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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 binds to HLA-B7 and HLA-B27 free heavy chains.A. Isotype control (IgG2b), anti-FLAG (αFLAG) and eGFP staining of 293T (upper panels) and RBL cells (lower panels) transduced with LILRB5 lentivirus. B Left hand panel. Representative western blot of immunoprecipitates from cell lysates of 221B27 cells transduced with LILRB5 with isotype control MAb (IgG2a) or the class I heavy chain MAb HC10. Blots were probed with HRP conjugated anti-FLAG MAb. Right hand panel. Representative western blot of immunoprecipitates from cell lysates of RBLB27 and RBLB7 cells transduced with LILRB5 with isotype control MAb (IgG2a) or the class I heavy chain MAb HC10. Blots were probed with HRP conjugated anti-FLAG MAb. C. Upper panel. Representative western blot of immunoprecipitates with rabbit anti-FLAG antisera from cell lysates from parental RBL or RBL cells transduced with B27 and RBL cells transduced with LILRB5 and HLA-B27. Western blots were probed with the class I heavy chain MAb HC10 and HRP-conjugated-anti-mouse Igs. The asterisked band is non-specific and the position of monomeric class I heavy chain (M) is indicated. D. Upper panels. Representative western blots of anti-FLAG immunoprecipitates or cell lysates from RBLB27 and RBLB7 cells transduced with LILRA5 or LILRB5. Western blots of immunoprecipitates were probed with the class I heavy chain MAb HC10 and HRP-conjugated-anti-mouse Igs. Centre panels. Western blots of cell lysates were probed with HRP-conjugated mouse anti-FLAG antibody as indicated. Lower panel. Representative western blot of with HC10 MAb of cell lysates from RBL cells or RBL cells transduced with LILRB5 and HLA-B27 or HLA-B7. Representative blots from 1 of 2 independent experiments. All immunoprecipitates were resolved by SDS-PAGE performed under reducing conditions.
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pone.0129063.g005: LILRB5 binds to HLA-B7 and HLA-B27 free heavy chains.A. Isotype control (IgG2b), anti-FLAG (αFLAG) and eGFP staining of 293T (upper panels) and RBL cells (lower panels) transduced with LILRB5 lentivirus. B Left hand panel. Representative western blot of immunoprecipitates from cell lysates of 221B27 cells transduced with LILRB5 with isotype control MAb (IgG2a) or the class I heavy chain MAb HC10. Blots were probed with HRP conjugated anti-FLAG MAb. Right hand panel. Representative western blot of immunoprecipitates from cell lysates of RBLB27 and RBLB7 cells transduced with LILRB5 with isotype control MAb (IgG2a) or the class I heavy chain MAb HC10. Blots were probed with HRP conjugated anti-FLAG MAb. C. Upper panel. Representative western blot of immunoprecipitates with rabbit anti-FLAG antisera from cell lysates from parental RBL or RBL cells transduced with B27 and RBL cells transduced with LILRB5 and HLA-B27. Western blots were probed with the class I heavy chain MAb HC10 and HRP-conjugated-anti-mouse Igs. The asterisked band is non-specific and the position of monomeric class I heavy chain (M) is indicated. D. Upper panels. Representative western blots of anti-FLAG immunoprecipitates or cell lysates from RBLB27 and RBLB7 cells transduced with LILRA5 or LILRB5. Western blots of immunoprecipitates were probed with the class I heavy chain MAb HC10 and HRP-conjugated-anti-mouse Igs. Centre panels. Western blots of cell lysates were probed with HRP-conjugated mouse anti-FLAG antibody as indicated. Lower panel. Representative western blot of with HC10 MAb of cell lysates from RBL cells or RBL cells transduced with LILRB5 and HLA-B27 or HLA-B7. Representative blots from 1 of 2 independent experiments. All immunoprecipitates were resolved by SDS-PAGE performed under reducing conditions.

Mentions: B lymphoblasts and mast cells have been shown to express mRNA for LILRB5. Thus, we transduced the human B lymphoblast cell line LCL.721.221 and the rat basophil RBL line with LILRB5 lentivirus. We also transduced 293T cells with LILRB5. Surface expression of LILRB5 was assessed by staining with anti-FLAG antibody. Although LILRB5 was expressed at the cell surface of transduced 293T cells (Fig 5A), we did not detect cell surface expression of LILRB5 by staining transduced RBL or 221 cells with anti-FLAG antibody (Fig 5A and results not shown). This was despite high levels of expression of LILRB5 in these cells as assessed by western blotting of immunoprecipitates and eGFP expression by flow cytometry (Fig 5A and 5B and results not shown).


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 to HLA-B7 and HLA-B27 free heavy chains.A. Isotype control (IgG2b), anti-FLAG (αFLAG) and eGFP staining of 293T (upper panels) and RBL cells (lower panels) transduced with LILRB5 lentivirus. B Left hand panel. Representative western blot of immunoprecipitates from cell lysates of 221B27 cells transduced with LILRB5 with isotype control MAb (IgG2a) or the class I heavy chain MAb HC10. Blots were probed with HRP conjugated anti-FLAG MAb. Right hand panel. Representative western blot of immunoprecipitates from cell lysates of RBLB27 and RBLB7 cells transduced with LILRB5 with isotype control MAb (IgG2a) or the class I heavy chain MAb HC10. Blots were probed with HRP conjugated anti-FLAG MAb. C. Upper panel. Representative western blot of immunoprecipitates with rabbit anti-FLAG antisera from cell lysates from parental RBL or RBL cells transduced with B27 and RBL cells transduced with LILRB5 and HLA-B27. Western blots were probed with the class I heavy chain MAb HC10 and HRP-conjugated-anti-mouse Igs. The asterisked band is non-specific and the position of monomeric class I heavy chain (M) is indicated. D. Upper panels. Representative western blots of anti-FLAG immunoprecipitates or cell lysates from RBLB27 and RBLB7 cells transduced with LILRA5 or LILRB5. Western blots of immunoprecipitates were probed with the class I heavy chain MAb HC10 and HRP-conjugated-anti-mouse Igs. Centre panels. Western blots of cell lysates were probed with HRP-conjugated mouse anti-FLAG antibody as indicated. Lower panel. Representative western blot of with HC10 MAb of cell lysates from RBL cells or RBL cells transduced with LILRB5 and HLA-B27 or HLA-B7. Representative blots from 1 of 2 independent experiments. All immunoprecipitates were resolved by SDS-PAGE performed under reducing conditions.
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Related In: Results  -  Collection

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

pone.0129063.g005: LILRB5 binds to HLA-B7 and HLA-B27 free heavy chains.A. Isotype control (IgG2b), anti-FLAG (αFLAG) and eGFP staining of 293T (upper panels) and RBL cells (lower panels) transduced with LILRB5 lentivirus. B Left hand panel. Representative western blot of immunoprecipitates from cell lysates of 221B27 cells transduced with LILRB5 with isotype control MAb (IgG2a) or the class I heavy chain MAb HC10. Blots were probed with HRP conjugated anti-FLAG MAb. Right hand panel. Representative western blot of immunoprecipitates from cell lysates of RBLB27 and RBLB7 cells transduced with LILRB5 with isotype control MAb (IgG2a) or the class I heavy chain MAb HC10. Blots were probed with HRP conjugated anti-FLAG MAb. C. Upper panel. Representative western blot of immunoprecipitates with rabbit anti-FLAG antisera from cell lysates from parental RBL or RBL cells transduced with B27 and RBL cells transduced with LILRB5 and HLA-B27. Western blots were probed with the class I heavy chain MAb HC10 and HRP-conjugated-anti-mouse Igs. The asterisked band is non-specific and the position of monomeric class I heavy chain (M) is indicated. D. Upper panels. Representative western blots of anti-FLAG immunoprecipitates or cell lysates from RBLB27 and RBLB7 cells transduced with LILRA5 or LILRB5. Western blots of immunoprecipitates were probed with the class I heavy chain MAb HC10 and HRP-conjugated-anti-mouse Igs. Centre panels. Western blots of cell lysates were probed with HRP-conjugated mouse anti-FLAG antibody as indicated. Lower panel. Representative western blot of with HC10 MAb of cell lysates from RBL cells or RBL cells transduced with LILRB5 and HLA-B27 or HLA-B7. Representative blots from 1 of 2 independent experiments. All immunoprecipitates were resolved by SDS-PAGE performed under reducing conditions.
Mentions: B lymphoblasts and mast cells have been shown to express mRNA for LILRB5. Thus, we transduced the human B lymphoblast cell line LCL.721.221 and the rat basophil RBL line with LILRB5 lentivirus. We also transduced 293T cells with LILRB5. Surface expression of LILRB5 was assessed by staining with anti-FLAG antibody. Although LILRB5 was expressed at the cell surface of transduced 293T cells (Fig 5A), we did not detect cell surface expression of LILRB5 by staining transduced RBL or 221 cells with anti-FLAG antibody (Fig 5A and results not shown). This was despite high levels of expression of LILRB5 in these cells as assessed by western blotting of immunoprecipitates and eGFP expression by flow cytometry (Fig 5A and 5B and results not shown).

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