Antigen targeting to endosomal pathway in dendritic cell vaccination activates regulatory T cells and attenuates tumor immunity by Mikael Maksimow, Mari Miiluniemi, Fumiko Marttila-Ichihara, Sirpa Jalkanen, and Arno Hänninen Blood Volume 108(4):1298-1305 August 15, 2006 ©2006 by American Society of Hematology

Plasmids used for protein expression in DCs Plasmids used for protein expression in DCs. Plasmids were constructed using standard recombinant DNA techniques, and all constructs were sequenced for potential errors. Plasmids used for protein expression in DCs. Plasmids were constructed using standard recombinant DNA techniques, and all constructs were sequenced for potential errors. Plasmid pG encodes GFP alone. Plasmids pOG and pGO encode for OVA-GFP and GFP-OVA fusion proteins, respectively. To obtain more efficient CD4 T-cell activation, plasmids pGOL2tail and psigGOL1tail were done: pGOL2 has a carboxyl-terminal sequence of LIMP-2 protein attached to GFP-OVA fusion, whereas psigGOL1tail has both amino- and carboxyl-terminal signal sequences of LAMP-1 protein connected to GFP-OVA fusion. Mikael Maksimow et al. Blood 2006;108:1298-1305 ©2006 by American Society of Hematology

DCs nucleofected with OVA-encoding plasmids have a highly activated phenotype. DCs nucleofected with OVA-encoding plasmids have a highly activated phenotype. At 20 hours after the nucleofection, DCs were collected and analyzed for viability, green fluorescence, and activation markers. (A) Viable (7-AAD–) DCs nucleofected with GFP-encoding plasmid or without any plasmid were analyzed for green fluorescence. (B) The surface expression levels of CD11c, B7.2 (CD86), MHC class II, and CD40 (gray open histograms) were analyzed from viable cells. Appropriate staining controls are shown as black histograms. (C) DCs nucleofected with pGO become more brightly green fluorescent than DCs expressing pOG. Mikael Maksimow et al. Blood 2006;108:1298-1305 ©2006 by American Society of Hematology

DCs nucleofected with OVA-encoding plasmids activate antigen-specific T cells. DCs nucleofected with OVA-encoding plasmids activate antigen-specific T cells. At 20 hours after the nucleofection, DCs were collected and used to activate either OT-I (CD8) or OT-II (CD4) T cells. OT-I (A-C) and OT-II (D-F) T-cell activation was measured using thymidine incorporation (panels A and D) or IFN-γ production (panels B and E), respectively. n.d. indicates not detectable. Number of DCs used was 1000, 2500, or 5000, from top to bottom in each panel. (C) In vitro proliferation of CFSE-labeled OT-I T cells was analyzed at day 6. DCs (5000) were nucleofected with pGO (black histogram, top panel) or with psigGOL1tail (black histogram, bottom panel) or with pG (gray open histogram, both panels). (F) In vivo proliferation of CFSE-labeled OT-II T cells at day 6 stimulated by 5000 DCs nucleofected with psigGOL1tail (black histogram) or with pG (gray open histogram) was also analyzed using CFSE dilution assay at day 6. (G) In vivo proliferation of adoptively transferred CFSE-labeled OT-I (top panels) and OT-II (bottom panels) cells 5 days after vaccination with 1 × 106 DCs nucleofected with pGO (left panels) or psigGOL1tail (right panels). In panels A, B, D, and E, results are the mean ± standard error of mean of 3 individual experiments. Mikael Maksimow et al. Blood 2006;108:1298-1305 ©2006 by American Society of Hematology

Nucleofected DCs induce strong antigen-specific immune responses in vivo. Nucleofected DCs induce strong antigen-specific immune responses in vivo. (A) Percentage of OVA-reactive T cells was analyzed from naive mice and from mice vaccinated with DCs nucleofeted with pG or pGO. OVA-tetramer-positive CD8 T cells can be found in the spleens of C57BL/6 mice 5 days after the vaccination, only if the DCs used encoded OVA. (B) Spleen cells also exhibit OVA-specific CTL activity when OVA-encoding DCs were used in the vaccination. Results from 3 independent mice per group are shown. Mikael Maksimow et al. Blood 2006;108:1298-1305 ©2006 by American Society of Hematology

Nucleofected DCs induce strong primary and memory antitumor immunity. Nucleofected DCs induce strong primary and memory antitumor immunity. (A) When tumor cells and DC vaccination are given at the same time, mice vaccinated with DCs encoding GFP-OVA fusion protein (pGO) are protected against tumor cells, whereas mice vaccinated with DCs encoding for GFP alone (pG) are not. The protective primary response was not affected even if the CD4 T cells were depleted before DC vaccination (pGO CD4 dep). (B) When memory response against tumor cells was measured by vaccinating the mice 3 weeks before tumor challenge, mice vaccinated using DCs nucleofected with pGO were almost completely protected against the tumor cells, whereas CD4 T-cell depletion abrogated the protection. P values were calculated using the Student t test. Mikael Maksimow et al. Blood 2006;108:1298-1305 ©2006 by American Society of Hematology

CD4 T-cell activation by DC vaccination induces CD25+ cells capable of suppressing OVA-specific responses. CD4 T-cell activation by DC vaccination induces CD25+ cells capable of suppressing OVA-specific responses. (A) C57Bl/6 mice vaccinated with DCs nucleofected with psigGOL1tail developed large tumors within 2 weeks. If mice were depleted of CD25+ cells before vaccination (psigGOL1tail CD25dep), protection was significantly more efficient. P values were calculated using the Student t test. (B) Numbers of CD25+Foxp3+ cells were equal in mice vaccinated with DCs nucleofected with either construct. The percentages shown are the average of 6 mice per group. (C) OVA-specific suppression of T-cell proliferation by CD4+CD25+ T cells isolated from mice vaccinated with psigGOL1tail-, but not pGO-nucleofected DCs. CD4+CD25+ T cells were pooled from 3 mice per group and coincubated with splenocytes from PPD- or OVA-primed mice. PPD- or OVA-induced proliferation is shown as white or black bars, respectively, plus or minus the standard error of mean. (D) DCs nucleofected with either construct had no effect on the protective response against luciferase-expressing B16-F10-luc-G5 melanoma cells in vivo. White bars show the bioluminescence from unimmunized control mice (n = 2) plus or minus the standard error of mean, light gray bars show bioluminescence from mice immunized with tumor-cell lysate (n = 4), dark gray bars show bioluminescence from mice similarly immunized and then vaccinated with DCs nucleofected with pGO (n = 6), and black bars show bioluminescence from mice vaccinated with DCs nucleofected with psigGOL1tail (n = 6). Mikael Maksimow et al. Blood 2006;108:1298-1305 ©2006 by American Society of Hematology

DC vaccination against endogenously expressed tumor antigen is enhanced by depletion of CD25+ cells before vaccination. DC vaccination against endogenously expressed tumor antigen is enhanced by depletion of CD25+ cells before vaccination. RIP-OVAlo mice on C57Bl/6 background expressing OVA under the control of rat insulin promoter were vaccinated using DCs nucleofected with pGO plasmid (pGO) (A). A group of mice were depleted of CD25+ cells 5 days before vaccination (pGO CD25dep). (B) Depletion of CD25+ cells 5 days before immunization with subcutaneous OVA in IFA does not increase lymphocyte infiltration into pancreatic islets within 2 weeks. Pancreases from 4 RIP-OVAlo mice in each group were analyzed in a blinded fashion for the level of lymphocytic infiltration (insulitis) in individual islets (each graded from 0 to 3 to count a score for each pancreas) using hematoxylin and eosin staining and light microscopy. At least 50 islets per pancreas were analyzed. P values were calculated using the Student t test. Black bars show insulitis score in untreated mice plus or minus the standard error of mean, white bars indicate OVA immunized mice, and gray bars indicate OVA-immunized and CD25-depleted mice. Mikael Maksimow et al. Blood 2006;108:1298-1305 ©2006 by American Society of Hematology