In vitro DC migration assays and DC activation. A DC line, JAWSII (ATCC, Manassas, VA) was used for in vitro experiments

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1 Supplemental Material Supplemental Methods: In vitro DC migration assays and DC activation. A DC line, JAWSII (ATCC, Manassas, VA) was used for in vitro experiments and was maintained in -MEM (Invitrogen, Carlsbad, CA) supplemented with 20% FBS (Invitrogen, Carlsbad, CA) and 5ng/ml of GM-CSF. Migration assays were performed with 6.5 mm transwell dishes (Costar, Cambridge, MA) with a pore size of 5 μm. The effects of GM-CSF on the migration of JAWSII DCs was assessed by placing 0, 50 and 500 ng/ml of recombinant murine GM-CSF (Peprotech, Rocky Hill, NJ) in the bottom wells and 5x10 5 DCs in the top wells. To test whether local concentrations of GM-CSF may affect DC chemotaxis towards CCL19 (Peprotech, Rocky Hill, NJ), 5X10 5 DCs and 0, 50 and 500 ng/ml GM-CSF were placed in the top wells and 300 ng/ml of CCL19 was placed in the bottom well. After 12 hours the cells that migrated into the bottom wells of the chamber were harvested and counted using a coulter counter. The resultant gradients will dissipate over time as the GM-CSF diffuses, but the approximate diffusion time in this system (t~l 2 /D; l=0.6 cm, D~10-5 cm/s 2 ) is 10hr, which indicates gradients will exist over the time frame of the experiment. To assess the effects of GM-CSF on DC activation, JAWSII cells were cultured with the stimulatory factors, TNF- (10 ng/ml) (Peprotech, Rocky Hill, NJ) and LPS (10 ng/ml) (Sigma-Aldrich, St. Louis, MO), along with either 0, 50 or 500 ng/ml GM-CSF for 24 hours and then the cells were washed and fixed in 10% formulin. The cells were stained with the primary antibodies rat anti-mouse CCR7 (ebiosciences, San Diego, CA) and rat anti-mouse MHC class-ii (ebiosciences, San Diego, CA) and then FITC conjugated rabbit anti-rat secondary (Cell Sciences, Canton, MA) and examined using fluorescent microscopy (Olympus, Center Valley, PA).

2 Fabrication of Macroporous PLG matrices 16 mg of PLG microspheres were then mixed with 150 mg of the porogens, NaCl or sucrose (sieved to a particle size between 250 μm and 425 μm), and compression molded. The resulting disc was allowed to equilibrate within a high-pressure CO 2 environment, and a rapid reduction in pressure causes the polymer particles to expand and fuse into an interconnected structure 1. The NaCl was leached from the scaffolds by immersion in water yielding scaffolds that were 90% porous. Bioactivity of GM-CSF released from PLG matrices Scaffolds were prepared with iodinated GM-CSF and placed in 3 ml of Phosphate Buffer Solution (PBS) in an incubator (37 C). At various timepoints, the PBS release media was collected and replaced with fresh media. The amount of 125 I-hr-GM-CSF released from the scaffolds was determined at each time point by counting the radioactivity of the removed media in a gamma counter and normalizing the result to the total 125 I-GM-CSF incorporated into the scaffolds. To asses the retention of GM-CSF bioactivity, GM-CSF loaded scaffolds (1 μg) were placed in the top wells of 6.5 mm transwell dishes (Costar, Cambridge, MA) with a pore size of 3 μm and the proliferation of FDC-P1 cells cultured in the bottom wells was evaluated using cell counts from a hemacytometer. The theoretical amount of GM-CSF released into the media was calculated from in vitro release kinetics and the bioactivity was determined as a fraction of the cell proliferation induced by controls.

3 Cell Isolation from PLG matrices explanted from animals Scaffolds were excised at various time-points and the ingrown tissue was digested into single cell suspensions using a collagenase solution (Worthingtion, 250 U/ml) that was agitated at 37 C for 45 minutes. The cell suspensions were then poured through a 40μm cell strainer to isolate cells from scaffold particles and the cells were pelleted and washed with cold PBS. Analysis of DC recruitment to PLG matrices and emigration to lymph nodes APC-conjugated CD11c (dendritic cell marker), FITC-conjugated CD45 (leukoctye marker) and PE-conjugated CD86 (B7, costimulatory molecule) stains were conducted for DC and leukocyte recruitment analysis, and APC-conjugated CD11c, FITC-conjugated CCR7, and PE-conjugated MHCII stains were conducted for DC programming analysis. To further delineate the presence of specific DC subsets, cells were also stained with APC-conjugated CD11c, PE-conjugated PDCA-1 (plasmacytoid DC marker) and FITC-conjugated CD11b. Cells were gated according to positive FITC, APC and PE using isotype controls, and the percentage of cells staining positive for each surface antigen was recorded. To track in vivo DC emigration from scaffolds toward the inguinal lymph nodes, 250 μg of lyophilized fluoroscein isothiocyanate (FITC) (Molecular Probes, Carlsbad, CA) was incorporated into scaffolds by mixing with PLG microspheres before scaffold processing, and FITC was also applied by incubating scaffolds with 330 ul of 3% FITC solution for 30 min. FITC painted scaffolds were then implanted subcutaneously into the left flank of C57BL/6J mice and the inguinal lymph

4 nodes (LNs) were harvested at various time-points after scaffold implantation. Cell suspensions from LNs were prepared by mechanical disruption and pressing of the tissue through 70 μm cell strainers, and examined for CD11c(+)FITC(+) cell numbers by flow cytommetry. Assessment of T cell infiltrates into tumor tissue The primary antibodies used were GK 1.5 (CD4), and (CD8) and staining was developed using DAB+ substrate chromogen (DAKO, Carpinteria, CA). Sections from tumor samples (n=3 or 4) were visualized at 40x and 100x with a Nikon light microscope (Indianapolis, IN) and positively stained T-cells were counted manually. PLG vaccine performance in T cell knock-outs To assess PLG vaccine dependence on specific T-cell populations, B6.129S6- Cd4 tm1knw /J (CD4KO) and B6.129S2-Cd8atm1Mak/J (CD8KO) mice (Jackson Laboratory, Bar Harbor Maine) were also vaccinated using PLG scaffolds incorporating B16-F10 melanoma lysates and 3000ng GM-CSF prior to tumor cell inoculation. CD4KO mice have a targeted disruption in the CD4 gene and a significant block in CD4+ T-cell development 2. CD8KO mice also have a targeted mutation, but one that leads to a deficiency in functional cytotoxic T-cells; however, helper T-cell development and function is comparable to normal 3.

5 Statistical analysis. All values in the present study were expressed as mean ± S.D. The significant differences between the groups were analyzed by a, two-tailed, student s t test and a P value of less than 0.05 was considered significant. All animal studies were performed in accordance with NIH guidelines, under approval of Harvard University s Institutional Animal Care and Use Committee. Supplemental References 1. Harris, L.D., Kim, B.S., and Mooney, D.J. Open pore biodegradable matrices formed with gas foaming. J. Biomed.Mater. Res. 42, (1998). 2. Ganta R.R. Cheng C., Wilkerson M.J., and Chapes S.K. Delayed clearance of Ehrlicha chaffeensis infection in CD4+ T-cell knockout mice. Infect. Immun. 72(1), (2004). 3. Fung-leung W.P., et al. CD8 is needed for the development of cytotoxic T cells but not helper T cells. Cell. 65(3), (1991).

6 Supplemental Figure Legends: S1) (A) The total number of cells isolated from blank scaffolds (-- --) and GM-CSF (- -) loaded scaffolds (3μg) as a function of time post implantation. (B) The percentage of total cells residing in scaffolds that were positive for the DC markers CD11c and CD86, in blank (-- --) and GM- CSF (- -) loaded scaffolds as a function of time post implantation. (C) The total number of DCs isolated from blank (-- --) and GM-CSF (- -) loaded scaffolds as a function of time post implantation. (D) The percentage of total cells in scaffolds that were positive for the leukocyte marker CD45, in blank scaffolds (-- --) and GM-CSF (- -) loaded scaffolds as a function of time post implantation. (E) The total number of CD45(+) cells isolated from blank scaffolds (-- --) and GM-CSF (- -) loaded scaffolds as a function of time post implantation. Values represent the mean and standard deviation (n=4 or 5) and *P<0.05, ** P<0.01. S2) The percentage of CD11c(+)MHCII(+) host DCs isolated from scaffolds loaded with 0 ( ), 400 ( ),3000ng ( ), and 7000 ng of GM-CSF ( ) as a function of time after implantation into the backs of C57BL/6J mice. Scale bar 500 m. Values represent mean and standard deviation (n=4 or 5). * P<0.05. S3) FACS plots of CD11c(+)FITC(+) DCs isolated from inguinal lymph nodes after being programmed at FITC painted blank scaffolds and FITC painted GM-CSF loaded scaffolds. S4) Schematic displaying the anatomical location of PLG cancer vaccination and B16-F10 tumor cell inoculation (10 5 cells) in C57BL/6J mice. The approximate distances between the vaccine site, tumor inoculation site, and inguinal lymph nodes are indicated. S5) The percentage of mice that remained tumor free in C57BL/6J mice after PLG cancer vaccines were implanted to appropriately expose host DCs to B16-F10 tumor lysates and 40, 400, 3000, and 7000 ng of GM-CSF. At Day 14 after vaccination, C57BL/6J mice were challenged with 10 5 B16-F10 melanoma tumor cells and maintained until tumors reached a size (20-25mm in length) that required euthanization (n=9 or 10). Time 0 in curves corresponds to the time of tumor challenge. Variability in tumor free curves is approximately 20% as indicated by repeats of this experiment. S6) The percentage of mice that remained tumor free in CD4 and CD8 knock out (CD4KO and CD8 KO) mice after PLG cancer vaccines were implanted to appropriately expose host DCs to B16- F10 tumor lysates and 3000ng GM-CSF. Conditions include blank matrices (Blanks) in C57BL/6J mice and matrices containing antigen and 3000ng GM-CSF to vaccinate C57BL6/J mice (Lys+GM), CD8KO mice (Lys+GM, CD8KO) and CD4KO mice (Lys+GM, CD4KO). At Day 14 after vaccination (Day 0 on graph), C57BL/6J control mice and KO mice were challenged with 10 5 B16-F10 melanoma cells and the onset of tumor growth was monitored over time (n=9 or 10). S7) The cumulative release of PEI-CpG-ODN condensates from PLG scaffolds over time with incubation in PBS in vitro. Values represent mean and standard deviation (n=4). S8) Representative photomicrographs of tumor sections from mice vaccinated with PLG cancer vaccines that appropriately control the presentation of tumor lysates, 3000ng GM-CSF and 10μg CpG-ODN and blank (blank) scaffold controls. Sections were stained to detect for CD4(+) and CD8(+) Tcell infiltrates into tumor tissue that was explanted from mice that had developed tumors at days Scale bar 50 m.

7 S9) Digital photographs depicting patches of skin and hair depigmentation located on the backs of C57Bl/6 mice treated with PLG cancer vaccines (3000ng GM-CSF+100μg CpG+lysates) 14 days prior to B16-F10 tumor challenge. At 91 days after tumor inoculation, 33% of surviving mice developed skin and hair depigmentation starting at the sites of tumor challenge.

8 Supplemental Figures: S1 A Total cells recruited (cell #) ** ** ** Time (days) B C D E CD45(+) cells recruited ** ** ** CD45(+) cells recruited (% total cells) ** * Time (days) Time (days)

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11 S6 100 % tumor free Lys+GM, CD4 KO Lys+GM 20 Blanks Lys+ GM, CD8 KO Time (Days) S7 Cumulative ODN Release (% of total) Time(days)

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