Ig-like Receptor Research Articles

Peptide antagonism as a mechanism for NK cell activation

Inhibition of natural killer (NK) cells is mediated by MHC class I receptors including the killer cell Ig-like receptor (KIR). We demonstrate that HLA-C binding peptides can function as altered peptide ligands for KIR and antagonize the inhibition mediated by KIR2DL2/KIR2DL3. Antagonistic peptides promote clustering of KIR at the interface of effector and target cells, but do not result in inhibition of NK cells. Our data show that, as for T cells, small changes in the peptide content of MHC class I can regulate NK cell activity.

Natural killer-cell KIR repertoire reconstitution after haploidentical SCT

We studied killer-cell Ig-like receptor (KIR)/natural killer (NK)-cell group-2-Ag repertoires on donor-derived NK cells in 28 patients after haploidentical SCT in the first 6 months after SCT and correlated results with EFS. The reconstitution hierarchy of potentially alloreactive, single KIR+ NK cells was the following: HLA-C1 binding>HLA-Bw4 binding>HLA-C2 binding. The differences in reconstitution kinetics of the three potentially alloreactive NK cell subsets prompted an updated analysis of EFS in AML patients transplanted from haploidentical donors in our center. This analysis showed that in haploidentical transplantation for AML, HLA-C group 1 mismatching in the graft vs host direction not only provides a survival advantage over non-NK-alloreactive (KIR ligand-matched) transplants (5-year EFS 67±10% vs 17±5%) but, indeed, also provides the best EFS compared with C2 (35±10%) or Bw4 KIR ligand mismatches (44±17%). In conclusion, we show that the kinetics with which single KIR-expressing NK cells are generated after haploidentical SCT differ between individual KIR receptors and seem to influence survival after haploidentical SCT.

The beneficial impact of missing KIR ligands and absence of donor KIR2DS3 gene on outcome following unrelated hematopoietic SCT for myeloid leukemia in the Chinese population

The effect of natural killer (NK) cell alloreactivity on the outcome of unrelated hematopoietic SCT (HSCT) remains a topic of debate. NK cell alloreactivity after allogeneic HSCT is regulated by killer-cell Ig-like receptors (KIRs). To investigate the influence of KIRs on outcome after unrelated HSCT, we retrospectively analyzed the HLA and KIR genotypes of 116 donor-recipient pairs. We found that missing KIR ligands in recipients were significantly associated with a decreased leukemic relapse risk (P=0.019, HR=0.329), mainly in myeloid disease (P=0.003, HR=0.193). This beneficial effect was seen in AML/myelodysplastic syndrome and also in chronic myeloid leukemia. In myeloid disease, missing KIR ligands also improved 5-year OS (P=0.034, HR=0.430) and disease-free survival (DFS) (P=0.024, HR=0.445). Meanwhile, the presence of donor-activating KIR2DS3 gene was associated with increased relapse risk (P=0.003, HR=5.046), decreased OS (P=0.004, HR=3.181) and DFS (P=0.003, HR=2.919) in myeloid disease. No effect was seen in patients with lymphoid disease. Our study indicated that, in unrelated HSCT for myeloid leukemia, missing KIR ligands in recipients offered a lower relapse risk and a long-term survival advantage. The presence of KIR2DS3 in the donor was an important risk factor for myeloid leukemia.

Sequential recovery of NK cell receptor repertoire after allogeneic hematopoietic SCT

Alloreactivity of natural killer (NK) cells contributes to the GVL reaction after allogeneic hematopoietic SCT (allo-HSCT). However, various procedure-related factors may affect NK cell maturation and their ability to recognize and kill leukemic cells. In this study, we prospectively evaluated expression of NK cell inhibitory receptors in 83 adults treated with myeloablative, killer cell Ig-like receptor (KIR)-ligand-matched allo-HSCT. NK cell maturation was evaluated by comparing the phenotypic patterns after allo-HSCT with the donor ones. The frequencies of KIR3DL1 were comparable to the donor ones on day +28, while they decreased significantly starting from day +100. The expression of KIR2DL2/3 was significantly lower in patients compared with donors up to day +100. The expression of KIR2DL1, despite continues growth, remained significantly decreased for 1 year after allo-HSCT. NKG2A was over-expressed up to day +180. Within 1 year after allo-HSCT, the NK cell phenotypic pattern tended to recapitulate the donor type. The process was disturbed by the use of steroids with significant differences observed on days +56 (P=0.01) and +100 (P=0.04). Up to day +100, reconstitution of NK cell receptor repertoire correlated with the absolute numbers of circulating CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells. Our observations should be taken into account when trying to predict potential benefit from NK cell alloreactivity.

Expression of the HLA-C2-specific activating killer-cell Ig-like receptor KIR2DS1 on NK and T cells

Killer Ig-like receptors (KIRs) are MHC class I-specific receptors expressed by Natural Killer (NK) and T cell subsets. KIRs either inhibit (KIR-L) or activate (KIR-S) lymphocyte functions. Inhibitory KIR2DL1 and activating KIR2DS1 share ligand specificity for the HLA-C2 group, consistent with their almost identical extracytoplasmic domain. This homology hampered the distinction between KIR2DL1 and KIR2DS1. We report here the characterization of the KIR2DS1(+) subsets among primary human NK and T cells. Regardless of the host HLA-C genotype, around 10% of circulating NK cells expressed KIR2DS1 in absence of KIR2DL1. In HLA-C2 individuals, KIR2DS1 was not able to induce NK cell education (i.e., the acquisition of NK cell competence) nor to interfere with KIR2DL1-induced NK cell education. KIR2DS1 was also present on rare oligoclonal TCRalphabeta(+)CD8alpha(+) and TCRalphabeta(+)CD4(-)CD8(-) subsets. As KIR2DS1 has been associated with autoimmunity and hematopoietic stem cell transplantation, these results pave the way to dissect the function of KIR2DS1 in these clinical conditions.

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![Figure][1]</img> The killer-cell Ig-like receptors (KIRs) are encoded by a highly variable family of genes that has evolved extremely rapidly in primates. Four lineages of KIR have been identified, but the dominant lineage varies among primate species, and the KIR loci of smaller apes like

The impact of KIR2DS4 alleles and the expression of KIR in the development of acute GVHD after unrelated allogeneic hematopoietic SCT

The role of killer Ig-like receptors (KIR) in SCT was analyzed. A total of 75 Chinese patients were transplanted with T-depleted hematopoietic stem cells from unrelated donors. Among the 75 donor-recipient pairs, 60 were HLA 10/10 matched and 15 had some mismatches at HLA-C. Transplants from KIR haplotype B/x group donors showed significantly higher overall survival rates compared with those from KIR haplotype A/A donors (relative risk (RR) 3.1 (95% confidence interval (CI) 1.1-8.6), P=0.007). In the haplotype A/A group, a higher risk of acute GVHD (aGVHD) (RR 9.0 (95% CI 1.2-66.9), P=0.01), especially grade III-IV aGVHD (P=0.006), was observed when the donor was homozygous for the full-length expressed KIR2DS4*00101 allele. Real-time PCR showed that a high expression of inhibitory KIR (2DL1 and 3DL1) in the early stages (<90 days) after transplantation correlated with the development of aGVHD (z=2.558, P=0.011). Our findings indicated a significant association of full-length KIR2DS4 or KIR2DL1/3DL1 expression with the occurrence of aGVHD. In aggregate these results suggested that combining KIR and HLA genotyping could help in the selection of transplant donors and improve the outcome of transplantation. Dynamic detection of KIR2DL1/3DL1 expression would be beneficial for prediction of aGVHD after transplantation.

HIV Protective KIR3DL1 and HLA-B Genotypes Influence NK Cell Function Following Stimulation with HLA-Devoid Cells

Epidemiological studies in humans have implicated carriage of combinations of genes encoding certain KIR3DL1 (killer Ig-like receptor 3DL1) alleles and their HLA-Bw4 ligands in slower progression to AIDS, lower viral load and protection from infection. Given that the KIR3DL1*h/*y/HLA-B*57 genetic combination is strongly associated with favorable HIV outcomes, we measured responses from NK cells isolated from these individuals by multiparametric flow cytometry for cytokine secretion and degranulation in response to stimulation with HLA-devoid cells to assess whether the KIR/HLA compound genotypes linked to better HIV outcome favor increased NK cell functional potential. Our results indicate that NK cells from these individuals had increased functional potential, particularly in the KIR3DL1(+) NK cell subset. These results support a link between KIR/HLA genotypes and NK cell function and could provide an explanation for the observation that some KIR/HLA combinations are associated protective phenotypes in the context of host-HIV interactions.

Modulation of Toll-Like Receptor Activity by Leukocyte Ig-Like Receptors and Their Effects during Bacterial Infection

Toll-like receptors (TLRs) are a potent trigger for inflammatory immune responses. Without tight regulation their activation could lead to pathology, so it is imperative to extend our understanding of the regulatory mechanisms that govern TLR expression and function. One family of immunoregulatory proteins which can provide a balancing effect on TLR activity are the Leukocyte Ig-like receptors (LILRs), which act as innate immune receptors for self-proteins. Here we describe the LILR family, their inhibitory effect on TLR activity in cells of the monocytic lineage, their signalling pathway, and their antimicrobial effects during bacterial infection. Agents have already been identified which enhances or inhibits LILR activity raising the future possibility that modulation of LILR function could be used as a means to modulate TLR activity.

Genome-wide comparison of FGFRL1 with structurally related surface receptors

FGFRL1 (FGFR5) is a member of the fibroblast growth factor receptor family, which lacks the intracellular tyrosine kinase domain required for signal transduction by trans-phosphorylation. Since it still contains a cytoplasmic domain of 100 amino acid residues with a peculiar histidine-rich element, it might be involved in an alternative pathway of downstream signaling. To get a clue about a possible mechanism, we compared the overall structure of FGFRL1 with all proteins from the UniProt databank. We found that the human genome encodes 42 structurally related proteins with a signal peptide, three Ig-like domains and a transmembrane domain. These proteins can be grouped in seven families, fibroblast growth factor receptors (FGFRs), Fc receptor-like proteins, IL-1 receptor-like proteins, killer cell Ig-like receptors (KIRs), nectin-like proteins, sialic acid binding lectins (SIGLECs) and signal regulatory proteins (SIRPs). The 7 families utilize four different strategies for signaling, namely a protein tyrosine kinase domain, a TIR (Toll/IL-1 receptor) domain, ITIM/ITAM motifs as well as carboxy-terminal peptides that interact with the PDZ domain of an adaptor protein. It remains to be determined whether FGFRL1 might also utilize one of these strategies for signaling.

Inhibition of Th2 Adaptive Immune Responses and Pulmonary Inflammation by Leukocyte Ig-Like Receptor B4 on Dendritic Cells

We previously established that the inhibitory receptor LILRB4 mitigates LPS-induced, neutrophil-dependent pathologic effector mechanisms in inflammation. We now report that LILRB4 on dendritic cells (DCs) counterregulates development of an adaptive Th2 immune response and ensuing inflammation in a model of allergic pulmonary inflammation, initiated by inhalation sensitization with OVA and LPS followed by airway challenge with OVA. We found that Lilrb4(-/-) mice had significantly exacerbated eosinophilic pulmonary inflammation, as assessed in bronchoalveolar lavage and lung tissue, as well as elevated levels of OVA-specific IgE and Th2 cytokines produced by OVA-restimulated lymph node cells. LILRB4 was preferentially expressed on MHC class II(high)CD86(high) OVA-bearing DCs in lung-draining lymph nodes after sensitization or challenge. Moreover, the lymph nodes of Lilrb4(-/-) mice had significantly more of these mature DCs after challenge with OVA, which was accompanied by significantly more IL-4-producing lymphocytes, compared with Lilrb4(+/+) mice. Sensitization of naive Lilrb4(+/+) mice by transfer of OVA-LPS-pulsed Lilrb4(-/-) bone marrow-derived DCs was sufficient to confer exacerbated allergic lung pathology upon challenge with OVA, compared with mice that received Lilrb4(+/+) bone marrow-derived DCs. Our findings establish that maturation and migration of pulmonary DCs to lymph nodes in response to Ag and an innate immune stimulus is associated with upregulated expression of LILRB4. In addition, this receptor attenuates the number of these mature DCs and attendant IL-4-producing lymphocytes in the lymph nodes, and accordingly, the ability of DCs to elicit pathologic Th2 pulmonary inflammation.

Natural Killer Group 2 Member D, Its Ligands, and Liver Disease: Good or Bad?

Liver lymphocytes are enriched in natural killer (NK) cells, which play an important role in host defenses against microbial infection and tumor transformation in the liver.1 Generally, it is believed that the cytotoxicity of NK cells against target cells is controlled by the opposing signals from inhibitory and stimulatory receptors on NK cells interacting with their corresponding ligands expressed on target cells.2, 3 The NK cell inhibitory receptors include CD94/NKG2, Ly49A, and the Ig-like killer inhibitory receptor, which interact with inhibitory ligands (eg, self MHC class I molecules) expressed on target cells and inactivate NK cell function. The stimulatory receptors include NKp46, NKp30, NKp44, NKG2D, and DNAX accessory molecule 1 (CD226). Among these, the best-defined receptor is NKG2D (natural-killer group 2, member D), a highly conserved C-type lectin-like membrane glycoprotein that is also one of the major activating receptors on NK cells. 2, 3 Expressed on essentially all NK cells, as well as on γδ-TcR+ T cells and αβ-TcR+ CD8+ T cells, NKG2D is found in both humans and mice. In humans, known NKG2D ligands include major histocompatiblity complex (MHC) class I-related chain A and B (MICA/B) and UL16-binding protein 1, 2, 3, 4 (ULBP1, 2, 3, 4). In mice, known NKG2D ligands include retinoic acid early inducible gene-1 (RAE-1), minor histocompatibility H60, and murine UL16-binding protein-like transcript 1 (MULT1). The expression of these NKG2D ligands is usually upregulated on microbe-infected, transformed, or stressed cells. The interaction between these ligands and NKG2D on NK cells leads to NK cell activation, thereby playing an important role in host defenses against viral infection and tumor transformation. In addition, CD8+ T cells also express NKG2D, which serves as a co-stimulatory signal to activate CD8+ T cells. 2, 3

A Small, Variable, and Irregular Killer Cell Ig-Like Receptor Locus Accompanies the Absence of MHC-C and MHC-G in Gibbons

The killer cell Ig-like receptors (KIRs) of NK cells recognize MHC class I ligands and function in placental reproduction and immune defense against pathogens. During the evolution of monkeys, great apes, and humans, an ancestral KIR3DL gene expanded to become a diverse and rapidly evolving gene family of four KIR lineages. Characterizing the KIR locus are three framework regions, defining two intervals of variable gene content. By analysis of four KIR haplotypes from two species of gibbon, we find that the smaller apes do not conform to these rules. Although diverse and irregular in structure, the gibbon haplotypes are unusually small, containing only two to five functional genes. Comparison with the predicted ancestral hominoid KIR haplotype indicates that modern gibbon KIR haplotypes were formed by a series of deletion events, which created new hybrid genes as well as eliminating ancestral genes. Of the three framework regions, only KIR3DL3 (lineage V), defining the 5' end of the KIR locus, is present and intact on all gibbon KIR haplotypes. KIR2DL4 (lineage I) defining the central framework region has been a major target for elimination or inactivation, correlating with the absence of its putative ligand, MHC-G, in gibbons. Similarly, the MHC-C-driven expansion of lineage III KIR genes in great apes has not occurred in gibbons because they lack MHC-C. Our results indicate that the selective forces shaping the size and organization of the gibbon KIR locus differed from those acting upon the KIR of other hominoid species.

Leukocyte Ig-like Receptor B4 (LILRB4) Is a Potent Inhibitor of FcγRI-mediated Monocyte Activation via Dephosphorylation of Multiple Kinases

The leukocyte immunoglobulin-like receptor (LILR) B4 belongs to a family of cell surface receptors that possesses cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LILRB4 is believed to down-regulate activation signals mediated by non-receptor tyrosine kinase cascades through the recruitment of SHP-1. However, the exact mechanisms of LILRB4-mediated inhibition are not fully elucidated. In this study, we demonstrate high level surface expression of LILRB4 on THP-1 cells and primary peripheral blood monocytes, which profoundly inhibited production of a key pro-inflammatory cytokine (TNFalpha) induced by FcgammaRI (CD64). We also report that LILRB4 aggregated to sites of activation upon co-ligation with CD64 and that this may enhance its inhibitory effects. Cross-linking of CD64 on THP-1 cells markedly increased phosphorylation of multiple proteins including tyrosine kinases and signaling molecules (Lck, Syk, LAT, and Erk), an adaptor protein that targets protein-tyrosine kinases for degradation (c-Cbl) and a protein involved in the formation of actin cytoskeletal rearrangement (alpha-actinin-4). Co-ligation of LILRB4 considerably reduced CD64-mediated phosphorylation of Lck, Syk, LAT, Erk, and c-Cbl but not alpha-actinin-4, suggesting selective inhibition of signaling molecules. Treatment of cells with a broad-spectrum phosphatase inhibitor, sodium pervanadate (SP), significantly reversed LILRB4-mediated inhibition of TNFalpha production and protein tyrosine phosphorylation. In comparison, treatment with an SHP-1 specific inhibitor, sodium stibogluconate (SS) has no effects indicating involvement of phosphatase(s) other than SHP-1 in LILRB4 signaling. Collectively, our data show LILRB4 is a potent inhibitor of monocytes activation. This may provide a new potential therapeutic strategy for inflammatory conditions characterized by excessive TNFalpha production.

Soluble HLA‐I‐mediated secretion of TGF‐β1 by human NK cells and consequent down‐regulation of anti‐tumor cytolytic activity

Soluble HLA class I (sHLA-I) molecules can regulate survival of NK cells and their anti-tumor killing activity. Herein, we have analysed whether interaction of sHLA-I with CD8 and/or different isoforms of killer Ig-like receptors (KIR) induced secretion of transforming growth factor (TGF)-beta1. CD8+KIR- NK cell clones secreted TGF-beta1 upon the interaction of sHLA-I with CD8 molecule. sHLA-Cw4 or sHLA-Cw3 alleles engaging inhibitory isoforms of KIR, namely KIR2DL1 or KIR2DL2, strongly downregulated TGF-beta1 production elicited through CD8. On the other hand, sHLA-Cw4 or sHLA-Cw3 alleles induced secretion of TGF-beta1 by ligation of stimulatory KIR2DS1 or KIR2DS2 isoforms. TGF-beta1 strongly reduced NK cell-mediated tumor cell lysis and production of pro-inflammatory cytokines such as TNF-alpha and IFN-gamma. Also, TGF-beta1 inhibited NK cell cytolysis induced by the engagement of stimulatory receptors including NKG2D, DNAM1, 2B4, CD69, NKp30, NKp44 and NKp46. The IL-2-dependent surface upregulation of some of these receptors was prevented by TGF-beta1. Furthermore, TGF-beta1 hampered IL-2-induced NK cell proliferation but not IL-2-mediated rescue from apoptosis of NK cells. Depletion of TGF-beta1 restored all the NK cell-mediated functional activities analysed. Taken together these findings suggest that sHLA-I antigens may downregulate the NK cell-mediated innate response by inducing TGF-beta1 release.

Siglec-E Is Up-Regulated and Phosphorylated Following Lipopolysaccharide Stimulation in Order to Limit TLR-Driven Cytokine Production

Although production of cytokines by TLR is essential for viral and bacterial clearance, overproduction can be detrimental, thus controlling these responses is essential. CD33-related sialic acid binding Ig-like lectin receptors (Siglecs) have been implicated in the control of leukocyte responses. In this study, we report that murine Siglec-E is induced by TLRs in a MyD88-specific manner, is tyrosine phosphorylated following LPS stimulation, and negatively regulates TLR responses. Specifically, we demonstrate the Siglec-E expression inhibits TLR-induced NF-kappaB and more importantly, the induction of the antiviral cytokines IFN-beta and RANTES. Siglec-E mediates its inhibitory effects on TIR domain containing adaptor inducing IFN-beta (TRIF)-dependent cytokine production via recruitment of the tyrosine [corrected] phosphatase SHP2 and subsequent inhibition of TBK1 activity as evidenced by enhanced TBK1 phosphorylation in cells following knockdown of Siglec-E expression. Taken together, our results demonstrate a novel role for Siglec-E in controlling the antiviral response to TLRs and thus helping to maintain a healthy cytokine balance following infection.

Alleviation of Acute Graft-Verus-Host Disease Following Regulatory Dendritic Cell Treatment in Young and Older Mice.

Abstract 3550Poster Board III-487Older patients experience increased severity of graft-versus-host disease (GVHD) following allogeneic bone marrow transplant and host dendritic cells (DC) are a vital initiator of this disease.To evaluate the effect of age on GVHD severity and establish a model that can be used to study treatment of GVHD, a complete MHC mismatched bone marrow transplant (BMT) model was used to induce GVHD. Older (≥14 month) and young (3-4 month) mice succumbed to GVHD, but older mice experienced faster onset of weight loss and earlier mortality. Prophylactic treatment with syngeneic (host strain-derived) regulatory DC (DCreg) has been shown to decrease mortality of GHVD in young mice (Immunity 2003 18: 367-379). To determine whether this treatment also decreases the morbidity from GVHD in young mice and to evaluate its efficacy in older mice, day +2 BMT mice were treated with DCreg (hereafter referred to as DCreg treated BMT mice). As previously described, DCreg were generated by culture of bone marrow cells (BM) with IL-10, GM-CSF, and TGF-β1 (Immunity 2003 18: 367-379). All DCreg treated mice exhibited a brief period of GVHD and survived at least 45 days post-BMT; overall, older mice had increased morbidity and decreased long-term survival than young mice. In vivo analysis of regulatory and co-stimulatory DC molecules (PD-L1, PD-L2, CD103, CD40, CD80, and CD86) on host and donor-derived DC revealed no statistical differences between young and older DCreg treated BMT mice. Paired Ig-like receptor B (PIR B) is an inhibitory receptor expressed on DC and GVHD is exacerbated in PIR B−/− mice (Nat. Immunol. 2004 5:623-629). An antibody that recognizes both PIR A and PIR B was used to look at expression on DC in DCreg treated BMT mice. PIR A/B expression was increased in vivo on young host DC compared to older host DC. Transferred DCreg are host-strain derived and it is unclear if the PIR A/B expression in these experiments was on residual host DC or the transferred DCreg. Future utilization of green fluorescent protein positive (GFP+) DCreg will determine the origin of upregulated PIR A/B expression in vivo. In contrast to cultured stimulatory DC, young cultured DCreg revealed increased PIR A/B expression and were negative for co-stimulatory DC molecule expression. This difference in PIR A/B expression potentially provides an explanation for the disparate outcome of older DCreg treated BMT mice.Previous studies have implicated Treg in the induction of tolerance in DCreg treated mice (Immunity 2003 18: 367-379). However, the mechanism of tolerance maintenance has not been investigated. Abrogation of Treg mediated suppression in vivo with anti-GITR antibody (DTA-1) did not result in recurrence of GVHD or mortality in young mice, implying maintenance of the GVHD-free state is not Treg mediated in young mice.Human transplant recipients have a compromised immune system from both the transplant and immunosuppressive drug regimen that results in increased susceptibility to infection. Upon infection, GVHD recurrence often occurs. A murine influenza infection model was used to assess the donor immune system's capacity to respond to infection and remain free of GVHD in young DCreg treated BMT mice. Forty-five days or more post-BMT, once completely recovered from GVHD and demonstrated to be allo-reconstituted, the DCreg treated BMT mice were intra-nasally infected with 0.1LD50 of the H1N1 mouse adapted A/PR/8/34 influenza virus. DCreg treated BMT mice did not have a resurgence of GVHD following infection and displayed similar morbidity to control mice.In conclusion, DCreg treatment of BMT mice results in decreased morbidity and mortality from acute GVHD in both young and older mice relative to untreated BMT mice. However, the older mice still do not fully recover and exhibit increased morbidity relative to young mice. The maintenance of tolerance observed in young DCreg treated BMT mice does not appear to be Treg mediated. DCreg treated BMT mice experience similar morbidity and mortality as control mice during influenza infection and do not experience a resurgence of GVHD, implying an intact immune response to influenza infection.Future studies will specifically examine the requirement of PIR B on DCreg for successful inhibition of GVHD. Additional work will continue to investigate the mechanisms of induction and maintenance of tolerance in DCreg treated BMT mice.S.M.S. is supported by the National Institutes of Health (grant #5T32 AI07485). Disclosures:No relevant conflicts of interest to declare.

The amphibians Xenopus laevis and Silurana tropicalis possess a family of activating KIR-related Immunoglobulin-like Receptors

In this study, we searched the amphibian species Xenopus laevis and Silurana ( Xenopus) tropicalis for the presence of genes homologous to mammalian KIRs and avian CHIRs (KRIR family). By experimental and computational procedures, we identified four related ILR (Ig-like Receptors) genes in S. tropicalis and three in X. laevis. ILRs encode type I transmembrane receptors with 3–4 Ig-like extracellular domains. All predicted ILR proteins appear to be activating receptors. ILRs have a broad expression pattern, the gene transcripts were found in both lymphoid and non-lymphoid tissues. Phylogenetic analysis shows that the amphibian KRIR family receptors evolved independently from their mammalian and avian counterparts. The only conserved structural element of tetrapod KRIRs is the NxxR motif-containing transmembrane domain that facilitates association with FcRγ subunit. Our findings suggest that if KRIRs of various vertebrates have any common function at all, such a function is activating rather than inhibitory.

SHP-2 Expression Negatively Regulates NK Cell Function

Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) is required for full activation of Ras/ERK in many cytokine and growth factor receptor signaling pathways. In contrast, SHP-2 inhibits activation of human NK cells upon recruitment to killer cell Ig-like receptors (KIR). To determine how SHP-2 impacts NK cell activation in KIR-dependent or KIR-independent signaling pathways, we employed knockdown and overexpression strategies in NK-like cell lines and analyzed the consequences on functional responses. In response to stimulation with susceptible target cells, SHP-2-silenced NK cells had elevated cytolytic activity and IFN-gamma production, whereas cells overexpressing wild-type or gain-of-function mutants of SHP-2 exhibited dampened activities. Increased levels of SHP-2 expression over this range significantly suppressed microtubule organizing center polarization and granzyme B release in response to target cells. Interestingly, NK-target cell conjugation was only reduced by overexpressing SHP-2, but not potentiated in SHP-2-silenced cells, indicating that conjugation is not influenced by physiological levels of SHP-2 expression. KIR-dependent inhibition of cytotoxicity was unaffected by significant reductions in SHP-2 levels, presumably because KIR were still capable of recruiting the phosphatase under these limiting conditions. In contrast, the general suppressive effect of SHP-2 on cytotoxicity and cytokine release was much more sensitive to changes in cellular SHP-2 levels. In summary, our studies have identified a new, KIR-independent role for SHP-2 in dampening NK cell activation in response to tumor target cells in a concentration-dependent manner. This suppression of activation impacts microtubule organizing center-based cytoskeletal rearrangement and granule release.

Genome-wide comparison of FGFRL1 with structurally related surface receptors

FGFRL1 (FGFR5) is a member of the fibroblast growth factor receptor family, which lacks the intracellular tyrosine kinase domain required for signal transduction by trans-phosphorylation. Since it still contains a cytoplasmic domain of 100 amino acid residues with a peculiar histidine-rich element, it might be involved in an alternative pathway of downstream signaling. To get a clue about a possible mechanism, we compared the overall structure of FGFRL1 with all proteins from the UniProt databank. We found that the human genome encodes 42 structurally related proteins with a signal peptide, three Ig-like domains and a transmembrane domain. These proteins can be grouped in seven families, fibroblast growth factor receptors (FGFRs), Fc receptor-like proteins, IL-1 receptor-like proteins, killer cell Ig-like receptors (KIRs), nectin-like proteins, sialic acid binding lectins (SIGLECs) and signal regulatory proteins (SIRPs). The 7 families utilize four different strategies for signaling, namely a protein tyrosine kinase domain, a TIR (Toll/IL-1 receptor) domain, ITIM/ITAM motifs as well as carboxy-terminal peptides that interact with the PDZ domain of an adaptor protein. It remains to be determined whether FGFRL1 might also utilize one of these strategies for signaling.