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The Journal of Immunology, 2005, 175: 7125-7134.
Copyright © 2005 by The American Association of Immunologists

B Cells Productively Engage Soluble Antigen-Pulsed Dendritic Cells: Visualization of Live-Cell Dynamics of B Cell-Dendritic Cell Interactions

Ning-Na Huang, Sang-Bae Han, Il-Young Hwang and John H. Kehrl1

B Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interactions between B lymphocytes and Ag-bearing dendritic cells (DC) likely occur at inflammatory sites and within lymphoid organs. To better understand these interactions we imaged B cells (TgB) from hen egg lysozyme (HEL) transgenic mice and DC pulsed with HEL (DC-HEL) in collagen matrices. Analysis of live-cell dynamics revealed autonomous movements and repeated encounters between TgB cells and DC-HEL that are best described by a "kiss-run and engage" model, whereas control B cells had only short-lived interactions. Ag localized at contact sites between TgB cells and DC-HEL, and both cell types rearranged their actin cytoskeletons toward the contact zone. The interaction of a TgB cell with a HEL-bearing DC triggered strong Ca2+ transients in the B cells. Thus, B cells can productively interact with DC displaying their cognate Ag.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)2 function as the sentinels of the immune system (1). Immature DC traffic from the blood to inflamed tissues where they capture and process Ag, differentiate into mature DC, and move to the draining lymphoid nodes to prime naive T cells. In addition, resident immature DC associated with the conduit network in lymph nodes acquire Ag from the afferent lymph, differentiate into mature DC, and enter the T cell compartment to mingle with T cells (2). These T cell-DC interactions are necessary to generate effective T cell help for Ag-activated B cells. Besides their role in T cell activation, DC in draining lymph nodes may also provide a source of unprocessed Ag to stimulate specific B cells: either recirculating B cells entering into the lymph node or Ag-specific B cells already resident in lymph node follicles. B cell-DC interactions have been observed both in vitro and in vivo, and the transfer of Ag from DC to naive B cells has been documented (3, 4, 5, 6, 7). In addition, DC produce a soluble chemoattractant for B cells, which may promote their interactions (8). Blood DC can capture and transport particulate Ags such as invading bacteria to the spleen, where they promote the differentiation of marginal zone B cells into IgM secreting plasma blasts (9). This occurs through a pathway independent of T cell help, but dependent upon soluble factors secreted by DC such as BLyS (B lymphocyte stimulator) and APRIL (a proliferation-inducing ligand), which provide a crucial cosignal for plasma cell generation.

The interaction between B cells and DC has received significantly less study than T cell-DC interactions. The concept of the immunological synapse originally evolved from the study of the interactions of T cells with APC such as DC. It is known that the DC cytoskeleton is critical for forming this immunological synapse (10, 11). The specific interaction of T cells and APC is characterized by the accumulation of actin filaments in T cells at the contact point and by clustering of T cell signaling molecules and surface receptors at the contact region (12). In addition, T cells respond by raising their intracellular level of Ca2+, which assists in stabilizing their interaction with APC (13). Because less attention has been paid to the dynamics of B cell-DC interactions, particularly in primary culture systems, and because of the lack of information on the cytoskeletal changes and Ca2+ signaling following contact between DC and B cells, we have used several different approaches to specifically explore the interactions between Ag-specific B cells and Ag-pulsed DC.

Different from the conventional monolayer culture system, the fiber distribution and biophysical architecture of three-dimensional collagen lattices closely resemble tissues in vivo (14, 15, 16), therefore they have been widely used for studies in vivo-like culture of a variety of cells including DC and T cells (17, 18, 19, 20, 21, 22). In the present study we investigated the live-cell dynamics and functional consequences of direct interactions between Ag-specific B cells and Ag-pulsed DC in three-dimensional collagen matrix. The demonstration of stable complexes of Ag-specific B cells with Ag-pulsed DC led us to study the dynamics of their formation, evaluate the structure of the interaction zone between B cells and DC, and to analyze intracellular Ca2+ concentration [Ca2+]i signaling as well as other parameters of B cell activation. Similar to what has been observed in T cell-DC interactions, the cytoskeletal reorganization that occurs following the interaction of B cells with Ag-pulsed DC occurred at the interface of the B cell-DC physical contact region, where Ag clustered or accumulated. Interestingly, Ag-specific B cell-DC interactions occasionally induced rises of intracellular Ca2+ in a synchronized fashion. Overall, this study provides additional support for the functional importance of B cell-DC interactions in initiating and modulating B cell immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Isolation of splenic DC and B cells

DC were prepared from spleens of C57BL/6 mice as previously described by magnetic separation using the MACS system (Miltenyi Biotec) (23). The resulting DC were >90% CD11c+ by flow cytometry. DC were pulsed with or without hen egg lysozyme (HEL) or keyhole limpet hemocyanin (KLH) for 16 h before mixing with B cells. Nontransgenic B cells from spleens of C57BL/6 (B6) mice and Ag-specific transgenic (Tg) B cells (TgB) from C57BL/6-Tg(IghelMD4)4Ccg/J Tg mice (The Jackson Laboratory) were negatively selected using biotinylated Abs to CD4, CD8, GR-1, and CDllc (BD Pharmingen) and DynabeadsR M-280 Streptavidin (Dynal Biotech). This procedure yielded a population containing >90% B cells. Mice 6- to 16-wk-old were used for all studies. Animal care and use were in accordance with National Institute of Allergy and Infectious Diseases institutional guidelines.

Three-dimensional collagen matrix culture and cell collection

Purified B cells and DC were labeled with Vybrant green CFDA SE (carboxy-fluorescein diacetate, succinimidyl ester) or Vybrant red DiI (Molecular Probes), respectively, following the manufacturer’s protocols. Fluorescent-labeled or unlabeled cells were cultured in three-dimensional collagen matrix as previously described (19), but with minor modifications. In brief, 5–10 x 106 total cells were used per assay and suspended in 125 µl of RPMI 1640 with 10% FBS. In general, a ratio of DC to B cells of 1:5 was used. The cells were mixed with 250 µl of chilled RPMI 1640 medium collagen mix (pH 7) containing 2.5 mg/ml type I collagen (Vitrogen; Collagen). The final collagen concentration was 1.67 mg/ml. The cell mixture was transferred to a glass-bottom culture dish (MatTek) and allowed to polymerize for ~1 h at 37°C. After polymerization, 1000 µl of RPMI 1640 medium with 10% FBS was added and the three-dimensional cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. Cells cultured in the collagen matrix were observed by confocal microscopy system as specified. DC and B cells cultured in three-dimensional collagen matrix were harvested by digestion with collagenase and DNase as previously described (19). Collagenase A and DNase I were from Boehringer Mannheim. Collagen matrix containing DC and B cells was incubated with purified collagenase (final concentration, 7500 U/ml) for 5 min. Cells were washed with PBS and cultured in RPMI 1640 medium for further study.

Cell imaging and image processing

All fluorescent images were collected on inverted confocal microscopes as specified. The PerkinElmer Ultraview spinning wheel confocal system is mounted on Zeiss Axiovert 200 equipped with an argon/krypton laser, an Orca-ERII CCD camera (Hamamatsu), and filters suitable for the visualization of both green and red dyes. The Leica TCS-SP2 confocal microscope is equipped with an argon/krypton laser, and the samples were illuminated with either the 488- or 568-nm laser line. For live studies, the CO2 was maintained at 5% and the temperature of the sample was maintained at 37°C using an environmental controller system (Carl Zeiss). Time-lapse microscopy and computer-assisted single cell tracking was used to analyze the migration of cells within the three-dimensional collagen lattices. Images of dynamic cell interaction were recorded as vertical Z-stacks or as vertical Z-stacks over time. Images were collected using a x32 LD Achrostigmat Ph1 objective, a x10 Fluar objective, or a x63 Plan-Aprochromax 1.4 oil immersion objective as indicated. Paths of cell migration and the calculations for the velocity studies were performed in Excel. Adobe Photoshop (v. 6.0) was used to prepare composite images and for annotations. Imaris 4.0.4 (Bitplane), Ultraview 5.5 (PerkinElmer), Adobe Photoshop 6 (Adobe Systems), and IP Lab 3.6 (Scanalytics) were used for the image processing.

Analysis of actin fibers in DC and TgB following their interaction

The B cells and DC were cultured at a ratio of 5:1 in fibronectin-coated glass-bottom culture dishes (MatTek) in RPMI 1640 medium with 10% FBS for 3–4 h. Cells were fixed with methanol at –20°C before incubation with Alexa Fluor 594 phalloidin (Molecular Probes) for 30 min. The fluorescence signals were detected using the PerkinElmer Ultraview confocal microscope with the x63 oil objective. The cytoskeleton of both B cells and DC was defined as polarized when the intensity of F-actin was greater in the region adjacent to the binding partner cell than in nonbound areas.

[Ca2+]i imaging and trapping

Cells were cultured as indicated for analysis of actin dynamics. To measure cell [Ca2+]i during the interactions between DC and B cells, either the DC, B cells, or both were loaded with a combination of two long-wavelength Ca2+ indicators (Molecular Probes), fura red-AM (5 µM), and fluo-4-AM (5 µM) for 1 h at 37°C. When excited at 488 nm, fluo-4 exhibits an increase in green fluorescence (525 nm) on Ca2+ binding, whereas fura red shows a decrease in red fluorescence (640 nm). Because the emission ratio of fluo-4 to fura red is a ratiometric index, it is independent of changes in cell volume (24). The dye-loaded cells were scanned with a PerkinElmer Ultraview confocal argon/krypton laser, and the emission shifts were quantitated by dividing the fluorescence intensity signals from two emission bands of 525–550 nm (green) and 600–645 nm (red). Fluorescent signals were acquired from individual cells by alternately visualizing fluo-4 and fura red in the cell body every 5 s over the imaging time period. All images were collected with a Zeiss x63 oil objective. The montages and graphs of the fluorescent ratios as a function of time were generated using software provided with the Ultraview.

Assays for HEL-specific B cell activation by DC

The activation of Ag-specific B cells by DC was determined by measuring expression of activation-related molecules on B cells and cell proliferation. To analyze the expression of activation marker on TgB cells, cells collected from collagen matrix were incubated for 1 h with one of the following mAbs (BD Pharmingen) conjugated to FITC for direct staining: anti-CD86, anti-CD80, anti-CD69, or anti-IgM. PE anti-I-A/I-E was used for staining MHC class II molecules. IgG1 and IgG2 were used as isotype controls and were obtained from BD Pharmingen. Cells were washed with PBS and incubated in PBS containing 1% BSA (Sigma-Aldrich) with the appropriate concentration of labeled mAb for 1 h at 4°C. Cells were then washed and measured with a FACSCalibur flow cytometer. Data are analyzed with CellQuest software (BD Biosciences). Cell proliferation was assayed by [3H]thymidine incorporation. Briefly, the Ag-specific or wild-type B cells (106 cells/well) were cultured with DC pulsed with HEL (DC-HEL) or unpulsed at different ratios for 72 h in three-dimensional collagen matrix. The cells were collected from collagen as described. Cells harvested from collagen matrix were incubated with [3H]thymidine (1 µCi/well) for 18 h and the incorporated [3H]thymidine was then determined.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ag-dependent multicellular complexes form between TgB cells and DC-HEL in a three-dimensional collagen matrix

DC were prepared from mouse spleen and pulsed overnight or not with HEL. To examine the maturation status of the Ag-pulsed DC before coculture with B cells, the DC were examined for expression of CD11c, CD80, CD86, and MHC class II. We found that pulsing the DC with HEL very modestly increased the levels of expression of CD80 and CD86, but did not alter the expression of MHC class II (Fig. 1). Next, TgB cells and B cells freshly isolated from either the spleen of HEL Tg mice or wild-type mice, respectively, were embedded in collagen matrix with DC that had been pulsed for the previous 16 h with HEL or not pulsed. The cell combinations were cultured for 48 h and visualized by phase-contrast microscopy. Besides the various mixtures of cells, we also cultured each of the cell populations alone. We found that the HEL-specific B cells accumulated around the DC that had been pulsed with HEL to form complexes in the three-dimensional collagen matrix (Fig. 2A, top left). A higher power image of an individual cell complex is inserted in the corner of the panel. Culturing the TgB cells in the presence of DC not pulsed with Ag failed to produce visible cell complexes (Fig. 2A, top right). Furthermore, no visible complexes were observed between wild-type B cells and DC either pulsed with Ag or not, nor did any of the cells cultured alone in collagen form visible complexes (data not shown). To verify the Ag specificity of the B cell-DC interactions we cultured TgB cells with DC pulsed with either HEL or KLH. Again the TgB cells accumulated around the HEL-pulsed DC, but not around the KLH-pulsed DC (Fig. 2A, bottom panels). To better visualize the interactions of B cells with DC-HEL we fluorescently labeled the B cells with CSFE (green) and the pulsed DC with DiI (red) and collected images using an inverted confocal fluorescent microscope. A stable multicellular complex contained one or more DC-HEL and multiple B cells (Fig. 2B). Low and high power images of HEL TgB cells and DC-HEL cultured in a collagen matrix for 24 h are shown at two time points separated by 1 h (note the extensive remodeling of the complexes). Contrasting with the results using three-dimensional collagen matrices, few multicellular complexes between TgB cells and DC-HEL formed when we used standard culture tissue conditions (data not shown). These findings indicate that Ag-specific B cells in the appropriate environment interact for prolonged intervals with Ag-bearing DC.



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  		FIGURE 1. Effect of Ag pulsing on DC phenotype. DC prepared from spleen were pulsed overnight with HEL or not pulsed and the following day analyzed by flow cytometry. The expression of CD11c, CD80, CD86, and MHC class II on the pulsed cells (gray histogram) or unpulsed cells (black histogram) is shown.

 


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  		FIGURE 2. Formation of Ag-dependent multicellular complexes between B cells and DC in three-dimensional collagen matrix. A, Freshly isolated mouse spleen B cell, TgB cell, splenic DC-HEL or DC not pulsed, and various combinations of cells were embedded in a collagen matrix. Cells were imaged by phase-contrast microscopy (x10 objective) after culture in collagen for 48 h. TgB cells plus unpulsed DC or DC-HEL (top panels) are shown. Complexes are outlined with black squares, and an electronically zoomed image (magnification, x3) of a single complex is shown. TgB cells embedded in collagen matrices with DC previously pulsed with HEL (bottom left) or pulsed with KLH (bottom right) are shown. Cells were imaged by differential interference contrast microscopy (x63 objective). The data are representative of three independent experiments. B, Freshly isolated TgB cells and DC-HEL were fluorescently labeled by Vybrant green CFSE and Vybrant red DiI, respectively. Cells embedded in collagen matrix for 48 h were imaged 1 h apart using a spinning disk confocal x10 objective (top panels) and electronic zoom (bottom panels). Complexes are outlined with white squares and two complexes numbered 1 and 2. C, Confocal images of TgB cells and DC-HEL interactions in a collagen matrix. Fluorescent TgB (green) and DC-HEL (red) were embedded in collagen matrix for 16 h and then imaged by Leica confocal microscope with a x63 objective. A total of 63 images were collected in the z direction, separated by 0.3 µm. The location of the selected images in the image stack is indicated by the number of microns from the top of the stack. Three DC previously pulsed with HEL are identified. D, Confocal images of an individual DC interacting with several TgB cells. Fluorescent TgB (green) and DC-HEL (red) were embedded in collagen matrix for 24 h and then imaged by Zeiss confocal microscope (magnification, x63 objective) and electronically zoomed two times. Eight images were collected in the z direction, separation 0.3 µm, and four slices are shown. The location of the selected images in the image stack is indicated by the number of micrometers from the top of the stack.

 
We noted that these multicellular complexes remained relatively stable over a 24-h imaging period, although B cells and DC moved into and out of them. The initial complexes (<20 µm) were observed within 8 h after embedding the cells in collagen. Cells accumulated around the initial complex over ~1–3 days, developing to larger stable multicellular complexes (around 50–80 µm). The initial complex became more compact or enlarged by the new members of interactive TgB cells and DC-HEL. Both the TgB cells and the DC-HEL moved autonomously along apparently random paths. The TgB cells not present in cellular complexes moved at an average speed of 3.24 ± 1.19 µm/min, whereas the pulsed DC moved at a similar average speed of 3.94 ± 0.55 µm/min. The TgB cells approached and departed the complexes at approximately the same rate. Confocal images of such a complex are obtained with a scanning confocal microscope at various depths (Fig. 2C; HEL TgB cells are green and HEL-DC are red). Extensive cell-cell interactions are evident. We also examined a small complex between a single DC and several TgB cells in the z direction, which shows the intimate interactions between a single DC and at least two of the B cells (Fig. 2D). To look at the interactions between individual TgB cells and DC-HEL over time, we focused on a single DC interacting with an individual B cell. We acquired four-dimensional data sets over 30 min. Stereo four-dimensional and standard focal-plane four-dimensional viewing of the time-lapse recordings further confirmed the extensive membrane interactions between the pulsed DC and TgB cells (data not shown).

Stability and dynamics of the TgB-DC-HEL complexes

To better understand the development and stability of these multicellular complexes, we investigated the kinetics of cell-cell interactions using time-lapse microscopy of B cells and either DC or DC-HEL ~4 h after embedding them into a collagen matrix. Both the pulsed and unpulsed DC moved actively, demonstrating an extraordinary ability to extend long filopodia and to stretch and contort their cell bodies. Nontransgenic B cells engaged in transient, but not prolonged interactions with the pulsed DC (Fig. 3A) as shown on video (see supplemental video 1).3 In contrast, when the TgB cells were cocultured with the DC previously pulsed with HEL, both transient and prolonged interactions were observed with the latter likely leading to the development of the multicellular complexes. In a representative imaging experiment, three TgB cells variably interacted with a single DC-bearing HEL over 60 min (Fig. 3B) as shown on corresponding video (see supplemental video 2). The TgB cells variably displayed a polarized cell morphology and amoeboid-like cell movements with active membrane projections, whereas the DC extended processes in many directions. One polarized TgB cell interacted with the pulsed DC over the entire recording period even as the two other B cells approached and receded. Another B cell meandered for 48 min before showing an interest in the DC after which it polarized and remained associated with the DC until the time-lapse recording ended. The tracks of the cell bodies of the DC and this second B cell over the 1-h time-lapse study are shown (Fig. 3C). The two cells contact each other at the 54th minute of the study. The maximum speed of the DC noted during the recording period was 18 µm/min, whereas that of the B cell was 7 µm/min. The third B cell transiently interacted with the DC on three separate occasions each lasting <5 min; however, it did not become firmly associated with the DC during the course of the imaging. An analysis of fluorescent-labeled TgB cells (Fig. 3D, green) and DC-HEL (Fig. 3D, red) over a longer time period shows a DC meandering in the vicinity of a complex of TgB cells and DC-HEL and the eventual capture of a B cell and recruitment into the cell complex (video data not shown).



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  		FIGURE 3. Time-lapse analysis of the live-cell dynamics of TgB-DC-HEL complex formation. A, B cells and DC-HEL were prepared and embedded in collagen matrix. Cells were imaged at the time as shown by phase-contrast microscopy (x32 objective) after 4-h incubation in collagen matrix. The indicated times in each panel corresponds to the duration after the imaging had begun. A DC is identified with an arrow. B, TgB cells and DC-HEL were prepared and embedded in collagen matrix. Cells were imaged at the time as shown by phase-contrast microscopy after 4-h incubation in collagen matrix. The indicated time in each panel corresponds to the duration after the imaging had begun. A DC is identified with an arrow. C, Tracking of a TgB and DC-HEL cell interaction over 1 h. The DC and TgB cell shown to interact at 56 min in the previous panel were manually tracked during the 1 h recording. The locations of the cell body of the cells at 1-min intervals are shown with red (DC-HEL) and green (TgB cell) symbols. The origins of the two cells at the start of the recording are indicated with arrows. Because we tracked the cell bodies, the terminal tracks fail to show that the DC-HEL and TgB cells are engaged for the last 6 min of the tracking. D, Development of multicellular complexes. Fluorescent TgB (green) and DC-HEL (red) were embedded in collagen matrix for 18 h and then imaged every 10 min for 4 h using a PerkinElmer Ultraview confocal (x10 objective). Individual frames are shown taken at the time indicated in each panel. The DC is indicated by arrows. Scale bar is 15 µm.

 
Both actin and Ag localize at the TgB and DC-HEL cell contact region

Because of difficulties in obtaining high quality images of the cells in the collagen matrix, we also plated TgB cells and DC-HEL on fibronectin plates and determined whether we could observe long-term interactions between the cells. Although interactions between TgB cells and DC-HEL persisted for over 1 h on fibronectin-coated plates, the movements of the DC and TgB cells were not nearly as dynamic as in collagen matrices. Nevertheless the use of fibronectin plates allowed a more detailed study of the B cell-DC contacts. To determine whether TgB and DC-HEL cell contacts involve structures rich in F-actin, we plated the cells on fibronectin-coated tissue culture plates and imaged fixed cells after actin staining with phalloidin. The TgB cells engaged with DC-HEL often although not always polarized in their expression of actin toward the cell-cell interface (Fig. 4A, left and middle panels). No actin polarization occurred during the transient interactions between TgB cells and DC in the absence of HEL (Fig. 4A, right panel). Next, we examined whether we could image Ag in the context of the two cells. We labeled HEL with FITC (FITC-HEL) and verified that the FITC-HEL bound appropriately to the HEL TgB cells (Fig. 4B, left panel). We cocultured DC that had been previously pulsed with FITC-HEL overnight with HEL TgB cells (DiI labeled) on fibronectin-coated plates. Using live-cell imaging we identified a DC interacting with a B cell and imaged the event for varying durations. Three images separated by ~21/2 min are shown (Fig. 4B, two middle and right panels). It is evident that Ag accumulated at the interface between the DC and the right B cell in the image. Remodeling of the interface is shown by the differences in Fig. 4B (two middle and right panels). We also examined the distribution of Ag in relation to actin polymerization during the interaction between TgB cells and DC pulsed with FITC-labeled HEL. A confocal image of one closely juxtaposed TgB cell-DC pair is shown (Fig. 4C). Similar to the results discussed earlier, the TgB cell has acquired significant amounts of Ag, which is prominently localized toward the adjacent DC. Actin staining revealed that both of the B cells exhibited high amounts of polymerized actin most prominent at the interface, which colocalized with the FITC-HEL. Superimposition of the images revealed the colocalization of concentrated actin and FITC-HEL most prominently within the TgB cell.



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  		FIGURE 4. Actin cytoskeleton and Ag HEL are coupled to TgB and DC-HEL cell contact formation. A, Polarization of actin toward the interface of TgB cells and DC-HEL. TgB and DC-HEL or DC-HEL FITC cells were cultured on fibronectin-coated plates for 1 h, fixed, and stained with Alexa Red-phalloidin (left and right panels) or Alexa Red-phalloidin and 4',6'-diamidino-2-phenylindole (DAPI; middle panel). Samples were imaged using a Leica confocal microscopy, with x63 oil objective, electronically zoomed three times. Images of the contact were collected as Z-stacks, and individual images from the Z-stack are shown. Left and middle panels are images from conjugates between TgB cells and DC-HEL, whereas the right panel is from TgB cells and DC not pulsed with HEL. Results are representative of three independent experiments with at least 20 conjugates examined per experiment. B, Ag is focused at the TgB cell-DC interface. TgB plated on fibronectin plates (left panel) and pulsed with FITC-HEL was imaged immediately by spinning disk confocal microscope, x63 oil objective. The middle two and far right panels were collected from a time series of live-cell confocal imaging obtained using fluorescent-labeled TgB (red) incubated on fibronectin-coated plates with DC previously pulsed overnight with FITC-HEL. The time lapse between the images shown is indicated in seconds. The locations of two B cells interacting with a single DC are indicated. C, Both actin and HEL-FITC are focused at the interface of B cell and DC. TgB cells and DC previously pulsed with FITC-HEL were incubated on fibronectin-coated plates for 1 h, fixed, and stained with Alexa Red-phalloidin and DAPI and imaged as in A. The single optical section of a B cell and DC interaction is shown as FITC-HEL (left), actin (middle), and merged view (right).

 
TgB and DC-HEL cell contact increase [Ca2+]i

APC-T cell interactions trigger increases in T cell [Ca2+]i. Unstable contact patterns cause a weak rise in [Ca2+]i, whereas stable contact patterns generate more robust and sustained increases in [Ca2+]i (13). To analyze the consequence of B cell-DC interactions on [Ca2+]i levels, we used various combinations of two calcium-sensitive dyes, fura red and fluo-4, to label the HEL-pulsed DC and/or the TgB cells. First, we loaded the TgB cells with fluo-4 and the pulsed DC with fura red. As a TgB cell transiently interacted with the DC, the level of [Ca2+]i in the TgB cell rose, as visualized by the increase in green fluorescence (Fig. 5A). Subsequently the TgB cell disengaged for 40 s and then re-engaged, shortly after which the TgB cell flared its plasma membrane and more closely apposed itself with the DC. Thereafter the TgB cell remained in contact with the DC for the duration of the imaging (9 min). Shortly after re-engaging, the level of [Ca2+]i rose higher, peaking 3 min later and then slowly returned to basal levels. As the level of [Ca2+]i increased, the TgB cell adopted a more rounded morphology (see supplemental video 3) (Fig. 5A). A trace of the fluorescence level in gray scale units of the B cell is shown (Fig. 5A, bottom). An adjacent B cell, which only transiently interacted with the DC, did not significantly change its level of [Ca2+]i. We also examined changes in [Ca2+]i by labeling the TgB cell with both calcium sensitive dyes and by using a ratio between the two dyes displayed on a pseudo-colored scale. A stable complex of two B cells previously loaded with fura red and fluo-4 is shown interacting with three HEL-pulsed DC (Fig. 5B). Two TgB cells are shown together with three DC (Fig. 5B, visible in a brightfield image). Only the B cells are visible otherwise because the DC were unlabeled. The images acquired every 10 s over a 200-s interval reveal that the levels of [Ca2+]i increased in both B cells nearly simultaneously, although the magnitude of the response in the left B cell exceeded that in the right B cell. Below the montage is a tracing of the values obtained from the ratio imaging. Oscillations in the level of [Ca2+]i were occasionally noted during prolonged interactions between TgB cells and DC-HEL (Fig. 5C). Images from a fura red- and fluo-4-labeled B cell interacting with a DC (Fig. 5C, outlined) over 10 min. Below each montage are the results from the ratio imaging, which reveal the modulations in [Ca2+]i. Another pattern we noted was a rather slow rise in [Ca2+]i as the pulsed DC and TgB cell repetitively interacted (Fig. 5D). In this experiment we labeled both the TgB cells and DC-HEL with fura red and fluo-4 and relied on the morphology of the cells to separate the two cell types. Fig. 5D shows the montage as a RGB image rather than the ratio image. The DC is red whereas the B cell is reddish/green, but increasingly green as the level of [Ca2+]i rises (Fig. 5D). The B cell and DC interact for the first 4 min, disengage, and then re-engage. Fig. 5D (bottom) shows the graph of the ratio between fluo-4 and fura red only in the B cell. We failed to observe reproducible changes in the levels of [Ca2+]i in the pulsed DC. To determine how these patterns matched the response of TgB cells to soluble HEL in the absence of DC, we imaged TgB cells on fibronectin plates immediately after the addition of 100 ng of HEL (Fig. 5E). We found a rapid rise in [Ca2+]i that gradually returned to baseline, a pattern different from what we had observed during the B cell-DC interactions.



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  		FIGURE 5. Changes in [Ca2+]i following TgB and DC-HEL interactions. The results are from multiple experiments. All images were collected using a spinning disk confocal using a x63 oil objective. A, Repetitive interactions increase [Ca2+]i. The TgB cells were labeled with the calcium responsive dyes fluo-4 and the DC-HEL with fura red, and plated on glass-bottom dish coated with fibronectin. Cell interactions and calcium content were then dynamically imaged at 5-s interval. Individual RGB images are shown as a montage following the approach of a TgB cell to a DC. The timing of the images is indicated in seconds. A trace of the fluo-4 gray level over the region of the interacting B cell as a function of time is shown on bottom. B, Synchronous increase in intracellular calcium of TgB cells engaged with DC. B cells were coloaded with fluo-4 and fura red, whereas the DC were unlabeled. Cells were plated on fibronectin-coated plates and dynamically imaged at 5-s intervals. A transmitted light image (left corner) shows B cells indicated with arrows, whereas the rest of the montage shows the images from the time series as a ratio between fluo-4 and fura red. The ratio is directly proportional to [Ca2+]i and shown on a pseudo-colored scale and in the graph below as a function of time. C, Oscillations in [Ca2+]i. B cells were coloaded with fluo-4 and fura red, whereas the DC were unlabeled. Cells plated on fibronectin-coated plates were dynamically imaged at 5-s interval. A single DC (outlined) interacting with an individual B cell is shown as a montage of ratio images. The ratio is shown on a pseudo-colored scale and in the trace below as a function of time. D, Gradual increase in [Ca2+]i. DC-HEL and TgB cells were labeled with fura red, and fluo-4 were plated on fibronectin-coated plates and dynamically imaged at 5-s intervals. Results are shown as RGB images collected at the indicated times. The ratio of fluo-4 to fura red over the region of the B cell is shown in the tracing below as a function of time. E, Exposure of TgB cells to HEL increases [Ca2+]i. TgB cells labeled with fluo-4 and fura red were plated on fibronectin-coated plates. Immediately after the addition of HEL to the media, the cells were imaged every 5 s. Results of the ratio imaging of a highly responsive B cell are shown as a montage using a pseudo-colored scale, and actual ratios between the two dyes over the region of the TgB cell as a function of time are shown in the tracing to the right.

 
Activation of Ag-specific B cells cultured in three-dimensional collagen matrices in the presence of DC-HEL

We further studied whether the stable interaction between TgB cells and DC-HEL activated B cells by using flow cytometry to check B cell activation markers. TgB cells cultured in three-dimensional collagen matrices with splenic DC, which had previously been incubated with HEL or KLH and washed extensively, were recovered by collagen digestion with collagenase. The TgB cells cultured with DC-HEL greatly increased their expression of CD86, and moderately enhanced their expression of CD69, CD80, and MHC class II. In parallel, TgB cells cultured in collagen with DC unpulsed with HEL or pulsed with KLH expressed similar levels of these markers as TgB cultured alone (Fig. 6A and data not shown). TgB cells reduced their surface expression of IgM after culture with the DC-HEL reflecting the acquisition of HEL from the DC and internalization of the HEL-BCR complex by the TgB cell. The changes of surface expression of these markers indicate effective activation of TgB cells by DC-HEL. However, the changes in activation markers on the recovered B cells from the collagen matrices did not differ significantly from TgB cells cultured in standard culture media with HEL in the absence of DC. DC-HEL enhanced TgB cell proliferation as measured by [3H]thymidine incorporation after coculture in three-dimensional collagen matrices (Fig. 6B). When we compared fibronectin-coated plates and collagen matrices at the lowest ratio of TgB to pulsed DC, the TgB cells recovered from three-dimensional collagen matrices incorporated more [3H]thymidine than did TgB cultured at the same ratio on fibronectin plates.



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  		FIGURE 6. Analysis of TgB cells recovered from collagen matrices. A, Coculturing TgB cells with DC-HEL increases the levels of activation markers on TgB cells. TgB cells and DC pulsed or not pulsed with HEL were embedded in three-dimensional collagen matrices. Cells were cultured for 72 h, extracted from the collagen matrices, immunofluorescently stained for the indicated cell surface molecules, and analyzed by flow cytometry. Results from the analysis of CD69, CD80, CD86, MHC class II, and IgM are shown. The bottom right histogram shows the results of the analysis of CD69 expression on TgB cells extracted from collagen matrices that lacked DC (black line histogram), DC-HEL (patterned line histogram), or DC pulsed with KLH (gray line histogram). B, Culturing Ag-specific B cells and DC-HEL promotes B cell proliferation in three-dimensional collagen matrices. TgB cells (106 cells/well) were cultured with DC-HEL at different ratios as indicated for 72 h in three-dimensional collagen matrices or in two-dimensional tissue culture plates. The collagen matrices were digested with collagenase, and cells were recovered from the collagen. [3H]Thymidine was added for the last 18 h of the culture time period. Data shown are the mean of [3H]thymidine incorporation (cpm) from triplicate cultures and are representative of one experiment of two conducted.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Earlier data support using three-dimensional collagen matrix in the study of in vivo-like cell migration and DC-T cell interactions (14, 19, 20). In a collagen matrix, DC formed extended membrane processes that are characteristic of these cells. We describe in this study the direct action and formation of stable interactions between Ag-specific B cell and DC-HEL in three-dimensional collagen matrix. With this model system, we evaluate dynamic cell migration and cell-cell encounters, engaged cell-cell interactions on a single cell basis, and TgB-DC-HEL complex formation in real time with phase-contrast and fluorescence confocal microscopy. We suggest that autonomous movements and repeated encounters between TgB cells and DC-HEL lead to stable and prolonged interactions mimicking an interaction between a B cell and a mature DC bearing cognate Ag. The initial transient encounters appear to enhance the likelihood that long-term interactions occur. In addition we show that the engagement of the B cell with DC bearing HEL focuses Ag to the interface, orients actin polymerization in the B cell and DC, and triggers increases in [Ca2+]i in the B cells.

A "kiss-run and engage" mode of B cell-DC interaction

DC are essential to the initiation of an immune response due to their unique ability to capture and process Ags and meet and regulate Ag-specific T and B cells (1). DC and likely other APC can retain Ag in naive form for at least 48 h and release it for recognition by B cells (3, 25). To capture Ag presented by DC incorporated into a collagen matrix, B cells must directly interact with the DC. Typically, Ag-specific B cells that encountered HEL-presenting DC were able to extend and retract processes were generally polarized toward the contacting region of the cells, as shown by the real-time microscopy images. Our results revealed a kiss-run and engage mode of interaction, which differs from a previous study that examined the interactions of DC and T cells in a collagen matrix, where only repetitive transient interactions were observed (20). The HEL-specific TgB cells randomly encounter DC-HEL. TgB cell and DC-HEL interact (kiss) briefly and often depart (run). Frequently, they encounter again to process another kiss-run event. During kiss-run, the Ag-specific B cells may be exposed very transiently an activation signal, or a special configuration of the surface membrane or receptor leading to full engagement with DC-HEL. The active and dynamic kiss-run and engage events result in the formation of a stable B cell-DC interaction. One DC can engage multiple B cells resulting in the formation of the multicellular complexes that we observed. Kiss-run and engage is not a passive process, but depends on the presence of Ag. Consistently, only short-live interactions were observed between wild-type B cells and between DC-HEL or between TgB cells and unpulsed DC or previously pulsed DC with an irrelevant Ag. Thus the ability of B cells to form stable interactions requires Ag receptor engagement, but also likely depends on signals generated through the kiss-run and engage events. These signals may emanate from LFA-1-ICAM-1 interactions as well as from other adhesion molecules such as CD22 (26).

DC chemokine production could be important in facilitating B cell-DC interactions. Monocyte-derived DC have been reported to make CXCL13, which would attract B cells to the DC enhancing B cell-DC interactions (27). CXCL13 signaling via CXCR5 may enhance the affinity of LFA-1 and help drive prolonged interactions between B cells and DC. Consistent with this possibility, naive T cells displayed an augmented adhesion to bone marrow-derived DC, when chemokines were absorbed to the surface of the DC. However, chemokine-independent mechanisms also accounted for much of the T cell-DC adhesion (28). In our experiments DC chemokine production is unlikely to be sufficient to drive extensive B cell-DC interactions because TgB cells in the presence of unpulsed DC or DC pulsed with an irrelevant Ag failed to form long-term stable interactions.

Ag engagement and actin-rich zone at TgB-DC-HEL interface

The role of B cell actin cytoskeleton during interaction with DC has not been studied. It is known that B cells are able to contact APC directly to form a B synapse and acquire Ags (25). It has also been shown that T cell cytoskeleton rearrangement is critical for the formation of the immunological synapse and subsequent T cell activation (29). However, the cytoskeleton has been reported to play no active role in APC (30), although recent studies have shown that DC rearrange their actin cytoskeleton toward T cells to form an immunological synapse (11). Adding more confusion, no rearrangement of the B cell cytoskeleton has been observed when cells acted as APC interacting with T cells (31). Thus it was important to evaluate the distribution of actin in TgB cells and DC previously pulsed with HEL during their interaction. Different from when B cells act as APC, when B cells engage Ag-bearing DC they rearrange their actin cytoskeleton as do the DC. This results in cytoskeletal polarization toward the interface of the B cell and DC contact region, where Ag accumulation occurs. It has been suggested that B cells might be able to recognize Ag presented by DC (3). In this study we show that Ag-specific B cells likely acquire Ag directly from DC, and the Ag recognition correlates with the actin cytoskeletal rearrangement. Previous studies have demonstrated a crucial role for the DC cytoskeleton in stabilizing DC-T cell binding (10). Similarly, the changes in the B cell cytoskeleton likely serve to increase the contact between B cell and DC, to enhance Ag recognition, and to stabilize the interaction. The Ag specificity required for the actin cytoskeletal rearrangements helps ensure that only Ag-specific B cells receive activation signals.

Intracellular calcium elevation during B cell-DC interactions

The lymphocyte cytoskeleton is essential for lymphocyte activation and calcium signaling (29, 30). We examined the role of [Ca2+]i in TgB and DC-HEL cell contact by tracking the interaction of HEL-specific B cells and DC-HEL. A rise in intracellular Ca2+ can be detected following an initial interaction of a TgB cell with DC-HEL. High levels of [Ca2+]i correlated with the an eventual loss of cell polarity and "rounding up" of the cells as previously described for T cells interacting with APC (13). We noted several different patterns of [Ca2+]i following the interaction of a B cell with an Ag-pulsed DC. In some instances a sharp spike in [Ca2+]i occurred, in others a more gradual increase and decrease in the levels of [Ca2+]i, and in some instances the interaction resulted in oscillations of the [Ca2+]i level. Whether these different patterns have physiologic significance remains to be determined. Nevertheless, they differed from what we found following the exposure of B cells to soluble Ag, which caused a sharp spike in [Ca2+]i that gradually tapered back to baseline. In the absence of HEL, TgB cells did not form stable conjugates with DC and their basal levels of [Ca2+]i remained relatively constant. A single DC can trigger an increase in [Ca2+]i levels in two or more B cells. Surprisingly, our image analysis revealed a near synchronous rise of [Ca2+]i in two TgB cells complexed with three DC. Transients of [Ca2+]i have been found to be elicited by the neurotransmitters and appear synchronous not only in aggregates, but also in neighboring cells lacking physical contact (32, 33). It has also been suggested that rearrangement of the DC actin cytoskeleton may participate in the directional secretion of cytokines toward Ag-specific T cells and increases in intracellular calcium (34). Whether DC secrete mediators that can elicit increases in [Ca2+]i in B cells is unknown.

DC-mediated B cell activation

Ag recognition by BCR leads to B cell activation and can initiate intracellular signaling cascade that results in cell proliferation. We noted that in addition to triggering increases in [Ca2+]i, the culture of TgB cells with DC-HEL caused increased expression of CD69, CD80, CD86, and MHC class II on TgB cells. Not unexpectedly the surface expression of IgM on TgB cells was reduced indicating HEL Ag acquisition and internalization of the HEL-BCR complex. B cell proliferation was increased by coculturing with DC-HEL in three-dimensional collagen matrix. DC not loaded with HEL failed to increase the growth of B cells, supporting the idea that the B cell activation in this system is dependent upon Ag uptake by DC and its presentation to B cells.

The original formulation of Burnett’s clonal selection theory of B lymphocyte Ag specificity depended upon two assumptions, first that B lymphocytes differ from each other in the specificity of their Ag receptor and second that many structurally distinct Ags can transmit signals for the growth and differentiation of B cells (35). The first assumption has been validated by the discovery of a diverse set of V genes and their rearrangement with genes encoding constant regions of the Ig H and L chains. However, the molecular mechanism underlying the second assumption has not been elucidated. Because of the structural diversity of Ags that stimulate B cells, it has been argued that the BCR has a highly ordered oligomeric structure, which can be altered by Ags of differing configurations to allow associated signaling elements to become active (36). Our studies suggest that an additional consideration is that B cells may often not see soluble Ag in vivo, but rather Ag in the context of a DC. The surface of a DC may provide a means to convert a soluble, noncross-linking Ag incapable of eliciting B cells activation into a form capable of providing a stimulatory signal to a B cell.

In summary, we show that interactions between Ag-specific B cells and Ag-loaded DC provide signals for B cell activation. The development of stable B cell-DC complexes makes it possible to capture and stimulate Ag-specific lymphocyte cells with high efficiency. As B cells migrate into secondary lymphoid tissue, Ag-specific B cell may interact with DC bearing Ag in the lymph node cortex before entrance into the B cell follicle. Alternatively, Ag-reactive B cells within the follicle may interact with Ag-bearing DC that have entered into the lymph node and that line the region of the B cell follicle. Evidence of a productive interaction between an Ag-bearing DC and an Ag-reactive B cell includes alternations in the actin cytoskeleton of both cell types and oscillations or steady increases in [Ca2+]i. In some instances the interaction between an Ag-bearing DC and Ag-reactive B cell may provide sufficient stimuli to trigger B cell differentiation, whereas in others an additional interaction with an Ag-specific T cell is necessary.


   Acknowledgments
 
We thank Mary Rust for editorial assistance, Owen Schwartz and Juraj Kabat of the National Institute of Allergy and Infectious Diseases (NIAID) confocal facility for technical assistance, Susan Garfield and Stephen Wincovitch of the National Cancer Institute confocal facility for technical assistance, and Dr. Anthony S. Fauci and the NIAID intramural program for continued support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. John H. Kehrl, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 31 Center Drive, Bethesda, MD 20892. E-mail address: jkehrl{at}niaid.nih.gov Back

2 Abbreviations used in this paper: DC, dendritic cell; Tg, transgenic; HEL, hen egg lysozyme; DC-HEL, DC pulsed with HEL; [Ca2+]i, intracellular Ca2+ concentration; KLH, keyhole limpet hemocyanin. Back

3 The online version of this article contains supplemental material. Back

Received for publication April 14, 2005. Accepted for publication September 14, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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