1. Volume 15, Issue 12, Pages 2194-2202 (December 2007)
Rejection of Metastatic 4T1 Breast Cancer by Attenuation of Treg Cells in Combination With Immune Stimulation 
Li Chen, Tian-Gui Huang, Marcia Meseck, John Mandeli, John Fallon, Savio LC Woo 
Molecular Therapy 
Volume 15, Issue 12, Pages (December 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Treatment efficacy of Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs in 4T1 tumor-bearing mice(a) Schematic representation of the orthotopic 4T1 tumor model in syngeneic female Balb/c mice. Tumor cells were implanted into the mammary fat pads on day −10 to −7. Virus vectors and mIg-m4-1BBLs were administered on day 0 (as defined by a tumor size 3–4 mm in diameter) and day 3. The primary tumors were surgically resected on day 9. (b) Survival curves of 4T1 tumor-bearing mice treated with Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs (closed circle) or empty adenovirus vector (Ad.DL312) + control mouse IgG (open circle) (P= 0.025, n = 6). (c) Number of macroscopic metastatic nodules on the lung surface in treated mice that were killed on day 26. Solid bars represent the mean value for each treatment group, n = 6Ad. DL312, empty E1-depleted control adenovirus; Ad.mGM-CSF/mIL-12, replication-defective adenovirus expressing mouse GM-CSF and mouse IL-12 (p70) under direction of the cytomegalovirus promoter; GM-CSF, granulocyte macrophage colony stimulating factor;IL-12, interleukin-12; mIgG, mouse immunoglobulin G; mIg-m4-1BBLs: the extracellular domain of mouse 4-1BB ligand fused with the Fc fragment of mIgG.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
CD4+CD25+ Tregs in 4T1 tumor-bearing mice following Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs treatment. (a) Expression of CD4 and CD25 on freshly isolated splenocytes of the treated mice on day 15. Squarely gated (mean fluorescence intensity ≥ 50) criteria were used for sorting CD4+CD25+ cells. (b) Suppression of naïve T cell (3 × 104) proliferation in response to anti-CD3 (1 μg/ml) by increasing amounts of CD4+CD25+ T cells isolated from the treated mice on day 15. The suppressive activity of CD4+CD25+ T cells from treated mice was not significantly different from the cells isolated from the control mice at any of the T cells/CD4+CD25+ cell ratios used (P > 0.05). (c) Splenocytes (1 × 105) isolated from the experimentally treated mice on day 21 were cultured for 5 days with irradiated 4T1 cells in the presence or absence of CD4+CD25+ cells (2.5 × 104) isolated from the same animals. The proliferation was measured by 3H-thymidine incorporation. White bars: splenocytes without, black bars: withautologous CD4+CD25+ Treg. (d) Following in vitro CD4+CD25+ cell depletion, the proliferation of splenic T cells (3 × 104) isolated from the experimentally treated mice in response to 1 μg/ml of anti-CD3 was measured (P = 0.001). Data are presented as mean counts per minute (cpm) ± SEM of triplicates. These results are representative of at least two independent experiments. Ad.mGM-CSF/mIL-12, replication-defective adenovirus expressing mouse GM-CSF and mouse IL-12 (p70) under direction of the cytomegalovirus promoter; GM-CSF, granulocyte macrophage colony stimulating factor;IL-12, interleukin-12; mIgG, mouse immunoglobulin G; mIg-m4-1BBLs: the extracellular domain of mouse 4-1BB ligand fused with the Fc fragment of mIgG.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Molecular structure and functional activity of mIg-mGITRLs. (a) A schematic representation of the molecular structure of the mIg-mGITRLs expression cassette and the resulting fusion protein. The ectodomain of mouse glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (mGITRL) is fused to the mouse immunoglobulin G2a (mIgG2a) hinge and CH2-CH3 domains by overlapping polymerase chain reaction amplification. The chimeric preproinsulin/hIL-2 leader sequence was put in front of the mIgG2a hinge region. (b) Purified mIg-mGITRLs was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the presence of 2-mercaptoethanol (2-ME), the predominant band was ∼50 kd, the size of the monomer and in the absence of 2-ME, the predominant band was ∼100 kd, the size of the homodimer. (c) The binding of mIg-mGITRLs to GITR expressing cells. FITC-mIg-mGITRLs (1 μg) stained CD3/CD28-activated splenic T cells (1 × 106), without (left) or with (right) previous incubation with competitive inhibitors (10 μg of unlabeled mIg-mGITRLs) at 4 °C for 1 hour. Solid histograms, fluorescein isothiocyanate–conjugated mIg-mGITRLs staining; dashed lines, isotype staining. The number indicates the percentage of positive cells. (d) Proliferation of splenocytes isolated, on day 34, from mice that received Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs treatment. The addition of mIg-mGITRLs at 0.1, 1.0, and 10 μg/ml to these cultures significantly enhanced splenocyte proliferation in response to anti-CD3 stimulation over the IgG controls in a dose dependent manner. The x axis shows the concentration of mIg-mGITRLs or mIg used. Data are presented as mean counts per minute (cpm) ± SEM of triplicates. Results were reproduced in three independent experiments. Ad.mGM-CSF/mIL-12, replication-defective adenovirus expressing mouse GM-CSF and mouse IL-12 (p70) under direction of the cytomegalovirus promoter; GM-CSF, granulocyte macrophage colony stimulating factor;IL-12, interleukin-12; mIg-m4-1BBLs: the extracellular domain of mouse 4-1BB ligand fused with the Fc fragment of mIgG; mIg-mGITRLs: the extracellular domain of mouse GITR ligand fused with the Fc fragment of mIgG.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Therapeutic efficacy of mIg-mGITRLs in combination treatment with Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs. (a) Schematic representation of the combination treatment in 4T1 tumor-bearing mice. On a schedule consistent with that described in Figure 1a, mIg-mGITRLs was injected intraperitoneally starting on day 0 and repeated every 3 days for a total of eight injections. (b) Survival curves in a dose escalation study of mIg-mGITRLs treatment in addition to Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs in 4T1 tumor-bearing mice (n = 12). A dose-dependent effect of mIg-mGITRLs was observed (P values for the 3.3, 10, 33, and 100 μg groups relative to the mIg-mGITRLs-negative group were 0.295, 0.270, 0.029, and 0.006, respectively). (c) Survival curves comparing various treatment groups. Murine IgG (mIgG) and Ad.DL312 were used as controls. For all animals, the total number of virus particles and total amount of proteins used were the same and applied at the same intervals. Although 100 μg mIg-mGITRLs alone (open diamond) did not provide significant survival advantage over the control (closed square) (P = 0.114), addition of 100 μg mIg-mGITRLs to the Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs treatment regimen (open square) provided significant survival advantage over either treatment alone (P < against mIg-mGITRLs and P = against Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs/mIgG (closed triangle). (d) In a parallel experiment with panel c, a number of macroscopic metastatic nodules was observed on the lung surface of treated mice killed on day 26 (n = 6–14). Each data point represents the value for an individual animal. Solid bars represent the mean value for each treatment group. Ad. DL312, empty E1-depleted control adenovirus; Ad.mGM-CSF/mIL-12, replication-defective adenovirus expressing mouse GM-CSF and mouse IL-12 (p70) under direction of the cytomegalovirus promoter; GITR, glucocorticoid-induced tumor necrosis-factor-receptor-family-related protein; GITRL, GITR ligand; GM-CSF, granulocyte macrophage colony stimulating factor;IL-12, interleukin-12; mIgG, mouse immunoglobulin G; mIg-m4-1BBLs: the extracellular domain of mouse 4-1BB ligand fused with the Fc fragment of mIgG; mIg-mGITRLs: the extracellular domain of mouse GITR ligand fused with the Fc fragment of mIgG.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Changes in the CD4+CD25+ Treg cell profile following combination treatment with mIg-mGITRLs. (a) Glucocorticoid-induced tumor necrosis-factor-receptor-family-related protein (GITR), cytotoxic T lymphocyte–associated antigen 4 (CTLA4) and Foxp3 expression (black heavy histogram) in CD4+CD25+ cells. Histograms shown are gated on CD4+CD25+ cell population in splenocytes isolated from mice treated without (upper panel) or with (lower panel) mIg-mGITRLs in addition to Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs. Dashed lines, isotype control staining. Results shown are one of four independent experiments with similar results. (b) The suppressive function of splenic CD4+CD25+ cells following Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs combination treatment with mIg-mGITRLs. T cells (3 × 104) from naïve mice were co-cultured at the indicated ratios with splenic CD4+CD25+ cells isolated, on day 26, from 4T1 tumor-bearing mice that had received combination treatment, with (open bars) or without (filled bars) mIg-mGITRLs, in the presence of irradiated T cell–depleted splenocytes (1 × 105 cells) and anti-CD3 (1 μg/ml) for 5 days, with 3H-thymidine added during the last 6 hours of culture. Data shown are the mean values of six independent samples ± SEM. Ad.mGM-CSF/mIL-12, replication-defective adenovirus expressing mouse GM-CSF and mouse IL-12 (p70) under direction of the cytomegalovirus promoter; GITRL, GITR ligand; GM-CSF, granulocyte macrophage colony stimulating factor;IL-12, interleukin-12; mIgG, mouse immunoglobulin G; mIg-m4-1BBLs: the extracellular domain of mouse 4-1BB ligand fused with the Fc fragment of mIgG; mIg-mGITRLs: the extracellular domain of mouse GITR ligand fused with the Fc fragment of mIgG.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Augmentation of anti-tumor CD8+ T cell response and persistent antitumor immunity induced following mIg-mGITRLs combination treatment. (a) Immunohistochemical staining for CD8+ T cells in 4T1 tumors. 4T1 tumor sections on day 9 from mice treated with Ad.DL312 + mIgG (Group1); Ad.DL312 + mIgG/mIg-mGITRLs (Group2); Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs/mIgG (Group3); and Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs + mIg-mGITRLs (Group4) are shown from left to right. The brown color represents CD8+ T cells. Scale bar = 50 μm. (b) Cytotoxic T-lymphocyte (CTL) activity of splenic effectors (E) induced by the combination therapy against 4T1 cells (T). Data are presented as the mean specific lysis ± SEM of 4 mice each at different effector-to-target cell (E:T) ratios. The CTL activities from the splenocytes on day 26 of the Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs + mIg-mGITRLs treated mice (open square) was significantly higher overall than those from Ad.DL312 + mIg (closed diamond), mIg-mGITRLs + Ad.DL312 (open diamond) and Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs (closed square) treatment groups (P = by repeated measures analysis of variance). (c) Enzyme-linked immunospot assay of interferon-γ (IFN-γ) secreting CD8+ T cells. Splenic CD8+ T cells (1 × 105) from mice treated with Ad.mGM-CSF/mIL-12 + mIg-m4-1BBLs, ± mIg-mGITRLs on day 22 were cultured with or without irradiated 4T1 cells and the positive spots were counted 36 hours later. Data are represented as the mean ± SEM of quadruplicate wells. (d) Challenge experiment. 4T1 cells (2 × 104) and MCA26 cells (7 × 104) were injected subcutaneously in contra-lateral flanks of the animals that survived for the long-term (>125 days) treated with combination treatment containing mIg-mGITRLs (n = 6), or naïve animals as positive control (n = 6). N.S., not significant. P= Ad. DL312, empty E1-depleted control adenovirus; Ad.mGM-CSF/mIL-12, replication-defective adenovirus expressing mouse GM-CSF and mouse IL-12 (p70) under direction of the cytomegalovirus promoter; GITR, glucocorticoid-induced tumor necrosis-factor-receptor-family-related protein; GITRL, GITR ligand; GM-CSF, granulocyte macrophage colony stimulating factor;IL-12, interleukin-12; mIgG, mouse immunoglobulin G; mIg-m4-1BBLs: the extracellular domain of mouse 4-1BB ligand fused with the Fc fragment of mIgG; mIg-mGITRLs: the extracellular domain of mouse GITR ligand fused with the Fc fragment of mIgG.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions