Long-term antiviral antibody responses provide protection from re-infection and recurrence of persistent viruses. Using a polyomavirus (PyV) mouse model, our lab has shown that MyD88-deficient mice generate low levels of virus-specific IgG after the acute phase of infection and that these IgG responses have a skewed isotype distribution with low levels of IgG2a/c. Moreover MyD88-deficient mice have reduced numbers of long-lived plasma cells in the bone marrow. These studies suggest an important role of MyD88-mediated signaling in long-term antiviral responses. Our lab has shown that T cell-deficient mice can also maintain long-term virus-specific IgG responses following PyV infection. The goal of this thesis is to evaluate the role of innate signaling pathways in maintaining serological memory to persistent virus infection and to elaborate on how long-term antiviral responses can be maintained in an immunocompetent or partially immune compromised, T cell-deficient host.
Regarding T cell-dependent B cell responses, I set out to investigate the upstream and downstream components of the MyD88-mediated pathways required for normal antibody isotype and long-term humoral responses.
IgG2a is a predominant immunoglobulin isotype in most virus infections. Wild type mice, in response to PyV infection, primarily induce antiviral IgG2a with some IgG1. MyD88-deficient mice in response to PyV infection display attenuated levels of virus-specific IgG2a, but normal levels of IgG1. Using Unc93B1 mutant mice (3d mice), which are defective in TLRs 3, 7 and 9 signaling, I show that 3d mice also generated low levels of virus-specific IgG2a following PyV infection. Studies in individual TLR3-/-, TLR7-/- or TLR9-/- mice displayed PyV-specific IgG2a responses similar to wild type responses. TLR7 and TLR9 double deficient mice generated similar skewed antibody isotype responses, where virus-specific IgG2a was reduced compared to wild type mice. This shows that TLR7 and TLR9-MyD88 mediated pathways are important in regulating IgG2a responses during a PyV infection.
To investigate what components downstream of MyD88 are involved in mediating IgG2a responses, I worked with IRF5-deficient mice. IRF5 is a transcription factor that is activated upon stimulation of TLR7 or TLR9-MyD88-mediated pathways. Moreover, IRF5-deficient mice cannot generate autoantibodies specifically of the IgG2a isotype in a mouse lupus model, suggesting that IRF5 plays an important function in mediating class switching to IgG2a. In vitro studies where IRF5-/- B cells were stimulated with TLR7 or TLR9 ligands also generated low levels of γ2a germ-line transcripts, suggesting a B cell-intrinsic role for IRF5 in regulating γ2a germ-line transcription. PyV infection of IRF5-deficient mice resulted in similar skewed isotypes as observed in MyD88-deficient and 3d mice. To investigate a B cell-intrinsic role for IRF5 in regulating IgG2a responses in vivo upon PyV infection, I transferred IRF5-/- B cells and WT T cells into RAG KO mice prior to infection and compared the responses of these mice with mice reconstituted with wild type B6 B and T cells. Diminished numbers of IgG2a+ B cells and reduced levels of virus-specific IgG in mice reconstituted with IRF5-/- B cells were seen compared to mice reconstituted with wild type B cells.
Regarding the defect in long-term IgG production in MyD88-/- mice upon PyV infection, I conducted studies in IRF5-/-, 3d, single TLR3-/-, TLR7-/-, TLR9-/- and TLR7/9 double deficient mice. These studies reveal an important and redundant role for TLR7- and TLR9-MyD88 signaling in maintaining long-term anti-PyV IgG responses. To determine how MyD88 signaling affects the generation of long-lived plasma cells and memory B cells, I investigated germinal center (GC) responses in MyD88-deficient mice. A defect in GC B cell numbers is observed in MyD88-deficient mice after the acute phase of infection. The GC reaction is essential for the generation and maintenance of long-lived plasma cells and memory B cells. T follicular helper (TFH) cells are absolutely required to generate normal GC. l found reduced numbers of TFH cells in MyD88-deficient mice. Lower numbers of T FH cells suggests that poor T cell help may contribute to the diminished number of GC B cells. However, interaction with B cells is required for the formation of fully differentiated TFH cells. Along with B cell function, MyD88 signaling can affect T cell and dendritic cell function as well. Thus, it is not clear at this point whether the requirement for intact MyD88 signaling for the formation and maintenance of long-term B cell populations is completely B cell-intrinsic.
Some viruses can induce T cell-independent B cell responses, perhaps due to their complex arrays of repetitive antigenic epitopes on virions, coupled with the induction of innate cytokines. Nevertheless, T cell help is usually necessary for generating long-term antibody responses in the form of long-lived plasma cells and memory B cells. In contrast, our lab has found that T cell-deficient mice infected with PyV develop long-lasting, protective antiviral IgG responses. I questioned whether these mice could generate TI B cell memory cells or long-lived plasma cells. I show that long-lasting anti-PyV antibody in T cell-deficient mice was not due to the presence of long-lived plasma cells or memory B cell responses.
TCRβδ deficient mice, which lack both CD4 and CD8 T cells, had ~10 a times higher virus load persisting in various organs. Therefore, I hypothesized that the high level of persistent PyV antigen, in completely T cell-deficient mice, may activate naïve B cell populations continuously, thereby maintaining the long-lasting IgG responses. Prior to PyV infection, T cell-deficient mice received wild type CD8 T cells, which reduced PyV loads, and this was associated with decreased levels of antiviral serum IgG over time. As in TCRβδ deficient mice, high PyV loads were detected in the bone marrow, which is the site for B cell lymphopoiesis, I questioned how B cells develop in the presence of PyV antigen and still stay responsive to PyV, generating long-term antiviral IgG responses in the periphery. Studies have shown that self-antigens that trigger both B cell receptor signaling and TLR-MyD88 signaling pathways in the bone marrow lead to the breaking of B cell tolerance and production of autoantibody in the periphery. Thus, we hypothesized that high PyV levels in the bone marrow signal through both B cell-receptors and TLRs, allowing continuous antiviral antibody production by B cells. Using mice that are deficient in T cells and MyD88 signaling, I found that PyV-specific TI IgG levels gradually decreased, supporting this hypothesis. Thus, high PyV loads and innate signaling together can break B cell tolerance. During a persistent virus infection this can result in sustaining long-term protective T cell-independent IgG responses.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1637 |
| Date | 21 June 2012 |
| Creators | Raval, Forum M. |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved., select |
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