ARTICLE
Received 27 May 2016 | Accepted 27 Oct 2016 | Published 16 Dec 2016 DOI: 10.1038/ncomms13708 OPEN Interleukin-12 bypasses common gamma-chain signalling in emergency natural killer cell lymphopoiesis
Isabel Ohs1, Maries van den Broek2, Kathrin Nussbaum1, Christian Mu¨nz3, Sebastian J. Arnold4,5, Sergio A. Quezada6, Sonia Tugues1,* & Burkhard Becher1,*
Differentiation and homeostasis of natural killer (NK) cells relies on common gamma-chain (gc)-dependent cytokines, in particular IL-15. Consequently, NK cells do not develop in mice with targeted gc deletion. Herein we identify an alternative pathway of NK-cell development driven by the proinflammatory cytokine IL-12, which can occur independently of gc-signalling. In response to viral infection or upon exogenous administration, IL-12 is sufficient to elicit the emergence of a population of CD122 þ CD49b þ cells by targeting NK-cell precursors (NKPs) in the bone marrow (BM). We confirm the NK-cell identity of these cells by transcriptome- wide analyses and their ability to eliminate tumour cells. Rather than using the conventional pathway of NK-cell development, IL-12-driven CD122 þ CD49b þ cells remain confined to a NK1.1lowNKp46low stage, but differentiate into NK1.1 þ NKp46 þ cells in the presence of gc-cytokines. Our data reveal an IL-12-driven hard-wired pathway of emergency NK-cell lymphopoiesis bypassing steady-state gc-signalling.
1 Inflammation Research, Institute of Experimental Immunology, University of Zurich, 8057 Zurich, Switzerland. 2 Tumor Immunology, Institute of Experimental Immunology, University of Zurich, 8057 Zurich, Switzerland. 3 Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, 8057 Zurich, Switzerland. 4 Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, and BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, D-79104 Freiburg, Germany. 5 BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, D-79104 Freiburg, Germany. 6 Cancer Immunology Unit, Research Department of Hematology, University College London Cancer Institute, WC1E 6BT London, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to B.B. (email: [email protected]).
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s main components of the innate immune system, NK WT as well as Rag2 / Il2rg / mice (Fig. 1b). Moreover, cells play a key role in controlling infections and limiting neutralization of IL-12 prevented the expansion of CD122 þ Acancer progression1,2. Recognition of infected or CD49b þ cells in infected mice (both WT and Rag2 / transformed cells by NK cells involves a plethora of activating Il2rg / ; Fig. 1c), indicating a dependence of this population and inhibitory receptors, that in combination determine whether on IL-12. Altogether, these results highlight IL-12 as a key a target cell will be killed or spared3. The elimination of target cytokine to initiate the emergence of gc-signalling-independent cells is achieved via death receptor pathways or the release of CD122 þ CD49b þ cells (hereafter called emergency NK (eNK) cytotoxic granules containing perforin and granzyme4,5. cells) upon viral infection. In addition to their cytotoxic function, NK cells are a major source of proinflammatory cytokines such as tumour necrosis IL-12 generates unconventional but functional NK cells.We factor alpha (TNF-a) and interferon gamma (IFN-g), which next addressed whether IL-12 promotes the development of eNK activate the myeloid compartment to join the fight against cells in the absence of infection. Indeed, we found eNK cells, infections or cancer6. In turn, cytokines can modulate NK-cell which morphologically resembled lymphocytes, in lungs of responses7. More specifically, interleukin (IL)-15, which together Rag2 / Il2rg / mice treated with IL-12 (Fig. 2a,b). The with other cytokines (IL-2, IL-4, IL-7, IL-9 and IL-21) signals population of eNK cells was shown to express low levels of NK1.1 through the gc subunit, is critical for NK-cell development, (Fig. 2a). Of note, a subset of CD122 þ CD49b þ cells with low homeostasis and activation8. Once lineage committed, as seen by NK1.1 expression was also found in lungs of IL-12-treated WT acquiring IL-2/15Rb (CD122) expression, NK cells require mice (Fig. 2a), indicating that the IL-12-induced expansion of continuous IL-15R engagement for further differentiation and these cells also occurs in the face of physiological gc-signalling. maintenance9,10. Accordingly, mice deficient in IL-15, IL-15Ra or eNK cells expressed markers typically associated with conven- gc are devoid of NK cells11,12. One study reported an expansion tional NK (cNK) cells such as the transcription factor Eomeso- of lymphocytes with an NK-cell phenotype in Rag2 / Il2rg / dermin (Eomes) and the activating receptor NKG2D (Fig. 2c). In mice upon murine cytomegalovirus (MCMV) infection, comparison with the NK1.1 þ subset of CD122 þ CD49b þ cells suggesting that NK cells can expand in the absence of from control (NK) or IL-12-treated WT mice (NK þ IL-12), eNK IL-15 under highly inflammatory conditions13. Even though cells expressed low amounts of NKp46 (Fig. 2c) and the cytokine IL-12 was involved in this process, whether the unusually high levels of DNAM-1 (Fig. 2c), as well as a differ- expanded population represents a unique IL-15-independent entiated (CD11bhighCD27highKLRG1 þ ) phenotype (Supplemen- NK-cell subset remains elusive. tary Fig. 1a). Also, eNK cells did not express the IL-7 receptor IL-12 is secreted by myeloid cells during inflammation, and its (CD127; Supplementary Fig. 1b) and exhibited a high prolifera- primary effects on NK cells include increased IFN-g production, tion rate accompanied by reduced levels of Bcl-2 (Fig. 2d; proliferation and the expression of cytotoxic mediators14–20.So Supplementary Fig. 1c), an anti-apoptotic factor involved in far, the effect of IL-12 on NK cells has been reported for mature peripheral NK-cell survival by IL-15 (ref. 10). Importantly, NK (mNK) cells, but it is currently unknown whether IL-12 NK1.1low NK þ IL-12 cells from WT mice phenotypically closely contributes to NK-cell development in the BM. resembled the population of highly proliferative, less differen- Here we discovered that IL-12 drives a pathway of emergency tiated Eomes þ eNK cells (Fig. 2c,d; Supplementary Fig. 1a,c). NK-cell lymphopoiesis independently of gc-signalling. IL-12 We next investigated the ability of eNK cells to produce initiates the generation of a cell population expressing CD49b IFN-g, the main cytokine secreted by cNK cells upon IL-12 and CD122, but low levels of the lineage markers NK1.1 and stimulation20. IFN-g was secreted by lung CD122 þ CD49b þ NKp46. Using a comprehensive phenotypic and functional NK1.1low cells not only in IL-12-treated Rag2 / Il2rg / mice, characterization, we provide evidence that these cells represent but also in WT mice, where this population contributed to almost a population of NK cells with a unique cell-surface receptor 40% of the total IFN-g production (Fig. 2e). Furthermore, repertoire resembling immature NK cells. Mechanistically, IL-12- the responses of IL-12-induced eNK cells to YAC-1 targets driven CD122 þ CD49b þ NK cells differentiate from NKPs in the demonstrated their cytotoxic potential (Fig. 2f). Taken together, BM through an unconventional developmental pathway. We IL-12 bypasses the requirement of gc-signalling for NK-cell demonstrated the emergence of IL-12-induced NK cells during lymphopoiesis by inducing the generation of an unconventional viral infection as well their ability to clear tumour cells and limit population of eNK cells with cytotoxic properties. metastatic spread, reinforcing the relevance of this alternative process of NK-cell lymphopoiesis. eNK cells represent a distinct NK-cell subset. To further explore in which extent eNK cells mirror NK-cell features, we performed deep transcriptome analysis of sorted eNK cells as well as NK and Results NK þ IL-12 cells from WT mice. Principal component (PC) IL-12 induces cc-independent emergency NK-cell lymphopoiesis. analyses indicated that each group of cells formed individual NK cells were reported to expand independently of gc-signalling clusters in the PC space (Fig. 3a). Basic hierarchical clustering of in response to MCMV infection13, a hitherto unrecognized the three populations showed that eNK cells were more closely process whose underlying mechanism remains ill-defined. To related to NK þ IL-12 cells than they were to NK cells (Fig. 3b). investigate whether the expansion of such NK cells only occurs Similar to NK cells, eNK cells expressed high amounts of Eomes, during MCMV infection or is a general response to inflam- killer-cell lectin-like receptors (Klr), Fc receptors and adhesion mation, we infected wild-type (WT) and Rag2 / Il2rg / molecules, thus confirming the NK-cell identity of this population mice (lacking T, NKT and B cells as well as ILCs) with vaccinia (Fig. 3c; Supplementary Table 1). The transcripts differentially virus (VV), a poxvirus controlled by NK cells early after expressed in eNK compared with NK þ IL-12 cells revealed, infection21. In lungs of Rag2 / Il2rg / C57BL/6 mice, VV however, a gene signature reminiscent of immature NK cells, as infection induced the expansion of a population of lymphocytes shown by high expression of Cd27, Cd69, Cxcr3 and Cxcr6 expressing both CD122 and CD49b (Fig. 1a), two markers that (Fig. 3c; Supplementary Table 1). In contrast, transcripts mainly phenotypically define NK cells22. Viral infections trigger the confined to mNK cells24–28, such as the integrins CD49b (Itga2) release of inflammatory mediators such as IL-12 (ref. 23), and we and CD11b (Itgam), Cx3cr1, S1pr5 and several members of the detected elevated amounts of IL-12 in the serum of VV-infected Ly49 receptor family (Klra1, Klra3, Klra9 and Klra7), were
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a b WT Rag2 –/–Il2rg –/– 6
+ 2,500 105 16.2 105 0.061 2×10 ** 2,000 104 104 1×106 1,500 * 3 3 CD122 10 10 + 4 **
PBS 5×10 1,000 Abs. no. 0 0 ml serum 500 IL-12 (pg) per CD49b 0 0 103 104 105 0103 104 105 0
5 5 10 22.7 10 53.0 + VV + VV + PBS–/– + PBS–/– 104 104 WT +–/– VV WT +–/– VV WT + PBS rg WT + PBS Il2rg – Il2rg
3 3 VV – Il2 –/– – Il2rg–/ 10 10 –/ –/ 2 0 0 Rag2 Rag2 Rag Rag2 CD122 0103 104 105 0103 104 105 CD49b
c α-p40 6 + 1.5×10 * + 1×106 VV infection α-p40 Analysis 5×105 CD122 + 5,000 * Day 0 2 5 Abs. no. 2,500 –/– –/– –/– –/– Rag2 Il2rg Rag2 Il2rg CD49b –/– 0 + α-p40 /– WT Il12rb2 + Ctrl IgG – WT 105 31.7 105 26.2 105 5.91 105 0 α-p40 12rb2 + 4 104 104 104 Il + Ctrl–/– IgG 10 –/– 3 3 3 3 rg 10 10 10 10 2 Il2rg – Il –/– –/ 0 0 0 0
CD122 Rag2 Rag2 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 CD49b
Figure 1 | VV infection generates IL-12-dependent CD122 þ CD49b þ NK cells. (a) Representative plots and quantification of lung CD45 þ CD3 CD122 þ CD49b þ cells of uninfected versus VV-infected WTor Rag2 / Il2rg / mice. Data represent two similar experiments (n ¼ 3–5 mice per group per experiment). (b) IL-12 serum levels in VV-infected WT or Rag2 / Il2rg / mice were compared to controls. Data represent two independent experiments (n ¼ 3–5 mice per group per experiment). (c) Schedule of a-p40 administration in VV-infected mice. Representative plots and quantification of lung CD45 þ CD3 CD122 þ CD49b þ cells of VV-infected WT, Il12rb2 / or Rag2 / Il2rg / mice treated with Ctrl IgG or a-p40. Data represent two independent experiments with at least three mice per group. Statistical analysis was performed (*Po0.05, **Po0.01, unpaired Student’s t-test without (a) or with (b,c) Welch’s correction) and error bars represent±s.e.m. present albeit downregulated in eNK cells (Fig. 3c; Supplementary between NK1.1low NK þ IL-12 cells of WT mice and eNK cells Table 1). The immature profile of eNK cells was not associated reinforces the existence of an IL-12-induced pathway of alter- with decreased levels of Eomes and T-bet (Tbx21) (Fig. 3c; native NK-cell lymphopoiesis also when gc cytokines are present. Supplementary Table 1), but with changes in cytokine production and cytotoxic effectors. Thus, whereas NK þ IL-12 cells expressed higher amounts of granzyme (Gzma and Gzmb) and perforin IL-12 initiates a different pathway of NK-cell development. The (Prf1) compared to eNK cells, the latter upregulated GM-CSF unconventional phenotype observed in lung eNK cells raised the (Csf2), lymphotoxin-b (Ltb), TNF-a (Tnf) and TNF-Related question of how these cells originate in the BM. NK cells develop Apoptosis Inducing Ligand (TRAIL; Tnfsf10) (Fig. 3c; via four stages from (i) lineage negative (Lin ) CD127 þ Supplementary Table 1) usually acquired at earlier stages of CD135 þ common lymphoid progenitors (CLPs) into (ii) Lin NK-cell maturation29. CD244 þ CD127 þ CD135 pre-NK-cell progenitors (pre-NKPs) We examined the cell surface levels of several NK-cell receptors and further into (iii) Lin CD244 þ CD27 þ CD122 þ CD49b to further assess the maturation status of lung eNK cells. In NK1.1 NKPs, which acquire the expression of the IL-15R general, whereas NK1.1 þ NK þ IL-12 cells were phenotypically complex CD122 and ultimately give rise to mature (iv) similar to the NK-cell population from Ctrl-treated WT mice, the CD244 þ CD122 þ CD49b þ NK1.1 þ NK cells30,31. We used phenotype of eNK cells resembled that of NK1.1low NK þ IL-12 unsupervised non-linear dimensionality reduction (t-SNE)32 to cells from WT mice (Fig. 3d). Thus, eNK cells and NK1.1low identify and visualize these distinct NK-cell developmental NK þ IL-12 cells from WT mice expressed high levels of CD94, stages in the BM of IL-12-treated WT and Rag2 / Il2rg / NKG2A/C/E and TRAIL, but low amounts of Ly49D (Klra4) and mice (Fig. 4a). With this approach, the CD45 þ Lin CD122 þ Ly49I (Klra9) when compared with NK and NK1.1 þ NK þ IL-12 population could be subdivided into demarcated cluster sets cells from WT mice (Fig. 3d). Low levels of Ly49G2 (Klra7) and defined by differential expression of several NK-cell markers Ly49A (Klra1) were also observed in eNK cells (Fig. 3d). (Fig. 4a–c). Thus, cluster 1, negative for all NK-cell markers Importantly, this was not exclusive of eNK cells in the lung but for CD27 and present in both WT and Rag2 / Il2rg / microenvironment, since liver eNK cells showed a very similar mice, harbours NKPs (Fig. 4a–c). The population of mNK cells overall cell surface phenotype (Supplementary Fig. 2a,b). was clearly identified by clusters 3 and 6 in WT mice, represen- Altogether, these findings place eNK cells into a distinct ting less (CD27highCD11blow) and more differentiated NK-cell subset with immature characteristics. The resemblance (CD27lowCD11bhighKLRG1 þ ) mNK cells, respectively
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(Fig. 4a–c). Both in WT and Rag2 / Il2rg / mice, IL-12 Moreover, when transferred into lymphopenic mice, IL-12-driven induced the emergence of eNK cells (DX5 þ NK1.1 NKp46low CD122 þ CD49b þ NK1.1low eNK cells isolated from WT mice CD11b þ CD27lowKLRG1high cell in cluster 5), and an unexpec- developed into NK1.1 þ NKp46 þ cells, indicating that the presence ted population of DX5lowNK1.1 NKp46 CD11b CD27 of gc-cytokines is required for the acquisition of these NK-cell KLRG1 cells (cluster 4), probably representing an intermediate lineage markers (Fig. 4d; Supplementary Fig. 3b). precursor stage of NK-cell development (Fig. 4a–c). Also in the IL-12 expanded the populations of NKPs and eNK cells in the BM, we observed the expression of Eomes in eNK cells by using an BM of both WT and Rag2 / Il2rg / mice, whereas CLPs and Eomes fluorescent reporter mouse (Supplementary Fig. 3a). pre-NKPs in WT mice remained unaltered (Fig. 4e,f;
a Rag2 –/–Il2rg–/– Rag2 –/–Il2rg–/– b WT + Ctrl WT + IL-12 + Ctrl + IL-12 ** *** 105 15.1 105 19.2 105 0 105 24.9 8×105 + 104 104 104 104 6×105 103 103 103 103
CD122 5 102 102 102 102 + 4×10 CD122 WT + IL-12 WT 0 0 0 0 Abs. no. 2×105 3 4 5 3 4 5 3 4 5 3 4 5
0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 CD49b CD49b 0 5 0.034 99.9 5 95.4 5 0 0 5 85.9 12.6 10 10 4.18 10 10 –/– L-12 IL-12 4 4 4 4 + Ctrl 10 10 10 10 –/– + –/– WT + Ctrl g Il2rg 103 103 103 103 WT + I g Il2r –/– 2 –/– Il2r 102 102 102 10 –/– + IL-12 g2 0 0 0 0 a g2 R a Rag2 CD49b R 00103 104 105 103 104 105 0 103 104 105 0 103 104 105 NK1.1
c Isotype NK NK1.1+ NK + IL-12 NK1.1low NK + IL-12 Count Count Count Count eNK
0103 104 105 0103 104 105 0 103 104 105 0 103 104 105 Eomes NKG2D NKp46 DNAM-1
3,000 * 1,000 400 *** 15,000 *** ** ** * *** 800 300 2,000 10,000 600 200 400 1,000 5,000 200 100 Eomes (MFI) NKp46 (MFI) NKG2D (MFI) DNAM-1 (MFI) DNAM-1 0 0 0 0
d e f 105 5.08 105 104 104 5.08 Isotype 103 3 10 96.3 NK WT + Ctrl WT 0 0 1.88 only YAC-1 NK1.1+ NK + IL-12 0103 104 105 0103 104 105 NK1.1lowNK + IL-12 Count 5 5.75 5 eNK 10 10 33.2 104 104 3 4 5 6 103 3 0 10 10 10 10 10 56.1 NK:YAC-1 Ki-67 0 0 39.7 WT + IL-12 WT 0010103 104 105 3 104 105 19 –/– 105 0 105 104 4 Il2rg 10 Count eNK:YAC-1 –/– 3 3 Count
+ Ctrl 10 10 0 Topro 0 0 0 40 Rag2
0 103 104 105 0010103 104 105 3 104 105 30 Bcl-2 105 5.10 105 20 –/– 104 104 3 3 10 Il2rg 10 10
0.56 % specific lysis –/– 0 CD49b + IL-12 0 NK1.1 0 98.2 NK eNK
Rag2 0 103 104 105 0 103 104 105 IFN-γ NKp46
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Supplementary Fig. 3c,d). Accordingly, proliferation of NKPs as treatment of tumour-bearing WT mice led to a significant well as of eNK and mNK cells was increased upon IL-12 reduction of lung metastases (Fig. 6c). This was not due to a treatment (Supplementary Fig. 3e). Overall, these results point direct effect of IL-12 on 4T1 tumour cells, since these cells did not towards an hitherto unrecognized pathway of NK-cell develop- express IL-12Rb2 and IL-12Rb1 and their in vitro growth was ment regulated by IL-12, which bypasses canonical gc-chain not affected by IL-12 (Supplementary Fig. 4). Remarkably, signalling. IL-12 elicited tumour control also in tumour-bearing Rag2 / Il2rg / mice (Fig. 6c), demonstrating that IL-12 induces NKPs respond to IL-12 and differentiate into eNK cells.To protective immunity even in mice lacking all T, B and ILCs. determine the lymphoid precursor population of eNK cells, we In this setting, IL-12 treatment of 4T1 tumour-bearing sorted CLPs, pre-NKPs, NKPs, eNK and mNK cells from BM of Rag2 / Il2rg / mice led to the expansion of eNK cells WT mice and quantified Il12rb2 transcripts. Il12rb2 was expres- from the BM (Fig. 6d,e). These cells were identified as the main sed by NKPs, eNK and mNK cells, but not by CLPs and only at IL-12-responsive cell type, as they virtually produced all the low levels by pre-NKPs (Fig. 5a). Furthermore, we found higher IFN-g detected in Rag2 / Il2rg / mice (Fig. 6f). The depen- amounts of Il12rb2 transcripts in NKPs and eNK cells from dence of tumour rejection on IFN-g-producing eNK cells was IL-12-treated compared with Ctrl-treated WT mice (Fig. 5a), solidified by the finding that IL-12-treated IFN-g receptor indicating that IL-12 induces the expression of its own receptor deficient (Ifngr / ) mice failed to control lung metastasis complex in NKPs. Functional IL-12R engagement was further (Fig. 6g). Moreover, the depletion of eNK cells reversed IL-12- shown by the expression of Tbx21 and Ifng transcripts, which mediated suppression of metastasis in 4T1 tumour-bearing occurred only in the three NK-cell populations that express Rag2 / Il2rg / mice (Fig. 6h). Collectively, the contribu- Il12rb2 (Fig. 5b,c). tion of eNK cells to tumour control highlights the importance of To further assess direct effects of IL-12 on NKPs and eNK cells, gc-independent emergency NK-cell lymphopoiesis in tumour we isolated these cell populations from the BM of WT mice and protective immunity. treated them for 6 h with IL-12 in vitro. Ifng expression was increased upon IL-12 stimulation in both NKPs and eNK cells, Discussion indicating a direct signal through the IL-12R in both cell types Even though gc-cytokines regulate various aspects of NK-cell (Fig. 5d). IL-12 was sufficient to drive differentiation of NKPs biology, only IL-15 is essential for NK-cell development7,10. into eNK cells within whole BM suspension or highly purified Under physiological conditions, NK cells develop in the BM from NKPs kept on monolayers of OP-9 stromal cells (Fig. 5e,f). lymphoid precursor cells34, and this maturation pathway is Collectively, these data highlight a pivotal role of IL-12 in NK-cell aborted in the absence of IL-15R signalling11. Here, we differentiation by acting on NKPs, the stage at which IL-15 is demonstrated that IL-12 can bypass this developmental road required for the maturation of these cells during steady-state block and induce unconventional eNK cells independently of lymphopoiesis. gc-signalling. We propose that this pathway serves to combat infections or other threats to normal tissue homeostasis (for eNK cells display anti-tumour activity. The observed cytotoxic example, cancer) and can be exploited therapeutically. properties of eNK cells prompted us to test their role in tumour Early evidence of IL-15-independent NK-cell expansion upon surveillance. First, we used TRAIL-sensitive MC38 tumour cells CMV infection13 suggested that mammals might utilize in C57BL/6 mice. Intravenously inoculated MC38-GFP cells were alternative mechanisms to ensure the availability of NK cells efficiently eliminated from lungs of IL-12-treated Rag2 / during inflammatory responses. Our findings show that IL-12 is Il2rg / mice, in comparison with the high numbers of tumour sufficient for the generation of NK-like cells, a process that is also cells found in Ctrl-treated Rag2 / Il2rg / mice (Fig. 6a). The triggered when gc-signalling is functional. To date, IL-15 has dependence of this process on eNK cells was shown upon their been the only cytokine known to act on the progenitor stage to depletion with an anti-asialo GM1 antibody, which completely generate mNK cells in mice12. We now demonstrate that NKPs abolished the clearance of MC38-GFP target cells (Fig. 6a). also respond to IL-12, leading to the generation of eNK cells. We next tested the metastasis-controlling capacity of eNK cells Even though the signalling pathways downstream of IL-12 differ in the orthotopic 4T1 model of breast cancer 33. In this model, the to those triggered by IL-15, both cytokines induce T-bet inoculation of 4T1 cells in Rag2 / Il2rg / mice resulted in an expression and IFN-g-production by NK cells20,35. However, increased metastatic burden compared to WT and Rag1 / neither Tbx21 or Ifng are required for the generation of eNK cells mice (lacking T, NKT and B cells; Fig. 6b), thus indicating the (data not shown), and the search for additional factors that importance of ILCs in limiting metastatic dissemination. IL-12 control this process is ongoing.
Figure 2 | IL-12 induces eNK cells in lungs of naive Rag2 / Il2rg / mice. (a) Representative plots and absolute numbers of CD45 þ CD3 CD122 þ CD49b þ cells in lungs from IL-12 or Fc-treated (Ctrl) WT or Rag2 / Il2rg / mice. The expression of NK1.1 on CD45 þ CD3 CD122 þ CD49b þ cells in lungs from IL-12 or Ctrl-treated WT or Rag2 / Il2rg / mice is shown. Data represent five experiments (nZ4 mice per group per experiment). (b) Cytospin ( 100) of sorted CD45 þ CD3 CD122 þ CD49b þ cells from lungs of IL-12-treated WT or Rag2 / Il2rg / mice stained with May–Gru¨nwald–Giemsa solution. Two independent experiments were performed. Scale bar, 20 mm. (c) Expression levels of Eomes, NKG2D, NKp46 and DNAM-1 on the NK1.1 þ subsets of CD45 þ CD3 CD122 þ CD49b þ cells isolated from lungs of Ctrl (NK) and IL-12-treated WT mice (NK1.1 þ NK þ IL-12), the NK1.1low subset of CD45 þ CD3 CD122 þ CD49b þ cells isolated from lungs of IL-12-treated WT mice (NK1.1low NK þ IL-12) and eNK cells from Rag2 / Il2rg / mice. Three independent experiments were performed with at least three mice per group. (d) Ki-67 and Bcl-2 expression levels were quantified in lung NK, NK1.1 þ NK þ IL-12, NK1.1low NK þ IL-12 and eNK cells. Data shown represent three independent experiments (n ¼ 3–5 mice per group per experiment). (e) Leukocytes isolated from lungs of WT and Rag2 / Il2rg / mice either treated with IL-12 or Ctrl were cultured for 4 h with PMA/ionomycin and CD45 þ CD3 Gr-1 cells were analysed for their IFN-g production. NK1.1 and NKp46 expression was examined on IFN-g expressing cells. (f) NK cells and eNK cells were isolated from lungs of WTmice and Rag2 / Il2rg / mice, respectively. Their cytotoxicity was tested using YAC-1 cells at an effector:target ratio of 6:1. Data are representative of two independent experiments with three mice per group each. Data are expressed as mean±s.e.m. *Po0.05, **Po0.01, ***Po0.001 as determined by unpaired Student’s t-test with Welch’s correction or one-way analysis of variance with Bonferroni post-test.
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ac Fc receptors and Chemokines and Activating and adhesion molecules chemokine receptors inhibitory receptors 0.3 eNK Fcgrt Cxcr6 Klri1 NK Cd244 0.2 Fcgr2b Cxcr3 NK + IL-12 Fcer1g Ncr1 Cd9 Xcl1 Klrk1 0.1 S1pr4 Klri2 Fcgr3 Klre1 0.0 Cd2 Cx3cr1 Klra9 Cd7 Ccr5 Klra3 −0.1 Cd27 Ccr2 Klrd1 Cd69 Ccl5 Klra7
Principal component 2 −0.2 Itgax Klra1 Cd38 Ccl4 Klrb1c Ccl3 Cd96 −0.4 −0.2 0.0 0.2 0.4 0.6 Cd84 Icam1 Mif Klrb1f Cd160 Principal component 1 Cd53 S1pr1 Cd226 Itgb2 Cxcl15 Klrg1 Itgam Ccl6 Cd48 Itga2 b Cxcr4 Klrc2 Itgal Klrc1 Color key Itgb1 S1pr5 Klrc3 Itga1 Ccl9 Itgb7
NK NK NK eNK eNK eNK Color key −4 −2 0 2 4 Value NK + IL-12 NK + IL-12 NK + IL-12 −202 Cytokines and Value cytokine receptors Effector molecules Transcription factors Il2ra Tox Il1rl1 Gzmb Runx1 Csf2 Stat1 Tnf Tbx21 Ltb Nfkb2 Il18r1 Gzma Rora Il10 Nfkb1 Ifng Arntl Il18bp Il15ra Prf1 Id2 Il2rb Foxo1 Il12rb1 Stat4 Il27ra Foxp1 Il17ra Fasl Foxn2 Ifnar1 Ets1 Il12rb2 Tcf3 Tgfbr2 Tnfsf10 Klf13 Il10rb Klf2 Ifngr1 Eomes Il16 Nfil3 Il10ra Gzmk Nfatc3 Ifnar2 Gata3
NK NK NK NK + IL-12 eNK NK eNK eNK eNK
NK + IL-12 NK + IL-12 NK + IL-12 d Count Count Count Count Count
0 103 104 105 –103 0 103 104 105 0 103 104 105 0103 104 105 0103 104 105 CD94 NKG2A/C/E TRAIL Ly49D Ly49I
100 *** 100 *** 300 *** 80 *** 40 *** *** + *** *** *** ***
80 + + 80 60 30 200 60 60 40 20 40 40 100 % Ly49I % CD94 % Ly49D 20 10 20 20 TRAIL (MFI) % NKG2A/C/E 0 0 0 0 0
600 40 *** Isotype *** 30 NK 400 NK1.1+ NK + IL-12 20 low Count 200 Count NK1.1 NK + IL-12 % Ly49A 10 eNK Ly49G2 (MFI) 0 0 0 103 104 105 0 103 104 105 Ly49G2 Ly49A
Figure 3 | Spectrum of distinct and shared genes among different NK-cell subsets. (a) PCA of gene expression by sorted CD122 þ CD49b þ cells obtained from WT mice treated with IL-12 (NK þ IL-12) or Ctrl (NK) and from IL-12-treated Rag2 / Il2rg / mice (eNK). (b) Hierarchical clustering of gene expression by sorted NK þ IL-12, eNK and NK cells. (c) Heat maps showing differentially expressed genes by sorted eNK, NK þ IL-12 and NK cells, clustered to their indicated categories. Heat maps show representative data from one sample from each group. See also Supplementary Table 1 for detailed expression levels and significance. (d) Representative histograms and quantification of CD94, NKG2A/C/E, TRAIL, LY49D, Ly49I, Ly49G2 and Ly49A expression on lung NK, NK1.1 þ versus NK1.1low NK þ IL-12 and eNK cells. Two independent experiments were performed with at least 3–5 mice per group. Data are expressed as mean±s.e.m. One-way analysis of variance was used to determine significance (***Po0.001). PCA, principal component analyses.
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a WT + Ctrl WT + IL-12 Cluster b CD27 CD11b NK1.1
1
2 Expression 1.00 3 0.75
0.50 Rag2 –/–Il2rg–/– Rag2 –/–Il2rg–/– CD49b NKp46 KLRG1 4 + Ctrl + IL-12 0.25
0.00 5
6
c d Rag2 –/–Il2rg–/– Rag2 –/–Il2rg–/– Color key + NK1.1+ + NK1.1low and histogram WT Rag2 –/–Il2rg–/– CD49b+CD122+ CD49b+CD122+ 6 5 5 5 5 1 10 24.2 10 0.049 10 3.90 10 3.52 Count −1 0 1 104 104 104 104 Column Z−score 103 103 103 103 Cluster 3 0 0 0 0
Cluster 6 CD49b 3 4 5 3 4 5 3 4 5 3 4 5 Cluster 5 0 10 10 10 01010 10 01010 10 01010 10 CD122 Cluster 4 5 5 5 5 Cluster 1 10 97.8 10 0 10 76.0 10 97.1 4 4 4 4 Cluster 2 10 10 10 10 103 103 103 103 102 102 102 102
CD27 0 0 0 0 NK1.1 NKp46 CD11b CD49b NKp46 KLRG1 –102 –102 –102 –102 0103 104 105 0103 104 105 0103 104 105 0103 104 105 NK1.1
ef 1,500 * ** 45,000 *** *** 60,000 8,000 ** 6,000 1,000 30,000 40,000 4,000 500 15,000 20,000 2,000 Abs. no. eNK Abs. no. CLPs Abs. no. NKPs
0 0 0 Abs. no. pre-NKPs 0
+ Ctrl + Ctrl + Ctrl + Ctrl –/– + IL-12 –/– + IL-12 –/– + IL-12 –/– + IL-12 WT + Ctrl –/– WT + Ctrl –/– WT + Ctrl –/– WT + Ctrl –/– WT + IL-12 WT + IL-12 WT + IL-12 WT + IL-12 Il2rg Il2rg Il2rg Il2rg –/– Il2rg –/– Il2rg –/– Il2rg –/– Il2rg –/– –/– –/– –/– 2 2 2 Rag2 Rag2 Rag2 Rag2 Rag2 Rag Rag Rag Figure 4 | IL-12 induces expansion of eNK cells in the BM via an alternative developmental pathway. (a) tSNE dimensions 1 and 2 for CD45 þ Lin CD122 þ cells derived from the BM of Ctrl- or IL-12-treated WT or Rag2 / Il2rg / mice. (b) Annotated t-SNE maps depicting CD27, CD11b, NK1.1, CD49b, NKp46 and KLRG1 in the six identified clusters. (c) Heat map summary of average median expression of each cellular marker analysed for six clusters. (d) Adoptively transferred splenic CD45 þ CD3 CD122 þ CD49b þ NK1.1 þ and CD45 þ CD3 CD122 þ CD49b þ NK1.1low cells into Rag2 / Il2rg / mice were analysed in lungs at day 7 after transfer for expression levels of NK1.1. Two independent experiments were performed. (e) Quantification of NKPs and eNK cells in the BM of Ctrl- or IL-12-treated WT and Rag2 / Il2rg / mice. Data are pooled from two independent experiments. (f) Quantification of CLPs and pre-NKPs in the BM of Ctrl- or IL-12-treated WT and Rag2 / Il2rg / mice. Data are pooled from two independent experiments. Each dot represents one mouse. Data are expressed as mean±s.e.m. *Po0.05, **Po0.01, ***Po0.001 (unpaired Student’s t-test with Welch’s correction).
NK cells have been classically identified based on their lymphoid cells (ILC1)36,37 could place eNK cells into the latter expression of NK1.1 and NKp46 (ref. 22). Thus, the finding that category. We propose that eNK cells generated by IL-12 described eNK cells only express low levels of both lineage markers initially here are bona fide NK cells based on: (a) the expression of several raised doubts whether eNK cells are actually NK cells. Further- lineage defining NK-cell molecules (for example, Eomes), (b) the more, the similarities between NK cells and the group 1 innate lack of IL7-R expression and (c) their ability to kill target cells.
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P CD49b Count CD49b SSC-A Relative expression 100 200 300 10 10 10 o epesn Ksrsodt tmlto ihI-2addfeetaeit N cells. eNK into differentiate and IL-12 with stimulation to respond NKPs -expressing 100 K 150 K 200 K 250 K 10 10 10
50 K to Pol II 0 0 / 3 4 5 0 3 4 5 0.1 0.2 0.3 0.4 .5 *** 0.05,