Flt3 permits survival during infection by rendering dendritic cells competent to activate NK cells

Abstract

A previously unappreciated signal necessary for dendritic cell (DC)-mediated activation of natural killer (NK) cells during viral infection was revealed by a recessive N-ethyl-N-nitrosourea-induced mutation called warmflash (wmfl). Wmfl homozygotes displayed increased susceptibility to mouse cytomegalovirus (MCMV) infection. In response to MCMV infection in vivo, delayed NK cell activation was observed, but no intrinsic defects in NK cell activation or function were identified. Rather, coculture experiments demonstrated that NK cells are suboptimally activated by wmfl DCs, which showed impaired cytokine production in response to MCMV or synthetic TLR7 and TLR9 ligands. The wmfl mutation was identified in the gene encoding the Fms-like tyrosine kinase 3 (Flt3). Flt3 ligand (Flt3L) is transiently induced in the serum upon infection or TLR activation. However, antibody blockade reveals no acute requirement for Flt3L, suggesting that the Flt3L --> Flt3 axis programs the development of DCs, making them competent to support NK effector function. In the absence of Flt3 signaling, NK cell activation is delayed and survival during MCMV infection is markedly compromised.

Fig. 1.

Homozygous wmfl mice show increased susceptibility to MCMV infection. (A) Survival curve of C57BL/6J, BALB/c, and wmfl mice after infection with 2 × 10⁵ pfu of MCMV (n = 5 mice per genotype). Results are representative of three experiments. (B) Viral titer in spleen of C57BL/6J, BALB/c, and wmfl mice at day 5 postinfection with 10⁵ pfu of MCMV. **, P = 0.0046; Student's t test.

Fig. 2.

MCMV-induced cytokine production in wmfl homozygotes. C57BL/6J and wmfl mice were infected with 2 × 10⁵ pfu of MCMV (n = 3 mice per genotype). Production of (A) TNF-α, (B) IFN-γ, (C) IL-12p70, and (D) type I IFN was measured in the serum at 36 h postinfection. *, P < 0.05; Student's t test. Results are representative of three experiments. ns, not significant.

Fig. 3.

Intact intrinsic function of homozygous Flt3^wmfl/wmfl NK cells. (A) Percentage and absolute number of NK cells in the spleen of C57BL/6J (n = 3) and Flt3^wmfl/wmfl mice (n = 3). **, P = 0.0023; ^††, P = 0.0022; Student's t test. (B and C) Percentage of NK cells from the spleens of C57BL/6J and Flt3^wmfl/wmfl mice expressing CD69 (B) or producing IFN-γ (C) at the indicated time points after infection with 2 × 10⁵ pfu of MCMV (n = 3 mice per genotype per time point). Results are representative of three experiments. (D) Percentage of CFSE-labeled Tap1^−/− cells remaining in the blood of β2m^−/−, C57BL/6J, and Flt3^wmfl/wmfl mice 24 h after injection of a mixture of CFSE-labeled Tap1^−/− and WT splenocytes. (E) Serum concentration of IFN-γ 36 h after MCMV infection of Rag^−/−IL2Rγ^−/− mice reconstituted with WT or wmfl NK cells (n = 4 recipient mice per condition). nd, not detected; ns, not significant. –, Rag^−/−IL2Rγ^−/− mice reconstituted with medium only.

Fig. 4.

pDCs are reduced in Flt3^wmfl/wmfl mice. (A) Percentage of DCs (CD19^–CD3ε^–NK1.1^–CD11c⁺ cells) from spleen and bone marrow of C57BL/6J and Flt3^wmfl/wmfl mice. (B and C) Percentages of pDCs and cDCs from spleen (B) and bone marrow (C) of C57BL/6J and Flt3^wmfl/wmfl mice. (A–C) n = 3 mice per genotype; results are representative of three experiments. (B and C) **, P = 0.0022; Student's t test. ns, not significant.

Fig. 5.

Reduced cytokine production by Flt3^wmfl/wmfl DCs in response to TLR stimulation or MCMV infection. Shown are concentrations of (A) IL-12p40 or (B) type I IFN in the culture medium or (C) cell surface expression of IL-15Rα on enriched CD11c⁺ splenocytes of the indicated genotype 20 h following stimulation with ligands for TLR3, TLR4, TLR7, or TLR9 or after infection with MCMV. **, P < 0.005, Student's t test. nd, not detected; ns, not significant. Cells were isolated from n = 3 mice per genotype. Results are representative of two experiments.

Fig. 6.

Flt3^wmfl/wmfl DCs do not support NK cell activation. Shown is relative IFN-γ production of NK cells isolated from C57BL/6J or Flt3^wmfl/wmfl (wmfl) spleens and cocultured with C57BL/6J or Flt3^wmfl/wmfl (wmfl) CD11c⁺ splenocytes that were infected with MCMV or activated with ligands for TLR3, TLR7, or TLR9. IFN-γ production by WT NK cells cocultured with WT DCs was used as the reference. Results are representative of three experiments.

Fig. 7.

Flt3L is induced in the nonhematopoietic compartment after MCMV infection. (A) Concentration of Flt3L in the serum of C57BL/6J mice at the indicated times after infection with MCMV or injection i.p. with ligands for TLR3, TLR4, or TLR9 (n = 3 mice per condition). (B) Concentration of Flt3L in the serum of bone marrow transplant recipient mice of the indicated genotypes 36 h after MCMV infection (n = 3 mice per condition).