B cells express TLRs and have strong proliferative and differentiation responses to TLR stimulation in vitro
). Therefore, it seems likely that B-cell TLRs contribute importantly to antibody responses in vivo
, but dendritic cells, macrophages, and other cell types also express TLRs, so the relative contributions of different cell types to antibody responses is not immediately apparent. Investigators have used several different experimental approaches to address this issue. One approach has been to use adoptive transfer of B cells of different genotypes into B-cell deficient mice (for example, μMT or JH
T mutant mice) and compare antibody responses. In several reports using this approach, Myd88−/−
B cells made decreased antibody responses compared to wild type B cells, indicating an important role for TLR signaling in B cells (15
). This approach has the potential disadvantage that the normal architecture of secondary lymphoid organs is dependent on the lymphotoxin-β that is produced by B cells and therefore the recipient mice do not have a normal lymphoid structure of well separated T-cell zones and B-cell follicles (17
). Moreover, transfer of B cells into such mice does not correct this anatomical alteration. In one case, however, adoptive transfer of Ig transgenic wildtype or Myd88−/−
B cells into wildtype recipients was used, and a requirement for MyD88 for production of the transgene-encoded antibody was seen(18
). Taken together, these experiments demonstrate that B-cell TLRs are capable of enhancing antibody responses, and this is true of mice with normal lymphoid architecture in at least some circumstances.
A related approach is the use of mixed bone marrow chimeric mice in which one source of bone marrow is a B-cell-deficient genotype and the other source of bone marrow, which is the origin of all of the B cells, is wildtype or mutant for Myd88
or a particular TLR. In these chimeric mice, the majority of other cell types are normal, but the B cells are all derived from a particular mutant genotype, so a defect in the response is presumably due to the genetic alteration in the B-cell compartment. Experiments using this approach in the context of bacterial infection with Salmonella enterica
serovar Typhimurium have demonstrated that a protective T-helper 1 (Th1) response requires MyD88 signaling in B cells (19
), as does proper regulation of the innate response to the bacterial infection (20
). In both cases, these alterations were ascribed to a role of B cells in producing cytokines, rather than an effect on antibody production.
Studies in human have largely focused on using TLR agonists as adjuvant in vaccine studies (3
). In addition, there have been in vitro
studies examining the role of human B-cell maturation state in responsiveness to TLR stimulation (2
). While immature, transitional B cells and naive recirculating B cells do exhibit some responses to TLR ligands, especially to CpG-containing oligonucleotides (ODNs), TLR responses are considerably stronger for B cells simultaneously stimulated via the BCR and CD40 or for IgM+
memory B cells (2
), suggesting that physiologically, human B cell TLRs are most likely to promote antibody responses in the context of other signals promoting B-cell activation and/or in responses of IgM+
memory B cells. In addition, in human as in mouse, TLR stimulation of dendritic cells induces their production of cytokines that can promote antibody responses (21
), suggesting an alternative mechanism by which adjuvants based on TLR ligands may contribute to antibody responses.
As a general approach to examine the importance of TLR signaling in particular cell types in mice, we have created a conditional allele of the gene encoding MyD88 by using homologous recombination to place loxP sites on either side of exon 3 of the Myd88
allele). When combined with a CD11c-Cre
is deleted in approximately 98% of conventional dendritic cells in the spleen and other locations examined, in 80% of plasmacytoid dendritic cells, and in low percentages of other cell types (23
), with the exception of several macrophage subpopulations such as alveolar macrophages, which express CD11c and the transgenic Cre at a high level. Similarly, by combination of the Myd88fl
allele with a mb1-Cre
knockin allele (24
), mice are generated that are deficient for MyD88 in at least 98% of B cells. When these mice were immunized intraperitoneally (i.p.) with the protein antigen ovalbumin, either mixed with CpG-containing oligonucleotides (CpG ODNs) or chemically coupled to them, then it was found that the major adjuvant effect of the CpG ODNs was due to TLR signaling in dendritic cells and not in B cells (25
). This observation was surprising in the case of the ovalbumin-CpG ODN conjugate, as it would be expected that the B-cell TLR9 would be exposed to its ligand in this case. Nonetheless, a similar result was obtained with a second soluble protein-CpG ODN conjugate, containing the well characterized ragweed pollen allergen Amb a1. A similar result was also obtained after immunization with purified FliC
flagellin from Salmonella enterica
serovar Typhimurium, which is a ligand for TLR5, and measuring anti-flagellin IgG. In contrast, when MyD88-deficient B cells were adoptively transferred into μMT mice followed by immunization with flagellin, a defect in the anti-flagellin antibody response was seen (15
). The reason for the different results in these two systems is not known but may relate to an effect of the altered lymphoid architecture in the latter experimental system. In any case, the results in the cell type-specific MyD88 knockout mice argue that the adjuvant effect of CpG ODNs on the IgG response to a soluble protein antigen is primarily due to enhanced maturation and/or cytokine production by antigen-presenting dendritic cells. We hypothesize that TLR-stimulated dendritic cells induce robust activation of antigen-specific helper T cells, which then promote extrafollicular and GC antibody responses. These experiments did not distinguish between extrafollicular and GC antibody responses, although similar results were seen at day 7 and day 14 of the response and also in secondary responses (25
), which is consistent with an effect on the GC response but does not rule out an effect on the extrafollicular response.
In contrast to what was seen with soluble protein-CpG ODN conjugates, multivalent virus-like particles (VLPs) incorporating TLR ligands induced a robust antibody response that was enhanced substantially by MyD88 function in B cells (25
). Virus particles typically contain the viral genome inside them, and this nucleic acid can stimulate TLR7 or TLR9, depending on the type of virus. In addition, many virus particles have a highly repetitious structure in which a small number of epitopes are present in a polymeric array on the particle surface and hence have the ability to crosslink many BCRs and induce robust BCR signaling. Virus particles of this type are known to induce a rapid and robust T-cell-independent IgM response together with a T-cell-dependent IgG response (26
). Interestingly, the IgG response to the Qβ bacteriophage virus-like particle (VLP) was increased about 30-fold by inclusion of CpG ODNs inside it compared to an empty VLP mixed with CpG ODNs and coinjected i.p. (25
). The IgG response to VLPs containing nucleic acid was decreased by 1000 fold or more in Myd88−/−
mice compared to wildtype mice. Interestingly, deletion of MyD88 selectively in B cells decreased the IgG anti-VLP response by 30-fold, whereas deletion of Myd88 selectively in dendritic cells had no significant effect (25
). The anti-VLP IgG response was nevertheless highly dependent on the presence of T cells and of dendritic cells, as the response was approximately 1000-fold lower in mice lacking TCRα chain or mice in which dendritic cells were transiently depleted by use of a CD11c-driven diphtheria toxin receptor and injection of diphtheria toxin (25
). Presumably, the dendritic cells were needed to prime antigen-specific T cells, but they did not need to recognize the nucleic acid with their own TLRs to do so. We hypothesize that in this immunization, the dendritic cells received enough cytokines from other cells to promote their maturation. In support of this possible explanation, genetic deletion of the type 1 interferon receptor in this context greatly attenuated the IgG anti-VLP response, indicating that type 1 interferon, possibly produced by plasmacytoid dendritic cells, was responsible for inducing maturation of conventional dendritic cells (25
The ability of MyD88 in B cells to enhance the IgG anti-VLP response was accompanied by a 10-fold increase in the number of GC B cells that were specific for the VLPs (25
), indicating that MyD88 signaling in antigen-specific B cells enhanced their ability to participate in a GC response. These observations appear to be relevant to antibody responses to enveloped viruses as well: wild type mice or dendritic cells-specific Myd88−/−
mice immunized with chemically inactivated influenza virus had equivalent IgG anti-HA responses, whereas the response was decreased fivefold in mice lacking MyD88 selectively in B cells (25
One possible explanation for the different genetic requirements for the IgG responses to ovalbumin and Qβ VLPs is the different physical nature of these antigens, but there could also be something atypical about the Qβ epitopes. To examine these possibilities, we tried to dissociate the Qβ capsid into soluble monomers but were unable to do so. As an alternative approach, we immunized the various mouse strains lacking MyD88 in particular cell types with a soluble form of the cat dander allergen Fel d1 protein mixed with CpG ODNs or with chemical conjugates of VLPs and Fel d1 (26
). Soluble Fel d1 protein mixed with CpG induced a reasonable IgG response that was dependent on MyD88 expression in dendritic cells but was not affected by deletion of MyD88 selectively in B cells (25
). Thus, soluble Fel d1 behaved similarly to ovalbumin and the other low valency soluble protein antigens tested. When Fel d1 was conjugated to Qβ VLPs, it was now a much more potent antigen and, in this context, anti-Fel d1 IgG was substantially enhanced by the presence of MyD88 in B cells and did not require MyD88 expression in dendritic cells (25
). Thus, it is the VLP physical structure that enables TLR7 or TLR9 in the B cell to greatly enhance the magnitude of the GC response, resulting in much higher titers of IgG.
The results obtained with the Fel d1-Qβ VLP conjugates also provided additional insights into the role of polyvalency of these VLPs for enhancement of the IgG response. Two preparations of Fel d1-VLP conjugates with different amounts of Fel d1 attached to the VLPs were compared, and there was a clear correlation between epitope density and the degree of enhancement of the IgG anti-Fel d1 response by B cell MyD88. The particles with a higher Fel d1 epitope density showed a greater fold enhancement of IgG anti-Fel d1 in the presence of B-cell MyD88. The VLPs with a lower density of Fel d1 still showed an enhancement, but to a lesser extent. The reciprocal effect was seen with the anti-Qβ VLP IgG antibodies, presumably because Fel d1 protein coupled to the VLPs masked the Qβ epitopes, effectively decreasing the valency of the Qβ epitopes accessible to the BCRs on the antigen-specific B cells.
The results summarized above lead to a model whereby CpG ODNs can act as adjuvants for a T-cell-dependent antibody response in either of two ways (): (i) dendritic cells can respond to CpG via their TLR9, enhancing their maturation and ability to activate helper T cells, which then provide help for B cells to make antibody responses, or (ii) antigen-specific B cells take up antigen and internalize the attached CpG to late endosomes, where it comes in contact with B cell TLR9 and induces TLR9 signaling (27
), which then stimulates the ability of these B cells to participate in a GC response. In the case of a polyvalent virus particle, the ability to crosslink many BCRs and also induce TLR7 or TLR9 signaling evidently promotes a strong GC response of at least some of the antigen-specific B cells.
TLRs enhance GC IgG responses in two distinct ways
Many questions remain, however. It is unclear whether this synergy between the BCR and intracellular TLRs of the B-cell leading to an enhanced GC response occurs early, when B cells are choosing between extrafollicular and GC fates, or whether it promotes the response of the B cell after it has entered the GC. As described below, it is clear that TLR ligands can also promote extrafollicular antibody responses, so an important question is whether B-cell affinity for antigen influences the choice of fate between extrafollicular response and GC response.
B-cell intrinsic TLR signaling also influences isotype switching of the B cells, promoting class switch to IgG2c and IgG2b and away from IgG1 (25
). While the experiments with Salmonella enterica
serovar Typhimurium infection suggest that this could be an effect of B cells on T cell polarization (19
), there is also evidence to indicate that TLR9 directly induces expression in the responding B cell of the transcriptional activator T-bet, which in turn may promote class switch to IgG2c (32
). At this point, it is not clear which of these mechanisms is more important in vivo
and in situations where T-cell help is available.