Toll-like receptor 7 controls the anti-retroviral germinal center response

Abstract

The development of vaccines that can enhance immunity to viral pathogens is an important goal. However, the innate molecular pathways that regulate the strength and quality of the immune response remain largely uncharacterized. To define the role of Toll-like receptor (TLR) signaling in control of a model retroviral pathogen, Friend virus (FV), I generated mice in which the TLR signaling adapter Myd88 was selectively deleted in dendritic cell (DC) or in B cell lineages. Deletion of Myd88 in DCs had little effect on immune control of FV, while B cell specific deletion of Myd88 caused a dramatic increase in viral infectious centers and a significantly reduced antibody response, indicating that B cell-intrinsic TLR signaling plays a crucial role, while TLR signaling in DCs is less important. I then identified the single-stranded RNA sensing protein TLR7 as being required for antibody-mediated control of FV by analyzing mice deficient in TLR7. Remarkably, B cells in infected TLR7-deficient mice upregulated CD69 and CD86 early in infection, but failed to develop into germinal center B cells. CD4 T cell responses were also attenuated in the absence of TLR7, but CD8 responses were TLR7 independent, suggesting the existence of additional pathways for detection of retroviral particles. Together these results demonstrate 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.

Figure 1. B cell-intrinsic TLR signaling is required for control of FV.

Mice that were heterozygous (+/−) or knockout (−/−) for Myd88, and mice with Myd88 deleted in DCs (CD11c-Cre/Myd88^flox/−) or B cells (CD19-Cre/Myd88^flox/−) were infected with FV, and viral infectious centers were determined at 14dpi by measuring focus-forming units per spleen. Each bar represents an average of 6–10 mice.

Figure 2. B cell-intrinsic TLR signaling is required for an antibody response to FV.

Myd88 germline heterozygous mice (+/−) and mice with Myd88 deleted in DCs (CD11c-Cre/Myd88^flox/−) or B cells (CD19-Cre/Myd88^flox/−) were infected with FV, and FV-specific Ig levels in the serum determined at 14 dpi by staining of envelope expressing cells and flow cytometry. Units represent the mean fluorescence intensity (MFI) of cells stained with diluted serum, followed by allophycocyanin conjugated. anti-mouse IgG(H+L). Each dot represents an individual mouse.

Figure 3. TLR7 is required for an antibody response to FV.

A. Wild-type (WT) or TLR7 deficient mice (Tlr7 KO) were infected with FV. At 14 dpi, spleens from infected mice were harvested and the number of infectious focus-forming units per spleen was measured by focus-forming assay. Each bar represents the average of 6 mice. B. The levels of FV-specific Ig in the serum of infected and uninfected mice were measured by staining of an envelope expressing cell line and flow cytometry. Units represent the mean fluorescence intensity (MFI) of cells stained with diluted serum, followed by allophycocyanin conjugated. anti-mouse IgG(H+L). Each dot represents an individual mouse. The average is shown as a horizontal black bar.

Figure 4. TLR7 and B cell-intrinsic Myd88 are required for a neutralizing antibody response.

Serum from infected wild-type (WT) or TLR7-deficient mice (Tlr7 KO) (A), as well as mice with Myd88 deleted in B cells (CD19-Cre/Myd88^flox/−) (B), was diluted over a wide range and incubated with a defined quantity of infectious FV. The effect of the serum on the infectivity of the sample was then measured by focus-forming assay. The neutralizing antibody (NAb) titer was defined as the maximum serum dilution that reduced the infectivity of the virus sample by at least 50%. Each dot represents an individual mouse.

Figure 5. B cells upregulate CD86 and CD69 independently of TLR7.

Wild-type (WT) or TLR7-deficient mice were infected with FV. At 7 dpi, splenocytes were harvested and the levels of CD86 (A) and CD69 (B) expression determined for B cells (CD19+) by flow cytometry. A representative flow cytometry plot for the CD19+ gated cells for each group is shown. Each bar of the histogram represents the average of 5–6 mice. Control ‘naïve’ mice were inoculated with an equivalent dose of spleen homogenate without FV.

Figure 6. TLR7 is required for the development of germinal center B cells during FV infection.

Wild type (WT) or TLR7-deficient mice (Tlr7 KO) were infected with FV. At 14 dpi, splenocytes were harvested and analyzed for the presence of germinal center B cells by flow cytometry. The percentage of B cells that were (A) CD19+, GL7+ or (B) CD19+, IgM− were measured and plotted. A representative plot for the CD19+ gated cells for each group is shown. Each bar of the histogram represents the average of 5–6 mice.

Figure 7. Histological analysis of germinal center formation during FV infection.

Spleens were harvested from FV-infected wild-type (WT) or TLR7 deficient (Tlr7 KO) mice at 14 dpi, embedded in paraffin, and sectioned onto glass slides. Sections were stained with hematoxylin and eosin. Germinal centers (GCs) were identified by their characteristic staining pattern of a paler central circular area (indicated by arrows) surrounded by a darker mantle zone and marginal zone. Five mice for each group were analyzed, and four non-consecutive sections were analyzed for each mouse.

Figure 8. TLR7 is required for IFNγ expression in CD4 T cells.

Wild-type (WT) and TLR7 deficient (Tlr7 KO) mice were infected with FV. At 14 dpi, splenocytes were harvested, restimulated for 3 h with PMA/ionomoycin, and analyzed by flow cytometry for intracellular expression of IFNγ in (A) CD4 T cells (TCRβ+, CD4+) and (B) CD8 T cells (TCRβ+, CD8+). A representative flow cytometry plot for the T cell gated cells for each group is shown. Each bar of the histogram represents the average of 5–6 mice.

Figure 9. The FV-specific CD8 T cell response is independent of TLR7.

Wild-type (WT) or TLR7 deficient mice (Tlr7 KO) were infected with FV. At 14 dpi, splenocyte suspensions were analyzed by flow cytometry with an H2D^b-GagL tetramer (A), or with an antibody to detect PD1 expression (B). Flow cytometry plots are gated on TCRβ+, CD8+ T cells. Each bar of the histogram represents the average of 5–6 mice.

Figure 10. B cell-intrinsic Myd88 regulates germinal center B cells but not CD4 T cell IFNγ expression.

Heterozygous mice (CD19-Cre/Myd88^+/−) or mice with B cell specific deletion of Myd88 (CD19-Cre/Myd88^flox/−) were infected with FV, At 14 dpi, splenocytes suspensions were analyzed by flow cytometry for GL7 expression in CD19+ B cells (A) and for IFNγ expression in CD4+, TCRβ+ T cells (B). Each bar represents the average of four mice.