Mouse DCs were cocultured with ID8-ova lysates, matured and analyzed for CD11c. SIINFEKL-pentamer staining and mouse Treg cell phenoytyping

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1 Supplementary Materials and Methods Detection of SIINFEKL-MHC Class I complex on mouse DCs Mouse DCs were cocultured with ID8-ova lysates, matured and analyzed for CD11c (clone N418, Armenian hamster IgG, ebioscience), MHC Class II (I-A/I-E) [clone M , rat IgG2b, κ, ebioscience) and anti-mouse H-2K b bound to SIINFEKL (Biolegend). SIINFEKL-pentamer staining and mouse Treg cell phenoytyping Spleen cells were stained with H-2K b -SIINFEKL-pentamer (ProImmune Ltd, UK), anti- CD3 and anti-cd8. Treg cells in spleen were identified by CD3, CD4, CD25, and intracellular FOXP3 by flow cytometry. IFN- intracellular staining of mouse tumor-specific CD4 + T-cells Splenocytes of mice were cocultured with live ID8-ova tumor cells at 10 splenocytes to 1 tumor cell for 16 hours at 37 C, 5% CO 2. After the first 2 hours of incubation, brefeldin A (1000x dilution according to manufacturer s instructions; BD Pharmingen, San Jose, CA) was added to the cocultures for the remaining of the incubation period. At the end of the 12 hours incubation, cocultures were harvested and stained for CD3 (monoclonal, clone 17A2, ebioscience) and CD4 (monoclonal, clone GK1.5, ebioscience) followed by fixpermeabilization treatment (ebioscience, San Diego, CA) for 30 min at 4 C to stain intracellularly for IFN- (anti-ifn- monoclonal antibody, clone XMG1.2, ebioscience). Then the cells were washed twice with staining buffer (DPBS containing 2% heat-inactivated FBS), collected on the same day and interrogated using BD Canto flow cytometer (Becton Dickinson). 1

2 Data were analyzed with Pro CellQuest software. T-cells that were positive for CD3, CD4 and IFN- were expressed as a percentage of all the CD4 + T-cells. Measurement of stock HOCl concentration and preparation of autologous and allogeneic HOCl-oxidized whole tumor cell lysates Concentration of HOCl in the stock sodium hypochlorite reagent (NaOCl; Sigma- Aldrich) was determined by mixing 1 volume of 25 Mm Taurine solution (Sigma) and 1 volume of NaOCl (diluted 1 in 500 with DPBS) for 30 seconds by pipetting, and measuring the absorbance at 252 nm by spectrophotometer. The HOCl concentration was calculated with the following equation = [Absorbance 252nm /Extinction coefficient] x dilution factor, where the Extinction coefficient = 1/415. To prepare HOCl-oxidized tumour cell lysate, HOCl solution of 60 M was prepared by diluting the stock NaOCl reagent (Sigma-Aldrich) with DPBS (Cellgro) and adding immediately to the tumor cells to give a final cell density of 1 x 10 6 cells/ml. The tumor cell suspensions were incubated for 1 hour at 37 C, 5% CO 2 with gentle agitation after every 30 min to induce oxidation-dependent tumor cell death. At the end of the 1 hour treatment, tumor cells were harvested, washed twice with DPBS (3 volumes of DPBS to every volume of HOCl solution to ensure complete removal of HOCl) and subjected to 6 freeze-thaw cycles. Whole tumor cell lysates were frozen at -80 C for 1 hour or in dry ice for 20 min, and thawed completely at room temperature for 6 times to complete tumor cell fragmentation. Aliquots of the oxidized tumor lysate were evaluated as described above to ensure the complete removal of the HOCl solution before loading onto DCs. Quality control of OCDC vaccine 2

3 HOCl-oxidized autologous whole tumor lysates and the final OCDC vaccine must fulfilled the following criteria before being released for administration in the clinical trial 1) HOCl-oxidized whole tumor cell lysate had a cell viability of 0% by trypan blue exclusion before loading onto DCs; 2) OCDC vaccine was cultured and tested negative for mycoplasma, bacteria and fungi; 3) OCDC vaccine was tested by endotoxin gel clot test to contain <5 EU/ml endotoxin; 4) OCDC vaccine was evaluated for DC phenotype and >70% CD86 + HLA-DR +. Clinical grade LPS The purified LPS was processed by the Clinical Center/National Institute of Health by gel-filtration chromatography and -irradiation using the original bulk material extracted from Escherichia Coli O:113. It is as a US national reference standard in assays of pyrogenicity using the Limulus amebocyte lysate, and is vialed under Good Manufacturing Practice guidelines. Each vial contained 10,000 EU, 10 mg of lactose and 1 mg of polyethylene glycol A working LPS solution of 1 mg/ml was prepared by dissolving the sterile lyophilized endotoxin with Sterile Water for Injection, USP (SWI), and stored at -80 C until needed. Intact vials of lyophilized LPS endotoxin was stored at 4 C Mixed leukocyte reactions DCs from normal donor were cocultured with SKOV-3 lysates (HOCl-L, UVB-L or FLT) for 20 to 24 hours, and matured with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) for 16 hours. DCs were then seeded in 96-well round bottom plates at 2.5 x 10 3, 5 x 10 3 or 1 x 10 4 /well. Allogeneic CD3 + T-cells were added 1 x 10 5 cells/well, and cocultured for 7 days at 37 C. In the last 16 hours, 1 µci [ 3 H] (thymidine (ICN Biomedical, Costa Mesa, CA) was added. T-cell 3

4 proliferation was measured by liquid scintillation counting (Microbeta Systems; Immunotox, Richmond, VA). All assays were performed in triplicates. Control wells contained only unpulsed immature or mature DCs, or T-cells. Results were expressed as counts per minute (cpm). Autologous T-cell priming by lysate-loaded DCs DCs from normal donor were cocultured with SKOV-3 lysates (HOCl-L, UVB-L or FLT) and matured as described in Materials and Methods. The lysate-pulsed DCs were cocultured with autologous CD3 + T-cells at a ratio of 1 DC: 10 T-cell for 10 days at 37 C. Recombinant IL-2 (50 ng/ml; Peprotech) was added on the second day of culture. At the end of 10 days, viable T-cells were harvested and cocultured with live tumor lines (MDA-231 and OVCAR2), or T2 cells pulsed with HER-2/neu neu peptide at a ratio of 10 effector: 1 target for 24 hours at 37 C. Then culture supernatants were evaluated for IFN-γ production with ELISA (Biolegend). ELISA was performed according to the manufacturer s protocol. Human DC and Treg cell phenotyping After lysate-loading and maturation for 16 hours, normal donors DCs were stained for HLA-DR, CD11c, CD14, and a marker of DC maturation (i.e. CD80, CD86, or CD40), adhesion (ICAM-1) or migration (CCR7). Gated HLA-DR + CD11c + cells were selected for analysis. Treg cells in subjects PBMCs were identified by CD3, CD4, CD25 [all antibodies from BD Pharmingen, San Jose, CA] and intracellular FOXP3 (ebioscience, San Jose, CA) by flow cytometry. IFN- ELISPOT 4

5 IFN- ELISPOT assays were previously described (13-14) and performed with modifications. Cryopreserved subject PBMCs and OCDC were rapidly thawed, washed and cocultured at 10 PBMCs: 1 DC ratio for 40 hours at 37 C. For specificity, subject PBMCs were cocultured with unpulsed mature DCs or with media (i.e. no antigen [Ag]). PBMCs from HLA- A2 + subjects (S3 and S4) were also tested against various HLA-A2-restricted ovarian peptides (see supplemental data). Mouse splenocytes were cocultured with live ID8-ova or parent ID8 cells (10 splenocytes: 1 tumor cell), SIINFEKL peptide (10 g/ml) or media only for 20 hours at 37 C. Number of IFN- producing cells were counted with an automatic plate reader (Autoimmune Diagnostica GmbH, Strassberg, Germany) and expressed as number of IFN- spots per 1 x 10 5 human peripheral blood lymphocytes (PBLs), or number of IFN- spots per 1 x 10 6 mouse splenocytes. HLA-A2-restricted synthetic peptides HLA-A*0201-restricted peptides were derived from ovarian antigens: 1) HER-2/neu : KIFGSLAFL and HER-2/neu : IISAVVGIL (47-48); 2) MUC : STAPPVHNV (49; 3) NY-ESO : SLLMWITQC (50); 4) WT : RMFPNAPYL (51); 5) Mesothelin : VLPLTVAEV (52); 6) Survivin : LMLGEFLKL (53); 7) htert : ILAKFLHWL (54); and 8) p : LLGRNSFEV (55). Peptides were purchased from New England Peptide LLC (Gardner, MA). Purities were 79% as indicated by reverse-phase highperformance liquid chromatography and mass spectrometry. Tetramer staining and expansion of patients HER-2/neu-specific T-cells 5

6 PBMCs of subjects S3 and S4 at pre- and post-ocdc vaccinations were stained with HLA-A*0201-resticted HER-2/neu (KIFGSLAFL) tetramer (Beckman Coulter, Inc., Brea, CA), anti-cd3 and anti-cd8 (BD Pharmingin) and interrogated by Flow cytometry. Post-vaccine PBMCs were cultured in vitro for 10 days with HER-2/neu peptide (KIFGSLAFL), IL-7 and IL-15 (10 ng/ml each; Peprotech) to expand peptide-specific CD8 + T-cells. For specificity control, HER-2/neu peptide-stimulated PBMCs were stained for MART-1 tetramer specific for ELAGIGILTV (Beckman Coulter). Subject tumor immunohistochemistry (IHC) Tissue slides were stained for the followings: 1) HER-2/neu [rabbit polyclonal, Dako Cytomation, Carpinteria, CA] ; 2) NY-ESO-1 [mouse monoclonal, clone E978, Santa Cruz Biotechnology, Inc., Dallas, TX]; 3) Mesothelin [mouse monoclonal, clone 5B2, Thermo Scientific, Pittsburgh, PA]; and 4) WT1 [mouse monoclonal, clone F-6, Santa Cruz Biotechnology, Inc.]. All stains were performed with Dako Autostainer and counterstained with hematoxylin according to our laboratory s standard protocols. Human and mouse serum cytokines and chemokines measurements Sera from subjects were collected after first and third vaccines, and at EOS for Luminex cytokine array analysis according to the manufacturer s protocol. Sera samples from subject S1 were evaluated after the first and third OCDC vaccine. Mouse sera were collected 49 days (i.e. 2 weeks after third vaccine) post-tumor inoculation, and analyzed with BD Cytomteric Bead Array (CBA) [San Jose, CA]. 6

7 Supplementary Figure and Table Legends Supplementary Table 1S. Cytokine and chemokine productions from DCs of ovarian cancer subjects that were loaded with HOCl-oxidized autologous whole tumor lysate and matured with LPS and IFN-. Supplementary Table S2. Subject DC yield from leukapheresis. Supplementary Table S3. Toxicities observed in ovarian cancer subjects following OCDC administrations. Supplementary Figure S1. DCs pulsed with HOCl-L efficiently tumor-specific CD4 + and SIINFEKL-specific CD8 + T-cell responses in mice. A, representative dotplots showing the % of SIINFEKL-pentamer specific CD8 + T-cells (gated) in the spleens of mice following DC-lysate vaccinations. B, the % of CD3 + CD4 + IFN- secreting T-cells in response to live ID8-ova tumor cells was quantified in spleens 2 weeks after the third vaccine. Data was the mean of 3 independent experiments with 5 mice in each experiment. Asterisks indicate results that were significant at P<0.05. Supplementary Figure S2. Phenotype of DCs following lysate-pulsing (i.e. HOCl-L, UVB-L or FTL), and activation with LPS and IFN-. Unpulsed, mature (mdc) or immature DCs (idc) served as controls. Data was presented as mean ± SEM. 7

8 Supplementary Figure S3. OCDC manufacturing process. Elutriated monocytes were obtained from subjects after leukapheresis and cultured in cell factories as described in Materials and Methods. HOCl-oxidized autologous tumor lysate was pulsed onto DCs for 16 to 20 hours, followed by activation and maturation with LPS and IFN- for 6 to 8 hours. On day 5 of culture, matured lysate-pulsed DCs were harvested and cryopreserved until required. Supplementary Figure S4. A-B, Quantification of % Treg cells in subjects pre- and postvaccine PBMCs. C, fold-increase in serum cytokines and chemokines of subjects at EOS. Line indicated a 1 fold-increase from first OCDC vaccine to EOS. 8