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2 and Phospholipase C
1 in the Terminal Maturation of NK Cells1



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* Blood Research Institute, Milwaukee, WI 53226;
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing, Peoples Republic of China;
Dali University, Dali, Yunnan, Peoples Republic of China;
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226; and
¶ Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226
| Abstract |
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(PLC
)2, one of the key signal transducers, there are profound effects on the development and terminal maturation of NK cells. Lack of PLC
2 significantly impaired the ability of lineage-committed NK precursor cells to acquire subset-specific Ly49 receptors and thereby terminal maturation of NK cells. Overexpression of isozyme, PLC
1, in PLC
2-deficient NK cells resulted in the successful Ly49 acquisition and terminal maturation of the NK cells; however, it could only partially rescue NKG2D-mediated cytotoxicity with no cytokine production. Furthermore, PLC
2-deficient NK cells failed to mediate antitumor cytotoxicity and inflammatory cytokine production, displaying a generalized hyporesponsiveness. Our results strongly demonstrate that PLC
1 and PLC
2 play nonredundant and obligatory roles in NK cell ontogeny and in its effector functions. | Introduction |
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One of the receptors that activate NK cells to mediate cytotoxicity and cytokine secretion is the C-type lectin, NKG2D, which recognizes different stress-induced nonclassical MHC class I molecules such as MICA, MICB, and UL-16-binding proteins in humans (7) and minor histocompatibility Ag 60 (H60)3 (8), Rae-1 (9), and Mult-1 (10) proteins in mice. NKG2D noncovalently associates with DAP10 or DAP12, which are phosphorylated by membrane-associated Src family protein tyrosine kinases (PTKs). Phosphorylations of tyrosines in the ITAM of the adaptor proteins generate the docking sites for the intracellular Syk family PTKs. Activated PTKs in turn phosphorylate multiple signaling molecules that initiate gene transcription and lytic granule release, leading to cytotoxicity and cytokine release (11, 12). Although these intracellular signaling events seem to be highly complex, distinct pathways associated with different NK cell effector functions are emerging. Recent studies demonstrated that the signaling events processed through NKG2D/DAP10 are distinct from that of NKG2D/DAP12 (12). Both the human and murine NK cells have been shown to mediate cytotoxicity in the absence of DAP12 or the associated PTKs, Syk and Zap70 (11, 12). However, apart from being able to mediate cytotoxicity, the NKG2D/DAP12-Syk pathway was exclusively responsible for cytokine secretion (12). Thus, NKG2D/DAP10 complex, which recruits PI3K through a YINM motif, was capable of triggering only cytotoxicity and not cytokine secretion. Although triggering through NKG2D/DAP10 and NKG2D/DAP12 can result in different effector functions, both the activation pathways recruit phospholipase C
(PLC
) as their major signal transducer (13). PLC
play a vital role in the generation of secondary messenger molecules such as 1,2-diacylglycerol (DAG) and inositol phosphates through the hydrolysis of phosphatidylinositols (14). The DAG regulates the protein kinase (PKC) family members, and inositol 1,4,5-triphosphate (IP3) mediates calcium (Ca2+) mobilization. The PLC
family of enzymes has two isoforms, PLC
1 and PLC
2, which are 50% identical at the amino acid level; whereas the expression of PLC
1 is ubiquitous, PLC
2 is limited to hemopoietic tissues (15).
Despite our comprehensive understanding of receptor expression and effector functions of NK cells, the clear definition of developmental stages and the signaling events responsible are significantly lacking. Existence of common lymphoid progenitors (CLPs) that give rise to T, B, and NK cells in the bone marrow (BM) have been defined (16). Also, committed NK precursors (NKP) with specific cell surface markers have been identified in the fetal thymus and in adult BM, which exclusively mature into NK cells (17). This earliest developmental stage of NKP is defined by the expression of IL-2/IL-15 receptor common
-chain (CD122) (18). Expression of CD122 in NKPs and their dependence on a common
-chain and IL-15 generated from radio-resistant BM-derived stromal cells, demonstrate the vital role of IL-15-mediated activation during the development of committed NKPs (19). In the second stage, NKP cells acquire NK1.1, NKG2D, and the CD94/NKG2A/C/E in a sequential developmental process (20). The start of the expression of integrins such as CD49b, a pan NK cell marker that is identified as the integrin
2 subunit associated with
1 and the
v (CD51) are also part of this second stage of NK cell development (17, 20). Terminal maturation of NK cells is defined by the down-regulation of CD51, active proliferation, and a concomitant increase in the expression levels of both Mac 1 (CD11b) and CD43 receptors (21). At this stage, the immature NKP cells also start acquiring different Ly49 receptors, the expression of which defines a specific repertoire of NK cell subsets (17). The critical role of other factors such as IL-7, surface lymphotoxin (LT), and LT receptors in NK cell development is still being debated (22). Engagement of inhibitory Ly49 receptors with their respective MHC class I ligands on the target cells led to the activation of a negative signaling cascade with recruitment of protein tyrosine and lipid (SHIP) phosphatases to the ITIM in the cytoplasmic domains of Ly49 receptors (23).
Although these studies have provided tremendous insights regarding the receptors, adapter molecules, and early signaling events of NK cell activation, the signal transducers that translate the activation stimuli into effector functions have not been clearly understood. PLC
2 has a determinant role in the terminal maturation, differentiation, and function of B cells as a critical signal transducer (24). This is in contrast to T cells, where PLC
1 is the major signal transducer and lack of PLC
2 did not affect the normal development and functions of T cells (24). Although mature NK cells express detectable levels of PLC
1 (25) and abundant quantities of PLC
2 (13), the biochemical, functional, and nonredundant roles of these signal transducers in NK cell development and functions have not been determined. Recently, Caraux et al. (26) and Tassi et al. (27) have described the role of PLC
2 in NK cell functions using PLC
2 knockout animals; however, the relative contribution of PLC
1 and PLC
2 in NK cell development, maturation, and functions is still not understood.
In this study, using enzyme-deficient mice, we specifically investigated the role of PLC
2 in the development and function of NK cells. Our results show that although PLC
2 is a predominantly expressed signal transducer in NK cells, it is not an obligate signaling molecule during the early commitment phase of NK cells. Specifically, both the initiation of CD122 expression and the commitment of CD122+CD3 NKPs into NK1.1+NKG2D+ cells are unhindered in the absence of PLC
2. However, in PLC
2/ mice, peripheral and BM-derived NK1.1+NKG2D+ numbers were greatly increased, which appears to remain halted at a preterminal maturation stage. We further show PLC
2 plays a critical role in the terminal maturation of NK1.1+NKG2D+ NK cells by promoting the acquisition of subset-specifying Ly49 receptors and that the presence of PLC
2 in NK cells is imperative for executing effector functions such as cytotoxicity and cytokine or chemokine productions. Finally, retroviral transfection of PLC
1 into PLC
2-deficient BM cells promoted a partial maturation of NK cells that resulted in the successful acquisition of Ly49 receptors. However, overexpression of PLC
1 into PLC
2-deficient NK cells could rescue only a limited level of cytotoxicity and PLC
1-rescued NK cells completely failed to generate inflammatory cytokines. These results strongly indicate that the PLC
isoforms perform nonredundant and obligatory functions during the development and activation of NK cells.
| Materials and Methods |
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Mice deficient for the PLC
2 and JAK3 genes have been described through our earlier studies (24, 28). All mice used in this study were maintained in pathogen-free conditions at the Biological Resource Center at the Medical College of Wisconsin (Milwaukee, WI) and were used between 5 and 12 wk of age. All animal study protocols listed here are approved by the Biological Resource Center; WI.EL4, EL4 stably transfected with H60 (EL4H60) RMA/S, and YAC-1 were described earlier (29); and all cells were grown at 37°C, 5% CO2, in RPMI 1640 (Invitrogen Life Technologies) with L-glutamine, sodium pyruvate, 2-ME, antibiotics (penicillin/streptomycin), and 10% FCS.
NK cell preparation and cytotoxicity assays
NK cells from indicated mice strains were prepared from splenocytes using established methods (30). Briefly, single-cell suspensions from spleen were passed through nylon wool columns (Polysciences) for the depletion of adherent populations consisting of B cell and macrophages. Cells eluted from these columns were cultured with 1000 U of IL-2 (a gift from the National Cancer Institute-Biological Resources Branch, Preclinical Repository, Bethesda, MD). The purity of the NK cultures was checked, and preparations with >95% of NK1.1+ were used for the experiments. NK-mediated cytotoxicity was quantified using 51Cr-labeled target cells according to established protocols (31).
Flow cytometry and cell sorting
Hybridoma secreting anti-NK1.1 (PK136) was obtained from the American Type Culture Collection. anti-Ly49A (YE1/48), anti-Ly49C/I (SW5E6), anti-Ly49D (4E4), anti-Ly49G2 (4D11), anti-CD11b (M1/70), anti-CD43 (S7), anti-CD49b (DX5), anti-CD51 (H9.2B8), anti-CD69 (H1.2F3), anti-CD122, anti-NKG2D (A10), and anti-NKG2A/C/E (20d5) were obtained from either eBiosciences or BD Pharmingen. NK cells are stained with appropriate mAbs and subjected to cell sorting using FACSAria (BD Biosciences). FITC-conjugated rat anti-mouse Ig (eBiosciences) and PE-conjugated goat anti-human Fc-specific IgG (Jackson ImmunoResearch Laboratories) were used as secondary Abs. To avoid nonspecific binding through FcRs, NK cells were pretreated with Fc-Block (2.4G2, anti-CD16/CD32) mAb (BD Pharmingen), before adding specific mAb. Standard flow cytometry analysis was conducted in LSR-II using FACSDiva software (BD Biosciences).
Secretion and quantification of cytokines through ELISA and flow cytometry
IL-2-cultured, Fc-blocked NK cells were activated with titrated concentrations of plate-bound anti-NKG2D mAb, A10 (eBiosciences), or anti-NK1.1 mAb, PK136. Total concentrations of IFN-
and GM-CSF were quantified in the supernatants of NK cells using ELISA kits from eBiosciences, following the manufacturers instructions. Standard curves generated using recombinant murine IFN-
and GM-CSF were used to calculate the concentrations. MIP-1
, MIP-1ß, and RANTES were quantified using Bioplex kit from BioRad following the manufacturers suggested protocol. Intracellular cytokine were quantified using established methodologies. Briefly, NK cells were activated with 5 µg/ml plate-bound anti-NKG2D (A10) or anti-NK1.1 (PK136) mAbs for 16 h. Activated NK cells were Fc-blocked, stained for NK1.1, fixed, permeabilized and quantified for intracellular IFN-
using FITC-conjugated anti-IFN-
mAb through flow cytometry. Standard flow cytometry analysis was conducted in LSR-II using FACSDiva software (BD Biosciences).
Immunoprecipitations and Western blotting
NK cells were washed and lysed with buffer containing 10 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 1 mM PMSF, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% Triton X-100. Soluble proteins were resolved using 8% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with antisera specific for PLC
1 or PLC
2 (Santa Cruz Biotechnology). Western blotting for phosphorylated PLC
1 or PLC
2 proteins was performed as previously described (32). In brief, 10 x 106 purified NK cells were stimulated with soluble anti-NKG2D (20 µg/ml) followed by cross-linking with 10 µg of the appropriate secondary IgG Ab (Jackson ImmunoResearch Laboratories) per ml. After incubation at 37°C for 5 min, the cells were washed in PBS containing 10 mM Na3VO4, lysed in ice-cold lysis buffer, and immunoprecipitated with either anti-PLC
1 or anti-PLC
2 Ab (32). The precipitates were subjected to 8% SDS-PAGE and transferred to nitrocellulose membranes. Filters were incubated with the indicated Abs. The protein-Ab complexes were detected using the ECL detection system.
Retroviral transduction and BM transplantation
Retroviral transductions and BM transplantation were performed as described previously (33). Briefly, the rat PLC
1 or rat PLC
2 gene was cloned into a bicistronic retrovirus murine stem cell virus promoter-internal ribosome entry site (IRES)-GFP vector. The expression of the cloned gene and GFP is under the control of murine stem cell virus promoter, and the GFP was used as a marker for the identification of transduced cells. Conditioned media containing high-titer, amphotropic retrovirus particles were derived by cotransfection of 293T cells with the retrovirus vector expressing the cloned gene and GFP and a helper plasmid pEQPAM3 that contains the required gag, pol, and env retroviral genes driven by a Moloney leukemia virus long terminal repeat. This medium was filtered and used to transduce ecotropic packaging cells (GP + E86) with 6 µg/ml polybrene (Sigma-Aldrich) for a total of six times over 3 days. Cells exhibiting the highest levels of GFP expression were sorted under sterile conditions and subsequently expanded as virus-producing cells. Murine BM cells were transduced by retrovirus as follows: PLC
2-deficient mice (812 wk old) were injected i.p. with 150 mg/kg 5-fluorouracil 48 h before BM harvest. BM was isolated from both hind limbs and prestimulated with 20 ng/ml murine IL-3, 50 ng/ml human IL-6, and 50 ng/ml rat stem cell factor for 48 h. Cells were then cocultured on irradiated ecotropic producer cells (GP+E86) in the presence of IL-3, IL-6, stem cell factor, and polybrene (6 µg/ml). After 48 h, 25 x 106 nucleated BM cells were injected i.p. into sublethally irradiated JAK3-deficient recipient mice (300 rad); 4 wk later, the development of NK cells was analyzed through FACS and functional assays.
| Results |
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2/ affects the development of NK cells
To determine the role of PLC
2 on NK cell development, we first analyzed various tissues from wild-type (WT) littermate controls and PLC
2/ mice for cells expressing early NK cell marker, NK1.1 through flow cytometry. Fig. 1A demonstrates the status of NK cell numbers in spleen, BM, thymus, and liver, where there was a considerable increase in the percentages of the NK1.1+CD3 population (NK cells) exclusively in PLC
2/ mice compared with their WT controls. Thymi from these mice lacked sufficient number of NK1.1+CD3 cells for analysis, although there was a substantial decrease in the NK1.1+CD3+ NKT cells in the case of PLC
2/ mice compared with the WT littermates (Fig. 1A). The increases in the percentages were not due to a systemic effect on all lymphocyte populations, given that there were no statistical differences in the number of NK1.1CD3+ T cell populations (data not shown). Furthermore, in contrast to NK or T cells, the number of NK1.1+CD3+ NKT cells population drastically decreased in spleen, thymus, and liver (Fig. 1A). Collectively, these results indicate a consistent increase in the population of NK1.1+CD3 NK cells in PLC
2/ mice. The significant increases in the NK1.1+CD3 cells were also reflected in the absolute numbers in the spleen (Fig. 1B), BM, and liver (data not shown). On the basis of these observations, we conclude that the absolute numbers NK1.1+CD3 cells in PLC
2/ mice were higher (at least
2 fold) compared with that of WT controls. To further validate our observations with PLC
2/ animals, we also used another pan NK cell marker, CD49b, and obtained similar results indicating a specific increase in the number of NK cells (data not shown). Although for reasons presently not known, the concomitant decrease in the number of CD3+NK1.1+ cells independently validates our hypotheses that the PLC
2 plays selective and cell type-specific functions at different of their developmental stages.
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2 in the development and the maturation of NK cells, NK1.1+CD3 NK populations from the splenocytes were analyzed (Fig. 1C) with Abs against phenotypic markers such as CD122, NKG2D, NKG2A/C/E, CD51, CD49b, CD11b, CD43, and CD69 that are defined to reflect different developmental or functional stages of NK cells (17, 34). The single CD3+ (T cells) or the double NK1.1+CD3+ (NKT)+ cells were excluded from these analyses. We analyzed expression of CD122 (IL-2/IL-15 receptor common
-chain), which marks the earliest step in the developmental process of NK cells. Both IL-2 and IL-15 have been shown to play vital roles in the early stages of development, maturation, and homeostasis of lymphocytes including NK cells (35). Our results demonstrate that there was no difference in the expression levels of CD122 between the WT and PLC
2/-derived NK1.1+CD3 NK cells. NKG2D and NKG2A/C/E are acquired by the CD122+NK1.1+CD3 cell during early phase of differentiation (20). Our results also demonstrate that the expression of both activating NKG2D and inhibitory NKG2A/C/E receptors (Fig. 1C) were comparatively normal in the case of PLC
2-deficient NK cells. Thus, our results strongly argue that the commitment of CLPs into immature NKPs is unaffected in the PLC
2/ mice.
Other critical developmental markers such as CD51, CD49b, CD11b (integrins), and CD43 (leukosialin) have been shown to be sequentially acquired by the developing NK cells (20). Expression of CD49b is initiated in the committed CD122+NK1.1+ NKPs (36). However, levels of CD49b are known to be decreased in the activated NK cells, including lymphokine-activated killer cells. In the case of PLC
2/-derived fresh NK cells, the levels of CD49b were largely unaltered (Fig. 1C). In contrast, PLC
2-deficient NK cells have decreased levels of CD43 that are reported to be up-regulated during NK cell development (37). Levels of other developmental markers such as
v (CD51) are reported to be expressed at a higher level in the committed NKP but down-regulated upon NK cell maturation (20). In PLC
2-deficient NK cells, the levels of CD51 and the activation stage marker CD69 were unchanged (Fig. 1C). These results demonstrate an increase in NK1.1+CD3 NKPs and a partial halt in the NK cell developmental process in PLC
2/ mice.
Terminal maturation of PLC
2/ NK cells is impaired
Next, to assess the role of PLC
2 in the differentiation of NKPs into mature NK subsets, we studied the expression patterns of subset-defining Ly49 receptors (38, 39). Recent studies have also indicated that the acquisition of different Ly49 receptors by the respective NK subsets to be the final stage of NK cell maturation process (17, 20). Thus, the maturity of the NK cells can be determined through their expression levels of Ly49 receptors (40). Results presented in Fig. 1D show a considerable decrease in the expression levels of Ly49 receptors in fresh NK1.1+CD3 NK cells. Among Ly49 receptors, expression levels of Ly49A and Ly49G were most affected in PLC
2/ relative to WT NK cells (Fig. 1D). Thus, PLC
2 plays a vital role in the terminal maturation of the NK cells. Together, our results strongly demonstrate that: 1) the commitment of CLPs to become CD122+ and their expression of NKG2D, NKG2A/C/E and NK1.1 do not depend on PLC
2; 2) however, the terminal maturation in terms of acquiring Ly49 receptors is entirely dependent on the presence and functions of PLC
2.
An increase in the CD122+ immature NKPs is responsible for a higher NK cell number in PLC
2/ mice
Next we analyzed the number of CD122+CD3NK1.1CD49b immature NKPs in the BM of PLC
2/ animals. Immature NKPs are the earliest known NKPs to become NK cell lineage in the BM and are defined by the expression of CD122 (IL-2 and IL-15 receptor
-chain) and by the absence of mature NK cell markers such as NK1.1, CD49b, and Ly49 receptors (19). Defects in the NK cell development in the BM of PLC
2/ mice were evident for the following reasons. First, there was a consistent increase (
2-fold) in the population of CD122+CD3NK1.1CD49b immature NKPs in the BM of PLC
2/ relative to WT mice (Fig. 2, AC). Interestingly, the second stage CD122+NK1.1+CD49b cell population, which is an indicator of immature NK phenotype, was increased considerably (Fig. 2B). Alternatively, the expression of NKG2A/C/E is least affected in the CD122+NK1.1+CD3 population (Fig. 2D). Because the positivity for CD49b marks a third stage of NK cell maturation, we further analyzed the CD122+NKG2D+CD49b+ population (Fig. 2E). Consistent with our observations, there was an increase in the immature NKPs. Also there was an increase in the CD122+NKG2D+CD49b NK cells in the case of PLC
2-deficient mice compared with that of WT. These results indicate that, although the commitment of early NKPs into the CD122+NK1.1+NKG2A/C/D/E+CD49b+ developmental stage does not depend on the functions of PLC
2, this signaling molecule plays a critical role in the terminal maturation of the committed NKPs. Also, the PLC
2/-deficient NK cells possess the other isozyme, PLC
1, in normal quantities. The role of PLC
1 in the development of NKPs is yet to be determined. Our results presented here are parallel to B cell defects in the case of PLC
2 and Btk-deficient mice where the functional defects were observed only after pre-B cell commitment and development (33).
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2/ mice is due to augmented proliferation or increased survival rate (reduced cell death). Toward differentiating between these two possibilities, in the first set of experiments, we performed S phase fractionation and DNA content analyses on NK1.1+CD3 cells (Fig. 2F). IL-2-activated NK cells from WT and PLC
2/ mice were cultured with BrdU (a thymidine analog), followed by anti-BrdU mAb and the DNA-staining dye 7-aminoactinomycin (41). Our results, presented in Fig. 2F, show substantial differences indicating that NK cells from PLC
2/ mice have increased rate of cell proliferation compared with the WT counterparts. In particular, the number of NK cells that were in S and G2-M phase but yet to complete the cell cycle is markedly higher in PLC
2/ relative to WT mice (Fig. 2F). Interestingly, there were no detectable differences in the sub-G0-G1 cells, the population that is undergoing apoptosis (Fig. 2F). Thus, one possible reason for the increased NK1.1+CD3 NK cell numbers can be an augmented proliferation of these cells in PLC
2/ mice. To test whether an increased NK cell proliferation in vivo could be responsible for the higher number of NK1.1+CD3 cells, WT and PLC
2/ mice were injected i.p. with BrdU. Four days later, BM cells were isolated, and BrdU incorporation was analyzed specifically in the NK1.1+ cell populations. Our results indicate that there were no detectable differences in the levels of BrdU incorporation between WT and knockout animal-derived NK1.1+CD3 cells (Fig. 2G). These results exclude the possibility that the lack of PLC
2 resulted in an increased cell cycle in vivo. Although, the NK cells from the PLC
2/ mice proliferated more vigorously in vitro in response to IL-2, the probable reason(s) for the increased NK cell number in vivo are not clearly understood. It is possible that a homeostatic status in the absolute numbers of NK cells can be attained only after they successfully acquire Ly49 receptors. Therefore, in the absence of PLC
2, these developing NK cells failed to mature and could have accumulated without detectable levels of cell death or an augmented cell proliferation.
IL-2 and IL-15 cytokines fail to rescue the developmental defects in PLC
2/ NK cells
We next examined whether cytokine-mediated activation could drive PLC
2-deficient NK cells to mature in vitro. NK1.1+CD3 cells were generated from the spleen and activated with IL-2. Fig. 3 demonstrates that the levels of CD11b after IL-2 activation remained higher and were not reduced in the case of PLC
2/ compared with that of WT. The majority of the developing NK1.1+CD3 cells have been shown to express the integrin
v (CD51) in the BM (34). However, the levels of CD51 have been reported to be down-regulated during NK cell maturation (20). Our results show that although the majority of the NK cells were positive, the levels of CD51 remained significantly low on per cell basis in PLC
2-deficient NK cells compared with WT (Fig. 3A). Because the level of CD51 markers on the unstimulated, fresh NK1.1+CD3 cells from PLC
2/ animals were comparable with the WT subjects and the reduction is seen only in the case of IL-2-activated NK cells, we conclude that in the absence of PLC
2 the NK cells fail to increase the levels of CD51. The inability of PLC
2/-derived NK cells to augment CD51 integrins upon IL-2 activation further confirms an early developmental defect of NK cells. Also, no differences were observed in the level of either CD43 or CD122, confirming that the commitment of NKPs into NK lineage occurs at normal phase. Significantly, IL-2 activation did not increase the levels of all the Ly49 receptors (Ly49A, C, I, D, G) tested which remained unchanged in PLC
2-deficient NK cells (Fig. 3B). Staining for all these Ly49 receptors at the same time indicated tha only
23% of the NK1.1+ NK cells expressed one or more Ly49 receptors compared with
75% of NK1.1+ NK cells from the WT mice. Addition of IL-15, another key cytokine that has been shown to play an important role in the terminal maturation of NK cells (42) along with IL-2, did not alter the outcome of these results (data not shown). Therefore, activation of PLC
2-deficient NK cells with cytokines could not drive PLC
2-deficient NK cells to mature.
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2 in the downstream activation of NKG2D receptor
NKG2D is one of the important receptors that govern NK cell functions. Therefore, we next analyzed whether PLC
2 is involved in NKG2D-mediated activation in NK cells from the WT. Purified NK cells from WT mice were stimulated with anti-NKG2D mAb, A10, followed by cross-linking with secondary anti-hamster IgG-mediated mAbs (43). Fig. 4A demonstrates that engagement of NKG2D induced a considerable level of phosphorylation of PLC
2, an indication of PLC
2 activation. Thus, NKG2D receptor activates PLC
2 in NK cells.
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1 and PLC
2 isoforms perform similar cellular functions, we next examined expression of PLC
1 in NK cells. Although a considerable amount of PLC
1 was present (Fig. 4B, lower panel), activation of NK cells through NKG2D receptor resulted in rarely detectable levels of PLC
1 phosphorylation (Fig. 4B, upper panel). This demonstrates that in contrast to the observations with human NK cells (11), murine NK cells may have lower levels of NKG2D-induced PLC
1 phosphorylation. As described earlier (13, 25), both PLC
1 and PLC
2 proteins were present in WT NK cells, albeit in varying quantities (Fig. 4C). Moreover, the quantity of PLC
1 in the PLC
2/ NK cells was unchanged and was comparable with that of WT NK cells. As expected, NK cells from the knockout mice lacked PLC
2 protein (Fig. 4C). Collectively, murine NK cells express both isoforms of PLC
and PLC
2 deficiency does not lead to a compensatory increase in the levels of PLC
1. These experiments reveal a dominant role for PLC
2 in the signal transductions downstream of NKG2D receptor. However, the possibility that PLC
1 plays a role in activation receptor-mediated signaling (including NKG2D) during the development of NK cells still exists.
Tumor recognition including NKG2D-mediated cytotoxicity is defective in PLC
2/ NK cells
NKG2D is expressed by the majority of NK cells (44, 45, 46). Earlier studies illustrated that ectopic expression of NKG2D ligands, H60 molecules, on autologous tumor cells can render them susceptible to NK-mediated cytotoxicity through NKG2D receptor (8, 9). Although the impaired Ly49 receptor expression demonstrates incomplete maturation of PLC
2-deficient NK cells, the expression levels of NKG2D receptor was unaffected (Fig. 1C). To determine whether PLC
2 plays a role in NKG2D-mediated NK functions, we examined the abilities of PLC
2-deficient NK cells to lyse target tumor cells that are stably transfected with H60 encoding cDNA in conventional 51Cr release assays (47). Nontransfected, parental EL4 cells were used as negative controls. NK cells derived from WT mice mediated cytotoxicity against EL4H60 targets but not against parental EL4 cells; however, PLC
2-deficient NK cells were unable to recognize and lyse the EL4H60 targets (Fig. 5A). Furthermore, intracellular staining for perforin molecules demonstrated a comparable level of this important lytic agent present in both PLC
2/ and WT-derived NK cells (data not shown). Thus, the NKG2D-mediated cytotoxicity of NK cells requires the PLC
2-dependent activation pathway.
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2-deficient NK cells consistently failed to recognize and mediate cytotoxicity of these YAC-1 cells (Fig. 5A).
NK cells also mediate lysis of tumor cells that have lost or reduced their expression levels of MHC class I in a non-NKG2D receptor-dependent manner (48). RMA/S (H-2b) cells have been widely used as a MHC class I target cells to test this natural cytotoxicity of NK cells (38). In our assays, PLC
2-deficient NK cells were unable to lyse these MHC class I target cells, thereby demonstrating their inability to recognize missing-self (Fig. 5A). Although the specific receptor-ligand interaction that is responsible the lysis of target cells during the missing-self recognition is yet to be defined, these results demonstrate that this specific activation involves PLC
2-mediated signaling events. Thus, our results show a global hyporesponsiveness of PLC
2-deficient NK cells to various activation stimuli.
NK cells from PLC
2/ mice fail to generate cytokines and chemokines
In addition to cytotoxicity, NKG2D also mediates production of inflammatory cytokines, including IFN-
and GM-CSF, and chemokines in NK cells (49). To determine the role of PLC
2 in the NKG2D-mediated production of cytokines and chemokines, we first examined their secretion by WT and PLC
2-deficient NK cells following engagement of NKG2D with immobilized anti-NKG2D mAbs. Anti-NK1.1 mAbs were used as positive cytokine production controls in WT NK cells (50). As shown in Fig. 5B, in the case of NK cells derived from the WT, significant quantities of IFN-
(
2530 ng/ml) and GM-CSF (
3000 pg/ml) were generated with anti-NKG2D or anti-NK1.1 mAbs in a concentration dependent manner. To the contrary, the PLC
2-deficient NK cells were severely impaired in their ability to produce either IFN-
or GM-CSF (Fig. 5B). As well, the isotype matched control Abs had no or negligible effect on the cytokine secretion by the NK cells (data not shown). These results demonstrate that PLC
2 plays a critical role in the NKG2D as well as NK1.1-mediated cytokine production.
The reason for the inability of PLC
2/ NK cells to secrete cytokines upon anti-NKG2D or anti-NK1.1 mAbs-mediated activation could be due to defects either in the production or in secretion of these cytokines. Previous studies indeed have shown that PLC
2 deficiency affect cytokine secretion in monocytes (32). To differentiate between these two possibilities, we measured cytokine production by intracellular staining in PLC
2-deficient NK cells following either anti-NKG2D or anti-NK1.1 mAbs stimulation. Consistent with cytokine secretion results, the cytokine specific intracellular staining revealed the inability of the NK cells from PLC
2/ to generate these cytokines (Fig. 5C). Similar to that of cytokines, PLC
2/-derived NK cells also failed to generate chemokines such as MIP-1
, MIP-1
, and RANTES (Fig. 5D). These results demonstrate that PLC
2 plays an important role in effecting not only the cytotoxicity but also the cytokine and chemokine production of NK cells.
Both PKC and IP3 cascades govern NK activation and functions
Although the role of PLC
2 in effecting NK cell cytotoxicity and cytokine secretions was proven, the specific downstream pathway(s) has not been defined. Receptor-mediated phosphorylation of PLC
results in the activation of DAG and IP3. To understand which of these pathways was responsible for cytokine syntheses and cytotoxic granule release, we activated the NK cells from PLC
2/ animals with PMA and ionomycin. Activation of cells with these pharmacological agents can bypass the requirements of PLC
and can directly result in the activation of PKC and induction of calcium flux. Our results presented in Fig. 6A demonstrate that the NK cells from PLC
2/ mice were able to mediate cytotoxicity against the EL4H60 cells when stimulated with both PMA and ionomycin. However, PMA or ionomycin alone could not restore the ability of PLC
2-deficient NK cells to mediate cytotoxicity or cytokine secretions (Fig. 6A and data not shown). Irrespective of the concentration of specific stimulators, the NK cells from the WT mice mediated effective cytotoxicity against EL4H60. This is due to the fact that they already have normal levels of PLC
2 and are thereby able to mediate lysis even in the absence of these pharmacological agents (Fig. 6A). Intracellular staining of PLC
2/ NK cells also has demonstrated a comparable level of perforin available in both PLC
2/ and WT-derived NK cells (data not shown), indicating that the generation of lytic granules was not affected in the absence of PLC
2. Similar analyses for the generation of IFN-
demonstrated comparable levels of cytokine production between the PLC
2/ and WT-derived NK cells (Fig. 6B). Thus, our data demonstrate that the functional defects that we observed in the case of PLC
2-deficient NK cells were due to the lack of stimulation of both DAG/PKC and IP3/calcineurin-mediated activation cascades.
|
2 in NK cell activation are nonredundant and cell intrinsic
The microenvironment in the BM is speculated to play a major role in the development of the NK cells (51). Committed NKPs have been preferentially identified in the BM and in thymus (17, 52). Cytokines such as GM-CSF, IL-7, IL-15, and IL-21 generated in the microenvironment play a critical role in the initiation and sustenance of NK cell development through IL-15R
, IL-2/IL-15R
(CD122), and c-Kit (CD117) receptors (37). Furthermore, the cell surface-bound LT on the developing NKPs that binds to the LT
receptor on BM stromal cells have been specifically shown to play a role in the terminal maturation of NKPs to express Ly49 receptors (22). Therefore, we examined whether defects in PLC
2-deficient NK cells are due to hemopoietic cell intrinsic deficiencies or an impaired microenvironment of developing NKPs, such as stromal cells in the BM. In addition, NK cells express both isoforms of PLC
, PLC
1 and PLC
2. Endogenous PLC
1 fails to compensate for PLC
2 deficiency in NK cell maturation and functions; one explanation could be its inherent low level of expression. To determine whether PLC
2 indeed plays a unique and nonredundant role in NK cells, we overexpressed PLC
1 and analyzed its ability to restore the development and function of PLC
2-deficient NK cells.
To address the above questions, we used a previously developed retrovirus-mediated gene transfer with BM reconstitution strategy (33) First, PLC
2-deficient BM cells were infected in vitro with a retrovirus encoding PLC
1, an IRES, and GFP. Next, the retrovirally transduced BM cells were transplanted into sublethally irradiated JAK3-deficient mice, which inherently lack T, B, and NK cells (28, 53). These retrovirally transduced BM cells repopulated the different lymphocyte populations, including NK cells, in the recipient JAK3-deficient mice. Virus-transduced cells were identified by virtue of the GFP gene. A retrovirus encoding PLC
2 and GFP (PLC
2-IRES-GFP) was used as a positive control. A retrovirus encoding GFP alone was used as a negative control as well as to examine whether the defects of PLC
2/ NK cells are hemopoietically intrinsic.
First, the GFP+ NK cells from the JAK3/ recipients transplanted with PLC
2/ BM that were transduced with PLC
1-IRES-GFP virus exhibited higher levels of PLC
1 protein expression than the WT NK cells (Fig. 7A). Furthermore, the GFP+ NK cells from the recipient transplanted with PLC
2/ BM that were transduced with PLC
2-IRES-GFP virus exhibited normal levels of PLC
2 protein expression (Fig. 7A). Next, the retroviral vector-alone-transduced BM cells give rise to a number of GFP+ and GFP cells similar to that of the PLC
1 or PLC
2 rescued cells (Fig. 7, BD, leftmost panels, and data not shown) demonstrating comparable levels of viral transductions.
|
1-IRES-GFP or PLC
2-IRES-GFP retrovirus-transduced/BM-reconstituted animals were used to determine their maturation and ability to mediate cytotoxicity against EL4H60 target cells. First, the surface expressions of different developmental markers of NK cells from the reconstituted animals were analyzed. Our results demonstrate that the PLC
2-competent microenvironment including stromal cells in the BM of JAK3-deficient mice could not successfully support the development of empty vector-transduced, PLC
2-deficient NK cells. Therefore, we conclude that the NK cell developmental defects we observed in the PLC
2/ are not due to the environment in which these cells develop but rather cell intrinsic.
Our data show that, whereas the levels of CD43 were unchanged irrespective of the viral constructs transduced, the levels of CD51 were highest exclusively with PLC
2 reconstitution (Fig. 7C). Also, the levels of CD49b were down-regulated only in the case of NK cells that were transduced with PLC
2. Neither the GFPNK1.1+ NK cells from the same animals, nor NK cells transduced with PLC
1, nor the vector alone-transduced NK cells failed to show such a CD49b down-modulation upon IL-2 activation (Fig. 7, B and C). These data noticeably demonstrate that the requirement for PLC
2 and a substitution of PLC
2 with PLC
1 cannot compensate for the successful phenotypic maturation of NK cells. Interestingly, analyses of Ly49 receptors demonstrate that both PLC
2 and PLC
1 could rescue the expression of these receptors. NK cells from PLC
2/ animals do express a small but detectable level of PLC
1. Although, this level PLC
1 was evidently insufficient for the complete maturation process of NK cells, an overexpression of PLC
1 helped to rescue some of these phenotypic defects. Roles of PLC
1 in the early commitment phase of NKPs are exciting novel possibilities; however, the exact functions have yet to be defined. Therefore, we propose that the expression of receptors such as NKG2D, NK1.1, NKG2A/C/E, and CD122 may be dependent on the expression and function of PLC
1 but not PLC
2. Nevertheless, the later NK cell developmental processes are virtually dependent on the presence of PLC
2.
As expected, virally transduced, GFP+, PLC
2-overexpressing NK cells regained their ability to mediate the cytotoxicity of El4H60 target cells through NKG2D receptor. Fig. 8A demonstrates that the percentage of cytotoxicity mediated by the PLC
2-reconstituted NK cells increased significantly up to
60% (at an E:T ratio of 40:1), indicating that restoration of PLC
2 expression could correct the functional defects. Moreover, surprisingly, NK cells that were reconstituted with the PLC
1 could also partially regain their ability to mediate cytotoxicity (
40% at an E:T ratio of 40:1). These results demonstrate that although the comparatively low but normal levels of PLC
1 in PLC
2-deficient NK cells were insufficient to act as signal transducer, the overexpression of the PLC
1 could reconstitute some functions of PLC
2. The two isoforms of PLC
conforms around 50% amino acid homology; thus, it is biochemically possible that some functions of PLC
2 can be performed by the overexpression of PLC
1. However, the specific protein domain(s) and their secondary messengers that allow the PLC
1 to perform PLC
2-mediated function have yet to be defined.
|
1-IRES-GFP or PLC
2-IRES-GFP-reconstituted NK cells to generate these cytokines. Purified and IL-2-activated NK cells that are derived from reconstituted mice were activated with plate-bound anti-NKG2D or anti-NK1.1 mAbs and the quantity of IFN-
was measured in their culture supernatants. Results presented in Fig. 8B demonstrate that retrovirally transfected PLC
2 could generate IFN-
, thereby demonstrating a rescue of NK cells from their defects in transmitting NKG2D receptor-mediated signaling. However, unexpectedly, the GFP+, PLC
1-transduced NK cells failed to generate IFN-
. Thus, although the overexpression of PLC
1 could result in the partial rescue of the phenotypic defects, it cannot compensate for the requirement of PLC
2 in complete NK cell-mediated cytotoxicity or cytokine secretion. The failure of vector or PLC
1-transduced NK cells is not due to any technical reasons given that activation of these cells with PMA and ionomycin demonstrated a comparable level of IFN-
generation in all the cells tested (Fig. 8C). This includes the GFPNK1.1+CD3 cells, which are not retrovirally transduced but have the ability to generate IFN-
upon PMA and ionomycin stimulation. | Discussion |
|---|
|
|
|---|
2 in NK cell functions (11, 26, 27), our current study for the first time compares the nonredundant roles of PLC
1 and PLC
2 in NK cell development and functions.
One of the key observations is that there is significant increase in the numbers of CD3NK1.1+ NK cells in PLC
2/ mice. Our observations are different from that of Caraux et al. (26), where a transfer of PLC
2-deficient BM cells into Rag2/
c/ did not result in an overall increase in the CD3NK1.1+ cells. One possibility for this discrepancy could be the host animal backgrounds. Similar cell transfer experiments involving PLC
2-BM cells into JAK3/ mice also indicated an increase in the CD3NK1.1+ cells compared with that of WT-derived cell transfers (S. Malarkannan, unpublished observations). Therefore, we predict that although the increase in the CD3NK1.1+ cells in PLC
2/ mice could be cell intrinsic, there may be other unknown environmental factors that could regulate the total CD3NK1.1+ cell numbers.
On the basis of these results, we conclude that although the PLC
2 is predominantly expressed, it is not an obligate signaling molecule during the early developmental phase of NK cells. In particular, both the initiation of CD122 expression and the commitment of CD122+CD3 NKPs into NK1.1+NKG2D+ cells are unhindered in the absence of PLC
2. Nonetheless, NK1.1+NKG2D+ NK cells in PLC
2/ mice appear to remain halted at a preterminal maturation stage, which fail to down-modulate CD11b and to augment CD51 integrin expression upon IL-2 activation. Importantly, a lack of PLC
2 demonstrates that it plays a critical role in the terminal maturation of NK cells by promoting the acquisition of Ly49 receptors. Consistent with this notion, BM-derived NK cells from PLC
2/ mice have increased proliferation rates compared with those from WT mice. Previous studies from Yokoyamas laboratories have demonstrated that the heightened proliferation of NKPs at the preterminal stage (CD11blowCD49bhighCD43lowCD51high) is an inherent phenomenon of these immature cells (20). NK cells at this specific developmental stage start to acquire different Ly49 receptors and CD94/NKG2 complexes, and their ability to proliferate was also reported to be decreased upon further maturation (20). Therefore, our study indicates that the development of PLC
2/ NK cells halts at a postexpansion and prematuration stage. The exact mechanism by which lack of PLC
2 affects acquisition of the maturation markers and impairs maturation of NK cells is not clear. However, it is possible that PLC
2 deficiency could result in impaired activation of a number of transcription factors. Similar to the present study, an increase in the CD122+NK1.1+TCR
cells have been observed exclusively in the BM of transcription factor T-bet-deficient mice (40). However, in contrast to T-bet-deficient mice, PLC
2/ mice contained increased CD122+NK1.1+CD3 cells in the spleen, BM, and liver. The relationship between the activation PLC
2 and T-bet is not known and is worth investigating. In addition, lack of transcription factor GATA-3 results in the reduced expression of Ly49A, C/I, and D receptors (40). However, the expression of Ly49G2 and CD94NKG2A were relatively increased in GATA-3-deficient mice (56). GATA-3 is predicted to control the timing or accessibility of distinct Ly49 receptor genes and thereby their gene transcription (56). Thus, it is quite possible that the GATA-3 could be one possible target transcription factor downstream of the PLC
2 signaling cascade. Moreover, lack of SHIP phosphatase also results in the altered levels of several Ly49 and CD94 molecules (57). In the adult SHIP/ mice, the expressions of Ly49A, Ly49C were overrepresented, whereas Ly49D, Ly49G2, Ly49I, and CD94 were underrepresented (57). Lastly, PLC
2 deficiency might also affect activation of other specific transcription factors, including IFN-regulatory factor-1 (58), IFN-regulatory factor-2 (59), ID2 (60), Ets-1 (61), MEF (62), and PU-1 (63) that are involved in NK cell development. Investigation of a role of PLC
2 in the activation of these transcription factors may shed some light on the mechanism by which it mediates maturation of NK cells.
The other important finding is that PLC
2 deficiency disrupts all effector functions of NK cells that we have examined. Our results also demonstrate that engagement of NKG2D significantly induces activation of PLC
2. In agreement with this finding, PLC
2 deficiency completely disrupts NKG2D-mediated cytotoxicity and cytokine production. Furthermore, activation through NK1.1 receptors is dependent on PLC
2 since the PLC
2-deficient NK cells fail to generate cytokines/chemokines in response to anti-NK1.1 mAb. Taken together, these findings reveal PLC
2 as a master signal transducer for most, if not all, of the activation receptors in NK cells. The fact that neither PMA nor ionomycin alone fails to rescue functional defects of PLC
2-deficient NK cells, whereas PMA plus ionomycin successfully restore effector functions of mutant NK cells, demonstrate that both DAG/PKC and IP3/Ca2+ pathways are required for the cytotoxicity and cytokine production.
One possible explanation for the impaired cytotoxicity of PLC
2-deficient NK cells could be due to insufficient expression of Ly49 receptors. Low or nonexpression of Ly49 receptors have been strongly related to nonlytic NK cells (17). A subset of CD11blowLy49 NK cells that are present in the liver of normal mice do not have cytotoxic abilities (20). Furthermore, NK cells derived from osteoporotic mice with low levels of Ly49 do not possess the abilities to lyse target cells (64). Also, purified spleen or BM-derived NK cells that are Ly49 fail to mediate target cell lysis (17). Recent studies have strongly implicated the type and level of Ly49 receptor expression to the functional abilities of NK cells (65, 66). Collectively, these observations may be explained by two entirely different hypotheses. First, Ly49 expression may purely stand for the matured status of the NK cells. Thus, the absence or lower expression of Ly49 receptors designates an immature NK cell that cannot mediate effector functions.
The environment, including stromal cells, in the BM has been well documented to play a critical role in the successful development of NK cells (67, 68). Stromal cells contain at least two key factors that are essential for NK cell maturation, i.e., LT
R, which interacts with the membrane-bond form of LT (LT
1
2) (67) and the cytokine IL-15 (69). In particular, IL-15 secreted from the stromal cells has been implicated both in the early commitment phase of the NKPs and during the Ly49C acquisition at preterminal maturation phase. However, in the present study, the addition of IL-15 along with IL-2 to BM-derived PLC
2/ NK cells fails to rescue the expression of Ly49 receptors. Furthermore, the retroviral transduction of PLC
2/ BM and the ensuing BM transplantation into JAK3/ mice (PLC
2-sufficient) also fails to restore Ly49 expression on PLC
2/ NK cells and thereby their maturation. Thus, the developmental defects of NK cells observed in PLC
2/ mice are more hemopoietic cell intrinsic and independent of any possible defects in the stromal cells. In addition, whereas the absence of PLC
2 augmented the number of CD3NK1.1+ NK cells, the numbers of CD3+NK1.1+ NKT cells are considerably reduced in PLC
2-deficient animals. Because NK and NKT cells are thought to mature in different locations, it appears that lack of PLC
2 affects the ontogeny of these effector cell types in distinct ways. These results further validate the cell type-specific and intrinsic defects rather than a systemic impairment in PLC
2/ mice.
Studies using human NK cells have demonstrated presence of both PLC
1 and PLC
2, but in varying quantities (70). These studies also reveal that all the ITAM-containing receptors can use either PLC
1 or PLC
2, whereas the NKG2D/DAP10 complexes exclusively recruited PLC
2. The NKG2D/DAP12 complexes are undetectable in human NK cells. On the contrary, NKG2D in the murine NK cells associates with either DAP10 or DAP12, whereas the expression of NKG2D/DAP12 complexes become predominant following cytokine activation (45). Interestingly, in this study, anti-NKG2D-mediated activation specifically resulted in the phosphorylation of PLC
2, and the phosphorylation of PLC
1 was undetectable, which could be due to high and low expression levels of PLC
2 and PLC
1, respectively, in murine NK cells. Apart from these observations, the role of PLC
1 and PLC
2 in NK cell development and function has not been clearly understood. Our current studies form the first basis to indicate the nonredundant roles played by these two PLC
isozymes. The absence of PLC
2 does not alter the commitment of CD122+ cells into NK1.1+CD3NKG2D+ NKPs. It is quite possible that PLC
1, which is expressed throughout the life of NK cells, is mostly involved in the early NK cell commitment, whereas PLC
2 plays an important role in the subset specification and effector functions of NK cells. These notions are supported by precedence that similar observations for exclusive functions for PLC
1 and PLC
2 have been recently reported in B cell development (33). In addition, overexpression of PLC
1 in PLC
2/ NK cells by retroviral transduction rescues the expression of Ly49 receptors but fails to restore the expression patterns of CD43, CD49b, and CD51 molecules. However, PLC
1 partially restores ability of PLC
2/ NK cells to mediate cytotoxicity but completely fails to restore their ability to generate cytokines. Therefore, the two PLC
isoforms play nonredundant roles in the biology of NK cells. Our results are consistent with the recent observations regarding the functions of PLC
1 and PLC
2 in human NK cells by the Leibson laboratory (71). However, the exclusive roles of PLC
1 and PLC
2 in NK cells are far from completely understood. In particular, the effects of quantitative and qualitative variations between PLC
1 and PLC
2 in NK cells have yet to be defined, and further investigations are warranted.
| Disclosures |
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| Footnotes |
|---|
1 This work was supported in part by American Cancer Society Grants RSG-02-172-01-LIB (to S.M.) and RSG CCG-106204 (to D.W.); Roche Organ Transplant Research Foundation Grant 111662730 (to S.M.); and National Institutes of Health Grants U19 AI062627-01, NO1-HHSN26600 500032C, and R01 A1064826 (to S.M.), R01 AI52327 (to R.W.), and R01 HL073284 (to D.W.). J.R. is a Northwestern Mutual Postdoctoral Fellow, from the Cancer Center of the Medical College of Wisconsin. ![]()
2 Address correspondence and reprint requests to Dr. Subramaniam Malarkannan or Dr. Demin Wang, Blood Research Institute, Milwaukee, WI 53226. E-mail addresses: subra.malar{at}bcw.edu or demin.wang{at}bcw.edu ![]()
3 Abbreviations used in this paper: H60, minor histocompatibility Ag 60; PTK, protein tyrosine kinase; PLC
, phospholipase C
; DAG, 1,2-diacylglycerol; PKC, protein kinase C; IP3. inositol 1,4,5-triphosphate; CLP, common lymphoid progenitor; BM, bone marrow; NKP, NK precursor; IRES, internal ribosome entry site; WT, wild type; EL4H60, EL4 stably transfected with H60. ![]()
Received for publication October 12, 2005. Accepted for publication July 17, 2006.
| References |
|---|
|
|
|---|
receptor activation induces the tyrosine phosphorylation of both phospholipase C (PLC)-
1 and PLC-
2 in natural killer cells. J. Exp. Med. 176: 1751-1755.
-chain-dependent cytokines in the generation, differentiation, and maturation of NK cell precursors and peripheral NK cells in vivo. J. Immunol. 174: 1213-1221.
2 is essential in the functions of B cell and several Fc receptors. Immunity 13: 25-35. [Medline]
signaling complex in human natural killer (NK) cells. J. Exp. Med. 184: 2243-2250.
2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells. Blood 107: 994-1002.
2 is a critical signaling mediator for murine NK cell-activating receptors. J. Immunol. 175: 749-754.
2 is essential for specific functions of Fc
R and Fc
R. J. Immunol. 169: 6743-6752.
1 in pre-B-cell development and allelic exclusion. EMBO J. 23: 4007-4017. [Medline]
2 integrin, very late antigen-2). J. Immunol. 167: 1141-1144.
14i NKT cells. Immunity 20: 477-494. [Medline]
-chain-dependent cytokines in the generation, differentiation, and maturation of NK cell precursors and peripheral NK cells in vivo. J. Immunol. 174: 1213-1221.
production, and liver-specific homing of NK cells. Immunity 19: 701-711. [Medline]
R on bone marrow stromal cells is required for an early checkpoint of NK cell development. J. Immunol. 166: 1684-1689.
RIIIA on natural killer cells results in tyrosine phosphorylation of PLC
1 and PLC-
2. J. Immunol. 150: 2668-2674. [Abstract]
are differentially used by distinct human NK activating receptors. J. Immunol. 175: 213-218. This article has been cited by other articles:
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