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.

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 Ϫ /CD16 ϩ 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-1␤, and a significant impairment in cytolytic function associated with decreased killer immunoglobulin-like receptor 3DL1 expression. Genotyping patients (nϭ222) and controls (nϭ191) 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-␤, 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-␥ 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. (Circulation. 2012;126:1099-1109.)Key Words: immunology Ⅲ pulmonary hypertension Ⅲ natural killer cells Ⅲ transforming growth factor-␤ P ulmonary arterial hypertension (PAH) is characterized by a loss of pulmonary vasculature at the level of the precapillary arterioles. This pathological vascular remodeling is marked by excessive muscularization of the remaining pulmonary arteries and the formation of obstructive vascular lesions associated with the aberrant proliferation of endothelial cells, vascular smooth muscle cells, and fibroblasts. The resulting elevation of pulmonary arterial pressure leads to right ventricular hypertrophy and death from right heart failure. Recently, members of the transforming growth factor-␤ (TGF-␤) superfamily have been recognized as critical mediators of pulmonary vascular remodeling. Mutations in the gene encoding the bone morphogenetic protein type II receptor account for Ͼ70% of heritable PAH cases and 15% to 40% of cases of the sporadic form of the disease. 1 Reduced bone morphogenetic protein signaling and heightened TGF-␤ signaling have been reported in pulmonary vascular cells from PAH patients, whether or not...

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.

Salmonella typhimurium is a facultative pathogen capable of entering and replicating in both professional and non-professional antigen presenting cells. Control of infection requires MHC class II restricted CD4 T-helper cell responses. Here we show that Salmonella infection induced polyubiquitination of HLA-DR, a post-translational modification that led to removal of mature, peptide loaded, ␣␤ dimers from the cell surface. Immature ␣␤Ii complexes were unaffected. Surface expression of all class II isotypes, HLA-DP, -DQ, and -DR, was reduced in infected cells, but other cell-surface molecules that traffic through class II peptide loading compartments were unaffected. A Salmonella strain carrying a mutation in ssaV did not induce ubiquitination of class II, implicating Salmonella T3SS-2 effector proteins in the process. T3SS-2 effectors, with established or proposed roles in ubiquitination, were not required for class II down-regulation, suggesting that an additional T3SS-2 effector is involved in regulating MHC class II ubiquitination. Although recognized as a viral immune evasion strategy, here, we demonstrate that bacteria can control surface MHC expression through ubiquitination.

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

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 ¼ 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 ¼ 0.019). These results suggest that KIR genotype and HLA ligand interaction may contribute to the genetic susceptibility of UC.

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.

Restricted expression of human leucocyte antigen-G (HLA-G) to fetal extravillous trophoblast cells, which invade the decidua during implantation, suggests a role for HLA-G in placentation. In this study, we have investigated several aspects of HLA-G expression and function. Surface levels of HLA-G expression were measured in 70 normal pregnancies. We show the dimeric conformation that is unique to HLA-G forms after passage through the Golgi apparatus. Differences were found in the receptor repertoire of decidual natural killer (dNK) cells that express the leucocyte immunoglobulin-like receptor B1 (LILRB1), which binds dimeric HLA-G strongly. We then measured functional responses of dNK cells with LILRB1, when stimulated by HLA-G in both monomeric and dimeric conformations. Degranulation, interferon-γ and interleukin-8 production by dNK cells freshly isolated from the first trimester implantation site were either undetected or not affected by HLA-G. These findings should be considered when inferring the activity of tissue NK cells from results obtained with cell lines, peripheral NK or cultured dNK cells.