Batf3-dependent CD103+ dendritic cells are major producers of IL-12 that drive local Th1 immunity against Leishmania major infection in mice

Abstract

The role of different DC subsets in priming and maintenance of immunity against Leishmania major (L. major) infection is debated. The transcription factor basic leucine zipper transcription factor, ATF-like 3 (Batf3) is essential for the development of mouse CD103(+) DCs and some functions of CD8α(+) DCs. We found that CD103(+) DCs were significantly reduced in the dermis of Batf3-deficient C57BL/6 mice. Batf3(-/-) mice developed exacerbated and unresolved cutaneous pathology following a low dose of intradermal L. major infection in the ear pinnae. Parasite load was increased 1000-fold locally and expanded systemically. Batf3 deficiency did not affect L. major antigen presentation to T cells, which was directly exerted by CD8α(-) conventional DCs (cDCs) in the skin draining LN. However, CD4(+) T-cell differentiation in the LN and skin was skewed to nonprotective Treg- and Th2-cell subtypes. CD103(+) DCs are major IL-12 producers during L. major infection. Local Th1 immunity was severely hindered, correlating with impaired IL-12 production and reduction in CD103(+) DC numbers. Adoptive transfer of WT but not IL-12p40(-/-) Batf3-dependent DCs significantly improved anti-L. major response in infected Batf3(-/-) mice. Our results suggest that IL-12 production by Batf3-dependent CD103(+) DCs is crucial for maintenance of local Th1 immunity against L. major infection.

Keywords: Adaptive immune response ⋅ Batf3 ⋅ Dendritic cells ⋅ IL-12 ⋅ Leishmania major.

Figure 1

Batf3-deficient mice develop an exacerbated L. major cutaneous pathology with neutrophilia. (A) Pathology (the lesion diameter measured with a digital calliper) in WT and Batf3^−/− mice was tracked for 12 weeks after i.d. infection in the ear pinnae with 1000 L. major parasites. Data are shown as arithmetic mean ± SEM of 20 samples and are from one experiment representative of three independent performed. (B–D) WT and Batf3^−/− mice were i.d. infected in the ear with 5 × 10⁴ L. major parasites. (B) Parasite load in the ear, dLNs, and spleen in WT and Batf3^−/− mice at different time points p.i. Data are shown as arithmetic mean ± SEM (horizontal lines and whiskers) of individual data (n = 5 mice, each circle represents one sample) corresponding to one experiment representative of three performed. (C) Left: Representative plots showing analysis of myeloid cell infiltrates in the ear at day 21 p.i. Right: Analysis of frequency and absolute numbers of neutrophils (CD11b⁺ Ly6G⁺) in the ears at different time points p.i. Data are shown as arithmetic mean + SEM of ten samples and are pooled from three independent experiments. * p < 0.05; ** p< 0.01; *** p < 0.001 unpaired two-tailed Student's t test.

Figure 2

Priming of T-cell responses to L. major is mainly driven by Batf3-independent DCs. (A and B) WT and Batf3^−/− mice were transferred with (A) OTII (CD4⁺) or (B) OTI (CD8⁺) OVA-specific T cells labeled with cell violet and infected i.d. in the ear with 2 × 10⁵ L. major-OVA parasites. Cell violet dilution was analyzed in the transferred cells present in the dLNs (A) 4 days or (B) 3 days p.i. Left: Representative plots of three independent experiments performed. Right: Data are shown as arithmetic mean ± SEM of individual data (n = 11–14 samples) and are data pooled from three independent experiments. (C and D) CD8α⁻ cDCs, CD8α⁺ cDCs, and CD103⁺ mDCs were purified from WT dLNs 2 weeks p.i. and cocultured with polyclonal T cells from L. major infected and healed WT mice in different DC: T-cell ratios (2:1; 1:1; 0.5:1). IFN-γ production by (C) CD4⁺ and (D) CD8⁺ T cells was analyzed 4 h later by intracellular staining. Left: Representative plots from three independent experiments performed. DCs were pooled from the dLN of ten mice. Right: Data are shown as arithmetic mean + SEM. * p < 0.05; ** p< 0.01; unpaired ANOVA with Tukey post-hoc test.

Figure 3

Batf3 deficiency impairs local Th1 immunity and skews the adaptive response to L. major. (A) WT and Batf3^−/− mice were infected i.d. in the ear with 5 × 10⁴ L. major parasites. dLN cells (2 × 10⁶) obtained two and three weeks p.i. were restimulated with freeze-thawed (F/T) L. major, and IFN-γ, IL-10, and IL-4 were measured in the supernatant. (B, C) Ear cell suspensions obtained as in (A) 2, 3, and 7 weeks p.i. were restimulated with anti-CD3 and anti-CD28 and analyzed for (B) IFN-γ staining or (C) analyzed in steady state for FoxP3 expression. Representative plots of three independent experiments performed. (A–C) Data are shown as arithmetic mean + SEM (n = 5 mice (A) or 10 (B and C)) and are from a representative experiment of three performed. * p < 0.05; *** p < 0.001 unpaired two-tailed Student's t test.

Figure 4

Batf3 deficiency partially affects differentiation of dermal monocyte-derived DCs and macrophages during L. major infection. (A) Monocyte differentiation to P1 dermal monocytes (CD11b⁺ CD64^mid CCR2⁺ Ly6C^hiMHC-II⁻), monocyte-derived DCs (P2: CD11b⁺ CD64^hi CCR2⁺ Ly6C^mid MHC-II^lo and P3: CD11b⁺ CD64^hi CCR2⁺ Ly6C^lo MHC-II⁺) and dermal macrophages (P4: CD11b⁺ CD64^hi CCR2^lo MHC-II⁻ and P5: CD11b⁺ CD64^hi CCR2^lo Ly6C^lo MHC-II⁺) was tracked in ears of WT and Batf3^−/− mice 2 and 3 weeks p.i. with 5 × 10⁴ L. major parasites. (A) Representative plots and gating strategy are shown. (B) Right panels: Frequency of P1, P2, and P3 in the CD11b⁺ Ly-6C^lo-to-hi CD64^lo-to-hi CCR2 ⁺ subset; Left panel: P4, and P5 frequency in the CD11b⁺ Ly-6C^lo-to-hi CD64^lo-to-hi CCR2 ^– subset. (C) Absolute numbers of the populations in (B) per ear. (B, C) Data are shown as arithmetic mean + SEM (n = 10 samples) and are from one experiment representative of three performed. * p < 0.05; ** p< 0.01; *** p < 0.001 unpaired two-tailed Student's t test.

Figure 5

Batf-3-dependent CD103⁺ DCs are major IL-12 producers. (A–E) WT and Batf3^−/− mice were infected with 5 × 10⁴ L. major parasites i.d. in the ear and analyzed 2 weeks p.i. for IL-12p40 and IL-12p35 expression in (A) purified CD11c⁺ cells from the dLNs (B) CD45⁺ cells purified from the infected ears of WT and Batf3^−/− mice. RNA expression is standardized to the internal β-actin control and shown as fold induction to the WT average. (A and B) Data are shown as mean + SEM (n = 6 pooled samples analyzed in triplicate) from three independent experiments. (C–E) Five hours before sacrifice, mice were injected with Brefeldin A (250 μg i.p.). (C and D) dLN cells were stained for CD11c, MHC-class-II, CD8α, CD103, and intracellular IL-12p40. Plots show IL-12p40 staining in (C) CD8α⁺ CD11c⁺ MHC class II mid cDCs and (D) CD103⁺ CD11c⁺ MHC class II^hi DCs. (E) Ear dermal cells were extracted and stained for CD45, CD11c, MHC-class-II, CD103, and intracellular IL-12p40. Left: Plots showing IL-12p40 staining in CD11c⁺ MHC class II^hi CD103⁺ dermal DCs. Data in C–E are shown as arithmetic mean + SEM of frequency (upper panels) and absolute numbers (lower panels) in naive or infected mice (n = 5) and are from a representative experiment of three performed. (F) WT and Batf3^−/− mice were infected with 5 × 10⁴ L. major parasites i.d. in the ear and 10⁵ CD24^hi cells from Flt3L BMDC cultures were transferred locally every 3 days starting at day 4 p.i. Ears and dLNs were analyzed for parasite load 3 weeks p.i. Individual data and arithmetic mean ± SEM are shown for a representative experiment of two performed. * p < 0.05; ** p< 0.01; *** p < 0.001 unpaired ANOVA with Tukey's post-hoc test.