Follicular and Marginal Zone B Cells Fail to Cross-Present MHC Class I-Restricted Epitopes Derived from Viral Particles This information is current as of February 26, 2017.
Susanne A. Keller, Caroline E. von Allmen, Heather J. Hinton, Monika Bauer, Simone Muntwiler, Klaus Dietmeier, Philippe Saudan and Martin F. Bachmann
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 182:6261-6266; ; doi: 10.4049/jimmunol.0804035 http://www.jimmunol.org/content/182/10/6261
The Journal of Immunology
Follicular and Marginal Zone B Cells Fail to Cross-Present MHC Class I-Restricted Epitopes Derived from Viral Particles1 Susanne A. Keller, Caroline E. von Allmen, Heather J. Hinton, Monika Bauer, Simone Muntwiler, Klaus Dietmeier, Philippe Saudan, and Martin F. Bachmann2
T
he MHC class I-restricted pathway of Ag-presentation is classically fueled by endogenously synthesized Ags (1– 3). However, it has become evident that exogenous Ags may also enter this pathway in a process called cross-presentation (4 –7). Such cross-presented Ags may make an important contribution to the induction of CTL responses (8), in particular for nonreplicating Ags such as virus-like particles (VLPs)3 (9) or viruses that fail to infect professional APCs. The main cell types involved in cross-presentation are dendritic cells (DCs) as well as macrophages. Although B cells have been shown to be able to prime naive CD4⫹ T cells both in vitro (10, 11) and in vivo (12–14), the ability of B cells to cross- present Ag is less well characterized (15–17). One study demonstrated that in vitro loading of B cells with OVA in the presence of CpG oligonucleotides leads to cross-presentation of OVA, as determined by the activation of B3Z hybridoma cells (expressing a TCR specific for the H2-Kb-restricted, OVA-derived CD8⫹ T cell epitope SINFEKL) (17). However, DCs are known to present Ag far more efficiently in similar assays, questioning an important role for B cells in cross-presentation (18). Further evidence for an involvement of B cells in cross-priming has been obtained with hepatitis B core Ag (HBcAg)-derived VLPs. In this system, cross-
Cytos Biotechnology AG, Schlieren, Switzerland Received for publication December 3, 2008. Accepted for publication March 14, 2009. 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 funding under the Sixth Research Framework Program of the European Union, Project MUGEN (MUGEN LSHG-CT-2005-005203).
2
Address correspondence and reprint requests to Dr. Martin F. Bachmann, Cytos Biotechnology AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland. E-mail address:
[email protected]
3
Abbreviations used in this paper: VLP, virus-like particle; DC, dendritic cell; FCM, flow cytometry; FO, follicular; HBcAg, hepatitis B core Ag; MZ, marginal zone. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804035
priming could only be observed in vivo in the presence of B cells (19). However, the ability of B cells to cross-present exogenous Ags has never been addressed directly. There are three main types of B cells: follicular (FO) B cells, B1 B cells, and marginal zone (MZ) B cells. FO B cells are largely responsible for conventional B cell responses, including the germinal center reaction and the formation of long-lived plasma cells (20). B1 B cells are found predominantly in the peritoneum and are thought to be responsible for the production of natural Abs (21– 23). MZ B cells have a rather complex phenotype because they exhibit characteristics of both classical Ab-producing B cells as well as APCs. MZ B cells produce the first wave of Abs against bacteria and viruses (24, 25), characterizing them as B cells with a classical function, namely the production of Abs. In contrast, stimulation of MZ B cells with TLR ligands results in their migration to T cell areas, where they are thought to induce T cell responses (26). Indeed MZ B cells exhibit higher levels of costimulatory molecules than FO B cells and have increased capacity to activate T cells in vitro (26). This ability of MZ B cells to act as potent APCs therefore renders them likely candidates to contribute to cross-presentation of Ag to CTLs. To determine whether B cells are able to cross-present viral Ags, we assessed the capacity of FO B cells and MZ B cells to process VLPs for MHC class I- as well as MHC class II-associated presentation both in vitro and in vivo. We used the welldescribed lymphocytic choriomeningitis virus-derived peptides p33 (MHC class I restricted) and p13 (MHC class II restricted) genetically fused to VLPs derived from HBcAg. We found that DCs efficiently presented p13 and p33 to CD4⫹ T cells and CD8⫹ T cells, respectively. In contrast, although FO B cells as well as MZ B cells efficiently presented the MHC class II-restricted peptide p13 to Th cells, they failed to cross-present the MHC class I-restricted peptide p33 to CTLs both in vitro and in vivo. Thus, B cells are inefficient at cross-presenting MHC class I restricted peptides and are therefore unlikely to contribute significantly to cross-priming.
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Viruses and virus-like particles (VLPs) are known to be potent inducers of B cell as well as Th cell and CTL responses. It is well established that professional APCs such as dendritic cells (DCs) and macrophages efficiently process viral particles for both MHC class I- and MHC class II-associated presentation, which is essential for induction of CTL and Th cell responses, respectively. Less is known, however, about the ability of B cells to present epitopes derived from viral particles to T cells. Using two different VLPs, in this study we show in vitro as well as in vivo that DCs present VLP-derived peptides in association with MHC class I as well as class II. In contrast, although B cells were able to capture VLPs similarly as DCs and although they efficiently processed VLPs for presentation in association with MHC class II, they failed to process exogenous VLPs for presentation in association with MHC class I. Thus, in contrast to DCs, B cells are not involved in the process of cross-priming. This finding is of physiological importance because B cells with the ability to cross-present Ag to specific CD8ⴙ T cells may be killed by these cells, preventing the generation of neutralizing Ab responses. The Journal of Immunology, 2009, 182: 6261– 6266.
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Materials and Methods Mice Eight- to 12-wk old C57BL/6 mice were purchased from Harlan Netherlands. Transgenic mice expressing a TCR specific for peptide p13 in association with H-2 I-Ab (27) and mice expressing a TCR specific for peptide p33 in association with H-2 Db (28) have been described previously. Mice were bred and kept in a specific pathogen-free facility at Cytos Biotechnology.
Peptides and VLPs Lymphocytic choriomeningitis virus glycoprotein peptides p13 (GLNGP DIYKGVYQFKSVEFD) and p33 (KAVYNFATM) were synthesized by a solid-phase method and purchased from Eurogentec. Production and purification of recombinant HBcAg-p33 and HBcAg-p13 were previously described in detail (29).
Labeling of VLPs with Cy5 molecules or Alexa Fluor 488 HBcAg-p13 and HBcAg-p33 were labeled with Alexa Fluor 488 (Molecular Probes) or Cy5 (CyDye maleimide; GE Healthcare) according to the manufacturer’s instructions, and free Alexa Fluor 488 or Cy5 was removed by ultrafiltration (Millipore). VLPs were injected i.v. at a dose of 100 g.
Isolation and staining of DCs DCs were isolated from the spleen 1 day after i.v. injection as previously described (30). Briefly, spleens were collected and digested twice for 30 min at 37°C in RPMI 1640 supplemented with 5% FCS, 100 g/ml collagenase D (Boehringer Mannheim), and 4 g/ml DNase1 (Roche). Released cells were recovered and the CD11c population was isolated by magnetic bead isolation (anti-CD11c MACS beads; Miltenyi Biotec) according to the manufacturer’s instructions.
FIGURE 2. Detection of T cell proliferation after in vitro priming with different concentrations of virus-like particles (HBcAg-p13 or HBcAgp33) or free peptide. a, B cells were pulsed with the indicated molar concentrations of HBcAg-p13 or free peptide p13 and cultured with CD4⫹ T cells. b, B cells were pulsed with the indicated molar concentrations of HBcAg-p33 or free peptide p33 and cultured with CD8⫹ T cells. c, DCs were pulsed with the indicated molar concentrations of HBcAg-p13 or free peptide p13 and cultured with CD4⫹ T cells. d, DCs were pulsed with the indicated molar concentrations of HBcAg-p33 or free peptide p33 and cultured with CD8⫹ T cells. One representative experiment of three is shown.
Isolation and staining of B cells Splenic B cells were purified by magnetic cell sorting with CD19 MACS Microbeads (Miltenyi Biotec) according to the manufacturer’s protocol. Obtained cells were stained with CD23-PE and biotinylated anti-CD21 followed by streptavidin-APC Ab (BD Biosciences). Cells were sorted with a FACS Vantage apparatus.
CFSE labeling and in vitro stimulation of T cells CFSE was purchased from Molecular Probes. Transgenic CD4⫹ and CD8⫹ T cells were obtained by positive MACS microbead isolation according to the manufacturer’s instructions (Miltenyi Biotec) with a purity of at least 90%. Cells were labeled with CFSE (final concentration 0.5 M) by incubation for 7 min at 37°C. After labeling, cold RPMI 1640 plus 10% FCS was added and cells were subsequently washed with PBS at 4°C. T cells were stimulated with DCs and MZ or FO B cells pulsed with peptides or VLPs at the indicated concentrations. APCs were pulsed for 3 h at 37°C and extensively washed subsequently. Alternatively, T cells were
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FIGURE 1. Detection of T cell proliferation after in vitro priming with virus-like particles (HBcAg-p13 or HBcAg-p33) or free peptide. CFSElabeled transgenic T cells (1.4 ⫻ 105) with a TCR specific for MHC class I-restricted p33 peptide or MHC class II-restricted p13 peptide were cocultured with 4 ⫻ 105 syngeneic presenter cells. Cultures were stimulated with 10⫺8 M HBcAg-p13 or HBcAg-p33 (a) or 10⫺8 M p13 or p33 peptide (b) (calculated per VLP subunit). Five days later, TCR transgenic cells were analyzed by FACS by gating on CD4⫹ or CD8⫹ cells. One representative experiment of three is shown.
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stimulated with DCs and FO or MZ B cells isolated from mice injected with VLPs 1 day previously. For in vitro cultures, 4 ⫻ 105/well APCs were cocultured with 1.4 ⫻ 105/well Ag-specific transgenic CD4⫹ and CD8⫹ T cells. For in vivo primed APCs, 104 MZ B cells or 5 ⫻ 104 FO B cells were cultured with 10-fold more transgenic T cells. Proliferation was assessed 5 days later on a FACSCalibur flow cytometer and analyzed using FlowJo software.
Uptake of beads and VLPs by B cells and DCs DCs were obtained from spleens of naive mice by MACS purification as described above. DCs (5 ⫻ 105) were incubated with 5 ⫻ 105 rainbow calibration particles for 60 min at 37°C and analyzed by flow cytometry (FCM). From this, the relation between the fluorescence of DCs that had taken up particles in vitro and the molecules of the equivalent Cy5 was determined and used for subsequent experiments. To determine the number of VLPs interacting in vivo with DCs and B cells, mice were immunized with 100 g of Qb-Cy5 i.v. One day later, B cells and DCs were purified and the mean fluorescent intensities of these APCs were compared with the molecules of the equivalent Cy5 of rainbow calibration particles. Dividing the number of Cy5 molecules per APC by the number of Cy5 molecules bound to one hepatitis B core subunit resulted in the number of VLPs interacting with one APC.
Results DCs but not B cells cross-present peptide p33 in vitro To determine whether DCs and B cells are able to cross-present p33 genetically fused to HBcAg to CTLs in vitro, DCs and B cells were isolated by MACS purification from spleens of naive mice and incubated with ⌯〉cAg-p33. After 3 h of incubation at 37°C, APCs were extensively washed and cultured with CFSElabeled CD8⫹ T cells derived from mice transgenic for a TCR specific for peptide p33 in association with H2-Db. As expected, DCs efficiently presented VLP-derived p33 to transgenic T cells, leading to their proliferation (Fig. 1a). Similarly, DCs
pulsed with ⌯〉cAg-p13 induced proliferation of CD4⫹ T cells derived from mice transgenic for a MHC class II-restricted TCR specific for the peptide p13 (Fig. 1a). Thus, DCs were able to present MHC class I- and II-associated peptides derived from VLPs. In marked contrast, although B cells induced proliferation of p13-specific CD4⫹ T cells, they failed to induce proliferation of CD8⫹ T cells specific for p33 (Fig. 1a). The ability of B cells to present MHC-class II-restricted peptide p13 was expected, because B cells need to be able to present peptides in association with MHC class II molecules to receive T help. As a control, free peptide p33 and p13 were added to the cultures at molar concentrations corresponding to the respective peptide concentrations in ⌯〉cAg-p33 and ⌯〉cAg-p13 preparations (Fig. 1b). Both DCs and B cells presented free peptide p33 as well as p13 added to specific T cells and induced strong cellular proliferation (Fig. 1b). Note that B cells pulsed with 10 and even 100 times lower concentrations of free peptide p13 or p33 induced strong proliferation of specific CD4⫹ T cells (Fig. 2a) or CD8⫹ T cells (Fig. 2b), respectively. Similar results were found when pulsed DCs were cultured with CD4⫹ T cells (Fig. 2c) or CD8⫹ T cells (Fig. 2d). These data indicate that DCs efficiently cross-present peptide p33 to CTLs in vitro, whereas B cells fail to do so. Furthermore, the inability of B cells to cross-present peptide p33 to CD8⫹ T cells was due to a processing defect, because free peptide p33 was efficiently presented to T cells. Ag-specific B cells fail to cross-present peptide in vivo Next, we assessed the cross-presentation of Ag by B cells in vivo. In a first experiment, we quantified the percentage of B cells and
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FIGURE 3. A lower proportion but higher number of B cells than DCs interact with VLPs. Mice were immunized with 100 g of Alexa Fluor 488-VLPs i.v. a, Percentage of Alexa Fluor 488-VLP⫹ MZ B cells, FO B cells, and DCs in the spleen 1 day after immunization. b, Percentage of total MZ B cells, FO B cells, and DCs in the spleen. c, Total number of Alexa Fluor 488⫹ MZ B cells, FO B cells or DCs in the spleen. One representative experiment of four is shown.
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DCs that take up VLPs. For this purpose, we labeled VLPs with Alexa Fluor 488 and immunized mice i.v. with 100 g of labeled VLPs. Surprisingly, a four times higher percentage of MZ B cells captured Alexa Fluor 488-labeled VLPs than FO B cells (Fig. 3a). FIGURE 6. Molecules of the equivalent Cy5 in relation to the geometric mean of Cy5⫹ beads. Rainbow calibration particles display defined amounts of fluorochromes. The figure shows the relation between the fluorescence of DCs and B cells that had taken up particles and the molecules of the equivalent Cy5 of the particles. a, DCs (5 ⫻ 105) or B cells (not shown) were incubated with 5 ⫻ 105 beads for 60 min at 37°C. Cells were washed and analyzed by FACS. The peaks reflect the fluorescence intensity of a single particle taken up by DCs. b, The mean fluorescence of the peaks shows a linear correlation to the molecules of the equivalent Cy5 of the beads. Thus, the fluorescence intensity of these beads can be compared with that of APCs that had taken up VLP-Cy5 and used to determine the number of fluorochromes bound to VLPs taken up by APCs. One representative experiment of three is shown. MFI, Mean fluorescence intensity; SSC, side scatter.
FIGURE 5. DCs efficiently cross-present peptide p33 after in vivo interaction with VLPs. a, C57BL/6 mice were immunized with 100 g of HBcp33Alexa Fluor 488 i.v. and spleens were dissected 24 h postimmunization. Alexa Fluor 488⫹ DCs and Alexa Fluor 488⫺ DCs were sorted and 104 DCs were cultured with 105 CFSE-labeled TCR transgenic T cells. TCR transgenic cells were analyzed four days later by gating on CD8⫹ T cells. b and c, Cocultures with Alexa Fluor 488⫺ DCs (b) and DCs from nonimmunized mice (c) were used as control. One representative experiment of four is shown.
However, because FO B cells are a dominant cell population in the spleen, outnumbering both MZ B cells and DCs (Fig. 3b), they nevertheless were the dominant cell population interacting with VLPs in vivo (Fig. 3c). Because only a low percentage of FO B cells interacted with VLPs, we wanted to only use cells that had interacted with the VLPs for the stimulation of T cells. To do this, we immunized mice with labeled Alexa Fluor 488⫹ HBcAg-p13 or Alexa Fluor 488⫹ HBcAg-p33, and 1 day later FO B cells and MZ B cells as well as DCs were isolated from the spleen by MACS purification. Alexa Fluor 488⫹ cells were subsequently sorted by
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FIGURE 4. B cells fail to cross-present peptide p33 after in vivo interaction with VLPs. C57BL/6 mice were immunized with 100 g of HBcAg-p13Alexa Fluor 488 or HBcAg-p33-Alexa Fluor 488 i.v. and spleens were dissected 1 day later. Alexa Fluor 488⫹ and Alexa Fluor 488⫺ MZ B cells and FO B cells were sorted and 104 MZ B cells or 5 ⫻ 104 FO B cells were cultured with 10-fold more CFSE labeled p13-specific CD4⫹ TCR transgenic T cells (a) or p33-specific CD8⫹ TCR transgenic T cells (b). TCR transgenic cells were analyzed by FACS 4 days later by gating on CD4⫹ or CD8⫹ T cells. One representative experiment of two is shown. FSC, Forward scatter; SSC, side scatter.
The Journal of Immunology
FIGURE 7. B cells and DCs capture essentially similar numbers of VLPs in vivo. C57BL/6 mice were immunized with different amounts of Qb-Cy5 i.v. Twenty-four hours later DCs (F) and B cells (⽧) were purified from spleen and analyzed by FCM. The MFI of Cy5⫹ DCs and B cells were quantified, and the number of VLPs captured by the respective cells was determined as shown in Fig. 6. Note that VLPs have bound 520 Cy5 molecules on average. One representative experiment of three is shown.
B cells and DCs efficiently capture VLPs in vivo To determine whether B cells may be inferior in capturing large numbers of VLPs compared with DCs, we quantified the number of VLPs taken up by DCs and B cells. To do this we made use of rainbow calibration particles displaying defined amounts of fluorochromes. Thus, the fluorescent intensity of these beads could be compared with that of APCs that had taken up VLP-Cy5 and used to determine the number of VLPs taken up by APCs. To calibrate the system, B cells and DCs obtained from spleens of naive mice were incubated in vitro at 37°C for 1 h with the rainbow calibration particles and analyzed by FCM. To ensure that one APC only took up one single bead, we cultured APCs with beads at a ratio of APC to beads of 1:1, 3:1, and 10:1. At a higher bead to APC ratio more APCs took up beads. The mean fluorescence of each DC population, however, was the same for all three ratios (Fig. 6a). Thus, although more APCs take up beads if more beads are available, on average they take up only one single bead. In Fig. 6b the relation between the fluorescence of a DC that had taken up a particle in vitro and the number of molecules of the equivalent Cy5 of the particles are shown. The bead uptake by B cells looked similar to the bead uptake for DCs (data not shown), and the correlation between the fluorescence of B cells and number of molecules of the equivalent Cy5 of the particles was also similar to that of DCs (data not shown). These linear correlations were then used in subsequent experiments to calculate the number of Cy5-VLPs taken up by APCs. To do this, mice were immunized with 100 g of Cy5-VLPs i.v. 1 day later, B cells and DCs were purified, and the mean fluorescent intensities of these APCs were compared with the molecules of the equivalent Cy5 of rainbow calibration particles. We found that the numbers of VLPs taken up by one APC remained relatively independent of the amount of VLPs used for immunization when ⬎25 g VLPs were used for immunization. B
FIGURE 8. The frequency of B cells and DCs capturing VLPs as a function of dose. C57BL/6 mice were immunized with indicated amounts of Qb-Cy5. The next day B cells and DCs were purified from splenocytes and the percentage of B cells (dark gray bars) and DCs (light gray bars) that had captured Qb-Cy5 was measured by FCM. One representative experiment of two is shown.
cells took up an average of ⬃39 VLPs, whereas a DC took up ⬃122 VLPs (Fig. 7). In contrast to the number of VLPs captured per APCs, which was relatively dose independent, the frequency of VLP⫹ APCs was clearly dose dependent (Fig. 8) up to a dose of 100 g but did plateau at higher doses.
Discussion The main function of B cells is the production of Abs. In recent years, however, it has become evident that B cells may have additional functions, including the induction and/or maintenance of T cell responses (31). In addition, B cells have been shown to be important producers of regulatory cytokines, and such regulatory B cells may be able to blunt inflammatory T cell responses (32). It was therefore interesting to address the question of whether the function of B cells can be extended to cross-presentation and cross-priming. This could be a potentially important function of B cells, because cross-priming has been established as a crucial mechanism for the induction of CD8⫹ T cell responses (33). In the present study, we assessed the ability of B cells to present VLPderived epitopes to T cells. B cells efficiently presented Ags in association with MHC class II molecules and induced proliferation of CD4⫹ Th cells. This was to be expected, because B cells required cognate Th cells for isotype switching, one of the hallmarks of specific B cell responses (31). In contrast, both MZ and FO B cells failed to cross-present Ags to CD8⫹ T cells. Hence, B cells play a minor role in the induction of specific CD8⫹ T cell responses upon exposure to nonreplicating Ags. As shown previously, DCs were potent at cross-presenting Ags to CD8⫹ T cells, indicating that DCs, probably together with macrophages, are the key cells for cross-priming. Thus, the primary role of B cells is restricted to the induction/maintenance of Th cell responses as well as the production of Abs. An important role for B cells as APCs for the induction of CD8⫹ T cell responses is rendered unlikely by the present data. It has been shown in vitro that B cells cross-present OVA in the presence of the TLR9 ligand CpG (17). It is noteworthy that the VLPs used in the present study are loaded with RNA, a ligand for TLR7/8. Thus, TLR stimulation per se may not be sufficient to induce cross-presentation of Ags. We are currently testing whether VLPs loaded with a TLR9 ligand may be able to cross-present Ags in vivo. B cells may fail to cross-present VLP-derived Ags at several stages for the following reasons: 1) a failure to capture the VLPs; 2) a failure to shuttle them to the endosomes; 3) a failure to digest the VLPs within endosomes; or 4) some of the more complex
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FACS and used to stimulate specific T cells. Accordingly, only B cells that had taken up VLPs were used to stimulate T cells. Under these defined conditions, both FO B cells and MZ B cells efficiently presented peptide p13 to specific CD4⫹ T cells (Fig. 4a) while they failed to stimulate peptide p33-specific CD8⫹ T cells (Fig. 4b). As a control, sorted B cells from naive mice were cultured under the same conditions with TCR transgenic CD4⫹T cells and CD8⫹ T cells, respectively (data not shown). In contrast, DCs that had taken up VLPs efficiently presented peptides to specific CD8⫹ (Fig. 5a) as well as CD4⫹ T cells (not shown). In contrast, control Alexa Fluor 488⫺ DCs (Fig. 5b) and DCs from naive mice (Fig. 5c) failed to present Ag to specific CD8⫹ T cells.
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Acknowledgments We thank K. Schwarz for helpful discussions and S. Utzinger and P. Ja¨ger for excellent technical support.
Disclosures The authors have no financial conflict of interest.
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cellular machinery, which is required for cross-presentation of the Ags, may be missing. It is therefore interesting to note that the same B cells efficiently presented VLP-derived peptide p13 to Th cells, demonstrating that they are able to shuttle VLPs to endosomes and proteolytically digest them. Hence, it appears that B cells specifically lack cross-presentation machinery, a feature shared with CD8 ⫺ DCs (34), which also efficiently take Ag up and present on MHC class II molecules but fail to cross-present these Ags on MHC class I molecules (35). The observed failure of B cells to cross-present Ags may be of physiological importance because MHC class I-associated presentation of peptides could lead to the destruction of APCs by CTLs. Thus, B cells with the ability to cross-present Ags would be deleted by CTLs, leading to a failure to generate neutralizing Ab responses. The inability of B cells to cross-present Ags may therefore be a prerequisite for the induction of a protective B cell response. Indeed, it has been proposed that viruses selectively infecting B cells expressing neutralizing Abs may be deleted from the repertoire, precluding the generation of a neutralizing Ab response and facilitating viral persistence (36).
B CELLS FAIL TO CROSS-PRESENT VIRAL PARTICLES
