd (GL3; Thermo Fisher Scientific) for 2 d and subse- A broad number of studies using pLckCre mice have assumed quently cultivated with 100 U/ml recombinant human Il-2 (PeproTech). For that Cre expression should be similar in ab and gd T cells, and, intracellular staining, splenocytes were stimulated with 100 ng/ml PMA (Sigma-Aldrich) and 1 mg/ml ionomycin (Sigma-Aldrich) for 4 h at therefore, floxed genes would be deleted in both lineages (33–40). 37˚C in the presence of 1 ml/ml of GolgiPlug (BD Biosciences). http://www.jimmunol.org/ However, some isolated reports have already speculated that pLckCre might not delete in gd T cells (10, 41–44), but a detailed Flow cytometry analysis is currently missing. Single cell suspensions from thymus, spleen, and lymph nodes were ob- In this study, we show that crossing of four different floxed genes tained by mechanical disruption. Epidermal cells were prepared as previ- with the pLckCre deleter strain did not result in recombination in ously described (52). Dermal cells were prepared from backskin incubated dermal side down on 1% trypsin/PBS for 1.5 h at 37˚C. Upon separation, the majority of gd T cells in the adult mouse. Indeed, gd T cells dermis was incubated in RPMI 1640 containing 10% FBS, 2.5 mg/ml inefficiently transcribed endogenous Lck from its proximal pro- collagenase and 1 mg/ml DNase (both Roche) for 1 h at 37˚C. Lungs moter in the adult mouse thymus, providing an explanation to the were chopped and incubated in RPMI 1640 containing 1 mg/ml collage- absence of proximal Lck promoter–transcribed Cre in these cells. nase (Roche) and 0.1 mg/ml DNase for 1 h at 37˚C. The intestine was by guest on September 27, 2021 In contrast, in newborn mice transcription of both the Lck proxi- resected, washed, cut into pieces, and incubated in PBS/10% FCS/2 mM EDTA, shaking for 30 min at 37˚C to obtain intraepithelial mal promoter and pLckCre were significantly enhanced in devel- (IELs). Intestine pieces were subsequently incubated in RPMI 1640/15% oping thymic gd T cells. Consequently, perinatally generated gd FBS containing 0.2 mg/ml collagenase and 0.03 mg/ml DNase (both T cells recombined floxed genes more efficiently and could be Roche) for 40 min at 37˚C to prepare lamina propria cells. Livers were traced by floxed-stop– tandem dimeric dsRed (tdTomato) reporter mechanically disrupted. Solutions obtained were filtered through 70-mm strainers, and Percoll (GE Healthcare) gradients were performed; thereby, expression. Importantly, recombined and thus red (tdTomato- liver was supplemented with 100 U/ml heparin. Erythrocytes were lysed, expressing) gd T cells populated diverse tissues and gave rise to a and cells were stained. significant population of precommitted IL-17 and IFN-g effector All intracellular stainings were performed according to manufacturer cells. protocol (Cytofix/Cytoperm; BD Biosciences) or, alternatively, after fixa- tion in 2.9% paraformaldehyde (Sigma-Aldrich) for 30 min on ice and permeabilization using 87.7% methanol cooled to 220˚C for 30 min on ice Materials and Methods and upon FcR blocking. Flow cytometry was performed by conventional Mice methods with Gallios and Cyan flow cytometers (Beckman Coulter), and the data were analyzed with the FlowJo 6.1 software. Arylhydrocarbon receptor (Ahr)–deficient (AhR2/2; stock no. 002831) (45), floxed (AhRflox/flox; stock no. 006203) (46), pLckCre (3) [B6.Cg- Epidermal sheets Tg(Lck‐cre)548Jxm/J; stock no. 003802], Gt(ROSA)26Sor locus (ROSA26)flox-stop-tdTomato (47) [B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/ Epidermal sheets were obtained from ears as previously described (52). flox-stop-eYFP g J; stock no. 007914], and ROSA26 (48) [B6.129 3 1-Gt(ROSA) They were stained with anti-V 5-FITC and anti–I-A/I-E overnight at 4˚C tm1(EYFP)Cos and then developed with goat anti-rat IgG–TexasRed for 2 h at room 26Sor /J; stock no. 006148] mice were purchased from The flox/flox temperature. Epidermal sheets mounted on glass slides with Fluormount-G Jackson Laboratory. AhR mice were crossed with pLckCre mice to flox/flox + flox/flox 2 flox-stop-tdTomato (Thermo Fisher Scientific) were photographed and analyzed with a Leica obtain AhR pLckCre and AhR pLckCre . ROSA26 mice were crossed to pLckCre mice to obtain ROSA26flox-stop-tdTomatopLckCre+ DM2500 microscope (Leica) and AxioVision Rel.4.6 software (Zeiss). flox-stop-tdTomato 2 flox-stop-eYFP and ROSA26 pLckCre . ROSA26 mice were crossed PCR to pLckCre and Vav1Cre (49) mice to obtain ROSA26flox-stop-eYFPpLckCre+ and ROSA26flox-stop-eYFPpLckCre2 and ROSA26flox-stop-eYFPVav1Cre+ and The PCRs to detect recombination of the floxed AhR locus were done as ROSA26flox-stop-eYFPVav1Cre2, respectively. Double-heterozygous reporter described previously (46). For quantitative RT-PCR (qRT-PCR), thymic mice were analyzed. Kidins220+/flox mice (50) were crossed to pLckCre T cells of pLckCre+ mice were stained, and the following populations were mice. Mice were cohoused under specific pathogen–free conditions and sex sorted: CD4+CD8+ (ab T cells) and CD42CD82TCRb2TCRd+ (gd and age matched with litter controls whenever possible. Mice between 2 and T cells). In some experiments thymic gd T cells were further subdivided 5 d were considered newborn; adults were 6–20 wk of age. All animal protocols into CD24+ and CD242 cells. Total mRNA was prepared using the RNeasy (G12/64) were performed according to the German animal protection law with Mini Kit (QIAGEN) or TRIzol (Invitrogen). cDNA was prepared with permission from the Veterina¨r- und Lebensmittelu¨berwachungsbeho¨rde, oligo(dT) and random hexamer primers using the First Strand cDNA Freiburg, Germany. Experiments in Du¨sseldorf, Germany, were conducted Synthesis Kit (Thermo Fisher Scientific), including DNase I treatment. with permission from the Landesamt fu¨r Umwelt- und Naturschutz. Quantitative PCR was performed with the FastStart Universal SYBR The Journal of Immunology 571

Green Master Kit (Roche) on a qTower 2.2 (Analytik Jena) with specific as result of Cre expression from the proximal Lck promoter primers for the proximal and distal Lck transcripts (4) and the Cre-specific (Fig. 2B). primers 59-CGGTCTGGCAGTAAAAACTAT-39 and 59-CAGGGTGTTA- Because DETC are a unique gd T cell subset, we wondered how TAAGCAATCCC-39 (53). b-actin served as reference gene using the primers 59-CTAAGGCCAACCGTGAAAAG-39 and 59-ACCAGAGGCA- efficiently pLckCre turned on tdTomato expression in gd Tcells TACAGGGACA-39. generated in the adult thymus. Surprisingly, in the thymus the percentage of gd T cells that had expressed pLckCre, and thus were Statistical analysis tdTomato+, was even reduced to 6 6 5% (Fig. 2C). In contrast, All statistical analyses were performed using the Prism4 software. pLckCre expression resulted in efficient floxed gene deletion in the DN to CD4+CD8+ double-positive (DP) transition during ab thy- Results mocyte development (Fig. 2D), as previously reported, resulting in The AhRflox allele is not deleted by pLckCre in DETC 99 6 1% of TCRb+ thymic T cells expressing tdTomato (Fig. 2C). DETC are the main gd T cell subset in the murine epidermis. It During ab thymocyte development, tdTomato mean fluorescence has been suggested that AhR is crucial for DETC homeostasis, intensity (MFI) gradually increased, being intermediate in DP cells as demonstrated by strongly reduced DETC numbers in AhR- and maximal in single-positive ab T cells (Fig. 2D). In the adult + 2 deficient (AhR2/2) mice (52). To test for a cell-intrinsic role of thymus, immature CD24 CD73 gd T cells sequentially upregulate 2 + AhR in DETC, we aimed to generate a pan T cell–specific AhR CD73 and downregulate CD24 to become mature CD24 CD73 gd + ko. We crossed mice containing the floxed AhR gene (AhRflox/flox) T cells (54). Interestingly, the percentages of tdTomato gd 6 6 + 2 with the pLckCre deleter strain (AhRflox/floxpLckCre+) and ana- Tcellsincreasedfrom10 5% to 7 4% in CD24 CD73 and

2 2 + + 6 Downloaded from lyzed the abundance of DETC in epidermal sheets using AhR / CD24 CD73 , respectively, to 21 11% in the mature 2 + as controls. As published, AhR2/2 mice displayed a drastic re- CD24 CD73 stage (Fig. 2E). Similar to the gradual increase of the duction of DETC numbers compared with AhR+/+ mice (Fig. 1A, tdTomato MFI along ab T cell development, the tdTomato MFI of + 1B) (52). However, DETC abundance in AhRflox/floxpLckCre+ mice tdTomato gd T cells rose during development, reaching levels 2 + was indistinguishable from control AhRfloxfloxpLckCre2 mice comparable to single-positive ab T cells in the CD24 CD73 stage (Fig. 1A, 1C). To test whether the AhRflox allele was indeed deleted (Fig. 2E, 2F). This indicated that in the adult thymus pLckCre- in DETC, we performed a PCR distinguishing between the floxed and expressing gd T cells developed at low frequency. Furthermore, http://www.jimmunol.org/ + 2 the deleted AhR allele (46) on sorted DETC from AhRflox/floxpLckCre+ tdTomato cells accumulated among CD24 mature gd T cells. mice. Muscle cells and splenic CD3+ T cells served as controls Next, we analyzed mature peripheral gd T cells for tdTomato (Fig. 1D). Although the AhRflox allele was intact in muscle cells, expression. The proportion of peripheral gd T cells that had ex- ∼ we confirmed AhR deletion in splenic (mostly ab) T cells. perienced pLckCre activity varied from 6% in spleen, lymph ∼ Strikingly, only the AhRflox but not the deleted AhR allele was nodes, lung, and intestine (lamina propria and IELs) to 20% in detected in DETC. peritoneal cavity and liver (Fig. 3A, 3B). In the skin, tdTomato expression among TCRdhigh DETC was ∼20% and even 27% in pLckCre-mediated reporter expression is limited among gd dermal TCRdlow gd T cells (Fig. 3B, 3C) (55). As control, ∼99 6 T cells in the adult mouse 1% of ab T and 0 6 0% of B cells, were tdTomato+ (Fig. 3A, 3B). by guest on September 27, 2021 One possible explanation for the lack of a DETC phenotype in Together with gd T cells, specialized populations of innate-like AhRflox/floxpLckCre+ mice is that a few DETC would not express ab T cells that express semi-invariant TCRs compose the un- pLckCre and, therefore, would not recombine AhRflox. These AhR- conventional T cell pool. These cells are fast-responding T cells sufficient DETC would have an advantage and outcompete AhR- with important roles early during immune responses (reviewed in deficient DETC. To test this hypothesis, we crossed the pLckCre Ref. 56). We asked whether unconventional ab T cells expressed mice with the ROSA26floxed-stop-tdTomato reporter mouse. In the pLckCre. Using the ROSA26floxed-stop-tdTomatopLckCre+ reporter latter, Cre expression results in the deletion of a stop cassette and mice, we analyzed tdTomato expression in ab IELs, invariant the subsequent and irreversible expression of tdTomato, resulting NKT cells (iNKT), and MAIT cells. Similar to conventional ab in cells with a bright red fluorescence (47) (Fig. 2A). Flow T cells, 99.7 6 0.3% of the ab IELs expressed tdTomato (Fig. 3A, cytometry analysis of adult ROSA26floxed-stop-tdTomatopLckCre+ 3B). Likewise, at least 96 6 2% of the iNKT in thymus, spleen mice revealed that only 23 6 9% of DETC expressed tdTomato and liver (Fig. 3D) and 99 6 1% of lung MAIT cells were

FIGURE 1. The floxed AhR gene is not deleted in pLckCre+ DETC. (A) Epidermal sheets were prepared from AhR+/+, AhR2/2, and AhRflox/floxpLckCre+ mice stained with anti-Vg5-FITC (labeling DETC, green), anti–I-A/I-E and goat anti-rat IgG–TexasRed (labeling Langerhans cells, red), and imaged by fluorescence microscopy. Mice age: 11–12 wk. (B) The frequencies of DETC as percentage of skin cells from seven AhR+/+ and seven AhR2/2 mice prepared as in (A) were quantified. Mice age: 12–25 wk. (C) DETC abundance as percentage of skin cells prepared as in (A) were quantified for seven AhRflox/floxpLckCre2 and eight AhRflox/floxpLckCre+ mice at the age of 10–11 wk. (D) DETC, muscle cells and splenic CD3+ T cells were isolated from two independent 10-wk-old AhRflox/floxpLckCre+ mice and tested for the deletion of the floxed AhR exon by PCR. Arrows indicate the PCR products of 140 bp corresponding to AhRflox and of 180 bp corresponding to the deleted AhRD allele (n = 7). Statistical analysis by Mann–Whitney U test, error bars indicate SD. ns, not significant. 572 pLckCre INEFFICIENTLY DELETES FLOXED GENES IN gd T CELLS Downloaded from http://www.jimmunol.org/ FIGURE 2. pLckCre-mediated reporter gene expression is inefficient in adult DETC and adult thymic gd T cells. (A) Schematics of the ROSA26floxed-stop-tdTomato locus prior and after recombination by Cre [modified after (47)]. CAG denotes the exogenous ubiquitously expressed CAG promoter. Cre-mediated deletion of the floxed stop cassette leads to tdTomato transgene expression. (B) DETC were isolated from a total of 13 ROSA26floxed-stop-tdTomatopLckCre+ reporter mice and four ROSA26floxed-stop-tdTomatopLckCre2 controls in six independent experiments, stained with anti-CD3 and anti-TCRd Abs, and analyzed by flow cytometry. Representative plots and the frequencies of tdTomato+ cells among CD3+TCRd+ cells are depicted. Mice age: 14–22 wk. (C) Thy- mocytes from a total of 24 ROSA26floxed-stop-tdTomatopLckCre+ and 19 ROSA26floxed-stop-tdTomatopLckCre2 mice were stained with anti-TCRb and anti-TCRd Abs and analyzed by flow cytometry in 12 independent experiments. Representative plots and the frequencies of tdTomato+ cells within ab and gd T cell subsets are shown. Mice age: 7–22 wk. (D) Thymocytes from 10 ROSA26floxed-stop-tdTomatopLckCre+ mice were stained with anti-CD4 and anti-CD8 Abs and analyzed by flow cytometry in four independently performed experiments. A representative plot and the frequencies of tdTomato+ cells within each by guest on September 27, 2021 population are shown. Mice age: 11–20 wk. (E) Thymic gd T were gated as in (C) and analyzed for CD24 and CD73 maturation marker expression. Representative plots and the frequencies of tdTomato+ cells within the different maturation stages are shown. Mice age: 7–10 wk. (F) MFI of tdTomato+ gd T cells from each maturation stage analyzed in (E) are given. Statistical analysis in (B)–(E) by Mann–Whitney U test and in (F) by two-sided Student t test. Error bars indicate SD. tdTomato+ (Fig. 3E). Although innate-like in their function, un- ab and gd T cell pools, demonstrating that tdTomato was not toxic conventional ab T cells were similar to conventional ab T cells for gd T cells (Fig. 4C, 4D). We reproduced these findings using regarding pLckCre expression. tdTomato2 gd T cells from the ROSA26floxed-stop-tdTomatopLckCre+ The lack of pLckCre to mediate efficient recombination of reporter mouse (Supplemental Fig. 2B, 2C). Furthermore, using floxed genes in gd T cells appears to be a general limitation of this ROSA26floxed-stop-eYFPVav1Cre+ mice, which constitutively ex- deleter strain, as it also failed to delete the floxed AhR (Fig. 1C), press Cre under the control of the Vav1 promoter in hemato- floxed D–interacting substrate of 220 kDa (Kidins220)/ poietic cells and their progenitors, we confirmed that 99% of ankyrin-rich membrane spanning protein (ARMS) (Supplemental both gd Tcellsandab T cells expressed eYFP in the thymus Fig. 1A) and the floxed stop cassette in the ROSA26floxed-stop-eYFP (Fig. 4E). Reporter-labeled gd T cells developed unperturbed, reporter mouse (Supplemental Fig. 1B). despite constitutive Cre and eYFP expression, and were found in diverse tissues, including the skin and in different Vg subsets Adult thymic gd T cells mostly use the distal and not the (Supplemental Fig. 2D–G). Consequently, it is unlikely that Cre+ gd proximal Lck promoter T cells are counterselected in the ROSA26floxed-stop-tdTomatopLckCre+ The above findings could be explained by at least two possible but reporter mouse. Together with the absence of detectable levels of not mutually exclusive scenarios: 1) pLckCre is not expressed in Cre mRNA and protein, our data suggest that pLckCre expres- most gd T cells or/and 2) Cre and/or tdTomato expression is sion is not turned on in the majority of gd T cells, precluding toxic to gd T cells, removing them from the T cell pool. We first deletion of floxed genes. analyzed Cre mRNA and protein levels in thymocytes, in which How do gd T cells transcribe endogenous Lck during devel- both were detectable in ab T cells but not in gd T cells (Fig. 4A, opment, and could this explain the discrepancy between ab and 4B, Supplemental Fig. 2A). Second, we assessed survival of gd T cells in pLckCre expression? We assessed endogenous, splenic ab and gd TcellsfromROSA26floxed-stop-tdTomatopLckCre2 promotor-specific Lck transcript levels in FACS-sorted B6 mice upon ex vivo expansion and retroviral transduction with wildtype thymocytes (Fig. 4F). Thymic CD4+CD8+ ab T cells Cre.Bothab and gd T cells became equally well infected transcribed Lck from both the proximal and distal promoter (GFP+)andturnedontdTomato expression. tdTomato+ cells (proximal to distal Lck transcript ratio [p/d] 1.43), and proximal accumulated to a similar extent and were equally retained in both transcripts were detected with a mean 2^-dCT of 0.263 6 0.07. The Journal of Immunology 573 Downloaded from http://www.jimmunol.org/ FIGURE 3. Conventional and unconventional ab but not gd T cells efficiently express the pLckCre-mediated reporter gene in the adult. (A) Lymphocytes isolated from the indicated tissues of ROSA26floxed-stop-tdTomatopLckCre+ reporter mice were stained with anti-TCRb and anti-TCRd Abs and analyzed by flow cytometry. B cells were stained with anti-CD19. Two to twenty mice were analyzed per tissue in 2–10 independent experiments. Representative plots and frequencies of tdTomato+ cells within each population are shown. Mice age in weeks: 8–22 (spleen), 8–20 (lymph node, peritoneal cavity, lung), 7–10 (liver), 8–18 (intestinal IELs), 8 (lamina propria). (B) Statistical analyses of tdTomato+ frequencies within each cell population and tissue from the ex- periments shown in (A) and (C). “E” denotes epidermal and “D” dermal gd T cells. (C) Lymphocytes from dermal preparations of eight mice were stained with anti-CD3 and anti-TCRd Abs and analyzed by flow cytometry in two independent experiments and depicted as in (A). CD3lowTCRdlow cells were identified as dermal (“D”) and CD3highTCRdhigh as epidermal (“E”) gd T cells. Mice age: 8–10 wk. (D) ab iNKT were gated as TCRb+TCRd2NK1.1+ in the thymus and TCRb+TCRd2NK1.1+CD1d-PBS57+ in spleen and liver. Five to nine mice were analyzed in two to four independent experiments. A by guest on September 27, 2021 representative plot from spleen and the frequencies of tdTomato+ cells within each population are shown. Mice age: 7–22 wk. (E) Lymphocytes were prepared and MAIT cells gated as TCRb+TCRd2MR1-5-OP-RU+ cells in the lung of three mice. A representative plot and the frequency of tdTomato+ cells within the population are shown. Mice age: 7–8 wk. All statistical analyses were done using the Mann–Whitney U test. Error bars indicate SD.

In mature CD4+ ab T cells, proximal transcripts were decreased those gd T cells that might have diverged late from a common 10-fold (mean 2^-dCT of 0.027 6 0.008) and, preferentially, the ab/gd T cell precursor. distal promoter was used, reflected by a p/d of 0.22. Immature To understand whether tdTomato+ gd T cells were biased CD24+ gd T cells displayed 5-fold reduced proximal transcripts toward a specific gd T cell subset, we analyzed the abundance compared with DP with a mean 2^-dCT of 0.048 6 0.015. Lck of tdTomato+ cells among Vg1+,Vg4+,andVg12Vg42 gd transcript abundance was reduced for both promoters, and the T cells. All analyzed Vg-chain–expressing subsets encom- p/d was 0.96. In mature CD242 gd T cells proximal Lck tran- passed tdTomato+ cells to a similar extent in both thymus and scripts were virtually absent (mean 2^-dCT of 0.007 6 0.002), spleen. We noted a slight enrichment of tdTomato+ cells in whereas distal transcripts were similarlyabundantasinCD24+ Vg12Vg42 cells in the thymus. In contrast, in the spleen the gd T cells, giving a p/d of 0.13. Hence, differences in endoge- Vg4+ compartment contained the highest percentage of tdTo- nous Lck promoter usage in developing ab and gd T cells might mato+ cells (Fig. 5B). explain the differential expression of the pLckCre transgene Some gd T cell subpopulations are already committed to the promoter. production of either IL-17 or IFN-g during their thymic devel- opment (19, 23, 27–30). gd NKT cells are potent IFN-g producers tdTomato+ gd T cells are enriched in committed IL-17+ and + committed in the thymus, similar to iNKT of the ab lineage. IFN-g effector cells Analysis of the NK1.1+ gd T cell subset showed that in spleen 4 6 We next tested whether splenic tdTomato+ gd T cells which ex- 3% of gd NKTs expressed tdTomato similar to the total gd T cell press pLckCre during development might share features of the ab pool. In thymus and liver, gd NKTs were enriched in tdTomato+ lineage. In fact, it was reported that 15% of gd T cells express cells over the general gd T cell pool within the corresponding intracellular TCRb (icTCRb), have gone through TCRb-mediated organ (5 6 4% in thymus and 19 6 7% in liver, Fig. 3C), proliferation, and showed increased frequencies of pre-TCRa encompassing 10 6 7% and 30 6 7% tdTomato+ cells, respec- gene transcription (57, 58), thereby sharing some developmental tively (Fig. 5C, 5D). features with the ab lineage. Both thymic and splenic tdTomato+ Next, we analyzed the cytokine production by tdTomato2 and gd T cells contained a significantly higher proportion of icTCRb+ tdTomato+ gd T cells in the spleen upon ex vivo stimulation with cells compared with their tdTomato2 counterparts (Fig. 5A). PMA and ionomycin. TdTomato2 gd T cells encompassed 8 6 5% Thus, pLckCre-driven tdTomato expression preferentially labeled of IFN-g–producing cells, whereas tdTomato+ cells contained 19 6 574 pLckCre INEFFICIENTLY DELETES FLOXED GENES IN gd T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 4. Adult gd T cells lack pLckCre expression during development and show reduced transcription of the endogenous Lck promoters. (A) Cre transcripts were analyzed by qRT-PCR in FACS-sorted CD4+CD8+,CD4+CD82,andCD42CD8+ (ab) and TCRd+ (gd) T cells from thymi of adult pLckCre+ mice. Cre transcripts were normalized to the transcripts of the housekeeping gene b-actin. Mice age: 6–18 wk. The mean 2^-dCT values of four independent experiments are shown. Statistical analysis by two-sided Student t test. Only significant differences are depicted. (B) Thymocytes of 12 pLckCre+ and 11 pLckCre2 mice were stained on the surface with anti-CD4, anti-CD8, and anti-TCRd and intracellularly with anti-Cre Abs and measured by flow cytometry. The normalized fluorescence intensity of the anti-Cre staining in ab (gated on CD4+ and/or CD8+)andgd T cells (gated 2 2 + on CD4 CD8 TCRd ) is displayed; mice age: 8–13 wk. Statistical analysis was performed by two-sided Student t test. Error bars indicate SD (n =4). by guest on September 27, 2021 (C) Splenocytes from nine ROSA26floxed-stop-tdTomatopLckCre2 mice were expanded ex vivo and transduced with pMIGCreIRESGFP or pMI- GemptyIRESGFP plasmids in three independent experiments. Cells were gated on TCRb+d2 (ab T) and TCRb2d+ (gd T) and analyzed for GFP and tdTomato on day 5 after transduction. Mice age: 13–19 wk. (D) The relative frequencies of tdTomato2 (white) and tdTomato+ (gray) ab Tandgd T cells treated as in (C) on day 0, 2, and 5 after transduction are displayed. Statistical analysis was performed using the Wilcoxon signed-rank test for tdTomato+ subsets (n =3).(E) Thymocytes from three ROSA26floxed-stop-eYFPVav1Cre 2 and three ROSA26floxed-stop-eYFPVav1Cre+ mice were stained with anti-TCRb and anti-TCRd, and frequencies of eYFP+ cells were analyzed within each population by flow cytometry. Mice age: 11–12 wk. Statistical analysis was performed by Mann–Whitney U test. Error bars indicate SD. (F) Lck transcripts originating from the proximal (white) or distal (gray) promoter were assessed by qRT-PCR using sorted CD4+CD8+ and CD4+TCRbhigh (ab T) and TCRd+CD24+ and TCRd+CD242 (gd T) cells from the thymus of B6 wildtype mice. Lck transcripts were normalized to the transcripts of the housekeeping gene b-actin. The table depicts p/d ratios for the indicated subsets. Mice age: 7–10 wk. The mean 2^-dCT values from three independent experiments are shown. Two-sided Student t test was used to compare proximal Lck 2^-dCT values. Error bars indicate SD. ns, nonsignificant.

10%. Interestingly, 38 6 14% of the tdTomato+ gd T cells produced compose a significant proportion of the precommitted gd Tcell IL-17 upon stimulation, whereas only 4 6 2% of the tdTomato2 gd effector subset. T cells did so (Fig. 5E). The significant contribution of tdTomato+ gd T cells to the cytokine response was also reflected in total cell In newborn mice, tdTomato is expressed in the majority of gd numbers (Supplemental Fig. 3A, 3B). The PMA and ionomycin T cells stimulation did not alter the percentage of tdTomato+ gd Tcells Common to the gd T cell subsets enriched in tdTomato+ cells is (data not shown), showing that stimulation did not induce de novo their development early in ontogeny, suggesting that pLckCre pLckCre expression but detected tdTomato+ cells that had experi- might be expressed primarily by gd T cells during a discrete enced pLckCre expression already during development. developmental window early in life. To test this hypothesis, we Expression of pLckCre in ab T cells was shown to cause an analyzed newborn ROSA26floxed-stop-tdTomatopLckCre+ mice. In activated phenotype (41). To assess whether Cre expression in gd sharp contrast to adult mice, 58 6 7% of thymic gd Tcells T cells drives them toward an effector state, we compared IL-17 and expressed tdTomato (Fig. 6A). As a control, 99 6 1% of ab IFN-g production by gd T cells from ROSA26floxed-stop-eYFPVav1Cre + T cells expressed tdTomato, similar to the adult. During thy- mice, in which virtually all gd T cells express Cre (Fig. 4E, mic maturation, 44 6 10% of the CD24+CD732 gd Tcells Supplemental Fig. 2D), and ROSA26floxed-stop-eYFPVav1Cre 2 were tdTomato+ and 62 6 11 and 60 6 10% of the mature mice. The cytokine response by gd T cells was independent of CD242CD73+ and CD242CD732 subsets, respectively (Fig. constitutive Cre expression, arguing against a Cre expression– 6B). Likewise, the newborn spleen was strongly enriched in mediated acquisition of the effector phenotypes (Fig. 5F). Taken tdTomato+ gd Tcells(666 9%) compared with the adult together, tdTomato+ gd T cells are few in number, but they (Figs. 3C, 6C). The Journal of Immunology 575 Downloaded from http://www.jimmunol.org/ FIGURE 5. tdTomato+ gd T cells are enriched for icTCRb and for precommitted IL-17 and IFN-g producers. (A) gd T cells gated as TCRb2TCRd+ from a total of seven thymi and five spleens of ROSA26floxed-stop-tdTomatopLckCre+ mice were analyzed by flow cytometry for icTCRb in three independent experiments. Representative plots and the frequencies of icTCRb+ in each population are depicted. Mice age: 8–22 wk. (B) TCRb2TCRd+ gd T cells from a total of 11 thymi and spleens of ROSA26floxed-stop-tdTomatopLckCre+ mice were stained for Vg-chain expression (Vg1 and Vg4) and analyzed by flow cytometry for tdTomato in three independent experiments. Representative plots and the frequencies of tdTomato+ cells within each Vg subset is shown. Mice age: 7–10 wk. (C) gd NKT cells were gated as TCRb2TCRd+NK1.1+ cells from the liver. A representative plot and the frequency of tdTomato+ cells within gd NKT cells are depicted. (D) gd NKT cells from thymus, spleen, and liver of 5–10 ROSA26floxed-stop-tdTomatopLckCre+ mice were analyzed for tdTomato in two to four independent experiments. Frequencies of tdTomato+ cells are depicted. Mice age: 7–22 wk. (E) Splenocytes from six ROSA26floxed-stop-tdTomatopLckCre+ mice in three independent experiments were stimulated ex vivo with PMA and ionomycin. Cells were stained extra- cellularly for TCRb and TCRd and intracellularly for IFN-g and IL-17A. TCRb2TCRd+ cells were gated, and tdTomato2 and tdTomato+ cells were by guest on September 27, 2021 analyzed separately for IFN-g and IL-17A expression. Mice age: 8–22 wk. (F) Splenocytes from four ROSA26floxed-stop-eYFPVav1Cre2 and four ROSA26floxed-stop-eYFPVav1Cre+ mice were stimulated and stained as in (E). Mice age: 9–16 wk. Statistical analyses were done using the Mann–Whitney U test. Error bars indicate SD. ns, not significant.

Analysis of different Vg-chain–expressing gd T cell subsets in and prolonged transcription of the endogenous proximal Lck the newborn thymus indicated similarly high percentages of promoter. Consequently, the resulting tdTomato+ gd T cells con- tdTomato+ cells among all with a slight increase in the Vg1+ tributed substantially to the mature CD242 gd T cell pool, which subset (Fig. 6D). In the spleen, this shift was more pronounced encompasses long-lived effector gd T cells. (Fig. 6D). Of note, non-T cells did not express tdTomato, con- firming that pLckCre expression was also specific to the T cell Discussion lineage in newborn mice (data not shown). In adult thymi, Although gd T cells express Lck in the thymus (http://www. pLckCre expression correlated with the expression of the endog- immgen.org), we show in this study that gd T cells do not effi- enous proximal Lck promoter. Hence, we investigated endogenous ciently use the proximal Lck promoter during their development Lck promoter transcript levels in FACS-sorted thymocytes from in the adult thymus. Thus, the majority of gd T cells in the adult B6 wildtype newborn mice (Fig. 6E). Proximal Lck transcripts mouse did not express pLckCre to detectable and/or function- were highly abundant in the CD4+CD8+ stage, with a mean 2^-dCT ally relevant levels, as demonstrated by inefficient recombina- of 0.570 6 0.057, even 2-fold increased to the adult. Because tion of AhRflox/flox, Kidins220flox/flox, ROSA26floxed-stop-eYFP,and distal transcript levels were similar to adult DP, this resulted in an ROSA26floxed-stop-tdTomato genes. In contrast, gd T cells in the elevated p/d of 2.57. Mature CD4+ ab T cells had a mean 2^-dCT thymus of newborn mice showed enhanced expression of of 0.079 6 0.023 and a p/d of 0.38, indicating an enhanced pLckCre (visualized by tdTomato) and transcription of the en- preference for proximal Lck transcription compared with adult dogenous proximal Lck promoter. Indeed, we detected tdTomato CD4+. In newborns, proximal Lck transcripts were 3-fold in- expression in a relevant fraction of gd T cells generated early creased compared with adult in CD24+ gd T cells (mean 2^-dCT during ontogeny, such as DETC or IL-17 effector cells, which 2 of 0.155 6 0.046), resulting in a p/d of 2.08. In CD24 gd T cells, persisted in the adult animal. We have used the pLckCre mouse both proximal and distal Lck transcripts were strongly reduced, strain available from The Jackson Laboratory. A different and the mean 2^-dCT was 0.021 6 0.013 for the proximal tran- pLckCre strain is available from Taconic Biosciences (59) and 2 script. However, CD24 gd T cells still showed prevalence for the might show slightly different Cre expression. proximal Lck transcript and had a p/d of 1.24. Importantly, in case of the ROSA26floxed-stop-tdTomato reporter In summary, the majority of gd T cells in newborn thymi have mice, pLckCre activity induced solely tdTomato expression and experienced pLckCre expression. This correlated with enhanced not deletion of a potentially relevant gene for cell development 576 pLckCre INEFFICIENTLY DELETES FLOXED GENES IN gd T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 6. Most newborn gd T cells express pLckCre during their development. (A) Thymocytes from 12 newborn ROSA26floxed-stop-tdTomatopLckCre+ mice were stained with anti-TCRb and anti-TCRd Abs and analyzed by flow cytometry in six independent experiments. Frequencies of tdTomato+ cells within each population are shown. (B) Thymocytes from newborn ROSA26floxed-stop-tdTomatopLckCre+ mice as in (A) were stained with anti-TCRb, anti- TCRd, anti-CD24, and anti-CD73 Abs and analyzed by flow cytometry. Frequencies of tdTomato+ cells within each gd T cell maturation stage are shown. (C) Splenocytes from 11 newborn ROSA26floxed-stop-tdTomatopLckCre+ mice were stained with anti-TCRb and anti-TCRd Abs and analyzed by flow cytometry. Frequencies of tdTomato+ cells within each population are shown. (D) tdTomato2 and tdTomato+ TCRb2TCRd+ gd T cells from thymus and floxed-stop-tdTomato + A spleen of newborn ROSA26 pLckCre mice as in ( ) were analyzed by flow cytometry for Vg-chain expression (Vg1 and Vg4). Repre- by guest on September 27, 2021 sentative plots and the frequencies of tdTomato+ cells within each Vg subset are shown. (E) Lck transcripts originating from the proximal (white) or distal (gray) promoter were assessed by qRT-PCR using sorted CD4+CD8+ and CD4+TCRbhigh (ab T) and TCRd+CD24+ and TCRd+CD242 (gd T) cells from the thymus of newborn B6 wildtype mice. Lck transcripts were normalized to the transcripts of the housekeeping gene b-actin. The table depicts p/d for the indicated subsets. The mean 2^-dCT values of three independent experiments are shown; two-sided Student t test was used to compare proximal Lck 2^-dCT values. Error bars indicate SD. Statistical analyses in (A)–(C) were done using the Mann–Whitney U test; error bars indicate SD. ns, not significant. and/or maintenance. Analysis of pLckCre AhRflox mice revealed unaffected by the pLckCre-mediated conditional ko (1, 3, 33, 36, that they had normal levels of DETC in the skin. Interestingly, 38, 39, 62–65), and the authors concluded that the gene was not these DETC were AhR-sufficient as we were unable to detect Cre- important in the analyzed function for gd T cells. However, the mediated deletion of the floxed AhR allele by PCR in the adult data presented in this study strongly support that, in both sce- epidermis. This indicates that, although pLckCre is expressed in a narios, the gene of interest might have been inefficiently deleted in significant fraction of DETC and thus causes deletion of the gd T cells, and deleted cells might have even disappeared from the AhRflox allele, presumably, in pLckCre AhRflox mice, these AhR- cell pool, preventing accurate statements about the role of these deficient DETC might not be retained in the cell pool but replaced genes in gd T cells. by their nondeleted counterparts and escape analysis. This ex- As a prerequisite to study the role of a candidate gene in gd planation is congruent with the importance of AhR expression for T cell biology, a conditional ko strategy has to be used that ensures the maintenance of DETC (52). an efficient deletion in gd T cells, such as the Vav1 promoter– Our findings have important implications for the interpretation of driven Cre (Vav1Cre) that deletes throughout the entire hemato- past, current, and future data using the proximal Lck promoter to poietic compartment (66). We show, in this study, that Vav1Cre drive transgene expression. Multiple genes important for the de- deleted a floxed reporter gene very efficiently and in virtually all velopment of T cells, such as signaling , were deleted gd T cell subsets analyzed. Alternatively, the human Cd2 pro- using the pLckCre transgene in a large number of publications (for moter–driven Cre (hCd2Cre) targeting B and T cells (4–7, 49, 66, an analysis see Ref. 41). In an important fraction of these works, 67) or the RAG 1 promoter–driven Cre (Rag1Cre) (5, 68) might be the authors drew conclusions about the role of the targeted gene in used as deleter strains. Still, the expression and activity of gd T cells without testing specifically for successful deletion of hCd2Cre or Rag1Cre has not been specifically addressed in gd the floxed genes in the cells analyzed. In multiple cases, the ab T cells, and it remains unknown whether specific gd T cell subsets T cell numbers were diminished, but the relative, percentage will be efficiently targeted. A deleter line to exclusively delete fraction of gd T cells was increased, leading to the conclusion that genes in the T cell lineage is the PtcraCre knock-in (pTaiCre) the gene of interest had opposing functions in ab and gd T cells mouse. When PtcraCre mice were crossed with a reporter strain, (2, 34, 37, 40, 60, 61). In other examples, gd T cells were 90% of the gd T cells expressed the reporter (8–10, 58), and the The Journal of Immunology 577

10% nontargeted gd T cells might represent a functionally distinct T cells represented one third of the IL-17–producing gd T in the subset. Alternatively, a gd T cell–specific tamoxifen-inducible Cre spleen and, although to a lesser extent, a significant proportion of deleter strain, in which Cre expression is controlled by the Tcrd IFN-g producers, despite gd T cells being only 6% tdTomato+ in promoter (TCRdCreER), was described (69). this organ. Furthermore, gd T cells expressing the TCRb-chain It is worth mentioning that unconventional ab T cells, namely intracellularly were increased in tdTomato+ cells. It is important to iNKT, MAIT, and IELs, did efficiently express pLckCre. In con- note that intracellular expression of TCRb and cytokine produc- trast, a recent report using ROSA26floxed-stop-tdTomatopLckCre+ mice tion upon ex vivo stimulation did not considerably overlap (data showed that recombination in innate lymphoid cells 2 was inef- not shown). ficient and varied between tissues, being highest with 28% in the The specific accumulation of tdTomato+ gd T cells (e.g., in skin thymus (70). Several studies, including the one presented in this and among IL-17 producers in the adult mouse), is most probably study, clearly indicate that solely abTCR+ cells efficiently express attributed to their perinatal origin and longevity. In contrast, gd pLckCre and that the success of deletion has to be tested for every T cells, which leave the perinatal thymus as uncommitted cells, cell subset of interest in Cre-driven deletion studies. might be shorter lived in the periphery and get replaced over time Under the control of pLckCre, only 5–30% of the gd T cells in by tdTomato2 gd T cells, which are the predominant cells gen- adult mice had recombined the ROSA26floxed-stop-tdTomato locus. erated in the adult thymus. The increase of tdTomato+ gd T cells Analysis of developing gd T cells in the adult thymus confirmed in the mature CD242 subset of the adult thymus might be at- low frequency of tdTomato+ gd T cells being generated at steady- tributed to thymic-resident gd T cells generated early in ontogeny state. Interestingly, although secondary lymphoid organs con- (23, 75, 76). tained very low percentages of tdTomato+ gd T cells, several Taken together, past, current, and future data using the proximal Downloaded from tissues with elevated percentages of tdTomato+ gd T cells harbor Lck promoter to drive transgene expression in gd T cells should be gd T cells of fetal and perinatal origin (such as skin, liver, peri- re-evaluated in light of the developmentally controlled and inef- toneal cavity). ficient Cre expression in these cells. Further, we have identified a Indeed, analysis of newborn ROSA26floxed-stop-tdTomatopLckCre genetic approach, the ROSA26floxed-stop-tdTomatopLckCre, as a novel mice confirmed that in the first days after birth, more than half of tool to study a subset of gd T cells mainly produced during

the thymic gd T cells expressed tdTomato. Increased frequency of perinatal life, many of which are long-lived and precommitted to http://www.jimmunol.org/ tdTomato expression correlated with enhanced endogenous prox- IL-17 production. imal Lck transcript levels in thymic gd T cells. Indeed, whereas in the adult, Lck transcripts were low during gd T cell development Acknowledgments and the mean 2^-dCT, with 0.048 6 0.015 highest in immature We thank Iga Janowska, Michael Damrat, Kerstin Fehrenbach, Christiane cells, in newborns, Lck transcripts were elevated, and immature gd Happe, Simone von Lo¨wensprung, and Helena Brandt for technical assis- T cells showed a mean 2^-dCT value of 0.155 6 0.046. Of note, tance; Kristina Schachtrup and Elias Hobeika for material and discussions; newborn gd thymocytes preferentially used the proximal promoter Ingrid Haas, Julie Ribot, and Bruno Silva-Santos for discussions; Marco floxed-stop-tdTomato (p/d . 1). Both Lck promoters are highly conserved between Prinz for providing the ROSA26 ; and Fabrizia Cesca and flox mouse and human, and previous studies have shown that the Giampietro Sciavo for providing the Kidins220 mouse. The MR1 tetra- by guest on September 27, 2021 proximal and the distal promoters share little sequence similarity mer technology was developed jointly by Dr. James McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie, and the material was produced by the and give rise to mRNAs differing in the 59 untranslated region National Institutes of Health Tetramer Core Facility as permitted to be (7, 71, 72). Thus, both transcription and translation of the two Lck distributed by the University of Melbourne. This work benefitted from data transcripts are differentially controlled and impose additional assembled by the ImmGen Consortium. layers to adjust Lck expression in the course of T cell life. Sup- porting this rational, characteristic changes in proximal to distal promoter usage ratios have been described along ab thymocyte Disclosures development and thymic ontogeny (4, 5, 73). In this study, we The authors have no financial conflicts of interest. showed that, in developing gd T cells Lck as well as pLckCre, expression was 1) regulated during ontogeny and 2) heterogenous References among gd T cell subsets. This heterogeneity is in sharp contrast to 1. Orban, P. C., D. Chui, and J. D. Marth. 1992. Tissue- and site-specific DNA ab T cells, which homogenously expressed pLckCre and were recombination in transgenic mice. Proc. Natl. Acad. Sci. USA 89: 6861–6865. 2. Schmidt-Supprian, M., and K. Rajewsky. 2007. Vagaries of conditional gene consistently labeled by tdTomato. Future studies on single cells targeting. Nat. Immunol. 8: 665–668. might elucidate which molecular signatures determine to which 3. Hennet, T., F. K. Hagen, L. A. Tabak, and J. D. Marth. 1995. T-cell-specific extent an individual gd progenitor expresses Lck from the proxi- deletion of a polypeptide N-acetylgalactosaminyl- gene by site- directed recombination. Proc. Natl. Acad. Sci. USA 92: 12070–12074. mal or distal promoter. 4. Longo, N. S., X. Wang, R. S. Wildin, and K. M. Abraham. 1999. Regulation of We excluded the possibility that tdTomato, eYFP,orpLckCre Src-family protein tyrosine kinase transcription during lymphocyte ontogeny. expression was detrimental for gd T cells and impeded their de- Mol. Immunol. 36: 979–992. + 5. Reynolds, P. J., J. Lesley, J. Trotter, R. Schulte, R. Hyman, and B. M. Sefton. velopment because tdTomato gd T cells efficiently colonized 1990. Changes in the relative abundance of type I and type II lck mRNA tran- peripheral organs, as indicated by high rates of reporter labeling in scripts suggest differential promoter usage during T-cell development. Mol. Cell. Biol. 10: 4266–4270. newborn splenic gd T cells. This is in line with the observations that 6. Voronova, A. F., H. T. Adler, and B. M. Sefton. 1987. Two lck transcripts floxed-stop-tdTomato ROSA26 gd T cells transduced with Cre ex vivo containing different 59 untranslated regions are present in T cells. Mol. Cell. as well as that gd TcellsfromROSA26floxed-stopeYFPVav1Cre mice Biol. 7: 4407–4413. 7. Wildin, R. S., A. M. Garvin, S. Pawar, D. B. Lewis, K. M. Abraham, expressed the reporter and had no survival disadvantage. K. A. Forbush, S. F. Ziegler, J. M. Allen, and R. M. Perlmutter. 1991. Devel- Do gd T cells that have experienced pLckCre expression exhibit opmental regulation of lck gene expression in T lymphocytes. J. Exp. Med. 173: distinct characteristics in adult mice? Analysis of different Vg- 383–393. 8. Dose, M., I. Khan, Z. Guo, D. Kovalovsky, A. Krueger, H. von Boehmer, chain–expressing gd T cell subsets indicated a slight increase of K. Khazaie, and F. Gounari. 2006. c-Myc mediates pre-TCR-induced prolifer- tdTomato+ gd T cells in the Vg4+ subset of the adult spleen and ation but not developmental progression. Blood 108: 2669–2677. 2 2 9. Fischer, A. M., C. D. Katayama, G. Page`s, J. Pouysse´gur, and S. M. Hedrick. the Vg1 Vg4 subset in the adult thymus, which include a sig- 2005.Theroleoferk1anderk2inmultiple stages of T cell development. + + nificant fraction of Vg6 cells (74). Functionally, tdTomato gd Immunity 23: 431–443. 578 pLckCre INEFFICIENTLY DELETES FLOXED GENES IN gd T CELLS

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A Kidins220+lfoxpLckCre+ B mouse 1 mouse 2 thymus * p=0.028

αβ γδ αβ γδ 100 450 bp Kidins220+ cells + 75 250 bp Kidins220flox 50 20

100 bp % EYFP Kidins220Δ 10

0 αβ T γδ T αβ T γδ T pLckCre: − + 1

2 Supplemental Figure 1. In gd T cells the floxed Kidins220 gene and the ROSA26floxed-stop-eYFP cassette are

3 hardly recombined by pLckCre. (A) Kidins220+/floxpLckCre+ splenocytes from 2 independent mice were 4 ex vivo expanded with 1 µg/ml anti-TCRd (GL3) for 2 days and subsequently cultured with 100 U/ml

5 IL-2 (Preprotech) for 7 days. TCRb+TCRd- (ab), TCRb-TCRd+ (gd) T cells were sorted and the status of

6 Kidins220flox gene recombination was analyzed by PCR in purified cell populations. Arrows indicate the 7 100 bp PCR product corresponding to Kidins220D amplified as described (Cesca et al., 2012) and the

8 450 bp Kidins220+ and the 250 bp Kidins220flox PCR products amplified using the 5’- 9 AATTCGGCTTTCTGGCTACC-3’ and 5’-TCGACAGTTCAGCACAAAGG-3’ primers; n= 5. Mice

10 age: 10 weeks. (B) Thymocytes from 4 ROSA26floxed-stop-eYFPpLckCre- and 4 ROSA26floxed-stop-eYFPpLckCre+ mice were 11 stained with anti-TCRb and anti-TCRd antibodies and analyzed by flow cytometry. Frequencies of

12 eYFP+ cells within each population are shown. Mice age: 11 to 18 weeks. Statistical analysis by Mann- 13 Whitney test, error bars indicate standard deviation. 14 15

C ear skin not in text 40 ns δ 10 100 TCR cells

+ 80 CD3 60

40 % tdTomato 20 % of max epidermal γδ T 47 dermal T γδ 0 tdTomato dermal epidermal

1 Supp. S2

A Cre protein B ROSA26flox-stop-tdTomatopLckCre+ C ROSA26flox-stop-tdTomatopLckCre+ αβ T γδ T tdTomato- tdTomato+ pLckCre- pMIG Cre 38.4 0.3 pLckCre+ IRES GFP ns ns 100 αβ T γδ T 103 61.0 0.4 100 98 75 % of cells

102 83 50 101 pMIG empty 69.0 0.5 GFP- IRES GFP GFP+ CD4 + CD8 100 0.1 % of max 1.7 25

0 1 2 3

10 10 10 10 GFP : FL2δ INT LOG Cre % of max 100 3.6 0 31.0 0.5 after Cre: 0 2 5 0 2 5 (days) TCRδ tdTomato αβ T γδ T

D ROSA26flox-stop-eYFPVav1Cre+ E ROSA26flox-stop-eYFP F ROSA26flox-stop-eYFP G ROSA26flox-stop-eYFP αβ T γδ T B skin Vγ5+ ns ns ns ns Vγ1+ Vγ4+ 100 ** p=0.0039 100 100 80 100 80 80 cells cells cells

60 +

80 + + 60 60 40 60

% eYFP+ cells 40 40 % eYFP 20 40 % eYFP % eYFP 20 20 0 20 0 0 T T B T T B T T B T T αβ γδ αβ γδ αβ γδ αβ γδ 0 Vav1Cre: − + − + − + − + Vav1Cre: − + − + − + − + spleen lymph node peritoneal lung Vav1Cre: − + spleen lymph PC lung spleen lymph PC lung 16 cavity node node 17 Supplemental Figure 2. gd T cells can express Cre and recombine floxed genes.

18 Thymocytes of pLckCre+ and pLckCre- mice were stained on the surface with anti-CD4, anti-CD8 and 19 anti-TCRd and intracellularly with anti-Cre antibodies and measured by flow cytometry as in Fig. 4B.

20 The gating of ab (CD4+ and/or CD8+) and gd T cells (CD4-CD8-TCRd+) and representative histograms of

21 the intracellular Cre signals are shown. (B) Splenocytes from 5 ROSA26floxed-stop-tdTomatopLckCre+ mice were 22 expanded ex vivo and transduced with pMIGCreIRESGFP or pMIGemptyIRESGFP plasmids in 2

23 independent experiments. Cells were gated on TCRb+d- (ab T) and TCRb-d+ (gd T) and analyzed for 24 GFP and tdTomato expression on day 5 after transduction. Mice age: 13 to 19 weeks. (C) Splenocytes

25 from 5 ROSA26floxed-stop-tdTomatopLckCre+ mice were treated as in (B). The relative frequencies of tdTomato-

26 (white) and tdTomato+ (grey) ab T and gd T cells in culture on day 0, 2 and 5 after transduction are

27 displayed; ns=non-significant, statistical analysis using the Wilcoxon signed rank test for tdTomato+

28 subsets (grey); n=2. (D) Lymphocytes isolated from the indicated tissues of 3 ROSA26floxed-stop-eYFPVav1Cre+ 29 reporter mice were stained with anti-TCRb and anti-TCRd antibodies and analyzed by flow cytometry.

30 B cells were stained with anti-CD19. 4 mice were analyzed per tissue and frequencies of tdTomato+ 31 cells within each population are shown. Mice age: 11 to 16 weeks. (E) Lymphocytes were isolated

32 from 3 ROSA26floxed-stop-eYFPVav1Cre- and 3 ROSA26floxed-stop-eYFPVav1Cre+ mice and frequencies of eYFP+ TCRd+Vg5+ 33 DETCs are shown. Mice age: 11 to 12 weeks. (F) gd T cells isolated from the indicated tissues of

34 ROSA26floxed-stop-eYFPVav1Cre- and ROSA26floxed-stop-eYFPVav1Cre+ mice as in (D) were stained with anti-Vg1 35 antibodies and analyzed by flow cytometry. 3 to 4 mice were analyzed per tissue and frequencies of

36 tdTomato+ cells within Vg1+ cells are shown. Mice age: 11 to 16 weeks. (G) gd T cells as in (F) were

37 stained with anti-Vg4 antibodies and analyzed by flow cytometry. Frequencies of tdTomato+ cells

38 within Vg4+ cells are shown. Statistical analyses were done using the Mann-Whitney test, error bars 39 indicate standard deviations. 40

2 Supp. S3

A IL-17A B IFN-γ

60 70

50 40

30

20 # cell count (*10^3) # cell count (*10^3)

10 0 0 population: total IL-17A+ population: total IFN-γ+ IL-17A+ tdTomato+ IFN-γ+ tdTomato+ γδ T cells γδ T cells γδ T cells γδ T cells 41

42 Supplemental Figure 3. tdTomato+ gd T cells represent a substantial fraction of effector gd T cells.

43 (A) Splenocytes from a total of 6 ROSA26floxed-stop-tdTomatopLckCre+ mice in 3 independently performed 44 experiments were stimulated ex vivo with PMA and ionomycin for 4 hours. Cells were stained

45 extracellularly for TCRb and TCRd and intracellularly for IL-17A and IFN-g. TCRb-TCRd+ cells were

46 gated and tdTomato- and tdTomato+ cells were analyzed separately for IL-17A or IFN-g expression

47 (shown in Fig. 5E). Total cell counts of all IL-17A-producing gd T cells and of tdTomato+ IL-17A- 48 producing gd T cells are shown. (B) Total cell counts of all IFN-g-producing gd T cells and of

49 tdTomato+ IFN-g-producing gd T cells are shown as described in (A). 50 51 52 as Fig. 6D 53 ordered pooled SP Vg 54 ordered pooled THY Vg 100 100 similar figure as A 55 80 for newborn Vg? 56 80 57 60 60

58 40 40

59 20 20 60 0 0 61 62 63 64 65 66 67 68 69 70

3