Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor Emilie Narni-Mancinellia,b,c,1, Julie Chaixa,b,c,1,2, Aurore Fenisa,b,c, Yann M. Kerdilesa,b,c, Nadia Yessaada,b,c, Ana Reyndersa,b,c, Claude Gregoirea,b,c, Herve Luchea,b,c, Sophie Ugolinia,b,c, Elena Tomaselloa,b,c, Thierry Walzera,b,c,3, and Eric Viviera,b,c,d,4 aCentre d’Immunologie de Marseille-Luminy, Université de la Méditerranée UM 631, Campus de Luminy Case 906, 13288 Marseille, France; bInstitut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche S 631, 13009 Marseille, France; cCentre National de la Recherche Scientifique, Unité Mixte de Recherche 6102, 13288 Marseille, France; and dAssistance Publique, Hôpitaux de Marseille, Hôpital de la Conception, 13385 Marseille, France Edited* by Christophe Benoist, Harvard Medical School, Boston, MA, and approved September 19, 2011 (received for review July 26, 2011) NKp46 is a cell surface receptor expressed on natural killer (NK) the selectivity of expression of eGFP in NDE mice mirrored that of cells, on a minute subset of T cells, and on a population of innate endogenous NKp46 in a fraction of transgenic mice, variegation at lymphoid cells that produce IL-22 and express the transcription the transgenic locus led to unpredictable variation in the pene- factor retinoid-related orphan receptor (ROR)-γt, referred to as NK trance of the transgene expression in mouse littermates. Another cell receptor (NKR)+ROR-γt+ cells. Here we describe Nkp46iCre knock- transgenic mouse expressing the improved Cre (iCre) recombinase in mice in which the gene encoding the improved Cre (iCre) recom- under the control of the Nkp46 promoter was recently reported binase was inserted into the Nkp46 locus. This mouse was used to and crossed to eGFP reporter mice (29). However, on average noninvasively trace cells expressing NKp46 in vivo. Fate mapping only 80% of NKp46+ NK cells expressed eGFP, and no eGFP experiments demonstrated the stable expression of NKp46 on expression was detected in T cells, suggesting that the regulation NK cells and allowed a reappraisal of the sequential steps of NK of the iCre transgene expression in this model did not match with cell maturation. NKp46 genetic tracing also showed that gut the endogenous expression of NKp46, hence hampering the use NKR+ROR-γt+ and NK cells represent two distinct lineages. In addi- of these mice for selective gene targeting in NKp46+ cells. To tion, the genetic heterogeneity of liver NK cells was evidenced. circumvent these caveats of expression pattern that are classically Finally, Nkp46iCre mice also represent a unique mouse model of observed in transgenic mice (30), we generated a knock-in mouse conditional mutagenesis specifically in NKp46+ cells, paving the line in which the gene encoded the improved recombinase (icre) way for further developments in the biology of NKp46+ NK, T, and was inserted by homologous recombination at the 3′ end of Nkp46, NKR+ROR-γt+ cells. in an attempt to drive its expression by all endogenous Nkp46 regulatory elements. Here we report that iCre expression faithfully natural killer cell differentiation | NKp46 knock-in corresponds to the endogenous expression of NKp46, on bona fide NK cells, on a subset of gut ILCs, as well as on very discrete sub- atural killer (NK) cells are effector and regulatory lym- populations of T cells, allowing us to trace the fate of the het- Nphocytes of the innate immune system that contribute to erogeneous NKp46+ populations of cells. tumor surveillance, hematopoietic allograft rejection, control of microbial infections, and pregnancy (1). NK cells can be cyto- Results toxic and secrete an array of cytokines and chemokines, such as Characterization of Nkp46iCre Knock-in Mice. We generated a knock- IFN-γ and β-chemokines. in mouse line in which icre was inserted by homologous re- NKp46 (NCR1, CD335) is a marker of NK cells in all mam- combination at the 3′ end of the Nkp46 gene (Fig. 1A and Fig. S1). iCre/wt malian species tested so far, including human, nonhuman pri- Nkp46 mice were obtained at Mendelian frequencies, de- mates, mouse, rat, and cow (2–8). NKp46 is an Ig-like superfamily veloped normally, were fertile, and showed no significant varia- cell surface receptor, which is a member of a group of natural tions in the numbers of lymphoid and myeloid subsets compared cytotoxicity receptors (NCRs) with NKp44 (NCR2, CD336) and with wild-type (wt) littermates. NK cell counts, phenotype, and iCre/wt NKp30 (NCR3, CD337) (9). NKp46 is associated with immunor- in vitro effector function were not affected in Nkp46 mice, eceptor tyrosine-based activation motif-bearing polypeptides, such despite a down-regulation in the density of cell surface NKp46 that + as CD3-ζ and FcR-γ, which transduce potent activating signals did not alter the percentage of NKp46 NK cells (Fig. S2). eYFP/wt upon triggering (4, 10). NKp46 is involved in tumor cell recogni- Rosa26eYFP reporter mice (R26R ) carry under the fl tion via still-unidentified ligands (11, 12) and has also been de- Rosa26 promoter a loxP- anked STOP sequence that prevents the scribed as binding viral hemagglutinins (13, 14). Finally, it has been expression of the downstream eYFP gene. The STOP sequence is reported that NK cells contribute via NKp46 to type I diabetes removed and eYFP is expressed in cells where iCre is expressed through the destruction of pancreatic β-islets (15). NKp46 is found on all mature NK cells regardless of their anatomic localization in both human and mouse. The selective expression of NKp46 on Author contributions: E.N.-M., T.W., and E.V. designed research; E.N.-M., J.C., A.F., Y.M.K., N.Y., A.R., E.T., and T.W. performed research; J.C., A.F., C.G., H.L., and T.W. contributed NK cells has two reported exceptions: rare T-cell subsets (6, 16, new reagents/analytic tools; E.N.-M., S.U., and E.V. analyzed data; and E.N.-M. and + 17) and a mucosal population of NKp46 innate lymphoid cells E.V. wrote the paper. (ILCs) that express the transcription factor retinoid-related or- Conflict of interest statement: E.V. is a co-founder and shareholder of Innate-Pharma. γ phan receptor (ROR)- t and produce IL-22, a key cytokine for the *This Direct Submission article had a prearranged editor. – activation and defense of epithelial cells (18 28). 1E.N.-M. and J.C. contributed equally to this work. The NKp46 amino acid sequence is highly conserved in all 2Present address: Abramson Family Cancer Research Institute, Department of Medicine, mammals. The striking homologies in the regulatory regions of the University of Pennsylvania, Philadelphia, PA 19104. NKP46 gene from opossum to human prompted us to generate 3Present address: Université de Lyon, Institut National de la Santé et de la Recherche a transgenic mouse line, called NDE, in which the diphtheria toxin Médicale U851, F-69007 Lyon, France. receptor (DTR) and the enhanced green fluorescent protein 4To whom correspondence should be addressed. E-mail: [email protected]. (eGFP) expression were driven by a 450-bp conserved promoter This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. region (P1) upstream of Nkp46 (Ncr1) start codon (6). Although 1073/pnas.1112064108/-/DCSupplemental. 18324–18329 | PNAS | November 8, 2011 | vol. 108 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1112064108 Downloaded by guest on September 25, 2021 STOP Spleen Liver A A 4 3 — ) NKp46/Ncr1 IRES Cre ) 6 5 DT 3 2 Gated on splenic lymphocytes 2 B 1 1 NK cells (x10 NK cells (x10 *** *** 0 0 BM LN 2 6 ) ) 5 4 0 eYFP NK1.1 CD3 NKp46 4 1 2 Gated on NK1.1+/NKp46+/CD3- NK cells C BM BLOOD SPLEEN LNs NK cells (x10 *** NK cells (x1 *** 0 0 B Spleen Liver BM LN B LUNG THYMUS MLNs NKp46iCre T R26ReYFP NKp46iCre NKT NK 0 50 100 150 eYFP % of non-treated D B cells CD4+ αβT cells CD8+ αβT cells NKT Fig. 2. Specific depletion of NK cells in DT-treated NKp46iCreR26RDTR mice. Groups of mice were treated i.v. with DT, and cell recovery was analyzed 2 d later. (A) NK cell proportions among live leukocytes of nontreated (−)or DT-treated mice. (B) Lymphoid cell populations recovery expressed as per- cent of cell numbers in nontreated mice (mean ± SEM). n ≥ 12 per group and DCs neutrophils monocytes macrophages per organ. ***P < 0.001. induced a depletion of NK cells in blood, spleen, BM, and LN (Fig. 2A and Fig. S4). The depletion induced by DT treatment in eYFP NKp46iCreR26RDTR mice was restricted to NK cells (Fig. 2B), Fig. 1. Faithful expression of eYFP according to NKp46 expression in NKp46iCreR26ReYFP mice. (A) Schematic representation of the strategy used to generate NKp46iCre mice. (B) Flow cytometric measurement of eYFP and NKp46iCreR26ReYFP C D Gated on CD122+lin- NKp46 expressions on NK cells from spleen of .( and ) IMMUNOLOGY A BM cells Flow cytometric measurement of eYFP expression on gated NK cells (C)or indicated cell population (D) from indicated organs of NKp46iCreR26ReYFP (open histograms) and control NKp46iCre mice (gray histograms). Results are representative of three experiments. eYFP (31). Nkp46iCre/wt and R26ReYFP/wt mice were crossed to obtain NK1.1 iCre/wt eYFP/wt Nkp46 R26R mice heterozygous at both loci and re- Gated on CD122+NK1.1+lin- BM cells iCre eYFP iCre eYFP B Gated on CD122+lin-c-Kit- C ferred as to Nkp46 R26R thereafter. Nkp46 R26R CD11b- BM cells were generated to ensure the irreversible expression of the eYFP reporter gene in NKp46+ cells and their progeny, irrelevant of the 77% possible arrest in Nkp46 transcription.