NKR-P1, an activating molecule on rat natural killer cells, stimulates phosphoinositide turnover and a rise in intracellular calcium

NKR-P1 is a 60-kDa homodimer expressed on all rat NK cells. Previous studies by others suggest that NKR-P1 may play a role in NK cell activation because antibody to NKR-P1 stimulates the release of granules from NK cells, and anti-NKR-P1 causes redirected lysis by activated NK cells against targets that express FcR. To examine the mechanism of transmembrane signaling by NKR-P1, we studied the rat NK cell line, RNK-16. We here demonstrate that F(ab')2 antibody to NKR-P1 stimulates phosphoinositide turnover and a rise in intracellular calcium within RNK-16 cells. The response is augmented by cross-linking the F(ab')2 antibody. The phosphoinositide/calcium pathway is also stimulated by NKR-P1 in activated rat NK cells, although no response is detectable in polymorphonuclear cells, which also express NKR-P1. We also demonstrate that RNK-16 cells kill the anti-NKR-P1 (3.2.3) hybridoma and that exposure to the hybridoma target cells stimulates phosphoinositide turnover in RNK-16 cells. Both killing and phosphoinositide turnover are inhibited by F(ab')2 anti-NKR-P1, implicating NKR-P1 in both responses. In contrast, neither cytotoxicity nor phosphoinositide turnover is appreciably blocked by F(ab')2 anti-NKR-P1 in response to YAC-1 targets. Thus, with either target, killing is linked to phosphoinositide turnover, but killing of YAC-1 involves pathways that differ from those that direct killing of the anti-NKR-P1 hybridoma. Our studies support the hypothesis that NKR-P1 may serve as an activating cell-surface receptor on NK cells, and they clarify the mechanisms by which it activates NK cells.     

The NKR-P1B gene product is an inhibitory receptor on SJL/J NK cells

The mouse NKR-P1 family includes at least three genes: NKR-P1A, -B, -C. Neither surface expression nor function of the NKR-P1B gene product has previously been shown. Here, we demonstrate that the SJL/J allele of the NKR-P1B gene product is expressed on SJL/J NK cells, and is recognized by PK136 mAb. Interestingly, the same mAb does not recognize the NKR-P1B gene product of C57BL/6. We have also generated a novel mAb, 1C10, that recognizes an activation receptor on SJL/J NK cells. Activation of the NKR-P1B receptor-inhibited 1C10 mAb induced redirected lysis and recruited SHP-1, indicating that NKR-P1B is an inhibitory receptor. Therefore, the mouse NKR-P1 gene family, like the Ly49 family, includes both activation and inhibitory receptors.     

A novel NKR-P1B(bright) NK cell subset expresses an activated CD25(+)CX(3)CR1(+)CD62L(-)CD11b(-)CD27(-) phenotype and is prevalent in blood, liver, and gut-associated lymphoid organs of rats

The inhibitory NKR-P1B receptor identifies a subset of rat splenic NK cells that is low in Ly49 receptors but enriched for CD94/NKG2 receptors. We report in this study a novel NKR-P1B(bright) NK subpopulation that is prevalent in peripheral blood, liver, and gut-associated lymphoid organs and scarce in the spleen, peripheral lymph nodes, bone marrow, and lungs. This NKR-P1B(bright) NK subset displays an activated phenotype, expressing CD25, CD93, CX(3)CR1 and near absence of CD62-L, CD11b, and CD27. Functionally, NKR-P1B(bright) NK cells are highly responsive in terms of IFN-γ production and exert potent cytolytic activity. They show little spontaneous proliferation, are reduced in numbers upon in vivo activation with polyinosinic:polycytidylic acid, and have poor survival in ex vivo cytokine cultures. Our findings suggest that NKR-P1B(bright) NK cells are fully differentiated effector cells that rapidly die upon further activation. The identification of this novel rat NK cell subset may facilitate future translational research of the role of distinct NK cell subsets under normal physiological conditions and during ongoing immune responses.     

Association of human NK cell surface receptors NKR-P1 and CD94 with Src-family protein kinases

 Human natural killer (NK) cells express on their surface several members of the C-type lectin family such as NKR-P1, CD94, and NKG2 that are probably involved in recognition of target cells and delivery of signals modulating NK cell cytotoxicity. To elucidate the mechanisms involved in signaling via these receptors, we solubilized in vitro cultured human NK cells by a mild detergent, Brij-58, immunoprecipitated molecular complexes containing the NKR-P1 or CD94 molecules, respectively, by specific monoclonal antibodies, and performed in vitro kinase assays on the immunoprecipitates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and phospho-amino acid analysis revealed the presence of in vitro tyrosine phosphorylated proteins that were subsequently identified by re-precipitation (and/or by western blotting) as the respective C-type lectin molecules and Src family kinases Lck, Lyn, and Fyn. The NKR-P1 and the CD94-containing complexes were independent of each other and both very large, as judged by Sepharose 4B gel chromatography. Crosslinking of NKR-P1 on the cell surface induced transient in vivo tyrosine phosphorylation of cellular protein substrates. These results indicate involvement of the associated Src-family kinases in signaling via the NKR-P1 and CD94 receptors. Received: 4 February 1997 / Revised: 28 February 1997     

Characterization and utilization of a monoclonal antibody inhibiting porcine natural killer cell activity for isolation of natural killer and killer cells

A mAb, porcine NK-inhibitory mAb (PNK-I) that inhibits porcine NK activity without affecting antibody-dependent cellular cytotoxicity (ADCC) has been developed. PNK-I acts at the level of the effector cell and inhibition of NK activity is independent of complement. Inhibitory effects are seen against various human and murine NK-susceptible targets. Addition of PNK-I antibody up to 60 min after assay initiation was effective at inhibiting NK activity. Furthermore PNK-I does not inhibit E:T conjugation and inhibits during the Ca2(+)-dependent phase of NK cytolysis. PNK-I Ag is present on virtually all PBL showing a bimodal distribution with 74% "dim" and 15% "bright" by flow cytometry. Monocytes and granulocytes stain with an intermediate intensity with greater than 90% and 95% staining positively, respectively. F(ab')2 fragments of PNK-I antibody show identical staining and functional activity as the whole molecule indicating that PNK-I acts independently of FcR. PNK-I immunoprecipitates molecules of molecular mass of 166, 155, 95 kDa under reducing and nonreducing conditions. PNK-I appears to be recognizing an epitope on a CD18 molecule. The CD18 molecule (beta-chain of CD11a,b,c) is ubiquitous on the surface of leukocytes and is implicated in a variety of cellular functions. Dim and bright populations were sorted and assessed functionally for NK and ADCC activity. It is demonstrated that PNK-I+ bright lymphocytes contain all detectable NK and ADCC activity in porcine PBL. Furthermore PNK-I+ bright lymphocytes contain the cytokine responsive NK cells capable of stimulation by IL-2, porcine NK-activating factor, and porcine natural killer-enhancing mAb. PNK-I+ dim cells were devoid of all baseline as well as inducible NK and ADCC activity. Giemsa stain of sorted populations show PNK-I+ bright cells containing the large granular lymphocytes whereas dim are devoid of these. Two color analysis show that PT4+ cells are PNK-I+ dim whereas PT8+ lymphocytes are divided between PNK-I+ bright and dim populations. Our results indicate that we are able to isolate all active as well as inducible NK and ADCC effector cells from porcine PBL based on relative Ag expression of CD18. Therefore quantitative as well as qualitative antigen expression is important in NK/ADCC-mediated cytotoxicity.     

Phylogenetic and functional conservation of the NKR-P1F and NKR-P1G receptors in rat and mouse

Two clusters of rat Nkrp1 genes can be distinguished based on phylogenetic relationships and functional characteristics. The proximal (centromeric) cluster encodes the well-studied NKR-P1A and NKR-P1B receptors and the distal cluster, the largely uncharacterized, NKR-P1F and NKR-P1G receptors. The inhibitory NKR-P1G receptor is expressed only by the Ly49s3⁺ NK cell subset as detected by RT-PCR, while the activating NKR-P1F receptor is detected in both Ly49s3⁺ and NKR-P1B⁺ NK cells. The mouse NKR-P1G ortholog is expressed by both NKR-P1D⁻ and NKR-P1D⁺ NK cells in C57BL/6 mice. The rat and mouse NKR-P1F and NKR-P1G receptors demonstrate a striking, cross-species conservation of specificity for Clr ligands. NKR-P1F and NKR-P1G reporter cells reacted with overlapping panels of tumour cell lines and with cells transiently transfected with rat Clr2, Clr3, Clr4, Clr6 and Clr7 and mouse Clrc, Clrf, Clrg and Clrd/x, but not with Clr11 or Clrb, which serve as ligands for NKR-P1 from the proximal cluster. These data suggest that the conserved NKR-P1F and NKR-P1G receptors function as promiscuous receptors for a rapidly evolving family of Clr ligands in rodent NK cells.     

Functional requirements for signaling through the stimulatory and inhibitory mouse NKR-P1 (CD161) NK cell receptors

The NK cell receptor protein 1 (NKR-P1) (CD161) molecules represent a family of type II transmembrane C-type lectin-like receptors expressed predominantly by NK cells. Despite sharing a common NK1.1 epitope, the mouse NKR-P1B and NKR-P1C receptors possess opposing functions in NK cell signaling. Engagement of NKR-P1C stimulates cytotoxicity of target cells, Ca2+ flux, phosphatidylinositol turnover, kinase activity, and cytokine production. In contrast, NKR-P1B engagement inhibits NK cell cytotoxicity. Nonetheless, it remains unclear how different signaling outcomes are mediated at the molecular level. Here, we demonstrate that both NKR-P1B and NKR-P1C associate with the tyrosine kinase, p56(lck). The interaction is mediated through the di-cysteine CxCP motif in the cytoplasmic domains of NKR-P1B/C. Disrupting this motif leads to abrogation of both stimulatory and inhibitory NKR-P1 signals. In addition, mutation of the consensus ITIM (LxYxxL) in NKR-P1B abolishes both its Src homology 2-containing protein tyrosine phosphatase-1 recruitment and inhibitory function. Strikingly, engagement of NKR-P1C on NK cells obtained from Lck-deficient mice failed to induce NK cytotoxicity. These results reveal a role for Lck in the initiation of NKR-P1 signals, and demonstrate a requirement for the ITIM in NKR-P1-mediated inhibition.     

In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.     

Murine trophoblast can be killed by lymphokine-activated killer cells

The ability of fetal trophoblast cells in the placenta to resist cell-mediated lysis may be important for successful pregnancy. Previous studies in this laboratory demonstrated that cultured midterm mouse trophoblast cells are not susceptible to allospecific CTL generated by standard in vitro protocols, to antibody-dependent cell-mediated cytotoxicity, or to naive or IFN-activated NK cells, despite expressing the requisite target structures. However, we now report that murine trophoblast can be killed, in a non-MHC-specific manner, by LAK cells. Normal mouse spleen cells cultured for 4 days in IL-2-containing lymphokine preparations characteristically killed both NK-sensitive (YAC-1) and NK-resistant (EL4, P815) target cells, and mediated significant lysis of both cultured and freshly isolated trophoblast cells (35 to 55%, E/T 100/1). Pretreatment of the LAK cells with anti-ASGM1 antibody and C markedly reduced the lysis of trophoblast and YAC-1 targets, suggesting that the responsible cells belonged to the NK lineage. The ability of IL-2-activated NK cells to kill midterm murine trophoblast cells was confirmed using a population of highly lytic NK cells generated by culturing spleen cells from severe combined immunodeficiency mice in 500 U/ml rIL-2 for 5 days. These effector cells killed YAC-1, EL4 and P815 target cells at much lower E/T ratios than was achieved with the normal splenic LAK cells, and mediated significant lysis of both freshly isolated (45 to 50%, E/T 20/1) and cultured trophoblast cells (68 to 76%, E/T 20/1). The susceptibility of trophoblast to LAK cells and IL-2-activated NK cells supports the need for suppressor mechanisms regulating IL-2 activity at the maternal-fetal interface.     

Effects of cyclosporin on C57BL/6 splenocytes before and after culture with high-dose recombinant interleukin-2: Implications for immunosuppression with cyclosporin

Summary Lymphokine-activated killer cells appear to arise from precursor cells bearing natural killer (NK) cell antigens. Cyclosporin (CsA) is a well-known immunosuppressive agent that can down-regulate NK cell cytotoxicity. Studies were initiated to evaluate the effects of CsA on splenocytes before and after exposure to recombinant interleukin-2 (rIL-2). Normal C57BL/6 mice receiving CsA at a dose of 100 mg/kg demonstrated a decrease in NK cell lysis against the YAC-1 lymphoma target in a 4-h chromium-release assay. When splenocytes obtained from CsA-treated mice were cultured for 3 days in complete medium containing 1000 U rIL-2/ml, they demonstrated a return of NK cell lysis to normal (mean cytotoxicity = 65 LU versus 60 LU for control and CsA-exposed splenocytes respectively;P, NS, five consecutive experiments) but revealed a decrease in the lysis of a NK-resistant target: the MCA-102 sarcoma (mean cytotoxicity = 20 LU vs 12 LU for control and CsA-exposed splenocytes respectively;P <0.02, five consecutive experiments). Fresh splenocytes cultured in media containing rIL-2 and CsA demonstrated a decrease in proliferation, cell-cycle S-phase fraction and cell yields compared to splenocytes cultured in media containing rIL-2 alone. In addition, a decrease in tumor cell lysis for NK-cell sensitive (mean percentage lysis = 98% vs 60%, rIL-2 vs rIL-2 + CsA; effector-to-target ratio 100: 1) and resistant targets (mean percentage lysis = 68% vs 28%, rIL-2 vs rIL-2 + CsA; effector-to-target ratio 100: 1) was also seen. CsA had no effects on the phenotypic antigenic expression of splenocytes cultured with high-dose rIL-2 although activated T cell antigens were down-regulated when fresh splenocytes were evaluated after in vivo exposure to CsA. These studies support the down-regulating effects of CsA on NK cell lysis and suggest that the rIL-2-activated cell population is heterogeneous as demonstrated by the differential down-regulation and recovery of NK-resistant cell lysis versus NK-sensitive cell lysis.     

CD56brightCD16+ NK cells: a functional intermediate stage of NK cell differentiation

Human NK cells comprise two main subsets, CD56(bright) and CD56(dim) cells, which differ in function, phenotype, and tissue localization. To further dissect the differentiation from CD56(bright) to CD56(dim) cells, we performed ex vivo and in vitro experiments demonstrating that the CD56(bright)CD16(+) cells are an intermediate stage of NK cell maturation. We observed that the maximal frequency of the CD56(bright)CD16(+) subset among NK cells, following unrelated cord blood transplantation, occurs later than this of the CD56(bright)CD16(-) subset. We next performed an extensive phenotypic and functional analysis of CD56(bright)CD16(+) cells in healthy donors, which displayed a phenotypic intermediary profile between CD56(bright)CD16(-) and CD56(dim)CD16(+) NK cells. We also demonstrated that CD56(bright)CD16(+) NK cells were fully able to kill target cells, both by Ab-dependent cell cytotoxicity (ADCC) and direct lysis, as compared with CD56(bright)CD16(-) cells. Importantly, in vitro differentiation experiments revealed that autologous T cells specifically encourage the differentiation from CD56(bright)CD16(-) to CD56(bright)CD16(+) cells. Finally, further investigations performed in elderly patients clearly showed that both CD56(bright)CD16(+) and CD56(dim)CD16(+) mature subsets were substantially increased in older individuals, whereas the CD56(bright)CD16(-) precursor subset was decreased. Altogether, these data provide evidence that the CD56(bright)CD16(+) NK cell subset is a functional intermediate between the CD56(bright) and CD56(dim) cells and is generated in the presence of autologous T CD3(+) cells.     

Genomic structure and strain-specific expression of the natural killer cell receptor NKR-P1.

NK cells are able to lyse a variety of virally infected and neoplastic cells in an MHC-unrestricted manner. The cell-surface protein NKR-P1 is thought to play a key role in this process. NKR-P1, initially identified in rat IL-2 activated NK cells, is encoded in the mouse by at least three similar, but not identical, genes. We previously reported the isolation and characterization of three different NKR-P1 cDNA, termed cDNA 2, 34, and 40, from IL-2 activated mouse NK cells. This report describes the structure of the gene encoding NKR-P1 cDNA 2, the smallest of these three cDNA. Gene 2 is composed of six exons spanning approximately 14 kb of genomic DNA. The first exon encodes the N-terminal intracellular domain, and exons 4, 5, and 6 contain the sequences coding for the CRD. This organization is similar to that of other genes that encode C-type animal lectins. The expression of the NKR-P1 genes in A-LAK cells from 13 mouse strains was examined by Northern blot analysis. NKR-P1 expression appears to coincide with that of the NK1.1 Ag. This observation further supports the hypothesis that the NK1.1 Ag is encoded by one of the NKR-P1 genes. Nucleotide sequence analysis of the promoter region of the three NKR-P1 genes in BALB/c and C57BL/6 mice suggests that differences in the level of expression probably do not result from alterations in the upstream regions of these genes, but may be caused by the expression of strain-specific transacting factors.     

Cytotoxicity of CD56(bright) NK cells towards autologous activated CD4+ T cells is mediated through NKG2D, LFA-1 and TRAIL and dampened via CD94/NKG2A

In mouse models of chronic inflammatory diseases, Natural Killer (NK) cells can play an immunoregulatory role by eliminating chronically activated leukocytes. Indirect evidence suggests that NK cells may also be immunoregulatory in humans. Two subsets of human NK cells can be phenotypically distinguished as CD16(+)CD56(dim) and CD16(dim/-)CD56(bright). An expansion in the CD56(bright) NK cell subset has been associated with clinical responses to therapy in various autoimmune diseases, suggesting an immunoregulatory role for this subset in vivo. Here we compared the regulation of activated human CD4(+) T cells by CD56(dim) and CD56(bright) autologous NK cells in vitro. Both subsets efficiently killed activated, but not resting, CD4(+) T cells. The activating receptor NKG2D, as well as the integrin LFA-1 and the TRAIL pathway, played important roles in this process. Degranulation by NK cells towards activated CD4(+) T cells was enhanced by IL-2, IL-15, IL-12+IL-18 and IFN-α. Interestingly, IL-7 and IL-21 stimulated degranulation by CD56(bright) NK cells but not by CD56(dim) NK cells. NK cell killing of activated CD4(+) T cells was suppressed by HLA-E on CD4(+) T cells, as blocking the interaction between HLA-E and the inhibitory CD94/NKG2A NK cell receptor enhanced NK cell degranulation. This study provides new insight into CD56(dim) and CD56(bright) NK cell-mediated elimination of activated autologous CD4(+) T cells, which potentially may provide an opportunity for therapeutic treatment of chronic inflammation.     

cDNA cloning of mouse NKR-P1 and genetic linkage with LY-49. Identification of a natural killer cell gene complex on mouse chromosome 6.

NK cells lyse tumor cells and virally infected cells, but the molecular basis for this phenomenon has not been defined. A mAb specific for the rat cell surface molecule, NKR-P1, stimulates rat NK cell lytic activity and is reactive with all rat NK cells, suggesting that this molecule may play a significant role in NK cell function. We have previously described another NK cell-specific Ag, Ly-49, that belongs to a family of cross-hybridizing genes on distal mouse chromosome 6. The rat NKR-P1 Ag shares several features with the mouse Ly-49 Ag, including selective cell surface expression on NK cells, homology to the C-type lectins, expression as a type II integral membrane protein, and disulfide-linked homodimeric structure. To further examine the relationship of NKR-P1 to Ly-49, we have cloned the cDNA encoding a mouse homologue of NKR-P1 (mNKR-P1). The mouse and rat NKR-P1-deduced polypeptide sequences are highly conserved, suggesting a similar tertiary structure. By examination of DNA from informative recombinant inbred mice with Southern blot analysis, we have determined that mNKR-P1 is encoded by a distinct gene that is genetically linked to the Ly-49 locus, lying within 0.5 centi-Morgan (cM) of Ly-49. Although the deduced amino acid sequences of mNKR-P1 and Ly-49 reveal that these proteins are structurally similar, they are only 24% identical at the amino acid level and the cDNA sequences do not demonstrate significant nucleotide homology. Our studies suggest that we have identified a region on mouse chromosome 6 that includes distinct NK-specific genes that encode structurally related proteins (type II integral membrane proteins, C-type lectin super-gene family) but which demonstrate considerable heterogeneity. We have termed this genetic region the NK complex.     

The novel inhibitory NKR-P1C receptor and Ly49s3 identify two complementary, functionally distinct NK cell subsets in rats

The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors which in mice bind members of the genetically linked C-type lectin-related family, while the distal region encodes Ly49 receptors for polymorphic MHC class I molecules. Although certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique functional characteristics. NKR-P1C(+) NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and functionally recognize a nonpolymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHC-mismatched lymphoblast target cells. The NKR-P1C(+) NK cell subset also appears earlier during development and shows a tissue distribution distinct from its complementary Ly49s3(+) subset, which expresses a wide range of Ly49 receptors. These data suggest the existence of two major, functionally distinct populations of rat NK cells possessing very different killer cell lectin-like receptor repertoires.     

Natural killer cell lytic activity and CD56(dim) and CD56(bright) cell distributions during and after intensive training.

The purpose of this study was to examine the impact of intensive training for competitive sports on natural killer (NK) cell lytic activity and subset distribution. Eight female college-level volleyball players undertook 1 mo of heavy preseason training. Volleyball drills were performed 5 h/day, 6 days/wk. Morning resting blood samples were collected before training (Pre), on the 10th day of training (During), 1 day before the end of training (End), and 1 wk after intensive training had ceased (Post). CD3(-)CD16(bright)CD56(dim) (CD56(dim) NK), CD3(-)CD16(dim/-)CD56(bright) NK (CD56(bright) NK), and CD3(+)CD16(-)CD56(dim) (CD56(dim) T) cells in peripheral blood were determined by flow cytometry. The circulating count of CD56(dim) NK cells (the predominant population, with a high cytotoxicity) did not change, nor did the counts for other leukocyte subsets. However, counts for CD56(bright) NK and CD56(dim) T cells (subsets with a lower cytotoxicity) increased significantly (P < 0.01) in response to the heavy training. Overall NK cell cytotoxicity decreased from Pre to End (P = 0.002), with a return to initial values at Post. Lytic units per NK cell followed a similar pattern (P = 0.008). Circulating levels of interleukin-6, interferon-gamma, and tumor necrosis factor-alpha remained unchanged. These results suggest that heavy training can decrease total NK cell cytotoxicity as well as lytic units per NK cell. Such effects may reflect in part an increase in the proportion of circulating NK cells with a low cytotoxicity.