Journal: Immunogenetics

Article Title: Natural LILRB1 D1-D2 variants show frequency differences in populations and bind to HLA class I with various avidities

doi: 10.1007/s00251-022-01264-7

Figure Lengend Snippet: Primers used in the present study

Article Snippet: The pcDNA3.1-LILRB1-D1D2-Fc constructs were digested with XbaI and NotI (New England Biolabs, UK) and the target sequences were cloned into XbaI and NotI digested pAcGP67A vector to be used for protein production.

Techniques: Sequencing, Mutagenesis

Journal: Immunogenetics

Article Title: Natural LILRB1 D1-D2 variants show frequency differences in populations and bind to HLA class I with various avidities

doi: 10.1007/s00251-022-01264-7

Figure Lengend Snippet: Human LILRB1 is moderately polymorphic. The 46 LILRB1 alleles found in three or more individuals encode 28 LILRB1 allotypes. The amino acid changes are summarized by different domains, including the signal peptide (SP), D1-D2 domains, D3-D4 domains, STEM region, transmembrane region (TM), and the cytoplasmic tail (CYT)

Article Snippet: The pcDNA3.1-LILRB1-D1D2-Fc constructs were digested with XbaI and NotI (New England Biolabs, UK) and the target sequences were cloned into XbaI and NotI digested pAcGP67A vector to be used for protein production.

Techniques:

Journal: Immunogenetics

Article Title: Natural LILRB1 D1-D2 variants show frequency differences in populations and bind to HLA class I with various avidities

doi: 10.1007/s00251-022-01264-7

Figure Lengend Snippet: LILRB1 D1-D2 variants and their frequencies in populations. A Exon 5 and exon 6 encode the D1 and D2 domains respectively. B Nine natural LILRB1 D1-D2 variants revealed in the present study and corresponding allotypes. C Pie charts of global population LILRB1 variant incidence. Variants 1, 2, and 3 represent the three most frequent LILRB1 D1-D2 variants and they show frequency differences in populations

Article Snippet: The pcDNA3.1-LILRB1-D1D2-Fc constructs were digested with XbaI and NotI (New England Biolabs, UK) and the target sequences were cloned into XbaI and NotI digested pAcGP67A vector to be used for protein production.

Techniques: Variant Assay

Journal: Immunogenetics

Article Title: Natural LILRB1 D1-D2 variants show frequency differences in populations and bind to HLA class I with various avidities

doi: 10.1007/s00251-022-01264-7

Figure Lengend Snippet: LILRB1 D1-D2 variants bind to HLA class I with different avidities. A SDS-PAGE analysis of the eight successfully produced and purified LILRB1-D1D2-Fc proteins. B Representative integrity analysis of the D1-D2 portion of the produced proteins by flow cytometry using anti-human LILRB1 antibody. C Violin plots of the binding data of each of LILRB1 D1-D2 variants to 97 combined HLA class I tested by single antigen bead assay. The dashed line indicates there is no significant difference between variants, the single star indicates p ≤ 0.05, and the double stars indicate p ≤ 0.01. For all other comparisons, p ≤ 0.0001

Article Snippet: The pcDNA3.1-LILRB1-D1D2-Fc constructs were digested with XbaI and NotI (New England Biolabs, UK) and the target sequences were cloned into XbaI and NotI digested pAcGP67A vector to be used for protein production.

Techniques: SDS Page, Produced, Purification, Flow Cytometry, Binding Assay

Journal: Immunogenetics

Article Title: Natural LILRB1 D1-D2 variants show frequency differences in populations and bind to HLA class I with various avidities

doi: 10.1007/s00251-022-01264-7

Figure Lengend Snippet: Polymorphisms in the α3 domain of HLA class I affect the binding to LILRB1. Locus-specific polymorphisms at position 183 ( A ), 189 ( B ), and 268 ( C ) affect the binding to LILRB1. Dimorphisms at position 194 ( D ) in HLA-B and at positions 207 ( E ) and 253 ( F ) in HLA-A affect the binding to LILRB1. The star indicates p ≤ 0.05

Article Snippet: The pcDNA3.1-LILRB1-D1D2-Fc constructs were digested with XbaI and NotI (New England Biolabs, UK) and the target sequences were cloned into XbaI and NotI digested pAcGP67A vector to be used for protein production.

Techniques: Binding Assay

Journal: Immunogenetics

Article Title: Natural LILRB1 D1-D2 variants show frequency differences in populations and bind to HLA class I with various avidities

doi: 10.1007/s00251-022-01264-7

Figure Lengend Snippet: Positively charged residues at D1 domain of LILRB1 enhanced the binding to HLA class I. Electrostatic analysis of the LILRB1-HLA-A*02 structure complex ( A ), and the HLA class I ( B ), β2m ( C ), and D1-D2 of LILRB1 ( D ) are highlighted. Blue shading indicates positive charge and red indicates negative charge. In the ribbon diagrams, HLA class I is colored dark blue, β2m is red, and LILRB1 is orange. The effects of differently charged residues at positions 72 ( E ) and 103 ( F ) on the binding avidity of LILRB1 for HLA class I were determined by binding assay. Mean and SD are shown. Four stars indicate p ≤ 0.0001. G Ribbon diagram of LILRB1 and HLA-A2 complex, with positions 72 and 103 indicated by black arrows. Residues 101–106 were missing in the structure, indicated by the dashed line. The ribbon diagram shows a zoomed view of the LILRB1 D1 domain binding to HLA-A2 with residues 72 and 103 of LILRB1 indicated by arrows. Dark blue indicates the HLA-A2, red indicates the β2m, and orange indicates the LILRB1 D1 domain

Article Snippet: The pcDNA3.1-LILRB1-D1D2-Fc constructs were digested with XbaI and NotI (New England Biolabs, UK) and the target sequences were cloned into XbaI and NotI digested pAcGP67A vector to be used for protein production.

Techniques: Binding Assay

Journal: Cellular immunology

Article Title: In vitro ovarian tumor-conditioned CD163+ human macrophages retain phagocytic response to CD47 blockade.

doi: 10.1016/j.cellimm.2025.104932

Figure Lengend Snippet: Fig. 2. Tumor conditioning induces macrophage CD163 and IL-10 gene expression. Tumor-conditioned human monocyte-derived macrophages (M(A2780)) were generated and their gene expression was compared to control macrophages by RT-qPCR. A-F: Presents gene expression of polarization markers (CD163, IL-10, and TNF-α) and phagocytosis checkpoints (SIRPα, LILRB1, and Siglec-10) in tumor-associated macrophages compared to controls. Statistics: comparisons were made against controls using paired t-tests and a significance level of p = 0.05. When indicated by a logarithmic y-axis, comparisons were made on log-transformed data. Data are presented as individual data points with mean ± SD. N = 4.

Article Snippet: The plates were read at 450/ 620 nm and a standard curve ranging from 0.625 to 8 μg/L was prepared using recombinant human LILRB1 (R&D systems, 8989-T2). sCD206 (samples diluted 1:5) [34], and sSIRPα (samples diluted 1:10) [35] were measured as previously reported in detail.

Techniques: Gene Expression, Derivative Assay, Generated, Control, Quantitative RT-PCR, Transformation Assay

Journal: Cellular immunology

Article Title: In vitro ovarian tumor-conditioned CD163+ human macrophages retain phagocytic response to CD47 blockade.

doi: 10.1016/j.cellimm.2025.104932

Figure Lengend Snippet: Fig. 3. Tumor cell conditioning induces CD163, CD206, CD80 and LILRB1 on the membrane protein level. Tumor-conditioned human monocyte-derived macrophages (M(A2780)) were generated and their membrane protein expression compared to control macrophages by flow cytometry. The gating strategy is presented in Supplementary fig. 1. A-G: Presents median fluorescence intensities (MFI) of polarization markers (CD163, CD206, and CD80) and MFI values and percent macrophages positive for phagocytosis checkpoints (LILRB1, and Siglec-10). Statistics: comparisons were made against controls using paired t-tests and a significance level of p = 0.05. When indicated by a logarithmic y-axis, comparisons were made on log-transformed data. Data are presented as individual data points with mean ± SD. N = 9.

Article Snippet: The plates were read at 450/ 620 nm and a standard curve ranging from 0.625 to 8 μg/L was prepared using recombinant human LILRB1 (R&D systems, 8989-T2). sCD206 (samples diluted 1:5) [34], and sSIRPα (samples diluted 1:10) [35] were measured as previously reported in detail.

Techniques: Membrane, Derivative Assay, Generated, Expressing, Control, Flow Cytometry, Fluorescence, Transformation Assay

Journal: European journal of immunology

Article Title: Alternative mRNA splicing creates transcripts encoding soluble proteins from most LILR genes.

doi: 10.1002/eji.200839080

Figure Lengend Snippet: Figure 5. sLILRB1 transcript encodes a soluble protein, is present in DC supernatants and blocks the binding of HLA-class I molecules to mLILRB1. (A) LILRB1 was detected by Western blot in lysates of HEK293T cells transfected with sLILRB1 and mLILRB1 transcripts but not in mock-transfected cells. The sLILRB1 transcript encodes a secreted protein with an apparent molecular weight of 65 Kd, detected only in the supernatant of sLILRB1- transfected cells. (B) sLILRB1 protein was detected in concentrated supernatants of MDDC from two donors on days (d.) 5 and 7 during in vitro culturing but was absent in concentrated culture medium. For comparison, supernatants of sLILRB1 and mock-transfected HEK293T cells were loaded. (C) Increasing amounts (in microlitres) of concentrated supernatant from sLILRB1-transfected HEK293T cells induced a reciprocal decrease in HLA-B2705 pentamer binding of Hela cells expressing mLILRB1 or CD8a. The mean average and standard deviation from four experiments are shown. Similar results were obtained using a HLA-B0702 pentamer (data not shown). (D) Cytotoxicity assays were performed using YT-LILRB1 effector cells and either 721.221-HLA-G target cells or the parental 721.221 cell-line at E:T ratios of 10:1 and 0:1. Each reaction contained concentrated supernatant (sup.) from sLILRB1 or mock-transfected HEK293T cells. Each treatment was performed in triplicate. The figure displays a representative result from one of three experiments. NK-mediated lysis was greatly reduced by 721.221 expression of HLA-G. sLILRB1 induced a 1.5-fold increase in specific lysis of 721.221-HLA-G target cells.

Article Snippet: After overnight blocking with 5% non-fat milk in PBS/0.05% Tween, the membrane was incubated with a mouse anti-human LILRB1 antibody (clone 292319, R&D Systems) for 1 h, at a concentration of 1mg/mL in milk/PBS/Tween solution.

Techniques: Binding Assay, Western Blot, Transfection, Molecular Weight, In Vitro, Comparison, Expressing, Standard Deviation, Lysis