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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

B27 dimer binding to LILRB5 is inhibited by LILRB5 specific antiserum.A. FACS staining of LILRB5, LILRA1 and B2 transfected 293T cells with HLA-B27 FHC dimer tetramer or Extravidin PE with or without isotype control antisera (ISOT) or anti-LILRB5 antisera (αLILRB5) as indicated. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the respective fusion constructs. Representative FACS stain from 1 of 3 independent experiments. B. Representative FACS staining of 293T cells transfected with the indicated LILR constructs and stained with anti-LILRB5 or normal goat antisera (NGS). Representative stain from 1 of 3 independent experiments.
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pone.0129063.g003: B27 dimer binding to LILRB5 is inhibited by LILRB5 specific antiserum.A. FACS staining of LILRB5, LILRA1 and B2 transfected 293T cells with HLA-B27 FHC dimer tetramer or Extravidin PE with or without isotype control antisera (ISOT) or anti-LILRB5 antisera (αLILRB5) as indicated. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the respective fusion constructs. Representative FACS stain from 1 of 3 independent experiments. B. Representative FACS staining of 293T cells transfected with the indicated LILR constructs and stained with anti-LILRB5 or normal goat antisera (NGS). Representative stain from 1 of 3 independent experiments.

Mentions: We next addressed to what extent anti-LILRB5 antisera could specifically inhibit B27 dimer binding to LILRB5 transfected cells. Preincubation of LILRB5-transfected cells with anti-LILRB5 antisera inhibited B27 dimer staining of LILRB5-transfected 293T cells but did not affect B27 dimer staining of 293T cells transfected with LILRB2 or LILRA1 (Fig 3A). Control normal goat antiserum (NGS) had no effect on B27 dimer tetramer staining of LILRB5 transfected 293T cells (Fig 3A).


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)

B27 dimer binding to LILRB5 is inhibited by LILRB5 specific antiserum.A. FACS staining of LILRB5, LILRA1 and B2 transfected 293T cells with HLA-B27 FHC dimer tetramer or Extravidin PE with or without isotype control antisera (ISOT) or anti-LILRB5 antisera (αLILRB5) as indicated. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the respective fusion constructs. Representative FACS stain from 1 of 3 independent experiments. B. Representative FACS staining of 293T cells transfected with the indicated LILR constructs and stained with anti-LILRB5 or normal goat antisera (NGS). Representative stain from 1 of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4476610&req=5

pone.0129063.g003: B27 dimer binding to LILRB5 is inhibited by LILRB5 specific antiserum.A. FACS staining of LILRB5, LILRA1 and B2 transfected 293T cells with HLA-B27 FHC dimer tetramer or Extravidin PE with or without isotype control antisera (ISOT) or anti-LILRB5 antisera (αLILRB5) as indicated. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the respective fusion constructs. Representative FACS stain from 1 of 3 independent experiments. B. Representative FACS staining of 293T cells transfected with the indicated LILR constructs and stained with anti-LILRB5 or normal goat antisera (NGS). Representative stain from 1 of 3 independent experiments.
Mentions: We next addressed to what extent anti-LILRB5 antisera could specifically inhibit B27 dimer binding to LILRB5 transfected cells. Preincubation of LILRB5-transfected cells with anti-LILRB5 antisera inhibited B27 dimer staining of LILRB5-transfected 293T cells but did not affect B27 dimer staining of 293T cells transfected with LILRB2 or LILRA1 (Fig 3A). Control normal goat antiserum (NGS) had no effect on B27 dimer tetramer staining of LILRB5 transfected 293T cells (Fig 3A).

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