Recognition of CHO cells by inhibitory and activating Ly-49 receptors.

Upon ligand recognition, members of the murine Ly-49 receptor family can transmit inhibitory or activating signals that regulate NK cell function. Ly-49A, G, and D have been shown to recognize the murine class I molecule H-2Dd as a potential ligand. Recent studies also have demonstrated also that Ly-49D+ NK cells can lyse CHO cells, although the ligand responsible for this recognition was not identified. Because allorecognition by NK cells may be important in bone-marrow transplantation and because of the overlapping class I recognition by these receptors, recognition of CHO cells by Ly-49G and A was investigated. Our data suggest that Ly-49G and probably A transmit inhibitory signals in response to CHO cells. Receptor inhibition was assessed by examining NK lytic function, IFN-gamma secretion, and DAP12 phosphorylation in response to CHO cells by sorted subsets of Ly-49D vs. G B6 NK cells. Our results suggest that CHO cells may express a common ligand(s) that is capable of engaging Ly-49D, G, and possibly A in C576BL/6 NK cells. In addition to our findings that Ly-49 inhibitory receptors also recognize CHO cells, activating receptors other than Ly-49D are present in B6 mice that can lyse CHO cells.

Interaction of Ly-49D+ NK cells with H-2Dd target cells leads to Dap-12 phosphorylation and IFN-gamma secretion.

Murine Ly-49D augments NK cell function upon recognition of target cells expressing H-2Dd. Ly-49D activation is mediated by the immunoreceptor tyrosine-based activation motif-containing signaling moiety Dap-12. In this report we demonstrate that Ly-49D receptor ligation can lead to the rapid and potent secretion of IFN-gamma. Cytokine secretion can be induced from Ly-49D+ NK cells after receptor ligation with Ab or after interaction with target cells expressing their H-2Dd ligand. Consistent with the dominant inhibitory function of Ly-49G, NK cells coexpressing Ly-49D and Ly-49G show a profound reduction in IFN-gamma secretion after interaction with targets expressing their common ligand, H-2Dd. Importantly, we are able to demonstrate for the first time that effector/target cell interactions using Ly-49D+ NK cells and H-2Dd targets result in the rapid phosphorylation of Dap-12. However, Dap-12 is not phosphorylated when Ly-49D+ NK cells coexpress the inhibitory receptor, Ly-49G. These studies are novel in describing Ly-49 activation vs inhibition, where two Ly-49 receptors recognize the same class I ligand, with the dominant inhibitory receptor down-regulating phosphorylation of Dap-12, cytokine secretion, and cytotoxicity in NK cells.

Structure/function relationship of activating Ly-49D and inhibitory Ly-49G2 NK receptors.

Murine NK cells express Ly-49 family receptors capable of either inhibiting or activating lytic function. The overlapping patterns of expression of the various receptors have complicated their precise biochemical characterization. Here we describe the use of the Jurkat T cell line as the model for the study of Ly-49s. We demonstrate that Ly-49D is capable of delivering activation signals to Jurkat T cells even in the absence of the recently described Ly-49D-associated chain, DAP-12. Ly-49D signaling in Jurkat leads to tyrosine phosphorylation of TCRzeta and requires Syk/Zap70 family kinases and arginine 54 of Ly-49D, suggesting that Ly-49D signals via association with TCRzeta. Coexpression studies in 293-T cells confirmed the ability of Ly-49D to associate with TCRzeta. In addition, we have used this model to study the functional interactions between an inhibitory Ly-49 (Ly-49G2) and an activating Ly-49 (Ly-49D). Ly-49G2 blocks activation mediated by Ly-49D in an immunoreceptor tyrosine-based inhibitory motif (ITIM)-dependent manner. In contrast, Ly-49G2 was incapable of inhibiting activation by the TCR even though human killer cell inhibitory receptor (KIR) (KIR3DL2(GL183)) effectively inhibits TCR. Both the ability of Ly-49G2 to block Ly-49D activation and the failure of Ly-49G2 to inhibit TCR signaling were confirmed in primary murine NK cells and NK/T cells, respectively. These data demonstrate the dominant effects of the inhibitory receptors over those that activate and suggest an inability of the Ly-49 type II inhibitory receptors to efficiently inhibit type I transmembrane receptor signaling in T cells and NK cells.

Cloning and characterization of a novel activating Ly49 closely related to Ly49A.

The majority of the known Ly49 family members have been isolated from either C57BL/6 (B6) or BALB/c mice. Interestingly, the anti-Ly49 Ab reactivities observed in 129/J mice are different from those of B6 mice. Furthermore, immunoprecipitation of 129/J NK cell lysates with YE1/32 and YE1/48, Abs specific for the inhibitory Ly49A in B6, resulted in detection of the activation-associated DAP12 molecule. These results indicated a need for a more detailed study of this strain. Therefore, a cloning strategy was devised to isolate Ly49 cDNAs from 129/J mice. An immunoreceptor tyrosine-based inhibitory motif-containing, Ly49D-related clone was discovered that we have named Ly49O, and one immunoreceptor tyrosine-based inhibitory motif-lacking, Ly49A-related clone was discovered that we have named Ly49P. No anti-Ly49 mAb reacted with Ly49O, whereas the molecule encoded by the Ly49P cDNA was found to react with YE1/32 and YE1/48. Ly49P was found to associate with mouse DAP12, and Ab-mediated cross-linking of Ly49P resulted in mouse DAP12 phosphorylation and Ca2+ mobilization, indicating that Ly49P is a competent activation receptor. Ly49P, therefore, represents a novel member of the Ly49 activating receptor subfamily.

Ly-49 receptor expression and functional analysis in multiple mouse strains.

We present data on the strain distribution and functional characteristics of the Ly-49 receptors A, C/I, D, and G2 on DX5+ natural killer (NK) cells. We have examined tyrosine phosphorylation of the Ly-49 molecules, regulation of NK cytotoxic functions, and in vivo marrow rejection capability. The flow cytometry results demonstrate a diverse and complex pattern of expression of the Ly-49 receptors in the 11 strains examined. The vast majority of NK cells express Ly-49s, although some NK1.1+ CD3+ cells also express these receptors. The results of our functional analysis indicate that H-2Dd was able to inhibit the function of Ly-49G2+ NK cells, not only in B6 mice, but also by NK cells derived from several haplotypes. The examination of Ly-49 receptor tyrosine phosphorylation, which is a biochemical measure of inhibitory function, was consistently observed in the 11 mouse strains examined. In contrast, analysis of Ly-49D function suggests its expression appears to be more restricted and that H-2Dd is an activating ligand for this receptor. In addition, the in vivo examination of both inhibitory (Ly-49G2) and activating (Ly-49D) receptors demonstrated regulatory roles of these class I binding receptors in marrow transplantation.

Induction of DAP12 phosphorylation, calcium mobilization, and cytokine secretion by Ly49H.

The ability of several Ly49 family members to inhibit natural killer (NK) cell functions through recruitment of SHP-1 phosphatase has been reported. In contrast, the mechanisms underlying the activating signal generated by Ly49D are poorly understood. A homodimeric phosphoprotein (pp16) that physically and functionally associates with Ly49D has been described. In this study, a rabbit anti-mouse pp16 antiserum was generated and used to demonstrate that pp16 corresponds to the recently described DAP12 molecule. In addition, we show that a second Ly49 family member that lacks an immunoreceptor tyrosine-based inhibitory motif and contains a charged residue in the transmembrane domain, Ly49H, also associates with DAP12. Furthermore, we show that engagement of the Ly49H/DAP12 complex results in phosphorylation of DAP12, intracellular calcium mobilization, and tumor necrosis factor secretion in transfected cells. These results thus provide evidence that Ly49H is an activating receptor that associates with DAP12, previously described as a pp16 component of the Ly49D receptor complex.

The natural killer gene complex genetic locus Chok encodes Ly-49D, a target recognition receptor that activates natural killing.

Previously, we established that natural killer (NK) cells from C57BL/6 (B6), but not BALB/c, mice lysed Chinese hamster ovary (CHO) cells, and we mapped the locus that determines this differential CHO-killing capacity to the NK gene complex on chromosome 6. The localization of Chok in the NK gene complex suggested that it may encode either an activating or an inhibitory receptor. Here, results from a lectin-facilitated lysis assay predicted that Chok is an activating B6 NK receptor. Therefore, we immunized BALB/c mice with NK cells from BALB.B6-Cmv1(r) congenic mice and generated a mAb, designated 4E4, that blocked B6-mediated CHO lysis. mAb 4E4 also redirected lysis of Daudi targets, indicating its reactivity with an activating NK cell receptor. Furthermore, only the 4E4(+) B6 NK cell subset mediated CHO killing, and this lysis was abrogated by preincubation with mAb 4E4. Flow cytometric analysis indicated that mAb 4E4 specifically reacts with Ly-49D but not Ly-49A, B, C, E, G, H, or I transfectants. Finally, gene transfer of Ly-49DB6 into BALB/c NK cells conferred cytotoxic capacity against CHO cells, thus establishing that the Ly-49D receptor is sufficient to activate NK cells to lyse this target. Hence, Ly-49D is the Chok gene product and is a mouse NK cell receptor capable of directly triggering natural killing.

Positive recognition of MHC class I molecules by the Ly49D receptor of murine NK cells.

Members of the murine Ly49 family of receptors have been shown to inhibit and activate NK cell function. Subsets of Ly49-expressing NK cells mediate the rejection of bone marrow cell allografts and the lysis of allogeneic lymphoblasts. In this report we have studied Ly49-mediated positive and negative signaling in an in vitro cytotoxicity assay using sorted NK cell subsets as effectors and a panel of 51Cr-labeled Con A lymphoblasts as targets in the presence or the absence of Abs to Ly49 and/or class I molecules. Our results demonstrate that the activating receptor Ly49D delivers stimulatory signals for target cell lysis upon interacting with H2-Dd, Dr, and Dsp2, but not H2b or H2k class I Ags. On the other hand, the inhibitory receptor Ly49G2 delivers negative signals for target cell lysis upon interacting with Dd, Dr, and H2k, but not H2b or Dsp2, class I Ags. Furthermore, Ly49-mediated negative signaling dominates Ly49D-mediated positive signaling. Thus, lysis of class I MHC-bearing targets by NK cells is not merely the consequence of the absence of an Ly49-mediated negative signal, but also requires positive recognition of class I molecules by certain Ly49 receptors. Activation of NK cells by nonself class I molecules was not predicted by the missing self hypothesis.

Mouse Ly-49D recognizes H-2Dd and activates natural killer cell cytotoxicity.

Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are activated by target MHC I. The mouse Ly-49D receptor has been shown to activate NK cytotoxicity, although recognition of MHC class I has not been demonstrated previously. To define Ly-49D-ligand interactions, we transfected the mouse Ly-49D receptor into the rat NK line, RNK-16 (RNK.mLy-49D). As expected, anti- Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor- bearing rat target YB2/0 by RNK.mLy-49D transfectants. RNK.mLy-49D effectors were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb. This augmented lysis of H-2Dd targets was specifically inhibited by F(ab')2 anti-Ly-49D (12A8) and F(ab')2 anti-H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2(d) mice (BALB/c, B10.D2, and DBA/2) but not from H-2(b) or H-2(k) mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.

DAP12-mediated signal transduction in natural killer cells. A dominant role for the Syk protein-tyrosine kinase.

The murine Ly49 family contains nine genes in two subgroups: the inhibitory receptors (Ly49A, B, C, E, F, G2, and I) and the noninhibitory receptors (Ly49D and H). Unlike their inhibitory counterparts, Ly49D and H do not contain immunoreceptor tyrosine-based inhibitory motifs but associate with a recently described co-receptor, DAP12, to transmit positive signals to natural killer (NK) cells. DAP12 is also expressed in myeloid cells, but the receptors coupled to it there are unknown. Here we document the signaling pathways of the Ly49D/DAP12 complex in NK cells. We show that ligation of Ly49D results in 1) tyrosine phosphorylation of several substrates, including phospholipase Cgamma1, Cbl, and p44/p42 mitogen-activated protein kinase, and 2) calcium mobilization. Moreover, we demonstrate that although human DAP12 reportedly binds the SH2 domains of both Syk and Zap-70, ligation of Ly49D leads to activation of Syk but not Zap-70. Consistent with this observation, Ly49D/DAP12-mediated calcium mobilization is blocked by dominant negative Syk but not by catalytically inactive Zap-70. These data demonstrate the dependence of DAP12-coupled receptors on Syk and suggest that the outcome of Ly49D/DAP12 engagement will be regulated by Cbl and culminate in the activation of transcription factors.

Characterization of an associated 16-kDa tyrosine phosphoprotein required for Ly-49D signal transduction.

Ly-49D is an activating receptor on NK cells that does not become tyrosine phosphorylated upon activation. This report demonstrates that immunoprecipitation of Ly-49D, following pervanadate treatment or specific Ab cross-linking, coprecipitates a 16-kDa tyrosine-phosphorylated protein (pp16). Immunoblotting experiments and data from TCR-zeta/Fc epsilonRIgamma double knockout mice confirm that pp16 is not TCR-zeta, TCR-eta, or Fc epsilonRIgamma. Association of pp16 with Ly-49D involves a transmembrane arginine since mutation to leucine (Ly-49D[R54L]) abolishes association with pp16 in transfected P815 cells. In addition, Ly-49D(R54L) transfectants fail to mediate Ca2+ mobilization following Ab cross-linking. Therefore, signaling through Ly49D on NK cells depends on association with a distinct tyrosine phosphoprotein (pp16) in a manner analogous to that of TCR and FcR. Expression of this novel signaling peptide in both the NK and myeloid lineages indicates that pp16 is likely involved in the signal transduction cascade of additional receptor families.

The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells.

The Ly-49 gene families are class I-recognizing receptors on murine NK cells. Most Ly-49 receptors inhibit NK cell lysis upon recognizing their target class I ligands. In this report we have examined the ability of Ly-49A and Ly-49G2 to regulate T cell functions on CD3+ cells, primarily the subset that also expresses NK-1.1 and/or DX5. The majority (>50%) of T cells that express Ly-49 molecules also coexpress NK-1.1 and/or DX5, although some NK-1.1- and/or DX5-/CD3+ cells express Ly-49 molecules. Lysis of target cells by IL-2-cultured T cells expressing Ly-49A and G2 was enhanced by Abs specific for Ly-49A and G2 as well as by Abs to class I (H-2Dd alpha1/alpha2). Murine T cells also were cultured in the presence of targets that express (H-2Dd) which is inhibiting for the Ly-49A and G2 receptors. These cells were examined for a coincident increase in cytokine production (IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF). Abs to Ly-49A and G2 or their respective class I ligands blocked the negative signals mediated via the Ly-49 receptors and increased IFN-gamma and granulocyte-macrophage CSF production after interaction of these T cells with H-2Dd-expressing tumor targets. Furthermore, an EL-4 T cell line expressing both Ly-49A and G2, when treated with mAb YE148 and 4D11, demonstrated reduced cytokine production and calcium mobilization. These results demonstrate for the first time that Ly-49 class I binding receptors, previously thought to be restricted to mouse NK cells, can mediate important physiological functions of T cell subsets.

The Ly-49 family: regulation of cytokine production in murine NK cells.

Most proteins encoded by members of the Ly-49 gene family are class I-recognizing receptors on murine natural killer (NK) cells. Class I recognition by Ly-49 receptors usually results in inhibition of NK cell lysis of target cells. However, NK cells function not only in a lytic capacity, but also can mediate cytokine production. In this report we have demonstrated the ability of Ly-49A and Ly-49G2 to inhibit production of cytokines by NK cells by showing that specific antibodies against these gene products stimulate cytokine production. Murine NK cells were cultured in the presence of P815 (H2-Dd), and supernatants were analyzed for the production of interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocytemacrophage colony-stimulating factor (GM-CSF). NK cell populations were sorted for Ly-49A+ or Ly-49G+ subsets, and these subsets were analyzed for their ability to alter cytokine induction by target cell interaction. In the presence of target cells expressing the appropriate class I molecules, Ly-49A and G2 were found to inhibit cytokine induction by NK cells. Examination of mRNA for IFN-gamma and GM-CSF indicated that Ly-49 receptors increased mRNA levels of NK cells. These results demonstrate that class I binding of these NK receptors can inhibit production of important physiological cytokines, in addition to the regulation of cytotoxic activity.

Differential tyrosine phosphorylation of inhibitory versus activating Ly-49 receptor proteins and their recruitment of SHP-1 phosphatase.

Killer cell inhibitory receptors represent a family of p58/70-Ig-like proteins expressed on the surface of human NK cells. Engagement of class I MHC by killer cell inhibitory receptors turns off the lytic machinery of NK cells. This receptor/ligand interaction results in phosphorylation of intracellular tyrosine residues of p58/70 proteins. Murine NK cells express surface receptors of an unrelated family of type II lectin-like proteins, Ly-49, that have similar functions. Ly-49A, -C, and -G2 represent murine inhibitory receptors. However, Ly-49D functions as an activation receptor on the surface of NK cells. This dichotomy of function between Ly-49 family members suggested different signaling events upon receptor/ligand interaction. Here we demonstrate that: 1) in transfected Cos7 and murine NK cells, Ly-49A, -C, and -G2 are phosphorylated following pervanadate stimulation, whereas Ly-49D is not; 2) mAb-induced receptor ligation mediates tyrosine phosphorylation of Ly-49A and -G2, but not Ly-49D; 3) SHP-1 coprecipitates with Ly-49A and -G2 following receptor phosphorylation; and 4) tyrosine phosphorylation of Ly-49 inhibitory receptors depends on tyrosine residues restricted to the immunoreceptor tyrosine-based inhibitory motif. Our data further support the involvement of immunoreceptor tyrosine-based inhibitory motifs as crucial sequences regulating receptor-mediated inhibitory functions in NK cells.

Selective inhibition of human and mouse natural killer tumor recognition using retroviral antisense in primary natural killer cells: involvement with MHC class I killer cell inhibitory receptors.

The natural killer tumor recognition (NK-TR) protein has been shown to be a necessary component for the killing of NK-sensitive and virus-infected targets by the rat RNK-16 cell line. Class I-recognizing killer cell inhibitory receptors (KIR) have been found in the human (p58; NKAT family) and mouse (Ly-49 family). The principal functional characteristic of these receptors is their ability to block NK cell lysis by recognition of selected class I molecules on target cells. In the present study, we examined whether abrogation of NK-TR expression by retroviral infection of primary human or mouse NK cells with virus-producing antisense NK-TR also would demonstrate loss of non-MHC-restricted killing and whether the NK-TR was associated with KIR function in humans or with Ly-49 in the mouse. Using short term culture of fresh human or mouse NK cells, antisense NK-TR-treated NK cells demonstrated strong selective reduction of NK cytotoxicity. NK-TR was necessary for lytic activity even when KIR function was blocked by Ab in experiments involving NK3.3 lysis of HLA.cw3-expressing targets or killing of Dd targets by Ly-49A+ or Ly-49G2+ mouse NK cells. These studies extend our previous studies in rat NK cell lines to demonstrate that primary mouse and human NK cells require NK-TR for non-MHC-restricted lysis of tumor and virus-infected targets. In addition, the reversal of KIR or Ly-49 inhibition of NK cell lysis requires NK-TR expression for cellular killing in both human and mouse.

The Ly-49D receptor activates murine natural killer cells.

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immuno-precipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I-bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc gamma R+ target cells in a reverse antibody-dependent cellular cytotoxicity-type assay and induces apoptosis in Ly-49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

Cloning and functional characteristics of murine large granular lymphocyte-1: a member of the Ly-49 gene family (Ly-49G2)

Large granular lymphocyte (LGL) 1 is a cell surface glycoprotein expressed on a subset (50%) of C57BL/6 natural killer (NK) cells. Immunoprecipitation experiments reveal that the LGL-1 protein exists as a disulfide-linked 40-kD homodimer. Functional studies of LGL-1+ cells indicate that selected H-2d target cells are not lysed efficiently by these interleukin (IL)-2-cultured NK cells. These findings suggested that LGL-1 may be a member of the Ly-49 gene family. Here we report the molecular cloning of the LGL-1 cDNA from a severe combined immunodeficient-adherent lymphokine-activated killer cell library transfected into Cos-7 cells and find LGL-1 to be homologous to the Ly-49 gene at both the nucleotide (85%) and amino acid levels (73%). Sequencing of our LGL-1 cDNA has revealed it to be nearly identical to the Ly-49G2 cDNA recently isolated by cross-hybridization with an Ly-49 probe. LGL-1 represents a type II transmembrane protein of 267 amino acids with its carboxyl end exposed extracellularly. The LGL-1 protein contains 11 highly conserved cysteine residues and a 25-amino acid transmembrane region. Southern blot analysis demonstrates that there are a number of homologous genes in mouse DNA that hybridize strongly to LGL-1. Northern analyses using poly A+ RNA from LGL-1+ NK cells indicate that LGL-1 is expressed as a 1.4 kb mRNA. Two-color flow cytometry analysis (FCA) of C57BL/6 splenic NK cells demonstrates that LGL-1 and Ly-49 label overlapping subsets of cells. FCA identifies four subsets of NK cells as defined by LGL-1 versus Ly-49 staining. We have sorted these individual subsets, expanded them in IL-2, and performed cytotoxicity experiments to determine their target cell profiles in relation to class I expression. Results of these studies are complex, but indicate that Ly-49 may not be the only molecule that recognizes class I as an inhibitory signal for cytotoxicity. LGL-1+ cells also fail to lyse several H-2d-expressing tumor targets and concanavalin A lymphoblasts from BALB/c but not C57BL/6 mice. This inhibition of lysis by LGL-1+ NK cells is negated by addition of monoclonal antibody (mAb) 4D11 that recognizes the LGL-1 protein. When mAbs to the class I molecules H-2Dd and H-2Ld (alpha 1 alpha 2 domains only) are added to cytotoxicity assays, LGL-1+ cells lyse H-2d targets very effectively.(ABSTRACT TRUNCATED AT 250 WORDS)

LGL-1: a potential triggering molecule on murine NK cells.

Natural killer (NK) cells mediate non-major histocompatibility complex-restricted lysis of tumor cells, lymphokine-activated killing (LAK), antibody-dependent cellular cytotoxicity (ADCC), and reverse ADCC (RADCC). LGL-1+ cells identify a major subset (50%) of murine NK cells. Here we demonstrate that monoclonal antibodies (mAbs) to LGL-1 consistently induce interleukin-2-cultured, and Corynebacterium parvum (in vivo)-activated NK cells to induce RADCC. LGL-1 triggering of activated NK cells coincides with enhanced LGL-1 expression. Testing of murine mAbs to epitopes of CD2 only appears to augment RADCC induced by mAb NK-1.1 on fresh NK cells. Immunoprecipitation of the LGL-1 antigen reveals a highly disulfide-linked 40-kDa homodimer subunit that is N-glycosylated. Therefore, LGL-1 may be similar to other recently characterized NK-associated antigens such as NK-1.1, Ly-49, and NKR-PI. We conclude that although LGL-1 is expressed on "resting" NK cells, enhanced surface expression following activation is usually required for it to act as a signaling molecule.

Substrate-specific proteases (BLT-esterase) are localized predominantly in the natural killer cells of unprimed mice.

In leukocytes isolated from unprimed mice, the levels of extractable N alpha-Cbz-Lys-thiobenzylesteresterase (BLT-esterase) closely correlated with the number of natural killer (NK) cells. The spleens of mice that exhibit severe combined immunodeficiency (SCID) contained much higher levels of this enzyme than other mouse strains. Treatments that resulted in a local accumulation of NK cells (as assessed by lytic activity) produced a concomitant increase in BLT-esterase activity. However, short-term in vitro treatment of spleen cells with interferon (IFN)-alpha/beta indicated that BLT-esterase levels correlated more closely with absolute numbers of NK cells than with their lytic capacity. There was a very good correlation between the numbers of cells bearing the NK phenotype (NK-1.1+) and BLT-esterase levels. Cells positively sorted using the NK-specific antibodies NK-1.1 and LGL-1 had high enzymatic activity. The BLT-esterase levels were high in both the NK-1.1+/LGL-1- and NK-1.1+/LGL-1+ subsets. Highly purified CD4+ and CD8+ T cells and sIg+ B cells demonstrated negligible enzyme, as did populations of cells highly enriched for macrophages or neutrophils. However, it should be stressed that the inbred mice used on this study have been maintained in a pathogen-free facility. It would be anticipated that mice maintained under less stringent conditions could exhibit appreciable levels of BLT-esterase activity in their T cells. Nonetheless, BLT-esterase is present at high levels in NK cells and cannot be regarded as a T cell-specific enzyme.

Natural killer (NK) cell subsets in the mouse. NK-1.1+/LGL-1+ cells restricted to lysing NK targets, whereas NK-1.1+/LGL-1- cells generate lymphokine-activated killer cells.

Our laboratory has recently identified a novel Ag, LGL-1, that is expressed on a major population of mouse NK cells. Two color immunofluorescence analysis has demonstrated that spleen cells consist of two major subsets of NK cells. We have identified an NK-1.1+/LGL-1+ subset that consists of 50% of the total NK cells and an NK-1.1+/LGL-1- subset comprising the remaining 50%. Because numerous reports have identified NK cells as the major cell type mediating lymphokine-activated killing (LAK), the NK-1.1+/LGL-1+ and NK-1.1+/LGL-1- subsets were examined for their contribution toward LAK generation, as defined by their ability to lyse P815 tumor targets. Antibody plus C depletion experiments with the use of anti-LGL-1 indicated that LGL-1+ cells were not found on LAK precursor or effector cells. Two-color cell sorting experiments were also performed to separate freshly isolated NK-1.1+/LGL-1+ spleen cells from the NK-1.1+/LGL-1- subset. It was found that the vast majority of LAK activity (greater than 95%) is derived from the NK-1.1+/LGL-1- cells. Cell sorting of LAK effectors also demonstrated that the NK-1.1+/LGL-1- cells mediated the vast majority of lysis against P815 targets. Similar results were obtained when NK cell subsets were analyzed for their contribution toward ADCC. These findings may prove important in understanding and further elucidating the contribution of NK cells to the LAK phenomenon. Our data also indicates that subsets of NK cells exist that may function differently in response to stimulation by various lymphokines and cytokines.