Commonly used prophylactic vaccines as an alternative for synthetically produced TLR ligands to mature monocyte-derived dendritic cells by Gerty Schreibelt, Daniel Benitez-Ribas, Danita Schuurhuis, Annechien J. A. Lambeck, Maaike van Hout-Kuijer, Niels Schaft, Cornelis J. A. Punt, Carl G. Figdor, Gosse J. Adema, and I. Jolanda M. de Vries Blood Volume 116(4):564-574 July 29, 2010 ©2010 by American Society of Hematology

Vaccines contain TLR ligands. Vaccines contain TLR ligands. Vaccines were added (10× diluted) to recombinant HEK293 cell lines, functionally expressing a given TLR protein and a reporter gene driven by an nuclear factor-κB–inducible promoter. TLR stimulation was measured as activation of the reporter gene. The negative control value for each clone is the background signal. Positive control ligands used are: PAM2 for the HEK293-hTLR2 cell line, LPS K12 for the HEK293-hTLR4 cell line, and Flagellin for the HEK293-hTLR5 cell line. Data are expressed as optical density (OD) values. Gerty Schreibelt et al. Blood 2010;116:564-574 ©2010 by American Society of Hematology

Vaccines induce DC maturation Vaccines induce DC maturation. imDCs were incubated with the conventional cytokine cocktail (TNF-α, IL-6, IL-1β, and PGE2) or with different preventive vaccines for 48 hours. Vaccines induce DC maturation. imDCs were incubated with the conventional cytokine cocktail (TNF-α, IL-6, IL-1β, and PGE2) or with different preventive vaccines for 48 hours. (A) Viability was analyzed by Trypan blue exclusion. Data are mean ± SD of 3 independent experiments performed with DCs from different donors. **P < .01 compared with cDCs. (B-D) The expression of maturation markers HLA-DR/DP, CD80 (B), CD83 (C), and CD86 (D) was measured by flow cytometry. Results are shown as fold increase of mean fluorescence intensity relative to imDCs. Data are mean ± SEM of 3 experiments with different donors. *P < .05. **P < .01. The dotted line indicates fold increase 1.0 (unchanged fluorescence intensity compared with imDCs). (E) Example of expression of HLA-DR/DP, CD80, CD83, and CD86 (bold line) on cDCs and DCs treated with BCG. The thin line represents the isotype control. (F) At 48 hours after addition of the vaccines, IL-12p70 secretion was measured in the supernatant by ELISA. Per condition, each symbol represents 1 donor. Mean values are shown for each vaccine. **P < .01 compared with cDCs. Gerty Schreibelt et al. Blood 2010;116:564-574 ©2010 by American Society of Hematology

Combined vaccines have a synergistic effect on DC maturation. Combined vaccines have a synergistic effect on DC maturation. DCs were matured for 48 hours with the conventional cytokine cocktail (TNF-α, IL-6, IL-1β, and PGE2), preventive vaccines (BCG, Typhim, and Influvac), or vaccines with PGE2, and the expression of maturation markers and IL-12p70 production was evaluated. (A) The expression of maturation markers HLA-DR/DP, CD80, CD83, and CD86 (bold line) was measured by flow cytometry. The thin line represents the isotype control. (B) IL-12p70 production was measured by ELISA in the supernatant of DC cultures 48 hours after maturation. Per condition, each symbol represents 1 donor. Mean values are shown for each maturation cocktail. *P < .05, **P < .01 compared with cDCs. Gerty Schreibelt et al. Blood 2010;116:564-574 ©2010 by American Society of Hematology

PGE2 is essential for migration of vaccine DCs PGE2 is essential for migration of vaccine DCs. (A) Random migration on fibronectin. cDCs, vaccine DCs, and vaccine PGE2 DCs were added to a fibronectin-coated plate, and migration of individual cells was monitored for 60 minutes. PGE2 is essential for migration of vaccine DCs. (A) Random migration on fibronectin. cDCs, vaccine DCs, and vaccine PGE2 DCs were added to a fibronectin-coated plate, and migration of individual cells was monitored for 60 minutes. Data represent the percentage of migrating cells (mean ± SEM) of 3 experiments with cells from different donors. For each experiment, migration of 50 cells per condition was monitored. *P < .05. **P < .01. (B) The expression of CCR7 (bold line) on cDCs, vaccine DCs, and vaccine PGE2 DCs was measured by flow cytometry. The thin line represents the isotype control. (C) CCR7-mediated chemotaxis of cDCs, vaccine DCs, and vaccine PGE2 DCs was determined by the number of cells that had migrated into the lower compartment of a transwell system containing 10 or 100 ng/mL CCL21, counted by flow cytometry. To measure spontaneous migration, cells were incubated in a transwell without CCL21 in the upper and lower compartment (medium) or with 100 ng/mL CCL21 in both compartments (kinesis). Migration of cDCs in the presence of 100 ng/mL CCL21 was regarded as 100% (100% corresponds to 26 190 ± 10 636 migrated cells). The graph represents means ± SEM from 3 experiments (with cells from different donors) performed in duplicate. *Significant difference (P < .05) compared with medium. #Significant difference (P < .05) compared with vaccine DCs. Gerty Schreibelt et al. Blood 2010;116:564-574 ©2010 by American Society of Hematology

Vaccine PGE2 DCs have a high stimulatory capacity. Vaccine PGE2 DCs have a high stimulatory capacity. (A) The profile of cytokines secreted by PBLs on contact with allogeneic cDCs, vaccine DCs, and vaccine PGE2 DCs was measured by cytokine bead array. The graph represents the fold change in cytokine production of vaccine DCs and vaccine PGE2 DCs relative to cDCs of 3 different donors. The table presents the mean concentration (picograms per milliliter) of each cytokine in absolute numbers for all conditions. (B) IFN-γ production by PBLs cocultured with vaccine DCs or vaccine PGE2 DCs in the absence () or presence () of neutralizing anti–IL-12 antibody. The graph represents the mean ± SD from 2 experiments (with cells from different donors) of relative IFN-γ production compared with control vaccine DCs (100% corresponds to 48 ± 5 pg/mL). *P < .05. (C) KLH-specific proliferation of PBLs from a patient vaccinated with KLH-loaded DCs. PBLs were cocultured with autologous DCs matured with the cytokine cocktail, vaccines, or vaccines with PGE2 with or without KLH. Proliferation was measured by incorporation of tritiated thymidine. The graph represents mean ± SEM counts per minute relative to cDCs + KLH (100% corresponds to 1201 ± 818 cpm) of 3 experiments with different donors, performed in triplicate. represents DCs loaded with KLH; and represents DCs without KLH. *P < .05. ***P < .001. ≠Significant difference (P < .05) with cDCs with KLH. #Significant difference (P < .05) with cDCs without KLH. Gerty Schreibelt et al. Blood 2010;116:564-574 ©2010 by American Society of Hematology

Vaccine PGE2 DCs induce antigen-specific effector CD8+ T cells. Vaccine PGE2 DCs induce antigen-specific effector CD8+ T cells. Naive CD45RA+CD8+ T cells specific for gp100:280-288 (50 000 per well) were cocultured with autologous cDCs, TLR DCs, TLR PGE2 DCs, vaccine DCs, or vaccine PGE2 DCs (7000 per well) that were loaded with either gp100:280-288 or an irrelevant peptide. After 16 hours, antigen-specific activation of CD8+ T cells was analyzed by measurement of CD69 surface expression (A) and secretion of IFN-γ in the supernatant (B). T-cell proliferation was analyzed by 3H-thymidine incorporation after 4 days (C). Granzyme B expression was measured by intracellular FACS staining after 5 days (D). Antigen-specific degranulation was measured by CD107a surface expression on gp100;280 TCR-expressing PBLs cocultured for 5 hours with differently matured DCs in the presence of Golgi-stop and PE-Cy5–labeled anti-CD107a (E). represents DCs loaded with specific peptide (gp100:280-288); and represents DCs loaded with irrelevant peptide. (A-B,D) Mean ± SEM values of the relative mean fluorescence intensity (A,D) or relative IFN-γ production (B) compared with cDCs of 3 (B) or 4 (A,D) independent experiments performed with cells from different donors; 100% corresponds to 40% ± 27% CD69-positive cells (A), 834 ± 697 pg/mL IFN-γ (B), and a mean fluorescence intensity of 238 ± 363 (D). Panel C shows mean ± SEM of 1 representative experiment of 4 performed. (E) Mean ± SEM values of the percentage of cells expressing CD107a. *P < .05. **P < .01. ***P < .001. Gerty Schreibelt et al. Blood 2010;116:564-574 ©2010 by American Society of Hematology

Vaccine-PGE2 DCs do not express viral sensors and effector molecules and express and present tumor antigens after mRNA electroporation. Vaccine-PGE2 DCs do not express viral sensors and effector molecules and express and present tumor antigens after mRNA electroporation. (A) DCs were matured for 48 hours with the conventional cytokine cocktail (cDCs), combined vaccines (BCG, Typhim, and Influvac; vaccine DCs), poly(I:C), or with separate preventive vaccines. mRNA levels of PKR, RIG-I, and 2,5-OAS were determined using quantitative PCR 48 hours after maturation. (B) DCs were matured for 48 hours with the conventional cytokine cocktail or combined vaccines (BCG, Typhim, and Influvac; vaccine DCs) with PGE2. DCs were electroporated with mRNA encoding gp100 or tyrosinase. After 4 hours, gp100 or tyrosinase protein expression was determined by FACS analysis. Filled curves represent staining with specific antibody; and thin-lined curves represent the isotype control. Numbers indicate percentage of cells expressing the antigen. Data are a representative experiment of 3 performed. Average antigen expression (mean ± SEM) of 3 experiments was 73 ± 7 for gp100 and 76 ± 3 for tyrosinase on vaccine PGE2 DCs, and 65 ± 7 for gp100 and 73 ± 4 for tyrosinase on cDCs. (C) A total of 50 000 gp100:280-specific CD8+ T cells were coincubated with 7000 cDCs or vaccine PGE2 DCs 4 hours after electroporation with gp100 mRNA or tyrosinase mRNA as a control, and T-cell activation was analyzed by measurement of CD69 surface expression (left), IFN-γ production (middle), and IP-10 production (right). Data are mean ± SEM of 1 representative experiment of 3 performed in triplicate (CD69 and IFN-γ) or 1 representative experiment (IP-10). *P < .05. Gerty Schreibelt et al. Blood 2010;116:564-574 ©2010 by American Society of Hematology