Supporting Information for. Bongseo Choi, 1, Hyojin Moon, 1, Sung Joon Hong, 1 Changsik Shin, 1 Yoonkyung Do, 1 Seongho Ryu, 2,* Sebyung Kang 1,*

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1 Supporting Information for Effective Delivery of Antigen-Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection Bongseo Choi, 1, Hyojin Moon, 1, Sung Joon Hong, 1 Changsik Shin, 1 Yoonkyung Do, 1 Seongho Ryu, 2,* Sebyung Kang 1,* 1 Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, , Korea and 2 Soonchunhyang Institute of Medi-bio Science (SIMS), Soonchunhyang University, Cheonan, , Korea 1

2 Figure S1. Characterization of Encap containing OT-1 peptides at the N-termini (OT-1-Encap-N). (A) Schematic representation of OT-1 peptide addition to the N-termini of Encap. (B) Molecular mass measurements of the dissociated subunits of Encap (bottom) and OT-1-Encap-N (top). Calculated and observed molecular masses were indicated. (C) Size exclusion elution profiles (280 nm) of Encap (bottom) and OT-1-Encap-N (top). (D) Transmission electron micrographic (TEM) image of 2 % uranyl acetate stained Encap (left) and OT-1-Encap-N (right). Scale bars (100 nm) were indicated. 2

3 Figure S2. Characterization of Encap containing OT-1 peptides at the loop region (OT-1-Encap-L). (A) Schematic representation of OT-1 peptide insertion to the loop region of Encap. (B) Molecular mass measurements of the dissociated subunits of Encap (bottom) and OT-1-Encap-L (top). Calculated and observed molecular masses were indicated. Molecular mass of the dissociated subunits of OT-1-Encap-L was smaller than that of Encap, because the residues of loop region were substituted with OT-1 peptides. (C) Size exclusion elution profiles (280 nm) of Encap (bottom) and OT-1-Encap-L (top). (D) Transmission electron micrographic (TEM) image of 2 % uranyl acetate stained Encap (left) and OT-1-Encap-L (right). Scale bars (100 nm) were indicated. 3

4 Figure S3. Encap variants and OVA protein were efficiently phagocytosed by DCs. (A) DCs harvested from naïve C57BL/6 mice were incubated with indicated proteins labeled with phrodo either at 4 C (light shaded histograms) or 37 C (dark shaded histograms) for 2 hours and data was analyzed by flow cytometry. (B) DCs were matured with fluorescein-labeled OT-1-Encap-C in the presence of poly (I:C) at 37 C for 18 hours. Subsequently, they were incubated with 50 nm of lysotracker for additional 2 hours and fluorescence cell images of them were obtained from confocal microscope. Scale bars (20 μm) were indicated. 4

5 Figure S4. The maturation of DCs were efficiently induced by OT-1-Encap-C. Immature DCs harvested from naïve C57BL/6 mice were incubated with indicated proteins in the presence of poly (I:C) at 37 C for 18 hours and the DC maturation markers (CD80, CD86 or MHC II) was stained with PE-conjugated antibodies. The degree of DC maturation was evaluated with flow cytometry. All the DC maturation markers were successfully observed in OT-1-Encap-C treated DCs similar to those of OVA protein or OT- 1 peptide treated DCs. 5

6 Figure S5. Dose-dependent OT-1 specific CD8 + T cell proliferations. DCs harvested from naïve C57BL/6 mice were pulsed with indicated amounts of Encap variants carrying OT-1 peptides and OVA protein for 3 hours. The pulsed DCs were washed and co-cultured with CFSE-labeled OT-1 CD8 + T cells at a ratio of 1:3. Four days later, different degrees of OT-1 specific CD8 + T cell proliferation were measured by flow cytometry. 6

7 Figure S6. OT-1-Encap-C treated mature DCs formed stable conjugates with OT-1 specific CD8 + T cells. DAPI stained mature OT-1-Encap-C treated mature DCs (blue) were co-cultured with 5 μm CFSE labelled CD8 + T cells (green) derived from OT-1 transgenic mice with 1:3 ratio at 37 C for 2 hours. Stable cognate interactions between OT-1-Encap-C treated mature DCs and CD8 + T cells (red arrows) were observed with confocal fluorescence microscope. Scale bars (40 μm) were indicated. 7

8 Figure S7. OT-1 peptides delivered to DCs by OT-1-Encap-C induce the differentiation of functional effector CD8 + T cells in lymph nodes. (A) Naïve C57BL/6 mice were immunized subcutaneously either with PBS, OVA protein, Encap or OT-1-Encap-C in the presence of poly (I:C) as an adjuvant. Mice were intravenously injected with CFSE-labeled syngeneic splenocytes pulsed with (CFSE hi ) or without (CFSE low ) OT-1 peptide seven days later. Next day, cytotoxicity of OT-1 specific CD8 + T cell was measured by Flow cytometry. (B) Single cells were isolated from lymph nodes and stimulated again with OT-1 peptides for 2 days. The amounts of IFN-γ produced were measured with CBA from cultured supernatants. The P values < 0.05 were considered significant (*). 8

9 Figure S8. Simple pulsing of OT-1 peptide onto Encaps does not efficiently proliferate OT-1 specific CD8 + T cells as those of OVA protein or OT-1-Encap-C in vivo. Equivalent amount of OT-1 peptide as OT-1- Encap-C were pulsed with Encaps in 37 C for 2 hours. PBS, OVA protein, OT-1 peptide pulsed Encaps and OT-1-Encap-C were primarily immunized to naïve C57Bl/6 mice. Mice were intravenously injected with CFSE-labeled syngeneic splenocytes pulsed with (CFSE hi ) or without (CFSE low ) OT-1 peptide seven days later. Next day, cytotoxicity of OT-1 specific CD8 + T cell was measured by flow cytometry. 9

10 Figure S9. OT-1 peptides delivered to DCs by OT-1-Encap-C efficiently generate functional OT-1 peptide specific CD8 + T cells. (A) Naïve C57BL/6 mice were primed and boosted subcutaneously either with PBS, OVA protein, Encap or OT-1-Encap-C in the presence of poly (I:C) as an adjuvant. CD8 + T cells were isolated from whole splenocytes and subsequently treated with PE conjugated SIINFEKL-MHC I tetramers for 30 minutes. The population of OT-1 peptide specific CD8 + T cells was analyzed by flow cytometry. (B) Bar graph represents % of total cells, which were positive to PE conjugated SIINFEKL-MHC I tetramers. OVA protein or OT-1-Encap-C immunized mice exhibited increased populations of OT-1 peptide specific CD8 + T cells compared to those of PBS, Encap, or OT-1 peptide immunized mice. 10

11 Figure S10. Prophylactic vaccination with OT-1-Encap-C cannot suppressed B16-F10, OT-1 peptide nonexpressing melanoma, tumor growth. (A) Mice were primed intraperitoneally either with PBS, OVA protein, Encap or OT-1-Encap-C in the presence of poly (I:C) as an adjuvant and boosted with the same antigens 14 days later. At day 21, of B16-F10 melanoma cells were subcutaneously injected onto the right flank. After 10 day of tumor challenge, tumor bearing mice were intravenously injected with CFSE-labeled syngeneic splenocytes pulsed with (CFSE hi ) or without (CFSE low ) OT-1 peptide. OVA-specific CD8 + T cell cytotoxicity was measured by flow cytometry. (B) Tumor sizes were measured afterwards with a caliper every two or three days (n=7). (C) Mice were sacrificed at day 23 after tumor challenges and tumor masses were isolated presented. 11

12 Figure S11. Isolation of tumor-infiltrating lymphocytes (TILs) and the CD8/CD4 cell ratio in TILs. (A) Mice were primed intraperitoneally either with PBS, OVA protein, Encap or OT-1-Encap-C in the presence of poly (I:C) as an adjuvant and boosted with the same antigens 14 days later. At day 21, of B16- OVA melanoma cells were subcutaneously injected on right flank. Mice were sacrificed at day 21 after tumor challenges and tumor masses were isolated. (B) The populations of CD8 and CD4 cells were analyzed by flow cytometry and CD8/CD4 cell ratios in TILs isolated from each group were plotted. (C) Isolated TILs were stimulated again with 1 μm of OT-1 peptides and total IFN-γ productions of each group determined by cytometric beads assay (CBA). 12

13 Figure S12. Therapeutic vaccination with OT-1-Encap-C cannot suppressed B16-F10, OT-1 peptide nonexpressing melanoma, tumor growth. (A) Mice were subcutaneously injected with of B16-F10 melanoma onto the right flank. 7 days later, the mice were therapeutically treated with either with PBS, OVA protein, Encap or OT-1-Encap-C in the presence of poly (I:C) as an adjuvant intraperitoneally. Tumor sizes were measured afterwards with a caliper every two or three days for 23 days (n=7). (B) Mice were sacrificed at day 23 after tumor challenges and tumor masses were isolated presented. (C) Mice were subcutaneously injected with of B16-F10 melanoma onto the right flank. 10 days of therapeutic vaccination, tumor bearing mice were intravenously injected with CFSE-labeled syngeneic splenocytes pulsed with (CFSE hi ) or without (CFSE low ) OT-1 peptide. OVA-specific CD8 + T cell cytotoxicity was measured by flow cytometry. 13