Supplementary Figures - Nature · CFSE-labeled OT-I CD8 T cells (1x106 total cells) were adoptively...
Transcript of Supplementary Figures - Nature · CFSE-labeled OT-I CD8 T cells (1x106 total cells) were adoptively...
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.