In this study, we obtained evidence that TLR ligands can engage TLR signaling in both B cells and DCs resulting in augmentation of TD antibody responses, and that a dominant determinant of the cellular pathway engaged and the extent of augmentation is the physical context in which the TLR ligand is presented to the immune system. In particular, TLR-MyD88 signaling in DCs but not in B cells was required to enhance the IgG response to a soluble protein antigen either mixed with a soluble TLR9 ligand or chemically conjugated to it. In contrast, VLPs that contained a TLR9 or TLR7 ligand or inactivated influenza virus were able to engage TLR and MyD88 signaling within the antigen-specific B cells to strongly enhance a primary germinal center response.
The selective utilization of B cell TLR signaling by TLR7 or TLR9 ligands contained within VLPs but not by soluble TLR ligand or by TLR ligand chemically coupled to soluble antigen for the enhancement of T cell-dependent IgG production is the most remarkable finding of this study. TLR ligands are potent activators of murine B cells in vitro (Bekeredjian-Ding and Jego, 2009
) (Shlomchik, 2009
), and thus, it has been presumed that TLRs in B cells play an important role in promoting antibody responses in vivo. Indeed, Pasare and Medzhitov provided evidence that B cell TLRs were responsible for enhancing the magnitude of the TD antibody response. Subsequent studies have either provided evidence for a role for TLRs in promoting antibody responses or failed to see a role, depending on the circumstance examined, illustrating that much needs to be learn about the role of TLRs in antibody response. We examined antibody responses to several soluble protein antigens with different apparent immunogenicity mixed a TLR ligand as adjuvant, and found that deletion of Myd88 selectively in B cells did not decrease the magnitude of the IgG responses. In addition, when we used a TLR ligand that had been physically-linked to an antigen to increase delivery of the TLR ligand to B cell TLRs, we observed a contribution of TLR signaling in B cells only to the early IgM response but not to the TD IgG response. Instead, in all these circumstances, we found that MyD88 signaling in DCs accounted for most of the effect of TLR stimulation, suggesting that signals from helper T cells are sufficient to promote a strong B cell response, provided the CD4 T cells are activated by TLR-stimulated DCs. Similarly, MyD88 signaling in DCs was recently shown to be important for the IgA response to intranasal immunization (Bessa et al., 2009
). Our results argue against the hypothesis that proteins of low immunogenicity enable TLRs to boost the response, whereas highly immunogenic proteins, such as haptenated proteins, do not (Palm and Medzhitov, 2009
). Rather, regardless of the strength of the TD antibody response to a soluble protein antigen, it was MyD88 in DC, not B cell that boosted the response.
In contrast to what was seen with soluble protein antigens and soluble TLR ligands, we found that similar TLR ligands contained within VLPs had the unique ability to engage TLR signaling in B cells to strongly enhance TD IgG production. The magnitude of the IgG response to VLPs was typically approximately 30-fold greater in wild type mice compared to mice with deletion of Myd88 selectively in B cells. The low IgG response in B cell-Myd88−/− mice was similar to what was seen upon immunization of wild type mice with VLPs depleted of nucleic acid and mixed with free CpG ODNs, and this latter response was not affected by deletion of Myd88 in B cells. Thus, incorporation of the TLR ligand within the VLP was necessary for enabling B cell TLR signaling to boost the IgG response. This ability of VLPs to induce a robust IgG response by engaging B cell TLR signaling was a property of the physical form of the VLPs, not the inherent immunogenicity of the protein epitopes exposed on the particle. For example, a weakly immunogenic protein, the cat allegen Fel d1, when used as a soluble antigen mixed with CpG ODN behaved like the other soluble protein antigens tested in that the IgG response was decreased by deletion of Myd88 in DCs but was unaffected by deletion of Myd88 in B cells. In contrast, when Fel d1 was conjugated to the VLPs containing nucleic acid ligands for TLRs, these VLPs induced a robust anti-Fel d1 IgG response that benefited substantially from Myd88 expression in B cells.
Earlier studies designed to distinguish the contribution of B cell MyD88 from the contribution of MyD88 in other cell types (Pasare and Medzhitov, 2005
) used adoptive transfer of MyD88-deficient B cells into μMT mice, which have deletion of exon encoding the transmembrane domain of IgM and therefore are genetically defective in B cell development. Such mice are known to have substantial alterations in the structure of their secondary lymphoid organs due to the role of B cells in expressing lymphotoxin-β (Chaplin, 2002
). These mice may also have elevated amounts of the cytokine B cell activating factor belonging to the TNF family (BAFF) due to B cell lymphopenia, and either of these changes may alter the timing or other aspects of the B cell response. Thus, it is possible that the activation of CD4 helper T cells is suboptimal in this experimental system. In this respect, the use of Cre-lox technology for deletion of MyD88
selectively in B cells in situ, as was done in the experiments presented here, has obvious advantages.
The mechanism by which virus particles engage B cell TLR7 or TLR9 signaling to enhance the TD IgG response deserves further investigation, but the results presented here provide some insights. The major component of the TD response that was stimulated was the germinal center response, which is responsible for producing high affinity antibody, long-lived plasma cells, and memory B cells (King et al., 2008
). Moreover, the magnitude of the enhancement seen in wild type mice compared to B cell-Myd88−/−
mice was consistently affected by the epitope density on the VLPs: unmanipulated VLP-ssRNA or VLP-CpG exhibited the strongest enhancement by B cell MyD88, whereas low density Fel d1 conjugation resulted in a lower enhancement of the anti-Fel d1 IgG response compared to the anti-Qβ IgG response. When Fel d1 was conjugated onto the VLPs at a higher density, then a larger enhancement of the anti-Fel d1 IgG response was seen. Thus, there was a consistent trend toward greater enhancement by B cell MyD88 when the epitope density on the surface of the VLP was higher, indicating that greater engagement of BCRs and presumably stronger BCR signaling enabled B cell TLR enhancement of the response. Another feature of VLPs that may enable B cell MyD88 to enhance the antibody response may be their likely ability to induce strong TLR signaling by virtue of the amount of TLR7 or TLR9 ligand contained within a single VLP. For example, the VLP-CpG contained an estimated 60–80 ODNs per virus particle. Additionally, it is possible that soluble and particulate antigens are processed differently in B cells after being taken-up by BCRs; the latter may be more efficiently trafficked to the location where nucleic acid-recognizing TLRs encounter their cognate ligands. Similar cell biological contributions to the nature of TLR responses have been described in plasmacytoid DCs (Honda et al., 2005
It is striking that particles with two of the characteristic features of most virus particles, and lacking in many other types of particles, namely high density of particular epitopes and internal ligands for TLR7 or TLR9, uniquely engage this mechanism to strongly enhance the germinal center IgG response. Given the well-established function of neutralizing antibodies for defense against many viruses (Zinkernagel et al., 2001
), this property of B cells is likely to be an evolutionary advantageous regulatory mechanism to promote rapid and robust production of high affinity antibodies to protect against virus infection. This phenomenon may also explain the common observations that immunization with attenuated or inactivated complete pathogens typically induces stronger antibody responses than immunization with the components of pathogen mixed with a TLR ligand or with other commonly used adjuvants. For example, Geeraedts et al have reported that vaccination with inactivated whole H5N1 influenza virus induced better protective antibody responses than vaccination with split virus or a viral subunit vaccine (Geeraedts et al., 2008
). Our results indicate that activation of MyD88 signaling in B cells by a TLR ligand presented in the physical context of a virus particle may be an important mechanism to account for the superior immunogenicity of this type of antigen.
The ability of VLPs to promote vigorous IgG responses by TLR signaling in B cells is intriguing in light of previous work implicating B cell TLR signaling in spontaneous production of anti-nuclear antibodies and anti-RNA antibodies, the autoantibodies characteristic of the human autoimmune disease systemic lupus erythematosus (Marshak-Rothstein and Rifkin, 2007
). Based on our results, we propose that the autoantigens in these cases may be exposed to B cells in the form of apoptotic blebs, since many antibodies of this type will bind to those structures (Cline and Radic, 2004
). Apoptotic blebs resemble VLPs in their particulate nature and in their inclusion of a substantial amount of ligands for TLR7 and/or TLR9. Interestingly, the role of MyD88, TLR7 and TLR9 in augumenting spontaneous or induced autoantibody production in a few mouse models has been attributed to promoting extrafollicular antibody responses (Shlomchik, 2009
), whereas we found that it was the germinal center response that was promoted by VLPs and TLR signaling in B cells. Therefore, it would be worth examining the contribution of the germinal center B cell response to spontaneous anti-DNA or anti-ribonucleoparticle autoantibody production in other mouse models of SLE.
In summary, our experiments clearly demonstrate that TLRs of DCs and B cells can both augment antibody responses, depending on the physical nature of the antigen. In particular, immunizations with soluble protein antigens mixed with or conjugated to TLR ligands as the major adjuvant demonstrated a requirement for TLR signaling in DCs but not B cells for a high titer IgG response to soluble protein antigens, whereas immunization with VLPs containing TLR ligands within them had a requirement for B cell MyD88 for a robust IgG response to the repeating epitopes of the VLP. Pathogens such as viruses contain nucleic acid that serves as ligands for TLRs such as TLR7 and TLR9, but it was not previously appreciated that such particles are especially able to engage B cell TLRs to enhance TD antibody responses. Thus, the results presented here suggest that B cells are hard-wired to respond strongly to virus particles and use their TLRs for this purpose, a property that likely has considerable advantage for defense against viral infection. These insights may be useful in the development of more effective vaccines.