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

Quah et al. 10.1073/pnas.0800259105.

Supporting Information

Files in this Data Supplement:

SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Figure 11
SI Figure 12
SI Figure 13
SI Table 1
SI Text

SI Figure 6

Fig. 6. Confocal microscopy of B cells expressing nonendogenous IgM. Spleen cells from EGFP-Tg mice expressing EGFP and IgM^b were cultured for 3 days alone or in the presence of MD4 spleen cells with LPS (10 mg/ml). Cells were stained with anti-B220-Cy-Chrome and anti-IgM^a-PE conjugated mAbs and then fixed and analyzed by confocal microscopy. A single field from cocultured EGFP-Tg and MD4 cells is depicted, showing the three separate fluorescence channels, namely B220⁺ B cells (Cy-Chrome, all B cells blue), MD4 splenocytes (red IgM^a-PE⁺, green EGFP^-, indicated by D) surrounded by green EGFP⁺ B cells expressing low to medium levels of red IgM^a. A single field of B220⁺ EGFP-Tg cells cultured alone is also shown and depicts green EGFP⁺ B cells (Cy-Chrome⁺; data not shown) that do not bind red anti-IgM^a-PE. Data are representative of four independent experiments

SI Figure 7A
SI Figure 7B

Fig. 7. B cells exchange BCRs following activation by danger signals and T helper cell signals.

(A) CD40L stimulation induces BCR transfer to bystander B cells. B6.CD45.1 splenocytes (expressing both IgM^b and CD45.1) were labeled with CFSE (5 mM) and then cocultured with MD4 splenocytes (expressing both a HEL-specific IgM^a, IgD^a transgene on a C57BL/6 background and CD45.2) for 3 days with CD40L (prepared from CD40L-baculovirus-infected Sf9 cell membranes, as described in SI Text, and used at amounts generating maximal B cell proliferation), IL-4 (50 ng/ml; Peprotech) and HEL (1 mg/ml). Both B6.CD45.1 and MD4 splenocytes were also cultured alone as controls. B cell blasts were then analyzed by flow cytometry for IgM^a, with regions gated on the basis of single color control samples as shown. Note the substantial increase in IgM^a expression on B6.CD45.1 (CFSE⁺) B cells after coculture with MD4 B cells. (B) Activation of B cells by T helper cells can induce BCR transfer to bystander B cells. EGFP⁺ spleen cells from HEL-specific BCR-Tg (MD4/EGFP-Tg) mice (2 ´ 10⁶ per milliliter) were cultured with spleen cells from OVA-specific TCR-Tg OT-II mice (2 ´ 10⁶ per milliliter) in the presence of the conjugated antigen HEL-OVA. B cells from each population were then monitored for MD4 (Donor)-derived IgM^a expression after 3 days of coculture (expressed as a percentage relative to fluorescent controls). B cells from OT-II spleen cells (Recipient B cells) cultured alone were used as a control to monitor background levels of IgM^a expression. These cultures showed that, in the presence of increasing concentrations of specific antigen, which resulted in increasing activation of the OT-II T cells and MD4 B cells (data not shown), there was a dramatic increase in transfer of IgM^a from the MD4 (Donor) B cells to the OT-II bystander (Recipient) B cells.

SI Figure 8

Fig. 8. Viable cells, but not cell debris, apoptotic cells, or dead cells, are responsible for BCR transfer. Day-3 LPS-activated MD4 spleen cell cultures were separated by flow cytometry into viable, dead, and apoptotic cells as well as a cell debris fraction based on Hoechst 33258 staining and forward-scatter properties (dot plots). These fractions and the total MD4 spleen cell culture were then incubated with day-3 LPS-activated B6.CD45.1 spleen cells for 1 h at 4°C. B6.CD45.1 B cells were then assessed for IgM^a expression after incubation either in the presence of the various MD4 spleen cell culture fractions (black open histogram) or in the absence of the fractions (gray filled histogram), with IgM^a expression on the different MD4 B220⁺ fractions also assessed (gray open histogram). The percentage of IgM^a+ B6.CD45.1 B cells, after incubation with the various MD4 fractions, is indicated in each histogram. Data are representative of four independent experiments.

SI Figure 9

Fig. 9. BCR-specific antigen increases BCR transfer. This figure shows data from Fig. 2D but as histograms, revealing the extent to which BCR-specific antigen can increase BCR transfer to recipient B cells. Data show transfer of IgM^a from 3-day LPS-activated MD4 spleen cells to LPS-activated B6.CD45.1 spleen cells in the presence of HEL or OVA (both 0.1 ng/ml) after a 1-h incubation at 4°C. MIX refers to IgM^a expression by B6.CD45.1 B cells coincubated with MD4 B cells, whereas ALONE refers to IgM^a expression by B6.CD45.1 B cells incubated alone. B cell populations were distinguished based on CD45 allotype expression. Data are representative of three independent experiments.

SI Figure 10

Fig. 10. Electron microscopy of cell-cell interactions. Day-3 LPS-activated MD4 spleen cells were mixed with freshly isolated B6.CD45.1 spleen cells for 40 min on 0.1% poly-L-lysine-coated plastic coverslips and then prepared for EM analysis as described in Methods. Activated lymphocytes (MD4 B cells) in contact with resting lymphocytes (B6.CD45.1 cells) were chosen for analysis by SEM (i-iv) and TEM (v and vi). ii, iv, and vi are magnifications of membrane contacts between cells depicted in i, iii, and v, respectively. The larger cell in each panel, which exhibits extensive membrane ruffling by SEM, is a LPS-activated B cell, whereas the smaller cell represents a resting B lymphocyte. Note the close association between the plasma membranes of the interacting lymphocytes and contact between the cells by short nanotube-like structures.

SI Figure 11

Fig. 11. Enhancement and inhibition of BCR transfer by various reagents. Day-3 LPS-activated MD4 spleen cells were incubated with freshly isolated B6.CD45.1 spleen cells for 1-2 h in the continuous presence (C) of various reagents, and surface expression of IgM^a (i.e., MD4-derived IgM) was then assessed on B220⁺ B6.CD45.1 cells by flow cytometry. In some cases, MD4 cells were pretreated (P) with the various reagents as indicated and washed twice before incubation with B6.CD45.1 cells. The percentage of IgM^a+ B6.CD45.1 B220⁺ cells resulting from the treatment incubations was then divided by the percentage of IgM^a+ B6.CD45.1 B220⁺ cells from untreated incubations and multiplied by 100 to give percentage of normal IgM^a transfer. The shaded region indicates the 95% confidence interval around the mean IgM^a transfer observed in control cultures (based on controls from untreated incubations from 12 samples across four independent experiments). Each data value is the mean of 1-10 experiments.

Refs. in Fig. 11 relevant to cell membrane exchange:

1. Vignery A (2000) Osteoclasts and giant cells: Macrophage-macrophage fusion mechanism. Int J Exp Pathol 81:291-304.

2. Takeda Y, et al. (2003) Tetraspanins CD9 and CD81 function to prevent the fusion of mononuclear phagocytes. J Cell Biol 161:945-956.

3. Tachibana I, Hemler ME (1999) Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance. J Cell Biol 146:893-904.

4. Proulx A, Merrifield PA, Naus CC (1997) Blocking gap junctional intercellular communication in myoblasts inhibits myogenin and MRF4 expression. Dev Genet 20:133-144.

5. Poupot M, Fournie JJ (2003) Spontaneous membrane transfer through homotypic synapses between lymphoma cells. J Immunol 171:2517-2523.

6. Sauer I, Dunay IR, Weisgraber K, Bienert M, Dathe M (2005) An apolipoprotein E-derived peptide mediates uptake of sterically stabilized liposomes into brain capillary endothelial cells. Biochemistry 44: 2021-2029.

7. Driesen RB, et al. (2005) Partial cell fusion: A newly recognized type of communication between dedifferentiating cardiomyocytes and fibroblasts. Cardiovasc Res.

8. Hong W. (2005) SNAREs and traffic. Biochim Biophys Acta 1744:493-517.

9. Gaus K, et al. (2003) Visualizing lipid structure and raft domains in living cells with two-photon microscopy. Proc Natl Acad Sci U S A 100: 15554-15559.

10. Harroun TA, Balali-Mood K, Gourlay I, Bradshaw JP (2003) The fusion peptide of simian immunodeficiency virus and the phase behavior of N-methylated dioleoylphosphatidylethanolamine. Biochim Biophys Acta 1617:62-68.

11. Chung HA, Kato K, Itoh C, Ohhashi S, Nagamune T (2004) Casual cell surface remodeling using biocompatible lipid-poly(ethylene glycol)(n): Development of stealth cells and monitoring of cell membrane behavior in serum-supplemented conditions. J Biomed Mater Res A 70:179-85.

12. Rhodes J (1990) Erythrocyte rosettes provide an analogue for Schiff base formation in specific T cell activation. J Immunol 145:463-469.

SI Figure 12A
SI Figure 12B

Fig. 12. Bystander B cells that acquire an antigen-specific BCR gain the ability to present antigen to CD4+ T cells but the acquired BCR is unable to generate an intracellular Ca2+ signal following ligation. (A) 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 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) were cultured with 1 ´ 10⁵ purified CFSE-labeled CD4⁺ 3A9 TCR-Tg T cells specific for a HEL-peptide presented by I-A^k. After 3 days, CD4⁺ T cells were also assessed for CFSE expression, with the numbers in each histogram referring to the percentage of T cells that have entered one or more divisions based on CFSE dilution. Data are representative of 3 independent experiments. (B) Transferred BCRs do not transduce signals required for normal Ca²⁺ mobilization. Day 3 LPS-activated cocultures of B6.CD45.1 and MD4 spleen cells or separate cultures of LPS-activated B6.CD45.1 splenocytes were loaded with Indo-1 for internal Ca²⁺ flux studies. B6.CD45.1 (CD45.1⁺ CD45.2^- B220⁺) B cells were then assessed for IgM^a and IgM^b expression and for their ability to produce a Ca²⁺ flux in response to the addition of 1 mg/ml anti-IgM^a or anti-IgM^b antibody. "Mix" refers to B6.CD45.1 from B6.CD45.1 and MD4 B cells cocultured for 3 days with LPS before flow cytometry experiments, and "alone" refers to B6.CD45.1 B cells cultured alone with LPS. Data are representative of 7 independent experiments.

SI Figure 13

Fig. 13. Antigen-specific B cells transfer their BCR to bystander B cells in vivo during antigen-specific immune reactions and convert bystander B cells to antigen-specific APCs. Freshly isolated CFSE-labeled MD4 spleen cells, together with 10 mg of HEL-OVA and 10 mg of LPS, were injected i.v. into B6.CD45.1 recipient mice 2 h after the i.v. injection of CFSE-labeled OT-II lymphocytes. On day 4, animals were challenged i.v. with a 30-mg bolus of either HEL or HEL-OVA 1 h before spleen harvest. Spleen cells were then labeled with IgM^a-, B220-, and CD45.2-specific mAbs and B6.CD45.1 B cells (B220⁺, CD45.2^-) sorted into populations expressing low levels (Low) and medium levels (Med) of IgM^a by flow cytometry as indicated in the dot-plot profile. Graded numbers of the different B cell populations, from both HEL- and HEL-OVA-challenged mice, were then assessed for their capacity to activate (CD69⁺ and/or divided based on CFSE dilution) a constant number (1.5 ´ 10⁵) of CFSE-labeled CD4⁺ OT-II lymphocytes after 3 days of coincubation. Data are representative of three independent experiments.

Table 1. Primary antibodies used for marker analysis

Specificity	Clone of origin	Preparation^ab	Source
CD4	GK1.5	APC conjugated	1
CD16/CD32	2.4G2	Purified	2
B220(CD45R)	RA3-6B2	Cy-Chrome, PerCP-Cy5.5 conjugated	2
CD45.1	A20	Biotinylated, PE and PE-Cy7 conjugated	2
CD45.2	104	Biotinylated, FITC conjugate	2
CD69	H1.2F3	PE conjugated	2
Thy1.2 (CD90.2)	53-2.1	Biotinylated, PE conjugated	2
IgM^a	DS-1	Biotinylated, FITC and PE conjugate	2
IgM^b	AF6-78	Purified, Biotinylated and PE conjugate	2

^aAntibodies were used at the minimum amount required for saturating binding to splenocytes (LPS-activated splenocytes in the case of CD69 antibody binding).

^bFITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin-chlorophyll-protein; APC, allophycocyanin.

1. Caltag.

2. Pharmingen.

SI Text

Antibodies. MAbs used in the study are described in SI Table 1.

Doublet Discrimination. Where possible, cell fluorescence pulse area and width (normally of the FSC and SSC parameters) were used to exclude cell doublet. In cases where pulse area and width were not available, doublets were eliminated from the data by excluding "recipient" events that had the same level of fluorescence as "donor" events (i.e., CD45 allotypes or EGFP expression).

Confocal Microscopy. Confocal microscopy was performed with a Radiance Confocal Microscope (Bio-Rad) using the Argon 488 nm laser as the sole source of excitation. For assessment of IgM^a transfer, cells were labeled with a Cy-Chrome-conjugated B220-specific mAb and a PE-conjugated mAb specific for IgM^a, then fixed in 1% paraformaldehyde/PBS and mounted on slides in reagents from the SlowFade antifade kit (Molecular Probes) as per instructions.

Electron Microscopy. Activated MD4 spleen cells (3-day LPS-activated) were mixed with freshly isolated B6.CD45.1 RBC-depleted spleen cells at a concentration of 4 ´ 10⁷ cells per milliliter each in 0.3 ml of sDMEM and left to attach to plastic coverslips coated with 0.1% poly-L-lysine for 40 min at 37°C. Cells were then immediately prepared for transmission electron microscopy (TEM) or scanning EM (SEM). For TEM, samples were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h, washed three times in 0.1 M sodium cacodylate buffer, and postfixed in 1% OsO₄ in 0.1 M sodium cacodylate for 20 min. After two washes in buffer, the samples were dehydrated in a graded ethanol series to 100% ethanol and embedded in Spurrs resin at 70°C. Samples were photographed by using a Hitachi H7000 electron microscope at 75 kV. For SEM, samples were prepared by using the same method as for TEM until the 100% ethanol step. They were then transferred into 100% amyl acetate (three washes) and critical point dried. The samples were coated with gold-palladium and photographed by using a Hitachi S4500 FESEM at 10 kV.

CD40L Preparation. SF9 cells were infected with 10% vol/vol recombinant CD40L baculovirus stock (kindly provided by Phil Hodgkin, Walter and Eliza Hall Institute, Melbourne). After 5 days at 27°C in air, infected cells were harvested and sedimented at 300 × g for 5 min. Cells from the equivalent of 1,000 ml of 5-day culture were then resuspended in 7 ml of homogenisation buffer (20 mM Tris·HCL (pH 7.4), 10 mM NaCl, 0.1 mM MgCl₂ 0.5 mM CaCl₂, 0.1 mM PMSF) and homogenized with a Polytron homogenizer (Kinematica). Membranes containing CD40L were then enriched from the cell homogenate by density centrifugation using a 3-ml 41% sucrose underlay and sedimentation at 96,000 × g for 1 h at 4°C. The membrane-containing interface was then collected and washed twice, by diluting the membranes up to 10 ml in PBS and centrifugation at 100,000 × g for 1 h at 4°C and removing the supernatant. The final membrane pellet was resuspended in PBS (10 ml/1,000 ml of infected SF9 cells originally harvested), passed through an 18-gauge needle 15 times, aliquoted, and stored at -70°C until use.

Ca²⁺ Flux Assay. Intracellular Ca²⁺ analysis was performed by using the ratiometric indicator Indo-1 (Molecular Probes). Day-3 LPS-activated cocultures of MD4 and B6.CD45.1 spleen cells and separate control cultures of MD4 and B6.CD45.1 spleen cells were harvested, washed, and resuspended to 1 ´ 10⁷ cells per milliliter in sDMEM/10%FCS containing 1 mM Indo-1. Cells were incubated for a total of 30 min at 37°C, the last 10 min in the presence of anti-B220-Cy-Chrome, anti-CD45.1-PE, and anti-CD45.2-FITC-conjugated mAbs (Pharmingen). Cells were then washed twice, resuspended in sDMEM/10%FCS at 5 ´ 10⁶ cells per milliliter, and prewarmed to 37°C immediately before analysis by flow cytometry for Ca²⁺ flux. Stimuli included anti-IgM^a or anti-IgM^b mAbs (Pharmingen) at a final concentration of 1 mg/ml.

Comments on SI Fig. 11: Additional Data on the Molecular Basis of BCR Transfer Between B Cells. In an attempt to define the molecular basis of BCR transfer, a large number of inhibitors of cell adhesion, membrane integrity, and membrane fusion were assessed for their ability to inhibit BCR donation to bystander cells. The culture system entailed mixing LPS-activated transgenic (MD4) B cells with freshly isolated splenic B cells and assessing BCR/membrane transfer after coincubation for 2 h at 37°C. This system most closely mimics the in vivo situation, where there is rapid unidirectional transfer of BCR from activated B cells to bystander naïve B cells.

A total of 26 mAbs or mAb combinations were examined for their ability to modify BCR transfer, mAbs being chosen that recognize cell-surface molecules on B cells. Particular attention was given to investigating molecules that have been shown to be involved in cell adhesion and cell fusion [reviewed in Chen EH, Olsen EN (2005) Unveiling the mechanisms of cell-cell fusion. Science 308:369-373] with the same mAb clones being used that have been reported previously to block function. Also all mAbs were used at saturating concentrations. None of the mAbs tested inhibited BCR transfer, although a few mAbs significantly enhanced BCR transfer, namely anti-CD2, CD19, CD44, CD48, and CD62L. The effect of the CD19-specific mAb is not surprising because CD19 is a signaling molecule that can associate with the BCR and BCR engagement by antigen (HEL) also enhances transfer. CD2 and CD48 represent a receptor-ligand pair that may facilitate transfer, CD44 has been implicated in cell fusion, whereas the role of CD62L requires further investigation. Metabolic inhibitors (sodium azide, 4°C), protein kinase C inhibitors (Rottlerin, Gu6976), modifiers of plasma membrane lipid organization (annexin V, apolipoprotein-E, methyl b-cyclodextrin, CBZ-D-FFG, polyethleneglycol-lipid), cytoskeleton/microtubule disrupters (cytochalasin-B, latrunculin-B, colchicine), broad-spectrum ion-channel blockers (hexamethylamiloride, amantadine), gap junction blockers (1-octanol), and inhibition of protein secretion (Brefeldin A) had little or no effect on transfer. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, which facilitate intracellular membrane fusion, appeared to be not involved because N-ethylmaleimide slightly enhanced rather than inhibited transfer. Fixation of the donor MD4 B cells with glutaraldehyde or paraformaldehyde prevented transfer. Schiff base formation has been reported to stabilize cell adhesion [Rhodes J (1996) Covalent chemical events in immune induction: Fundamental and therapeutic aspects. Immunol Today 17:436-439], but the continual presence of high concentrations (170 mM) of lysine, an inhibitor of Schiff base formation, actually enhanced BCR transfer. Furthermore, reduction of cell-surface aldehydes by pretreatment of donor MD4 B cells with sodium borohydride enhanced the rate of BCR transfer (data not shown), a result consistent with Schiff base formation interfering with BCR/membrane exchange between B cells.