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The Journal of Immunology, 2007, 179, 5877 -5885
Copyright © 2007 by The American Association of Immunologists, Inc.
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B Cell Activation State-Governed Formation of Germinal Centers following Viral Infection1

Katja Fink2,3,4,*, Nataly Manjarrez-Orduño3,5,*, Anita Schildknecht*, Jacqueline Weber*, Beatrice M. Senn{dagger}, Rolf M. Zinkernagel* and Hans Hengartner*

* Institute of Experimental Immunology, University Hospital, Zurich, Switzerland; and {dagger} Intercell AG, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Germinal centers are structures that promote humoral memory cell formation and affinity maturation, but the triggers for their development are not entirely clear. Activated extrafollicular B cells can form IgM-producing plasmablasts or enter a germinal center reaction and differentiate into memory or plasma cells, mostly of the IgG isotype. Vesicular stomatitis virus (VSV) induces both types of response, allowing events that promote each of these pathways to be studied. In this work, extrafollicular vs germinal center responses were examined at a cellular level, analyzing VSV-specific B cells in infected mice. We show that VSV-specific germinal centers are transiently formed when insufficient proportions of specific T cell help is available and that strong B cell activation in cells expressing high levels of the VSV-specific BCR promoted their differentiation into early blasts, whereas moderate stimulation of B cells or interaction with Th cells restricted extrafollicular responses and promoted germinal center formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Antibody production after an antigenic challenge is a complex event whose outcome depends on the nature of the signals that B cells receive. Upon specific antigenic encounter, B cells are activated and migrate to the T-B cell border of secondary lymphoid organs, where they interact with Th cells. B cells then proliferate and differentiate into plasmablasts or become germinal center (GC)6 founder cells (reviewed in Refs. 1 and 2). The GC response is characterized by a phase of extensive proliferation of B cells followed by selection by Ag presented on follicular dendritic cells (1, 3, 4).

Studies on the GC reaction have mainly used haptens as immunogens. This approach may not faithfully represent immune responses following natural infections, since protein Ags often elicit transient GCs (5), whereas long-lived GCs are observed during viral infections (6). Besides, the established models of GC have only partially been extended to Ags that also exert T-independent reactions (7, 8, 9, 10). For example, many bacterial and viral pathogens elicit GCs, but are also able to trigger potent extrafollicular responses in the absence of T cells, even though a positive impact of (specific) Th cells has been described (11, 12, 13). Thus, these agents represent a good model to address which factors determine the fate of an early- activated B cell in the context of a natural infection. Using sheep RBC conjugates of mutant hen egg lysozyme proteins with different affinity as immunizing Ag, Paus et al. (14) recently demonstrated that the extrafollicular B cell response became weaker with decreasing Ag affinity and density, whereas GC formation did not depend on the strength of B cell activation. These data clearly demonstrated an impact of Ag affinity for the extent of an extrafollicular response, at least in the context of an antihapten response.

In this work, we analyzed the impact of B cell activation and T cell availability on GC formation in a model with vesicular stomatitis virus (VSV)-specific B and Th cells. B cells were derived from mice expressing the rearranged VHDJH region of a VSV-neutralizing Ab (VI10) (19). As Th cell donors, we used TCR-transgenic (tg) mice with CD4 cells specific for a VSV glycoprotein epitope presented on I-Ab (L7) (15). We compared hetero- and homozygous VI10 donor B cells because we had found earlier that Ab production by homozygous B cells was earlier and higher compared with heterozygous B cells, probably due to different expression levels of specific BCRs on the cell surface.

Our data suggest that sufficient T cell help and the degree of B cell activation decide the fate of an activated B cell toward an extrafollicular or a follicular pathway: strong B cell activation drives differentiation into early blasts, whereas moderate stimulation or interaction of B cells with Th cells restricts extrafollicular responses and promotes GC formation.


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

Homo- and heterozygous VI10 mice (16) and L7 mice (15) were kept at the Biologische Zentrallabor, University Hospital Zurich (Zurich, Switzerland), under specific pathogen-free conditions. C57BL/6 mice were bred by the Institute of Laboratory Animals, University of Zurich.

Virus

VSV, Indiana strain (VSV-IND, Mudd-Summers isolate) was originally obtained from Prof. D. Kolakofsky (University of Geneva, Geneva, Switzerland).

VSV neutralization assay

Titers of VSV-neutralizing Abs in sera were determined as described previously (6). Briefly, sera were diluted in MEM/2% FCS, and the dilution that gave a 50% reduction of virus plaques on Vero cells was taken as the neutralizing titer of all isotypes. To measure 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 to inactivate complement.

VSV-specific ELISPOT

VSV-specific Ab-secreting cell numbers were determined as described previously (17), with slight modifications. Twenty-four-well plates were coated overnight with 15 µg/ml polyethylene glycol-precipitated VSV-IND in PBS, then blocked with 2% BSA and washed. Single-cell suspensions were serially diluted 1/5 in serum-free medium and incubated for 5 h at 37°C. Cells were washed off, and plates were incubated successively with rat anti-mouse IgM (BD Pharmingen) and donkey anti-rat alkaline phosphatase-conjugated Fab (Jackson ImmunoResearch Laboratories) or with goat anti-mouse IgG {gamma}-chain(Sigma-Aldrich) and donkey anti-goat alkaline phosphatase-conjugated Ab (Jackson ImmunoResearch Laboratories). Color was developed by adding 5-bromo-4-chloro-3-indolyl phosphate in alkaline phosphatase buffer (Sigma-Aldrich) containing 0.6% agarose. Spots were counted using a reflected light microscope.

Sorting of cells

Splenic B cells from VI10 animals were sorted with anti-B220 magnetic beads, while splenic Th cells from L7 spleens were sorted with CD4-specific magnetic beads (Miltenyi Biotec) for adoptive transfers. Before sorting, erythrocytes were lysed in 0.83% NH4Cl. Purity of B220+ and CD4+ cells was between 85 and 95%.

Flow cytometry

Anti-idiotypic Ab 35.61 specific for the VSV-neutralizing H and L chain variable region of VI10 BCR and anti-idiotypic Ab IIIC4.1 specific for the LCMV-neutralizing H chain variable region of KL25 have been described previously (16) and were labeled with FITC (Sigma-Aldrich) or Cy5 (Amersham Biosciences). All other Abs were purchased from BD Pharmingen. For flow cytometry analysis, surface molecules were stained in FACS buffer (PBS, 2% FCS, 1 mM EDTA, and 0.1 mg/ml sodium azide) for 30 min at 4°C. For intracellular staining of plasma cells, cells were fixed and permeabilized with Cytofix/Perm Solution obtained 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/1% formalin and data were acquired in a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Histology

Freshly removed spleens 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. For fluorescent histology, cryosections were blocked for 30 min with 1 µg per sample of the Fc-blocking Ab 2.4G2, 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, nuclei were counterstained with 4',6-diamino-2-phenylindole (Sigma-Aldrich) and sections were mounted with fluorescence mounting solution (DakoCytomation). Fluorescence was acquired in a Zeiss Axiophot microscope (Zeiss). Color channels were assembled automatically with the analySIS software (Soft Imaging System).

Images were processed using Adobe Photoshop, without nonlinear operations.

Ca2+ mobilization in activated B cells

In brief, 3 x 106 splenic cells were incubated with 1 µM Fluo-4 (Molecular Probes) in 700 µl of RPMI 1640 medium/5% FCS for 25 min at 37°C. Cells were then diluted 1/1 with RPMI 1640 medium/10% FCS and incubated for another 10 min at 37°C. Cells were stained with anti-B220 (BD Pharmingen) in RPMI 1640 medium containing 5% FCS and washed twice with Krebs-Ringer solution (10 mM HEPES (pH 7.0), 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM glucose). Cells were resuspended in 400 µl of Krebs-Ringer solution with 0.5 mM EDTA and no CaCl2 for data acquisition in a FACSCalibur (BD Biosciences). After 30 s, 5 µM anti-IgM F(ab')2 (Jackson ImmunoResearch Laboratories) or 107 PFU of UV-VSV were added in Krebs-Ringer solution containing 0.5 mM EDTA. Analysis was performed with FlowJo software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
GC exclusion of tg VI10 cells

In heterozygous H chain-targeted VI10 mice, 15–20% of all B cells were VSV neutralizing (16). Following infection, both VSV-neutralizing IgM and IgG Abs were produced rapidly (Fig. 1a), and immense numbers of extrafollicular foci were observed in the spleen, peaking between days 2 and 3 after infection (18). These early extrafollicular plasma cells produced mostly IgM, although lower numbers of IgG Ab-secreting cells (ASCs) could also be detected by a VSV-specific ELISPOT (Fig. 1b). Flow cytometric analysis using intracellular staining with an anti-idiotypic Ab (35.61) to identify 35.61bright, neutralizing Ab-producing cells showed that these extrafollicular Ab-producing cells were CD138+ (Fig. 1c) and B220– (data not shown). The formation of transgene-derived ASCs in VI10 mice was fast and plateaued quickly, so that after day 4 CD138+ cells were mostly 35.61– and ASCs detected at days 8 and 20 by ELISPOT (Fig. 1b) were not of tg origin. The VSV-specific response in wild-type mice was also transient, albeit with a later peak at day 4. In both VI10 and wild-type mice, the short-lived appearance of ASCs in the bone marrow was observed 2 days following ASC peaks in the spleen, where high numbers of ASCs were established at day 20 (data not shown).


Figure 1
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  		FIGURE 1. GC exclusion of tg B cells in VI10 mice. A, VI10 and C57BL/6 mice were infected with 2 x 106 PFU of VSV-IND i.v., and the neutralizing capacity of total serum Abs or IgG Abs was analyzed at the indicated time points. Symbols, Means ± SD, n = 3–4. B, VI10 mice were infected with 2 x 106 PFU of VSV-IND, and VSV-specific ASCs in spleens were analyzed at different time points after infection by ELISPOT. Bars, Means ± SD, n = 3–4. C, Spleen cells of infected VI10 mice were stained with anti-CD138 and 35.61 mAb (intracellular stain) to detect 35.61brightCD138+ plasma cells. Graphs represent one of three infected animals. D, Splenic histology from VI10 mice infected with 2 x 106 PFU or 2 x 109 PFU of VSV-IND i.v. 5 days earlier. Sections were stained with Abs against B220 (red), 35.61 (anti-VI10; green), and the GC B cell marker GL7 (blue). Micrographs are shown with and without GL7 stain to illustrate the absence of VI10 cells in GCs. Sections are representative for one mouse of four independent experiments.

 
To determine whether the efficient class switch observed in serum Ab titers (Fig. 1a) involved the formation of GCs, VI10+/– mice were infected with a low (2 x 106 PFU) or a high dose (109 PFU) of VSV-IND, and histology was performed at day 5. As shown in Fig. 1d, GCs, identified with the marker GL7 (19) seemed to consist exclusively of non-tg B cells, since GCs were free of Id-specific B cells (yellow cells), even after 20 days following infection (data not shown).

Thus, in VI10 mice, preexisting VSV-specific B cells (35.61+) followed an extrafollicular path, whereas these cells were absent in the GCs.

Low B cell frequencies and increased T cell help promote GC formation

Since the interaction of B cells with Th cells is required for GC maintenance (20, 21, 22, 23), we hypothesized that the imbalance between specific B and T cell frequencies could underlie the inefficient GC formation and maintenance in VI10 mice. To decrease specific B cell precursor frequencies, we adoptively transferred VI10 splenocytes into wild-type recipients and analyzed GC formation at day 9 after infection. Flow cytometry analysis showed that a small population of transferred GL7+ specific B cells was responsible for GC formation (Fig. 2a). Yet, when specific T cell help was cotransferred using splenocytes from L7 mice, the population of VSV-specific cells that was part of a GC was significantly larger (Fig. 2b). This corresponded with a change in the behavior of the transferred VI10 cells, since the CD138+ population of plasmablasts was formed at day 4, later than in VI10 mice, and lasted until day 8 after infection (Fig. 2c). Although 35.61+CD138+ cells were largely IgM+ at day 4, >90% of 35.61+CD138+ cells had switched to IgG by day 6 (Fig. 2d).


Figure 2
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  		FIGURE 2. VSV-specific GC formation as a result of sufficient Th cell frequencies during GC induction. A and B, In brief, 6 x 106 splenocytes from VI10 (A) and from VI10 and L7 donors (B) were adoptively transferred into B6 recipients. Nine days following infection with 2 x 106 PFU of VSV-IND, splenocytes of recipient mice were stained with anti-CD19, -GL7, and 35.61 for flow cytometry analysis. CD19+ gated cells (gray area) were analyzed for GL7 and 35.61 expression to identify specific GC B cells. Graphs are representative for one mouse of three, and the experiment was repeated twice, giving similar results. C and D, In brief, 3 x 106 B220+ sorted VI10 splenocytes and 1.5 x 106 CD4+ sorted L7 splenocytes were cotransferred into B6 recipients and recipients were infected 24 h later. C, At indicated days after infection, splenic plasma cells were detected as described for Fig. 1C. IgM production of plasma cells in the indicated gates was analyzed after intracellular anti-IgM staining (D). E, In brief, 3 x 106 B220+ sorted VI10 splenocytes and 1.5 x 106 CD4+ sorted L7 splenocytes were cotransferred into B6 recipients, and recipients were infected 24 h later = time point day 0 ({blacksquare}) or 1.5 x 106 CD4+ sorted L7 splenocytes from an infected L7 donor (infection at day 0) were transferred at day 4 ({square}). Total numbers of GL7+ and CD138+ plasma cells among 35.61+ cells were analyzed by flow cytometry in the spleens of recipient mice. Bars, Means ± SD, n = 3, p > 0.05. F, Donor-derived IgG titers of the mice shown in E were determined by a 35.61-specific ELISA (p = 0.055).

 
The specific B:T cell ratio was decisive for the entrance of VI10 B cells to the GCs. To address the impact of proper timing of T cell help and to simulate the slower Th activation in a natural situation, we transferred VSV-specific Th cells at day 4 after infection (Fig. 2, e and f). Although there was no difference in total VI10 and CD138+ numbers found at day 7, whenever activated Th cells were available at the time of infection, GC formation was increased (Fig. 2e). Yet, the slightly higher IgG titers in a situation where T cell help was available at the time point of B cell activation suggested that T cell help also contributed to an early and transient, most likely extrafollicular, B cell response (shown later in Fig. 3a, and Ref. 24).


Figure 3
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  		FIGURE 3. Th cell-dependent GC formation and isotype switch of tg cells. In brief, 3 x 106 B220+ sorted VI10 splenocytes were adoptively transferred into B6 recipients with or without cotransfer of 1.5 x 106 CD4+ sorted L7 splenocytes. Twenty-four hours after cell transfer, recipient mice were infected with 2 x 106 PFU of VSV-IND. A, VSV-neutralizing titers in the sera of recipient mice were measured at different time points after infection. p < 0.05 for all time points (unpaired t test). B, Total 35.61+ cell numbers recovered in recipient mice with or without cotransfer of L7 Th cells were determined by flow cytometry at the indicated time points following infection. C, Specific GC formation was evaluated in the same mice by quantifying 35.61+GL7+ lymphocytes. Each symbol represents one mouse. D, Percentages of intracellular 35.61+CD138+IgM+ (IgM plasma cells) or IgM– (switched plasma cells, E) were analyzed by flow cytometry at indicated time points after infection (mean ± SD, n = 3). All experiments were repeated, giving similar results. *, p < 0.05; **, p < 0.005 (unpaired t test). F–K, Splenic histology of recipient mice 12 days after infection and 13 days after transfer of VI10 B cells (F) or VI10 B and L7 T cells (G–K). Ab 35.61 was used to detect VSV-specific tg B cells of donor origin (F–H), a peanut agglutinin (PNA) stain was used to show GCs (J), or slides were incubated with virus and anti-VSV-specific polyclonal Ab for the staining of VSV-specific B cells of both donor and recipient origin.

 
In an earlier study with quasi-monoclonal mice expressing a high-affinity (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific BCR, it was shown that GCs were formed following immunization with the TI Ag NP-Ficoll in the absence of T cell help. These T cell-independent GCs were short-lived, suggesting that the maintenance of GC required Th cells (8). Because T cell help seemed to be required for the formation of GCs with VSV-specific B cells, we wondered whether the amount of specific T cell help would affect the kinetics of a GC response. We therefore transferred VI10 B cells with or without cotransfer of L7 Th cells into wild-type recipients and analyzed serum Ab titers and splenic GC formation at different time points following infection.

IgM and switched VSV-neutralizing titers in the serum were higher in the presence of T cell help, showing that T cell help supported both early extrafollicular formation of IgM and the subsequent formation of IgG (Fig. 3a). To address whether IgG was produced from GC-derived plasma cells or from isotype-switched extrafollicular blasts, GL7+35.61+ cells with or without cotransfer of specific T cell help in recipient mice were quantified. Analysis of total recovered B cells in recipient mice showed that in the presence of specific L7 Th cells, VI10 B cells expanded strongly until day 6 and remained in the spleen at high numbers until day 8 (Fig. 3b), where GC analysis of specific GC B cells (Fig. 3c) also showed that with cotransfer of T cell help, more specific GC B cells were recovered at day 6 after infection than with B cell transfer alone.

Furthermore, T cell help prolonged IgM production by CD138+ plasma cells, because IgM+ plasma cells could only be detected with cotransfer of L7 cells at day 6 following infection, whereas the IgM plasma cell response of VI10 B cells transferred without L7 T cell help was short-lived and not detectable after day 4 (Fig. 3d; 35.61+ IgM plasma cells). Moreover, cotransfer of T cell help efficiently induced switching (i.e., IgM–) of plasma cells after day 4 following infection, whereas very few switched plasma cells were recovered in the absence of L7 cells (Fig. 3e). The formation of GCs by endogenous, 35.61– B cells was not altered significantly by the cotransfer of Th cells, and few endogenous switched plasma cells were detected at all time points (data not shown), suggesting that the VSV-specific response was largely performed by the transferred cells.

The relatively short-lived GC response of tg cells detected by flow cytometry may suggest that an endogenous GC response, which presumably starts with lower-affinity B cells, would persist longer. When comparing 35.61-binding vs VSV-binding cells in histology at day 12 after infection, almost all GCs stained weakly for 35.61 in areas we consider as light zones (Fig. 3, f and g). This observation suggested that the vast majority of the GCs formed were of tg origin and that the endogenous VSV-specific (and 35.61-negative) GC formation was negligible (Fig. 3, h–k).

These experiments demonstrated that early specific T-B cell interaction promoted GC formation following viral infection, whereas most VSV-specific B cells differentiated into early IgM-producing plasma cells when specific (endogenous) T cell help was in a very low proportion compared with the number of specific B cells. Formation of switched VSV-specific plasma cells depended on the presence of specific Th cells and coincided with the appearance of specific GCs, providing evidence that the isotype switch occurred largely in early VSV- induced GCs.

Efficient GC formation with specific B cells expressing reduced BCR density

Thus, T cell help seemed critical for the extent and the duration of GC responses and for the generation of isotype-switched Abs. We had assumed before that the relatively low availability of specific T help compared with the high frequency of specific B cells in VI10 mice could have prevented formation of GCs. However, when we crossed VI10 mice with H chain gene-targeted KL25 mice expressing the rearranged VHDJH region of a LCMV-neutralizing Ab (16), we found 1) that B cells in VI10 x KL25 mice had dual BCR specificities and 2) that B cells formed specific GCs following infection with VSV (Fig. 4a). This result was surprising because the frequency of 35.61+, VSV-specific cells was comparable between VI10 and KL25 x VI10 mice (Fig. 4b). Consequently, endogenous T cell help in KL25 x VI10 mice should be insufficient for early GC formation of specific B cells as in VI10 mice. We hypothesized that a B cell-intrinsic effect accounted for the observed difference in GC formation of VI10 and VI10 x KL25 B cells. Because B cells in VI10 x KL25 mice coexpressed both BCR specificities (Fig. 4b), it seemed reasonable that the BCR density for one Ag on B cells with both specificities would be lower than on single tg B cells. We analyzed the mean fluorescence of B cells from VI10 and VI10 x KL25 cells stained with Ab 35.61 and found that the density of the VSV-specific BCR was indeed lower on VI10 x KL25 B cells compared with VI10 B cells (Fig. 4c). Therefore, a lower BCR density might affect the cross-linking of BCRs by Ag, resulting in a weaker BCR-mediated signal (25, 26).


Figure 4
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  		FIGURE 4. VI10 cells with low BCR density form GCs even at high B cell frequencies. A, VI10 x KL25 mice were infected i.v. with 109 PFU of VSV-IND. Five days later, spleens were removed for histology and stained as described in Fig. 1. Upper graphs are shown without GL7 stain (blue) for a clearer illustration of 35.61+ B cells in GCs. B, VI10 x KL25 mice generated comparable numbers of VSV-specific (35.61+) B cells. Splenocytes were stained with anti-CD19, 35.61 (anti-VI10), and IIIC4.8 (anti-KL25) and were gated on lymphocytes. Bold numbers in the upper graphs are percentages of VSV-specific 35.61+ cells of CD19+ cells. B cells (gray areas) were analyzed for the expression of 35.61 and IIIC4.8. C, BCR densities of VI10 x KL25 CD19+ splenocytes were compared with those of VI10 and KL25 mice by overlaying fluorescent intensities of 35.61+ or IIIC4.8+ B cells from single- and double-tg mice.

 
Extrafollicular or follicular response as a B cell- intrinsic decision

The results with VI10 x KL25 mice suggested a mechanism to drive GC formation with specific B cells irrespective of the low frequency of specific T cell help. To investigate the influence of the BCR-mediated activation on the formation of GC vs extrafollicular response further, we compared heterozygous and homozygous VI10 mice. Homozygous VI10 mice (VI10+/+) had higher numbers of VSV-specific B cells (Fig. 5a) and higher preimmune titers of VSV-neutralizing Abs compared with heterozygous VI10 mice (VI10+/–) (data not shown). Following infection, B cells in homozygous VI10 mice up-regulated activation marker CD86 earlier than B cells in heterozygous VI10 mice, showing faster activation of homozygous, compared with heterozygous B cells (Fig. 5b). BCR density, as expressed by the mean fluorescent intensity of 35.61+ cells, was indeed comparable in VI10+/+ and VI10+/– mice. However, the 35.61–/low population was shifted toward higher fluorescent intensity in VI10+/+ mice, which can be explained by the fact that mAb 35.61 binds with lower affinity to non-neutralizing B cells expressing the VH chain in combination with an L chain containing a V region other than the "neutralizing" V{kappa} gene family (data not shown).


Figure 5
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  		FIGURE 5. Different responses of VI10+/– and VI10+/+ B cells. A, B220 and 35.61 stain of naive spleen cells from VI10+/– and VI10+/+ mice. Percentages of 35.61+ cells among B220+ cells are indicated in the upper right quadrants (mean ± SD, n = 4). B, Expression of activation marker CD86 was analyzed on B cells from VI10+/– and VI10+/+ mice at 1 and 2 days after infection with 109 PFU of VSV-IND. Shaded areas are the expression levels of naive VI10+/– B cells. Naive VI10+/+ and VI10+/– and VI10+/+ B cells expressed the same levels of CD86. C–E, In brief, 3 x 106 B cells from VI10+/– or VI10+/+ mice were adoptively transferred into B6 recipients and recipients were infected i.v. with 2 x 106 PFU of VSV-IND. Graphs represent total numbers of VSV-specific B cells (C), absolute numbers of specific GC B cells (D), and percentages of VSV-specific GC cells of lymphocytes (E) recovered in recipients at the indicated time points following infection. Each symbol represents one mouse, and bar height indicates the mean of these values. *, p = 0.01; **, p < 0.01; ns, Not significant (unpaired t test). F, VSV-neutralizing titers of recipient mice following transfer of 3 x 106 B cells from VI10+/– or VI10+/+ mice (mean ± SD, n = 3). All experiments were repeated, showing similar results. G, Spleen histology of recipient mice after transfer of 3 x 106 VI10+/– or VI10+/+ B cells and 2 days after infection with 2 x 106 PFU of VSV-IND. Sections were stained with mAb 35.61. Histology is representative for one of three mice.

 
Next, we compared the responses of heterozygous and homozygous VI10 B cells following transfer to wild-type recipients. Strikingly, 10 times fewer VI10+/+ B cells could be recovered in recipient mice at day 4 following infection compared with transfer of VI10+/– B cells (Fig. 5c). VI10+/– B cells had expanded and had formed GCs at day 4 (Fig. 5, d and e). As shown in Fig. 5a, VI10+/+ mice had higher numbers of specific B cells, which could account for a frequency-dependent GC exclusion in the recipient as well as in VI10-tg mice (Fig. 1b). However, when only 1.5 x 106 instead of 3 x 106 VI10+/+ B cells were transferred, the same absolute numbers of total and GC-specific VI10+/+ B cells were recovered, demonstrating that transferred VI10+/+ B cells expanded and generated mostly extrafollicular responses independent of the initial input of B cells (data not shown). When measuring VSV-neutralizing titers in the sera of recipient mice, we observed that IgG titers were at least 10-fold lower in recipients of VI10+/+ donor cells (Fig. 5f). Of note, VI10+/+-derived Abs at day 4 were mostly IgMs. The lower IgG titer was in line with significantly reduced numbers of GL7+VI10+/+ compared with GL7+VI10+/– B cells at day 4 (Fig. 5e), suggesting that the isotype switch was supported in early GCs following viral infection. Splenic histology of recipients of VI10+/+ or VI10+/– B cells at day 2 after infection showed that the majority of VI10+/+ B cells had differentiated into plasmablasts, whereas most VI10+/– B cells had a naive phenotype (Fig. 5g). When L7 CD4+ T cells were cotransferred with VI10+/+ or VI10+/– B cells, B cells from both donors formed transient GCs, peaking at day 7 and disappearing by day 11 following infection (data not shown).

These results suggested that B cell-intrinsic mechanisms could determine whether an activated B cell takes the extrafollicular or follicular route. Yet, experiments with cotransfer of B and Th cells showed that T cell help dominated over B cell-intrinsic factors in driving the onset of a GC response.

Stronger activation of extrafollicular VI10+/+ B cells

Different capacities of VI10+/+ and VI10+/– B cells to enter GC responses suggested that these cells were activated with different efficiencies, determining the fate of the B cell response (14, 27). Intracellular mobilization of Ca2+ is a sensitive method to measure the magnitude of B cell activation after stimulation. Comparing VI10+/+, VI10+/–, and C57BL/6 B spleen cells, the release of intracellular Ca2+ was measured after stimulation with anti-IgM F(ab')2 and UV-inactivated VSV (Fig. 6, a and b) by flow cytometry. When cells were stimulated specifically with UV-VSV, VI10+/+ B cells showed a higher signal than VI10+/– and C57BL/6 B cells (Fig. 6b). Because mAb 35.61 activates specific B cells, it could not be used as a marker to gate on the VSV-specific population. However, the absence of activated cells in C57BL/6 and higher numbers of activated VI10+/+ compared with VI10+/– B cells was evidence that the observed Ca2+ signal derived from the VSV-specific population (Fig. 6b).


Figure 6
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  		FIGURE 6. Increased Ca2+ mobilization in extrafollicular-biased VI10+/+ B cells. A, Spleen cells from VI10+/–, VI10+/+, and C57BL/6 mice were stimulated with anti-IgM F(ab')2 or UV-VSV. The mean Fluo-4 intensity of B220+ gated cells is shown over time. B, Spleen cells were stimulated with UV-VSV and the Fluo-4 signal for B220+ cells was analyzed for 5 min. The partial activation of VI10+/– and VI10+/+ B cells showed that only VSV-specific were stimulated.

 
Strong stimulation of B cells as detected by high intracellular Ca2+ mobilization would thus qualify a B cell to form an extrafollicular blast, whereas a more moderate activation would potentially lead to the onset of a GC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The mechanisms that drive an activated B cell to become a founder GC cell have been investigated mainly using TD protein Ags (1, 28) as models. In this study, we analyzed the events leading to an extrafollicular or a GC response in the context of a biologically active Ag with the ability to induce both T cell-dependent and -independent responses.

When adoptively transferred to wild-type recipients, we found that most VSV-specific B cells entered the extrafollicular path when VSV-specific T cell help was available only in the form of endogenous, i.e., non-tg Th, cells. The functional consequence was early production of neutralizing IgM, which can prevent systemic spread and persistence of the virus (29). Most specific B cells were exhausted in the early extrafollicular response and only a few cells acquired a GC phenotype. In contrast, when VSV-specific Th cells were cotransferred with specific B cells, extrafollicular plasma cell formation was strongly reduced and GCs were efficiently induced. This finding supports a balancing influence of Th cells on early B cell activation and generation of extrafollicular blasts, keeping more B cells to seed GCs. B cell exhaustion in the absence of sufficient T cell help makes sense to curtail formation of affinity-matured autoantibodies in GCs (30). In contrast, this strategy is potentially dangerous for the host if the virus is not cleared by the first wave of Abs. We assume that the high-affinity (K = 109 M–1) of the VSV-specific BCR in VI10 mice (31) was decisive for the observed efficient activation of B cells (32). Ab VI10 contains hypermutations, yet germline Abs against VSV also reach a very high affinity (33), probably as a result of adaptation of the immune system to lethal pathogens, which need to be cleared early to ensure survival of the host. In our viral system and in contrast to immunization with haptens, unspecific B cell stimulation mechanisms such as triggering of pattern recognition receptors (34) and induction of inflammatory cytokines (35) may also predispose for an early extrafollicular response due to efficient B cell activation.

In recipients of specific B and T cells, GCs started to form by day 4 after infection and class-switched plasma cells appeared at day6, a time course that corresponds to the endogenous formation of neutralizing Abs (Fig. 1a). This implies that isotype switching occurred in these early GCs (Fig. 3). In contrast, extrafollicular plasma cells, which were efficiently induced in the absence of cotransferred specific T cell help, were found to produce mostly IgM by flow cytometry and in histology (Fig. 3d and data not shown). The same inverse relation between specific extrafollicular B cell response and specific Th cell frequency was observed in the LCMV-specific system (36). Although an isotype switch can occur outside GCs (9, 37), this did not seem to happen when sufficient specific T cell help was available.

Regardless of T cell help, our results with VI10 x KL25 mice suggested that B cell-intrinsic factors such as BCR density critically influenced the outcome of a B cell response independently of B cell frequencies. In addition, our data with VI10+/+ and VI10+/– cells pointed to a critical role of initial B cell activation determining the outcome of the immune response. A number of studies (14, 38) have recently addressed the question how the extent of B cell activation, thus the affinity of a BCR-Ag interaction, would determine the outcome of a humoral response. Whereas Paus et al. (14) studied hen egg lysozyme Ags of different affinities for a given BCR, O’Connor et al. (38) adoptively transferred BCR-tg B cells with different affinities for (4-hydroxy-3-nitrophenyl)acetyl (NP). Both studies concluded that high-affinity BCR-Ag interactions predisposed to extrafollicular responses.

The experiments described here demonstrate that the same principle of a B cell activation-dependent outcome of the B cell immune response seems to apply after a viral infection, which induces a strong inflammatory response. When adoptively transferred into wild-type recipients, the avidity of B cells from heterogeneous VI10 mice may predispose to GC formation, whereas higher-affinity B cells from homogenous VI10 mice are largely activated without follicle formation. However, specific Th cells can induce GC formation with both high- and low-affinity B cells (Figs. 2a and 6). Thus, the B cell affinity-driven differentiation program for plasma-blast formation might be overruled by the interaction with specific Th cells. This may be important after secondary infection, possibly allowing high-affinity B cells to remain in the memory pool, rather than being consumed in an extrafollicular response.

In summary, we find that at least two factors contribute to the differentiation of virus-activated B cells and the quality of the Ab response: first, the availability of specific T cell help, which seems to support GC formation and reduce extrafollicular responses, and, second, the efficiency of B cell activation. When B cells are activated early by a strong stimulus, B cells preferentially enter the extrafollicular response and are consumed within a few days, whereas a more moderate B cell activation also leads to the formation of GCs. Yet, T cell help plays a dominant role and restricts B cell autonomy and determines B cell survival in GCs.


   Acknowledgments
 
We thank Silvia Behnke and André Fitsche for their work in preparing histology. We thank Michael Engelke for his help with designing Ca2+-signaling experiments.


   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 was supported by Swiss National Foundation Grants 3100AO-100068/1 and 3100AO-100779/1. Back

2 Current address: Novartis Institute for Tropical Diseases, 10 Biopolis Road, 138670 Singapore. Back

3 K.F. and N.M.-O. contributed equally to this study. Back

4 Address correspondence and reprint requests to Dr. Katja Fink, Novartis Institute for Tropical Diseases, 10 Biopolis Road, 05-01 Chromos, 138670 Singapore. E-mail address: katja.fink{at}novartis.com Back

5 Current address: Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642. Back

6 Abbreviations used in this paper: GC, germinal center; ASC, Ab-secreting cell; VSV, vesicular stomatitis virus; VSV-IND, VSV, Indiana; LCMV, lymphocytic choriomeningitis virus; NP, (4-hydroxy-3-nitrophenyl)acetyl; tg, transgenic. Back

Received for publication February 22, 2007. Accepted for publication August 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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