Research ArticleIMMUNOLOGY

Fc receptor–like 1 intrinsically recruits c-Abl to enhance B cell activation and function

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Science Advances  17 Jul 2019:
Vol. 5, no. 7, eaaw0315
DOI: 10.1126/sciadv.aaw0315
  • Fig. 1 FcRL1 deficiency in CH27 cells impairs B cell activation induced by antigen binding in the absence of FcRL1 ligation.

    Left: Representative TIRFM images showing the synaptic accumulation of BCRs (A) and the synaptic recruitment of pSyk (C), pBLNK (E), and pPI3K (p85α) (G) in CH27-WT, CH27-FcRL1-KO, and CH27-FcRL1-Rescue cells. Scale bars, 1.5 μm. Right: Statistical quantification of the synaptic accumulation of BCRs (B) (n = 24 to 27 cells) and signaling molecules of pSyk (D) (n = 38 to 75 cells), pBLNK (F) (n = 39 to 44 cells), and pPI3K (p85α) (H) (n = 49 to 59 cells) in CH27-WT, CH27-FcRL1-KO, and CH27-FcRL1-Rescue cells. Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SEM. Two-tailed Student’s t tests were used for statistical comparisons.

  • Fig. 2 FcRL1 deficiency impairs antigen binding–induced calcium mobilization and the in vitro proliferation of primary B cells.

    (A and B) Normalized Ca2+ mobilization indicated by Fluo-4–to–Fura Red ratio in primary B cells stimulated with F(ab′)2 goat anti-mouse IgM (A) and in CH27 B cells stimulated with PC10-BSA (B). The arrows indicate the time of antigen stimulation. Experiments were repeated three times, and data from a representative experiment are shown. (C to E) Representative proliferation profiles of mouse primary B cells at 72 hours after stimulation indicated by the decreased intensity of the proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE). Mouse WT and FcRL1-KO primary B cells were stimulated via BCR ligation (C). Mouse FcRL1-deficient primary B cells expressing HA-FcRL1(WT)-mCherry or HA-FcRL1(Y281F)-mCherry were stimulated via FcRL1 ligation (D). Mouse FcRL1-deficient primary B cells expressing HA-FcRL1(WT)-mCherry were treated with imatinib or dimethyl sulfoxide (DMSO) via stimulated by FcRL1 ligation (E). Experiments were repeated three times, and data from a representative experiment are shown.

  • Fig. 3 FcRL1 is passively recruited into B cell immunological synapses upon BCR engagement in primary spleen B cells.

    (A) Representative TIRFM images showing the synaptic accumulation of BCR or FcRL1 in primary B cells stimulated with 30 nM F(ab′)2 goat anti-mouse MHC I, 30 nM F(ab′)2 rabbit anti-HA, 30 nM F(ab′)2 goat anti-mouse IgM, or 30 nM F(ab′)2 goat anti-mouse IgM + 30 nM F(ab′)2 rabbit anti-HA. Scale bars, 1.5 μm. (B) Colocalization analyses of BCR and FcRL1 microclusters within B cell immunological synapses (n = 33 to 36 cells). Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SEM. Two-tailed Student’s t tests were used for the statistical comparisons. (C and D) Statistical quantification of accumulated FcRL1 (C) (n = 31 to 34 cells) and BCR (D) (n = 31 to 35 cells) within B cell immunological synapses. Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SD. Each symbol represents one cell. Two-tailed Student’s t tests were used for statistical comparisons.

  • Fig. 4 FcRL1 recruits c-Abl via the phosphorylated Y281ENV motif.

    (A) Representative TIRFM images showing the synaptic recruitment of the c-Abl–SH2 domain within FcRL1 clusters in CH27 cells stimulated via FcRL1 or MHC I ligation. Scale bars, 1.5 μm. (B) Colocalization analyses of the c-Abl–SH2 domain and FcRL1 microclusters in CH27 cells stimulated via FcRL1 or MHC I ligation (n = 30 cells). Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SEM. Two-tailed Student’s t tests were used for statistical comparisons. (C) Detection of the interaction between the c-Abl–SH2 domain and FcRL1 via immunoprecipitation in CH27 cells stimulated via FcRL1 ligation. Immunoblots of glutathione S-transferase (GST) and GST-c-Abl–SH2 were probed with mouse anti-GST antibody. Immunoblots of FcRL1 chimeric receptors were probed with rabbit anti-YFP antibody. WT, HA-FcRL1(WT)-YFP; M1, HA-FcRL1(Y281F)-YFP; M2, HA-FcRL1(Y293F)-YFP; M3, HA-FcRL1(Y339F)-YFP. Experiments were repeated three times, and data from a representative experiment are shown. (D) Binding of different concentration of synthetic peptides of the FcRL1 intracellular domain to the c-Abl–SH2 domain as tested using ELISA. Binding was indicated by the optical density (O.D.) at 490 nm. Experiments were repeated three times, and data from a representative experiment are shown. Data are shown as means ± SD from triplicate replicates. (E) Tyrosine phosphorylation within HA-FcRL1(WT)-YFP and HA-FcRL1(Y281F)-YFP chimeric receptors expressed in CH27-FcRL1-KO cells stimulated via FcRL1 or BCR ligation. Immunoblots of phosphorylated tyrosine were probed with phosphorylated tyrosine-specific antibody 4G10. Immunoblots of FcRL1 chimeric receptors were probed with rabbit anti-YFP antibody. Mu, HA-FcRL1(Y281F)-YFP. Experiments were repeated three times, and data from a representative experiment are shown. (F) Given are representative TIRFM images showing the synaptic recruitment of the whole c-Abl kinase within FcRL1 clusters in CH27 B cells stimulated via FcRL1 or MHC I ligation. Scale bars, 1.5 μm. (G) Colocalization analyses of the whole c-Abl kinase and FcRL1 microclusters in CH27 B cells stimulated via FcRL1 or MHC I ligation. (n = 40 to 56 cells). Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SEM. Two-tailed Student’s t tests were used for statistical comparisons. (H and I) Quantitative analysis of the mFI of FcRL1 microclusters (H) (n = 42 to 51 cells) and the c-Abl participated in FcRL1 microclusters (I) (n = 37 to 41 cells) in CH27 B cells stimulated via FcRL1 or MHC I ligation. Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SEM. Two-tailed Student’s t tests were used for the statistical comparisons. (J) Detection of the interaction between the c-Abl–mCherry-GST and FcRL1 by immunoprecipitation in CH27 B cells stimulated via FcRL1 or BCR ligation. Immunoblots of c-Abl–mCherry-GST were probed with mouse anti-GST. Immunoblots of FcRL1 chimeric receptors were probed with rabbit anti-YFP antibody. Ct, control group without ligation. Experiments were repeated three times, and data from a representative experiment are shown. (K) Detection of the interaction between the endogenous c-Abl kinase and FcRL1 by immunoprecipitation in CH27 B cells stimulated via FcRL1 ligation. Immunoblots of endogenous c-Abl were probed with anti-c-Abl antibody. Immunoblots of FcRL1 chimeric receptors were probed with rabbit anti-YFP antibody. Experiments were repeated three times, and data from a representative experiment are shown.

  • Fig. 5 Mutation of Y281ENV motif in FcRL1 impairs BCR engagement–induced B cell activation in the absence of FcRL1 ligation.

    Left: Representative TIRFM images showing the synaptic recruitment of BCRs (A), pSyk (C), pBLNK (E), and pPI3K (p85α) (G) in FcRL1-KO primary B cells expressing HA-FcRL1(WT)-YFP and HA-FcRL1(Y281F)-YFP chimeric receptors. Scale bars, 1.5 μm. Right: Quantitative analysis of the mFI of synaptic BCRs (B) (n = 34 to 36 cells), pSyk (D) (n = 72 to 77 cells), pBLNK (F) (n = 76 to 96 cells), and pPI3K (p85α) (H) (n = 60 to 75 cells). Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SEM. Two-tailed Student’s t tests were used for the statistical comparisons.

  • Fig. 6 FcRL1 deficiency impairs the germinal center formation and antibody responses.

    (A and B) Representative flow cytometric gating (A) and quantitative analysis of the percentage of GC B cells (B) in the spleens of FcRL1-KO and WT mice (n = 6 mice per group) immunized with sheep red blood cells for 7 days. Each symbol represents one mouse. Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SD. Two-tailed Student’s t tests were used for statistical comparisons. SSC-H, side scatter–height. (C to E) Antibody titers from FcRL1-KO and WT mice (n = 6 mice per group) at different times after immunization with 5 μg of NP32-KLH and Imject Alum Adjuvant. Sequential serum ELISA was performed to detect NP-specific antibody. NP8-BSA for IgM (C), NP8-BSA for IgG (D), and NP30-BSA for IgG (E) were used as the coating antigens. Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SEM. Two-tailed Student’s t tests were used for statistical comparisons. (F to H) Representative enzyme-linked immunosorbent spot assays (F) and quantitative analysis of NP-specific IgM (G) and IgG antibody-forming cells (H) in the spleens of FcRL1-KO and WT mice (n = 9 mice per group). Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SEM. Two-tailed Student’s t tests were used for statistical comparisons. (I and J) Antibody titers from FcRL1-KO and WT mice (n = 6 mice per group) at different times after immunization with 20 μg of NP50-Ficoll and Imject Alum Adjuvant. Sequential serum ELISA was performed to detect the NP-specific antibody. NP30-BSA for IgM (I) and NP30-BSA for IgG3 (J) were used as the coating antigens. Experiments were repeated three times, and data from a representative experiment are shown. Bar represents means ± SEM. Two-tailed Student’s t tests were used for statistical comparisons.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw0315/DC1

    Fig. S1. Deletion of FcRL1 gene in CH27 cells and mouse with CRISPR-Cas9.

    Fig. S2. Flow cytometry analysis of the expression level of BCR and FcRL1 on plasma membrane and intracellular c-Abl fusion protein.

    Fig. S3. Off-target detection of the sgRNA used for knocking out FcRL1 in CH27 cells.

    Fig. S4. Off-target detection of the upstream sgRNA used for knocking out FcRL1 in mouse.

    Fig. S5. Off-target detection of the downstream sgRNA used for knocking out FcRL1 in mouse.

    Fig. S6. FcRL1 deficiency in primary B cell intrinsically impaired the antigen binding–induced B cell activation in the absence of FcRL1 ligation.

    Fig. S7. FcRL1 is passively recruited into the B cell immunological synapse upon BCR engagement in CH27-FcRL1-KO B cells expressing HA-FcRL1-YFP.

    Fig. S8. Sequence alignment of the cytoplasmic tail of FcRL1.

    Fig. S9. Disruption of FcRL1 gene had no effect on B cell development in FcRL1-KO mice.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Deletion of FcRL1 gene in CH27 cells and mouse with CRISPR-Cas9.
    • Fig. S2. Flow cytometry analysis of the expression level of BCR and FcRL1 on plasma membrane and intracellular c-Abl fusion protein.
    • Fig. S3. Off-target detection of the sgRNA used for knocking out FcRL1 in CH27 cells.
    • Fig. S4. Off-target detection of the upstream sgRNA used for knocking out FcRL1 in mouse.
    • Fig. S5. Off-target detection of the downstream sgRNA used for knocking out FcRL1 in mouse.
    • Fig. S6. FcRL1 deficiency in primary B cell intrinsically impaired the antigen binding–induced B cell activation in the absence of FcRL1 ligation.
    • Fig. S7. FcRL1 is passively recruited into the B cell immunological synapse upon BCR engagement in CH27-FcRL1-KO B cells expressing HA-FcRL1-YFP.
    • Fig. S8. Sequence alignment of the cytoplasmic tail of FcRL1.
    • Fig. S9. Disruption of FcRL1 gene had no effect on B cell development in FcRL1-KO mice.

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