Saponin-based adjuvants induce cross-presentation in dendritic cells by intracellular lipid body formation

Saponin-based adjuvants (SBAs) are being used in animal and human (cancer) vaccines, as they induce protective cellular immunity. Their adjuvant potency is a factor of inflammasome activation and enhanced antigen cross-presentation by dendritic cells (DCs), but how antigen cross-presentation is induced is not clear. Here we show that SBAs uniquely induce intracellular lipid bodies (LBs) in the CD11b+ DC subset in vitro and in vivo. Using genetic and pharmacological interference in models for vaccination and in situ tumour ablation, we demonstrate that LB induction is causally related to the saponin-dependent increase in cross-presentation and T-cell activation. These findings link adjuvant activity to LB formation, aid the application of SBAs as a cancer vaccine component, and will stimulate development of new adjuvants enhancing T-cell-mediated immunity.


A B
SS ISCOMs C Supplementary figure 3. B3Z reaction to externally pulsed peptide dilutes normally and is not influenced by SBA addition. In vitro cross-presentation assay using B3Z cells. GM-CSF BMDCs were exposed for 5 hrs to ISCOMs (400 or 800 ng/ml) or medium. In this period, cells received medium (A and C) or 80 µg/ml OVA protein (B). After washing, cells were externally pulsed for 30 minutes with the indicated amounts of OVA or HPV K b peptide. After washing, cells were incubated o/n with the B3Z cells. Data represent single values in titration.

Supplementary figure 4. Saponins in the ISCOM structures are the active component inducing cross-presentation.
Quantification of cross-presentation in GM-CSF BMDCs following 5 hrs exposure to solubilized cholesterol, 'Fraction C' saponin, or 'Matrix C' following 5 hrs exposure to solubilized cholesterol, Fraction C saponin, or Matrix C ISCOMs (made with Fraction C saponin) (all 400 ng/ml). Other components of ISCOMs are water-insoluble. Similar data were obtained in two independent experiments.

Supplementary figure 5. SBA-induced cross-presentation is not caused by endosomal ROS.
In vitro cross-presentation assay in the presence of the indicated amounts of three ROS scavengers. Compounds were added during the 5 hr exposure period to g p g p p ISCOMs. Data represent single values in titration. Similar data were obtained in two independent experiments.

. ISCOM adjuvant induces cross-presentation only in GM-CSFcultured BMDCs. (A and B)
In vitro cross-presentation after 5 hrs exposure of GM-CSF BMDCs or Flt3-L BMDCs to wide concentration ranges of OVA and ISCOMs. External peptide pulsing was used to control for viability and/or MHC-I levels. Data represent single values in titration. Similar results were obtained in two independent experiments. (C) FACS analysis of GM-CSF BMDCs or Flt3-L BMDCs. Cells were exposed for 5 hrs to medium, ISCOMs or 0.25 µg/ml OVA coupled to the fluorophore Alexa647. Cross-presentation assay on ex vivo isolated DC subsets following in situ tumor ablation. Established B16F10 melanomas (5-8mm DC subsets following in situ tumor ablation. Established B16F10 melanomas (5 8mm diameter) were treated with cryo ablation and 50 µg peritumorally injected CpG-ODN1668. 12 hours later draining lymph nodes were harvested and subjected to FACS sorting of four cell populations using the indicated markers. Different from the Flt3-L tumor assay in Fig. 5e, ablation with CpG does not induce a prominent pDC population (CD11c pos CD11b neg ), but does create an influx of inflammatory monocytes (CD11c neg CD11b hi Ly6C hi ) The populations were exposed in vitro for 5 hrs to the (CD11c g CD11b Ly6C ). The populations were exposed in vitro for 5 hrs to the indicated compounds (ISCOMs 800 ng/ml, OVA 300 µg/ml), after which crosspresentation was analyzed using the B3Z cell readout. Statistical analysis was done using two way ANOVA with post hoc Bonferroni tests.
Supplementary figure 11. SBA-exposed IGTP -/-and +/+ BMDCs show equal levels of CD206, MHC-I (K b /D b ), and takeup/degradation of OVA. FACS analysis of GM-CSF DCs generated from bone-marrow from IGTP -/-and +/+ mice. Cells were given medium, generated from bone marrow from IGTP / and / mice. Cells were given medium, 0.25 µg/ml OVA coupled to the fluorophore Alexa647, or 1 µg/ml DQ-OVA, during the 5 hrs exposure time to medium or ISCOMs. DQ-OVA will start to fluoresce once degraded. Next, cells were processed for FACS stainings with anti CD206 (mannose receptor), or anti MHC-I antibodies. The filled black lines show the medium-treated cells, while the open red lines are the ISCOM treated samples.

Supplementary figure 12. ADRP-/-BMDCs show lower levels of LBs and crosspresentation under stimulation with SBAs. (A) FACS analysis of GM-CSF DCs generated
from bone-marrow from ADRP-/-and +/+ mice. Cells were stimulated for 5 hrs with from bone marrow from ADRP / and / mice. Cells were stimulated for 5 hrs with ISCOMs, subsequently stained for LBs with the Bodipy dye, and analyzed by FACS. (B) ADRP or IGTP -/-cells were stimulated similar as above with ISCOMs, 250 ng/ml IFNγ, and OVA. Next, cross-presentation was determined using the B3Z assay. Statistical analyses were done using one way ANOVA with post hoc Bonferroni tests. Similar results were obtained in two independent experiments.

B
Supplementary figure 13. Endosomal acidification and the proteasome are needed for SBA-aided cross-presentation. In vitro cross-presentation assays after 5 hrs exposure of GM-CSF BMDCs to ISCOMs or the indicated concentrations of inhibitors of exposure of GM CSF BMDCs to ISCOMs or the indicated concentrations of inhibitors of endosomal acidification ((A) Chloroquine and Bafilomycin A1) and the proteasome ((B) Epoxomicin). External peptide pulsing with OVA K b peptide was used to control for viability and/or MHC-I levels. Statistical analysis was done using two way ANOVA with post hoc Bonferroni tests. Similar results were obtained in two independent experiments.

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Supplementary figure 14. In vivo cross-presentation in ADRP or IGTP knockout mice, or after pharmacological LB inhibition. In vitro cross-presentation assays on ex vivo isolated lymph node DCs using B3Z cells as a readout. (A) Wild-type, IGTP-/-, or ADRP-/ k k i i j d i h 300 d f OVA i 40 OVA K b /-knockout mice were injected with 300 µg endofree OVA protein or 40 µg OVA K b peptide, with or without 30 µg ISCOMs. 12 hrs later, draining lymph nodes were harvested from which CD11c+ cells were isolated that entered the cross-presentations assays. B3Z cells were analyzed after 2 days. (B) Wild-type mice were injected s.c. on the femur with LB inhibitors (Xanthohumol: 500 µg, or A922500: 150 µg), or DMSO vehicle control. Four hrs later, identical injections were given, this time combined with 300 µg endofree OVA protein or 40 µg OVA K b peptide, with or without 30 µg ISCOMs. After 12 hrs, CD11c+ cells from lymph nodes were isolated and analyzed similar as under (A). Statistical analysis was done using two way ANOVA with post hoc Bonferroni tests.