Studies in mouse models of retroviral infection have provided new insights into the mechanisms by which the innate immune system regulates the adaptive immune response to and control of retroviral infections such as HIV. A series of experiments by Edward P. Browne, Ph.D., at the Massachusetts Institute of Technology (MIT) Koch Institute for Integrative Cancer Research, indicates that the vertebrate immune system detects retroviruses in vivo via TLR7 and that this pathway regulates a key checkpoint controlling development of germinal center B cells.
Reporting in PloS Pathogens, Dr. Browne reports that the findings suggest manipulating TLR7 and its signaling pathway in B cells could be a viable strategy for enhancing immunity to viruses. His published paper is titled “Toll-like Receptor 7 Controls the Anti-Retroviral Germinal Center Response.”
Innate sensors of microbial infection fall into three basic classes, Dr. Browne explains: NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and Toll-like receptors (TLRs). Research has identified the innate immune system as an important contributor to the activation and fine-tuning of adaptive immune responses to pathogens, but the mechanisms by which innate immune response pathways are involved is, for most pathogens, still unclear.
More specifically, investigating the role of innate sensing pathways in regulating the adaptive immunity to HIV-1 has been hampered by the lack of a genetically modifiable animal model for HIV-1 infection, Dr. Browne continues. Friend virus (FV), however, is a murine gammaretrovirus that has been widely used to investigate basic principles of retroviral immunology.
FV consists of a replication-competent virus (F-MLV) and a defective spleen focus-forming virus (SFFV). In mice, FV infection leads to an acute viremia that peaks at 7–8 days post infection, but is brought under control by potent CD8 T cell and B cell responses by day 14 dpi in resistant strains of mice. Mice deficient in the TLR signaling adapter Myd88 have a markedly reduced ability to effect immune control of FV, and mount a reduced antibody response to the virus, indicating that a member of the TLR family is involved in antibody-mediated immune control.
Dendritic cells (DCs) are required for the development of FV-specific antibodies, and express a number of different TLRs. Murine B cells also express high levels of a number of TLRs. To investigate role of TLR signaling in controlling FV, Dr. Browne generated mice in which Myd88 was selectively deleted in either DC or B-cell lineages.
Supporting previous research findings, the mouse models confirmed that germline deletion of Myd88 led to much higher numbers of viral foci by two weeks. However, while mice with Myd88 selectively deleted in DCs (CD11c-Cre/Myd88flox/−) showed only a small increase in infectious centers, animals in which Myd88 was deleted selectively in B cells had dramatically higher numbers of infectious centers than nondeleted mice and were much less able to control FV infection.
To investigate whether Myd88 deletion in B cells directly affected the antibody response, or whether the role of Myd88 in B cells was antibody-independent, total FV-specific Ig levels in the serum of infected mice with Myd88 deleted in DCs or in B cells was measured at 14 days post infection. This confirmed that while animals with Myd88 deleted in DCs only demonstrated small reductions in antibody titers in comparison with their control littermates, mice with Myd88 deleted in B cells showed a strong reduction in antibody levels. “These data demonstrate that B cell-intrinsic Myd88 plays a direct role in the regulation of the antibody response to FV,” Dr. Browne states.
Myd88 mediates signaling from all members of the TLR family except TLR3, as well as signaling from IL1R and IL18R. And because retroviruses have single-stranded RNA genomes that could stimulate TLR7, and TLR7 is abundantly expressed in B cells, a reasonable hypothesis is that TLR7 regulates the antibody response to FV.
Indeed, while wild-type mice infected with FV demonstrated only low numbers of infectious centers at 14 days, TLR7-deficient mice demonstrated much higher numbers of infectious centers. Moreover, while wild-type mice exhibited a robust serum FV-specific Ig response to FV at 14 days post infection, TLR7-deficient mice exhibited a profoundly attenuated antibody response to FV. This observation, combined with the finding that animals with Myd88 deleted in B cells also showed strong reductions in antibody levels, indicated that a lack of TLR7 signaling is likely to account for the defect observed in Myd88 knockout mice.
Supporting evidence came from analyses that showed that while serum from wild-type mice was able to potently neutralize FV samples in vitro, serum from TLR7-deficient mice or mice with Myd88 deleted in B cells exhibited significantly attenuated neutralizing ability.
Subsequent analysis of the development of germinal center B cells during FV infection demonstrated that B cells in wild-type mice had upregulated the germinal center marker GL7, and there was a significant population of non-IgM B cells, “indicating that class switching and germinal center reactions were occurring,” Dr. Brown continues. By contrast, in TLR7-deficient mice the levels of GL7+ and non-IgM B cells were significantly lower. Indeed, wild-type infected mice exhibited abundant germinal center structures in the spleen by 14 days post infection, while in TLR7-deficient mice germinal centers were significantly reduced.
To examine whether TLR7 was required for T cell responses during FV infection, wild-type or TLR7-deficient mice were infected with FV, and the expression of IFNγ in CD4 and CD8 T cells measured at 14 days post infection. Wild-type infected mice strongly upregulated IFNγ expression in CD4 and CD8 T cells, indicating a robust T cell response to FV. However, Results from TLR7-deficient mice showed that IFNγ upregulation in CD4 T cells was dependent on TLR7, while IFNγ expression in CD8 T cells and the development of GagL-specific CD8 T cells were independent of TLR7.
Antibody responses to viral infection are regulated by a network of cells and signaling pathways, including dendritic cells, CD4 T cells, and B cells, Dr. Browne continues. Naïve B cells interact with viral antigen through the B cell receptor (BCR), and with CD4 T cells through cell surface molecules including CD40. Activation initially results in the upregulation of markers such as CD69 and CD86. Interestingly, B cells in both wild-type and TLR7-deficient mice infected with FV upregulated expression of CD69 and CD86, indicating both that B cell activation, and that activation of B cells in vivo, doesn’t require TLR7.
This suggested that TLR7 may regulate a later step in the antibody response: possibly the development of germinal center B cells, given the previous findings that FV infection in TLR7-deficient mice led to reduced numbers of germinal center structures and lower than expected levels of GL7+ and non-IgM B cells.
Continuing along this line of investigation, Dr. Browne also found that GL7 expression was significantly reduced in infected mice with B cell-deleted Myd88, while IFNγ expression was unaffected. This demonstrated that B cell-intrinsic Myd88 is required for germinal center responses but not for IFNγ expression in CD4 T cells. “Thus it is possible that TLR7 signaling in a cell lineage other than B cells regulates CD4 T cell expression of IFNγ,” he writes.
Although the issue of whether TLRs contribute to B cell responses has been controversial, “The results presented in this study strongly support the hypothesis that B cell-intrinsic TLR signaling can regulate antibody responses, and also suggest that this process specifically regulates the development of germinal centers during viral infection.” These findings, combined with those from previous research, suggest that a requirement for B cell-intrinsic TLR7 signaling may be a general feature of the antibody response to viruses with RNA genomes.
“Determining how TLR7 regulates the development of germinal center B cells during viral infection should be an area of further investigation,” he concludes. “The finding that TLR7 and B cell-intrinsic Myd88 regulate the antibody response to a retroviral pathogen by promoting the development of germinal center B cells has potentially significant implications for understanding mechanisms behind the antibody response to human retroviral pathogens such as HIV-1 and HTLV-1.”