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The Leukocyte Immunoglobulin-Like Receptor Family Member LILRB5 Binds to HLA-Class I Heavy Chains.

Zhang Z, Hatano H, Shaw J, Olde Nordkamp M, Jiang G, Li D, Kollnberger S - PLoS ONE (2015)

Bottom Line: LILRB5 binding to HLA-class I heavy chains was confirmed by co-immunoprecipitation.Our findings show that class I free heavy chains are ligands for LILRB5.The unique binding specificity of LILRB5 for HLA-class I heavy chains probably results from differences in the D1 and D2 immunoglobulin-like binding domains which are distinct from other LILR which bind to β2m-associated HLA-class I.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom.

ABSTRACT

Objective: The leukocyte immunoglobulin-like receptor (LILR) family includes inhibitory and stimulatory members which bind to classical and non-classical HLA-class I. The ligands for many LILR including LILRB5 have not yet been identified.

Methods: We generated C-terminal eGFP and N-terminal FLAG-tagged fusion constructs for monitoring LILR expression. We screened for LILR binding to HLA-class I by tetramer staining of 293T cells transfected with LILRA1, A4, A5 A6 and LILRB2 and LILRB5. We also studied HLA class I tetramer binding to LILRB5 on peripheral monocyte cells. LILRB5 binding to HLA-class I heavy chains was confirmed by co-immunoprecipitation.

Results: HLA-B27 (B27) free heavy chain (FHC) dimer but not other HLA-class I stained LILRB5-transfected 293T cells. B27 dimer binding to LILRB5 was blocked with the class I heavy chain antibody HC10 and anti-LILRB5 antisera. B27 dimers also bound to LILRB5 on peripheral monocytes. HLA-B7 and B27 heavy chains co-immunoprecipitated with LILRB5 in transduced B and rat basophil RBL cell lines.

Conclusions: Our findings show that class I free heavy chains are ligands for LILRB5. The unique binding specificity of LILRB5 for HLA-class I heavy chains probably results from differences in the D1 and D2 immunoglobulin-like binding domains which are distinct from other LILR which bind to β2m-associated HLA-class I.

No MeSH data available.


Related in: MedlinePlus

LILRB5 is expressed on the surface of peripheral monocytes.B27 heavy chain dimers bind to monocyte LILRB5. A. Forward scatter (FSC-A) and side scatter (SSC-A) FACS plot of peripheral blood mononuclear cells (PBMC) showing gating strategy for monocytes (Mθ) and lymphocyte (Lθ) populations. The relative proportions of monocyte and lymphocyte populations are indicated to the side of each gate. B. FACS staining of peripheral CD14+ (CD3-, CD19-, CD56-) monocytes with anti-LILRB5 antiserum (bold line) or normal goat serum (NGS, shaded histogram). C. FACS staining of (left panel) CD19+ (CD3-,CD14-CD56-) peripheral B, (centre panel) CD56+ (CD3-,CD14-,CD19-) natural killer (NK) or (right panel) CD3+ (CD14-,CD19-) T cell populations with anti-LILRB5 antiserum (bold line) or NGS (shaded histogram). Representative staining from 1 of 3 independent experiments. D. B27 dimer tetramer staining of peripheral monocytes without antiserum (dashed line) or in the presence of NGS (light line) or LILRB5 antiserum (bold line). FACS staining with extravidin PE (ExPE, shaded histogram) is included as a background staining control. The respective geometric mean fluorescent intensities (MFI) for staining without antiserum, with NGS and with anti-LILRB5 antiserum were 2498, 2827 and 881 respectively. The geometric MFI for staining with extravidin PE was 236. Representative staining from 1 of 3 independent experiments.
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pone.0129063.g004: LILRB5 is expressed on the surface of peripheral monocytes.B27 heavy chain dimers bind to monocyte LILRB5. A. Forward scatter (FSC-A) and side scatter (SSC-A) FACS plot of peripheral blood mononuclear cells (PBMC) showing gating strategy for monocytes (Mθ) and lymphocyte (Lθ) populations. The relative proportions of monocyte and lymphocyte populations are indicated to the side of each gate. B. FACS staining of peripheral CD14+ (CD3-, CD19-, CD56-) monocytes with anti-LILRB5 antiserum (bold line) or normal goat serum (NGS, shaded histogram). C. FACS staining of (left panel) CD19+ (CD3-,CD14-CD56-) peripheral B, (centre panel) CD56+ (CD3-,CD14-,CD19-) natural killer (NK) or (right panel) CD3+ (CD14-,CD19-) T cell populations with anti-LILRB5 antiserum (bold line) or NGS (shaded histogram). Representative staining from 1 of 3 independent experiments. D. B27 dimer tetramer staining of peripheral monocytes without antiserum (dashed line) or in the presence of NGS (light line) or LILRB5 antiserum (bold line). FACS staining with extravidin PE (ExPE, shaded histogram) is included as a background staining control. The respective geometric mean fluorescent intensities (MFI) for staining without antiserum, with NGS and with anti-LILRB5 antiserum were 2498, 2827 and 881 respectively. The geometric MFI for staining with extravidin PE was 236. Representative staining from 1 of 3 independent experiments.

Mentions: We next stained peripheral blood mononuclear cells with anti-LILRB5 or control antisera to determine surface expression of this receptor by lymphocyte and monocyte populations by multiparameter flow cytometry (Fig 4). Lymphocyte and monocyte gates for flow cytometry were drawn on the basis of forward and side scatter of the different leukocyte populations (Fig 4A). Monocytes were defined by staining with CD14 and lack of staining with CD3 and CD19 lineage markers (Fig 4B). B lymphocytes were defined by staining with CD19 and lack of staining with CD3 and CD14 antibodies (Fig 4C left hand panel). Natural killer (NK) lymphocytes were defined by staining with CD56 and lack of staining with CD3, CD14 and CD19 antibodies (Fig 4C, centre panel). T lymphocytes were distinguished by staining with anti-CD3 and by their lack of staining with anti-CD14 and anti-CD19 antibodies (Fig 4C, right hand panel). Significant LILRB5 expression was only detected on the surface of CD14+ monocyte cells (Fig 4B). We did not detect significant expression of LILRB5 by B, NK, and T lymphocytes (Fig 4C).


The Leukocyte Immunoglobulin-Like Receptor Family Member LILRB5 Binds to HLA-Class I Heavy Chains.

Zhang Z, Hatano H, Shaw J, Olde Nordkamp M, Jiang G, Li D, Kollnberger S - PLoS ONE (2015)

LILRB5 is expressed on the surface of peripheral monocytes.B27 heavy chain dimers bind to monocyte LILRB5. A. Forward scatter (FSC-A) and side scatter (SSC-A) FACS plot of peripheral blood mononuclear cells (PBMC) showing gating strategy for monocytes (Mθ) and lymphocyte (Lθ) populations. The relative proportions of monocyte and lymphocyte populations are indicated to the side of each gate. B. FACS staining of peripheral CD14+ (CD3-, CD19-, CD56-) monocytes with anti-LILRB5 antiserum (bold line) or normal goat serum (NGS, shaded histogram). C. FACS staining of (left panel) CD19+ (CD3-,CD14-CD56-) peripheral B, (centre panel) CD56+ (CD3-,CD14-,CD19-) natural killer (NK) or (right panel) CD3+ (CD14-,CD19-) T cell populations with anti-LILRB5 antiserum (bold line) or NGS (shaded histogram). Representative staining from 1 of 3 independent experiments. D. B27 dimer tetramer staining of peripheral monocytes without antiserum (dashed line) or in the presence of NGS (light line) or LILRB5 antiserum (bold line). FACS staining with extravidin PE (ExPE, shaded histogram) is included as a background staining control. The respective geometric mean fluorescent intensities (MFI) for staining without antiserum, with NGS and with anti-LILRB5 antiserum were 2498, 2827 and 881 respectively. The geometric MFI for staining with extravidin PE was 236. Representative staining from 1 of 3 independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476610&req=5

pone.0129063.g004: LILRB5 is expressed on the surface of peripheral monocytes.B27 heavy chain dimers bind to monocyte LILRB5. A. Forward scatter (FSC-A) and side scatter (SSC-A) FACS plot of peripheral blood mononuclear cells (PBMC) showing gating strategy for monocytes (Mθ) and lymphocyte (Lθ) populations. The relative proportions of monocyte and lymphocyte populations are indicated to the side of each gate. B. FACS staining of peripheral CD14+ (CD3-, CD19-, CD56-) monocytes with anti-LILRB5 antiserum (bold line) or normal goat serum (NGS, shaded histogram). C. FACS staining of (left panel) CD19+ (CD3-,CD14-CD56-) peripheral B, (centre panel) CD56+ (CD3-,CD14-,CD19-) natural killer (NK) or (right panel) CD3+ (CD14-,CD19-) T cell populations with anti-LILRB5 antiserum (bold line) or NGS (shaded histogram). Representative staining from 1 of 3 independent experiments. D. B27 dimer tetramer staining of peripheral monocytes without antiserum (dashed line) or in the presence of NGS (light line) or LILRB5 antiserum (bold line). FACS staining with extravidin PE (ExPE, shaded histogram) is included as a background staining control. The respective geometric mean fluorescent intensities (MFI) for staining without antiserum, with NGS and with anti-LILRB5 antiserum were 2498, 2827 and 881 respectively. The geometric MFI for staining with extravidin PE was 236. Representative staining from 1 of 3 independent experiments.
Mentions: We next stained peripheral blood mononuclear cells with anti-LILRB5 or control antisera to determine surface expression of this receptor by lymphocyte and monocyte populations by multiparameter flow cytometry (Fig 4). Lymphocyte and monocyte gates for flow cytometry were drawn on the basis of forward and side scatter of the different leukocyte populations (Fig 4A). Monocytes were defined by staining with CD14 and lack of staining with CD3 and CD19 lineage markers (Fig 4B). B lymphocytes were defined by staining with CD19 and lack of staining with CD3 and CD14 antibodies (Fig 4C left hand panel). Natural killer (NK) lymphocytes were defined by staining with CD56 and lack of staining with CD3, CD14 and CD19 antibodies (Fig 4C, centre panel). T lymphocytes were distinguished by staining with anti-CD3 and by their lack of staining with anti-CD14 and anti-CD19 antibodies (Fig 4C, right hand panel). Significant LILRB5 expression was only detected on the surface of CD14+ monocyte cells (Fig 4B). We did not detect significant expression of LILRB5 by B, NK, and T lymphocytes (Fig 4C).

Bottom Line: LILRB5 binding to HLA-class I heavy chains was confirmed by co-immunoprecipitation.Our findings show that class I free heavy chains are ligands for LILRB5.The unique binding specificity of LILRB5 for HLA-class I heavy chains probably results from differences in the D1 and D2 immunoglobulin-like binding domains which are distinct from other LILR which bind to β2m-associated HLA-class I.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, United Kingdom.

ABSTRACT

Objective: The leukocyte immunoglobulin-like receptor (LILR) family includes inhibitory and stimulatory members which bind to classical and non-classical HLA-class I. The ligands for many LILR including LILRB5 have not yet been identified.

Methods: We generated C-terminal eGFP and N-terminal FLAG-tagged fusion constructs for monitoring LILR expression. We screened for LILR binding to HLA-class I by tetramer staining of 293T cells transfected with LILRA1, A4, A5 A6 and LILRB2 and LILRB5. We also studied HLA class I tetramer binding to LILRB5 on peripheral monocyte cells. LILRB5 binding to HLA-class I heavy chains was confirmed by co-immunoprecipitation.

Results: HLA-B27 (B27) free heavy chain (FHC) dimer but not other HLA-class I stained LILRB5-transfected 293T cells. B27 dimer binding to LILRB5 was blocked with the class I heavy chain antibody HC10 and anti-LILRB5 antisera. B27 dimers also bound to LILRB5 on peripheral monocytes. HLA-B7 and B27 heavy chains co-immunoprecipitated with LILRB5 in transduced B and rat basophil RBL cell lines.

Conclusions: Our findings show that class I free heavy chains are ligands for LILRB5. The unique binding specificity of LILRB5 for HLA-class I heavy chains probably results from differences in the D1 and D2 immunoglobulin-like binding domains which are distinct from other LILR which bind to β2m-associated HLA-class I.

No MeSH data available.


Related in: MedlinePlus