RNK-16 Cells Research Articles

Whole Genome Sequencing of Spontaneously Occurring Rat Natural Killer Large Granular Lymphocyte Leukemia Identifies JAK1 Somatic Activating Mutation.

Large granular lymphocyte (LGL) leukemia arises spontaneously in elderly Fischer (F344) rats. This rodent model has been shown to emulate many aspects of the natural killer (NK) variant of human LGL leukemia. Previous transplantation of leukemic material into young F344 rats resulted in several strains of rat NK (RNK) primary leukemic cells. One strain, RNK-16, was adapted into the RNK-16 cell line and established as an aggressive NK-LGL leukemia model. Whole genome sequencing of the RNK-16 cell line identified 255,838 locations where the RNK16 had an alternate allele that was different from F344, including a mutation in Jak1. Functional studies showed Jak1 Y1034C to be a somatic activating mutation that mediated increased STAT signaling, as assessed by phosphoprotein levels. Sanger sequencing of Jak1 in RNK-1, -3, -7, and -16 found only RNK-16 to harbor the Y1034C Jak1 mutation. In vivo studies revealed that rats engrafted with RNK-16 primary material developed leukemia more rapidly than those engrafted with RNK-1, -3, and -7. Additionally, ex vivo RNK-16 spleen cells from leukemic rats exhibited increased STAT1, STAT3, and STAT5 phosphorylation compared to other RNK strains. Therefore, we report and characterize a novel gain-of-function Jak1 mutation in a spontaneous LGL leukemia model that results in increased downstream STAT signaling.

Acid Ceramidase Inhibition Impairs Tumor Progression in a Rat Model of LGL Leukemia

Natural killer cell large granular lymphocytic (NK-LGL) leukemia is a disorder characterized by the expansion of clonal CD3-, CD56+ cells in the peripheral blood and marrow. NK-LGL leukemia could manifest in both aggressive and chronic forms. Currently, no curative treatment is available for patients with NK-LGL leukemia. The pathogenetic mechanisms of NK-LGL leukemia are also poorly understood, which highlights the need to increase our understanding of the disorder in order to develop novel therapeutic targets. Sphingolipid dysregulation has been shown to promote tumor growth and survival in several types of cancers, including LGL leukemia. In cancer cells, the accumulation of pro-survival sphingosine-1-phosphate (S1P) throws the S1P/ceramide rheostat off balance, which leads to tumor growth and activated signaling pathways. Our laboratory previously discovered that the reversal of sphingolipid dysregulation in NK-LGL leukemia, either through targeting sphingolipid enzymes or by the addition of antiproliferative ceramide, leads to programmed cell death in leukemic cells.Acid ceramidase, a cysteine hydrolase, catalyzes the breakdown of ceramide into sphingosine and fatty acid. We found that cells from chronic NK-LGL patients have lower pro-death ceramide levels and higher S1P levels when compared to normal NK donors. Furthermore, the mRNA expression and activity of acid ceramidase (AC) were elevated in leukemic NK cells compared to normal NK cells. We further demonstrate the importance of AC in NK-LGL leukemic cells through AC knockdown in human (NKL) and rat (RNK-16) NK-LGL leukemic cell lines. AC knockdown decreased cell viability and increased ceramide levels, significantly ceramide species C16 and C24 (p<0.05), showing that AC is essential for the growth of leukemic NK cells. We further confirm that the accumulation of both C16 and C24 ceramide mediated the decreased viability of NKL and RNK-16 cells by treating the cells with C16 and C24 ceramide (6.25µM, 12.5µM and 25µM) for 24 hours.As our data showed that AC mediates the survival of NK-LGL, we did further analysis on the effects of AC knockdown on survival signals. Protein expression of AC knockdown- NKL and RNK-16 cell lines showed that AC knockdown decreased survival signaling mediators, including Bcl-2, Mcl-1 and survivin. Conversely, pro-apoptotic puma increased with AC knockdown in both cell lines. We further extended our in vitro findings in an established in vivo NK-LGL Fischer rat model. RNK-16 cells stably expressing AC shRNA (RNK-16/shRNA) or stably expressing scrambled shRNA (RNK-16/scr) were transplanted into 6-week old male rats (7 per group). The rats engrafted with RNK-16/shRNA survived significantly longer than those engrafted with RNK-16/scr (Mantel-Cox test, p<0.05). Interestingly, RNK-16/shRNA rats did not exhibit organomegaly compared to their leukemic counterparts. Splenocytes isolated from RNK-16/shRNA engrafted animals showed 50 percent reduction in AC expression compared to splenocytes from RNK-16/scr. Lipid analysis on splenocytes isolated from both RNK-16/scr and RNK-16/shRNA showed that RNK-16/shRNA cells had significant increases in total ceramide, C16 and C24 ceramide species, and a significant decrease in S1P. Collectively, our data show that targeting AC slows the progression of NK-LGL leukemia by reversing sphingolipid dysregulation and warrants further investigation as a therapeutic target in this incurable disease. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4112: Inositol hexaphosphate (IP6) and inositol against large granular lymphocytic leukemia (LGLL)

Abstract Introduction: The anticancer activity of dietary IP6 and its parent compound inositol, either alone or in combination, has been shown in many different experimental models. Using rat leukemia RNK-16 cells, we investigated the in vitro efficacy of IP6 and inositol in large granular lymphocytic leukemia (LGLL) that develops spontaneously in male Fisher 344 rats and is very similar to human disease. RAS and ERK are constitutively active in LGLL in both humans and rats, and the PI3K/Akt pathway plays a major role in proliferation and survival of these leukemic cells. Therefore, we also investigated the effect of IP6 and inositol on these pathways. Methods: We have previously shown that after in vitro or in vivo administration, exogenous IP6 rapidly dephosphorylates to inositol. In this study, we first implemented the assay for the measurement of unlabeled intracellular myo-inositol by LC-MS/MS after in vitro exposure to IP6 and inositol. Cytotoxicity of IP6 and inositol against Fisher 344 RNK-16 cells alone and in combination with 4-OH-cyclophosphamide was measured by MTT cytotoxicity assay. Western blot analysis was used for protein expression. Results: Uptake of inositol increased more than 5-fold following exposure to 2 mM inositol, and no differences were observed between unsynchronized and synchronized cells. Inositol was relatively non-toxic (30% cytotoxicity at 100 mM), while IP6 had an IC50 of approximately 2 mM. 4-OH-cyclophosphamide had an IC50 of 180 µg/mL, and co-administration of either IP6 or inositol decreased its IC50 by 3-fold. While the expression and phosphorylation status of ERK1/2 were unaffected, p-Akt was completely inhibited between 5 and 60 min after 2 mM IP6 and between 15 and 60 min after inositol, by 2-fold. Furthermore, IP6 treatment caused a significant increase in the expression of the Cdk inhibitor p27Kip1 in RNK-16 cells (1.9-fold) 5 and 15 min. Conclusion: Our data data suggest the use of inositol and IP6 as chemopreventive agents in control of LGLL in humans or as adjuvant therapies. Further studies are justified and warranted. Support: P30-CA-47904 Citation Format: Ivana Vucenik, Jan H. Beumer, Julie L. Eiseman. Inositol hexaphosphate (IP6) and inositol against large granular lymphocytic leukemia (LGLL). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4112. doi:10.1158/1538-7445.AM2014-4112

Identification of an MHC Class I Ligand for the Single Member of a Killer Cell Lectin-like Receptor Family, KLRH1

Natural killer cells are able to recognize and kill target cells according to differences in MHC class I expression. In rodents, the Ly49 receptors are primarily responsible for this MHC differentiation. We previously described the cloning of a novel C-type lectin-like receptor, KLRH1, encoded in the NK complex adjacent to the Ly49 genes and expressed by subsets of NK and NKT cells. MHC influence on selection of KLRH1(+) NK cells in congenic strains suggested that KLRH1 may have an MHC ligand, although we were unable to identify any such ligand. In this study, we have used a sensitive reporter system and Fc fusion protein to demonstrate that KLRH1 binds specifically to the classical MHC class I molecule RT1-A2 of the RT1(n) haplotype. Cytolytic activity of KLRH1-transfected RNK-16 cells was also inhibited by target cells expressing RT1-A2(n). Thus, KLRH1 represents a novel family of MHC allele-specific inhibitory receptors expressed by NK cells.

Opiate Antagonist Prevents μ- and δ-Opiate Receptor Dimerization to Facilitate Ability of Agonist to Control Ethanol-altered Natural Killer Cell Functions and Mammary Tumor Growth

In the natural killer (NK) cells, δ-opiate receptor (DOR) and μ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.

The activating receptor, NKp30, is important in the interactions between rat natural killer cells and dendritic cells (102.20)

Abstract There is considerable interest in using dendritic cells (DC) to induce transplant tolerance. DC and natural killer (NK) cells have been found to interact in a bi-directional manner. DCs can activate human NK cells to more effectively kill, while NK cells can mediate both maturation and killing of DCs via an NK cell activation receptor, NKp30. NK cells expressing this receptor are elevated post-transplantation prompting us to examine NK cell interactions with DC in a rodent experimental system. Wildtype and rNKp30-transfected RNK-16 cells (rat NK cell line) were co-cultured with bone marrow-derived DCs (BMDC). There was a significant increase in IFNγ production when rNKp30+, but not rNKp30−, NK cells were co-cultured with both immature and mature BMDCs. This NK-DC interaction required cell contact. Flow cytometric analysis of BMDCs with the rNKp30-Fc fusion protein (as compared to a control Fc fusion protein) indicated the presence of rNKp30 ligand(s) on the surface of DCs. Several tumor cell lines also specifically bound rNKp30-Fc. rNKp30-Fc binding was abolished by pretreatment of the tumor cells with trypsin suggesting the ligand is a protein. Furthermore, we established that the expression level of putative rNKp30 ligands correlated with the ability to activate NK cells, as indicated by the levels of IFNγ. Understanding NK cell interactions with DC may suggest potential new targets for tolerance induction.

NKp30 is a functional activation receptor on a subset of rat natural killer cells

NKp30 is a stimulatory receptor on human NK cells implicated in tumor immunity, and is capable of promoting or terminating dendritic cell maturation. To gain a better understanding of NKp30 biology, we have investigated the expression and function of rat NKp30 (rNKp30). We generated stable transfectants of rNKp30 in RNK16 cells, a rat NK lymphoma line, and used a novel panel of mAb against rNKp30 to study this receptor. Using agonistic rNKp30 mAb, we demonstrated that rNKp30 mediates robust IFN-gamma production and cytolytic responses from rNKp30-transfected RNK16 cells. We determined by flow cytometry that rNKp30 is expressed by a subset of primary NK cells isolated from the blood and spleen, and to a lesser extent also on liver NK cells. Stimulation of rNKp30 on primary NK cells led to IFN-gamma production. Liver NK cells expressed low levels of NKp30 and had reduced rNKp30-mediated IFN-gamma responses. During an alloimmune response in vivo, the proportion of the rNKp30(+) NK cell subset in the peripheral blood significantly increased, suggesting that rNKp30 may play an important role during alloactivation. Thus, our data demonstrate that NKp30 is indeed expressed in rodents and is a functional stimulatory receptor in a subset of rat NK cells.

Mutational Analysis of the Human 2B4 (CD244)/CD48 Interaction: Lys68 and Glu70 in the V Domain of 2B4 Are Critical for CD48 Binding and Functional Activation of NK Cells

Interaction between receptors and ligands plays a critical role in the generation of immune responses. The 2B4 (CD244), a member of the CD2 subset of the Ig superfamily, is the high affinity ligand for CD48. It is expressed on NK cells, T cells, monocytes, and basophils. Recent data indicate that 2B4/CD48 interactions regulate NK and T lymphocyte functions. In human NK cells, 2B4/CD48 interaction induces activation signals, whereas in murine NK cells it sends inhibitory signals. To determine the structural basis for 2B4/CD48 interaction, selected amino acid residues in the V domain of the human 2B4 (h2B4) were mutated to alanine by site-directed mutagenesis. Following transient expression of these mutants in B16F10 melanoma cells, their interaction with soluble CD48-Fc fusion protein was assessed by flow cytometry. We identified amino acid residues in the extracellular domain of h2B4 that are involved in interacting with CD48. Binding of CD48-Fc fusion protein to RNK-16 cells stably transfected with wild-type and a double-mutant Lys(68)Ala-Glu(70)Ala h2B4 further demonstrated that Lys(68) and Glu(70) in the V domain of h2B4 are essential for 2B4/CD48 interaction. Functional analysis indicated that Lys(68) and Glu(70) in the extracellular domain of h2B4 play a key role in the activation of human NK cells through 2B4/CD48 interaction.

Expression of regulator of G protein signalling proteins in natural killer cells, and their modulation by Ly49A and Ly49D

The small GTPase accelerators regulator of G protein signalling (RGS) proteins are important regulators of proximal signalling from G protein coupled receptors. Although natural killer (NK) cells express a number of G-protein coupled receptors, expression of RGS proteins has not been investigated. We analysed the expression of RGS proteins in rat NK cells, and detected mRNA for RGS1, RGS2, RGS5, RGS8, RGS16, and RGS18. Interestingly, when we included a panel of different leucocyte subsets, we found that RGS8 was selectively expressed by NK cells. NK cells are under control of both activating and inhibitory receptors and, utilizing a xenogeneic system where the mouse activating Ly49D or inhibitory Ly49A receptors were transfected into the rat RNK-16 cell line, the potential regulation of RGS proteins by single NK cell receptors was studied. We found that ligation of Ly49D led to a rapid and transient increase in message for RGS2, while Ly49A ligation up-regulated RGS2, RGS16, and RGS18 mRNA. Both receptors also induced a prolonged increase in RGS2 endogenous protein levels. These findings suggest that RGS proteins may be influenced by or involved in NK cell receptor events, suggesting a crosstalk between G-protein coupled receptors and NK cell receptors.

The murine NK receptor 2B4 (CD244) exhibits inhibitory function independent of signaling lymphocytic activation molecule-associated protein expression.

2B4 (CD244) is a receptor belonging to the CD2-signaling lymphocytic activation molecule family and is found on all murine NK cells and a subset of NKT and CD8+ T cells. Murine 2B4 is expressed as two isoforms (2B4 short and 2B4 long) that arise by alternative splicing. They differ only in their cytoplasmic domains and exhibit opposing function when expressed in the RNK-16 cell line. The ligand for 2B4, CD48, is expressed on all hemopoietic cells. Previous studies have shown that treatment of NK cells with a 2B4 mAb results in increased cytotoxicity and IFN-gamma production. In this report, we used CD48+/- variants of the P815 tumor cell line and 2B4 knockout mice to show that engagement of 2B4 by its counterreceptor, CD48, expressed on target cells leads to an inhibition in NK cytotoxicity. The addition of 2B4 or CD48 mAb relieves this inhibition resulting in enhanced target cell lysis. This 2B4-mediated inhibition acts independently of signaling lymphocytic activation molecule-associated protein expression. Imaging studies show that 2B4 preferentially accumulates at the interface between NK and target cells during nonlytic events also indicative of an inhibitory receptor. This predominant inhibitory function of murine 2B4 correlates with increased 2B4 long isoform level expression over 2B4 short.

Molecular and functional characterization of a CS1 (CRACC) splice variant expressed in human NK cells that does not contain immunoreceptor tyrosine-based switch motifs.

CS1 (CRACC, novel Ly9) is a novel member of the CD2 family expressed on natural killer (NK), T and stimulated B cells. Although the cytoplasmic domain of CS1 contains immunoreceptor tyrosine-based switch motifs (ITSM), which enables to recruite signaling lymphocyte activation molecule (SLAM)-associated protein (SAP/SH2D1A), it activates NK cells in the absence of a functional SAP. CS1 is a self ligand and homophilic interaction of CS1 regulates NK cell cytolytic activity. Here we have identified a novel splice variant of CS1 (CS1-S), which lacks ITSM. Human NK cells express mRNA for both wild-type CS1 (CS1-L) and CS1-S and their expression level remained steady upon various stimulations. To determine the function of each isoform, cDNA for CS1-L and CS1-S were transfected into the rat NK cell line RNK-16 and functionally tested using redirected cytotoxicity assays and calcium flux experiments. CS1-L was able to mediate redirected cytotoxicity of P815 target cells in the presence of monoclonal antibody against CS1 and a rise in intracellular calcium within RNK-16 cells, suggesting that CS1-L is an activating receptor, whereas CS1-S showed no effects. Interestingly, SAP associated with unstimulated CS1-L and dissociated upon pervanadate stimulation. These results indicate that CS1-L and CS1-S may differentially regulate human NK cell functions.

The rat RT1-A1c MHC molecule is a xenogeneic ligand recognized by the mouse activating Ly-49W and inhibitory Ly-49G receptors.

Mouse Ly-49 receptors are known to recognize xenogeneic ligands from hamster and rat. However, until now, there has been no description of a specific rat xenogeneic ligand for any mouse Ly-49 receptor. In this report, we identify RT1-A1c, a rat classical class I MHC molecule, as a ligand for the Ly-49G(BALB/c) inhibitory receptor and the closely related activating receptor, Ly-49W. Xenogeneic class I recognition of targets from PVG but not DA strain rats was mapped to the classical region of the RT1c haplotype by using Con A blasts from RT1c/RT1av1 intra-MHC recombinant rats as targets for RNK-16 cells expressing either Ly-49W or Ly-49G(BALB/c) receptors. Individual expression of class I molecules from PVG and DA rat strains in YB2/0 target cells demonstrate the xenogeneic recognition to be allele specific, because other class I molecules of the RT1c haplotype, RT1-A2c and RT1-U2c, and a classical class I molecule encoded by the RT1av1 haplotype, RT1-Aa, are not recognized by Ly-49W and -G(BALB/c). Furthermore, specificity for RT1-Ac can be transferred from Ly-49W to Ly-49P, which is normally unable to recognize RT1-Ac, by substitution of three residues shared by Ly-49W and -G(BALB/c) but not Ly-49P. These residues are located in the Ly-49 beta4-beta5 loop, which can determine class I allele specificity in mouse Ly-49 receptor interactions with mouse class I ligands, suggesting that mouse Ly-49 recognition of rat class I molecules follows similar principles of interaction. These findings have implications for xenotransplantation studies and for discerning Ly-49 recognition motifs present in MHC molecules.

Activating and inhibitory Ly49 receptors modulate NK cell chemotaxis to CXC chemokine ligand (CXCL) 10 and CXCL12.

NK cells can migrate into sites of inflammatory responses or malignancies in response to chemokines. Target killing by rodent NK cells is restricted by opposing signals from inhibitory and activating Ly49 receptors. The rat NK leukemic cell line RNK16 constitutively expresses functional receptors for the inflammatory chemokine CXC chemokine ligand (CXCL)10 (CXCR3) and the homeostatic chemokine CXCL12 (CXCR4). RNK-16 cells transfected with either the activating Ly49D receptor or the inhibitory Ly49A receptor were used to examine the effects of NK receptor ligation on CXCL10- and CXCL12-mediated chemotaxis. Ligation of Ly49A, either with Abs or its MHC class I ligand H2-D(d), led to a decrease in chemotactic responses to either CXCL10 or CXCL12. In contrast, Ly49D ligation with Abs or H2-D(d) led to an increase in migration toward CXCL10, but a decrease in chemotaxis toward CXCL12. Ly49-dependent effects on RNK-16 chemotaxis were not the result of surface modulation of CXCR3 or CXCR4 as demonstrated by flow cytometry. A mutation of the Src homology phosphatase-1 binding motif in Ly49A completely abrogated Ly49-dependent effects on both CXCL10 and CXCL12 chemotaxis, suggesting a role for Src homology phosphatase-1 in Ly49A/chemokine receptor cross-talk. Ly49D-transfected cells were pretreated with the Syk kinase inhibitor Piceatannol before ligation, which abrogated the previously observed changes in migration toward CXCL10 and CXCL12. Piceatannol also abrogated Ly49A-dependent inhibition of chemotaxis toward CXCL10, but not CXCL12. Collectively, these data suggest that Ly49 receptors can influence NK cell chemotaxis within sites of inflammation or tumor growth upon interaction with target cells.

Activation of multiple caspases and modification of cell surface fas (CD95) in proteasome inhibitor-induced apoptosis of rat natural killer cells.

The proteasome is a multi-subunit protease complex that is involved in intracellular protein degradation in eukaryotes. Previously, we have reported that selective, synthetic chymotryptic proteasome inhibitors inhibit A-NK cell-mediated cytotoxicity by approximately 50%; however, the exact role of the proteasome in NK cell-mediated cytotoxicity remains unknown. Herein, we report that proteasome inhibitors, MG115 and MG132, decreased the proteasome chymotrypsin-like activity in the rat natural killer cell line RNK16 by 85% at a concentration of 5 microM. The viability of RNK16 cells was also reduced in the presence of these inhibitors. Both inhibitors induced the apoptosis of RNK16 cells, as shown by DNA fragmentation, caspase-3 activation and the appearance of sub-G-cell populations. An increase in the fraction of apoptotic cells was observed in a dose- and time-dependent manner in our studies. In addition, the activity of caspase-1, -2, -6, -7, -8, and -9, was increased following the treatment of RNK16 cells with these inhibitors. Further investigation revealed that the expression of Fas (CD95) protein on the RNK16 cell surface was increased after the treatment by MG115 or MG132, indicating that apoptosis induced by proteasome inhibitors in RNK16 cells might be mediated through the Fas (CD95)-mediated death pathway as well. Our studies indicate, for the first time, that proteasomal chymotryptic inhibitors can reduce natural killer cell viability and therefore indirectly inhibit cell-mediated cytotoxicity via the apoptosis-inducing properties of these agents.

Ly49i2 is an inhibitory rat natural killer cell receptor for an MHC class Ia molecule (RT1-A1c).

We have exploited strain-specific differences in the NK allorecognition repertoires to generate rat monoclonal antibodies against receptors involved in the control of allogeneic responses by rat NK cells. The monoclonal antibody STOK2 binds to a homodimeric glycoprotein that has been implicated as an inhibitory receptor for an MHC molecule in the PVG strain. In the present study, we haveidentified this glycoprotein as a novel rat Ly49 receptor (Ly49i2) containing an immunoreceptor tyrosine-based inhibitory motif. Ligation of the Ly49i2 receptor induces inhibitory signals, and Ly49i2 coprecipitates with the inhibitory tyrosine phosphatase SHP-1 in stably transfected RNK-16 cells. Moreover, it inhibits natural killing of lymphoblast targets and transfected fibroblast targets expressingthe classical MHC class Ia allele RT1-A1(c). Ly49i2, therefore, is an inhibitory receptor for specific MHC class Ia molecules, similar to inhibitory members of the mouse Ly49 family.

Granzyme A activates an endoplasmic reticulum-associated caspase-independent nuclease to induce single-stranded DNA nicks.

The cytotoxic T lymphocyte protease granzyme A (GzmA) initiates a novel caspase-independent cell death pathway characterized by single-stranded DNA nicking. The previously identified GzmA substrate SET is in a multimeric 270-420-kDa endoplasmic reticulum-associated complex that also contains the tumor suppressor protein pp32. GzmA cleaved the nucleosome assembly protein SET after Lys(176) and disrupted its nucleosome assembly activity. The purified SET complex required only GzmA to reconstitute single-stranded DNA nicking in isolated nuclei. DNA nicking occurred independently of caspase activation. The SET complex contains a 25-kDa Mg(2+)-dependent nuclease that degrades calf thymus DNA and plasmid DNA. Thus, GzmA activates a DNase (GzmA-activated DNase) within the SET complex to produce a novel form of DNA damage during cytotoxic T lymphocyte-mediated death.

Ly-49W, an activating receptor of nonobese diabetic mice with close homology to the inhibitory receptor Ly-49G, recognizes H-2D(k) and H-2D(d).

The diversity and ligand specificity of activating Ly-49 receptors expressed by murine NK cells are largely unknown. We cloned a new Ly-49-activating receptor, expressed by NK cells of the nonobese diabetic mouse strain, which we have designated Ly-49W. Ly-49W is highly related to the known inhibitory receptor Ly-49G in its carbohydrate recognition domain, exhibiting 97.6% amino acid identity in this region. We demonstrate that the 4D11 and Cwy-3 Abs, thought to be Ly-49G specific, also recognize Ly-49W. Rat RNK-16 cells transfected with Ly-49W mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49W can activate NK-mediated lysis. We further show that Ly-49W is allo-MHC specific: Ly-49W transfectants of RNK-16 only lysed Con A blasts expressing H-2(k) or H-2(d) haplotypes, and Ab-blocking experiments indicated that H-2D(k) and D(d) are ligands for Ly-49W. Ly-49W is the first activating Ly-49 receptor demonstrated to recognize an H-2(k) class I product. Ly-49G and Ly-49W represent a new pair of NK receptors with very similar ligand-binding domains, but opposite signaling functions.

Molecular characterization of the rat NK cell receptor 2B4

2B4 (CD244) is a cell surface glycoprotein of the immunoglobulin superfamily involved in the regulation of natural killer and T lymphocyte function. It is the high affinity counter-receptor for CD48. In mouse and human NK cells, crosslinking of 2B4 with a specific monoclonal antibody or with CD48 can trigger cell-mediated cytotoxicity, IFN-γ secretion, phosphoinositol turnover and NK cell invasiveness. Recent reports of defective 2B4 signaling and NK cell function in X-linked lymphoproliferative syndrome suggest that this may contribute to the progression of this human disease. Here we describe the molecular characterization of the rat 2B4 gene. The cDNA encodes a protein of 395 amino acid residues that contain two Ig domains in the extracellular region and three unique tyrosine motifs (TxYxxV/I/A) in the cytoplasmic region. The predicted protein has 81 and 68% similarity with mouse 2B4 and human 2B4, respectively. Additionally, it has 94 and 89% similarity at the protein level with the recently reported rat 2B4 related genes, r2B4R-tm and r2B4R-se respectively. Northern blot analysis indicated the presence of multiple transcripts in rat LAK cells and RNK-16 cells. Immunoprecipitation and deglycosylation studies showed that rat 2B4 is glycosylated to similar extent as that of mouse and human 2B4. The cloning of r2B4 in the light of the availability of rat NK cell lines should facilitate in vitro and in vivo experiments to decipher the functional role of 2B4 in NK cell biology.

Uterine natural killer cells are targets for a trophoblast cell-specific cytokine, prolactin-like protein A.

PRL-like protein A (PLP-A) is a member of the PRL family expressed in trophoblast cells coincident with establishment of the chorioallantoic placenta. The purpose of this investigation was to identify targets for PLP-A. Using an alkaline phosphatase-tagging strategy, we show that PLP-A specifically interacts with a population of natural killer (NK) lymphocytes within the mesometrial compartment of decidua from pregnant and pseudopregnant rats. These observations are supported by the codistribution of PLP-A targets with cells expressing the rat NK cell surface marker, gp42, the absence of PLP-A binding in conceptuses from NK cell-deficient tg epsilon26 mice, and the specific interaction of PLP-A with a rat NK cell line, RNK-16. We have further demonstrated that PLP-A effectively suppresses RNK-16 cell cytolytic activities. Our results provide evidence for a new paradigm of embryonic-maternal communication involving a PLP-A signaling pathway between trophoblast cells and uterine NK lymphocytes.

Uterine Natural Killer Cells Are Targets for a Trophoblast Cell-Specific Cytokine, Prolactin-Like Protein A

PRL-like protein A (PLP-A) is a member of the PRL family expressed in trophoblast cells coincident with establishment of the chorioallantoic placenta. The purpose of this investigation was to identify targets for PLP-A. Using an alkaline phosphatase-tagging strategy, we show that PLP-A specifically interacts with a population of natural killer (NK) lymphocytes within the mesometrial compartment of decidua from pregnant and pseudopregnant rats. These observations are supported by the codistribution of PLP-A targets with cells expressing the rat NK cell surface marker, gp42, the absence of PLP-A binding in conceptuses from NK cell-deficient tgε26 mice, and the specific interaction of PLP-A with a rat NK cell line, RNK-16. We have further demonstrated that PLP-A effectively suppresses RNK-16 cell cytolytic activities. Our results provide evidence for a new paradigm of embryonic-maternal communication involving a PLP-A signaling pathway between trophoblast cells and uterine NK lymphocytes.