Fig 1.
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.
Fig 2.
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.
Fig 3.
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.
Fig 4.
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.
Fig 5.
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.