Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface

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 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 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 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 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 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 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 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. 66% 100% We monitored the expression of eYFP to analyze the distribu- 23% iCre eYFP tion of iCre in Nkp46 R26R mice. The cell surface expres- eYFP − sion of NKp46 on NK1.1+CD3 splenic cells matched with the NK1.1 expression of YFP expression (Fig. 1B). NK cells isolated from bone marrow (BM), peripheral blood, spleen, parietal lymph 88% nodes (LNs), mesenteric LNs, lungs, and thymus uniformly expressed eYFP (Fig. 1C). Besides NK cells, discrete subsets of 98% 12% − − + CD27 CD4 CD8 NK1.1 T cells also express NKp46 (6, 16, 17). Con- CD11b NKp46 sistent with these data, minute percentages of eYPF+αβTandγδT cells were detected in the spleen (Fig. S3). Reciprocally, splenic − NKp46 cells, such as B cells, CD4 and CD8 αβT cells, CD1d- 0% 88%

100% restricted NKT cells, dendritic cells, neutrophils, and macrophages 100% 10% 0% did not express eYFP (Fig. 1D). Thus, the expression of eYFP in iCre eYFP Nkp46 R26R mice corresponded to the endogenous cell eYFP eYFP surface expression of NKp46, showing the faithful expression of Fig. 3. Early expression of NKp46 during NK cell maturation. (A) Flow iCre driven by Nkp46 regulatory regions. + − iCre DTR cytometric measurement of NK1.1 and eYFP on gated CD122 lin NK cells in We also generated NKp46 R26R mice by crossing NKp46iCreR26ReYFP B iCre lsl-DTR/lsl-DTR BM of mice. ( ) Flow cytometric measurement of eYFP Nkp46 to Rosa26 mice, which carry under the + − − − − fl and NKp46 expression on gated CD122 lin c-Kit CD11b NK1.1 NK pre- Rosa26 promoter a loxP- anked STOP sequence that prevents the cursors and NK1.1+ immature NK cells in BM of NKp46iCreR26ReYFP mice. (C) − expression of the downstream diphtheria toxin receptor (DTR) gene eYFP expression is shown on CD122+lin NK1.1+ BM NK cells according to iCre DTR (32). Diphtheria toxin (DT) treatment in NKp46 R26R mice their expression of CD27 and CD11b markers.

Narni-Mancinelli et al. PNAS | November 8, 2011 | vol. 108 | no. 45 | 18325 Downloaded by guest on September 25, 2021 Gated on CD122+NK1.1+lin- A BM cells CD27 CD16 CD11b CD16

eYFP CD16 CD16

BEGated on CD122+CD3- BM cells C Gated on NKp46+CD3- BM cells DFGated on NKp46+CD3- BM cells Gated on NKp46+CD3- BM cells Gated on NKp46+CD11b-CD3- BM cells

NK1.1 NKG2D DX5 CD11b c-Kit

CD94 CD16 NKG2D c-Kit Ly49

DX5

− Fig. 4. CD16 is expressed on immature NK cells. (A) CD16 expression is shown in CD122+lin NK1.1+ BM NK cells of NKp46iCreR26ReYFP mice according to CD27 and CD11b expression. eYFP expression is also shown on immature CD27+CD11b−/lowCD16− and CD16+ NK cells. (B–F) Flow cytometric measurement of NK cell maturation marker expression (open histograms) or control isotype (shaded histograms) on indicated cells from BM of NKp46iCreR26ReYFP mice.

reinforcing the demonstration of the selectivity of iCre expression Following the same track, we then focused on CD16 assessed in NKp46iCreR26ReYFP mice. (FcγRIIIA). Indeed, the cell surface expression of CD16 defines a checkpoint in human NK cell differentiation that is associated Expression of NKp46 During NK Cell Differentiation. It has been with NK cell maturation, as evidenced by the perforin-dependent proposed that NK cell differentiation is initiated at a precursor cytotoxic function of CD16+CD56dim NK cells in contrast to − stage (stage I) defined by the cell surface expression of CD122, the CD56brightCD16 /low human NK cells (38). In addition, recent data − β-chain of IL-2/IL-15 receptors, and the lack of the lineage (lin) support a model in which CD56brightCD16 /low NK cells differen- markers (33). The acquisition of surface NK1.1 was used to define tiate into CD16+CD56dim NK cells (39). Although mouse NK cells stage II of NK cell differentiation. Stage III NK cells was defined also express CD16, the kinetics of CD16 surface expression on by the expression of c- (the stem cell factor receptor CD117), mouse NK cells is still unknown, in part due to the lack of reagent stage IV by the CD49 β1 integrin DX5, and stage V by the CD11b able to discriminate CD16 from CD32 (FcγRIIB). We addressed β this issue using a recently described anti-CD16 antibody (40). As 2 integrin (34). On fully mature NK cells (stage V), c-kit and − shown in Fig. 4A, CD16 was induced at the CD27+CD11b /low CD27 expression are lost, whereas KLRG-1 and CD43 are ex- − − pressed (35). One of the caveats of this classification resides stage, but after NKp46, because CD16 cells contain both eYFP and eYFP+ NK cells in comparable amounts, whereas CD16+ in the use of mouse markers that are not conserved in human + (e.g., DX5). In contrast to these molecules, NKp46 is conserved cells are uniformly eYFP . We then analyzed the kinetics of in- iCre eYFP duction of CD94, NKG2D, DX5, CD11b, c-Kit, and Ly49 recep- across species (7). We took advantage of the Nkp46 R26R tors (using a pan Ly49 mAb). We found that CD94 was expressed mice to revisit the stages of NK cell differentiation. Stage I + − + − very early in CD122 NK1.1 cells (Fig. 4B). Because the majority (CD122 NK1.1 ) BM NK cells were negative for both NKp46 and − of NKG2D /low cells expressed CD16, the data indicated that eYFP, whereas NKp46 and eYFP were expressed on a majority of + CD16 induction preceded that of NKG2D (Fig. 4C). NKG2D was NK1.1 BM NK cells (Fig. 3 A and B). Furthermore, after in vitro − + + − − − expressed before DX5, because DX5 cells contain NKG2D culture in IL-15 of sorted CD122 CD3 CD19 NKp46 BM cells, + + − cells, whereas all DX5 cells are uniformly NKG2D (Fig. 4D). As + − we observed the induction of NKp46 selectively on NK1.1 CD3 for c-Kit, despite its low cell surface expression in both CD11b /low − NK cells (Fig. S5). Thus, the cell surface expression of NKp46 was and CD11b+ cells, the c-Kit cells contained a majority of DX5+ acquired on BM NK cells after NK1.1. The monitoring of NK cell cells, indicating that c-Kit expression occurs after DX5 induction − maturation using CD27 and CD11b markers further supported (Fig. 4E). Finally, regarding Ly49 expression, most c-Kit cells are − this kinetics of differentiation (36, 37), because the acquisition of Ly49 , whereas most c-Kit+ cells are Ly49+ (Fig. 4F), suggesting − NKp46 occurred at the CD27+CD11b /low stage and remained coinduction of these molecules. stable (Fig. 3C and Fig. S6). Similar data were obtained when NK We then analyzed whether the acquisition of the cell surface cells were isolated from spleen, LNs, and lung. expression of NKp46 and CD16 was associated with modifications

18326 | www.pnas.org/cgi/doi/10.1073/pnas.1112064108 Narni-Mancinelli et al. Downloaded by guest on September 25, 2021 A 783 1525 9448 370

eYFPhiNK1.1- Gated on CD3-CD19- gut cells

1721 1579 10412 286

eYFPhiNK1.1dim

2632 432 17318 57

eYFP+NK1.1+ eYFP NK1.1 NKp46 CD127 CD45 c-Kit

B YFP ROR t Merge

Sytox

− Fig. 5. Characterization of gut NKp46+eYFP+ and NKp46dim/loweYFPhigh cells. (A) Flow cytometric characterization of CD3-CD19-eYFPhiNK1.1 , eYFP- hiNK1.1dim, eYFP+NK1.1+ cells from small intestine of NKp46iCreR26ReYFP mice for indicated makers (open histograms) or control isotype (gray histograms). Results are representative of two experiments. (B) Immunofluorescence histology of small intestine frozen sections from NKp46CreR26ReYFP mice stained with anti-eYFP (green) and anti-RORt (red). Nuclei were counterstained with Sytox (grey). Scale bars, 10 mm. Data are representative of at least three experiments.

in NK cell function. The induction of NKp46 expression was as- induction of NKp46 as a key step in the acquisition of NK cell sociated with a higher ability to secrete IFN-γ upon stimulation reactivity to IL-12 and -18. − + with IL-12 and -18, although the response of NKp46 NK1.1 NK Together these data converged to propose the acquisition of IMMUNOLOGY cells and NKp46+NK1.1+ NK cells to phorbol 12-myristate 13- NKp46 expression as a useful and stable tool to mark a previously acetate (PMA) and ionomycin was comparable (Fig. S7A). This undescribed stage 2 in NK cell differentiation. These findings also higher reactivity of NKp46+NK1.1+ NK cells to IL-12 and -18 lead us to propose a revised model of NK cell differentiation based stimulation was associated with the induction of cell surface IL-12 on the sequential and stable acquisition of cell surface molecules and -18 receptors (Fig. S8). In contrast, the response of as follows: CD122 (stage 1), NK1.1 (stage 2), NKp46 (stage 3), − CD16 NK1.1+ NK cells and CD16+NK1.1+ NK cells to IL-12 CD16 (stage 4), and CD11b (stage 5) (Table 1). Stage 6 of NK cell and -18 or PMA and ionomycin was similar (Fig. S7B). Un- maturation is still defined by the disappearance of CD27 in −/low + + fi expectedly, the IFN-γ production of CD11b NK1.1 and CD11b mature NK cells, awaiting the identi cation of a cell CD11b+NK1.1+ NK cells was also comparable (Fig. S7C). Thus, surface molecule that would be selectively and stably induced at despite the reported acquisition of NK cell functional response to this stage. Indeed, although KLRG-1 and CD43 are preferentially various stimulations at the CD11b+ stage (34), our data define the expressed in stage 6 NK cells (35), their cell surface expression is initiated at stage 5. This model is fully compatible with the pre- vious classification (34) but refines the early stages of NK cell development, i.e., between stage II, as defined earlier by the in- Table 1. NK cell maturation duction of NK1.1 and CD94, and stage III, as defined by the in- Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Stage 6 duction of Ly49 and c-kit.

CD122 + +++++Gut NKp46+ Cells. NKp46+ cells comprise not only bona fide NK NK1.1 − + ++++cells and a minute subset of T cells, but also a population of mu- NKp46 −−+ +++cosal cells. We thus took advantage of the Nkp46iCreR26ReYFP CD16 Low Low Low + ++mice to revisit the heterogeneity of NKp46+ cells in various organs CD11b Low Low Low Low + + by comparing for the expression of eYFP and the cell surface of − −− CD27 Low + + +/ NKp46. In BM, spleen, LNs, and lungs, NKp46 and eYFP were NKG2D Low Low Low + + + coexpressed (Fig. S9A Upper). Less than 10% of splenic and BM −−− DX5 +++eYFP+ lymphocytes expressed low surface density of NKp46, but −−− −− − c-Kit ++/ they did not correspond to a population of eYFP+NKp46 cells −−− Ly49 +++because they appeared as a continuum with NKp46+ cells (Fig. CD94 Low + + + + + S9A Upper) and were uniformly NK1.1+ (Fig. S9A Lower). Con- CD43 −−−−Low + fi −−−− rmation of this interpretation was obtained by using a brighter KLRG1 Low + revelation of the NKp46 fluorescence using an anti-NKp46 goat A revised model of NK cell maturation is proposed based on the sequen- antiserum and Alexa 647-conjugated secondary reagent instead of tial expression of CD122, NK1.1, NKp46, CD16, and CD11b at NK cell surface. an anti-NKp46 mAb (Fig. S10).

Narni-Mancinelli et al. PNAS | November 8, 2011 | vol. 108 | no. 45 | 18327 Downloaded by guest on September 25, 2021 Gated on CD3- liver cells 357 134 294 361

eYFPhi

801 923 44 1861

eYFP+ eYFP

NK1.1 NKp46 CD11b TRAIL DX5

Fig. 6. Characterization of liver NKp46+eYFP+ and NKp46dim/loweYFPhigh cells. Flow cytometric characterization of NK1.1+CD3−eYFPhi and NK1.1+CD3−eYFP+ liver cells from NKp46iCreR26ReYFP mice for indicated makers (open histograms) or control isotype (gray histograms). Results are representative of two to four experiments.

A distinct picture emerged from the analysis of gut lymphocytes mor necrosis factor-related apoptosis-inducing (TRAIL) because two populations of cells expressing eYFP were detected in (45). A similar phenotype was observed for the eYFPhi liver the small intestine (Fig. 5ALeft). Indeed, besides gut eYFP+ cells NK cells (Fig. 6), indicating that they corresponded to the − that uniformly express NK1.1 and the same level of eYFP com- CD11b /lowDX5dimTRAIL+ NK cell subset previously described in pared with NKp46+ BM, splenic, LN, and lung cells, a subset of the same organ. The cell surface expression of Ly49 molecules was gut eYFPhi cells with lower cell surface expression of NK1.1 was mostly restricted to the eYFP+ liver NK cells as in the spleen (Fig. detected. Further analysis revealed that eYFP+NK1.1+ cells were S12B), which is consistent with the bona fide NK cell phenotype − − CD3 NKp46+CD127dimCD45hic-Kit /dim (Fig. 5A Right)and of eYFP+ liver NK cells and the more immature phenotype of corresponded to bona fide small intestine NK cells (28). In con- eYFPhi liver NK cells. Genetic tracing revealed that these two liver − trast, eYFPhiNK1.1 and eYFPhiNK1.1dim cells corresponded to a subsets are quite divergent, because the level of eYFP expression in − continuum of NKp46dimCD127+c-Kit+ expressing intermediate CD11b /low and CD11b+ BM NK cells was similar, in contrast to − levels of CD45 (Fig. 5A Right). These cells corresponded to the the distinct eYFP levels in CD11b /low and CD11b+ liver NK cells NKp46+ROR-γt+ subset that produces IL-22 (22, 23, 26, 28, 41), (Fig. S13). Thus, the levels of YFP are not merely associated with consistent with the coexpression of eYFP and ROR-γt in gut cells various stages of NK cell maturation, but with a more profound from isolated lymphoid follicules (Fig. 5B) and their ability to lineage commitment of NK cell subsets, highlighting the use of produce IL-22 at steady state and upon IL-23 stimulation (Fig. Nkp46iCreR26ReYFP mice for in vivo fate-mapping experiments. S11). In contrast, only the eYFP+NK1.1+ cells produced IFN-γ upon IL-12 and -18 stimulation, consistent with their bona fide Conclusions small intestine NK cell identity (Fig. S11). We report here the fate mapping of NKp46+ cells in vivo through Because the expression of iCre induced the irreversible ex- the generation and characterization of an unprecedented model of pression of eYFP in Nkp46iCreR26ReYFP mice, the distinct levels Nkp46iCre knock-in mice. Earlier attempts to create a mouse of eYFP observed at a single cell level could not result from model selectively targeting NKp46+ cells by using nontargeted a distinct regulation at the Nkp46 locus but, rather, indicated transgenesis have failed due to a complex and still poorly un- a distinct regulation of the Rosa26 locus in eYFPhi vs. eYFP+ derstood regulation of the NKp46 locus (6, 29). To visualize iCre cells, as shown in other reporter mouse models (42). Thus, the activity in Nkp46iCre mice, we crossed them with R26ReYFP re- genetic tracing in Nkp46iCreR26ReYFP mice indicated that the porter mice. In Nkp46iCreR26ReYFP mice, the fluorescent reporter Rosa26 locus was differently regulated in bona fide NK cells and permanently labeled cells that had switched on the expression of in NKp46+ROR-γt+ cells, supporting by another approach that the NKp46 gene. Using these mice, we have shown that the ex- NK cell receptor (NKR)+ROR-γt+ and NK cells represent two pression of iCre faithfully corresponded to the endogenous ex- distinct lineages (19, 24, 28). pression of NKp46. The genetic tracing of NKp46+ cells in vivo allowed us to reveal the stability of NKp46 cell surface expression. Liver NKp46+ Cells. The analysis of liver lymphocytes in In addition, the acquisition of NKp46 marked a checkpoint of NK Nkp46iCreR26ReYFP mice also revealed an unexpected complex- cell maturation. Based on these data, we propose a unique model ity within eYFP+ cells. As in the gut, two populations of eYFPhi of NK cell differentiation, which also includes CD16 as a marker of and eYFP+ lymphocytes were detected. eYFPhi cells were NK cell maturation. One advantage of this unique model resides in NK1.1+ and comprised a continuum of NKp46dim and NKp46+ the use of cell surface molecules that are conserved in both mouse cells (Fig. 6), whereas eYFP+ cells consisted of a more homo- and human, with the exception of mouse NK1.1. Along this line, geneous NK1.1+NKp46+ cell population. Besides the cell sur- preliminary data obtained on CD34+ hematopoietic cell progeni- face expression of NKp46, eYFPhi and eYFP+ cells expressed tors from human cord blood indicate that the induction of surface a panel of cell surface receptors present on NK cells, such as CD56 precedes that of NKp46 in an in vitro NK cell differentiation CD122, DX5, 2B4, NKG2D, and CD16, indicating that they assay, supporting the hypothesis that CD56 could be positioned in corresponded to two distinct subsets of liver NK cells (Fig. 6 and the human NK cell differentiation pathway as NK1.1 in the mouse. Fig. S12A). Of note, it has recently been shown that liver NK Furthermore, the differential expression of YFP in Nkp46iCre cells comprised a population of CXCR6+ NK cells capable of R26ReYFP mice showed that gut NKR+ROR-γt+ and NK cells mediating hapten- and virus-specific memory responses (43, 44). represent two distinct lineages. In addition, fate mapping experi- However, the cell surface expression of CXCR6 did not correlate ments revealed the genetic heterogeneity of the two subsets − − with eYFP levels in liver NK cells from Nkp46iCreR26ReYFP.A of CD11b /lowDX5dimTRAIL+ and CD11b+DX5+TRAIL liver noticeable difference between liver eYFPhi and eYFP+ lympho- NK cells. It is puzzling that besides bona fide eYFP+ − − cytes resided in the high percentage of CD11b /dim cells within CD3 NKp46+NK1.1+ NK cells, subsets of NKp46dim/loweYFPhigh the eYFPhi NK cell subset. This sign of immaturity in a subset cells were present in gut and liver. Besides their common of liver NK cells has been reported and associated with the low NKp46dim/loweYFPhigh phenotype, gut and liver eYFPhi cells did surface expression of DX5 and the constitutive expression of tu- not appear to be directly related, because gut eYFPhi cells were

18328 | www.pnas.org/cgi/doi/10.1073/pnas.1112064108 Narni-Mancinelli et al. Downloaded by guest on September 25, 2021 − NK1.1 /dim and corresponded to NKR+ROR-γt+ cells, whereas used for experiments. Unnicked Diphtheria Toxin from Corynebacterium liver eYFPhi cells were NK1.1+ and expressed TRAIL. Together, diphtheria (Calbiochem, 500 ng/mouse) diluted in PBS was injected i.p. fi iCre these results de ne Nkp46 mice as a unique mouse model of SI fi + Cell Preparation. Cells were prepared as described (22). For details, see speci c targeting in NKp46 cells, allowing the generation of Materials and Methods. unique mouse strains based on the crossing of Nkp46iCre mice to a + variety to floxed mice to dissect the biology of NKp46 NK, T, and Flow Cytometry. Flow cytometric analysis was done on a FACS Canto (Becton + + gut NKR ROR-γt cells. Dickinson, San Diego, CA). For details on reagents, see SI Materials and Methods.

Material and Methods Tissue Immunofluorescence. Portions of small and large intestine were iso- Mice and Crosses. We have generated a Nkp46iCre knock-in mouse line in lated from mice and treated as previously described (22). For staining details, which the gene encoded the improved recombinase (icre) was inserted by see SI Materials and Methods. homologous recombination at the 3’ end of the Nkp46 gene (see SI Materials and Methods). Recombinant offspring of chimeric mice was ACKNOWLEDGMENTS. We thank C. Cognet for help and advice and the ’ crossed with transgenic flipase (FLP)-expressing mice (46). Nkp46iCre mice Centre d Immunologie de Marseille-Luminy (CIML) mouse house, cytome- R26ReYFP try, and knockout/knock-in facilities. E.V. and S.U. are supported by Euro- were crossed to (31) (CDTA, Orleans, France). All mice used in pean Research Council Advanced Grants, grants from Agence Nationale de iCre eYFP iCre these studies were Nkp46 R26R , Nkp46 , or WT control litter- la Recherche (ANR) and Ligue Nationale Contre le Cancer (Equipe Labellisée mates. All mice were bred in pathogen-free breeding facilities at Centre “La Ligue”), and institutional grants from Institut National de la Santé et d’Immunologie de Marseille-Luminy and used between 6 and 10 weeks de la Recherche Médicale, Centre National de la Recherche Scientifique, of age. Experiments were conducted in accordance with institutional and Université de la Méditerranée (to CIML). E.V., E.T., and N.Y. are sup- Nkp46iCre ported by ANR Grant N°R08063AS. E.V. is a scholar from Institut Universi- guidelines for animal care and use. mice were crossed to taire de France. E.N.-M. is a recipient of Agence pour la Recherche Contre lsl-DTR/lsl-DTR iCre/wt lsl-DTR/wt Rosa26 mice (32) to obtain NKp46 ; Rosa26 mice le Cancer. J.C. was supported by Région Provence Alpes Côte d’Azur and referred as to Nkp46iCreR26RDTR. Six- to eight-week-old offspring mice were Innate-Pharma.

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Narni-Mancinelli et al. PNAS | November 8, 2011 | vol. 108 | no. 45 | 18329 Downloaded by guest on September 25, 2021