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

Abstract

B cell activation is regulated by the stimulatory or inhibitory co-receptors of B cell receptors (BCRs). Here, we investigated the signaling mechanism of Fc receptor-like 1 (FcRL1), a newly identified BCR co-receptor. FcRL1 was passively recruited into B cell immunological synapses upon BCR engagement in the absence of FcRL1 cross-linking, suggesting that FcRL1 may intrinsically regulate B cell activation and function. BCR cross-linking alone led to the phosphorylation of the intracellular Y₂₈₁ENV motif of FcRL1 to provide a docking site for c-Abl, an SH2 domain-containing kinase. The FcRL1 and c-Abl signaling module, in turn, potently augmented B cell activation and proliferation. FcRL1-deficient mice exhibited markedly impaired formation of extrafollicular plasmablasts and germinal centers, along with decreased antibody production upon antigen stimulation. These findings reveal a critical BCR signal-enhancing function of FcRL1 through its intrinsic recruitment to B cell immunological synapses and subsequent recruitment of c-Abl upon BCR cross-linking.

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.