Limits...
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 binds specifically to HLA-B27 free heavy chain dimers but does not bind to β2m and peptide associated HLA-A3, HLA-B7 and HLA-B27 heterodimers A. FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRB2 and LILRB5 and stained with Extravidin-PE, orExtravidin-PE conjugated tetramers of HLA-A3, HLA-B7, and HLA-B27 heterodimer or HLA-B27 free heavy chain (FHC) dimers. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. Representative FACS stain from 1 of 3 independent experiments B. FACS staining of LILRB5 transfected 293T cells with HLA-B27 FHC dimer tetramer or Extravidin PE with or without isotype control antibody (IgG2a) or free heavy chain antibody HC10 as indicated. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. Representative FACS stain from 1 of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4476610&req=5

pone.0129063.g002: LILRB5 binds specifically to HLA-B27 free heavy chain dimers but does not bind to β2m and peptide associated HLA-A3, HLA-B7 and HLA-B27 heterodimers A. FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRB2 and LILRB5 and stained with Extravidin-PE, orExtravidin-PE conjugated tetramers of HLA-A3, HLA-B7, and HLA-B27 heterodimer or HLA-B27 free heavy chain (FHC) dimers. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. Representative FACS stain from 1 of 3 independent experiments B. FACS staining of LILRB5 transfected 293T cells with HLA-B27 FHC dimer tetramer or Extravidin PE with or without isotype control antibody (IgG2a) or free heavy chain antibody HC10 as indicated. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. Representative FACS stain from 1 of 3 independent experiments.

Mentions: Both B27 dimers and β2microglobulin-associated HLA-class I heterodimers bind to LILRB2. It is unknown whether β2microglobulin-associated HLA-class I bind to LILRB5. Thus, we next stained LILRB2-, and LILRB5-transfected 293T cells with extravidin PE, or HLA-A3, HLA-B7, and HLA-B27 heterodimer and B27 homodimer tetramers. LILRB2 transfected 293T cells stained with both heterodimer and homodimer tetramers stained LILRB5 transfected 293T cells (Fig 2A).


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 binds specifically to HLA-B27 free heavy chain dimers but does not bind to β2m and peptide associated HLA-A3, HLA-B7 and HLA-B27 heterodimers A. FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRB2 and LILRB5 and stained with Extravidin-PE, orExtravidin-PE conjugated tetramers of HLA-A3, HLA-B7, and HLA-B27 heterodimer or HLA-B27 free heavy chain (FHC) dimers. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. Representative FACS stain from 1 of 3 independent experiments B. FACS staining of LILRB5 transfected 293T cells with HLA-B27 FHC dimer tetramer or Extravidin PE with or without isotype control antibody (IgG2a) or free heavy chain antibody HC10 as indicated. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. Representative FACS stain from 1 of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129063.g002: LILRB5 binds specifically to HLA-B27 free heavy chain dimers but does not bind to β2m and peptide associated HLA-A3, HLA-B7 and HLA-B27 heterodimers A. FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRB2 and LILRB5 and stained with Extravidin-PE, orExtravidin-PE conjugated tetramers of HLA-A3, HLA-B7, and HLA-B27 heterodimer or HLA-B27 free heavy chain (FHC) dimers. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. Representative FACS stain from 1 of 3 independent experiments B. FACS staining of LILRB5 transfected 293T cells with HLA-B27 FHC dimer tetramer or Extravidin PE with or without isotype control antibody (IgG2a) or free heavy chain antibody HC10 as indicated. FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. Representative FACS stain from 1 of 3 independent experiments.
Mentions: Both B27 dimers and β2microglobulin-associated HLA-class I heterodimers bind to LILRB2. It is unknown whether β2microglobulin-associated HLA-class I bind to LILRB5. Thus, we next stained LILRB2-, and LILRB5-transfected 293T cells with extravidin PE, or HLA-A3, HLA-B7, and HLA-B27 heterodimer and B27 homodimer tetramers. LILRB2 transfected 293T cells stained with both heterodimer and homodimer tetramers stained LILRB5 transfected 293T cells (Fig 2A).

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