Slc15a4, a gene required for pDC sensing of TLR ligands, is required to control persistent viral infection

Abstract

Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.

Figure 1. Slc15a4 is required to control chronic viral infection.

Serum and organs were collected at the indicated times post LCMV infection and infectious virus was titered. WT and feeble mice were infected i.v. with 2×10⁶ pfu of the acute LCMV Armstrong (Arm) strain or the persistent LCMV Clone 13 (Cl13) strain (A). Serum was then harvested at the indicated time points for >4 months to enumerate viremia. After Cl13 infection, organs were harvested from WT and feeble mice 5 dpi (B), 8 dpi (C), and 2 mpi (D), homogenized and titered for infectious virus. Individual replicates and means are shown. Feeble, Slc15a4^{feeble/feeble} mice; l.o.d., limit of detection. Representative data of 2 independent experiments are shown; n> = 5 per group. Unless marked, p>0.05 between WT and feeble and not statistically significantly different.

Figure 2. Slc15a4 is required for antigen specific T cell responses in vivo.

8 days after LCMV Cl13 infection, splenocytes from WT and feeble mice were cultured ex vivo with the indicated peptides at 10⁻⁷ M, 10⁻⁸ M (“lo” concentration) or without peptide for 5 hours and then stained with antibodies to quantitate T cell antigen specific production of IFN-γ (A) and TNF-α (B). In (C) mice were primed with TAP1−/− 5E1 fibroblasts expressing the model antigen from Adeno E1B. Seven days after i.p. injection splenocytes from WT, feeble, and inept mice were cultured ex vivo with the immunodominant antigen for 5 hours. Then IFN-γ expression was quantitated in CD8⁺ T cells by flow cytometry. Mean and standard error of the mean are shown. feeble, Slc15a4^{feeble/feeble} mice; inept, IRF7^inept/inept mice. Representative data of 2 independent experiments are shown. n≥4 per group of mice. Unless marked, p>0.05 between WT and feeble and not statistically significantly different.

Figure 3. Defect in antigen specific feeble T cell responses is extrinsic.

Equal numbers of bone marrow progenitors were harvested from WT and feeble mice and transferred into feeble recipients to generate mixed bone marrow chimeras. >8 weeks post irradiation, mixed bone marrow chimeras were challenged with LCMV Cl13. Eight days post infection splenocytes were harvested, counted, cultured with the indicated peptides for 5 hours, stained with antibodies, and analyzed by flow cytometry IFN-γ production in CD8⁺ (A) and CD4⁺ (B) antigen specific T cells. In (C) mice were primed with TAP1−/− 5E1 fibroblasts expressing the model antigen from Adeno E1B. Seven days after i.p. injection splenocytes from mixed bone marrow chimeras (prepared as above) were cultured ex vivo with the immunodominant antigen for 5 hours. Then IFN-γ expression was quantitated in CD8⁺ T cells by flow cytometry. Mean and standard error of the mean are shown. Feeble, Slc15a4^{feeble/feeble} mice. n = 5 per group of mice. 1 of 2 similar experiments is shown. Unless marked, p>0.05 between WT and feeble and not statistically significantly different.

Figure 4. Slc15a4 is required for specific acute inflammatory cytokine production.

One and 8 days post LCMV Cl13 infection serum was collected and the cytokines displayed were quantitated from WT and feeble mice. Individual replicates, mean and standard error of the mean are shown. Representative data of 2 independent experiments are shown; n = 5 per group of infected mice. Unless marked, p>0.05 between WT and feeble and not statistically significantly different.

Figure 5. Prophylactic treatment with CpG-DOTAP prevents persistent viral infection.

WT mice were treated with vehicle or i.v. 2 µg CpG-ODN in DOTAP (“Rx”) either 4 h prior, 4 h post or 3 days post infection with LCMV Cl13. Serum was collected at the indicated times post infection and titered for infectious virus. CpG-DOTAP: CpG-A plus DOTAP (Roche); l.o.d., limit of detection. Mean and standard error of the mean are shown for 5 mice per group of infected mice. 1 of 2 similar experiments is shown. Unless marked, p>0.05 between treatment groups and not statistically significantly different.