Supplementary Figures

 

 

Supplementary Figure 1 | The experimental outcome of matched versus

mismatched antigen-presenting DC and TLR adjuvant

Experimental outcomes of this study: “Matched” refers to an antigen-presenting

DC stimulated by its corresponding TLR agonist. “Mismatched” refers to an

antigen-presenting DC not stimulated by its corresponding TLR agonist, or a DC

stimulated by its corresponding TLR agonist but not presenting the antigen. CTL =

Cytotoxic T cells.

Supplementary Figure 2 | Batf3-/- mice lack pulmonary CD103+ DCs

a, FACS analysis of DCs in the lung of WT and Batf3-/- mice. b, Twenty-four hours

post intranasal delivery of OVA-FITC, all OVA-FITC+ cells (black) in the LLN,

observed as CD11c+MHCIIhi, overlay all LLN cells (yellow).

Supplementary Figure 3 | Endogenous T cell response to OVA in Batf3-/-

mice

a, Analysis in the LLN of antigen-bearing DC subsets for SIINFEKL (OVA peptide)

loaded on MHC I, 24 h post intranasal delivery of OVA. Dots represent the MFI of

one DC within the LLN. Data are representative of 2 independent experiments with

3 mice per group. ***p< 0.0001, t-test.. b, Endogenous OVA-specific T cell in the

LLN 4 days post intranasal delivery of 100 µg sOVA +/- 100 µg Poly I:C (PIC).

Data are representative of 2 independent experiments with 4 mice per group

Supplementary Figure 4 | TLR ligation is required for induction of CTL but

not for T cell proliferation

CFSE-labeled OT-I CD8 T cells (1x106 total cells) were adoptively transferred into

WT mice 1d prior to i.n. delivery of 1 µg sOVA +/- 10 µg Poly I:C (PIC). T cell

proliferation and intracellular staining of TNFα, GranzymeB, and IFNγ was

assessed 3d later by FACS. Gates were determined based on isotype controls.

Data are representative of 3 independent experiments with 4-5 mice per group.

Supplementary Figure 5 | Monocytes and plasmacytoid DCs are not

necessary for the induction of CTL

WT and Batf3-/- mice were given i.v. OT-I CD8 T cells and i.p. 400 µg anti-Gr-1

antibody or isotype control 1 day prior to i.n. instllation of 1 µg sOVA +/- 50µg

R848. 5d after immunization, mice received PBSE-labeled OVA— and OVA+

targets cells. 24h later spleens were assessed for target cell frequency. Each dot

represents one mouse. *p< 0.0001, t-test.. Data are representative of 3

independent experiments.

Supplementary Figure 6 | IFNγ production by antigen-specific T cells

CFSE-labeled OT-I CD8 T cells (1x106 total cells) were adoptively transferred into

WT 1d prior to i.n. delivery of OVA+ apoptotic cells +/- 10 µg Poly I:C (PIC) and 1

µg sOVA (used as a control to examine IFNγ production in non-activated

proliferating OT-I T cells). T cell proliferation and intracellular IFNγ was assessed

3d later by FACS. Data are representative of 3 independent experiments with 4-5

mice per group.

Supplementary Figure 7 | Identification of migratory DCs, epithelial and

endothelial cells in the lungs of WT and TLR3-/- mice

a, Migratory DC subsets in the lungs were gated on CD45+ low SSC CD11c+MHC

II+ cells, which displayed both DC subsets: CD103 and CD11b present in WT and

TLR3-/- mice. b, Non-hematopoietic cells in the lungs were gated on CD45- and

then further gated on EpCam for epithelial cells and CD31 for endothelial cells.

Supplementary Figure 8 | OT-I T cell proliferation in WT and Batf3-/- mice

immunized with soluble OVA and ligands for TLR3 (PIC) and TLR7 (R848)

CFSE-labeled OT-I CD8 T cells (1x106 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) or 50 µg R848. 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 3 mice per group.

Supplementary Figure 9 | Preventive treatment model for metastatic

melanoma

a, Immunization schematic of intranasal deliveries of PBS or apoptotic B16F10

melanoma cells +/- 10 µg Poly I:C (PIC) or 50 µg R848 at days -14 and -7 prior to

i.v. challenge with 2x105 live B16F10 melanoma cells at day 0. Control mice

receive apoptotic B16F10 cells alone and were not challenged i.v. with live

B16F10 cells. Lungs were isolated from mice on day 15 and samples were blinded

and surface tumor metastases were counted. b, Mice were inoculated i.n. with

CFSE-labeled apoptotic B16F10 melanoma cells. Lung-draining lymph node cells

were isolated 24h later and analyzed by FACS. Apoptotic cell-bearing DCs were

identified as CFSE+, while all migratory DCs were identified as CD11c+MHC IIhi.

Overlay shows CFSE+ cells (blue) and total migratory DCs (yellow). Number

indicates frequency of CFSE+ DCs that express CD103. c, Whole lungs were

inflated with 1% PFA/1% agarose and are displayed by immunization group. Data

are representative of 3 independent experiments with 4-5 mice per group.

Supplementary Figure 10 | OT-I T cell proliferation induced by Poly I:C or

R848.

Quantitative analysis of OT-I T cell proliferation in the LLN of WT mice 3 days post

i.n. delivery of 1 µg soluble OVA + 1, 10, or 50 µg Poly I:C (PIC) or 5, 50, 100 µg

R848. SEM, bar graphs. Data are representative of 3 independent experiments

with 3 mice per group.