Dendritic cell subsets require cis-activation for cytotoxic CD8 T-cell induction

Abstract

Dendritic cells (DCs) are required for the induction of cytotoxic T cells (CTL). In most tissues, including the lung, the resident DCs fall into two types expressing the integrin markers CD103 and CD11b. The current supposition is that DC function is predetermined by lineage, designating the CD103(+) DC as the major cross-presenting DC able to induce CTL. Here we show that Poly I:C (TLR3 agonist) or R848 (TLR7 agonist) do not activate all endogenous DCs. CD11b(+) DCs can orchestrate a CTL response in vivo in the presence of a TLR7 agonist but not a TLR3 agonist, whereas CD103(+) DCs require ligation of TLR3 for this purpose. This selectivity does not extend to antigen cross-presentation for T-cell proliferation but is required for induction of cytotoxicity. Thus, we demonstrate that the ability of DCs to induce functional CTLs is specific to the nature of the pathogen-associated molecular pattern (PAMP) encountered by endogenous DC.

Figure 1. CD11b⁺ DCs induce cytotoxic T cells in the presence of a TLR7 agonist

a, Pulmonary DCs were isolated for microarray analysis. Bar graph shows relative mRNA expression of the indicated genes for CD103⁺ DCs (black) and CD11b⁺ DCs (gray). Error bars represent S.E.M., n=3 b, Gating of migratory CD11c⁺MHCII^hi DCs in the lung-draining LN (LLN) of WT and Batf3^-/- mice c, LLN 24h post IN delivery of FITC-labeled soluble OVA. Left, live cells plotted as empty channel (no stain) vs FITC to identify antigen-bearing FITC⁺ cells Right, antigen-bearing DCs plotted as CD103 versus CD11b to display the antigen⁺ DCs present in the LLN. d, Top, experimental set up. OT-I cells (1×10⁶ total cells) were transferred into WT and Batf3^-/- mice 1d prior to i.n. immunization with PBS or 1μg soluble OVA +/- 10 μg Poly I:C (PIC) or 50 μg R848. 5d later, mice were i.v. injected with (1:1) PBSE-labeled OVA^—(CD45.1⁺) and OVA⁺ (CD45.2⁺) target cells. 24h post target cell transfer, cytotoxicity was assessed by flow cytometry (above). Dot plots display the frequencies of transferred target cells: OVA^—(open) versus OVA⁺ (closed) cells. Each dot represents one mouse. Data are representative of at least 4 independent experiments with 3-4 mice per group.*** p<0.0001, * p<0.03, t-test..

Figure 2. Mice deficient of TLR3⁺CD103⁺ DCs cannot induce antigen-specific cytotoxic T cells in the presence of Poly I:C

a, CFSE-labeled OT-I CD8 T cells (1×10⁶ total cells) were adoptively transferred into WT and Batf3^-/- mice 1d prior to i.n. delivery of PBS or 1 μg sOVA +/- 10 μg Poly I:C (PIC). T cell proliferation was assessed 3d later by FACS (above). Bar graphs show total numbers of proliferating OT-I CD8 T cells (below). Data are representative of three independent experiments of 3 mice per group. b, CD45.1⁺ OT-I CD8 T cells were adoptively transferred into WT and Batf3^-/- mice 1d prior to i.n. delivery of 1μg sOVA + 10 μg Poly I:C. 5d after instillation, CD45.1⁺ OT-I CD8 T cells were purified from spleens of 8 WT and 10 Batf3^-/- mice. 1×10⁶ CD45.1⁺ OT-I CD8 T cells purified from either WT or Batf3^-/- mice were adoptively transferred into naïve WT mice. 24h later PBSE-labeled OVA^— and OVA⁺ target cells were transferred. 24h later spleens were assessed for target cell frequency. Data shown is a representative experiment of two independent experiments of 4 mice per group. c, By ELISA, Type I IFNs were detected in the lungs of WT and Baft3^-/- mice 10 hours after i.n. delivery of PBS or 10 μg Poly I:C. Error bars, SEM. Data shown is a representative experiment of two independent experiments with 5 mice per group. d, In the LLN, migratory DCs were analyzed in WT and Batf3^-/- mice for the presence of CD103⁺ DCs 1 and 2 days after intranasal delivery of 10 μg Poly I:C (PIC) or 50 μg R848. CD11c⁺MHCII^hiLy6C^- migratory DCs were plotted as CD103 versus CD11b. Data shown is a representative experiment of two independent experiments.

Figure 3. Antigen-bearing DC require matched TLR agonist to induce CTL

a, FACS analysis of cells within the LLN 24h post IN delivery of CFSE-labeled OVA⁺ apoptotic thymocytes. Left, live cells plotted as empty channel (no stain) vs CFSE to identify antigen-bearing CFSE⁺ cells. Right, antigen-bearing DCs plotted as CD103 versus CD11b to display the antigen⁺ DCs in the LLN. b, OT-I CD8 T cells were adoptively transferred into WT mice 1 day prior to i.n. instillation of WT(OVA^-) or OVA⁺ apoptotic cells +/- 10 μg Poly I:C (PIC) or 50 μg R848. Five days after apoptotic cell administration, PBSE-labeled OVA^— and OVA⁺ target cells were transferred. 24h later spleens were assessed for target cell frequency. Each dot represents one mouse. ***p< 0.0001, *p < 0.02, t-test..

Figure 4. Cell-associated and soluble antigens are processed differently in CD103⁺ DCs

a, TLR3 protein expression of hematopoietic (CD45⁺) and non-hematopoietic (CD45^-) cells isolated from the lungs of WT and TLR3^-/- mice. Dendritic cells were identified as low SSC CD11c⁺MHCII^hi cells prior to being plotted as CD103 versus TLR3. b,d, OT-I CD8 T cells were adoptively transferred into WT, TLR3^-/-, MDA5^-/-, MAVS^-/- or TLR3^-/-MAVS^-/- mice 1d prior to i.n. delivery of PBS, soluble OVA or OVA⁺ apoptotic cells +/- 10μg Poly I:C (PIC). Five days after immunization, mice were given PBSE-labeled target (OVA^— or OVA⁺) cells. 24h later spleens were assessed for target cell frequency. Each dot represents one mouse. Data represents at least 3 independent experiments with 3-4 mice per group. ***p< 0.0001 t-test. c, CFSE-labeled OT-I CD8 T cells (1×10⁶ total cells) were adoptively transferred into WT and TLR3^-/- mice 1d prior to i.n. delivery of OVA⁺ apoptotic cells +/- 10 μg Poly I:C (PIC). T cell proliferation was assessed 3d later by FACS. e, Microscopy analysis of isolated pulmonary CD103⁺ DCs 2 hours after intranasal co-delivery of CFSE-labeled apoptotic cells (green) and soluble OVA-Alexa 647 (red); DAPI (blue). f, Microscopy analysis of isolated pulmonary CD103⁺ DCs 2 hours after intranasal co-delivery of CFSE-labeled apoptotic cell (green) or soluble OVA-FITC (green) with Rhodamine-labeled Poly I:C (red); DAPI (blue). Scale bar represents 5μm. Data represents at least 4 independent experiments with 10-20 CD103⁺ antigen-bearing DCs analyzed per slide.

Figure 5. TLR7 ligation activates CD11b⁺ DCs to induce CTL

a, OT-I CD8 T cells were adoptively transferred into WT or TLR7^-/-, mice 1d prior to i.n. delivery of PBS, soluble OVA +/- 10 μg Poly I:C (PIC) or 50 μg R848. Five days after immunization, mice were given PBSE-labeled target (OVA^— or OVA⁺) cells. 24h later spleens were assessed for target cell frequency. Each dot represents one mouse. Data represents at least 3 independent experiments with 3-4 mice per group. ***p< 0.0001, t-test.. b, Enriched CD11c⁺ cells from the lung were plated overnight in triplicate +/- 10 mg per ml horse cyt. c with 10 μg per ml Poly I:C or 50 μg per ml R848. FACS plots show ratios of isolated DAPI^- MHC II⁺ DCs (upper) and total DC cell counts (below). Data is representative of three independent experiments.

Figure 6. IL-27 and not IL12 is a key mediator for the induction of CTL

a, DC subsets were isolated from PBS-perfused lung tissue for microarray analysis. Bars show relative mRNA expression of the indicated genes in pulmonary CD103⁺ DCs and CD11b⁺ DCs treated +/- PIC or R848. b, FACS analysis of migratory DCs isolated from LLN of IL-12p40 reporter mice, 24h post i. n. instillation of 10 μg Poly I:C (PIC) or 50 μg R848. c,d, OT-I CD8 T cells were adoptively transferred into WT, IL-12^-/-, IL-27^-/-, or IL-12^-/-IL-27^-/- mice 1d prior to i.n. delivery of PBS, soluble OVA or OVA⁺ apoptotic cells +/- 10 μg Poly I:C (PIC) or 50 μg R848. Five days after immunization, mice were given PBSE-labeled target (OVA^— or OVA⁺) cells. 24h later spleens were assessed for target cell frequency. Each dot represents one mouse. Data represents at least 3 independent experiments with 3-4 mice/group. **p< 0.0015, *p<0.015, t-test..

Figure 7. Endogenous melanoma-specific CTLs are induced through the selective activation of antigen-bearing pulmonary DCs

a, Mice were delivered i.n. PBS or 10×10⁶ apoptotic B16F10 +/- 10 μg Poly I:C (PIC) or 50 μg R848, 14 and 7 days prior to an i.v. challenge of 2×10⁵ live B16F10 melanoma cells. WT mouse lungs were inflated 15 days after i.v. challenge. Control mice receive i.n. instillation of apoptotic B16F10 cells alone and were not given an i.v. challenge of live B16F10 cells. Samples were blinded and scored accordingly. Total surface metastases per lung were enumerated and shown by dot plots, each dot representing one mouse (right). Data are representative of 3 independent experiments with 4-5 mice per group. ***p< 0.0001. b, WT and Batf3^-/- mice were delivered i.n PBS or 100 μg of each long-tumor peptide PMEL, TRP-1 and TRP-2 +/- 10 μg Poly I:C (PIC) or 50 μg R848. Mice were immunized, challenged and scored as described above. Data are representative of 3 independent experiments with 4-5 mice per group. ***p< 0.0001, **p< 0.005, t-test.