Lise Kveberg

Sphingosine 1 phosphate induces the chemotaxis of human natural killer cells. Role for heterotrimeric G proteins and phosphoinositide 3 kinases

We have examined the effect of sphingolipids on the chemotaxis of human natural killer (NK) cells. Messenger RNA for Edg‐1, Edg‐6 and Edg‐8 but not Edg‐3, are expressed in these cells. Sphingosine 1 phosphate (SPP), dihydro SPP (DHSPP) or the CC chemokine RANTES (CCL5), but not sphingosine induces the chemotaxis of these cells. Pertussis toxin inhibits the chemotaxis induced by these ligands. Permeabilization of NK cells with streptolysin O (SLO) and introduction of blocking antibodies to the heterotrimeric G proteins, showed that Gαi2, Gαs, Gαq/11 or Gα13 mediate the chemotaxis of SPP, whereas Gαi2, Gαo or Gαq/11 mediate the chemotaxis of DHSPP. Gαi2, Gαo, Gαs, Gαq/11, Gαz or Gα12 mediates RANTES‐induced NK cell chemotaxis. Further analysis showed that phosphoinositide 3 kinase (PI3K) inhibitors wortmannin and LY294002 inhibit NK cell chemotaxis induced by SPP, DHSPP or RANTES. Blocking antibody to PI3Kγ inhibits the chemotaxis induced by the three ligands, whereas anti‐PI3Kβ was without effect. In contrast, SPP and DHSPP recruit PI3Kβ isozyme into NK cell membranes, suggesting that although this isoform is not involved in chemotaxis, it is activated by these phospholipids.

Ly-49s3 Is a Promiscuous Activating Rat NK Cell Receptor for Nonclassical MHC Class I-Encoded Target Ligands

Previous studies of the rapid rejection of MHC-disparate lymphocytes in rats, named allogeneic lymphocyte cytotoxicity, have indicated that rat NK cells express activating receptors for nonclassical MHC class I allodeterminants from the RT1-C/E/M region. Using an expression cloning system that identifies activating receptors associated with the transmembrane adapter molecule DAP12, we have cloned a novel rat Ly-49 receptor that we have termed Ly-49 stimulatory receptor 3 (Ly-49s3). A newly generated anti-Ly-49s3 Ab, mAb DAR13, identified subpopulations of resting and IL-2-activated NK cells, but not T or B lymphocytes. Depletion of Ly-49s3-expressing NK cells drastically reduced alloreactivity in vitro, indicating that this subpopulation is responsible for a major part of the observed NK alloreactivity. DAR13-mediated blockade of Ly-49s3 inhibited killing of MHC-congenic target cells from the av1, n, lv1, and c haplotypes, but not from the u or b haplotypes. A putative ligand was mapped to the nonclassical MHC class I region (RT1-C/E/M) using intra-MHC recombinant strains. Relative numbers of Ly-49s3+ NK cells were reduced, and surface levels of Ly-49s3 were lower, in MHC congenic strains expressing the putative Ly-49s3 ligand(s). In conclusion, we have identified a novel Ly-49 receptor that triggers rat NK cell-mediated responses.

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 subsets in man and rodents.

NK cells are important contributors to the early immune defence against infected or transformed cells. They are rapidly activated in response to cytokines, whereby they exert their effector functions. NK cell responses are controlled by a multitude of receptors, which are expressed by subpopulations of NK cells with distinct phenotypes and functionalities. Direct comparisons between species are often difficult because of differences in the expression of NK cell receptors and other markers. In addition, NK cells change their phenotype and effector functions during differentiation, by tissue-specific factors, or upon activation, complicating interpretations. We will here review the similarities and differences between the major NK cell subsets in man and two well-characterized rodent models.

Two major groups of rat NKR-P1 receptors can be distinguished based on chromosomal localization, phylogenetic analysis and Clr ligand binding.

A major subset of non‐alloreactive NK cells in PVG strain rats is generally low in Ly49 receptors, but expresses the rat NKR‐P1BPVG receptor (previously termed NKR‐P1C). The NKR‐P1B+ NK subset is inhibited by a non‐polymorphic target cell ligand, which we have shown here to be a C‐type lectin‐related molecule (Clr). Clr11 ligates two divergent NKR‐P1B alleles as judged by an NFAT‐driven reporter assay, and inhibits NK‐cell cytotoxicity of NKR‐P1B+ NK cells. Clr11 also interacts with the prototypic NKR‐P1A receptor and exerts a stimulatory influence on NK lysis. NKR‐P1A and B are encoded by adjacent genes in the proximal part of the NK gene complex and show close sequence homology in their extracellular region. They diverge from another pair, NKR‐P1F and ‐G, which is encoded by a second, distal Nkrp1 gene cluster. NKR‐P1F and ‐G bind an overlapping panel of Clr ligands, but not Clr11. Rat Clr molecules appear to be constitutively expressed by hematopoietic cells; expression in tumor cell lines is more variable. The data show the existence of two phylogenetic groups of NKR‐P1 molecules, which demonstrate conservation of ligand‐binding properties independent of signaling function.

Two Structurally Related Rat Ly49 Receptors with Opposing Functions (Ly49 Stimulatory Receptor 5 and Ly49 Inhibitory Receptor 5) Recognize Nonclassical MHC Class Ib-Encoded Target Ligands

The Ly49 family of lectin-like receptors in rodents includes both stimulatory and inhibitory members. Although NK alloreactivity in mice is regulated primarily by inhibitory Ly49 receptors, in rats activating Ly49 receptors are equally important. Previous studies have suggested that activating rat Ly49 receptors are triggered by polymorphic ligands encoded within the nonclassical class Ib region of the rat MHC, RT1-CE/N/M, while inhibitory Ly49 receptors bind to widely expressed classical class Ia molecules encoded from the RT1-A region. To further investigate rat Ly49-mediated regulation of NK alloreactivity, we report in this study the identification and characterization of two novel paired Ly49 receptors that we have termed Ly49 inhibitory receptor 5 (Ly49i5) and Ly49 stimulatory receptor 5 (Ly49s5). Using a new mAb (mAb Fly5), we showed that Ly49i5 is an inhibitory receptor that recognizes ligands encoded within the class Ib region of the u and l haplotypes, while the structurally related Ly49s5 is an activating receptor that recognizes class Ib ligands of the u haplotype. Ly49s5 is functionally expressed in the high NK-alloresponder PVG strain, but not in the low alloresponder BN strain, in which it is a pseudogene. Ly49s5 is hence not responsible for the striking anti-u NK alloresponse previously described in BN rats (haplotype n), which results from repeated alloimmunizations with u haplotype cells. The present studies support the notion of a complex regulation of rat NK alloreactivity by activating and inhibitory Ly49 members, which may be highly homologous in the extracellular region and bind similar class Ib-encoded target ligands.

Characterization of a Novel Killer Cell Lectin-Like Receptor (KLRH1) Expressed by Alloreactive Rat NK Cells

NK cells have the ability to recognize and kill MHC-mismatched hemopoietic cells. In the present study, strain-specific differences in the rat NK allorecognition repertoire were exploited to generate Abs against receptors that may be involved in allogeneic responses. A mAb termed STOK9 was selected, and it reacted with subsets of NK cells and NKR-P1+ T cells from certain rat strains possessing highly alloreactive NK cells. The STOK9+ NK subset was broadly alloreactive and lysed Con A lymphoblast targets from a range of MHC-mismatched strains. The mAb STOK9 precipitated a 75-kDa dimeric glycoprotein from NK lysates. Expression cloning revealed that each monomer consisted of 231 aa with limited homology to other previously characterized killer cell lectin-like receptors (KLRs). This glycoprotein therefore constitutes a novel KLR branch, and it has been termed KLRH1. A gene in the central region of the natural killer gene complex on rat chromosome 4 encodes KLRH1. A mouse homolog appears to be present as deduced from analyses of genomic trace sequences. The function of KLRH1 is unknown, but it contains an immunoreceptor tyrosine-based inhibitory motif, suggesting an inhibitory function. The MHC haplotype of the host appears to influence KLRH1 expression, suggesting that it may function as an MHC-binding receptor on subsets of NK cells and T lymphocytes.

Two complementary rat NK cell subsets, Ly49s3+ and NKR-P1B+, differ in phenotypic characteristics and responsiveness to cytokines

Two major subsets of rat NK cells can be distinguished based on their expression of the Ly49s3 or the NKR‐P1B lectin‐like receptor. Ly49s3+ NK cells, but not NKR‐P1B+ NK cells, express a wide range of Ly49 receptors. Here, we have examined differences between these two subsets in their expression of certain NK cell‐associated molecules as well as their responses to cytokines. A microarray analysis suggested several differentially expressed genes, including preferential expression of NKG2A/C receptors by NKR‐P1B+ NK cells. This was confirmed by staining with tetramers of RT.BM1, the putative ligand of CD94/NKG2, indicating that Ly49 and CD94/NKG2 receptors separate into distinct NK cell compartments. Further, expression of CD25 by Ly49s3+ NK cells was associated with more rapid proliferation in response to IL‐2 as compared with NKR‐P1B+ NK cells. Thus, certain inflammatory situations may preferentially expand the Ly49s3+ NK cells. Moreover, freshly isolated Ly49s3+ and NKR‐P1B+ NK cells produce similar amounts of cytokines, and a minor Ly49s3–NKR‐P1B– double‐negative NK subset appears to be hyporesponsive based on its significantly lower IFN‐γ production. Collectively, our data demonstrate divergent profiles of NKR‐P1B+ and Ly49s3+ NK cells, indicating distinct tasks in vivo.

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.

Strain-dependent expression of four structurally related rat Ly49 receptors; correlation with NK gene complex haplotype and NK alloreactivity

Natural killer (NK) cells from certain rat strains promptly kill MHC allogeneic lymphocytes in vivo, a rejection phenomenon termed allogeneic lymphocyte cytotoxicity (ALC). ALC can be reproduced in vitro, and is preferentially mediated by a subset of NK cells expressing the Ly49 stimulatory receptor 3 (Ly49s3) in PVG strain rats. Functional studies have suggested that Ly49s3 triggers NK cell alloreactivity, but its importance relative to other Ly49 receptors has not been investigated. In this study, we have characterized three rat Ly49 receptors with close sequence similarity to Ly49s3 in the extracellular region, i.e., Ly49s4, Ly49 inhibitory receptor 3 (Ly49i3), and Ly49i4. Similar to Ly49s3, Ly49s4 mediated cellular activation while Ly49i4 inhibited NK cytolytic function. Ly49s4, -i3, and -i4 all reacted with a previously described anti-Ly49s3 monoclonal antibody (mAb) (DAR13), but not a novel mAb (STOK6), which was shown to be specific for Ly49s3. Expression of these Ly49 receptors varied markedly between inbred strains, in patterns related to their NK gene complex (NKC) haplotype, and ability to mediate ALC. Three major groups of NKC haplotypes could be discerned by restriction fragment length polymorphism analysis. Ly49s3 was present in strains from one of the groups, which corresponded with the “high” ALC responders. Ly49s3 surface expression was also markedly reduced in the presence of its putative MHC class Ib ligand(s) in MHC congenic strains. These data support the notion that Ly49s3 functions as a triggering MHC receptor both in vitro and in vivo. MHC ligands for the other three Ly49 receptors remain to be determined.