Toll-like receptor 7 is required for effective adaptive immune responses that prevent persistent virus infection

Abstract

TLR7 is an innate signaling receptor that recognizes single-stranded viral RNA and is activated by viruses that cause persistent infections. We show that TLR7 signaling dictates either clearance or establishment of life-long chronic infection by lymphocytic choriomeningitis virus (LCMV) Cl 13 but does not affect clearance of the acute LCMV Armstrong 53b strain. TLR7(-/-) mice infected with LCMV Cl 13 remained viremic throughout life from defects in the adaptive antiviral immune response-notably, diminished T cell function, exacerbated T cell exhaustion, decreased plasma cell maturation, and negligible antiviral antibody production. Adoptive transfer of TLR7(+/+) LCMV immune memory cells that enhanced clearance of persistent LCMV Cl 13 infection in TLR7(+/+) mice failed to purge LCMV Cl 13 infection in TLR7(-/-) mice, demonstrating that a TLR7-deficient environment renders antiviral responses ineffective. Therefore, methods that promote TLR7 signaling are promising treatment strategies for chronic viral infections.

Figure 1. TLR7^−/− mice eliminated an acute LCMV Arm infection, but failed to terminate a persistent LCMV Cl 13 infection

TLR7^+/+ (black circles) or TLR7^−/− (shaded circles) mice were infected with 2×10⁶ PFU of LCMV Arm or LCMV Cl 13 i.v. (A) Virus burden in the liver of LCMV Arm and Cl 13 infected mice at specified time points. Circles represent individual mice and the bars indicate the average. (B) LCMV Cl 13 titers were assayed in sera at specified time points. 4–10 mice per group were used per time point and data represents the average ± SEM. *, p < 0.05; **, p < 0.005; ***, p < 0.0005. See also figure S1.

Figure 2. T cells from TLR7^−/− mice have diminished anti-viral functionality that is caused by T cell extrinsic factors

TLR7^+/+ and TLR7^−/− mice were infected with 2×10⁶ PFU of LCMV Arm or Cl 13 i.v. (A) Quantification of LCMV-specific CD8⁺ (LCMV GP_33–41-specific) and (B) CD4⁺ (LCMV GP_67–77-specific) T cells in the spleen by tetramer staining at specified time points. (C) GP_33–41 peptide stimulation of TLR7^+/+ and TLR7^−/− splenocytes harvested on day 9 p.i. with LCMV Arm or Cl 13. The left panel displays representative dot plots of CD8⁺ IFN-γ⁺ cells producing TNF-α and IL-2 with respective frequencies. The right panel exhibits the average frequencies of CD8⁺ IFN-γ only producers, IFN-γ-TNF-α dual producers and IFN-γ-TNF-α-IL-2 triple producers from LCMV Arm and Cl 13-infected mice. (D) Frequencies of intracellular granzyme B⁺ GP_33–41 tetramer⁺ CD8⁺ T cells in the spleens of LCMV Cl 13-infected TLR7^+/+ and TLR7^−/− mice. (E) Quantification of adoptively transferred TLR7^+/+ Thy1.1⁺ TCR tg CD8⁺ T cells (P14) in the spleens of Thy1.2⁺ TLR7^+/+ and TLR7^−/− mice on day 9 p.i. with LCMV Arm or Cl 13 i.v. Frequencies of cytokine producing LCMV GP_61–80 or GP_33–41 peptide stimulated TLR7^+/+ Ly5.1⁺ TCR tg CD4⁺ (SMARTA, F) and Thy1.1⁺ TCR tg CD8⁺ (P14) T cells (G), respectively, in the spleens of Thy1.2⁺ Ly5.2⁺ TLR7^+/+ and TLR7^−/− mice 8 days following infection with LCMV Cl 13 i.v. (A–G) Bar graphs represent the average ± the standard of the mean. 4–5 mice per group. *, p < 0.05; **, p < 0.005; ***, p < 0.0005. See also figure S2.

Figure 3. LCMV-specific CD8⁺ T cells from TLR7^−/− mice have enhanced expression of NIRs

TLR7^+/+ and TLR7^−/− mice were infected with 2×10⁶ PFU LCMV Cl 13 i.v. (A) Normalized PD-1 expression on the surface of GP_33–41 tetramer⁺ CD8⁺ T cells. Normalization of expression was performed by dividing the MFI of PD-1 on GP_33–41 tetramer⁺ CD8⁺ T cells by the MFI of PD-1 on the PD-1⁻ CD8⁺ T cell population. Data represents 3–5 mice per group from 3 separate experiments. Bar graphs represent the average ± SEM. *, p < 0.05, ***, p < 0.0005. (B) Expression of the NIRs on CD44⁺ GP_33–41 tetramer⁺ CD8⁺ T cells from the spleens of TLR7^+/+ and TLR7^−/− mice at day 225 p.i. Histogram data generated from a single TLR7^+/+ mouse that had cleared LCMV-Cl 13 systemically and a representative TLR7^−/− mouse from a group of 3 that were viremic. MFI of specific NIR is displayed on the top right of the panels for each individual mouse.

Figure 4. B cell intrinsic defects in plasma cell formation and the generation of LCMV-specific antibody in TLR7^−/− mice

TLR7^+/+ and TLR7^−/− mice were infected with 2×10⁶ PFU LCMV Cl 13. The frequencies of CD138⁺ (A) and IgM producing (B) splenic plasma cells on day 20 p.i. The left panels are representative dot plots and the right panels are calculated averages ± SEM. (C) The frequencies of splenic germinal center B cells (GL-7⁺ and CD38⁻ based on the B220⁺, CD19⁺, IgD⁻ parent population) were determined at specified time points. (D) Quantification of the number of LCMV-specific IgG secreting B cells from the spleens of TLR7^−/− and TLR7^+/+ mice by ELISpot on day 33 p.i. Serum from LCMV Cl 13 infected TLR7^−/− and TLR7^+/+ mice was harvested at days 35 (E) and 74 (F) p.i. and evaluated by ELISA to calculate the titer of anti-LCMV specific antibody. (G) Frequencies of TLR7^+/+ (Ly5.1⁺ C57Bl/6) and TLR7^−/− (Ly5.2⁺ TLR7^−/−) germinal center B cell formation ion the spleens of bone marrow chimeric mice. The left panel depicts representative histograms; the right panel is the calculated average ± SEM for frequencies. (H) Enumeration by ELISpot of anti-LCMV antibody producing TLR7^+/+ (Ly5.1⁺) and TLR7^−/− (Ly5.2⁺) B cells (CD19⁺, B220⁺) purified by FACS from bone-marrow chimeras. (A–F) Bar graphs represent the average ± SEM. Each group contained 4–5 mice and data are representative of two independent experiments. *, p < 0.05; **, p < 0.005, ***, p < 0.0005. See also figure S3.

Figure 5. Adoptive transfer of TLR7^+/+ memory cells fails to clear LCMV Cl 13 infection from TLR7^−/− mice

TLR7^+/+ and TLR7^−/− mice were infected i.v. with 2×10⁶ PFU LCMV Cl 13. (A) 2×10⁷ purified TLR7^+/+ immune memory splenic B cells were adoptively transferred i.p. into TLR7^+/+ and TLR7^−/− mice on day 15 p.i. Serum virus burden was calculated at specified time points. The ratios listed in the right side of the figure represent the number of non-viremic mice/viremic mice. (B and C) 3×10⁷ total immune memory splenocytes from TLR7^+/+ or TLR7^−/− mice were adoptively transferred as mentioned above. (B) Kinetic analysis of virus titer in the serum. (C) Analysis of virus burden in sera and tissues of recipient mice 73 days post-transfer. Symbols represent individual mice, and black bars signify mean values. (A and B) TLR7^+/+ mice that did not receive cells (black diamonds) were used as controls for normal LCMV Cl 13 clearance. Data points represent the average ± the standard error of the mean. 3–10 mice were used per group per time point. Data are representative of 2–4 independent experiments. See also figure S4.