1. Volume 8, Issue 4, Pages 552-558 (October 2003)
Interleukin (IL)-21 and IL-15 genetic transfer synergistically augments therapeutic antitumor immunity and promotes regression of metastatic lymphoma 
Tsunao Kishida, Hidetsugu Asada, Yoshiki Itokawa, Feng-De Cui, Masaharu Shin-Ya, Satoshi Gojo, Kakei Yasutomi, Yuji Ueda, Hisakazu Yamagishi, Jiro Imanishi, Osam Mazda 
Molecular Therapy 
Volume 8, Issue 4, Pages (October 2003)
DOI: /S (03)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
A combination of IL21 and IL15 gene transfer protected mice from tumor metastasis. Mice were transfected with pGEG.mIL-21 and/or pGEG.mIL-15 as described under Materials and Methods. Control mice were transfected with the empty vector alone (pGEG.4). Twelve hours after transfection, 104 RLmale1 lymphoma cells were injected intravenously into the mice via the tail vein (day 0). (A) Mice were sacrificed on day 14, and the numbers of metastatic foci in the liver were counted. Bars, means ± SD (n = 5). (B) The survival rates of each group of mice were plotted as a function of time (n = 5).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Suppression of preestablished tumors by IL-21 and IL-15 combination therapy. Aliquots of 104 RLmale1 lymphoma cells were transplanted intravenously into mice (day 0), and 5 days later the mice were transfected with pGEG.4 (control), pGEG.mIL-15, or both pGEG.mIL-15 and pGEG.mIL-21 as described in the legend to Fig. 1. The survival rates of each group of mice were plotted as a function of time (n = 5).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Influence of IL21 and IL15 gene transfer on NK and CTL cytolytic activities. (A and B) Balb/c mice were transfected intravenously with pGEG.mIL-21 and/or pGEG.mIL-15. Control mice were transfected with the control plasmid alone (pGEG.4). (A) Forty-eight or (B) 72 h later, mice were sacrificed, and spleen cells were subjected to 51Cr release assays using YAC-1 cells as the target. Bars, means ± SD (three mice in each group). (C) Mice were inoculated intravenously with RLmale1 cells and transfected with pGEG.mIL-21 and/or pGEG.mIL-15. Ten days later, spleen cells were subjected to 51Cr release assay against RLmale1 as described under Materials and Methods. Bars, means ± SD (three mice in each group).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Hepatic NK cells increased in number after IL-15 treatment. Mice were transfected with pGEG.4 (control) or plasmids carrying the indicated cytokine genes as described in the legend to Fig. 1. Twenty-four hours later, the mice were sacrificed and the HNPCs were harvested. The cells were stained with DX5- and CD45-specific antibodies followed by flow cytometric analysis. The numbers in each panel indicate the percentages of cells in the rectangular gate (means ± SD).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Assessment of immune effector cells induced by IL-21 and IL-15. (A) Mice were inoculated intravenously with RLmale1 cells and cotransfected with pGEG.mIL-21 and pGEG.mIL-15 as for Fig. 3C (diamonds and squares). Control mice received the tumor cell inoculation, but they were not transfected (circles). Ten days later, mice were sacrificed and spleen cell suspensions were obtained. Whole spleen cells (diamonds and circles) or spleen cells deprived of CD8+ cells (squares) were subjected to 51Cr release assay against RLmale1 as for Fig. 3C. Bars, means ± SD (three mice in each group). (B) Mice were deprived of CD4+ (lane 2), CD8+ (lane 3), or NK (lane 4) cells as described under Materials and Methods. These mice as well as normal mice (lane 5) were cotransfected with pGEG.mIL-21 and pGEG.mIL-15, and 12 h later RLmale1 cells were intravenously inoculated as for Fig. 1A (day 0) (n = 4 in each group). As a control, a group of mice was inoculated with the tumor cells, but not transfected with the cytokine genes (lane 1) (n = 3). Mice were sacrificed on day 14, and the numbers of metastatic foci in the liver were counted. Bars, means ± SD.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
IL-21 and IL-15 transfection did not induce strong systemic Th1 or Th2 responses. (A–C) Mice were transfected with the control plasmid (pGEG.4) or the indicated plasmids as described for Fig. 4. Forty-eight hours after transfection, mice were sacrificed and sera were collected. Serum concentrations of (A) IFN-γ, (B) GM-CSF, or (C) IL-4 were evaluated by ELISA. Bars, means ± SD (three mice in each group). (D) Transfection was performed as above, and 24 h later, serum concentrations of IFN-γ were evaluated by ELISA. Bars, means ± SD (three mice in each group).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions