Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor

Abstract

NKp46 is a cell surface receptor expressed on natural killer (NK) cells, on a minute subset of T cells, and on a population of innate lymphoid cells that produce IL-22 and express the transcription factor retinoid-related orphan receptor (ROR)-γt, referred to as NK cell receptor (NKR)⁺ROR-γt⁺ cells. Here we describe Nkp46^iCre knock-in mice in which the gene encoding the improved Cre (iCre) recombinase was inserted into the Nkp46 locus. This mouse was used to noninvasively trace cells expressing NKp46 in vivo. Fate mapping experiments demonstrated the stable expression of NKp46 on NK cells and allowed a reappraisal of the sequential steps of NK cell maturation. NKp46 genetic tracing also showed that gut NKR⁺ROR-γt⁺ and NK cells represent two distinct lineages. In addition, the genetic heterogeneity of liver NK cells was evidenced. Finally, Nkp46^iCre mice also represent a unique mouse model of conditional mutagenesis specifically in NKp46⁺ cells, paving the way for further developments in the biology of NKp46⁺ NK, T, and NKR⁺ROR-γt⁺ cells.

Natural killer (NK) cells are effector and regulatory lymphocytes of the innate immune system that contribute to tumor surveillance, hematopoietic allograft rejection, control of microbial infections, and pregnancy (1). NK cells can be cytotoxic and secrete an array of cytokines and chemokines, such as IFN-γ and β-chemokines.

NKp46 (NCR1, CD335) is a marker of NK cells in all mammalian species tested so far, including human, nonhuman primates, mouse, rat, and cow (2–8). NKp46 is an Ig-like superfamily cell surface receptor, which is a member of a group of natural cytotoxicity receptors (NCRs) with NKp44 (NCR2, CD336) and NKp30 (NCR3, CD337) (9). NKp46 is associated with immunoreceptor tyrosine-based activation motif-bearing polypeptides, such as CD3-ζ and FcR-γ, which transduce potent activating signals upon triggering (4, 10). NKp46 is involved in tumor cell recognition via still-unidentified ligands (11, 12) and has also been described as binding viral hemagglutinins (13, 14). Finally, it has been reported that NK cells contribute via NKp46 to type I diabetes 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 NK cells has two reported exceptions: rare T-cell subsets (6, 16, 17) and a mucosal population of NKp46⁺ innate lymphoid cells (ILCs) that express the transcription factor retinoid-related orphan receptor (ROR)-γt and produce IL-22, a key cytokine for the activation and defense of epithelial cells (18–28).

The NKp46 amino acid sequence is highly conserved in all mammals. The striking homologies in the regulatory regions of the NKP46 gene from opossum to human prompted us to generate a transgenic mouse line, called NDE, in which the diphtheria toxin receptor (DTR) and the enhanced green fluorescent protein (eGFP) expression were driven by a 450-bp conserved promoter region (P1) upstream of Nkp46 (Ncr1) start codon (6). Although the selectivity of expression of eGFP in NDE mice mirrored that of endogenous NKp46 in a fraction of transgenic mice, variegation at the transgenic locus led to unpredictable variation in the penetrance of the transgene expression in mouse littermates. Another transgenic mouse expressing the improved Cre (iCre) recombinase under the control of the Nkp46 promoter was recently reported and crossed to eGFP reporter mice (29). However, on average only 80% of NKp46⁺ NK cells expressed eGFP, and no eGFP expression was detected in T cells, suggesting that the regulation of the iCre transgene expression in this model did not match with the endogenous expression of NKp46, hence hampering the use of these mice for selective gene targeting in NKp46⁺ cells. To circumvent these caveats of expression pattern that are classically observed in transgenic mice (30), we generated a knock-in mouse line in which the gene encoded the improved recombinase (icre) was inserted by homologous recombination at the 3′ end of Nkp46, in an attempt to drive its expression by all endogenous Nkp46 regulatory elements. Here we report that iCre expression faithfully corresponds to the endogenous expression of NKp46, on bona fide NK cells, on a subset of gut ILCs, as well as on very discrete subpopulations of T cells, allowing us to trace the fate of the heterogeneous NKp46⁺ populations of cells.

Results

Characterization of Nkp46^iCre Knock-in Mice.

We generated a knock-in mouse line in which icre was inserted by homologous recombination at the 3′ end of the Nkp46 gene (Fig. 1A and Fig. S1). Nkp46^iCre/wt mice were obtained at Mendelian frequencies, developed normally, were fertile, and showed no significant variations in the numbers of lymphoid and myeloid subsets compared with wild-type (wt) littermates. NK cell counts, phenotype, and in vitro effector function were not affected in Nkp46^iCre/wt mice, despite a down-regulation in the density of cell surface NKp46 that did not alter the percentage of NKp46⁺ NK cells (Fig. S2).

Fig. 1.

Faithful expression of eYFP according to NKp46 expression in NKp46^iCreR26R^eYFP mice. (A) Schematic representation of the strategy used to generate NKp46^iCre mice. (B) Flow cytometric measurement of eYFP and NKp46 expressions on NK cells from spleen of NKp46^iCreR26R^eYFP. (C and D) Flow cytometric measurement of eYFP expression on gated NK cells (C) or indicated cell population (D) from indicated organs of NKp46^iCreR26R^eYFP (open histograms) and control NKp46^iCre mice (gray histograms). Results are representative of three experiments.

Rosa26eYFP reporter mice (R26R^eYFP/wt) carry under the Rosa26 promoter a loxP-flanked STOP sequence that prevents the expression of the downstream eYFP gene. The STOP sequence is removed and eYFP is expressed in cells where iCre is expressed (31). Nkp46^iCre/wt and R26R^eYFP/wt mice were crossed to obtain Nkp46^iCre/wtR26R^eYFP/wt mice heterozygous at both loci and referred as to Nkp46^iCreR26R^eYFP thereafter. Nkp46^iCreR26R^eYFP were generated to ensure the irreversible expression of the eYFP reporter gene in NKp46⁺ cells and their progeny, irrelevant of the possible arrest in Nkp46 transcription.

We monitored the expression of eYFP to analyze the distribution of iCre in Nkp46^iCreR26R^eYFP mice. The cell surface expression of NKp46 on NK1.1⁺CD3⁻ splenic cells matched with the expression of YFP expression (Fig. 1B). NK cells isolated from bone marrow (BM), peripheral blood, spleen, parietal lymph nodes (LNs), mesenteric LNs, lungs, and thymus uniformly expressed eYFP (Fig. 1C). Besides NK cells, discrete subsets of CD4⁻CD8⁻NK1.1⁺ T cells also express NKp46 (6, 16, 17). Consistent with these data, minute percentages of eYPF⁺αβT and γδT cells were detected in the spleen (Fig. S3). Reciprocally, splenic NKp46⁻ cells, such as B cells, CD4 and CD8 αβT cells, CD1d-restricted NKT cells, dendritic cells, neutrophils, and macrophages did not express eYFP (Fig. 1D). Thus, the expression of eYFP in Nkp46^iCreR26R^eYFP mice corresponded to the endogenous cell surface expression of NKp46, showing the faithful expression of iCre driven by Nkp46 regulatory regions.

We also generated NKp46^iCreR26R^DTR mice by crossing Nkp46^iCre to Rosa26^{lsl-DTR/lsl-DTR} mice, which carry under the Rosa26 promoter a loxP-flanked STOP sequence that prevents the expression of the downstream diphtheria toxin receptor (DTR) gene (32). Diphtheria toxin (DT) treatment in NKp46^iCreR26R^DTR mice induced a depletion of NK cells in blood, spleen, BM, and LN (Fig. 2A and Fig. S4). The depletion induced by DT treatment in NKp46^iCreR26R^DTR mice was restricted to NK cells (Fig. 2B), reinforcing the demonstration of the selectivity of iCre expression assessed in NKp46^iCreR26R^eYFP mice.

Fig. 2.

Specific depletion of NK cells in DT-treated NKp46^iCreR26R^DTR 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 percent of cell numbers in nontreated mice (mean ± SEM). n ≥ 12 per group and per organ. ***P < 0.001.

Expression of NKp46 During NK Cell Differentiation.

It has been proposed that NK cell differentiation is initiated at a precursor stage (stage I) defined by the cell surface expression of CD122, the β-chain of IL-2/IL-15 receptors, and the lack of the lineage (lin) markers (33). The acquisition of surface NK1.1 was used to define stage II of NK cell differentiation. Stage III NK cells was defined by the expression of c-kit (the stem cell factor receptor CD117), stage IV by the CD49 β1 integrin DX5, and stage V by the CD11b β2 integrin (34). On fully mature NK cells (stage V), c-kit and CD27 expression are lost, whereas KLRG-1 and CD43 are expressed (35). One of the caveats of this classification resides in the use of mouse markers that are not conserved in human (e.g., DX5). In contrast to these molecules, NKp46 is conserved across species (7). We took advantage of the Nkp46^iCreR26R^eYFP mice to revisit the stages of NK cell differentiation. Stage I (CD122⁺NK1.1⁻) BM NK cells were negative for both NKp46 and eYFP, whereas NKp46 and eYFP were expressed on a majority of NK1.1⁺ BM NK cells (Fig. 3 A and B). Furthermore, after in vitro culture in IL-15 of sorted CD122⁺CD3⁻CD19⁻NKp46⁻ BM cells, we observed the induction of NKp46 selectively on NK1.1⁺CD3⁻ NK cells (Fig. S5). Thus, the cell surface expression of NKp46 was acquired on BM NK cells after NK1.1. The monitoring of NK cell maturation using CD27 and CD11b markers further supported this kinetics of differentiation (36, 37), because the acquisition of NKp46 occurred at the CD27⁺CD11b^−/low stage and remained stable (Fig. 3C and Fig. S6). Similar data were obtained when NK cells were isolated from spleen, LNs, and lung.

Fig. 3.

Early expression of NKp46 during NK cell maturation. (A) Flow cytometric measurement of NK1.1 and eYFP on gated CD122⁺lin⁻ NK cells in BM of NKp46^iCreR26R^eYFP mice. (B) Flow cytometric measurement of eYFP and NKp46 expression on gated CD122⁺lin⁻c-Kit⁻CD11b⁻NK1.1⁻ NK precursors and NK1.1⁺ immature NK cells in BM of NKp46^iCreR26R^eYFP mice. (C) eYFP expression is shown on CD122⁺lin⁻NK1.1⁺ BM NK cells according to their expression of CD27 and CD11b markers.

Following the same track, we then focused on CD16 (FcγRIIIA). Indeed, the cell surface expression of CD16 defines a checkpoint in human NK cell differentiation that is associated with NK cell maturation, as evidenced by the perforin-dependent cytotoxic function of CD16⁺CD56^dim NK cells in contrast to CD56^brightCD16^−/low human NK cells (38). In addition, recent data support a model in which CD56^brightCD16^−/low NK cells differentiate into CD16⁺CD56^dim NK cells (39). Although mouse NK cells also express CD16, the kinetics of CD16 surface expression on mouse NK cells is still unknown, in part due to the lack of reagent able to discriminate CD16 from CD32 (FcγRIIB). We addressed this issue using a recently described anti-CD16 antibody (40). As shown in Fig. 4A, CD16 was induced at the CD27⁺CD11b^−/low stage, but after NKp46, because CD16⁻ cells contain both eYFP⁻ and eYFP⁺ NK cells in comparable amounts, whereas CD16⁺ cells are uniformly eYFP⁺. We then analyzed the kinetics of induction of CD94, NKG2D, DX5, CD11b, c-Kit, and Ly49 receptors (using a pan Ly49 mAb). We found that CD94 was expressed very early in CD122⁺NK1.1⁻ cells (Fig. 4B). Because the majority of NKG2D^−/low cells expressed CD16, the data indicated that CD16 induction preceded that of NKG2D (Fig. 4C). NKG2D was expressed before DX5, because DX5⁻ cells contain NKG2D⁺ cells, whereas all DX5⁺ cells are uniformly NKG2D⁺ (Fig. 4D). As for c-Kit, despite its low cell surface expression in both CD11b^−/low and CD11b⁺ cells, the c-Kit⁻ cells contained a majority of DX5⁺ cells, indicating that c-Kit expression occurs after DX5 induction (Fig. 4E). Finally, regarding Ly49 expression, most c-Kit⁻ cells are Ly49⁻, whereas most c-Kit⁺ cells are Ly49⁺ (Fig. 4F), suggesting coinduction of these molecules.

Fig. 4.

CD16 is expressed on immature NK cells. (A) CD16 expression is shown in CD122⁺lin⁻NK1.1⁺ BM NK cells of NKp46^iCreR26R^eYFP 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 NKp46^iCreR26R^eYFP mice.

We then analyzed whether the acquisition of the cell surface expression of NKp46 and CD16 was associated with modifications in NK cell function. The induction of NKp46 expression was associated with a higher ability to secrete IFN-γ upon stimulation with IL-12 and -18, although the response of NKp46⁻NK1.1⁺ NK cells and NKp46⁺NK1.1⁺ NK cells to phorbol 12-myristate 13-acetate (PMA) and ionomycin was comparable (Fig. S7A). This higher reactivity of NKp46⁺NK1.1⁺ NK cells to IL-12 and -18 stimulation was associated with the induction of cell surface IL-12 and -18 receptors (Fig. S8). In contrast, the response of CD16⁻NK1.1⁺ NK cells and CD16⁺NK1.1⁺ NK cells to IL-12 and -18 or PMA and ionomycin was similar (Fig. S7B). Unexpectedly, the IFN-γ production of CD11b^−/lowNK1.1⁺ and CD11b⁺NK1.1⁺ NK cells was also comparable (Fig. S7C). Thus, despite the reported acquisition of NK cell functional response to various stimulations at the CD11b⁺ stage (34), our data define the induction of NKp46 as a key step in the acquisition of NK cell reactivity to IL-12 and -18.

Together these data converged to propose the acquisition of NKp46 expression as a useful and stable tool to mark a previously undescribed stage 2 in NK cell differentiation. These findings also lead us to propose a revised model of NK cell differentiation based on the sequential and stable acquisition of cell surface molecules as follows: CD122 (stage 1), NK1.1 (stage 2), NKp46 (stage 3), CD16 (stage 4), and CD11b (stage 5) (Table 1). Stage 6 of NK cell maturation is still defined by the disappearance of CD27 in CD11b⁺ mature NK cells, awaiting the identification of a cell surface molecule that would be selectively and stably induced at this stage. Indeed, although KLRG-1 and CD43 are preferentially expressed in stage 6 NK cells (35), their cell surface expression is initiated at stage 5. This model is fully compatible with the previous classification (34) but refines the early stages of NK cell development, i.e., between stage II, as defined earlier by the induction of NK1.1 and CD94, and stage III, as defined by the induction of Ly49 and c-kit.

Table 1.

NK cell maturation

	Stage 1	Stage 2	Stage 3	Stage 4	Stage 5	Stage 6
CD122	+	+	+	+	+	+
NK1.1	−	+	+	+	+	+
NKp46	−	−	+	+	+	+
CD16	Low	Low	Low	+	+	+
CD11b	Low	Low	Low	Low	+	+
CD27	−	Low	+	+	+/−	−
NKG2D	Low	Low	Low	+	+	+
DX5	−	−	−	+	+	+
c-Kit	−	−	−	+	+/−	−
Ly49	−	−	−	+	+	+
CD94	Low	+	+	+	+	+
CD43	−	−	−	−	Low	+
KLRG1	−	−	−	−	Low	+

A revised model of NK cell maturation is proposed based on the sequential expression of CD122, NK1.1, NKp46, CD16, and CD11b at NK cell surface.

Gut NKp46⁺ Cells.

NKp46⁺ cells comprise not only bona fide NK cells and a minute subset of T cells, but also a population of mucosal cells. We thus took advantage of the Nkp46^iCreR26R^eYFP mice to revisit the heterogeneity of NKp46⁺ cells in various organs by comparing for the expression of eYFP and the cell surface of NKp46. In BM, spleen, LNs, and lungs, NKp46 and eYFP were coexpressed (Fig. S9A Upper). Less than 10% of splenic and BM eYFP⁺ lymphocytes expressed low surface density of NKp46, but they did not correspond to a population of eYFP⁺NKp46⁻ cells because they appeared as a continuum with NKp46⁺ cells (Fig. S9A Upper) and were uniformly NK1.1⁺ (Fig. S9A Lower). Confirmation of this interpretation was obtained by using a brighter revelation of the NKp46 fluorescence using an anti-NKp46 goat antiserum and Alexa 647-conjugated secondary reagent instead of an anti-NKp46 mAb (Fig. S10).

A distinct picture emerged from the analysis of gut lymphocytes because two populations of cells expressing eYFP were detected in the small intestine (Fig. 5A Left). Indeed, besides gut eYFP⁺ cells that uniformly express NK1.1 and the same level of eYFP compared with NKp46⁺ BM, splenic, LN, and lung cells, a subset of gut eYFP^hi cells with lower cell surface expression of NK1.1 was detected. Further analysis revealed that eYFP⁺NK1.1⁺ cells were CD3⁻NKp46⁺CD127^dimCD45^hic-Kit^−/dim (Fig. 5A Right) and corresponded to bona fide small intestine NK cells (28). In contrast, eYFP^hiNK1.1⁻ and eYFP^hiNK1.1^dim cells corresponded to a continuum of NKp46^dimCD127⁺c-Kit⁺ expressing intermediate levels of CD45 (Fig. 5A Right). These cells corresponded to the NKp46⁺ROR-γt⁺ subset that produces IL-22 (22, 23, 26, 28, 41), consistent with the coexpression of eYFP and ROR-γt in gut cells from isolated lymphoid follicules (Fig. 5B) and their ability to produce IL-22 at steady state and upon IL-23 stimulation (Fig. S11). In contrast, only the eYFP⁺NK1.1⁺ cells produced IFN-γ upon IL-12 and -18 stimulation, consistent with their bona fide small intestine NK cell identity (Fig. S11).

Fig. 5.

Characterization of gut NKp46⁺eYFP⁺ and NKp46^dim/loweYFP^high cells. (A) Flow cytometric characterization of CD3^-CD19^-eYFP^hiNK1.1⁻, eYFP^hiNK1.1^dim, eYFP⁺NK1.1⁺ cells from small intestine of NKp46^iCreR26R^eYFP 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 NKp46^CreR26R^eYFP 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.

Because the expression of iCre induced the irreversible expression of eYFP in Nkp46^iCreR26R^eYFP mice, the distinct levels of eYFP observed at a single cell level could not result from a distinct regulation at the Nkp46 locus but, rather, indicated a distinct regulation of the Rosa26 locus in eYFP^hi vs. eYFP⁺ cells, as shown in other reporter mouse models (42). Thus, the genetic tracing in Nkp46^iCreR26R^eYFP mice indicated that the Rosa26 locus was differently regulated in bona fide NK cells and in NKp46⁺ROR-γt⁺ cells, supporting by another approach that NK cell receptor (NKR)⁺ROR-γt⁺ and NK cells represent two distinct lineages (19, 24, 28).

Liver NKp46⁺ Cells.

The analysis of liver lymphocytes in Nkp46^iCreR26R^eYFP mice also revealed an unexpected complexity within eYFP⁺ cells. As in the gut, two populations of eYFP^hi and eYFP⁺ lymphocytes were detected. eYFP^hi cells were NK1.1⁺ and comprised a continuum of NKp46^dim and NKp46⁺ cells (Fig. 6), whereas eYFP⁺ cells consisted of a more homogeneous NK1.1⁺NKp46⁺ cell population. Besides the cell surface expression of NKp46, eYFP^hi and eYFP⁺ cells expressed a panel of cell surface receptors present on NK cells, such as CD122, DX5, 2B4, NKG2D, and CD16, indicating that they corresponded to two distinct subsets of liver NK cells (Fig. 6 and Fig. S12A). Of note, it has recently been shown that liver NK cells comprised a population of CXCR6⁺ NK cells capable of mediating hapten- and virus-specific memory responses (43, 44). However, the cell surface expression of CXCR6 did not correlate with eYFP levels in liver NK cells from Nkp46^iCreR26R^eYFP. A noticeable difference between liver eYFP^hi and eYFP⁺ lymphocytes resided in the high percentage of CD11b^−/dim cells within the eYFP^hi NK cell subset. This sign of immaturity in a subset of liver NK cells has been reported and associated with the low surface expression of DX5 and the constitutive expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (45). A similar phenotype was observed for the eYFP^hi liver NK cells (Fig. 6), indicating that they corresponded to the CD11b^−/lowDX5^dimTRAIL⁺ NK cell subset previously described in the same organ. The cell surface expression of Ly49 molecules was mostly restricted to the eYFP⁺ liver NK cells as in the spleen (Fig. S12B), which is consistent with the bona fide NK cell phenotype of eYFP⁺ liver NK cells and the more immature phenotype of eYFP^hi liver NK cells. Genetic tracing revealed that these two liver subsets are quite divergent, because the level of eYFP expression in CD11b^−/low and CD11b⁺ BM NK cells was similar, in contrast to the distinct eYFP levels in CD11b^−/low and CD11b⁺ liver NK cells (Fig. S13). Thus, the levels of YFP are not merely associated with various stages of NK cell maturation, but with a more profound lineage commitment of NK cell subsets, highlighting the use of Nkp46^iCreR26R^eYFP mice for in vivo fate-mapping experiments.

Fig. 6.

Characterization of liver NKp46⁺eYFP⁺ and NKp46^dim/loweYFP^high cells. Flow cytometric characterization of NK1.1⁺CD3⁻eYFP^hi and NK1.1⁺CD3⁻eYFP⁺ liver cells from NKp46^iCreR26R^eYFP mice for indicated makers (open histograms) or control isotype (gray histograms). Results are representative of two to four experiments.

Conclusions

We report here the fate mapping of NKp46⁺ cells in vivo through the generation and characterization of an unprecedented model of Nkp46^iCre knock-in mice. Earlier attempts to create a mouse model selectively targeting NKp46⁺ cells by using nontargeted transgenesis have failed due to a complex and still poorly understood regulation of the NKp46 locus (6, 29). To visualize iCre activity in Nkp46^iCre mice, we crossed them with R26R^eYFP reporter mice. In Nkp46^iCreR26R^eYFP mice, the fluorescent reporter permanently labeled cells that had switched on the expression of the NKp46 gene. Using these mice, we have shown that the expression of iCre faithfully corresponded to the endogenous expression of NKp46. The genetic tracing of NKp46⁺ cells in vivo allowed us to reveal the stability of NKp46 cell surface expression. In addition, the acquisition of NKp46 marked a checkpoint of NK cell maturation. Based on these data, we propose a unique model of NK cell differentiation, which also includes CD16 as a marker of NK cell maturation. One advantage of this unique model resides in the use of cell surface molecules that are conserved in both mouse and human, with the exception of mouse NK1.1. Along this line, preliminary data obtained on CD34⁺ hematopoietic cell progenitors from human cord blood indicate that the induction of surface CD56 precedes that of NKp46 in an in vitro NK cell differentiation assay, supporting the hypothesis that CD56 could be positioned in the human NK cell differentiation pathway as NK1.1 in the mouse.

Furthermore, the differential expression of YFP in Nkp46^iCreR26R^eYFP mice showed that gut NKR⁺ROR-γt⁺ and NK cells represent two distinct lineages. In addition, fate mapping experiments revealed the genetic heterogeneity of the two subsets of CD11b^−/lowDX5^dimTRAIL⁺ and CD11b⁺DX5⁺TRAIL⁻ liver NK cells. It is puzzling that besides bona fide eYFP⁺ CD3⁻NKp46⁺NK1.1⁺ NK cells, subsets of NKp46^dim/loweYFP^high cells were present in gut and liver. Besides their common NKp46^dim/loweYFP^high phenotype, gut and liver eYFP^hi cells did not appear to be directly related, because gut eYFP^hi cells were NK1.1^−/dim and corresponded to NKR⁺ROR-γt⁺ cells, whereas liver eYFP^hi cells were NK1.1⁺ and expressed TRAIL. Together, these results define Nkp46^iCre mice as a unique mouse model of specific targeting in NKp46⁺ cells, allowing the generation of unique mouse strains based on the crossing of Nkp46^iCre mice to a variety to floxed mice to dissect the biology of NKp46⁺ NK, T, and gut NKR⁺ROR-γt⁺ cells.

Material and Methods

Mice and Crosses.

We have generated a Nkp46^iCre knock-in mouse line in which the gene encoded the improved recombinase (icre) was inserted by homologous recombination at the 3’ end of the Nkp46 gene (see SI Materials and Methods). Recombinant offspring of chimeric mice was crossed with transgenic flipase (FLP)-expressing mice (46). Nkp46^iCre mice were crossed to R26R^eYFP (31) (CDTA, Orleans, France). All mice used in these studies were Nkp46^iCreR26R^eYFP, Nkp46^iCre, or WT control littermates. All mice were bred in pathogen-free breeding facilities at Centre d'Immunologie de Marseille-Luminy and used between 6 and 10 weeks of age. Experiments were conducted in accordance with institutional guidelines for animal care and use. Nkp46^iCre mice were crossed to Rosa26^{lsl-DTR/lsl-DTR} mice (32) to obtain NKp46^iCre/wt; Rosa26^lsl-DTR/wt mice referred as to Nkp46^iCreR26R^DTR. Six- to eight-week-old offspring mice were used for experiments. Unnicked Diphtheria Toxin from Corynebacterium diphtheria (Calbiochem, 500 ng/mouse) diluted in PBS was injected i.p.

Cell Preparation.

Cells were prepared as described (22). For details, see SI Materials and Methods.

Flow Cytometry.

Flow cytometric analysis was done on a FACS Canto (Becton Dickinson, San Diego, CA). For details on reagents, see SI Materials and Methods.

Tissue Immunofluorescence.

Portions of small and large intestine were isolated from mice and treated as previously described (22). For staining details, see SI Materials and Methods.