1. Volume 17, Issue 2, Pages 380-388 (February 2009)
Local IL-21 Promotes the Therapeutic Activity of Effector T cells by Decreasing Regulatory T Cells Within the Tumor Microenvironment 
Seunghee Kim-Schulze, Hong Sung Kim, Qing Fan, Dae Won Kim, Howard L Kaufman 
Molecular Therapy 
Volume 17, Issue 2, Pages (February 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
B16/mIL-21 melanoma cells exhibit normal growth kinetics and produce functional IL-21. (a) Growth curve of stably transfected B16 melanoma secreting IL-21 in vitro. (b) IL-21 dose-dependent proliferation of mouse naive splenocytes. Proliferation assay was performed triplicates in an anti CD3 precoated plate, increasing doses of supernatant containing mIL-21 (conditioned medium) were added at the time of stimulation as described in Materials and Methods. Anti CD3 and CD28 antibodies are used to stimulate cells. Bars, means ± SD.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Local IL-21 results in delayed tumor growth. Tumor growth of B16, B16/pcDNA, and B16/mIL-21 in syngeneic mice is represented as average tumor size (mean ± SD) of each experimental group (n = 5), as detailed in Materials and Methods. Tumor growth data shown are representative pattern of tumor growth in one of three experiments. Statistical analysis with t-test showed that the difference in tumor size between the control group and B16/mIL-21 was significant. *P < 0.05, **P <
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Local IL-21 results in an increase in the frequency of effector and memory CD8+ T cells. (a) IL-21 strongly enhances infiltration of CD3+ T cells in tumor. The percentage of CD4+, CD8+ T cells in tumor tissue was determined by flow cytometry and based on CD45+ cells. (b) Specific increases in the frequency of CD8+ T cells in TIL. (c) Single-cell suspension of tumor isolated from each group at day 23 was stained with CD137, CD62L, and CCR7 for activated memory T cells phenotypes followed by flow cytometric analysis. (d) Splenocytes isolated from each group at day 23 were stimulated for 24 hours with phorbol myristate acetate/Ionomycin A followed by intracellular staining of IL-2 and IFN-γ and flow cytometric analysis in CD4+ T cells and CD8+ from the lymphocyte populations. The unshaded histogram in the plot indicates the isotype antibody control. (e) Splenocytes were carboxyfluorescein succinimidyl ester (CFSE) labeled and stimulated with suboptimal dose of anti-CD3 mAb (0.02 µg/ml) for 72 hours followed by surface staining of CD4 and CD8 and flow cytometry analysis of CFSE dilution. CFSE profiles gated on CD4+ and CD8+ in the plot are shaded histogram and the dotted line indicates the unstimulated cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Local IL-21 prevents the accumulation of Tregs in the tumor microenvironment. (a) Representative staining for CD4+FOXP3+ and CD25+FOXP3+ cells in B16/mIL-21 tumor infiltrating cells. Gated CD4+ T cells stained with CD25 and FOXP3. Percentage of CD25+FOXP3+ cells is indicated in the upper right quadrant. (b) Graph shows the proportion of CD4+CD25+FOXP3+ Tregs in tumor infiltrating CD4+ T cells of each animal group. *P < (c) Whole splenocytes were isolated and Treg cells were enriched by microbead cell isolation method and purity of 92–95% was determined by flow cytometry. CD4+CD25− T cells were cocultured with CD4+CD25+ Treg cells at 1:1 ratio for 3 days in anti-CD3 mAb precoated plate with anti-CD28 mAb stimulation.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Local IL-21 enhances therapeutic responses of adoptively transferred T cells and acts synergistically with IL-2. (a) Tumor response to adoptive transfer of 5 × 106 activated gp100-specific T cells (pmel-1), gp100 peptide vaccination and systemic IL-2 treatment as described in Materials and Methods (n = 5). (b) Enhanced tumor response to the adoptive transfer of pmel-1 cell and gp100 peptide vaccination in mice bearing IL-21 secreting tumor (n = 5). Error bars represents ± SDs.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Local IL-21 enhances therapeutic responses of adoptively transferred T cells through expansion of CD8+ T cells. (a) IL-21 induced accumulation of Thy1.1+TCRvβ13+T cells in tumor. The percentage of Thy1.1+TCRvβ13+ T cells is indicated in the oval gate of each dot plot. The plots shown were gated on small lymphocytes infiltrating tumor. Bar graph on the right shows the average percentages of Thy1.1+TCRvβ13+ T cells and the error bar represents ± SDs. The bar graph shows the % of average IFN-γ producing cells. (b) Effector cell function was analyzed by production of IFN-γ by whole splenocytes by stimulation with gp100 peptide in vitro. The percentage of IFN-γ+ Thy1.1+ T cells is indicated in the box gate of each plot determined by intracellular cytokine staining analyzed by flow cytometry. Bar graph on the right shows the average percentages of Thy1.1+IFN-γ+ T cells and the error bar represents ± SDs. (c) IL-21 and IL-2 induced tumor antigen specific-T cells proliferation in vivo. All Vβ13+ T cells were analyzed for BrdU staining in lymphocyte populations of whole splenocytes.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Local IL-21 prevents the accumulation of Tregs in the tumor microenvironment following adoptive T-cell transfer. (a) Treatment of IL-21 and combination with IL-2 induced decrease in the frequency of Treg cells in spleen at day 5 while no difference was observed at day 10. The percentage of Treg cells were from CD4+ T cells. (b) In contrast, Treg cells in the tumor displayed low frequency at day 5 and maintained low at day 10. The percentage of Treg is based on CD4+ T cells of the small lymphocytes in tumor single-cell suspension.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions