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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

HLA-B27 free heavy chain dimers bind to LILRB5.FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRA1, LILRA4, LILRA5, LILRA6, LILRB2 and LILRB5 stained with Extravidin-PE (left panels) or Extravidin PE-conjugated HLA-B27 free heavy chain dimer tetramers (centre panels). FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. (Right panels) FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRA1, LILRA4, LILRA5, LILRA6, and LILRB5 stained with allophycocyanin (APC) conjugated anti-FLAG antibody. FACS plots show APC fluorescence from anti-FLAG staining plotted against eGFP expression of each of the fusion constructs. Representative FACS stain from 1 of 4 independent experiments.
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pone.0129063.g001: HLA-B27 free heavy chain dimers bind to LILRB5.FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRA1, LILRA4, LILRA5, LILRA6, LILRB2 and LILRB5 stained with Extravidin-PE (left panels) or Extravidin PE-conjugated HLA-B27 free heavy chain dimer tetramers (centre panels). FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. (Right panels) FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRA1, LILRA4, LILRA5, LILRA6, and LILRB5 stained with allophycocyanin (APC) conjugated anti-FLAG antibody. FACS plots show APC fluorescence from anti-FLAG staining plotted against eGFP expression of each of the fusion constructs. Representative FACS stain from 1 of 4 independent experiments.

Mentions: We and others have previously reported that HLA-B27 β2microglobulin (B27) free heavy chain dimers bind to the leukocyte immunoglobulin-like receptors LILRA1 and LILRB2 [9, 10, 21]. We extended these studies to investigate binding of B27 dimers to other LILR members. 293T cells were transfected with FLAG and eGFP-tagged B5, A1, A4, A5 and A6 or HA and eGFP tagged LILRB2 expression constructs and stained with extravidin PE or extravidin PE-conjugated B27 dimer tetramer (Fig 1). Good levels of expression of all constructs used in this study were observed as indicated by detection of eGFP fluorescence by FACS (Fig 1). B27 dimer tetramers stained LILRA1 and LILRB2 transfected cells in agreement with previous results [9, 21]. B27 dimer tetramers also stained LILRB5 transfected cells (Fig 1). We have previously shown cell surface expression of HA and eGFP tagged LILRB2 in transfected 293 T cells [10]. LILRA1, A4, A5 and A6 and LILRB5 constructs were expressed at the surface of transfected cells as assessed by staining with anti-FLAG antibody (Fig 1).


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)

HLA-B27 free heavy chain dimers bind to LILRB5.FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRA1, LILRA4, LILRA5, LILRA6, LILRB2 and LILRB5 stained with Extravidin-PE (left panels) or Extravidin PE-conjugated HLA-B27 free heavy chain dimer tetramers (centre panels). FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. (Right panels) FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRA1, LILRA4, LILRA5, LILRA6, and LILRB5 stained with allophycocyanin (APC) conjugated anti-FLAG antibody. FACS plots show APC fluorescence from anti-FLAG staining plotted against eGFP expression of each of the fusion constructs. Representative FACS stain from 1 of 4 independent experiments.
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Related In: Results  -  Collection

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

pone.0129063.g001: HLA-B27 free heavy chain dimers bind to LILRB5.FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRA1, LILRA4, LILRA5, LILRA6, LILRB2 and LILRB5 stained with Extravidin-PE (left panels) or Extravidin PE-conjugated HLA-B27 free heavy chain dimer tetramers (centre panels). FACS plots show PE fluorescence from tetramer or Extravidin staining plotted against eGFP expression of each of the fusion experiments. (Right panels) FACS staining of 293 T cells transfected with eGFP and FLAG-tagged constructs of LILRA1, LILRA4, LILRA5, LILRA6, and LILRB5 stained with allophycocyanin (APC) conjugated anti-FLAG antibody. FACS plots show APC fluorescence from anti-FLAG staining plotted against eGFP expression of each of the fusion constructs. Representative FACS stain from 1 of 4 independent experiments.
Mentions: We and others have previously reported that HLA-B27 β2microglobulin (B27) free heavy chain dimers bind to the leukocyte immunoglobulin-like receptors LILRA1 and LILRB2 [9, 10, 21]. We extended these studies to investigate binding of B27 dimers to other LILR members. 293T cells were transfected with FLAG and eGFP-tagged B5, A1, A4, A5 and A6 or HA and eGFP tagged LILRB2 expression constructs and stained with extravidin PE or extravidin PE-conjugated B27 dimer tetramer (Fig 1). Good levels of expression of all constructs used in this study were observed as indicated by detection of eGFP fluorescence by FACS (Fig 1). B27 dimer tetramers stained LILRA1 and LILRB2 transfected cells in agreement with previous results [9, 21]. B27 dimer tetramers also stained LILRB5 transfected cells (Fig 1). We have previously shown cell surface expression of HA and eGFP tagged LILRB2 in transfected 293 T cells [10]. LILRA1, A4, A5 and A6 and LILRB5 constructs were expressed at the surface of transfected cells as assessed by staining with anti-FLAG antibody (Fig 1).

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