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.     

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.     

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.     

CD161 (human NKR-P1A) signaling in NK cells involves the activation of acid sphingomyelinase

NK and NKT cells play a major role in both innate immunity and in influencing the development of adaptive immune responses. CD161 (human NKR-P1A), a protein encoded in the NK gene complex, is a major phenotypic marker of both these cell types and is thought to be involved in the regulation of NK and NKT cell function. However, the mechanisms of action and signaling pathways of CD161 are poorly understood. To identify molecules able to interact with the cytoplasmic tail of human CD161 (NKR-P1A), we have conducted a yeast two-hybrid screen and identified acid sphingomyelinase as a novel intracellular signaling pathway linked to CD161. mAb-mediated cross-linking of CD161, in both transfectants and primary human NK cells, triggers the activation of acid, but not neutral sphingomyelinase. The sphingomyelinases represent the catabolic pathway for N-acyl-sphingosine (ceramide) generation, an emerging second messenger with key roles in the induction of apoptosis, proliferation, and differentiation. These data therefore define a novel signal transduction pathway for the CD161 (NKR-P1A) receptor and provide fresh insights into NK and NKT cell biology.     

Mouse NKR-P1B, a novel NK1.1 antigen with inhibitory function

The mouse NK1.1 Ag originally defined as NK cell receptor (NKR)-P1C (CD161) mediates NK cell activation. Here, we show that another member of the mouse CD161 family, NKR-P1B, represents a novel NK1.1 Ag. In contrast to NKR-P1C, which functions as an activating receptor, NKR-P1B inhibits NK cell activation. Association of NKR-P1B with Src homology 2-containing protein tyrosine phosphatase-1 provides a molecular mechanism for this inhibition. The existence of these two NK1.1 Ags with opposite functions suggests a potential role for NKR-P1 molecules, such as those of the Ly-49 gene family, in regulating NK cell function.     

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.     

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.     

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     

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells,B Cells and NKT Cells

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450–463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment with low-dose interleukins themselves or in combination with hsp70 derived (TKD) peptide.     

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.