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. 2008 Jun 15;14(12):3966-74.
doi: 10.1158/1078-0432.CCR-08-0356.

Transforming growth factor-beta receptor blockade augments the effectiveness of adoptive T-cell therapy of established solid cancers

Africa Wallace  1 , Veena Kapoor, Jing Sun, Paul Mrass, Wolfgang Weninger, Daniel F Heitjan, Carl June, Larry R Kaiser, Leona E Ling, Steven M Albelda
Affiliations

Transforming growth factor-beta receptor blockade augments the effectiveness of adoptive T-cell therapy of established solid cancers

Africa Wallace et al. Clin Cancer Res. .

Abstract

Purpose: Adoptive cellular immunotherapy is a promising approach to eradicate established tumors. However, a significant hurdle in the success of cellular immunotherapy involves recently identified mechanisms of immune suppression on cytotoxic T cells at the effector phase. Transforming growth factor-beta (TGF-beta) is one of the most important of these immunosuppressive factors because it affects both T-cell and macrophage functions. We thus hypothesized that systemic blockade of TGF-beta signaling combined with adoptive T-cell transfer would enhance the effectiveness of the therapy.

Experimental design: Flank tumors were generated in mice using the chicken ovalbumin-expressing thymoma cell line, EG7. Splenocytes from transgenic OT-1 mice (whose CD8 T cells recognize an immunodominant peptide in chicken ovalbumin) were activated in vitro and adoptively transferred into mice bearing large tumors in the presence or absence of an orally available TGF-beta receptor-I kinase blocker (SM16).

Results: We observed markedly smaller tumors in the group receiving the combination of SM16 chow and adoptive transfer. Additional investigation revealed that TGF-beta receptor blockade increased the persistence of adoptively transferred T cells in the spleen and lymph nodes, increased numbers of adoptively transferred T cells within tumors, increased activation of these infiltrating T cells, and altered the tumor microenvironment with a significant increase in tumor necrosis factor-alpha and decrease in arginase mRNA expression.

Conclusions: We found that systemic blockade of TGF-beta receptor activity augmented the antitumor activity of adoptively transferred T cells and may thus be a useful adjunct in future clinical trials.

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Figures

Figure 1
Figure 1. TGF-β Receptor Blockade and adoptive transfer of activated OT-1 splenocytes causes tumor regression
A. Representative Experiment. EG7 tumor cells were injected on the flanks of C57/B6 mice on Day 0 (n=8 in each group). On Day 9, established EG7 tumors were left on control chow (control and adoptive transfer groups) or begun on SM16 chow (solid arrow: SM16 and combo group). On Day 12, the adoptive transfer group and the combination group (combo) received an intravenous injection of 10 million activated OT-1 cells (AT- dashed arrow). Both SM16 and activated OT-1 cells caused a slowing of tumor growth (tumors were significantly smaller than control from Day 20 onwards). However, all animals treated with the combination had complete tumor regression up till Day 32. Tumor size in the combination group was significantly smaller (*p<0.05 after Bonferroni correction for multiplicity) than all of the other groups from Day 20 onwards. B. Combined Data. This experiment was repeated 3 more times with similar results. This panel shows the data combined from the four different experiments (N=26 in each group). To control for minor differences in tumor size at the beginning of each experiment, the average tumor volume at each time point was normalized to the volume at the start of treatment. In this panel, Day 0 represents the day SM16 chow was started and adoptive transfer was performed three days later (arrow). These data are similar to the individual study shown above, with significant regression in the combination group compared to all others (* = p<0.05 after Tukey correction).
Figure 2
Figure 2. TGF-β Receptor Blockade Increases the Number of Transferred T-cells in spleen and lymph node
Twenty million activated GFP-expressing OT-1 splenocytes were injected into tumor-bearing animals on control diets or SM16 diets. Three days after transfer, spleen and lymph nodes were isolated and subjected to FACS analysis to identify transferred CD8+/GFP+ T-cells. Panel A shows results from Spleen and Panel B from lymph nodes. The upper panels show representative FACS tracings (CD8 on the X-axis and GFP on the Y axis) from control animals (no adoptive transfer), animals on control chow who received adoptive transfer of T cells, and animals on SM16 chow who received adoptive transfer of T cells. The transferred cells (CD8+/GFP+) are visualized in the right upper quadrant of each tracing. The lower graph shows the average values of the CD8+/GFP+ populations in each group for three independent experiments. *= p<0.05 from control and SM16 + AT; ** = p<0.05 from control and Control chow plus adoptive transfer.
Figure 3
Figure 3. TGF-β Receptor Blockade Increases the Number of Transferred T-cells in Tumors
Twenty million activated GFP-expressing OT-1 cells were injected into tumor-bearing animals on control diets or SM16 diets. Three days after transfer, the tumors were isolated, digested, subjected Ficoll purification, and analyzed by FACS to identify transferred CD8+/GFP+ T-cells. The upper panels show representative FACS tracings (GFP on the X-axis and Side Scatter on the Y axis) from control animals (no adoptive transfer), animals on control chow who received adoptive transfer of T cells, and animals on SM16 chow who received adoptive transfer of T cells. Transferred cells (GFP+) are visualized in the boxes. The lower graph shows the average number of GFP+ cells in the tumors in each group for three independent experiments. *= p<0.05 from control and control chow plus AT; ** = p<0.05 from control and Control chow plus adoptive transfer.
Figure 4
Figure 4. TGF-β Receptor blockade augments the effector function of adoptively transferred T-cells in tumor
Twenty million activated GFP-expressing OT-1 cells were injected into tumor-bearing animals on control diets or SM16 diets. Three days after transfer, tumors were isolated and digested. Due to the very small numbers of T-cells within tumors, we pooled cells from 4 tumors, did a Ficoll purification, stimulated the cells with IL-2, PMA/ionomycin, or IL-2 plus SIINFEKL peptide, and then performed FACS to detect intracellular IFN-γ staining using PE-labeled anti-IFN-γ antibody (X-axis is GFP, Y-axis is IFN-γ). The two left hand panels were treated with an unlabeled anti-IFN–γ antibody (blocking antibody) before addition of PE-labeled antibody and were used to identify non-specific binding and define our stringent gates. Each row shows an independent experiment. The percent of activated cells (GFP+/IFN-γ+) of the total GFP+ (CD8+) cells are shown in the upper right hand quadrant of each FACS panel.
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