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Blood, 15 June 2004, Vol. 103, No. 12, pp. 4573-4580. Prepublished online as a Blood First Edition Paper on February 5, 2004; DOI 10.1182/blood-2003-08-2975. This Article Abstract Full Text (PDF) All Versions of this Article: 2003-08-2975v1 103/12/4573    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Samson, S. I. Articles by Di Santo, J. P. Search for Related Content PubMed PubMed Citation Articles by Samson, S. I. Articles by Di Santo, J. P. Related Collections Immunobiology Apoptosis Cell Adhesion and Motility Gene Expression Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  IMMUNOBIOLOGY Combined deficiency in IB and IB reveals a critical window of NF-B activity in natural killer cell differentiation Sandrine I. Samson, Sylvie Mémet, Christian A. J. Vosshenrich, Francesco Colucci, Odile Richard, Delphine Ndiaye, Alain Israël, and James P. Di Santo From the Unité des Cytokines et Développement Lymphoïde and Unité de Biologie Moléculaire de l'Expression Génique, Institut Pasteur, Paris, France.    Abstract Top Abstract Introduction Materials and methods Results Combined deficiency in... Discussion References   Nuclear factor B (NF-B) transcription factors are key regulators of immune, inflammatory, and acute-phase responses and are also implicated in the control of cell proliferation and apoptosis. While perturbations in NF-B activity impact strongly on B- and T-cell development, little is known about the role for NF-B in natural killer (NK) cell differentiation. Inhibitors of NF-B (IBs) act to restrain NF-B activation. We analyzed the cell-intrinsic effects of deficiencies in 2 IB members (IB and IB) on NK cell differentiation. Neither IB nor IB deficiency had major effects on NK cell generation, while their combined absence led to NF-B hyperactivation, resulting in reduced NK cell numbers, incomplete NK cell maturation, and defective interferon (IFN-) production. Complementary analysis of transgenic mice expressing an NF-B-responsive reporter gene showed increased NF-B activity at the stage of NK cell development corresponding to the partial block observed in IB x IB-deficient mice. These results define a critical window in NK cell development in which NF-B levels may be tightly controlled. (Blood. 2004;103:4573-4580)    Introduction Top Abstract Introduction Materials and methods Results Combined deficiency in... Discussion References   Natural killer (NK) cells are an essential component of the innate and adaptive immune responses. Their major weapons include secreted cytolytic proteins (perforin, granzymes) and proinflammatory cytokines (tumor necrosis factor [TNF-] and interferon [IFN-]). NK cells provide a first line of defense against infections by viral and bacterial pathogens and play important roles in the immunosurveillance of tumors (reviewed in Trinchieri1 and Colucci et al2). NK cells distinguish between healthy and diseased cells via activating and inhibitory receptors that sense the quality and quantity of self-major histocompatibility complex (MHC) class I molecules expressed at the cell surface (reviewed in Ljunggren and Karre3). While the intracellular pathways that control NK cell activation are being unraveled,4 the molecular signatures that drive their development are still unclear. Soluble factors including growth factors (Flt3L and stem cell factor [SCF]) and cytokines like interleukin 15 (IL-15) appear important for NK cell generation from hematopoietic precursors.2 On the other hand, the role for transcription factors (TFs) in orchestrating T-cell, B-cell, and NK cell development is less well defined: deficiencies in several TFs affect all lymphoid lineages (Ikaros, purine rich box-1 [PU.1], E2b transformation specific [Ets-1]), whereas others (such as inhibitors of DNA binding-2 [Id2]) affect NK cell generation.2 The nuclear factor B (NF-B) family of transcription factors is composed of several DNA binding proteins that play pleiotropic roles in the regulation of immunity, cell proliferation, and death (reviewed in Caamano and Hunter5 and Li and Verma6). The NF-B family is characterized by the presence of a Rel homology domain and comprises 5 evolutionary conserved and structurally related members, including c-Rel, RelA (p65), RelB, NF-B1 (p50/p105), and NF-B2 (p52/p100). NF-B/Rel members associate to form homodimers or heterodimers. NF-B dimers are sequestered in an inactive form in the cytoplasm through interactions with inhibitors of NF-B (IB) proteins, which include IB, , , , , and as well as the NF-B1 and NF-B2 precursors.5,6 In response to various stimuli (bacteria, viruses, proinflammatory cytokines, or stress), the activation of IB kinase (IKK) complex (which includes the kinases IKK and IKK and the regulatory subunit IKK/NF-B essential modulator [NEMO]) results in IB phosphorylation, leading to its ubiquitinylation and subsequent degradation. As a result, released NF-B dimers translocate into the nucleus to activate or repress target genes. Since different stimuli can activate distinct or overlapping combinations of NF-B/Rel dimers, the number of potential NF-B-dependent target genes is extensive and includes cytokines, adhesion molecules, and regulators of apoptosis (reviewed in Pahl7). NF-B members are expressed in a cell- and tissue-specific pattern: NF-B1 and RelA are ubiquitous, whereas NF-B2, RelB, and c-Rel are expressed abundantly in lymphocytes. Gene targeting experiments have helped to elucidate the roles of NF-B pathways in T- and B-cell differentiation. Considering the well-characterized antiapoptotic role for NF-B, it is not surprising that deficiencies in NF-B members are associated with defects in B- and T-cell development. For example, mice deficient in NF-B1, NF-B2, and c-Rel develop normally, but all manifest defects in lymphocyte activation.8-11 RelB-deficient mice die after birth from a multiorgan inflammatory disorder that has been traced to defects in dendritic cell development.12 Mice deficient in RelA die in utero due to massive TNF-mediated liver apoptosis.13 Nevertheless, hematopoietic chimeras made using RelA-deficient (RelA°) stem cells have shown that RelA is also required for normal lymphocyte activation.14 Given the structural similarities between different NF-B members (NF-B1 vs NF-B2 and RelA vs c-Rel), some level of functional redundancy may operate during NF-B activation. Indeed, the combined deficiency in multiple NF-B proteins often (NF-B1°NF-B2°, NF-B1°RelA°, NF-B1°RelB°), but not always (NF-B1°c-Rel°), results in a more severe phenotype.6,15,16 Inactivation of the regulatory IKK, IKK, or NEMO subunits also results in a block in NF-B activation; IKK- and NEMO-deficient mice die in utero due to TNF-dependent liver apoptosis (similar to RelA° mice).17,18 Mice deficient in IKK are viable but show severe developmental defects and an absence of mature B cells.19 Lastly, expression of a degradation-resistant form of IB (which cannot be phosphorylated and is thereby incapable of dissociating from NF-B family members) results in global inactivation of the NF-B pathway. When this IB inhibitor is expressed in the T lineage, thymic cellularity is decreased due to enhanced apoptosis of early thymocytes.20 Collectively, the data clearly indicate that inhibition of NF-B activation has severe and detrimental effects on lymphocyte differentiation. Analysis of mice deficient in IB members demonstrates that hyperactivation of NF-B signaling can likewise have a dramatic impact on lymphopoiesis. Mice deficient in IB die about 10 days after birth and appear immunodeficient with extensive inflammatory dermatitis and secondary granulocytosis due to constitutive NF-B activation.21 In contrast, IB-deficient mice exhibit a subtle immunologic phenotype but demonstrate an up-regulation of IB and IB, suggesting that IB members may functionally compensate for one another.22 This hypothesis has been recently confirmed through the generation and analysis of mice deficient in both IB and IB (IB°°23). In contrast to single IB or IB mutants, IB°° mice die at birth, lack mature B and T cells, and their lymphocyte precursors exhibit increased apoptosis due to NF-B hyperactivation.23 Taken together, it appears that a relatively narrow range of NF-B activation must be controlled during B- and T-cell development: either hypoactivation or hyperactivation of NF-B can perturb both B and T lymphopoiesis. Little is known about the roles of different NF-B family members in NK cell development. While the ubiquitous expression of RelA and NF-B1 has been confirmed in human NK cells,24 data relating to expression patterns of other NF-B or IB proteins has not been reported. Hypohidrotic ectodermal dysplasia with immune deficiency patients with point mutations in NEMO have NK cells with reduced cytotoxic potential against certain target cells.25 T and NK cells from mice deficient in IL-1 receptor-associated kinase (IRAK) have a partial impairment in IL-18-induced NF-B activation with a decrease in IFN- production.26 However, NK cell development or function has not been studied in the context of other perturbations in the NF-B signaling pathways. In this report, we characterize steady-state NF-B activity in NK cells and analyze the cell-intrinsic effects of IB and IB deletion on NK cell differentiation.    Materials and methods Top Abstract Introduction Materials and methods Results Combined deficiency in... Discussion References   Mice and generation of hematopoietic chimeras Alymphoid mice deficient in the recombinase-activating gene-2 (Rag°) and the common cytokine receptor chain (°c) on the H-2k background have been described.27 Mice deficient in IB (129Sv x C57Bl/6 x DBA/2, H-2b/d) or IB (C57Bl/6 x 129Sv, H-2b) have been described21,22 and were intercrossed to generate embryos deficient in both IB and IB expressing H-2b and H-2d. Fetal livers (FLs; embryonic day 14.5 or 15.5 [E14.5/15.5]) were genotyped by polymerase chain reaction (PCR) and used to generate hematopoietic chimeras in Rag°°c mice (H-2k) as previously described.23,28 Analysis of endogenous NF-B activity Transgenic mice carrying an NF-B reporter comprising the immunoglobulin (Ig) promoter (containing NF-B binding sites) fused to LacZ gene have been previously described.29,30 The -galactosidase activity (an indicator of NF-B activity) can be monitored using the substrate fluorescein-di- galactopyranoside (FDG; Sigma, St Louis, MO), which upon cleavage generates fluorescein. Cell suspensions (10 x 106/mL) were prepared in prewarmed complete medium (RPMI-1640 with 10% fetal calf serum [FCS], 10-5 M - mercaptoethanol [-ME], 100 µg/mL streptomycin, 100 U/mL penicillin) and an equal volume was mixed with a solution of FDG (2 mM in distilled water) for 1 minute at 37°C. FDG loading was stopped by adding a 10-fold excess of cold medium containing 300 µM chloroquine to inhibit lysosomal degradation of FDG. After 30 minutes of incubation on ice, cells were stained for cell surface markers and analyzed by flow cytometry. Cell culture FL cells (E14.5/15.5) were cultured on irradiated (25 Gy [2500 rad]) OP9 stromal cells in complete medium supplemented with IL-7, SCF, Flt3-ligand, and IL-2 as previously described.28,31 Lymphokine-activated killer cells were generated using unfractionated splenocytes cultured in complete medium containing human IL-2 (huIL-2; 1000 U/mL) or murine IL-15 (muIL-15; 20 ng/mL) as described.28 At various time points, cells were harvested and percentages of viable NK cells (DX5+CD3-CD19-) quantified by fluorescence-activated cell sorter (FACS) analysis. Flow cytometric analysis Single-cell suspensions were prepared from various organs (spleen, bone marrow [BM], and liver) as described,27 and red blood cells were lysed using hypotonic ammonium chloride. Cells were resuspended in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.01% sodium azide. Monoclonal antibodies (Pharmingen, San Diego, CA) were directly coupled to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll A protein (PerCP), PerCP-Cy5.5, allophycocyanin (APC), or biotin (revealed with streptavidin-PerCP-Cy5.5; Pharmingen). Analysis of murine B-cell lymphoma-2 (Bcl-2) was performed using FITC-labeled antibodies (3F11) and isotype controls (Pharmigen) as previously described.32 Stained cells were analyzed using a FACScalibur flow cytometer running Cell Quest 3.3 software (Becton Dickinson, Mountain View, CA). Dead cells were excluded by means of their forward and side scatter profiles and using propidium iodide staining. We analyzed the mitochondrial membrane potential as an early marker of entry into the apoptotic process.33 Cells were incubated with 3,3'-dihexyloxacarbocyanine iodide (DIOC6; Molecular Probes, Leiden, Netherlands; 40 nM in RPMI 1640 with 5% FCS) and analyzed using a FACScalibur flow cytometer. Ex vivo cytokine production Splenocytes or liver cells (5 x 105 cells/200 µL) were cultured in U-bottom microtiter plates in complete medium containing huIL-2 (1000 U/mL) and stimulated with mIL-12 (5 ng/mL; Peprotech, Rocky Hill, NJ) and/or IL-18 (25 ng/mL; R&D Systems, Minneapolis, MN) overnight. Intracellular staining for IFN-, IL-13 (BAF13; R&D Systems), or granulocyte-macrophage colony-stimulating factor (GM-CSF; MP1-22E9; Pharmingen) was performed as described34 except cells were incubated in Brefeldin A (20 ng/mL; Sigma) for the last 4 hours of culture. Stimulation with phorbol myristate acetate (PMA) and ionomycin (4 hours) served as a positive control. Cytokine levels in culture supernatants were quantified by a cytokine-specific sandwich enzyme-linked immunosorbent assay (ELISA) as previously described.35 NK cell lytic activity A standard 51Cr-release assay was used to measure NK lytic activity ex vivo as described.27 YAC-1 cells (mouse thymoma; H-2a) and Chinese hamster ovary (CHO) cells were used as targets and maintained in complete medium. Effector cells were either freshly isolated splenic cells or cells treated overnight with IL-2, IL-12, and IL-18. Equivalent NK cell numbers were used after normalization based on FACS staining. Statistical analysis Data were analyzed with the Microsoft Excel software (Redmond, WA) applying the 2-tailed Student t test. The null hypothesis was rejected and difference assumed significant when P is less than .05.    Results Top Abstract Introduction Materials and methods Results Combined deficiency in... Discussion References   Visualizing NF-B activity in NK cells In order to assess NF-B activity in murine NK cells under steady-state conditions, we analyzed transgenic mice harboring the lacZ reporter gene under the control of the B sites from the light chain gene enhancer (B-lacZ mice22,29). In these mice, NF-B activity is reflected by -galactosidase activity and can be visualized in target cell populations by flow cytometry using the vital fluorescent substrate fluorescein-di--galactopyranoside. As previously shown,22 a large proportion of BM and splenic CD19+ B cells from B-lacZ mice exhibit constitutive NF-B activation (Figure 1A). We further investigated NF-B expression in NK cells from different organs. Most BM NK cells (defined as CD3-NK1.1+IL-2R+) and mature peripheral splenic NK cells showed evidence of NF-B activation (Figure 1A). These results suggest that the NF-B pathway is engaged in freshly isolated NK cells from normal mice. View larger version (40K): [in this window] [in a new window]   Figure 1.. Steady-state NF-B activation in BM and splenic NK cells. (A) NF-B activation was assessed using B-lacZ transgenic mice having the -galactosidase gene driven by a B responsive promoter.29,30 Increasing NF-B activity is correlated with increasing fluorescence. NF-B activity is shown on BM and splenic B (CD19+) and NK (CD3-NK1.1+) cells of B-lacZ mice (bold lines) and nontransgenic littermate controls (shaded histograms). (B) Steady-state NF-B activity in NK cell subsets. Splenic NK cells (CD3-NK1.1+) were electronically gated and analyzed for expression of CD43, CD45R, or CD117. NF-B activity is shown on NK cells gated from B-lacZ mice (bold lines) and nontransgenic littermate controls (shaded histograms) gated for -/lo versus +/hi expression of the indicated markers. Mean fluorescence intensities (MFIs) of cells are indicated (background levels in nontransgenic mice/levels in B-lacZ mice).   We next analyzed NF-B activation in several splenic NK cell subsets in B-lacZ mice. Phenotypic changes occur as NK cells develop and mature in the BM prior to their seeding of the peripheral lymphoid tissues.36 For example, expression levels of the Ly49 family of inhibitory and activating receptors, the sialophorin CD43 and the integrin CD11b (macrophage antigen-1 [Mac-1]), increase on NK cells as they mature. In contrast, the receptor for stem cell factor (c-kit or CD117) is expressed by NK precursors but is largely extinguished on mature NK cells (although a minor NK cell subset remains CD117+).28,36,37 Steady-state NF-B activity was modulated in NK cell subsets depending on their expression of CD43, CD45R, and CD117 (Figure 1B). NK cells (CD3-NK1.1+) with the highest constitutive NF-B activity were CD43lo, CD45R+, and CD117+ (Figure 1B). Thus, NF-B activity is regulated during NK cell differentiation and appears highest in NK cells with an immature phenotype.    Combined deficiency in IB and IB perturbs NK cell development Top Abstract Introduction Materials and methods Results Combined deficiency in... Discussion References   Previous results suggested that normal B and T lymphopoiesis can tolerate only a relatively restricted level of NF-B: hyperactivation or hypoactivation of NF-B appears detrimental to B- and T-cell development.23,38 In contrast, whether modulating NF-B activity will influence NK cell differentiation is not known. Since various NK cell subsets exhibit differential steady-state NF-B activity (Figure 1A-B), we assessed the effects of constitutive activation of NF-B pathways on NK cell differentiation by analyzing NK cells in mice deficient in the NF-B inhibitors IB (IB°) or IB (IB°) or lacking both IB and IB (IB°°). We generated hematopoietic chimeras by reconstituting alymphoid Rag°°c mice27 with fetal liver hematopoietic stem cells (FL-HSCs) from IB° or IB° mutant mice or from IB°° double-mutant mice. This strategy (i) eliminated any confounding effects of abnormal NF-B activation on nonhematopoietic cells and (ii) bypassed the limitations imposed by early death due to the IB mutation. Using this approach, we could assess the cell-intrinsic roles of IB and IB in the development of NK cells from HSCs. As observed in previous work,23 wild-type (WT), IB°, and IB° FL-HSCs could give rise to B cell and T cells after reconstitution of alymphoid mice. IB° and IB° mice were on a mixed genetic background (including 129Sv and DBA/2), and most mice tested did not express the NK1.1 allelic marker (data not shown). We therefore quantified NK cells using a combination of markers including CD3, CD122 (IL-2R), and the pan-NK marker DX5.39 We found equivalent numbers of splenic NK cells (CD3-CD122+DX5+) in WT and IB° chimeras, whereas they were slightly decreased in IB° chimeras (2-fold; Table 1). The cytotoxic potential of NK cells from IB° and IB° chimeras were similar to that of NK cells derived from WT FL-HSCs (data not shown). These results indicate that IB and IB are not individually essential to generate the normal pool of mature lytic NK cells. View this table: [in this window] [in a new window]   Table 1.. Lymphocyte development in the absence of IB and/or IB   In marked contrast, FL-HSCs from IB°° double mutants demonstrated a differential capacity to generate B, T, and NK cells. Splenic B and T cells were essentially absent (300-fold and 50-fold reduced, respectively, compared with controls; Figure 2A; Table 1). In contrast, NK cells (CD3-CD122+DX5+) were only 3- to 4-fold reduced in the BM and liver and 10-fold reduced in the spleen in the IB°° chimeras compared with WT chimeras (Figure 2A-B). These results suggest that developing NK cells, unlike B and T cells,23 are more tolerant of the combined absence of IB and IB. View larger version (17K): [in this window] [in a new window]   Figure 2.. Lymphoid reconstitution in HSC chimeras. FL-derived HSCs were transferred into alymphoid mice (Rag°g°c) to generate hematopoietic chimeras as described in "Materials and methods." (A) Flow cytometric analysis of splenocytes stained for B (CD19+) and T (CD3+) cells. NK cells (CD122+DX5+) were detected in the CD19-CD3- fraction. (B) Absolute numbers of NK cells (CD3-CD122+DX5+) in WT () and IB°° () chimeras. Each circle represents values obtained from a single mouse and a gray bar indicates the mean values. There is a significant decrease in BM, liver, and splenic NK cells (P < .001) in the absence of IB and IB compared with controls.   The reduction in NK cell numbers in IB°° chimeras suggested a cell-intrinsic defect in developing NK cells in the absence of IB and IB. To address this more directly, we used an in vitro culture system whereby NK cells are generated from fetal liver precursors in the presence of cytokines and OP9 stromal cells.28,31 We found that NK cells were present in such cultures derived from either WT or IB°° FL precursors (expressing normal levels of CD2, CD94, and CD122; data not shown), although the absolute numbers of NK cells were reduced by a factor of 5 in the absence of IB and IB (WT, 175 000 ± 95 600 versus IB°°, 35 000 ± 61 500; P = .013). These results demonstrate a cell-intrinsic defect in NK cell generation in IB°° FL precursors. Survival, proliferation, apoptosis of IB°° NK cells Transcriptional targets of NF-B activation include genes controlling cellular homeostasis, ie, survival, proliferation, and apoptosis (reviewed in Pahl7). Curiously, NF-B activation can promote or protect against cell death depending on the cell type and on the pathway of induction. For example, NF-B inhibits apoptosis by up-regulation of molecules including Bcl-xl, Bfl1/A1, cellular inhibitors of apoptosis protein 1 (c-IAP-1), and -2.40-42 However, in thymocytes, NF-B activation may up-regulate expression of Fas/FasL molecules and result in increased apoptosis.43,44 The developmental block in B and T lymphopoiesis in IB°° double-mutant mice results from apoptosis of B- and T-cell precursors as they receive antigen receptor signals.23 In order to understand the mechanism for reduced NK cell numbers in IB°° chimeras, we analyzed different parameters of NK cell survival. We assessed NK cell death by several cytofluorometric assays using WT and IB°° BM and splenic NK cells. There was no difference in the percentage of annexin V-positive NK cells or in the mitochondrial membrane potential between BM or splenic NK cells from the different genotypes (data not shown; Figure 3), suggesting that the IB°° NK cells were not programmed for apoptosis. Cell cycle analysis demonstrated a similar percentage of splenic NK cells in S-G2/M phase (approximately 4%) in WT and IB°° chimeras, making it unlikely that a block in the cell cycle caused reduced NK cell numbers in IB°° chimeras (data not shown). Interestingly, expression of the antiapoptotic molecule Bcl-2 was clearly up-regulated in IB°° NK cells compared with controls (Figure 3). View larger version (20K): [in this window] [in a new window]   Figure 3.. NK cells from IB°° chimeras have normal mitochondrial membrane potential but overexpress Bcl-2. (Left) Assessment of mitochondrial membrane potential using DIOC6 staining of splenic NK cells in WT (shaded histogram) and IB°° (bold line) chimeras. (Right) Bcl-2 expression on splenic NK cells from WT (shaded histograms) and IB°° (bold line) chimeras. Dotted line shows staining with isotype control antibody.   Since the ex vivo analysis of NK cells from IB°° chimeras failed to reveal a major defect in cell survival, we next assessed the proliferative capacity of IB°° NK cells in vitro. Cytokines, including IL-2 or IL-15, can expand murine NK cells in vitro generating lymphokine-activated killer cells with potent cytolytic and cytokine production capacity (reviewed in Colucci et al2). We therefore analyzed the fate of WT and IB°° splenic NK cells at various time points in culture with or without IL-2 or IL-15 (Figure 4). Both WT and IB°° NK cells were rapidly lost in culture in the absence of exogenous cytokines, whereas IL-2 could maintain (at day 1) and expand WT NK cells. In contrast, NK cells from IB°° chimeras survived normally in IL-2 but did not expand (Figure 4). Similar results were obtained with IL-15 (Figure 4). By the end of the culture period (day 6), WT cells had expanded 5-fold, while absolute numbers of IB°° NK cells were slightly reduced (1.5-fold). Thus IB°° NK cells manifest a cell-intrinsic defect in their capacity to respond to homeostatic signals that are required to maintain normal steady-state NK cell levels.45 View larger version (15K): [in this window] [in a new window]   Figure 4.. IL-2 or IL-15 maintain but do not expand NK cells from IB°° chimeras. Splenocytes (2 x 105) from WT (Rag° mice, triangles) or IB°° chimeras (circles) were cultured in complete medium with or without IL-2 or IL-15 and percentages of NK evaluated daily. NK cells were rapidly lost in cultures lacking exogenous cytokines (open symbols). IL-2 (or IL-15, ) maintained but did not expand IB°° NK cells (absolute NK cell number was 27 000 on day 5). WT NK cells proliferated vigorously over the same time period (NK cell number was 8 x 105 at day 5).   Phenotype of NK cells in IB°° chimeras We next analyzed levels of expression of molecules known to be modulated following NF-B activation. Classical NF-B target genes, including MHC class I molecules and the CD69 activation marker, were clearly overexpressed on NK cells from IB°° chimeras compared with WT controls (Figure 5; Table 2), consistent with NF-B hyperactivation in the absence of IB and IB. View larger version (21K): [in this window] [in a new window]   Figure 5.. NK cells from IB°° chimeras express increased levels of MHC class I and CD69 molecules. Expression of H-2b,d and CD69 on splenic CD3-CD122+DX5+ NK cells from WT (shaded histograms) and IB°° (bold lines) chimeras.   View this table: [in this window] [in a new window]   Table 2.. Mean fluorescence intensity of surface markers on NK cells from IB°° chimeras   We further characterized the phenotype of the NK cells that developed in the absence of IB and IB. Previous studies have demonstrated that immature BM NK cells differ in the expression of several surface markers (CD11b, CD43, CD45R, CD117, and Ly49 family members) compared with their splenic counterparts.36 For example, BM NK cells are CD11bloCD43lo, while splenic NK cells are mostly CD11bhiCD43hi. In the absence of IB and IB, splenic NK cells expressed low levels of CD11b and CD43 and elevated levels of CD117 (Figure 6A-B). CD45R expression was also higher and more uniform in intensity. In this way, splenic IB°° NK cells more closely resembled immature BM NK cells.36 Nevertheless, expression of inhibitory (Ly49G2) and activating (Ly49D) members of the Ly49 receptor family, and that of CD2, was normal (Figure 6A-B). Collectively, the phenotypic analysis suggests that the maturation of NK cells in the absence of IB and IB may not be complete. View larger version (34K): [in this window] [in a new window]   Figure 6.. Splenic NK cells from IB°° chimeras have an abnormal phenotype. (A) Expression of CD2, CD11b, CD43, and CD45R on splenic CD3-CD122+DX5+ NK cells from WT (shaded histograms) and IB°° (bold line) chimeras. (B) Expression of CD117, Ly49D, and Ly49G2 on splenic CD3-CD122+DX5+ NK cells from and IB°° chimeras. Percentages of positive cells are indicated.   IB°° NK cells demonstrate normal cytotoxic activity We next determined whether the abnormal phenotype in NK cells from IB°° chimeras was associated with defective NK cell effector functions. One of the best-characterized NK cell activities is natural cytotoxicity: the capacity to lyse target cells without prior sensitization (reviewed in Trinchieri1). NK cells use a variety of receptors to monitor the level of MHC expression on target cells and to detect stress- or infection-induced molecules.3 We found that the prototypical NK cell target YAC-1 was lysed with the same efficiency by WT and IB°° splenic NK cells (Figure 7A). Moreover, IB°° NK cells were cytolytic against CHO targets (Figure 7B), consistent with expression of a functional Ly49D receptor.46 We also cultured splenocytes from IB°° chimeras overnight in the combination of IL-2, IL-12, and IL-18; this cytokine cocktail has been shown to stimulate NK cell IFN- production and cytolytic potential.47 We found that IL-12/IL-18 stimulation of both WT and IB°° NK cells resulted in enhanced lysis of YAC-1 targets compared with their fresh counterparts (Figure 7C), suggesting that IB°° NK cells express functional receptors for IL-2, IL-12, and IL-18. The collective results suggest that despite an apparent immature phenotype, IB°° NK cells had clearly attained cytotoxic potential. View larger version (13K): [in this window] [in a new window]   Figure 7.. Natural cytotoxicity in the absence of IB°°. (A) Freshly isolated splenocytes from WT () and IB°° () chimeras showed similar lytic activity against Cr51-labeled YAC-1 target cells. NK cell-target cell ratios were normalized using flow cytometric analysis. One representative experiment of 2 is shown. (B) Freshly isolated splenocytes from WT () and IB°° () chimeras showed similar lytic activity against CHO target cells. (C) WT (black symbols) and IB°° (gray symbols) splenocytes were stimulated for 12 hours with IL-2 alone (circles) or cytokine combination (IL-2+IL-12+IL-18) (triangles) prior to analysis of lytic activity against YAC-1 target cells. Error bars represent SEM.   IB°° NK cells have a selective defect in cytokine production NK cells are potent and prompt producers of multiple cytokines, most notably IFN-.1 Previous reports identified a role for NF-B in modulating CD4+ T-cell differentiation and cytokine production. NF-B members (including RelB) stimulate T-helper 1 (Th1) responses (IFN- production) by directly increasing promoter activity.48,49 In contrast, NF-B1 appears necessary for efficient Th2 responses.50 We therefore analyzed whether NF-B hyperactivation modified NK cell cytokine production capacity. Freshly isolated splenic NK cells were cultured in the presence of IL-2 and stimulated with a combination of IL-12 plus IL-18. This protocol results in a synergistic secretion of IFN- from control NK cells (Figure 8A). Surprisingly, splenic NK cells from IB°° chimeras were clearly defective in IFN- production after either IL-12 or combined IL-12/IL-18 stimulation; levels of secreted IFN- were 7-fold reduced compared with controls (Figure 8A). The defective IFN- production from IB°° NK cells was confirmed by intracellular cytokine staining: IB°° splenic NK cells produced quantitatively (3-fold percentage) and qualitatively (2-fold mean fluorescence intensity) less IFN- compared with control NK cells (Figure 8B). Since IB°° NK cells could survive in the presence of IL-2 (Figure 4) and respond to IL-12/IL-18 stimulation with increased cytotoxicity (Figure 7A), the abnormal IFN- responses in mutant NK cells are not due to defective cytokine signaling through these receptors. View larger version (21K): [in this window] [in a new window]   Figure 8.. IB°° NK cells have a defect in IFN- production. (A) Splenic NK cells (104 cells) from Rag° mice or IB°° chimeras were stimulated for 24 hours with the indicated combinations of IL-12 and/or IL-18 and IFN- release measured by ELISA. Error bars represent SEM. (B) Splenocytes from WT or IB°° chimeras were stimulated for 7 hours with a combination of IL-12 and IL-18 (for IFN-) or with PMA and calcium ionophore (for IL-13, GM-CSF) and were analyzed by intracellular staining. Analysis was performed on electronically gated CD3-DX5+ cells. Percentages of positive cells are shown in each panel. One representative experiment of 3 to 6 is shown.   To assess whether IB°° NK cells had a global inability to produce cytokines, these cells were stimulated with the pharmacologic activators PMA plus calcium ionophore. Previous studies have shown that this type of stimulation (albeit nonphysiologic) can reveal the capacity of human and mouse NK cells to produce IL-5, IL-13, or GM-CSF (reviewed in Colucci et al2). We were unable to detect IL-5 or IL-13 production from WT or IB°° splenic NK cells after stimulation with PMA/ionomycin (Figure 8B; data not shown). In contrast, both WT and IB°° NK cells were capable of GM-CSF production (Figure 8B).    Discussion Top Abstract Introduction Materials and methods Results Combined deficiency in... Discussion References   NF-B pathways are pleiotropic in nature, impacting on cell survival, apoptosis, and effector functions. Moreover, since NF-B signals are ubiquitous, identifying roles for NF-B members in cellular differentiation remains a challenge. In this report, we have begun to assess the roles for NF-B in NK cell development and function. We observed that a substantial proportion of NK cells show constitutive NF-B activation and that hyperactivation of NF-B secondary to the combined absence of IB and IB results in (1) reduced NK cell numbers and (2) defective NK cell maturation, affecting IFN- secretion but not lytic capacity. These results offer new insights into the role of NF-B pathways in controlling lymphocyte differentiation and define a crucial stage in early NK maturation where cells appear sensitive to overall NF-B activity. Since all cells use the NF-B pathway, we used a hematopoietic reconstitution system whereby the effects on NF-B signaling would be confined to cells of the lymphoid lineage.6 This allowed us to evaluate the cell-intrinsic roles of NF-B in NK cells. This approach was necessary since previous studies had shown that NF-B pathways are implicated in relaying the proper cellular and soluble signals derived from the nonhematopoietic (stromal cell) microenvironment to developing lymphocytes. For example, NF-B pathways are involved in the regulation of interferon regulatory factor 1 (IRF-1)-controlled IL-15 expression and in the signaling through lymphotoxin- receptors; both are key elements in NK cell development.51-54 By exploiting hematopoietic chimeras generated in alymphoid Rag°°c mice, we found that NK cells, in contrast to B and T cells, could develop in the absence of IB and IB. Nevertheless, overall NK cell numbers were reduced in IB°° chimeras; this phenomenon was recapitulated using an in vitro system of NK cell generation from FL precursors. Thus NF-B hyperactivation has differential effects on B/T versus NK development; the former cells are exquisitely sensitive, while the latter are more tolerant. We observed increased Bcl-2 expression in IB°° NK cells but not in IB°° B or T cells (S.I.S. and J.P.D., unpublished observations, January 2004). Bcl-2 is a target of NF-B transcriptional activation55,56; the increased Bcl-2 expression in the absence of IB and IB may reflect a compensatory mechanism that helps to maintain these IB°° NK cells in face of NF-B hyperactivation. In contrast, NEMO mutant NK cells develop to normal levels in human blood,25 suggesting that hypoactivation versus hyperactivation of NF-B differentially affect NK cell development. NK cell numbers were decreased in all tested organs (BM, spleen, liver) of IB°° chimeras, but the degree of reduction varied according to the site (BM and liver less than spleen). Interestingly, these sites have been shown to harbor varying ratios of immature to mature NK cells (using low versus high expression of CD11b and CD43 as markers of immaturity and maturity, respectively36). Taking this into account, it is striking that the reduction in NK cell number in IB°° chimeras is most pronounced in tissues (such as the spleen) having the highest proportion of CD11bhiCD43hi NK cells. In fact, the residual NK cells in the BM, spleen, and liver of mutant mice are similar in absolute terms to the number of immature (CD11bloCD43lo) NK cells found in control chimeras. These observations are consistent with the hypothesis that NK cell maturation (in terms of CD11b and CD43 expression) is partially blocked upon constitutive NF-B activation. The inability to establish normal peripheral NK cell numbers in the absence of IB and IB suggests a defect in cellular homeostasis of this lymphoid subset. We did not observe any increased susceptibility of mature NK cells to apoptosis when assessed ex vivo. In contrast, IB°° NK cells did not proliferate in vitro in response to high doses of IL-2, which can robustly expand WT NK cells. Although IB°° NK cells express normal levels of IL-2R and c (data not shown), we found that the IL-2R/c complex did not provide adequate proliferative signals to IB°° NK cells, although IL-2 or IL-15 could maintain their survival in culture. This defect in response to homeostatic signals could explain the failure of IB°° NK cells to achieve normal cell numbers in vivo. A partial defect in IL-2/IL-15 signaling would be predicted to have detrimental effects on NK cell development.57 An alternative mechanism to account for the defective IL-2 responses in IB°° NK cells could involve induction of apoptotic mechanisms. It is known that IL-2 can stimulate NF-B activation.25 Since NF-B activity is already elevated in IB°° NK cells, additional NF-B stimulation might not be well tolerated and could induce cell death after long-term culture. The IL-2 response of NK cells in the context of NF-B hyperactivation (as in IB x IB deficiency) contrasts sharply with that of NF-B hypoactivation, as in the case of NEMO mutations, in which treatment with IL-2 normally expands NK cells and allows for a partial recovery of their functional defects.25 Several reports have demonstrated that an inability to normally activate NF-B pathways results in abnormal development of NK cell effector functions. Patients with mutations in NEMO develop normal numbers of NK cells, however, ex vivo natural cytotoxicity is reduced. This defect can be corrected in vitro following expansion in IL-2, which can activate NF-B pathways.25 Whether NEMO mutant NK cells have other defects in cytokine production is not known. Tato and colleagues48 recently observed that NK cell cytotoxicity and IFN- production were defective in transgenic mice expressing a dominant-negative mutant form of IB. These effects were also apparent under physiologic conditions in the context of Toxoplasma gondii infection. Finally, defects in IRAK affect IL-18-induced NK cell cytotoxicity and cytokine production in vitro as well as during the course of an infection with Propionibacterium acnes.26 The collective results indicate that adequate NF-B activation is essential for the acquisition of NK cell effector functions. In this work, we found that NF-B hyperactivation results in selective defects in NK cell effector functions. Residual IB°° NK cells develop a normal cytolytic program but have reduced capacity for IFN- production. Since NK cell cytotoxicity does not require new gene transcription, this effector function may be less sensitive to NF-B levels. Although the molecular mechanism for this selective defect remains unclear, we can speculate that an excess of NF-B might activate a repressor of IFN- transcription or diminish transcription factors implicated in IFN- synthesis, such as T-box expressed in T cells (T-bet) or H2.0-like homeo box (Hlx) (reviewed in Szabo et al58). Alternatively, IB°° NK cells may manifest an imbalance between different NF-B members. For example, an excess of NF-B1 relative to RelB would be predicted to cause a defect in IFN- production (the former inhibits IFN- transcription in the contrast to the latter). In any event, NK cell IFN- expression appears tightly regulated by overall NF-B levels, since both hypoactivation and hyperactivation have the same negative effect. In contrast, NK cell production of GM-CSF was not affected by the combined absence of IB and IB, ruling out a generalized defect in cytokine production by these cells. Our results support the notion that NF-B levels may be critically regulated during NK cell maturation. Uncontrolled NF-B activation during lymphopoiesis (as in the case in IB°° chimeras) results in the development of NK cells with an immature phenotype, expressing low levels of CD11b and CD43 but high levels of CD117 and CD45R. As this phenotype has been previously described for BM NK cells,36 we further analyzed NF-B activity in various NK cell subsets using B-lacZ mice. These results identified elevated NF-B activity in NK cells expressing low levels of CD43 and high levels of CD45R and CD117. These observations are consistent with a critical stage in early NK cell development defined by NK cells bearing the CD43loCD45hiCD117hi phenotype. We would propose that NF-B levels during this developmental window would need to be maintained within a tolerable range. In this model, developing NK cells would normally receive signals that up-regulate NF-B levels during their differentiation. In the context of excessive NF-B activation (IB°°), a threshold may be passed with detrimental consequences including arrest of maturation and/or induction of apoptosis. Immature NK cells would therefore need to fine-tune NF-B activity in order to traverse this critical checkpoint.    Acknowledgements   We would like to thank Erwan Corcuff and Thomas Ranson for help in the realization of this work.    Footnotes   Submitted August 29, 2003; accepted January 25, 2004. Prepublished online as Blood First Edition Paper, February 5, 2004; DOI 10.1182/blood-2003-08-2975. Supported by grants from the Institut Pasteur, Institut National de la Santé et la Recherche Medicale (Inserm), the Ligue National Contre le Cancer (LNCC), and the Association pour le Recherche contre le Cancer (ARC). S.I.S. was supported by fellowships from the MENRT, the LNCC, and the ARC. C.A.J.V. was supported by a Poste Vert fellowship from Inserm. 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