1. Volume 22, Issue 12, Pages 2107-2117 (December 2014)
Co-Expression of Tumor Antigen and Interleukin-2 From an Adenoviral Vector Augments the Efficiency of Therapeutic Tumor Vaccination 
Benjamin Anderschou Holbech Jensen, Maria Abildgaard Steffensen, Karen Nørgaard Nielsen, Jan Pravsgaard Christensen, Allan Randrup Thomsen, Peter Johannes Holst 
Molecular Therapy 
Volume 22, Issue 12, Pages (December 2014)
DOI: /mt
Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Co-expression of IL-2 augments the Ag-specific T cell response in WT mice. WT mice were vaccinated in the hind footpad with 2 × 107 IFU Ad5-IiGP ± IL-2 in 30 μl PBS. (a) At the indicated time points subsequent to vaccination, spleens were harvested, and numbers of GP33-specific CD8+ T cells was quantified by intracellular IFNγ staining. (b) As in a, but the fraction of antigen-specific memory precursor effector cells (MPEC), double positive (DP) cells, double negative (DN) cells and short-lived effector cells (SLEC) are depicted. MPECs are characterized as CD127highKLRG-1low. DPs are CD127highKLRG-1high. DNs are CD127lowKLRG-1low, and SLECs are CD127lowKLRG-1high.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Analyzing the action range of vector produced IL-2. (a) WT mice were vaccinated in the hind footpad with 2 × 107 IFU Ad5-IiGP ± IL-2 in 30 μl PBS. After 11, 14, and 21 days, spleens were harvested, and numbers of Tregs (FoxP3+CD25+ CD4+) were quantified by flow cytometry. (b,c) groups of WT mice were co-infected in the same footpad with two different antigen-expressing constructs (NP or GP), only one of which co-expressed IL-2 at a time; controls were mice vaccinated with both antigens in the absence of vector encoded IL-2. Eleven days later numbers of GP- and NP-specific CD8 T cells were determined by intracellular IFNγ staining. Data are pooled from three independent experiments.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Prolonged CD25 expression on antigen-specific CD8 T cells in draining lymph nodes. (a) WT mice were vaccinated in the hind footpad with 2 × 107 IFU Ad5-IiGP ± IL-2 in 30 μl PBS. Nine days later spleens and popliteal lymph nodes were harvested and cells were stained with CD8 and CD44 specific Abs in addition to GP33- and GP34-specific tetramers (to define antigen-specific CD8 T cells) followed by staining for the apoptotic marker Annexin V, the anti-apoptotic marker BCL-2 or CD25; cellular distribution with regard to the indicated markers is depicted. Filled graph: Ad5-IiGP-IL-2; full line: Ad5-IiGP. (b) 107 splenocytes from CD8+ or CD4+ TCR transgenic donors (TCR318 and SMARTA, respectively) were adoptively transferred into naive recipients subsequent to CFSE labeling. One day after cell transfer, the mice were vaccinated with 2 × 107 IFU Ad5-IiGP ± IL-2. Seventy hours later the popliteal lymph nodes were harvested, and donor cell distribution with regard to CFSE dilution was analyzed. Full line: Ad5-IiGP-IL-2; filled graph: Ad5-IiGP. Results are representative data from two to four independent experiments with four to six animals per group per experiment.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Co-expression of IL-2 reduces the requirement for CD4 T-cell help and increases the expansion of CD25 expressing cells. (a) WT or MHC-II-/- mice were vaccinated with Ad5-GP, either with or without IL-2. Fourteen days after vaccination GP33-specific splenic T cells were quantified by intracellular cytokine staining. (b) Lethally irradiated B6.SJL (CD45.1) mice were reconstituted with a mixture of bone-marrow cells from CD25-/- (CD45.2) and B6.SJL (CD25+/+) mice. Ten weeks after irradiation and cell transfer, the mice were vaccinated with the indicated construct, and 11 days later, GP33-specific splenic CD8+ T cells were quantified by intracellular cytokine staining. The data represent one out of two independent experiments.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Inclusion of IL-2 in the vaccine construct improves both tumor control and overall survival. (a) WT mice were challenged with 106 B16.F10-GP melanoma cells at day 0. Five days later, all mice were vaccinated with vectors expressing either a relevant (GP) or an irrelevant (NP) Ag together with or without IL-2. (b) WT mice were challenged with 106 B16.F10 melanoma cells at day 0. Five days later mice were vaccinated with either Ad5-IiGP or Ad5-NP, both irrelevant antigens, or with Ad5-IiGP-IL-2. (c) WT mice were challenged with 105 B16.F10-GP melanoma cells at day 0. Five days later, all mice were vaccinated the Ad5-IiGP construct, either with or without inclusion of IL-2. (a–c) Mortality of tumor bearing mice as a function of time. (d) As in c, except that tumor volume as a function of time is depicted; data are presented as mean ± SEM. Representative data of two to three independent experiments; all with 10 mice per group. Mantel-Cox Log-Rank test were used for statistical evaluation of survival curves; two-way analysis of variance is used for comparison of tumor volume.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
IL-2 expression from the vaccine construct dramatically increases immune competence in CD80/86-/- mice. (a,b) WT and CD80/86-/- mice were vaccinated with Ad5-IiGP, either with or without IL-2. (a) Eleven days or (b) 14 days after vaccination, GP33-specific splenic CD8+ T cells were quantified by intracellular staining. (c) CD80/86-/- mice were challenged with 105 B16.F10-GP melanoma cells at day 0. Five days later, all mice were vaccinated as indicated, tumor-induced mortality is depicted. (d) Comparison of the IL-2 encoding vaccine's potential in WT vs. CD80/86-/- mice. The WT mice are from the same experiment as depicted in Figure 2b. Representative data of two to three independent experiments, 5–10 mice per group. Statistic evaluation: (a,b) Mann–Whitney U-test; (c,d) Mantel-Cox Log-Rank Test.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
IL-2 inclusion in the vaccine construct increases anti-tumor function in tumor bearing mice. (a) WT mice were challenged with 106 B16.F10-GP melanoma cells in the right flank at day 0. Five days later, the mice were vaccinated as indicated, and after further 10 days, the spleens were harvested, and numbers of tumor-specific, GP33-specific T cells were determined by intracellular IFNγ staining. (b) As in A, except that the ratio between Tregs and GP33-specific Tconv cells are depicted. (c) Mice were vaccinated as indicated in the figure, followed by intravenous injection of 5 × 105 B16.F10-GP melanoma cells 11 days after vaccination. Forty-six hours later, the lungs were isolated, and numbers of tumor-specific, GP33-specific CD8+ T cells recovered were determined by intracellular IFNγ staining.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 8
Vaccine encoded IL-2 does not impair Ag-specific CD8+ T-cell memor. (a) WT mice were vaccinated with Ad5-IiGP either with or without IL-2, and 60 days later some of the vaccinated mice and naive controls were challenged with 2 × 106 VV-GP i.p. Six days post challenge, numbers of GP33-specific T cells in the spleen were determined by intracellular IFNγ staining. (b) WT mice were vaccinated with Ad5-IiGP either with or without IL-2, and 120 days later 104 GP-specific CD8+ splenocytes were adoptively transferred into naive B6.SJL recipients. Prior to transfer the splenocytes from 5 donor mice per group were pooled and enriched for CD8+ T cells. One day after adoptive transfer the mice were challenged with 2 × 106 VV-GP. Six days after challenge, the spleens were isolated and the number of GP-specific T cells was quantified by tetramer staining. (c) 104 GP-specific splenocytes from day 60 Ad5-IiGP-IL-2 vaccinated mice were adoptively transferred into naive B6.SJL recipients. Subsequent procedures mirrors b, except that in this case the donor mice were either WT or CD80/86-/- as indicated in the figure.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions