Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) SDS-PAGE analysis of rLILRA5-His, used for immunisation of Balc/c mice. (B) Binding of anti-LILRA5 P4-11A mAb to magnetic beads coated with recombinant LILR or control proteins. mAb binding was detected using anti-IgG mAb and flow cytometric analysis. Mean ± SD of n = 3 independent experiments is shown. Paired t -test between anti-LILRA5 P4-11A and isotype control, where * p < 0.05. (C) U937 cells transfected with DNA vectors expressing LILRA5 protein, or control cells, were analysed for binding of anti-LILRA5 clone P4-11A mAb. mAb binding was detected using anti-IgG mAb and flow cytometric analysis. n = 3 from 3 independent experiments, one representative experiment is shown. (D) A schematic of the LILRA5CD3ζ reporter 2B4 T cell line, expressing a surface protein composed of extracellular and transmembrane LILRA5 domains fused to the cytoplasmic tail of CD3ζ. The cross-linking of CD3 or the fusion LILRA5CD3ζ protein induces phosphorylation of the ITAM (immunoreceptor tyrosine-based activation motif) domains in CD3ζ by Src kinases, and a signalling cascade that activates the NFAT (nuclear factor of activated T-cells) transcription factor. NFAT subsequently induces the expression of green fluorescent protein (GFP). (E) Cross-linking capacity of plate-bound anti-LILRA5 P4-11A mAb assessed using a LILRA5CD3ζ reporter cell line. GFP expression by LILRA5CD3ζ+ 2B4T cells or control 2B4T cells was assessed by flow cytometric analysis after incubation in wells containing plate-bound anti-LILRA5 P4-11A or isotype mAb. The % of GFP+ cells were quantified. Mean ± SD of n = 4 independent experiments are shown. One-way ANOVA, where **** p < 0.0001. (F) Development of an ELISA for detecting sLILRA5. anti-LILRA5 P4-11A mAb or isotype control were used to capture rLILRA5-His. A titration curve ( n = 3 ) of rLILRA5-His detected by anti-LILRA5 P4-11A mAb is shown.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: SDS Page, Binding Assay, Magnetic Beads, Recombinant, Control, Transfection, Expressing, Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Titration

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) SDS-PAGE analysis of recombinant (r) proteins, used for assessing the specificity of anti-LILRA5 P4-11A mAb. (B) Specificity of anti-LILRA5 P4-11A antibody for LILRA5. Magnetic beads coated with rLILR or control proteins and analysed for binding of anti-LILRA5 P4-11A mAb. mAb binding was detected using anti-IgG mAb and flow cytometric analysis. n = 3 from 3 independent experiments, one representative experiment is shown. (C) Concentration-dependent binding of anti-LILRA5 P4-11A mAb to rLILRA5-coated dynabeads. mAb binding was detected using anti-IgG mAb and flow cytometric analysis. n = 3 from 3 independent experiments.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: SDS Page, Recombinant, Magnetic Beads, Control, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) The normalised transcripts per million (nTPM) from the HPA dataset and the Monaco dataset are shown. nTPM values give a quantification of the transcript abundance which is comparable across samples and genes. nTPM values for classical monocytes that have been characterised to express LILRA5 is shown as a control. Mean ± SD are shown. (B) Representative example showing the gating strategy used to identify granulocyres in human whole blood using anti-CEACAM8 (CC8; clone 6/40c) and anti-LILRA5 (clone P4-11A). A first gate was set on physical parameters of SSC-A vs. FSC-A, then on SSC-A and SSC-H to eliminate doublets, then granulocytes and monocytes were gated on CD14+ and CEACAM8+, then on CEACAM8+ events to identify granulocytes. (C, D and E) Expression of LILRA5 on human monocytes and neutrophils analysed by flow cytometry analysis. C shows a representative FACS staining indicating LILRA5 expression in neutrophils and monocytes from a healthy donor. D and E show quantification of the geometric mean of monocytes ( n = 8 independent donors) and neutrophils ( n = 12 independent donors) stained with anti-LILRA5 clone P4-11A or isotype control, respectively. Paired t-test, where ** p < 0.01 and *** p < 0.001.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: Control, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) Representative example showing the gating strategy used to identify monocytes in human whole blood using anti-CD14 and anti-LILRA5 (clone P4-11A). A first gate was set on physical parameters of SSC-A vs. FSC-A, then on SSC-A and SSC-H to eliminate doublets (not shown, but similarly presented in ), then monocytes and granulocytes were gated on CD14+ and CEACAM8+ (not shown), then on CD14+ events to identify monocytes. (B) Representative flow cytometry histogram showing LILRA5 expression on CD14+ monocytes. (C) Representative flow cytometry histogram showing LILRA5 expression on CEACAM8+ neutrophils.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A and B) Stimulating LILRA5 on PBMCs induces reactive oxygen species (ROS) production, as measured using Amplex Red. A representative plot is shown in A. Mean ± SD of n = 7 independent donors is shown in B. A paired-sample t -test was used to compare Area Under the Curve (AUC) after stimulating PBMCs with anti-LILRA5 P4-11A or isotype IgG1 control, where * p < 0.05. (C and D) Stimulating LILRA5 on neutrophils induces ROS production, as measured using Amplex Red. A representative plot is shown in C. Mean ± SD of n = 8 independent donors is shown in D. A paired-sample t -test was used to compare AUC after stimulating neutrophils with anti-LILRA5 P4-11A or isotype IgG1 control, where * p < 0.05.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: Control

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) LILRA5 expression in whole blood from healthy donors after 4-hour ex vivo infection with E. coli ( n = 5, control n = 8) or S. aureus ( n = 5, control n = 8) (GSE65088). Mean ± SD are shown. Statistics tested by limma adjusted *** p < 0.001. (B) Expression of LILRA5 on human monocytes after ex vivo infection of whole blood by E. coli or S. aureus , analysed by flow cytometry analysis. One-way ANOVA was used to compare the mean fluorescence intensity (MFI) from anti-LILRA5 P4-11A staining (after subtracting of MFI isotype IgG1) for infection vs control, where ** p < 0.01 and * p < 0.05, is shown for n = 3 independent donors. (C) Surface LILRA5 expression on human monocytes from healthy donors ( n = 8) and sepsis patients ( n = 26), upon hospital admission, analysed by flow cytometry analysis. Each data point represents the MFI from anti-LILRA5 P4-11A stained cells after subtracting the MFI of isotype IgG1 stained cells. Mean ± SD are shown. (D) Surface LILRA5 expression on human monocytes from sepsis patients ( n = 8) at day 1, day 7 and day 14 of hospital admission, analysed by flow cytometry analysis. Each data point represents the MFI from anti-LILRA5 P4-11A stained cells after subtracting the MFI of isotype IgG1 stained cells. (E) Comparison of sLILRA5 in serum from sepsis patients ( n = 128) or healthy donors ( n = 60). Mean ± SD are shown. Statistics were tested by student t-test, where **** p < 0.0001.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: Expressing, Ex Vivo, Infection, Control, Flow Cytometry, Fluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: LILRA5 functions to induce ROS production on innate immune cells

doi: 10.1101/2025.03.16.643524

Figure Lengend Snippet: (A) LILRA5 expression by monocytes purified from healthy donors that were cultured ± LPS for 18 hours (GSE147310). Data from n = 5 donors are shown. Statistics tested by limma , where adjusted * p < 0.05. (B) Surface LILRA5 expression on human monocytes purified from healthy donors ( n = 3), analysed by flow cytometry analysis. Each data point represents the signal of anti-LILRA5 P4-11A relative to isotype IgG1 control. Statistics tested by paired t-test, where * p < 0.05. (C) sLILRA5 in culture supernatants from PBMCs purified from healthy donors ( n = 4), after 18 hours culture ± LPS. Statistics were tested by paired t-test, where * p < 0.05. (D) Production of reactive oxygen species (ROS) by PBMCs in response to LILRA5 ± LPS stimulation, as measured using Amplex Red. ROS production was quantified as the area under the curve (AUC). The ROS production induced by anti-LILRA5 P4-11A relative to IgG1 was calculated. Data is shown from n = 5 independent donors. Paired t -test was relative ROS production induced by anti-LILRA5 P4-11A for control vs LPS-treated cells, where ** p < 0.01.

Article Snippet: To quantify ROS production, wells of a white 96-well plate was coated overnight at 4°C with 50 µl of 5 µg/ml anti-LILRA5 clone P4-11A mAb, anti-CD89 (MCA1824; Bio Rad) or IgG1 Isotype control (0102-01; Southern Biotech) mAbs diluted in NaHCO₃ buffer, pH8.6.

Techniques: Expressing, Purification, Cell Culture, Flow Cytometry, Control

Journal: European Journal of Immunology

Article Title: LILRA5 Functions to Induce ROS Production on Innate Immune Cells

doi: 10.1002/eji.70079

Figure Lengend Snippet: A highly specific anti‐LILRA5 antibody with cross‐linking capacity. (A) SDS‐PAGE analysis of rLILRA5‐His. (B) Binding of anti‐LILRA5 P4‐11A mAb to magnetic beads coated with rLILR or control protein, detected using anti‐IgG mAb and flow cytometric analysis. Mean ± SD of n = 3 independent experiments. Paired t ‐test. (C) Binding of anti‐LILRA5 clone P4‐11A mAb to U937 cell lines, detected using anti‐IgG mAb and flow cytometric analysis. One representative experiment from n = 3 independent experiments. (D) A schematic of the LILRA5CD3ζ reporter 2B4 T cell line, expressing a surface protein composed of extracellular and transmembrane LILRA5 domains fused to the cytoplasmic tail of CD3ζ. Cross‐linking of CD3 or the fusion LILRA5CD3ζ protein induces ITAM phosphorylation, NFAT activation and GFP expression. (E) Reporter cells stimulated with anti‐CD3 mAb, anti‐LILRA5 P4‐11A mAb, or isotype control. Mean ± SD of n = 4 independent experiments. (F) Detection of rLILRA5‐His by ELISA using anti‐LILRA5 P4‐11A mAb or isotype control. Mean and SD of n = 3 experiments. In all, * p < 0.05 and **** p < 0.0001.

Article Snippet: 45 μL of 5×106 cells/mL were incubated with 3 μg/mL IgG1 isotype (0102‐01; Southern Biotech), PE‐conjugated IgG1 Isotype control, or anti‐LILRA5 P4‐11A mAb or PE‐conjugated anti‐LILRA5 P4‐11A mAb for 30 min at 4°C.

Techniques: SDS Page, Binding Assay, Magnetic Beads, Control, Expressing, Phospho-proteomics, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: European Journal of Immunology

Article Title: LILRA5 Functions to Induce ROS Production on Innate Immune Cells

doi: 10.1002/eji.70079

Figure Lengend Snippet: LPS activation reduces LILRA5‐dependent ROS production. (A) LILRA5 expression by monocytes cultured ± LPS for 18 h ( GSE147310 ). Data from n = 5 donors. Statistics tested by limma . (B) Surface LILRA5 expression on human monocytes from healthy donors ( n = 3). Student t ‐test. (C) sLILRA5 in culture supernatants from PBMCs from healthy donors ( n = 4), after 18 h culture ± LPS. Student t‐test. (D, E) Production of ROS by PBMCs in response to LILRA5 ± LPS stimulation. Data from n = 5 independent donors. Raw ROS production values are shown in (D). Student t‐ test. The ROS production induced by anti‐LILRA5 P4‐11A relative to IgG1 is compared through area under the curve (AUC), as shown in (E). In all, ** p < 0.01, * p < 0.05.

Article Snippet: 45 μL of 5×106 cells/mL were incubated with 3 μg/mL IgG1 isotype (0102‐01; Southern Biotech), PE‐conjugated IgG1 Isotype control, or anti‐LILRA5 P4‐11A mAb or PE‐conjugated anti‐LILRA5 P4‐11A mAb for 30 min at 4°C.

Techniques: Activation Assay, Expressing, Cell Culture