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Supplementary Table 1Suppl. Table 1 related to materials and methods

Supplementary Figures S1-S7Supplementary Figures S1-S7

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1–expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1–induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.

Supplementary Materials and Methods, and Supplementary Figures

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1− T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells.Significance:The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1− T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. Significance: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy. See related commentary by Burton and Tawbi, p. 1008. This article is highlighted in the In This Issue feature, p. 995

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1–expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1–induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor., A panel of LILRB3-specific monoclonal antibodies was generated and used to unravel the immunoregulatory functions of LILRB3 on human myeloid cells using relevant preclinical models.

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin (Ig)-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress for this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAb) was generated. LILBR3-specific mAb bound to discrete epitopes in either Ig-like domain two or four. LILRB3 ligation on primary human monocytes by agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppresive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.

Leukocyte immunoglobulin-like receptors (LILR) are expressed mostly on myelomonocytic cells where they are mediators of immunological tolerance. Two LILR genes, LILRA3 and LILRA6, exhibit marked copy number variation. We assessed the contribution of these genes to atopic dermatitis (AD) by analysing transmission in 378 AD families. The data indicated that copies of LILRA6 were over-transmitted to affected patients. They are consistent with a contribution of LILR genes to AD. They could affect the equilibrium between activating and inhibitory signals in the immune response.

Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) has been reported to interact with a wide spectrum of HLA class I (HLA-I) molecules, albeit with different affinities determined by allelic polymorphisms and conformational features. HLA-G dimerization and the presence of intracellular Cys residues in HLA-B7 have been shown to be critical for their recognition by LILRB1. We hypothesized that dimerization of classical HLA class Ia molecules, previously detected in exosomes, might enhance their interaction with LILRB1. A soluble LILRB1-Fc fusion protein and a sensitive cellular reporter system expressing a LILRB1-ζ chimera were employed to assess receptor interaction with different HLA class Ia molecules transfected in the human lymphoblastoid 721.221 cell line. Under these conditions, intracellular Cys residues and HLA-I dimerization appeared associated with increased LILRB1 recognition. On the other hand, a marginal interaction of LILRB1 with primary monocytic cells, irrespective of their high HLA-I expression, was enhanced by type I interferon (IFN). This effect appeared disproportionate to the cytokine-induced increase of surface HLA-I expression and was accompanied by detection of HLA class Ia dimers. Altogether, the results support that a regulated assembly of these noncanonical HLA-I conformers during the immune response may enhance the avidity of their interaction with LILRB1.

We identified a novel, evolutionarily conserved receptor encoded within the human leukocyte receptor complex and syntenic region of mouse chromosome 7, named T cell–interacting, activating receptor on myeloid cells-1 (TARM1). The transmembrane region of TARM1 contained a conserved arginine residue, consistent with association with a signaling adaptor. TARM1 associated with the ITAM adaptor FcRγ but not with DAP10 or DAP12. In healthy mice, TARM1 is constitutively expressed on the cell surface of mature and immature CD11b+Gr-1+ neutrophils within the bone marrow. Following i.p. LPS treatment or systemic bacterial challenge, TARM1 expression was upregulated by neutrophils and inflammatory monocytes and TARM1+ cells were rapidly recruited to sites of inflammation. TARM1 expression was also upregulated by bone marrow–derived macrophages and dendritic cells following stimulation with TLR agonists in vitro. Ligation of TARM1 receptor in the presence of TLR ligands, such as LPS, enhanced the secretion of proinflammatory cytokines by macrophages and primary mouse neutrophils, whereas TARM1 stimulation alone had no effect. Finally, an immobilized TARM1-Fc fusion protein suppressed CD4+ T cell activation and proliferation in vitro. These results suggest that a putative T cell ligand can interact with TARM1 receptor, resulting in bidirectional signaling and raising the T cell activation threshold while costimulating the release of proinflammatory cytokines by macrophages and neutrophils.

Killer cell immunoglobulin-like receptors (KIR) are highly polymorphic members of the immunoglobulin superfamily, which influence the response of natural killer cells and some T-lymphocyte subsets. Analysis of a cohort of previously human cytomegalovirus (HCMV)-negative patients, who developed primary HCMV infection following HCMV-positive renal transplant (n=76), revealed an increase in the frequency of KIR genes located on the telomeric region of B haplotypes (Tel B). The presence of Tel B in combination with the KIR ligand HLA-C2 was significantly more frequent in this subgroup. These genetic factors were associated with resistance to HCMV infection in a second cohort (n=65), where the Tel B genes KIR2DL5, -2DS1, 2DS5 and -3DS1 were all significantly associated with high viral loads. Furthermore, the KIR haplotype Tel A when in combination with the KIR ligand HLA-C1 was significantly protective against the development of severe infection. Our results suggest that KIR are a significant factor in the control of primary HCMV infection, and that determination of KIR gene repertoire may help in detection of renal transplant patients who were most at risk.

Background— Beyond their role as innate immune effectors, natural killer (NK) cells are emerging as important regulators of angiogenesis and vascular remodeling. Pulmonary arterial hypertension (PAH) is characterized by severe pulmonary vascular remodeling and has long been associated with immune dysfunction. Despite this association, a role for NK cells in disease pathology has not yet been described. Methods and Results— Analysis of whole blood lymphocytes and isolated NK cells from PAH patients revealed an expansion of the functionally defective CD56 − /CD16 + NK subset that was not observed in patients with chronic thromboembolic pulmonary hypertension. NK cells from PAH patients also displayed decreased levels of the activating receptor NKp46 and the killer immunoglobulin-like receptors 2DL1/S1 and 3DL1, reduced secretion of the cytokine macrophage inflammatory protein-1β, and a significant impairment in cytolytic function associated with decreased killer immunoglobulin-like receptor 3DL1 expression. Genotyping patients (n=222) and controls (n=191) for killer immunoglobulin-like receptor gene polymorphisms did not explain these observations. Rather, we show that NK cells from PAH patients exhibit increased responsiveness to transforming growth factor-β, which specifically downregulates disease-associated killer immunoglobulin-like receptors. NK cell number and cytotoxicity were similarly decreased in the monocrotaline rat and chronic hypoxia mouse models of PAH, accompanied by reduced production of interferon-γ in NK cells from hypoxic mice. NK cells from PAH patients also produced elevated quantities of matrix metalloproteinase 9, consistent with a capacity to influence vascular remodeling. Conclusions— Our work is the first to identify an impairment of NK cells in PAH and suggests a novel and substantive role for innate immunity in the pathobiology of this disease.

Leucocyte Ig-like receptors (LILR) are a family of innate immune receptors expressed on myeloid and lymphoid cells that influence adaptive immune responses. We identified a common mechanism of alternative mRNA splicing, which generates transcripts that encode soluble protein isoforms of the majority of human LILR. These alternative splice variants lack transmembrane and cytoplasmic encoding regions, due to the transcription of a cryptic stop codon present in an intron 5' of the transmembrane encoding exon. The alternative LILR transcripts were detected in cell types that express their membrane-associated isoforms. Expression of the alternative LILRB1 transcript in transfected cells resulted in the release of a soluble approximately 65 Kd LILRB1 protein into culture supernatants. Soluble LILRB1 protein was also detected in the culture supernatants of monocyte-derived DC. In vitro assays suggested that soluble LILRB1 could block the interaction between membrane-associated LILRB1 and HLA-class I. Soluble LILRB1 may act as a dominant negative regulator of HLA-class I-mediated LILRB1 inhibition. Soluble isoforms of the other LILR may function in a comparable way.

The human leukocyte receptor complex (LRC) encompasses several sets of genes with a common evolutionary origin and which form a branch of the immunoglobulin superfamily (IgSF). Comparisons of LRC genes both within and between species calls for a high degree of plasticity. The drive for this unprecedented level of variation is not known, but it relates in part to interaction of several LRC products with polymorphic human leukocyte antigen (HLA) class I molecules. However, the range of other proposed ligands for LRC products indicates a dynamic set of receptors that have adapted to detect target molecules relating to numerous cellular pathways. Several receptors in the complex bind a molecular signature in collagenous ligands. Others detect a variety of motifs relating to pathogens in addition to cellular stress, attesting to the opportunistic versatility of LRC receptors.

Antigen presenting cells (APC) are critical components of innate immunity and consequently shape the adaptive response. Leukocyte Ig Like Receptors (LILR) are innate immune receptors predominantly expressed on myeloid cells. LILR can influence the antigen presenting phenotype of monocytic cells to determine the nature of T cell responses in infections including Mycobaterium leprae. We therefore investigated the relevance of LILR in the context of Mycobacterium tuberculosis. Real-time PCR studies indicated that the transcriptional profile of the orphan receptor LILRB5 was significantly up-regulated following exposure to mycobacteria. Furthermore, LILRA1 and LILRB5 were able to trigger signalling through direct engagement of mycobacteria using tranfectant cells incorporating a reporter system. We describe for the first time the expression of this receptor on T cells, and highlight the potential relevance to mycobacterial recognition. Furthermore, we demonstrate that crosslinking of this receptor on T cells increases proliferation of cytotoxic, but not helper, T cells.

KIR3DL3 is a framework gene of the Leukocyte Receptor Complex, present in all individuals and haplotypes analysed to date. We describe 17 novel KIR3DL3 alleles, including seven single nucleotide polymorphic (SNP) positions within the coding region. Sequence variation within introns included a VNTR within intron 1. As KIR3DL3 mRNA is known to be expressed in decidual NK cells, we investigated the impact of KIR3DL3 allelic variation on pre-eclampsia. No statistical difference in allele frequency or polymorphism was observed between pre-eclampsia patient and control cohorts. Linkage disequilibrium (LD) analysis of exonic SNPs suggested that recombination may be a mechanism of generating sequence diversity within KIR3DL3. A potential recombination hotspot was located within intron 5. A strong LD was detected between polymorphism in exon 6 of KIR3DL3 and the KIR gene -2DL3 or -2DS2 loci, which define the centromeric end of two main haplotypes (A and B) of the KIR cluster. Comparison of primate KIR sequences indicated that the Ig domains of KIR3DL3 are highly conserved between chimpanzee, gorilla and humans. Investigation of KIR3DL3 dN/dS ratios indicated a greater level of synonymous mutations consistent with purifying selection, although positive selection was detected acting on two sites within the stem region.

Alloimmunization to human platelet alloantigens (HPAs) is responsible for neonatal alloimmune thrombocytopenia (NAIT), post-transfusional purpura (PTP) and platelet transfusion refractoriness. HPAs may also have a role as histocompatibility antigens in transplantation as well as associations with cardiac disease. We have developed a polymerase chain reaction-sequence- specific primer (PCR-SSP) assay capable of detecting 15 HPA allelic variants. As part of the validation of the assay, 134 UK renal donors were genotyped to determine HPA allele frequencies in the UK population. The HPA allele frequencies obtained are consistent with those of the other European studies: GP1A*1 (HPA-5a) and GP1A*2 (HPA-5b), 0.914 and 0.086, respectively; GP1BA*1 (HPA-2a) and GP1BA*2 (HPA-2b), 0.925 and 0.075; GP2B*1 (HPA-3a) and GP2B*2 (HPA-3b), 0.627 and 0.373; GP3A*1 (HPA-1a) and GP3A*2 (HPA-1b), 0.840 and 0.161. The rare alleles GP2B*3 (HPA-9bw) and GP3A*3 to *8 (HPA-4b, -6b, -7bw, -8bw, -10bw and -11bw, respectively) were all absent. This comprehensive HPA genotyping assay allows rapid, accurate and reproducible results at low cost.

Natural killer (NK) lymphocytes are potent effector cells that are controlled by the expression of a variety of cell surface receptors with either inhibitory or activating functions. The genetic and functional diversity of this receptor repertoire and the role of HLA class I molecules as a major group of NK receptor ligands create an innate alloreactive capacity in this cell type. Both animal models and in vitro studies have implicated NK cells as contributors to the pathology of clinical transplantation. However, recent clinical studies have indicated the potential benefit of exploiting NK cell alloreactivity in mismatched haematopoietic stem cell transplantation. Further investigations of NK cell alloreactivity will undoubtedly reveal additional applications of this fundamental cell type in clinical transplantation.

Natural progression of HIV-1 infection depends on genetic variation in the human major histocompatibility complex (MHC) class I locus, and the CD8+ T cell response is thought to be a primary mechanism of this effect. However, polymorphism within the MHC may also alter innate immune activity against human immunodeficiency virus type 1 (HIV-1) by changing interactions of human leukocyte antigen (HLA) class I molecules with leukocyte immunoglobulin-like receptors (LILR), a group of immunoregulatory receptors mainly expressed on myelomonocytic cells including dendritic cells (DCs). We used previously characterized HLA allotype-specific binding capacities of LILRB1 and LILRB2 as well as data from a large cohort of HIV-1-infected individuals (N = 5126) to test whether LILR-HLA class I interactions influence viral load in HIV-1 infection. Our analyses in persons of European descent, the largest ethnic group examined, show that the effect of HLA-B alleles on HIV-1 control correlates with the binding strength between corresponding HLA-B allotypes and LILRB2 (p = 10−2). Moreover, overall binding strength of LILRB2 to classical HLA class I allotypes, defined by the HLA-A/B/C genotypes in each patient, positively associates with viral replication in the absence of therapy in patients of both European (p = 10−11–10−9) and African (p = 10−5–10−3) descent. This effect appears to be driven by variations in LILRB2 binding affinities to HLA-B and is independent of individual class I allelic effects that are not related to the LILRB2 function. Correspondingly, in vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of DCs. Thus, we propose an impact of LILRB2 on HIV-1 disease outcomes through altered regulation of DCs by LILRB2-HLA engagement., Author Summary Leukocyte immunoglobulin-like receptors B1 and B2 (LILRB1 and LILRB2) bind HLA class I allotypes with variable affinities. Here, we show that the binding strength of LILRB2 to HLA class I positively associates with level of viremia in a large cohort of untreated HIV-1-infected patients. This effect appears to be driven by HLA-B polymorphism and demonstrates independence from class I allelic effects on viral load. Our in vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of dendritic cells (DCs). Thus, we propose an impact of LILRB2 on HIV-1 immune control through altered regulation of DCs by LILRB2-HLA engagement.

A revised version of Table S1 with additional information can be viewed here: Click here for additional data file.(479K, tif) [^]

Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and\ud adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbourjoining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.

Leukocyte Ig-like receptors (LILRs) are a family of innate immune receptors predominantly expressed by myeloid cells that can alter the Ag presentation properties of macrophages and dendritic cells. Several LILRs bind HLA class I. Altered LILR recognition due to HLA allelic variation could be a contributing factor in disease. We comprehensively assessed LILR binding to >90 HLA class I alleles. The inhibitory receptors LILRB1 and LILRB2 varied in their level of binding to different HLA alleles, correlating in some cases with specific amino acid motifs. LILRB2 displayed the weakest binding to HLA-B*2705, an allele genetically associated with several autoimmune conditions and delayed progression of HIV infection. We also assessed the effect of HLA class I conformation on LILR binding. LILRB1 exclusively bound folded β2-microglobulin–associated class I, whereas LILRB2 bound both folded and free H chain forms. In contrast, the activating receptor LILRA1 and the soluble LILRA3 protein displayed a preference for binding to HLA-C free H chain. To our knowledge, this is the first study to identify the ligand of LILRA3. These findings support the hypothesis that LILR-mediated detection of unfolded versus folded MHC modulates immune responses during infection or inflammation.

Testis-specific gene antigen 10 (TSGA10) encodes an 82-kDa protein expressed during development, and in testis and brain tissues. We report its expression in human monocyte-derived dendritic cells (DC) and macrophages in vitro and in murine spleen CD11c(+) cells ex vivo. An interaction between DC/macrophage-derived TSGA10 and vimentin, as well as a few other major cytoskeletal proteins (e.g., actin-?1), was identified by pull-down and mass spectroscopy assays. The interaction between TSGA10 and vimentin was further confirmed by immunoprecipitation and immunolocalisation in transfected RAW267 and HEK293 cell lines. TSGA10 formed filamentous structures in transfected COS-1 cells and was observed in cellular projections. We propose that TSGA10 could influence the function of antigen presenting cells (APC) via its interaction with cytoskeletal proteins such as vimentin.

KIR3DL1 is one of the best-characterised inhibitory NK cell receptors. Unusually, one common allele at the 3DL1 locus encodes an activating receptor known as 3DS1. There is genetic evidence for a protective role of 3DS1 in certain viral diseases, but there has been uncertainty about expression of the 3DS1 protein. Using transfection, we show that surface expression of 3DS1 is reliant on the adaptor protein DNAX-activating protein 12 (DAP12). KIR3DS1 was recognised by the antibody Z27, a reagent that also detects KIR3DL1 but no other killer immunoglobulin-like receptor (KIR) molecule. Z27 stained 3DS1 on the surface of fresh circulating NK cells from 3DS1/3DS1 homozygotes. By double-staining with Z27 and DX9, an antibody specific for 3DL1, we obtained evidence that in 3DS1/3DL1 heterozygous donors significant numbers of NK cells express 3DS1 without co-expressing 3DL1 and that NK cells expressing both alleles are difficult to detect.

Killer immunoglobulin-like receptors (KIRs) are expressed on natural killer cells and some T-cell subsets and produce either activation or inhibitory signals upon binding with the appropriate human leucocyte antigen (HLA) ligand on target cells. Recent genetic association studies have implicated KIR genotype in the development of several inflammatory conditions. Ulcerative colitis (UC) is an inflammatory disorder of the colonic mucosa that results from an inappropriate activation of the immune system driven by host bacterial flora. We developed a polymerase chain reaction-sequence specific primer (SSP)-based assay to genotype 194 UC patients and 216 control individuals for 14 KIR genes, the HLA-Cw ligand epitopes of the KIR2D receptors and a polymorphism of the lectin-like-activating receptor NKG2D. Initial analysis found the phenotype frequency of KIR2DL2 and -2DS2 to be significantly increased in the UC cohort (P=0.030 and 0.038, respectively). Logistic regression analysis revealed a protective effect conferred by KIR2DL3 in the presence of its ligand HLA-Cw group 1 (P=0.019). These results suggest that KIR genotype and HLA ligand interaction may contribute to the genetic susceptibility of UC.

Leukocyte immunoglobulin-like receptors (LILR) are cell surface molecules that regulate the activities of myelomonocytic cells through the balance of inhibitory and activation signals. LILR genes are located within the leukocyte receptor complex (LRC) on chromosome 19q13.4 adjacent to KIR genes, which are subject to allelic and copy number variation (CNV). LILRB3 (ILT5) and LILRA6 (ILT8) are highly polymorphic receptors with similar extracellular domains. LILRB3 contains inhibitory ITIM motifs and LILRA6 is coupled to an adaptor with activating ITAM motifs. We analysed the sequences of the extracellular immunoglobulin domain-encoding regions of LILRB3 and LILRA6 in 20 individuals, and determined the copy number of these receptors, in addition to those of other members of the LILR family. We found 41 polymorphic sites within the extracellular domains of LILRB3 and LILRA6. Twenty-four of these sites were common to both receptors. LILRA6, but not LILRB3, exhibited CNV. In 20 out of 48 human cell lines from the International Histocompatibility Working Group, LILRA6 was deleted or duplicated. The only other LILR gene exhibiting genomic aberration was LILRA3, in this case due to a partial deletion. Electronic supplementary material The online version of this article (doi:10.1007/s00251-013-0742-5) contains supplementary material, which is available to authorized users.