Bystander B cells rapidly acquire antigen receptors from activated B cells by membrane transfer

Abstract

The B cell antigen receptor (BCR) efficiently facilitates the capture and processing of a specific antigen for presentation on MHC class II molecules to antigen-specific CD4(+) T cells (1). Despite this, the majority of B cells are thought to play only a limited role in CD4(+) T cell activation because BCRs are clonotypically expressed. Here, we show, however, that activated B cells can, both in vitro and in vivo, rapidly donate their BCR to bystander B cells, a process that is mediated by direct membrane transfer between adjacent B cells and is amplified by the interaction of the BCR with a specific antigen. This results in a dramatic expansion in the number of antigen-binding B cells in vivo, with the transferred BCR endowing recipient B cells with the ability to present a specific antigen to antigen-specific CD4(+) T cells.

Fig. 1.

B cells exchange BCRs after prolonged activation by LPS. (A) B6.CD45.1 and MD4 spleen cells were cocultured in the presence of LPS. Dot plots show expression of IgM^a and IgM^b on live (Hoechst 33258^low) CD45.1⁺ (red) and CD45.1⁻ (black) B220⁺ cells over 3 days. Values are percentages of B6.CD45.1 B cells expressing IgM^a (red) or MD4 B cells expressing IgM^b (black) relative to background expression on B cells cultured independently (represented by quadrant gates). (B) Transfer of IgM^a to B6.CD45.1 B220⁺ cells and B220⁻ cells after 3 days of culture with MD4 cells (red open histograms). Gray filled histogram represents background IgM^a staining of B6.CD45.1 splenocytes cultured alone, and black open histogram depicts IgM^a expression on MD4 B cells.

Fig. 2.

Activated B cells rapidly transfer BCRs to both resting and activated B cells through nonsoluble factors in a process enhanced by specific antigens. (A) Fractions of day-3 LPS-activated MD4 spleen cell cultures were added to 3-day LPS-activated B6.CD45.1 spleen cells and incubated at 37°C for up to 2 h or at 4°C for 1 h. The percentage of B6.CD45.1 B cells expressing IgM^a was assessed as in Fig. 1A and IgM^a transfer represented as a proportion (%) of IgM^a transfer over 3 days of continuous coculture. MD4 cell culture fractions included whole culture, MD4 cells washed to remove soluble factors, MD4 culture supernatant (CSN) depleted of cells by centrifugation, and the cell-depleted CSN passed through an 800-nm filter. (B) The capacity of B cells from μ_s^−/− mice to transfer IgM^a to B6 B cells after continuous coculture for 3 days at 37°C or after brief (3 h) coincubation at 4°C. For 3-day incubations, B6 spleen cells (IgM^b) were cocultured with spleen cells from μ_s^−/− mice (IgM^a) or 129sv (μ_s^+/+) mice (the founder strain for the IgM^a expressed in μ_s^−/− mice but that can secrete IgM^a) in the presence of LPS. For brief incubations, 3-day LPS-activated spleen cells were incubated together for ≈3 h. B6 B cells were then assessed for IgM^a surface expression. Numbers without parentheses are the percentages of B6 B cells expressing IgM^a after coculture with donor B cells relative to IgM^a expression by B6 B cells cultured alone. Numbers in parentheses are mean fluorescence intensities of the respective histograms. The histogram depicts the concentration of IgM in the culture supernatants of either μ_s^−/− or μ_s^+/+ splenocytes cultured for 3 days with LPS. (C) Day-3 LPS-activated MD4 spleen cells were incubated with either 3-day LPS-stimulated (activated) or freshly isolated (resting) B6.CD45.1 spleen cells for 1 h at 4°C. B6.CD45.1 B cells were then assessed for IgM^a surface expression (Left) and MD4 B cells assessed for IgM^b surface expression (Right). Data are expressed as the percentage of B cells expressing nonendogeneous IgM. (D) Day-3 LPS-activated MD4 spleen cells were incubated with LPS-activated B6.CD45.1 spleen cells in the presence of HEL or OVA for 1 h at 4°C. B6.CD45.1 B cells were then assessed for IgM^a surface expression represented as a proportion (%) of IgM^a transfer over 3 days of continuous coculture. Data in A, C, and D are representative of at least three independent experiments.

Fig. 3.

BCR transfer occurs concomitantly with transfer of other membrane components. (A) (Upper) Simultaneous expression of IgM^a and CD45.2 on B6.CD45.1 B cells cultured with MD4 B cells for 3 days with LPS. The percentage of B6.CD45.1 B cells positive for IgM^a and CD45.2 after coculture with MD4 B cells is shown adjacent to the respective axis (and arrow) for each marker. The boxed area represents background staining for CD45.2 and IgM^a of B6.CD45.1 spleen cells cultured alone. (Lower) Simultaneous transfer of IgM^a and prelabeled MD4 surface molecules to resting B6.CD45.1 B cells. Day-3 LPS-activated MD4 spleen cells were covalently cell-surface labeled with fluorescein and prelabeled with antibodies to IgM^a and CD45.2. After extensive washing, cells were incubated for 1 h at 37°C with freshly isolated B6.CD45.1 spleen cells prelabeled with antibodies to B220 and CD45.1 and immediately analyzed by flow cytometry. The percentage of B6.CD45.1 B cells positive for IgM^a and fluorescein is shown as in Upper. The boxed area represents background staining for IgM^a and background fluorescein fluorescence of freshly isolated B6.CD45.1 spleen cells. (B) Day-3 LPS-activated MD4 spleen cells, labeled with PKH-26, were incubated with day-3 LPS-activated B6.CD45.1 spleen cells for 1 h at 4°C in the presence of various concentrations of HEL. B cells were then assessed for expression of PKH-26 and IgM^a. Numbers are the percentage of B6.CD45.1 B cell expressing each marker. (C) Day-3 LPS-activated MD4 spleen cells were stained with PKH-26 and an anti-B220 mAb, incubated on a cooling stage set at 4°C with anti-B220 mAb-labeled day-3 LPS-activated EGFP-Tg spleen cells, and analyzed by confocal microscopy. Red, PKH-26; green, EGFP. (Scale bars, 5 μm.) (i and ii) Green and red channel merging of simultaneous confocal images of B cells, showing many membrane adhesions between the two B cell populations within 60 min of coincubation (i, 59 min; ii, 17 min). Arrows show overlap of EGFP and PKH-26 fluorescence within the same focal plane. Data in A–C are representative of at least three independent experiments.

Fig. 4.

Bystander B cells that acquire an antigen-specific BCR gain the ability to present antigen to CD4⁺ T cells. B cells purified from CBA/H (H-2^k) spleen were cultured with LPS in either the absence or presence of purified splenic B cells from MD4/EGFP-Tg (H-2^b) mice. After 3 days of culture, EGFP⁺ MD4 B cells were depleted from CBA/H-MD4 B cell cocultures by flow cytometry. CBA/H B cells purified from the cocultures (CBA/H +/- MD4), as well as CBA/H and MD4 B cells cultured alone, were then pulsed with HEL for 20 min at 4°C and washed and equal numbers (1.5 × 10⁵) cultured with 1 × 10⁵ purified CFSE-labeled CD4⁺ 3A9 TCR-Tg T cells specific for a HEL-peptide presented by I-A^k. After 0.5 days, CD4⁺ T cells were assessed for CD69 expression. Data are representative of three independent experiments.

Fig. 5.

Antigen-specific B cells transfer their BCR to bystander B cells in vivo during antigen-specific immune reactions. (A) CFSE-labeled MD4 spleen cells, together with 10 μg of HEL–OVA and 10 μg of LPS, were injected i.v. into B6.CD45.1 mice 2 h after the i.v. injection of CFSE-labeled OT-II lymphocytes. Spleen cells from the mice were analyzed over the next 7 days by flow cytometry. (i) Dot plots show B220 and IgM^a expression on host (CD45.2⁻, black events) and donor (CD45.2⁺, red events) cell populations, with numbers referring to the percentage of splenocytes (boxed region) that are MD4 B cells (B220⁺, CD45.2⁺, IgM^ahigh). Histograms show IgM^a expression on host B cells (B220⁺, CD45.2⁻, CFSE⁻) and MD4 B cells (B220⁺, CD45.2⁺, IgM^ahigh) and isotype control mAb binding to host B cells, with numbers showing the percentage of host cells expressing IgM^a relative to the isotype control. (ii) Viable MD4, OT-II, and recipient (host) B cells were assessed for CFSE expression. (B) An identical experiment to that described in A, except that on day 3 after commencement of the experiment, one animal was challenged i.v. with a 10-μg bolus of HEL 1 h before spleen cell harvest and marker analysis. (C) As in A, except that on day 4 after commencement of the experiment, one animal was challenged i.v. with a 10-μg bolus of HEL 1 h before spleen harvest. In addition, spleen cells from a non-HEL-injected animal were incubated at 4°C for 1 h in the presence of various amounts of HEL before measurement of marker expression. Analysis involved assessing expression of IgM^a and CD45.2 on B6.CD45.1 host B cells compared with autofluorescence and isotype control mAb binding (boxed region). Numbers are the percentages of B6.CD45.1 B cells expressing CD45.2 or IgM^a above background levels, with MD4 B cells that express high levels of IgM^a and CD45.2 being gated out of the analysis.