The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Scandella, E.
Right arrow Articles by Ludewig, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scandella, E.
Right arrow Articles by Ludewig, B.
The Journal of Immunology, 2007, 178: 1468-1476.
Copyright © 2007 by The American Association of Immunologists, Inc.

Dendritic Cell-Independent B Cell Activation During Acute Virus Infection: A Role for Early CCR7-Driven B-T Helper Cell Collaboration1

Elke Scandella2,3,*, Katja Fink2,{dagger}, Tobias Junt{dagger}, Beatrice M. Senn{dagger}, Evelyn Lattmann*, Reinhold Förster{ddagger}, Hans Hengartner{dagger} and Burkhard Ludewig3,*

* Research Department, Kantonal Hospital St. Gallen, St. Gallen, Switzerland, {dagger} Institute of Experimental Immunology, University Hospital Zurich, Zurich, Switzerland, {ddagger} Hannover Medical School, Institute of Immunology, Hannover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
This study provides a detailed spatiotemporal interaction analysis between B cells, Th cells, and dendritic cells (DC) during the generation of protective antiviral B cell immunity. Following vesicular stomatitis virus (VSV) infection, conditional ablation of CD11c-positive DC at the time-point of infection did not impair extrafollicular plasma cell generation and Ig class switching. In contrast, the generation of Th and B cell responses following immunization with recombinant VSV-glycoprotein was DC-dependent. Furthermore, we show that the CCR7-dependent interplay of the three cell-types is crucial for virus-neutralizing B cell responses in the presence of limiting amounts of Ag. An immediate event following VSV infection was the CCR7-mediated interaction of VSV-specific B and Th cells at the T cell-B cell zone border that facilitated plasma cell differentiation and Th cell activation. Taken together, these experiments provide evidence for a direct, CCR7-orchestrated and largely DC-independent mutual activation of Th cells and Ag-specific B cells that is most likely a critical step during early immune responses against cytopathic viruses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection with cytopathic viruses demands an early and efficient immune response to limit hematogenic spread of the virus and to minimize tissue damage (1). Cytopathic viruses therefore generally elicit rapid neutralizing Ab responses in a T cell-independent (TI)4 manner (2, 3). In contrast, B cell responses to most protein Ags are T cell dependent (TD) (4). It is thus conceivable that the cellular and molecular events during the activation of Ab responses against nonviral model Ags such as OVA (5), hen egg lysozyme (6), or 4-hydroxy-3-nitrophenyl acetyl (7) differ significantly from those engaged in the generation of protective immune responses against replicating pathogens.

The spleen is essential for limiting the systemic spread of pathogens from the circulation (1). Blood-borne Ags enter the spleen through sinuses in the marginal zone (MZ) where they are filtered and captured by MZ macrophages (8) or MZ B cells (9). In addition, blood dendritic cells (DC) may capture bacteria and subsequently transport their pathogen load to the spleen for Ag presentation (10). Up to now, the involvement of DC in the priming of TI or TD B cell responses, and their participation in the Ig class switching process have merely been investigated in adoptive transfer studies using Ag-loaded DC (11, 12, 13). Furthermore, adoptive transfer or in vitro studies suggested that direct B cell-DC interactions are important for the activation and survival of B cells by providing stimulatory signals via molecules from the TNF family (14, 15) or particular cytokines (10). However, the question of whether intimate DC-B cell contacts are a prerequisite for B cell activation, differentiation, and Ig class switching in the context of a viral infection has remained elusive.

Chemokines are essential for the coordinated migration of activated lymphocytes in secondary lymphoid organs (16, 17). For example, stimulation of MZ B cells with LPS leads to down-regulation of sphingosin-1-phosphate receptors 1 and 3, which allows them to respond to the B cell zone chemokine CXCL13 and to migrate to B cell follicles (18). Expression of CXCR5 determines the proper positioning of both B and Th cells in follicles to provide efficient T cell help during class switching (19, 20). Immediately after their initial activation in the MZ, B cells migrate through the B cell follicle to the T cell-B cell zone interface and finally enter the T cell zone (6, 10, 21, 22). It has been shown previously that the binding of proteinaceous Ag to the BCR induces up-regulation of the chemokine receptor CCR7, thereby facilitating the migration of the sensitized B cells to the T cell-B cell border where the CCR7-ligands CCL19 and CCL21 are expressed (23). However, the functional consequences of the rapid CCR7-dependent relocation of B cells to the T cell zone, particularly for the generation of antiviral B cells responses, have not been explored.

This study presents a detailed analysis of the cellular interactions required for the rapid induction of neutralizing Ab responses against the cytopathic vesicular stomatitis virus (VSV). VSV infection in mice is controlled by the activation of B cells and the initiation of a rapid TI virus neutralizing Ab response during the first 48 h after infection (24). Furthermore, the TD Ig class switch provides long-term protection by generating a robust memory response (25, 26). We found that the early activation of VSV-specific B cells following exposure to live virus was largely independent of DC-mediated Ag presentation to B cells, whereas B cell responses induced by immunization with the recombinant VSV-glycoprotein (VSV-G) required the presence of DC. Furthermore, our analysis reveals the functional importance of CCR7-dependent migration of activated B cells to the T cell-B cell zone border: the mutual activation of VSV-specific B and Th cells provides an efficient amplification loop during the early production of antiviral neutralizing Abs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and viruses

C57BL/6 mice were obtained from Charles River. C57BL/6-IgHa-Thy1.1 mice were purchased from The Jackson Laboratory. CD11c-diphtheria toxin (DT) receptor-transgenic (DTR) mice were obtained from Prof. S. Jung (Weizmann Institute of Science, Rehovot, Israel). For systemic DC-ablation, CD11c-DTR mice were injected i.p. with DT at 4 ng/g body weight (in PBS; Sigma-Aldrich) that led to a 95–98% depletion of CD11c+ cells for >48 h. Mice bearing a knock-in construct for the VSV-neutralizing Ab VI10 in their Ig locus (VI10) (27) and TCR-transgenic mice for the T cell epitope p8 of VSV (L7) (28) have been described previously. For adoptive transfer experiments, VI10 and L7 mice were crossed to CCR7–/– mice. Mice were kept in ventilated cages under conventional conditions at the Research Department of the Kantonal Hospital (St. Gallen, Switzerland). All animal experiments were performed in accordance with the Swiss Federal legislation on animal protection.

The VSV Indiana strain (VSV-IND; Mudd-Summers isolate) was originally obtained from Prof. D. Kolakofsky (University of Geneva, Geneva, Switzerland). Recombinant VSV-G was obtained from a culture of Spodoptera frugiperda 9 (Sf9) cells after infection with a recombinant baculovirus (29).

VSV-neutralization assay

Titers of VSV-neutralizing Abs in sera were determined as described previously (26). Sera were diluted in MEM with 2% FCS, and the dilution that resulted in a 50% reduction of virus plaques was taken as the neutralizing titer. For IgG titers, sera were incubated with equal volumes of 0.1 M 2-ME in PBS for 1 h at room temperature before dilution. All mouse sera were heated at 56°C for 30 min for complement inactivation.

Sorting of cells

B cells from VI10 and VI10 x CCR7–/– spleens were sorted with B220-specific beads, and Th cells from L7 and L7 x CCR7–/– spleens were sorted with CD4-specific magnetic beads (Miltenyi Biotech). The purity of B220+ and CD4+ cells was between 90 and 96%. For adoptive transfer of Th and B cells into DC-ablated CD11c-DTR mice, DC were previously depleted from splenocytes using CD11c-specific magnetic beads (Miltenyi Biotech).

Flow cytometry

Anti-idiotypic Ab 35.61 specific for the VSV-neutralizing H and L chain variable region of VI10 (27) was labeled with FITC (Sigma-Aldrich) or Cy5 (Amersham Biosciences). For cell staining the following Abs were obtained from BD Pharmingen: anti-CD4-PerCp (clone RM4-5), anti-B220-allophycocyanin (clone RA3-6B2), anti-vbeta2 TCR-allophycocyanin (clone B20.6), anti-CD138-PE (clone 281-2), anti-Thy1.2-allophycocyanin (clone 53-2.1), anti-CD62L-PE (clone MEL-14), and anti-CD44-FITC (clone IM-7). The anti-CCR7 Ab was obtained from eBioscience. For flow cytometry analysis, surface molecules were stained in FACS buffer (PBS, 2% FCS, 1 mM EDTA, and 0.1 mg/ml azide) for 30 min at 4°C. For intracellular staining of plasma cells, cells were fixed and permeabilized with CytoFix/CytoPerm solution from BD Pharmingen. Fixed cells were then incubated with Abs in FACS buffer containing 0.1% saponin for 30 min at 4°C. For acquisition, cells were fixed in FACS buffer with 1% formalin and analyzed with a FACSCalibur flow cytometer (BD Biosciences) using CellQuest (BD Biosciences) or Flow Jo software (Tree Star).

Migration assay

For chemotaxis assays, 5 x 105 splenocytes were transferred into the upper chamber of Transwell plates with 5-µm filters (Costar; Corning) containing the indicated concentrations of CCL19 (R&D Systems) in the bottom chambers. After 4 h, transmigrated cells were stained with anti-B220 and anti-35.61 Abs and enumerated by flow cytometry. Transwell assays were performed in duplicate and repeated with splenocytes from three mice per group.

Proliferation assay

VI10 mice were infected with 109 PFU of VSV-IND and, 12 h later, CD11c+ and B220+ cells were sorted from splenocytes using MACS beads (Miltenyi Biotech). To avoid DC contamination within the B cell population, CD11c+ cells were previously depleted from splenocytes using CD11c-specific magnetic beads. The amount of VSV-specific 35.61+B220+ cells was determined by flow cytometry and graded numbers of 35.61+B220+ cells or CD11c+ cells were incubated with 1 x 105 MACS-sorted CD4+ cells from L7 mice for 48 h. One microcurie of [3H]thymidine was added to each well during the final 16 h of the incubation period and incorporation was determined in a scintillation counter (Packard Instrument). Proliferation assays were performed in duplicate and repeated with splenocytes from three different mice per group.

ELISA

For the detection of VSV-specific IgMb, IgG2ab, or IgG2bb, which were derived from adoptively transferred, purified B220+ cells of VI10 mice, plates were coated overnight with 5 mg/ml polyethylene glycol-precipitated VSV-IND particles, blocked with PBS with 3%BSA for 2 h at room temperature, washed, and incubated with 3-fold serum dilutions starting at a dilution of 1/30. After washing, biotin-labeled rat-anti-mouse IgG2ab or IgG2bb (BD Pharmingen) were added followed by streptavidin-HRP (Jackson ImmunoResearch Laboratories). ELISAs were developed with 0.1 mg/ml 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (Roche) in 0.1 M phosphate buffer (pH 4.0) plus 60{per thousand} freshly added H2O2. OD was measured at 405 nm. The dilution step at which the OD value was still higher than twice the background level was taken as the arbitrary titer. Each isotype was analyzed in one experiment including all time points for direct comparison of the values.

Fluorescence histology

Freshly removed organs were immersed in HBSS and snap frozen in liquid nitrogen. Five-micrometer tissue sections were air dried, fixed with acetone for 10 min, and stored at –70°C. Cryosections were blocked for 30 min with 1 µg of the Fc-blocking Ab 2.4G2 per sample, washed in PBS, and incubated with the respective fluorescent Abs for 1 h at 4°C. If necessary, streptavidin-Cy3 was added in a second step. After washing in PBS the nuclei were counterstained with 4',6'-diamino-2-phenylindoledihydrochloride (Sigma-Aldrich) and the sections were mounted with fluorescence mounting solution (DakoCytomation). Fluorescence was analyzed with a Zeiss Axiophot microscope (Carl Zeiss). Color channels were assembled automatically with analySIS software (Olympus Soft Imaging Solutions), and images were processed using Adobe Photoshop without nonlinear operations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
DC-independent activation of B cells in response to virus infection

To assess the role of DC for the induction of antiviral TI B cell responses, DC were ablated by the injection of DT into CD11c-DTR mice (30). DT treatment led to a transient depletion (95–98%) of CD11c+ cells for >48 h (Figs. 1 and 2A, right panel, days 0 and 1) as described previously (30). The depletion mainly affected CD4+-DC and CD8+-DC populations, whereas plasmacytoid PDCA-1+CD11cdim DC were only partially depleted by ~45% (Fig. 1). Sorted VSV-specific B cells from VHDJH-gene targeted (VI10) mice expressing the rearranged VH-region of a VSV-neutralizing Ab (27) and sorted CD4+ Th cells from mice expressing a transgenic TCR specific for the VSV-G (L7) (28) were adoptively transferred i.v. into CD11c-DTR mice that at the same time received DT i.p. Eighteen hours later, mice were infected with 2 x 106 PFU of VSV or immunized with soluble VSV-G protein. Furthermore, DT application did not influence the homing of the transferred cells to the spleen as confirmed by histology and flow cytometry (Fig. 2A, upper panel, and data not shown). Furthermore, the previously described depletion of some metallophilic and MZ macrophages in CD11c-DTR mice (31) did not affect the primary activation of B cells following VSV infection (see below).


Figure 1
View larger version (53K):
[in this window]
[in a new window]

  		FIGURE 1. Deletion of different DC populations by DT treatment in CD11c-DTR mice. A, CD11c-DTR mice were injected with DT (4 ng/g bodyweight) i.p. and splenocytes were analyzed by flow cytometry before (d0) and one day after (d1) DT application using the indicated surface markers. The values in the lower panels indicate the percentage of the remaining cells of the respective DC population. B, time course analysis of splenic DC-populations in CD11c-DTR mice following DT injection.

 

Figure 2
View larger version (77K):
[in this window]
[in a new window]

  		FIGURE 2. DC-independent activation of B cells in response to virus infection. CD11c-depleted B220+ B cells (5 x 105) from VI10 mice and CD4+ T cells from L7 mice were adoptively transferred i.v. into CD11c-DTR mice. DT (4 ng/g bodyweight; i.p. injection) was applied concomitantly. Eighteen hours later, mice received i.v. 2 x 106 PFU of VSV (A–E) or 20 µg of VSV-G (F–I). A, left panel, Spleen sections of DT-treated (+DT) and untreated (–DT) mice were stained with Abs against B220 (red), CD4 (blue), and VSV-specific B cells (35.61; green) to identify the localization of transferred B cells before (upper row), on day 1 (middle row), and on day 3 (lower row) post VSV-infection; right panel, DT-mediated DC-ablation was monitored by CD11c-staining (red) at the same time points. B and F, Quantification of proliferating VI10 B cells in spleens of recipient mice by flow cytometry (staining with anti-B220 and anti-35.61 antibodies). C and G, Assessment of plasma cell generation as determined by gating on CD138+ 35.61+ B220dim cells in the flow cytometric analysis. D and H, VSV-neutralizing serum Ab IgM and IgG titers. E and I, Proliferation of L7 T cells as determined by gating on CD4+Vbeta2+ cells. Data in all graphs represent means ± SD of one representative experiment of two. Each experiment was performed with three mice per group. Statistical analysis was performed using Student’s t test (*, p < 0.05; **, p < 0.005; ns, p > 0.05).

 
Within 24 h postinfection the VSV-specific B cells, which can be visualized by the monoclonal anti-idiotypic Ab 35.61 (27), had moved to the T cell zone border both in DT-treated and untreated recipient mice (Fig. 2A, middle row). Within 3 days, blasting cells were visible in bridging channels and the red pulp of the spleen, independent of the presence or absence of DC (Fig. 2A, bottom panels). Flow cytometric analysis on days 3 and 6 following VSV infection revealed that DC depletion had neither a significant impact on the proliferation (Fig. 2B) nor on the differentiation into CD138+ plasma cells (Fig. 2C) of VSV-specific B cells. Furthermore, the induction of neutralizing Abs following infection with live VSV was neither impaired under these conditions (Fig. 2D) nor in the DC-depleted CD11c-DTR mice that had not received VSV-specific B and Th cells (data not shown). This suggested that DC were not required for efficient priming of VSV-specific B cells. In contrast, DC were essential for efficient priming of Th cells following VSV infection (Fig. 2E).

Immunization of mice with 20 µg of VSV-G protein results in the induction of a strong TI anti-VSV neutralizing Ab response (25). However, B cell proliferation and differentiation, as well as Ab responses following immunization with 20 µg of recombinant VSV-G, were massively reduced in the absence of DC (Fig. 2, F–H). Proliferation of VSV-specific CD4+ T cells was diminished (Fig. 2I), similar to the decrease that could be observed after virus infection. Thus, DC-mediated Ag presentation to B cells appears to be dispensable for the early activation of B cells during cytopathic virus infection but not following protein immunization.

Rapid activation of VSV-specific B cells

To visualize the DC-independent response of Ag-specific B cells following an encounter with a live virus, VI10 mice were infected with 109 PFU of VSV and the migration pattern of VSV-specific B cells was analyzed. A pronounced reaction of VSV-specific B cells could already be recorded 30 min following Ag injection with an accumulation of the cells in the MZ. Later, between 4 and 12 h postinfection, VSV-specific B cells had migrated to the T cell-B cell interface and finally accumulated at the border of the T cell zone (Fig. 3A). The majority of VSV-specific B cells arrived in the T cell zone as large, blasting B220low cells that were detected by the exclusive and bright staining with the mAb 35.61 (Fig. 3A, lower right panel). An elevated CCR7 expression on VSV-specific B cells recovered from infected VI10 mice (Fig. 3B) and ex vivo chemotaxis assays of VSV-specific B cells at 6 and 12 h after VSV infection (Fig. 3C) strongly suggested that the observed B cell migration toward the T cell zone was a consequence of CCR7 engagement. Indeed, adoptive transfer experiments confirmed that only CCR7-competent, VSV-specific B cells were able to migrate to the T cell-B cell interface following VSV infection, whereas CCR7-deficient VSV-specific B cells were evenly distributed within B cell follicles (Fig. 3D). These results provide further evidence that B cells receive a full initial activation stimulus through direct contact with viral Ag in the MZ and that this process programs a CCR7-dependent migration toward the T cell zone.


Figure 3
View larger version (92K):
[in this window]
[in a new window]

  		FIGURE 3. CCR7-mediated migration of activated VSV-specific B cells. A, VI10 mice were infected with 109 PFU of VSV and spleens were isolated at the indicated time points and analyzed by fluorescent microscopy. Note that 35.61+B220+ double-positive cells appear yellow, whereas blasting cell have down-regulated B220 and appear as green cells. B, CCR7 expression on 35.61+B220+ double-positive cells from naive VI10 mice or VI10 mice infected 12 h previously with 109 PFU of VSV. C, Migratory capacity of activated VSV-specific B cells toward the T zone chemokine CCL19. Splenocytes from VSV-infected mice isolated 6 and 12 h after VSV infection from VI10 mice or naive VI10 splenocytes were used as input cells in a Transwell migration assay. Depicted values represent the mean percentage ± SD of migrated B220+35.61high (left panel) or B220+35.61– cells compared with the total numbers of the respective input cells. The assay was performed in duplicates using three mice per group. The mean values of duplicate measurements from three individual mice were used to calculate the mean percentages. One of two experiments with comparable results is shown. D, CCR7-dependent migration of 35.61+ B cells toward the T cell-B cell border. Sorted B220+ cells (5 x 106) from CCR7-competent (left panel) or CCR7-deficient (right panel) VI10 mice were adoptively transferred into C57BL/6 mice and spleens were analyzed by fluorescence histology 12 h postinfection with 2 x 106 PFU of VSV.

 
Antiviral Ab responses in CCR7-deficient mice

To investigate whether and how CCR7-expression on B cells affects their activation and Ab production, we analyzed VSV-specific Ab responses in CCR7-deficient (CCR7–/–) mice. CCR7–/– mice were able to rapidly generate VSV-neutralizing Abs, comparable to those obtained in wild-type C57BL/6 mice following VSV infection or VSV-G immunization (Fig. 4, A and B). Both CCR7–/– and C57BL/6 mice generated robust long-term Ig titers in response to live VSV (Fig. 4A). However, CCR7–/– mice showed a markedly impaired neutralizing Ab response following immunization with low amounts (5 µg) of recombinant VSV-G (Fig. 4C). This indicates that CCR7-mediated coordination of the three cell-type interaction between DC, B cells, and Th cells during antiviral B cell responses is of particular importance in the presence of limiting amounts of Ag, i.e., when Ab responses become dependent on T cell help (25).


Figure 4
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 4. VSV-neutralizing Ab response in CCR7-deficient mice. VSV-neutralizing IgM and IgG Ab titers were determined in the sera of mice that were infected with 2 x 106 PFU of VSV (A) or immunized with 20 µg (B) or 5 µg (C) of VSV-G. Data represent the mean ± SD of three mice per group.

 
CCR7-dependent B cell activation

The above results showed that the absence of CCR7 is not associated with an intrinsic impairment of the Ag-driven activation of VSV-specific B cells. In lymphoid organs, the absence of CCR7 is accompanied by an impaired lymphoid architecture with a lack of properly organized T cell and marginal zones (32) that may influence cellular interactions during an immune response. To exclude effects of the impaired lymphoid organization in CCR7–/– mice on early intercellular interactions, we used an adoptive transfer system of CCR7-competent or -deficient VSV-specific B and Th cells into mice with an intact lymphoid structure. To this end, VI10 B cells and L7 Th cells or VI10 x CCR7–/– B cells and L7 x CCR7–/– Th cells were cotransferred into congenic B6.Cg-IgHa Thy1.1 recipient mice facilitating the detection of Abs produced by transferred B cells via the congenic IgHb marker and the tracking of transferred Th cells and B cells via the Thy1.2 molecule and the 35.61 Ab, respectively. The homing efficiency of CCR7-deficient and -competent Th or B cells to the spleens of recipient mice was comparable (data not shown). Following infection with VSV, comparable amounts of IgM and IgG Abs were produced by CCR7-deficient and CCR7-competent B cells (Fig. 5A, left panel). However, following immunization with low amounts of VSV-G (5 µg), CCR7-competent VI10 donor cells produced significantly more IgM and IgG Abs than VI10 x CCR7–/– donor B cells (Fig. 5A, right panel).


Figure 5
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 5. Reduced extrafollicular plasma blast response by CCR7-deficient VSV-specific B cells. Sorted B220+ cells and CD4+ cells (5 x 105) from either CCR7-deficient or CCR7-competent VI10 and L7 mice, respectively, were adoptively transferred into B6.Cg-IgHa Thy1.1 mice. One day later, mice were either infected with 2 x 106 PFU of VSV (A–E) or immunized with 5 µg of VSV-G (A and D–E). A, Ab production by CCR7-deficient VI10 B cells following VSV infection (left panel) and VSV-G immunization (right panel). Serum Ab titers were determined by ELISA detecting VSV-specific IgM, IgG2a, and IgG2b Abs of the IgHb haplotype. B and C, Histological analysis of spleen sections on day 3 (B) and day 6 (C) after VSV infection. Ag-specific B cells (35.61, green) were blasting in bridging channels connecting the white and red pulp. Enlarged insets show physical association of CCR7-competent (left panels) but not CCR7-deficient (insets, right panel) 35.61+ blasting cells with transferred VSV-specific Thy1.2+ T cells. D and E, Total numbers of 35.61+ B cells (D) and the percentage of CD138+ plasma blasts (E) after VSV infection (left panels) or following VSV-G immunization (right panels). Following VSV-G immunization, 35.61+ B cells could not be detected (nd) by flow cytometry on day 3 after immunization. All graphs show the mean ± SD of one representative experiment of two. Each experiment was performed with three mice per group. Statistical analysis was performed using Student’s t test (*, p < 0.05; **, p < 0.005; ns, p > 0.05).

 
Despite the absence of an obvious impairment in neutralizing Ab responses following VSV infection, the localization of the VSV-specific cells within lymphoid organs was profoundly altered when CCR7-deficient B and Th cells were transferred. VI10 x CCR7–/– and VI10 B cells formed plasma blasts tht accumulated in bridging channels and in the red pulp on days 3 (Fig. 5B) and 6 (Fig. 5C) postinfection. However, CCR7-deficient VSV-specific Th cells were distributed in the B cell follicles but always excluded from the T cell zone (Fig. 5, B and C, right panel). Notably, only CCR7-competent B cells were blasting in physical association with VSV-specific Th cells (Fig. 5, B and C, enlarged insets).

Quantification of VSV-specific B cells after infection with VSV revealed that the early expansion of 35.61+ B cells was impaired when VSV-specific B and Th cells were CCR7-deficient (Fig. 5D, left panel). In addition, more VSV-specific VI10 B cells had up-regulated the plasma cell marker CD138 on day 3 postinfection compared with VI10 x CCR7–/– B cells (Fig. 5E, left panel). However, on day 6 postinfection equal numbers of plasma cells were generated by VI10 x CCR7–/– and VI10 B cells. An even more pronounced CCR7 dependence of B cell activation and differentiation could be observed following immunization with VSV-G (Fig. 5, D and E, right panels). Taken together, these data imply that the early extrafollicular plasma cell formation depends on CCR7-mediated migration of virus-specific B cells to the T cell zone and support the notion that CCR7-mediated T cell-B cell collaboration is particularly important for early B cell activation.

CCR7-mediated mutual amplification of Th and B cell responses

We next assessed the VSV-induced activation and expansion of VSV-specific CD4+ T cells in the presence or absence of CCR7 on specific B and T cells. Fig. 6A shows that it was predominantly the early Th cell response that was impaired if CCR7-mediated migration of both cell types was altered. T cell activation was not altered in the absence of CCR7, because both L7xCCR7–/– and L7 cells down-regulated the activation marker CD62L and up-regulated CD44 to a similar extent (Fig. 6B).


Figure 6
View larger version (42K):
[in this window]
[in a new window]

  		FIGURE 6. Impaired early expansion of CCR7-deficient Th cells. Sorted CD4+ cells and B220+ cells (5 x 105) from either CCR7-deficient or CCR7-competent L7 and VI10 mice, respectively, were adoptively transferred into B6.Cg-IgHa Thy1.1 mice. One day later, mice were infected with 2 x 106 PFU of VSV-IND. A, Total numbers of VSV-specific T cells in spleens as determined by flow cytometry using gates on CD4+Thy1.2+ cells. Data in the graph represent means ± SD from one representative experiment of two. Each experiment was performed with three mice per group. Statistical analysis was performed using Student’s t test (*, p < 0.05; **, p < 0.005; ns, p > 0.05). B, Representative FACS plots with the expression of the activation markers CD62L and CD44 on VSV-specific Th cells on day 3 after VSV infection. Histograms show populations gated on CD4+ T cells. Values in the upper quadrants indicate activation marker expression of CD4+Thy1.2+ cells. 

 
To further dissect the CCR7-dependence for T cell-B cell interaction early during antiviral immune responses, we adoptively transferred CCR7-deficient VI10 cells together with CCR7-competent L7 cells or CCR7-competent VI10 cells together with CCR7-deficient L7 cells into B6.Cg-IgHa Thy1.1 mice and infected the recipients with live VSV. As shown in Figs. 7A and 6B, only CCR7-competent VI10 B cells responded early with efficient proliferation and differentiation into CD138+ plasma cells, irrespective of the CCR7 competence of the cotransferred Th cells. Likewise, optimal early and late Th cell proliferation depended on the capacity of the Th cells to home to the T cell zone, i.e., on their CCR7-competence (Fig. 7C, gray bars). However, the early proliferation of CCR7-competent L7 Th cells was also impaired when they were cotransferred with CCR7-deficient VI10 B cells that were unable to migrate toward the T cell zone (Fig. 7C, filled bars). This provides further evidence for CCR7-mediated early T cell-B cell collaboration and additionally suggests that activated B cells located at the T cell-B cell zone border were capable of operating as APCs.


Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 7. CCR7-mediated mutual Th and B cell activation. B6.Cg-IgHa Thy1.1 mice received 5 x 105 sorted CD4+ cells and B220+ cells from either CCR7-deficient or CCR7-competent L7 and VI10 mice, as indicated. One day later, mice were infected with 2 x 106 PFU of VSV-IND. A–C, The proliferation of VSV-specific 35.61+ B cells (A) and Thy1.2+ Th cells (C) and the differentiation to CD138+ plasma cells (B) was determined by flow cytometry as described in the legends to Figs. 5 and 6. Data in the graph represent means ± SD from one representative experiment of two. Each experiment was performed with three mice per group. Statistical analysis was performed using Student’s t test (*, p < 0.05; **, p < 0.005; ns, p > 0.05). wt, Wild type. D, Ag presentation by VSV-specific B cells. VI10 mice were infected with 109 PFU of VSV-IND or left untreated and sacrificed 12 h later. CD11c+ and B220+ cells were sorted from spleens, irradiated, and used as stimulators for naive sorted CD4+ cells from L7 mice in a [3H]thymidine incorporation assay. The percentage of 35.61+ B220+ cells was assessed by flow cytometry and the amount of B220+ cells was adjusted to match the numbers of DC and VSV-specific B cells. The assay was performed in duplicates and mean values ± SD from sorted cells from three mice in each group are shown.

 
The ability of activated VSV-specific B cells to stimulate naive Th cells could be confirmed using in vitro proliferation assays. The Ag-presenting function of VSV-specific B cells was assessed by isolating CD11c+ DC and B220+ B cells from VI10 mice 12 h after infection with 109 PFU of VSV. MACS-purified cells were used as stimulators for CD4+ L7 cells in a [3H]thymidine incorporation assay. The results in Fig. 7D show that in vivo activated VSV Ag-presenting B cells can stimulate the proliferation of naive L7 T cells, although with a slightly reduced efficiency when compared with DC. Taken together, efficient B cell expansion and plasma cell differentiation as well as optimal T cell proliferation required the concomitant localization of both cell types at the T cell-B cell interface, suggesting that the reciprocal activation of Th and B cells is an important feature of the early VSV infection-associated immune response.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study provides new insight into the cellular interactions that shape TD and TI B cell responses following an encounter with a live pathogen. First, our study demonstrates that DC-mediated Ag presentation to B cells is dispensable for the early initiation of neutralizing antiviral Abs when a cytopathic virus spreads hematogenically. Second, the antigenic impact, that is, the integral of Ag dose and complexity (viral particles vs soluble protein), determines the DC dependency and, with this, the Th cell dependency of the B cell response. Third, CCR7-dependent mutual stimulation of Th and B cells at the T cell-B cell border ensures the rapid induction of antiviral B cell responses.

Blood-borne Ags such as those associated with infections transmitted by arthropods are efficiently cleared from the circulation by trapping in the spleen. Viruses, for example, can be fixed in the MZ by natural Abs (33) or complement components (34). The rapid trapping of viruses in the MZ is important not only to reduce dissemination of the infectious agent to different organs but also to enhance antiviral B cell responses (34). It has been suggested that the activation of MZ B cells following an encounter with TI Ags is dependent on Ag transport and display by DC (10). Our study shows that B cells, most likely MZ B cells, sense incoming pathogens and that a cytopathic virus such as VSV activates a full B cell response in the absence of DC. Likewise, a recent study by Hebel et al. (35) has shown the DC are dispensable for primary Ab responses against (4-hydroxy-3-nitrophenyl)acetyl-Ficoll. The high efficacy of the early processes during VSV infection is illustrated by the fact that VSV-specific B cells accumulate in the MZ as early as 30 min after VSV administration. It is most likely that the direct activation of VSV-specific B cells is mediated via BCR signaling, whereas other mechanisms, such as TLR ligation might later support B cell activation and differentiation (36). The importance of TLR ligation for the generation of TD and TI Ab responses has been shown recently (36, 37, 38, 39, 40, 41). In addition, it may well be that B cells receive additional early signals from plasmacytoid DC that can be activated by VSV via TLR-7 (42).

Under conditions of limiting antigenic impact, that is, following immunization with low amounts of soluble VSV-G, an important role of DC for the generation of neutralizing antiviral Ab responses became apparent. An insufficient DC-mediated Th cell activation that supports early antiviral Ab responses (43) is apparently one critical limiting factor in this multicellular process. Furthermore, it may well be that, under these conditions, a direct DC-B cell interaction is a second limiting factor. In this scenario, DC may provide critical survival factors to MZ B cells (10). Furthermore, DC may support more TD antiviral B cell responses via cell surface recycling of internalized Ag for the provision to B cells (44).

Most of the early TI neutralizing Abs are directed against VSV-G, which is present on the virus in a highly organized form (2). Furthermore, VSV-G shapes the TD antiviral Ab response because it contains efficiently presented Th cell epitopes that guarantee isotype switching and the generation of B cell memory (45). The presented data indicate that both TI and TD B cell responses are influenced by the expression of CCR7 on VSV-specific Th and B cells. Only CCR7-competent B cells were able to enter the T cell zone upon virus infection. This confirms previous results that were obtained using model Ags in a noninfectious situation (23, 46). It is noteworthy that the early CCR7-dependent B cell translocation to the T cell zone is not mandatory for isotype switching, because adoptively transferred CCR7-deficient B cells can produce IgG in response to live VSV as well as VSV-G. Thus, a situation where the isotype switch in B cells is programmed by these early CCR7-mediated cell contacts in the T cell zone is rather unlikely.

It is interesting to note that CCR7–/– mice mounted normal neutralizing Ab responses against replicating VSV or high doses of VSV-G but failed to do so in situations of limiting Ag availability. The reason for this differential reactivity could be that the altered lymphoid structure in CCR7–/– mice functionally compensates for the lack of CCR7-dependent migration in cases of highly abundant Ag. Likewise, it has been shown that CCR7–/– mice and plt/plt mice are able to mount protective immune responses against the lymphocytic choriomeningitis virus through ectopic priming of CTLs in the marginal zone (47, 48). Furthermore, CXCR5–/– mice generate normal Ab responses against VSV, provided that the amount of Ag is not limiting (19).

The fact that the early Th cell proliferation was impaired due to a lack of CCR7 expression on either Th or B cells implies an important role for B cells in the initial priming of Th cells. Indeed, in vivo activated and Ag-loaded B cells were able to stimulate naive VSV-specific Th cells in vitro. It is most likely that Ag is acquired in the MZ and that B cells receive the appropriate activation signal in the MZ, because MZ B cells are superior to follicular B cells in presenting Ag to Th cells (6). Given the fact that follicular VI10 B cells relocate rapidly to the MZ, it is difficult at the current stage to judge whether follicular or MZ VI10 B cells contribute more to the initial activation of Th cells. Although DC are, overall, the more potent APCs, B cells may contribute efficiently to the Th cell activation through the rapid remodeling of the MHC class II Ag-processing compartments (49). This process takes only 15 min, because cross-linking of the BCR enhances both the synthesis of MHC class II and its transit through the endocytic pathway (50, 51). In contrast, the remodeling of the Ag-processing compartments in DC takes several hours and requires DC maturation by proinflammatory stimuli (52, 53, 54).

Taken together, our study provides a high-resolution analysis of the interaction of Th cells, B cells, and DC during the immune response against a live pathogen and shows that antiviral B cell responses are coordinated in a context-dependent fashion; during the encounter with a rapidly spreading cytopathic virus, B cells respond initially in a DC and Th cell autonomous manner, thereby generating early plasma cells and neutralizing Abs. The CCR7-dependent interaction between Th and B cells serves as an amplification loop for the neutralizing Ab response, which is particularly important in situations of low antigenic impact.


   Acknowledgments
 
We thank Simone Miller, Antje Novotny, Silvia Behnke, and Andre Fitsche for technical assistance, and Rolf Zinkernagel for valuable discussions.


   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 This work received financial support from the Kanton St. Gallen, Switzerland. Back

2 E.S. and K.F. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Elke Scandella, Research Department, Kantonsspital St. Gallen, 9007 St. Gallen, Switzerland. E-mail address: elke.scandella{at}kssg.ch or Dr. Burkhard Ludewig, Research Department, Kantonsspital St. Gallen, 9007 St. Gallen, Switzerland. E-mail address: burkhard.ludewig{at}kssg.ch Back

4 Abbreviations used in this paper: TI, T cell independent; DC, dendritic cell; DT, diphtheria toxin; DTR, diphtheria toxin receptor transgenic; TD, T cell dependent; MZ, marginal zone; VSV, vesicular stomatitis virus; VSV-G, VSV-glycoprotein; VSV-IND, VSV Indiana. Back

Received for publication July 18, 2006. Accepted for publication November 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Griffin, D. E.. 2003. Immune responses to RNA-virus infections of the CNS. Nat. Rev. Immunol. 3: 493-502. [Medline]
  2. Bachmann, M. F., R. M. Zinkernagel. 1997. Neutralizing antiviral B cell responses. Annu. Rev. Immunol. 15: 235-270. [Medline]
  3. Zinkernagel, R. M., A. LaMarre, A. Ciurea, L. Hunziker, A. F. Ochsenbein, K. D. McCoy, T. Fehr, M. F. Bachmann, U. Kalinke, H. Hengartner. 2001. Neutralizing antiviral antibody responses. Adv. Immunol. 79: 1-53. [Medline]
  4. Parker, D. C.. 1993. T cell-dependent B cell activation. Annu. Rev. Immunol. 11: 331-360. [Medline]
  5. Cassese, G., S. Arce, A. E. Hauser, K. Lehnert, B. Moewes, M. Mostarac, G. Muehlinghaus, M. Szyska, A. Radbruch, R. A. Manz. 2003. Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals. J. Immunol. 171: 1684-1690. [Abstract/Free Full Text]
  6. Attanavanich, K., J. F. Kearney. 2004. Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells. J. Immunol. 172: 803-811. [Abstract/Free Full Text]
  7. Jacob, J., R. Kassir, G. Kelsoe. 1991. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I: the architecture and dynamics of responding cell populations. J. Exp. Med. 173: 1165-1175. [Abstract/Free Full Text]
  8. Aichele, P., J. Zinke, L. Grode, R. A. Schwendener, S. H. Kaufmann, P. Seiler. 2003. Macrophages of the splenic marginal zone are essential for trapping of blood-borne particulate antigen but dispensable for induction of specific T cell responses. J. Immunol. 171: 1148-1155. [Abstract/Free Full Text]
  9. Martin, F., J. F. Kearney. 2002. Marginal-zone B cells. Nat. Rev. Immunol. 2: 323-335. [Medline]
  10. Balazs, M., F. Martin, T. Zhou, J. Kearney. 2002. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 17: 341-352. [Medline]
  11. Colino, J., Y. Shen, C. M. Snapper. 2002. Dendritic cells pulsed with intact Streptococcus pneumoniae elicit both protein- and polysaccharide-specific immunoglobulin isotype responses in vivo through distinct mechanisms. J. Exp. Med. 195: 1-13. [Medline]
  12. Wykes, M., A. Pombo, C. Jenkins, G. G. MacPherson. 1998. Dendritic cells interact directly with naive B lymphocytes to transfer antigen and initiate class switching in a primary T-dependent response. J. Immunol. 161: 1313-1319. [Abstract/Free Full Text]
  13. Qi, H., J. G. Egen, A. Y. Huang, R. N. Germain. 2006. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312: 1672-1676. [Abstract/Free Full Text]
  14. Do, R. K., E. Hatada, H. Lee, M. R. Tourigny, D. Hilbert, S. Chen-Kiang. 2000. Attenuation of apoptosis underlies B lymphocyte stimulator enhancement of humoral immune response. J. Exp. Med. 192: 953-964. [Abstract/Free Full Text]
  15. Litinskiy, M. B., B. Nardelli, D. M. Hilbert, B. He, A. Schaffer, P. Casali, A. Cerutti. 2002. DCs induce CD40-independent immunoglobulin class switching through BLyS and APRIL. Nat. Immunol. 3: 822-829. [Medline]
  16. Cyster, J. G.. 2003. Homing of antibody secreting cells. Immunol. Rev. 194: 48-60. [Medline]
  17. Okada, T., V. N. Ngo, E. H. Ekland, R. Forster, M. Lipp, D. R. Littman, J. G. Cyster. 2002. Chemokine requirements for B cell entry to lymph nodes and Peyer’s patches. J. Exp. Med. 196: 65-75. [Abstract/Free Full Text]
  18. Cinamon, G., M. Matloubian, M. J. Lesneski, Y. Xu, C. Low, T. Lu, R. L. Proia, J. G. Cyster. 2004. Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone. Nat. Immunol. 5: 713-720. [Medline]
  19. Junt, T., K. Fink, R. Forster, B. Senn, M. Lipp, M. Muramatsu, R. M. Zinkernagel, B. Ludewig, H. Hengartner. 2005. CXCR5-dependent seeding of follicular niches by B and Th cells augments antiviral B cell responses. J. Immunol. 175: 7109-7116. [Abstract/Free Full Text]
  20. Hardtke, S., L. Ohl, R. Forster. 2005. Balanced expression of CXCR5 and CCR7 on follicular T helper cells determines their transient positioning to lymph node follicles and is essential for efficient B-cell help. Blood 106: 1924-1931. [Abstract/Free Full Text]
  21. Garside, P., E. Ingulli, R. R. Merica, J. G. Johnson, R. J. Noelle, M. K. Jenkins. 1998. Visualization of specific B and T lymphocyte interactions in the lymph node. Science 281: 96-99. [Abstract/Free Full Text]
  22. Pape, K. A., V. Kouskoff, D. Nemazee, H. L. Tang, J. G. Cyster, L. E. Tze, K. L. Hippen, T. W. Behrens, M. K. Jenkins. 2003. Visualization of the genesis and fate of isotype-switched B cells during a primary immune response. J. Exp. Med. 197: 1677-1687. [Abstract/Free Full Text]
  23. Reif, K., E. H. Ekland, L. Ohl, H. Nakano, M. Lipp, R. Forster, J. G. Cyster. 2002. Balanced responsiveness to chemoattractants from adjacent zones determines B-cell position. Nature 416: 94-99. [Medline]
  24. Bachmann, M. F., H. Hengartner, R. M. Zinkernagel. 1995. T helper cell-independent neutralizing B cell response against vesicular stomatitis virus: role of antigen patterns in B cell induction?. Eur. J. Immunol. 25: 3445-3451. [Medline]
  25. Ochsenbein, A. F., D. D. Pinschewer, B. Odermatt, A. Ciurea, H. Hengartner, R. M. Zinkernagel. 2000. Correlation of T cell independence of antibody responses with antigen dose reaching secondary lymphoid organs: implications for splenectomized patients and vaccine design. J. Immunol. 164: 6296-6302. [Abstract/Free Full Text]
  26. Bachmann, M. F., B. Odermatt, H. Hengartner, R. M. Zinkernagel. 1996. Induction of long-lived germinal centers associated with persisting antigen after viral infection. J. Exp. Med. 183: 2259-2269. [Abstract/Free Full Text]
  27. Hangartner, L., B. M. Senn, B. Ledermann, U. Kalinke, P. Seiler, E. Bucher, R. M. Zellweger, K. Fink, B. Odermatt, K. Burki, et al 2003. Antiviral immune responses in gene-targeted mice expressing the immunoglobulin heavy chain of virus-neutralizing antibodies. Proc. Natl. Acad. Sci. USA 100: 12883-12888. [Abstract/Free Full Text]
  28. Maloy, K. J., C. Burkhart, G. Freer, T. Rulicke, H. Pircher, D. H. Kono, A. N. Theofilopoulos, B. Ludewig, U. Hoffmann-Rohrer, R. M. Zinkernagel, H. Hengartner. 1999. Qualitative and quantitative requirements for CD4+ T cell-mediated antiviral protection. J. Immunol. 162: 2867-2874. [Abstract/Free Full Text]
  29. Bailey, M. J., D. A. McLeod, C. Y. Kang, D. H. Bishop. 1989. Glycosylation is not required for the fusion activity of the G protein of vesicular stomatitis virus in insect cells. Virology 169: 323-331. [Medline]
  30. Jung, S., D. Unutmaz, P. Wong, G. Sano, K. de los Santos, T. Sparwasser, S. Wu, S. Vuthoori, K. Ko, F. Zavala, et al 2002. In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 17: 211-220. [Medline]
  31. Probst, H. C., K. Tschannen, B. Odermatt, R. Schwendener, R. M. Zinkernagel, M. Van Den Broek. 2005. Histological analysis of CD11c-DTR/GFP mice after in vivo depletion of dendritic cells. Clin. Exp. Immunol. 141: 398-404. [Medline]
  32. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99: 23-33. [Medline]
  33. Ochsenbein, A. F., T. Fehr, C. Lutz, M. Suter, F. Brombacher, H. Hengartner, R. M. Zinkernagel. 1999. Control of early viral and bacterial distribution and disease by natural antibodies. Science 286: 2156-2159. [Abstract/Free Full Text]
  34. Ochsenbein, A. F., D. D. Pinschewer, B. Odermatt, M. C. Carroll, H. Hengartner, R. M. Zinkernagel. 1999. Protective T cell-independent antiviral antibody responses are dependent on complement. J. Exp. Med. 190: 1165-1174. [Abstract/Free Full Text]
  35. Hebel, K., K. Griewank, A. Inamine, H. D. Chang, B. Muller-Hilke, S. Fillatreau, R. A. Manz, A. Radbruch, S. Jung. 2006. Plasma cell differentiation in T-independent type 2 immune responses is independent of CD11c(high) dendritic cells. Eur. J. Immunol. 36: 2912-2919. [Medline]
  36. Ruprecht, C. R., A. Lanzavecchia. 2006. Toll-like receptor stimulation as a third signal required for activation of human naive B cells. Eur. J. Immunol. 36: 810-816. [Medline]
  37. Pasare, C., R. Medzhitov. 2005. Control of B-cell responses by Toll-like receptors. Nature 438: 364-368. [Medline]
  38. Viglianti, G. A., C. M. Lau, T. M. Hanley, B. A. Miko, M. J. Shlomchik, A. Marshak-Rothstein. 2003. Activation of autoreactive B cells by CpG dsDNA. Immunity 19: 837-847. [Medline]
  39. Leadbetter, E. A., I. R. Rifkin, A. M. Hohlbaum, B. C. Beaudette, M. J. Shlomchik, A. Marshak-Rothstein. 2002. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416: 603-607. [Medline]
  40. Lau, C. M., C. Broughton, A. S. Tabor, S. Akira, R. A. Flavell, M. J. Mamula, S. R. Christensen, M. J. Shlomchik, G. A. Viglianti, I. R. Rifkin, A. Marshak-Rothstein. 2005. RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement. J. Exp. Med. 202: 1171-1177. [Abstract/Free Full Text]
  41. Marshall-Clarke, S., L. Tasker, O. Buchatska, J. Downes, J. Pennock, S. Wharton, P. Borrow, D. Z. Wiseman. 2006. Influenza H2 haemagglutinin activates B cells via a MyD88-dependent pathway. Eur. J. Immunol. 36: 95-106. [Medline]
  42. Lund, J. M., L. Alexopoulou, A. Sato, M. Karow, N. C. Adams, N. W. Gale, A. Iwasaki, R. A. Flavell. 2004. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl. Acad. Sci. USA 101: 5598-5603. [Abstract/Free Full Text]
  43. Ludewig, B., K. J. Maloy, C. Lopez-Macias, B. Odermatt, H. Hengartner, R. M. Zinkernagel. 2000. Induction of optimal anti-viral neutralizing B cell responses by dendritic cells requires transport and release of virus particles in secondary lymphoid organs. Eur. J. Immunol. 30: 185-196. [Medline]
  44. Bergtold, A., D. D. Desai, A. Gavhane, R. Clynes. 2005. Cell surface recycling of internalized antigen permits dendritic cell priming of B cells. Immunity 23: 503-514. [Medline]
  45. Burkhart, C., G. Freer, R. Castro, L. Adorini, K. H. Wiesmuller, R. M. Zinkernagel, H. Hengartner. 1994. Characterization of T-helper epitopes of the glycoprotein of vesicular stomatitis virus. J. Virol. 68: 1573-1580. [Abstract/Free Full Text]
  46. Okada, T., M. J. Miller, I. Parker, M. F. Krummel, M. Neighbors, S. B. Hartley, A. O’Garra, M. D. Cahalan, J. G. Cyster. 2005. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol. 3: E150[Medline]
  47. Junt, T., H. Nakano, T. Dumrese, T. Kakiuchi, B. Odermatt, R. M. Zinkernagel, H. Hengartner, B. Ludewig. 2002. Antiviral immune responses in the absence of organized lymphoid T cell zones in plt/plt mice. J. Immunol. 168: 6032-6040. [Abstract/Free Full Text]
  48. Junt, T., E. Scandella, R. Forster, P. Krebs, S. Krautwald, M. Lipp, H. Hengartner, B. Ludewig. 2004. Impact of CCR7 on priming and distribution of antiviral effector and memory CTL. J. Immunol. 173: 6684-6693. [Abstract/Free Full Text]
  49. Siemasko, K., B. J. Eisfelder, E. Williamson, S. Kabak, M. R. Clark. 1998. Cutting edge: signals from the B lymphocyte antigen receptor regulate MHC class II containing late endosomes. J. Immunol. 160: 5203-5208. [Abstract/Free Full Text]
  50. Cheng, P. C., C. R. Steele, L. Gu, W. Song, S. K. Pierce. 1999. MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens. J. Immunol. 162: 7171-7180. [Abstract/Free Full Text]
  51. Zimmermann, V. S., P. Rovere, J. Trucy, K. Serre, P. Machy, F. Forquet, L. Leserman, J. Davoust. 1999. Engagement of B cell receptor regulates the invariant chain-dependent MHC class II presentation pathway. J. Immunol. 162: 2495-2502. [Abstract/Free Full Text]
  52. Trombetta, E. S., M. Ebersold, W. Garrett, M. Pypaert, I. Mellman. 2003. Activation of lysosomal function during dendritic cell maturation. Science 299: 1400-1403. [Abstract/Free Full Text]
  53. Kleijmeer, M., G. Ramm, D. Schuurhuis, J. Griffith, M. Rescigno, P. Ricciardi-Castagnoli, A. Y. Rudensky, F. Ossendorp, C. J. Melief, W. Stoorvogel, H. J. Geuze. 2001. Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells. J. Cell Biol. 155: 53-63. [Abstract/Free Full Text]
  54. Barois, N., B. de Saint-Vis, S. Lebecque, H. J. Geuze, M. J. Kleijmeer. 2002. MHC class II compartments in human dendritic cells undergo profound structural changes upon activation. Traffic 3: 894-905. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
L. Fahlen-Yrlid, T. Gustafsson, J. Westlund, A. Holmberg, A. Strombeck, M. Blomquist, G. G. MacPherson, J. Holmgren, and U. Yrlid
CD11chigh Dendritic Cells Are Essential for Activation of CD4+ T Cells and Generation of Specific Antibodies following Mucosal Immunization
J. Immunol., October 15, 2009; 183(8): 5032 - 5041.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
H. Y. Lee, D. J. Topham, S. Y. Park, J. Hollenbaugh, J. Treanor, T. R. Mosmann, X. Jin, B. M. Ward, H. Miao, J. Holden-Wiltse, et al.
Simulation and Prediction of the Adaptive Immune Response to Influenza A Virus Infection
J. Virol., July 15, 2009; 83(14): 7151 - 7165.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. H. Achtman, U. E. Hopken, C. Bernert, and M. Lipp
CCR7-deficient mice develop atypically persistent germinal centers in response to thymus-independent type 2 antigens
J. Leukoc. Biol., March 1, 2009; 85(3): 409 - 417.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. Fink, N. Manjarrez-Orduno, A. Schildknecht, J. Weber, B. M. Senn, R. M. Zinkernagel, and H. Hengartner
B Cell Activation State-Governed Formation of Germinal Centers following Viral Infection
J. Immunol., November 1, 2007; 179(9): 5877 - 5885.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Scandella, E.
Right arrow Articles by Ludewig, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scandella, E.
Right arrow Articles by Ludewig, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS