Loss of Canonical Notch Signaling Affects Multiple Steps in NK Cell Development in Mice

This information is current as Patricia Chaves, Alya Zriwil, Lilian Wittmann, Hanane of September 28, 2021. Boukarabila, Claudia Peitzsch, Sten Eirik W. Jacobsen and Ewa Sitnicka J Immunol published online 26 October 2018 http://www.jimmunol.org/content/early/2018/10/25/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 26, 2018, doi:10.4049/jimmunol.1701675 The Journal of Immunology

Loss of Canonical Notch Signaling Affects Multiple Steps in NK Cell Development in Mice

Patricia Chaves,*,† Alya Zriwil,*,† Lilian Wittmann,*,† Hanane Boukarabila,‡ Claudia Peitzsch,*,1 Sten Eirik W. Jacobsen,‡,x,{,|| and Ewa Sitnicka*,†

Within the hematopoietic system, the Notch pathway is critical for promoting thymic T cell development and suppressing the B and myeloid lineage fates; however, its impact on NK lymphopoiesis is less understood. To study the role of Notch during NK cell development in vivo, we investigated different NK cell compartments and function in Rbp-Jkfl/flVav-Cretg/+ mice, in which Rbp-Jk, the major transcriptional effector of canonical Notch signaling, was specifically deleted in all hematopoietic cells. Peripheral conventional cytotoxic NK cells in Rbp-Jk–deleted mice were significantly reduced and had an activated phenotype. Furthermore, the pool of early NK cell progenitors in the bone marrow was decreased, whereas immature NK cells were increased, leading to a

block in NK cell maturation. These changes were cell intrinsic as the hematopoietic chimeras generated after transplantation of Downloaded from Rbp-Jk–deﬁcient bone marrow cells had the same NK cell phenotype as the Rbp-Jk–deleted donor mice, whereas the wild-type competitors did not. The expression of several crucial NK cell regulatory pathways was signiﬁcantly altered after Rbp-Jk deletion. Together, these results demonstrate the involvement of canonical Notch signaling in regulation of multiple stages of NK cell development. The Journal of Immunology, 2018, 201: 000–000.

atural killer cells represent a distinct group of innate TNF and IFN, and chemokines that regulate adaptive immune http://www.jimmunol.org/ lymphocytes important for controlling viral infections responses as well as other innate populations, including dendritic N as well as cancer (1) and were first defined as cytotoxic cells, macrophages, and neutrophils (1). NK cell deficiency results effector cells able to kill target cells without specific immuniza- in an increased susceptibility to infection and correlates with el- tion. NK cells also produce proinflammatory cytokines, such as evated cancer incidence (2, 3). NK cell malignancies, although infrequent, are very aggressive and difficult to treat (4). Thus, understanding NK cell development has both basic biological and *Lund Research Center for Stem Cell Biology and Cell Therapy, Lund University, clinical significance. 221 84 Lund, Sweden; †Division of Molecular Hematology, Department of Labora- ‡ Mouse conventional cytotoxic NK cells are defined phenotyp-

tory Medicine, Lund University, 221 84 Lund, Sweden; Haematopoietic Stem Cell 2 + + by guest on September 28, 2021 Laboratory, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department ically as CD3 NK1.1 CD49b cells (5) and are mainly derived of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom; xMRC from progenitors in the bone marrow (BM) but can be found in Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, multiple different tissues, including the spleen, lymph nodes, and Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom; {Center for Hematology and Regenerative Medicine, Department of liver (6). Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, 141 86 NK cell differentiation from hematopoietic stem cells (HSC) and || Stockholm, Sweden; and Department of Cell and Molecular Biology, Karolinska their developmental niches as well as the cellular and regulatory Institutet, SE-171 77 Stockholm, Sweden pathways governing NK cell development are not fully understood. 1Current address: National Center for Tumor Diseases, Dresden German Cancer Research Center, Dresden, Germany. The current model of mouse NK cell development comprises ORCIDs: 0000-0003-1364-0130 (P.C.); 0000-0001-6262-7253 (A.Z.); 0000-0002- several major cellular stages (Fig. 1A) with the earliest step in- 5247-908X (C.P.); 0000-0002-1362-3659 (S.E.W.J.); 0000-0001-5332-7048 (E.S.). volving generation of a common lymphoid progenitor (CLP), Received for publication December 4, 2017. Accepted for publication September 27, giving rise to T, B, and NK cells (5). The transition from CLP to 2018. an early NK cell progenitor (pre-NKP) is marked by the down- This work was supported by the Swedish Research Council, the Swedish Cancer regulation of FLT3 (CD135) (pre-NKP) (7), followed by upregu- Foundation, a Clinical Research Award from Lund University Hospital (ALF), and lation of IL-2/IL-15R b-chain (CD122) (5) in fully restricted the Hemato-Linne and StemTherapy program. E.S. has an associate professor posi- tion supported by the Swedish Pediatric Cancer Foundation. NKPs (rNKPs) (7). NKPs express lower levels of IL-7R (CD127) P.C., A.Z., and E.S. conceptualized the research. P.C., A.Z., L.W., H.B., and C.P. compared with CLPs; however, the dynamics of IL-7R expression performed experiments. P.C. and A.Z. analyzed the data. P.C. and E.S. wrote the during early NK lymphopoiesis are not well understood (8). From manuscript. S.E.W.J. participated in the study design, data presentation, and writing the NKP stage, cells upregulate NK1.1 receptor [immature NK of the manuscript. (iNK) cell 2], and as they further mature, they acquire the ex- Address correspondence and reprint requests to Dr. Ewa Sitnicka, Division of Molecular Hematology, Department of Laboratory Medicine, Lund Univer- pression of Ly49 receptors (iNK3) and integrin a2 (CD49b) sity, BMC B12, Klinikgatan 26, 22184 Lund, Sweden. E-mail address: ewa. [mature NK (mNK) cell 4] (5), upregulate CD11b (Mac-1) [email protected] (mNK5) and CD43 molecules (9), and downregulate CD27 The online version of this article contains supplemental material. (mNK6) (5, 10, 11) (Fig. 1A). Thymic NK cells express IL-7R, Abbreviations used in this article: BM, bone marrow; CLP, common lymphoid pro- lack Ly49 receptor expression, and show low levels of CD43 and genitor; DN, double negative thymocyte; Eomes, eomesodermin; FMO, ﬂuorescence minus one; ILC, innate lymphoid cell; iNK, immature NK; IO, ionomycin; KLRG1, CD11b (12). killer cell lectin-like receptor G1; LMPP, lymphoid-primed multipotent progenitor; The identiﬁcation of bipotent T/NK progenitors in multiple fetal mNK, mature NK; NKP, NK cell progenitor; PB, peripheral blood; pre-NKP, early tissues (13–15), as well as in adult BM (16), and the fact that the NK cell progenitor; rNKP, restricted NKP; WT, wild-type. early T cell progenitors in adult thymus maintain the ability to Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 generate NK cells (17, 18) suggest a close developmental

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701675 2 ROLE OF NOTCH IN NK CELLS relationship between T and NK cells (18). Multiple transcription Competitive transplantation assay factors such as TCF-1, T-BET, eomesodermin (Eomes), and ID2 Lethally irradiated (900 cGy) 10–23-wk-old C57BL/6 CD45.1 WT re- are required for the development of both T and NK cells, cipient mice were transplanted with 2.5 3 106 unfractionated BM cells whereas Bc11b upregulation inhibits NK lineage potential from 10-wk-old CD45.2 Rbp-Jkfl/flVav-Cretg/+ or Rbp-Jkfl/flVav-Cre+/+ lit- 6 (5, 19, 20). termate controls together with 2.5 3 10 unfractionated BM cells from Notch signaling is essential during T cell commitment in the 10-wk-old WT CD45.1 mice. At 8–10 wk after transplantation, mice were analyzed for multilineage donor-derived reconstitution in PB, spleen, thymus, including suppressing alternative lineage fates of devel- thymus, and BM. oping lympho-myeloid progenitors (21) with B cell, myeloid, and dendritic cell potentials (22–24). In contrast, several in vitro Gene expression analysis by RT-PCR studies have suggested that NK cell development might not be Cells were sorted in duplicates at 25 cells per well into 96-well plates into restricted by Notch signaling (16, 17) and that Notch signaling 4 ml of lysis buffer containing 0.4% NP40, deoxynucleoside triphosphates, may be permissive or even supportive for NK lymphopoiesis DTT, and RNase OUT (Invitrogen) and were snap frozen. RT-PCR and 18 cycles of preamplification with a mix of TaqMan probes (at 0.43) were (reviewed by Kling et al. 25). In agreement with this, mice performed in each well following the One-Step qRT/PCR with ROX reconstituted with Rbpj-deleted BM cells showed no defect in protocol from Invitrogen. The product (diluted 1:5) and TaqMan probes mNK cell generation (26). However, the physiological role of were loaded into a 48.48 Dynamic Array IFC (Fluidigm) following the canonical Notch signaling during early NK cell development re- manufacturer’s protocol. The following genes were detected using re- mains unexplored. spective probes (Applied Biosystems): Blimp1 Mm00476128_m1; Elf4 Mm01321797_m1; Eomes Mm01351985_m1; Ets1 Mm01175819_m1; Gata3 Mm00484683_m1; Gzmb Mm00442834_m1; Hes1 Mm01342805_m1; Materials and Methods Hes5 Mm00439311_g1; Hprt Mm00446968_m1; Id2 Mm00711781_m1; Ikzf1 Downloaded from Mm00456421_m1; Il15ra Mm04336046_m1; Il2ra Mm00434261_m1; Il2rb Mice Mm00434268_m1; Il2rg Mm00442885_m1; Il7r Mm00434295_m1; Irf2 Rbp-Jkfl/flVav-Cretg/+ mice were generated by cross-breeding of Rbp-Jkfl/fl Mm00515206_m1; Nfil3 Mm00600292_s1; Notch1 Mm00435245_m1; (27) and Vav-Cretg/+ (28) mice; wild-type (WT) C57BL/6 CD45.1 (JBmsd) Notch2 Mm00803077_m1; Prf1 Mm00812512_m1; RBP-Jk Mm01217627_g1; mice were obtained from The Jackson Laboratory. All mice were main- Stat4 Mm00448890_m1; Stat5a Mm03053818_s1; Tbx21 Mm00450960_m1; tained under specific pathogen-free conditions at Lund University Animal Tcf1 Mm00493445_m1; Tox Mm00455231_m1; and Ubc Mm01201237_m1.

Facility. The Ethical Committee at Lund University approved all per- Chips were run in Biomark (Fluidigm), and data were analyzed through Real- http://www.jimmunol.org/ formed experiments. Time PCR Analysis Software (Fluidigm). Ubiquitin C and Hprt were both used as reference genes for double normalization. Tissues Statistics The BM, spleen, and thymus were obtained as previously described (16). 6 BM cells were extracted using mortar. Single-cell suspensions were pre- All results are expressed as means ( SEM). Graphs and statistical analysis pared by breaking up the tissues in PBS containing 5% FCS (Sigma- were performed using GraphPad Prism 6 (GraphPad Software, La Jolla, Aldrich) and ﬁltering through a 70-mm cell strainer (BD Biosciences) CA). The statistical signiﬁcances between groups were determined by the (16). Cells were counted with the Sysmex (KX-21N) Hematology analyzer Mann–Whitney U test for unpaired samples. or in a Neubauer chamber. WBCs were isolated from peripheral blood

(PB) on a dextran gradient (16). Results by guest on September 28, 2021 Rbp-Jk deletion leads to an impaired Abs, flow cytometry, and cell sorting hematopoietic development Cells were incubated with 2.4G2 (anti-FcRIII) Ab to block Fc receptors and then stained with specific mAbs (listed in Supplemental Table I). 7-ami- To address the potential involvement of the Notch pathway during noactinomycin D (7-AAD) (Sigma-Aldrich) was used to exclude dead NK cell development, we first investigated the expression pattern of cells from the analysis. Fluorescence minus one (FMO) and isotype con- the essential mediator of canonical Notch signaling Rbp-Jk and trols were used to determine the positive signal. Intracellular staining to other genes related to the Notch pathway, within different NK cell detect Eomes and IFN-g was performed using Foxp3 Staining Buffer Set (eBioscience) following the manufacturer’s instructions. Samples were compartments (Fig. 1A). mRNA for Rbp-Jk, as well as Notch1 and analyzed on LSR II (BD Biosciences), and analysis was done with FlowJo Notch2 receptors, and the transcription factor Tcf7, known to be software (TreeStar). induced by Notch signaling (30), were expressed at all NK cell All cell sorting was performed on FACSAria IIu (BD Biosciences) using developmental stages (Fig. 1B). The expression of the Notch di- Purity precision mode on 96-well plates. rect target gene Hes-1 (31) was also detected in nearly all NK cell Functional NK cell analysis/NK cell degranulation assay and compartments, whereas Hes-5 mRNA was found only in pre-NKP IFN-g production and rNKP but not at later stages (Fig. 1B). 3 6 To elucidate the role of the Notch pathway during NK cell Spleen cells were plated at 2 10 cells/ml in round-bottom 96-well fl/fl tg/+ plates in RPMI 1640 medium (PAA Laboratories) supplemented with development, we generated Rbp-J-k Vav-Cre mice in which 10% FCS (Sigma-Aldrich), 1% penicillin/streptomycin (Sigma-Aldrich), Rbp-Jk is specifically deleted in all hematopoietic cells after 24 fl/fl tg/+ 1% L-glutamine (Sigma-Aldrich), and 10 M 2-ME (Sigma-Aldrich). crossing Rbp-Jk with Vav-Cre mice (27, 28). NK cells were specifically activated with a 4-h incubation with a purified The distribution of mature cells in PB in Rbp-Jkfl/flVav-Cretg/+ anti-NK1.1 Ab at 37˚C. Cells incubated in medium alone were used as a fl/fl +/+ negative control, whereas those activated with PMA (Sigma-Aldrich) mice was significantly altered compared with Rbp-Jk Vav-Cre and ionomycin (IO) (Sigma-Aldrich) were a positive control. After- littermate controls (Fig. 1C, 1D); however, the total WBC counts wards, cells were harvested and the surface expression of CD107a were not changed (Fig. 1E). Interestingly, and not previously (lysosomal-associated membrane protein 1; LAMP-1) (29) was detected reported, Rbp-Jk–deleted mice showed a 67% decrease in the by flow cytometry. To measure IFN-g production, spleen cells were frequency of conventional NK1.1+CD49b+ NK cells in PB plated at 5 3 106 cells/ml in round-bottom 96-well plates in RPMI 1640 (PAA Laboratories), supplemented with 10% FCS (Sigma-Aldrich), 1% (Fig. 1C, 1D). As previously shown (22, 32), T cells were almost of penicillin/streptomycin (Sigma-Aldrich), and 1024 M2-ME(Sigma- undetectable, whereas B cells were slightly increased in PB of Aldrich), and cultured for 4 h in the presence of 60 ng/ml of recombinant Rbp-Jkfl/flVav-Cretg/+ mice (Fig. 1C, 1D). Cellularity was in- mouse IL-12 (BioLegend) and 30 ng/ml of recombinant mouse IL-18 creased 1.8-fold in the spleen, unchanged in the BM, and showed (BioLegend) with brefeldin A and monensin (BioLegend). Negative and positive control were treated as in the degranulation assay. Intracellular a reduction of 87% in the thymus (Fig. 1F). As previously IFN-g production within different NK cell populations was detected by reported, T cell development was impaired after Notch deletion flow cytometry. (32), including a loss of double negative thymocytes (DN) 2, DN3, The Journal of Immunology 3

FIGURE 1. RBPJ deletion leads to reduction in NK cell populations in PB. (A) Schematic representation of mouse NK developmental stages and the markers used to define each stage. LMPP = Lin2Sca-1hiKIThiFLT3high;CLP= 2 lo lo + + Lin Sca-1 KIT FLT3 CD127 ; pre-NKP = Downloaded from Lin2CD244+CD27+FLT32CD127+CD1222;stage1 (rNKP) = Lin2CD244+CD27+CD127+FLT32CD122+; iNK2 = NK1.1+Ly492 CD49b2; iNK3 = NK1.1+Ly49+CD49b2;mNK4=NK1.1+Ly49+ CD49b+CD27+CD11b2; mNK5 = NK1.1+Ly49+ CD49b+CD11b+CD27+CD43+;andmNK6= 2 NK1.1+Ly49+CD49b+CD11b+CD27 CD43+.(B) http://www.jimmunol.org/ Expression of Rbp-Jk– and Notch-related genes at the different stages of NK cell development and maturation. Results represent mean (6SEM) values of expression normalized against both Hprt and Ubiquitin from six individual mice analyzed in three independent experiments (four mice in two experiments in the case of the progenitors). (C–F) Adult 9–10-wk-old Rbp-Jkfl/flVav-Cre tg/+ mice (white bars) and Rbp-Jkfl/flVav-Cre +/+ littermate by guest on September 28, 2021 controls (gray bars) were analyzed. (C) Repre- sentative flow cytometry profiles of the different hematopoietic lineages in the PB. Numbers represent the mean percentage of total cells, and the gating is indicated above the plot. (D) Percentage of myeloid (Gr1+), B (Gr12CD19+), T (Gr12CD3+), and NK (Gr12CD192CD32NK1.1+CD49b+) cells in PB (from three to four mice). (E) Total WBC counts per milliliter in PB (from four to five mice). (F) Total cellularities in BM, spleen, and thymus. Data represent mean (6SEM) values of 18–27 age-matched mice from 10 different litters of each genotype.*p # 0.05, ***p # 0.001.

and double positive thymocytes as well as a significant decline in Fig. 1A–D). Although, as reported in previous studies (26), the DN1/early thymic progenitors (33) (Supplemental Fig. 1A–D). proportion of IL-7R+ thymic NK cells among thymocytes was not Also, single positive CD4+ and single positive CD8+ thymocytes altered after Rbp-Jk deletion (data not shown). When taking into were severely reduced in Rbp-Jk–deleted mice (Supplemental account reduced thymic cellularity (Fig. 1F), the total number of 4 ROLE OF NOTCH IN NK CELLS thymic NK cells was decreased (Supplemental Fig. 1E), whereas increases during their maturation (40). In Rbp-Jk–deleted mice, both the frequency (data not shown) and the total number of the level of KLRG1 expression and the number of KLRG1+ cells IL-7R2 conventional NK cells was increased (Supplemental Fig. were significantly reduced within the iNK pool and the mNK6 1F). Also, in the absence of Notch signaling, the total number of population (Fig. 3B, 3C). CD19+ B cells was expanded in the thymus (32), and the fre- These results together suggest that maturation of NK cells in quency of CD11b+CD11c+MHC class II+ dendritic cells was in- Rbp-Jk–deleted mice is delayed, and their phenotype is altered. creased (data not shown) (34), whereas the total dendritic cell number was not significantly altered (Supplemental Fig. 1G, 1H). Loss of canonical Notch signaling results in dysregulated NK cell regulatory pathways Rbp-Jk–deleted mice show a reduction in early BM NKPs and Next, we investigated whether and how Rbp-Jk deletion affects the a maturation block in splenic cytotoxic NK cells expression of genes controlling NK cell development (41). Next, we investigated whether the loss of canonical Notch signaling Transcription factors and cytokines govern NK lymphopoiesis, has an impact on NK cell differentiation at different hierarchical starting from the earliest progenitors. Although many are shared developmental stages (Fig. 1A). with the other lineages, some are unique to NK cells, and their The proportion of the earliest BM lymphoid progenitors are as specific deletion results in NK cell deficiencies (41). Transcription follows: lymphoid-primed multipotent progenitor (LMPP) and CLP factors ID2 (42, 43), STAT5 and STAT4 (44, 45), IRF-2 (46, 47), were not changed in Rbp-Jkfl/flVav-Cre tg/+ mice, in sharp contrast to ETS1 (48), NFIL3 (also called E4BP4) (49), and IKZF1 (50) are pre-NKP (Lin2CD27+CD244+CD127+FLT32CD1222)andrNKP known to drive early NK cell developmental stages, whereas 2 2 (Lin CD27+CD244+CD127+FLT3 CD122+) (7) that were signifi- Eomes and T-BET (encoded by Tbx21) (38, 51, 52), ELF4 (also Downloaded from cantly reduced (50 and 92.2% respectively; Fig. 2A, 2B). known as MEF) (53), TCF1 (8), GATA3 (12, 54), BLIMP1 (55), Conventional NK cells undergo their final maturation and and TOX (56, 57) are acting at the later stages involving differ- acquire cytotoxic functionality in the spleen (10, 11, 35–37); entiation, expansion, and functional maturation. therefore, we next examined whether Rbp-Jk deletion af- Consistent with the reduction in pre-NKPs and rNKPs in Rbp-Jk– fects different splenic NK cell populations (Fig. 1A). Nota- deleted mice (Fig. 2A, 2B), the expression of Ets-1 and Ikzf1 mRNA + 2 2 bly, the total number of iNK2 (NK1.1 Ly49 CD49b ), encoding the transcription factor IKAROS was significantly re- http://www.jimmunol.org/ mNK4 (NK1.1+Ly49+CD49b+CD27+CD11b2), and mNK5 duced in Rbp-Jk–deleted pre-NKPs (Fig. 4A). Also, the levels of (NK1.1+Ly49+ CD49b+CD11b+CD27+CD43+) cells was sig- Stat5b, Stat4,andIrf2 mRNA were reduced in Rbp-Jkfl/flVav-Cretg/+ nificantly increased, whereas the most mature cytotoxic pre-NKPs (Fig. 4A). In the absence of canonical Notch signaling, mNK6 (NK1.1+Ly49+CD49b+CD11b+CD272CD43+)cellswere Id2 expression was reduced in pre-NKPs but increased in iNK2 and reduced in Rbp-Jkfl/flVav-Cretg/+ mice (Fig. 2C, 2D), together iNK3 cells, whereas it was not changed at the later stages (Fig. 4A). suggesting a developmental defect resulting in severe reductions Nfil3 expression was not altered within any of the NK cell com- at the final maturation stage of mNK6. partments after Rbp-Jk deletion (Fig. 4A). Because Nfil3 is required Collectively, these results support the involvement of canonical only during early NK cell development (58), the NK cell impair- Notch signaling in regulation of the earliest NK cell developmental ment in Rbp-Jk–deleted mice is likely due to Notch signaling af- by guest on September 28, 2021 stages in the BM as well as their final maturation steps in the spleen. fecting other important NK cell regulatory pathways. IL-7 is a cytokine important during lymphoid development, and NK cell maturation is delayed, and the phenotype of IL-7R is expressed within pre-NKP and rNKP populations (7, 43). developing NK cells is changed in the absence of canonical Interestingly, the expression of Il-7r was reduced in pre-NKPs Notch signaling after Rbp-Jk deletion (Fig. 4A). To better understand the perturbed differentiation of Rbp-Jk–deleted In line with the perturbed and delayed maturation of Rbp-Jk– NK cells, we investigated the expression of specific receptors important deleted NK cells, the expression of several late-acting regulators for NK cell maturation and function (5). was changed. Eomes mRNA levels were reduced in Rbp-Jk– Functional cytotoxic NK cells express a diverse repertoire of deleted iNK2 and iNK3 cells, whereas the gene expression of Ly49 receptors required for NK cell tolerance and function. Ly49 Tbx21 as well as Tox and Blimp1 was not altered (Fig. 4B). The receptors start to be expressed at the iNK3 stage and Ly49 up- levels of Elf4 were lower in all of the NK cell developmental regulation, and expression is regulated by transcription factor stages in Rbp-Jkfl/flVav-Cretg/+ mice, whereas the expression of Eomes (38). Tcf1 was reduced in both iNK and mNK cells (Fig. 4B). The number of NK cells expressing diverse Ly49 receptors at the GATA3, which is directly regulated by Notch (21), is critical for early maturation stages of iNK and mNK4 was not significantly thymic NK cells; however, Gata3-deficient mice showed no major altered in Rbp-Jkfl/flVav-Cretg/+ mice (Fig. 3A), in line with no defects in conventional NK cells (12, 54). Upon Rbp-Jk deletion, significant changes in the level of expression and in the number of Gata3 was clearly expressed within the different NK cell com- Eomes+ cells (Fig. 3B, 3C). However, in the absence of Rbp-Jk, partments with reduced levels in pre-NKP and increased expres- the number of Ly49-expressing NK cells was reduced within the sion in iNK3 cells (Fig. 4B). more differentiated mNK5 and mNK6 populations (Fig. 3A), in- Collectively, these data suggest that the loss of canonical Notch dicating a delay in their functional maturation. signaling has an impact on the expression of multiple key genes TRAIL is a key NK cell effector molecule important for anti-tumor important for early and late NK cell developmental stages. activity. iNK cells upregulate TRAIL at the iNK2 stage (5), and the constitutive TRAIL expression represents a hallmark of cytotoxic NK cells in Rbp-Jk–deleted mice have a iNK cells (39). Importantly, after Rbp-Jk deletion, both the total hyperactivated phenotype number of NK cells expressing TRAIL and the level of receptor To establish whether the NK cells generated in the absence of expression were clearly increased at all of the differentiation stages, Rbp-Jk are functionally affected, we analyzed the expression of further supporting their delayed maturation (Fig. 3B, 3C). specific receptors involved in NK cell–mediated killing, cytotoxic Killer cell lectin-like receptor G1 (KLRG1) is an inhibitory activity using the degranulation assay, and cytokine release after receptor for MHC class I, and the fraction of KLRG1+ NK cells activation. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 FIGURE 2. RBPJ deletion produces a reduction in pre-NKPs and rNKPs in the BM and a block in the maturation in the spleen. (A–D) Adult 7–11-wk-old Rbp-Jkfl/flVav-Cretg/+ mice and age-matched Rbp-Jkfl/flVav-Cre+/+ littermate controls were analyzed. (A) Representative flow cytometry profiles of BM cells from Rbp-Jkfl/flVav-Cretg/+ mouse (lower panel) and Rbp-Jkfl/flVav-Cre +/+ littermate control (top panel). Numbers indicate the mean percentage of total BM, and the text above describes the gating. (B) Early lymphoid progenitor populations in the BM are as follows: Lin2Sca-1hiKIThiFLT3high LMPP; Lin2Sca-1loKITloFLT3+CD127+ CLP; Lin2CD244+CD27+FLT32CD127+CD1222 pre-NKP; and Lin2CD244+CD27+CD127+FLT32CD122+ rNKP from Rbp-Jkfl/flVav-Cre tg/+ mice (white bars) and Rbp-Jkfl/flVav-Cre +/+ littermate controls (gray bars). Data represent mean (6SEM) values of 8–15 age-matched mice from five to eight different litters of each genotype. Lin (pre-NKP, rNKP) = CD3, CD19, NK1.1, CD49b, Ter119, Gr1, and CD11b; Lin (LMPP, CLP) = CD3, B220, Gr1, Ter119, CD11b, CD4, CD8, and NK1.1. (C) Representative flow cytometry profiles of CD32 splenocytes from Rbp-Jkfl/flVav-Cre tg/+ mouse (lower panel) and Rbp-Jkfl/flVav-Cre +/+ littermate control (top panel) defining NK cell stages from 2 to 6 as shown in Fig. 1A. Numbers indicate the mean percentage in the spleen, and the text above describes the gating. (D) Total number of NK cells at each developmental stage in the spleen from Rbp-Jkfl/flVav-Cre tg/+ mice (white bars) and Rbp-Jkfl/flVav-Cre +/+ littermate controls (gray bars). iNK2 = NK1.1+Ly492CD49b2; iNK3 = NK1.1+Ly49+CD49b2; mNK4 = NK1.1+Ly49+CD49b+CD27+CD11b2; mNK5 = NK1.1+Ly49+CD49b+CD11b+CD27+CD43+; and mNK6 = NK1.1+Ly49+CD49b+CD11b+CD272CD43+. Results represent mean (6SEM) values of 6–15 age-matched mice from six litters of each genotype. *p # 0.05, **p # 0.005, ***p # 0.001.

Despite the significant reduction in the mature cytotoxic NK cell significantly higher in Rbp-Jk–deleted mice compared with littermate pool, Rbp-Jk–deleted NK cells maintained their ability to release controls, even without activation (Fig.5B, Supplemental Fig. 3B). cytolytic granules after specific activation with anti-NK1.1 Ab To further characterize NK cell functionality and spontaneous (59) that is marked by the upregulation of CD107a (LAMP-1) activation, we studied the expression of CD69 and IL-2Ra (CD25), molecule at the cell surface (Fig. 5A). Also, the expression of known NK cell activation markers (60), as well as DNAM-1 ad- NKp46, NK1.1, and IL-2Rb (CD122) receptors, known to be hesion molecule triggering NK-dependent anti-tumor activity engaged in NK cell–mediated killing, was not changed after RbpJ (61). Indeed, in Rbp-Jkfl/flVav-Cretg/+ mice, both the level of ex- deletion (Supplemental Fig. 2). However, the levels of CD107a pression and absolute number of iNK and mNK cells expressing were significantly higher within mNK4, mNK5, and mNK6 pop- CD69 were higher (Fig. 6A–C), and although the expression level ulations, not only in NK cells activated with anti-NK1.1 Ab but was not altered, the number of CD25 (Fig. 6B, 6C) and DNAM-1 even in nonactivated Rbp-Jk–deleted NK cells compared with (Fig. 6B, 6C) positive cells was increased, together indicating a controls (Fig. 5A, Supplemental Fig.3A). hyperactivation of NK cells. Cytokine production represents another functional NK cell activity However, the expression of Gzmb and Prf1, encoding cytolytic (1); therefore, we next investigated whether Rbp-Jk deletion has an proteins GRANZYME B and PERFORIN, released by NK cells impact on IFN-g production. The proportion of NK cells that re- during cytotoxic killing (1) was not altered after Rbp-Jk deletion leased IFN-g within iNK3, mNK5, and mNK6 populations was (Fig. 6D). 6 ROLE OF NOTCH IN NK CELLS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. RBPJ deletion disrupts the maturation signature of distinct NK developmental stages. (A) Total numbers of NK cells (within CD32 splenocytes) at the different developmental stages expressing different Ly49 isoforms in the spleen from adult 8–9-wk-old Rbp-Jkfl/flVav-Cretg/+ mice (white boxes) and Rbp-Jkfl/flVav-Cre+/+ littermate controls (gray boxes). iNK = NK1.1+CD49b2; mNK4 = NK1.1+Ly49+CD49b+CD27+CD11b2; mNK5 = NK1.1+Ly49+CD49b+CD11b+CD27+CD43+; and mNK6 = NK1.1+Ly49+CD49b+CD11b+CD272CD43+. Results represent mean (6SEM) values from 8–10 mice of each genotype in three independent experiments. (B) Representative flow cytometry histograms showing the surface expression of TRAIL and KLRG1 and the intracellular expression of Eomes in a Rbp-Jkfl/flVav-Cre tg/+ mouse (black line), a Rbp-Jkfl/flVav-Cre +/+ littermate control (dashed line), and in a FMO control (solid gray) at the different NK cell developmental stages in the spleen. (C) Total number of NK cells at the different developmental stages expressing surface TRAIL, KLRG1, and intracellular Eomes in the spleen from adult 8–9-wk-old Rbp-Jkfl/flVav-Cretg/+ mice (white boxes) and Rbp-Jkfl/flVav-Cre+/+ littermate controls (gray boxes). Results represent mean (6SEM) values from 8–10 mice of each genotype from five independent experiments. *p # 0.05, **p # 0.005, ****p # 0.0001.

Normally, liver-resident NK cells, unlike the conventional spleen Taken together, these results suggest that canonical Notch sig- NK cells, express CD49a while lacking CD49b expression (6). naling, in addition to an impact on the final NK cell maturation Interestingly, a significant fraction of splenic NK cells expressed stages, plays a role in limiting the activation status of NK cells. CD49a in Rbp-Jk–deleted mice (Fig. 6B, 6C). IL-15 is critical for the development, function, and survival of The effects of canonical Notch signaling on NK cell NK cells (5); however, prolonged stimulation with IL-15 leads to development effects are cell intrinsic and manifested at the chronic activation and phenotypic changes (62). The expression of early postnatal stages Il15ra, Il2rb, and Il2rg mRNA encoding IL-15Ra, IL-2Rb, and To determine whether the effects of the Rbp-Jk deletion are cell in- IL-2Rg subunits, all involved in IL-15 signaling, was elevated in trinsic, we performed competitive transplantations using unfractionated iNK cells in Rbp-Jkfl/flVav-Cretg/+ mice (Fig. 6D). BM cells from Rbp-Jkfl/flVav-Cre tg/+ mice or Rbp-Jkfl/flVav-Cre +/+ The Journal of Immunology 7

FIGURE 4. RBPJ deletion disrupts the expression of genes involved in regulation of NK cell development and effector functions. Downloaded from Relative gene expression in NK cells in the different developmental and maturation stages in BM and spleen of 8–11-wk-old Rbp-Jkfl/flVav-Cretg/+ mice (white bars) and Rbp-Jkfl/flVav-Cre +/+ littermate controls (gray bars). (A) Genes involved in regulation of the early stages of NK cell development. (B)Genes http://www.jimmunol.org/ involved in regulation of the maturation and effector functions of NK cells. Results represent mean (6SEM) values of expression normalized against both Hprt and Ubiq- uitin from seven individual Rbp-Jk– deleted mice and nine individual control mice analyzed in four independent experiments (data for pre-NKP were obtained from

ﬁve Rbp-Jk–deleted mice and six control mice by guest on September 28, 2021 in three independent experiments). pre-NKP = Lin2CD244+CD27+FLT32CD127+CD1222; iNK2 = NK1.1+Ly492CD49b2; iNK3 = NK1.1+Ly49+CD49b2 ; mNK5 = NK1.1+ Ly49+CD49b+CD11b+CD27+CD43+; and mNK6 = NK1.1+Ly49+CD49b+CD11b+CD272CD43+. *p # 0.05, **p # 0.005.

littermate controls. The hematopoietic reconstitution from Rbp- pre-NKPs were reduced by 23% (although not reaching statistical Jk–deleted BM was significantly lower in all the tissues compared significance; p = 0.071) and rNKPs were not produced from with controls (Fig. 7A). Notably, Rbp-Jk–deleted cells failed to transplanted Rbp-Jk–deleted BM cells (Fig. 7C), in agreement generate not only T cells, but also NK cells, whereas B and my- with the findings in the Rbp-Jkfl/flVav-Cretg/+ mice themselves. eloid cell regeneration was not impaired (Fig. 7B). Furthermore, The population of donor-derived mature splenic cytotoxic mNK6 although donor-derived reconstitution of CLPs was not altered, cells was significantly reduced, whereas the pool of mNK4/5 cells 8 ROLE OF NOTCH IN NK CELLS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. RBPJ deletion leads to increased functional activity of NK cells in the spleen. (A) Percentage of nonstimulated, PMA- plus IO-stimulated, and NK1.1-stimulated iNK2, iNK3, mNK4, mNK5, and mNK6 populations expressing surface CD107a in the spleen from 8–11-wk-old Rbp-Jkfl/flVav-Cretg/+ mice (white boxes) and Rbp-Jkfl/flVav-Cre+/+ littermate controls (gray boxes). (B) Percentage of nonstimulated, IL-12– plus IL-18–activated, and PMA- plus IO-stimulated iNK2, iNK3, mNK5, mNK5, and mNK6 populations with intracellular expression of IFN-g in the spleen from 8–11-wk-old Rbp-Jkfl/flVav-Cretg/+ mice (white bars) and Rbp-Jkfl/flVav-Cre+/+ littermate controls (gray bars). Data represent mean (6SEM) values of six age-matched mice of each genotype from two independent experiments. iNK2 = NK1.1+Ly492CD49b2; iNK3 = NK1.1+Ly49+CD49b2; mNK4 = NK1.1+Ly49+CD49b+CD27+CD11b2;mNK5= NK1.1+Ly49+CD49b+CD11b+CD27+CD43+; and mNK6 = NK1.1+Ly49+CD49b+CD11b+CD272CD43+.*p # 0.05. was increased (Fig. 7D), corroborating the maturation defect seen Discussion fl/fl tg/+ in the adult Rbp-Jk Vav-Cre nontransplanted mice (Fig. 2C, For decades after their discovery, conventional NK cells were 2D). Importantly, and in further support for cell-intrinsic defects thought to be the only innate effector lymphocytes. Recent studies induced by Rbp-Jk deletion, regeneration of NK cell compart- identifying new populations of innate lymphoid cells (ILCs) placed ments (both NK progenitors and mNK cells derived from WT NK cells within the ILC1 group. Conventional NK cells and CD45.1 BM cells cotransplanted with Rbp-Jk–deleted BM cells) noncytotoxic ILC1 cells represent different lineages (63); however, was not impaired (Fig.7E–G). their developmental origin partially overlaps (64). To establish whether the changes in NK cell compartments Within the hematopoietic system, Notch is critical for promoting induced by the loss of Notch occur also at early postnatal stages, T cell development (32). The Notch pathway has also been im- 2-wk-old Rbp-Jkfl/flVav-Cretg/+ mice were investigated. Similar to plicated in regulation of innate lymphocytes and in generation, adult mice, Rbp-Jk deletion resulted in a severe reduction of differentiation, and maturation of ILC2 and other ILCs cells (25, thymic cellularity, whereas the spleen and BM cellularities were 63). However, it has remained unclear to what degree canonical not changed (Supplemental Fig. 4A). The number of rNKPs in the Notch signaling has an impact on conventional NKPs and matu- BM was decreased in Rbp-Jk–deleted mice (Supplemental Fig. ration, because most experiments addressing the impact of Notch 4B, 4C), and the transition through mNK4–mNK6 stages was were limited to NK cell differentiation in vitro (16, 17, 25). perturbed as illustrated by the increased number of mNK4 cells, Previous in vivo studies with chimera mice generated after although the NK cell phenotype in 2-wk-old mice was less severe transplantation of Rbpj-deleted BM cells showed no significant compared with the adult. alterations in donor-derived conventional NK cell pool in the Collectively, these results support that the effects of the deletion spleen and no changes in thymic-dependent IL-7R+ NK cell of canonical Notch signaling are cell intrinsic, and the impact of the population, whereas, as expected, T cells were absent (21, 26). loss of Notch on NK cell development is already manifested early Interestingly and not previously reported, Rbp-Jk–deleted mice after birth. had a severely reduced pool of conventional NK cells in PB and an The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. RBPJ deletion leads to chronic activation of NK cells in the spleen. (A) Percentage of nonstimulated, PMA plus IO-stimulated, and NK1.1-stimulated Lin2CD122+DX5+NKp46+ NK cells expressing surface CD69 in the spleen from 9–11-wk-old Rbp-Jkfl/flVav-Cretg/+ mice (white boxes) and Rbp-Jkfl/flVav-Cre+/+ littermate controls (gray boxes). Data represent mean (6SEM) values of seven to nine age-matched mice of each genotype from three independent experiments. Lin = CD3, CD4, CD8a, CD19, Ter119, and Gr1. (B) Representative flow cytometry histograms showing CD25, CD69, DNAM-1, and CD49a surface expression within CD32 splenocytes at different NK cell developmental stages in a Rbp-Jkfl/flVav-Cretg/+ mouse (black line), Rbp-Jkfl/flVav-Cre+/+ littermate control (dashed line), and FMO control (solid gray). (C) Total number of CD25+, (Figure legend continues) 10 ROLE OF NOTCH IN NK CELLS altered phenotype of mature cytotoxic splenic NK cells showing a Rbp-Jkfl/flVav-Cretg/+ mice when competitively transplanted into sign of spontaneous hyperactivation with elevated levels of CD69, WT recipients, and Rbp-Jk–deleted cells in these chimera mice CD25, DNAM-1, and CD107a molecules as well as increased had no effect on generation of NK cell compartments from IFN-g production. The increased gene expression level of Il15ra, cotransplanted WT BM donor cells. Although previous studies Il2ra, Il2rb,andIL2rg, all part of the IL-15 signaling cascade, in reported (26) that chimera mice generated by transplanting Rbp-Jk–deleted NK cells suggests that this hyperactivation could Rbp-Jk–deleted BM cells showed no changes in splenic result from the altered response to IL-15, similar as in Ets12/2 mice CD32CD1272NKp46+ NK cells, the different NK cell develop- (48) or after chronic stimulation with IL-15/IL-15a complexes (62). mental stages were not investigated. In agreement with that work CD49a integrin is expressed on the surface of liver NK cells (6) (26), the frequency of CD127+ thymic NK cells was not altered in and ILC1 cells (64) but not on steady-state conventional splenic Rbp-Jk–deleted mice in our studies, although the observed in- CD49b+ NK cells. Because liver-resident NK cells do not migrate crease in the CD1272 conventional NK cells in the thymus, after to other tissues (6) and ILC1 cells lack CD49b, PERFORIN, and Rbp-Jk deletion, in this study was not previously reported. GRANZYME B expression, CD49a+ NK cells in the spleen Despite the almost undetectable rNKP pool in Rbp-Jk–deleted in Rbp-Jk–deleted mice most likely represent the conventional BM, functional NK cells were generated. This, together with the splenic NK cells with an activated phenotype, similar to super- high expression of NK lineage-specific genes in Rbp-Jk–deleted activated CD49a+ T lymphocytes (65). Furthermore, human PB pre-NKP and iNK cells, suggests that the CLP-rNKP path might cytotoxic NK cells have been shown to change their phenotype not be the only NK lineage pathway and that potentially LMPPs and upregulate CD49a after exposure to hypoxia, TGF-b1, and a and CLPs in the BM, and early thymic progenitors in the thymus demethylating agent (66). could support NK cell generation independently of rNKPs. Downloaded from To better depict the impairment in generation of mNK cells and To better understand the impact of canonical Notch signaling on their altered phenotype, we investigated in detail different NK cell the sequential NK cell differentiation stages, we investigated the compartments. The loss of canonical Notch signaling had no effect expression of genes involved in the NK cell regulatory circuits and on the earliest LMPP or CLP BM progenitors. In contrast, the pool found that Rbp-Jk deletion affected the expression of multiple of NK lineage-restricted progenitors pre-NKPs and in particular genes critical for NK cell development and function. In line with

rNKPs (7) was dramatically decreased, whereas the iNK cells were the severe reduction in pre-NKPs and rNKPs in Rbp-Jk–deleted http://www.jimmunol.org/ increased, suggesting a partial block at the early NK cell develop- mice, the expression of Ikzf1, Stat5b, and Ets1, known to be im- mental stages. Furthermore, compatible with perturbed NK cell portant for regulation of the very early NK developmental stages maturation, the number of cells expressing KLRG1 receptors within (41), was significantly reduced. the splenic iNK cells in Rbp-Jk–deleted mice was reduced, whereas The perturbation in NK cell maturation in the absence of ca- the number of TRAIL-positive cells increased and, similar to mature nonical Notch signaling can be, in part, due to the reduced level of cytotoxic NK cells, displayed an activated phenotype as indicated by Eomes that controls different aspects of NK (38, 41, 70) and T cell the increased number of DNAM1- and CD69-positive cells. differentiation (70). The Eomes expression in CD8+ T cells is The expression of IL-7R, CD69, DNAM1, TRAIL, and T-BET directly regulated by Notch signaling (71) and, in this study, we within CD32NK1.1+CD49b2 population defines a heterogeneous show that Notch deletion affects the Eomes expression in iNK by guest on September 28, 2021 pool that includes some subsets of ILC1. However, ILC1 cells are cells. Consistent with Eomes being required for the expression of rarely found in the spleen, and the CD49b2 splenic NK cells Ly49 and CD49b in differentiating NK cells (38, 67), the loss of found in RbpJ-deleted mice did not show an increase in IL-7R Notch led to a perturbed NK cell maturation. This is further expression compared with controls but clearly expressed PER- supported by studies showing that the reduced levels of Eomes in FORIN and GRANZYME B, cytolytic proteins not typically patients after BM transplantation affect NK cell differentiation produced by ILC1 cells. The increased levels of TRAIL detected and function (72). Reduced expression of transcription factor in the CD49b2 cells could be misleading; however, given the fact MEF,whichisknowntobeimportantforNKcellmaturation that the expression of TRAIL was maintained in more mature (53), could also contribute to the iNK cell status in Rbp-Jk– CD49b+ NK cells, these results collectively suggest that Rbp-Jk– deleted mice. deleted splenic iNK cells have a preserved NK lineage identity (67). T-BET regulates NK cell cytotoxic activity (51) and, together The final NK cell maturation stages were also affected by the loss with EOMES, is essential for generation of functional NK cells of Notch, leading to increased mNK4 and mNK5 compartments (38). The reduced Eomes expression and unchanged levels of and reduced pool of cytotoxic mNK6 cells. T-bet in RbpJk-deleted NK cells, most likely, contribute to their These findings are in agreement with the previous work im- immature phenotype. Consistent with the unaltered expression of plicating the involvement of the Notch pathway in promoting T-bet, NK cell cytotoxic activity seems not to be affected by the generation of human NK cells from hematopoietic progenitors, absence of Notch signaling. regulating production of IFN-g by NK cells, enhancing expression In agreement with the finding that TCF-1 regulated by canonical of KIR receptors, and functional NK cell maturation during se- Notch signaling (30, 73) is required for generation of NKPs and quential differentiation stages of human NK cells in vitro (68, 69). mNK cells (8), Tcf1 expression was significantly reduced after The requirements for Notch signaling were cell autonomous, as Rbp-Jk deletion, leading to a decrease in the number of NKPs as Rbp-Jk–deleted BM cells reconstituted the phenotype of donor well as mNK cells.

CD69+, DNAM-1+, and CD49a+ NK cells at the different stages of development in the spleen of 8–11-wk-old Rbp-Jkfl/flVav-Cretg/+ mice (white boxes) and Rbp-Jkfl/flVav-Cre+/+ littermate controls (gray boxes). Data represent mean (6SEM) values from six to eight age-matched mice of each genotype in six independent experiments. (D) Relative gene expression in NK cells at the different developmental and maturation stages in BM and spleen of Rbp-Jkfl/flVav- Cretg/+ mice (white bars) and Rbp-Jkfl/flVav-Cre+/+ littermate controls (gray bars). Results represent expression normalized against both Hprt and Ubiquitin expression from three independent experiments with four to nine mice analyzed. pre-NKP = Lin2CD244+CD27+FLT32CD127+CD1222; iNK = NK1.1+CD49b2; mNK5 = NK1.1+Ly49+CD49b+CD11b+CD27+CD43+; and mNK6 = NK1.1+Ly49+CD49b+CD11b+CD272CD43+.*p # 0.05, **p # 0.005, ***p # 0.001. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Impairment in lineage generation induced by RBPJ deletion is cell intrinsic. (A–G) Lethally irradiated (900 cGy) 10–23-wk-old CD45.1 WT recipient mice were transplanted with 2.5 3 106 unfractionated BM cells from 10-wk-old CD45.2 Rbp-Jkfl/flVav-Cre tg/+ mice (white bars) or Rbp- Jkfl/flVav-Cre +/+ littermate controls (gray bars), together with 2.5 3 106 unfractionated BM cells from 10-wk-old WT CD45.1 mice, and the donor- derived reconstitution was analyzed at 8–10 wk after transplantation. (A) Percentage of CD45.2 donor-derived reconstitution in the PB, BM, thymus, and spleen of recipient mice. (B) Percentage of CD45.2 donor-derived reconstitution of myeloid (Gr1+), B (Gr12CD19+), T (Gr12CD3+), and NK (Gr12CD192CD32NK1.1+CD49b+) cells in PB of recipient mice. (C) Percentage of CD45.2 donor-derived reconstitution of CLP (Lin2CD32CD244+CD27+FLT-3+IL-7R+), pre-NKP (Lin2CD32CD244+CD27+FLT-32IL-7R+CD1222), and rNKP (Lin2CD32CD244+CD27+FLT- 32IL-7R+CD122+) in the BM of recipient mice. (D) Donor (CD45.2)-derived reconstitution of the different NK developmental stages in the spleen of recipient mice. iNK2 = NK1.1+Ly492CD49b2; iNK3 = NK1.1+Ly49+CD49b2; mNK4 = NK1.1+Ly49+CD49b+CD27+CD11b2;mNK5= NK1.1+Ly49+CD49b+CD11b+CD27+CD43+;andmNK6=NK1.1+Ly49+CD49b+CD11b+CD272CD43+.(E) Percentage of CD45.1-derived reconstitution from cotransplanted WT BM cells of myeloid (Gr1+), B (Gr12CD19+), T (Gr12CD3+), and NK (Gr12CD192CD32NK1.1+CD49b+)cellsinPB of recipient mice. (F) Percentage of CD45.1-derived reconstitution from cotransplanted WT BM cells of CLP (Lin2CD32CD244+CD27+FLT-3+IL-7R+), pre-NKP (Lin2CD32CD244+CD27+FLT-32IL-7R+CD1222), and rNKP (Lin2CD32CD244+CD27+FLT-32IL-7R+CD122+) in the BM of recipient mice. (G) CD45.1-derived reconstitution from cotransplanted WT BM cells of the different NK developmental stages in the spleen of recipient mice. iNK2 = NK1.1+Ly492CD49b2; iNK3 = NK1.1+Ly49+CD49b2; mNK4 = NK1.1+Ly49+CD49b+CD27+CD11b2;mNK5= NK1.1+Ly49+CD49b+CD11b+CD27+CD43+; and mNK6 = NK1.1+Ly49+CD49b+CD11b+CD272CD43+. Data represent mean (6SEM) values from 6–14 recipient mice in each group from two independent experiments. Lin (BM) = CD3, CD19, NK1.1, CD49b, Ter119, Gr1, and CD11b. *p # 0.05, **p # 0.005, ***p # 0.001.

Identifying how exactly the loss of Rbp-Jk impacts NK cell Collectively, our data support a critical role of canonical Notch development at the molecular level will require further studies that signaling at multiple stages during NK cell lineage development are beyond the scope of this work. and for NK cell functionality. 12 ROLE OF NOTCH IN NK CELLS

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