Journal: JCI Insight

Article Title: LILRB3 (ILT5) is a myeloid cell checkpoint that elicits profound immunomodulation

doi: 10.1172/jci.insight.141593

Figure Lengend Snippet: ( A ) Schematic of antibody generation by phage-display via 3 independent “panning” techniques; (i) immobilized target (LILRB3), (ii) biotinylated target and excess nontarget (LILRB1), and (iii) LILRB3-transfected cell lines (from left to right). Biopanning was performed against generated target protein using an scFv library; “nontarget” cross-reactive scFv clones were removed by competition, and target-specific scFv clones were then eluted and converted to a soluble format, sequenced, and screened by various cell- and protein-based assays. ( B and C ) Screening of generated LILRB3 clones. ( B ) FMAT and ( C ) ELISA were performed and scFv clones screened against LILRB3 target– and LILRB1/LILRB2 nontarget–transfected CHO-S cells and extracellular LILRB1 protein, respectively. The relative binding to each target was calculated, with target-specific scFv clones depicted in yellow and the irrelevant isotype control shown in green. Nonbinding and cross-reactive scFv clones depicted in blue. ( D ) Screening of LILRB3 scFv clones by high-throughput flow cytometry. PBMCs (left plot) or LILR-transfected CHO-S (middle plot) cells were incubated with His-tagged scFv supernatants, followed by secondary anti-His staining. Where transfected CHO-S cells were used, LILRB1- and LILRB2-transfected cells were used as nontargets for LILRB3. Clones were compared against both gated CD14 + monocytes and target-transfected CHO-S cells (right plot). LILRB3-specific clones highlighted in yellow, nonspecific or nonbinding clones in red, and isotype control in green. ( E ) Specificity of LILRB3 clones against human LILR-transfected 2B4 cells. LILRB3 mAbs were tested against cells stably transfected with the indicated LILR family members by flow cytometry; a representative specific clone (A16; top panel) and a nonspecific cross-reactive clone (A30; bottom panel) are shown. ( F – G ) Testing the specificity of directly fluorochrome-labeled LILRB3 clones against primary cells by flow cytometry. ( F ) Fresh whole peripheral blood stained with either APC-labeled LILRB3 (represented by clone A16) or an irrelevant human (h) IgG1 isotype control as well as various leukocyte surface markers, as indicated. Dot plots and histograms are representative of multiple donors indicating gating of each leukocyte subset as indicated: T cells, B cells, NK cells, monocytes, and granulocytes. ( G ) Graph showing relative expression of LILRB3 on each leukocyte subset. One-way ANOVA test performed (* P < 0.05; ** P < 0.005); n = 5 independent donors (each color represents an individual donor). ( E – F ) Histogram pink and blue traces indicate staining with irrelevant isotype control or LILRB3 mAb, respectively.

Article Snippet: In panning 1, BioInvent n-CoDeR scFv were selected using biotinylated in-house–produced recombinant LILRB3-hFc fusion proteins (captured with streptavidin-coated Dynabeads, Thermo Fisher Scientific) with or without competition or LILRB1-hFc coated onto etched polystyrene balls (Polysciences) or plastic immunotubes.

Techniques: Transfection, Generated, Clone Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Control, High Throughput Screening Assay, Flow Cytometry, Incubation, Staining, Stable Transfection, Labeling, Expressing