The novel role of tyrosine kinase inhibitor in the reversal of immune suppression and modulation of tumor microenvironment for immune-based cancer therapies

Abstract

In tumor-bearing hosts, myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg) play important roles in immune suppression, the reversal of which is vitally important for the success of immune therapy. We have shown that ckit ligand is required for MDSC accumulation and Treg development. We hypothesized that sunitinib malate, a receptor tyrosine kinase inhibitor, could reverse MDSC-mediated immune suppression and modulate the tumor microenvironment, thereby improving the efficacy of immune-based therapies. Treatment with sunitinib decreased the number of MDSC and Treg in advanced tumor-bearing animals. Furthermore, it not only reduced the suppressive function of MDSCs but also prevented tumor-specific T-cell anergy and Treg development. Interestingly, sunitinib treatment resulted in reduced expression of interleukin (IL)-10, transforming growth factor-beta, and Foxp3 but enhanced expression of Th1 cytokine IFN-gamma and increased CTL responses in isolated tumor-infiltrating leukocytes. A significantly higher percentage and infiltration of CD8 and CD4 cells was detected in tumors of sunitinib-treated mice when compared with control-treated mice. More importantly, the expression of negative costimulatory molecules CTLA4 and PD-1 in both CD4 and CD8 T cells, and PDL-1 expression on MDSC and plasmacytoid dendritic cells, was also significantly decreased by sunitinib treatment. Finally, sunitinib in combination with our immune therapy protocol (IL-12 and 4-1BB activation) significantly improves the long-term survival rate of large tumor-bearing mice. These data suggest that sunitinib can be used to reverse immune suppression and as a potentially useful adjunct for enhancing the efficacy of immune-based cancer therapy for advanced malignancies.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1

Pharmacologic disruption of ckit receptor signaling through the use of a small-molecule inhibitor can prevent the accumulation of MDSCs and reduce the suppressive activity of MDSCs isolated from tumor-bearing mice. A, left, total MDSC number in the spleen was assessed. A dose-dependent decrease in the number of MDSC was observed in the spleen of tumor-bearing mice treated with sunitinib. Middle, total MDSC number in the bone marrow was assessed. A dose-dependent decrease in the number of MDSC was observed in the bone marrow of tumor-bearing mice treated with sunitinib. Right, total MDSC number per gram tumor tissue was assessed. A dose-dependent decrease in the number of MDSC was observed in the tumors of tumor-bearing mice treated with sunitinib. B, splenocytes from MCA26 tumor-bearing mice or naive mice with and without sunitinib (0.015 mg) treatment were stained for CD4+CD25+Foxp3+ Treg. C, the suppressive activities of MDSCs were assessed using HA peptide-mediated proliferation at various ratios of CD4 HA TCR transgenic splenocytes and MDSCs. Proliferation: CPM × 1,000. Representative of three experiments. Bars, SD (using different mice).

Figure 2

The effect of sunitinib treatment on the tumor microenvironment. A, the expression of Foxp3, various cytokines, and costimulatory molecules by TILs. Large (7 × 7 to 10 × 10 mm2) MCA26 tumor-bearing mice were treated with PBS or 0.015 mg/d sunitinib for 7 to 10 d. Left, mRNA was prepared from the isolated TILs and RT-PCR analysis was done. Top right, T cells in the tumors of treated mice (three mice per group) were isolated and the cytotoxic activity against parental MCA26 tumor cells was assessed in an ex vivo CTL assay. Columns, mean of triplicate wells; bars, SD. Bottom right, the concentration of IL-10 in the supernatants of anti-CD3/anti-CD28–stimulated T cells isolated from the spleen of naive or tumor-bearing mice treated with PBS or sunitinib for 1 wk. Data are from the representative of three reproducible experiments. Columns, mean of replicate wells; bars, SD. B, quantitation of the gene expression of Foxp3, various cytokines, and costimulatory molecules in TILs as determined by real-time PCR. The RNA preparations from A were also used to quantitate gene expression by quantitative real-time PCR. It is of note that the RT-PCR for TGF-β expression is saturated because of the abundance of TGF-β mRNA, resulting in no significant difference. However, a significant reduction in expression after sunitinib treatment was observed using quantitative real-time PCR.

Figure 3

Sunitinib treatment modulates the composition of TILs. Tumor-bearing mice were treated with sunitinib or PBS (four mice per group) for 10 d. TILs were isolated from treated tumor-bearing mice and stained with fluorochrome-conjugated antibodies followed by flow cytometric analysis. A, the percentages of CD8+ T cells, CD4+ T cells, MDSCs (Gr-1+CD115+), and pDC (PDCA+, CD11c+) were calculated. B, the cell number of various subsets was calculated. A significant increase in the percentage of CD8+ T cells and CD4+ T cells in the tumor was observed after sunitinib treatment, whereas the percentage of Treg, MDSC, and pDC was decreased. *, P < 0.05; **, P < 0.01, by unpaired Student’s t test, equal variance. Sunitinib treatment did not significantly affect the percentage of B cells in the tumor. C, MFI of negative costimulatory molecules (PD-1, CTLA4, and PDL-1) on various subpopulations (Treg, CD8, B cell, MDSC, and pDC) of TILs from tumor-bearing mice treated with sunitinib (white columns) or PBS (black columns). A significant decrease in the expression of PD-1 by Treg and B cells, CTLA4 by CD8 and Treg cells, and PDL-1 by MDSC and pDC was observed after sunitinib treatment. *, P < 0.05, by unpaired Student’s t test, equal variance. The average results of four separate experiments are presented.

Figure 4

Treatment with sunitinib prevents T-cell anergy in tumor-bearing mice. Thy-1.1 congenic mice were inoculated with HA-MCA26 tumor and adoptively transferred with HA-CD4 TCR T cells; mice were sacrificed and spleens were harvested after 10 d of treatment with PBS, sunitinib, or anti-ckit antibody. A, the tumor weight was determined in the treatment groups PBS, sunitinib, or anti-ckit antibody. B, stimulation index after sorting for Thy-1.2+ cells and HA peptide stimulation. C, the magnetically sorted Thy-1.2+ cells were also stained for CD4, CD25, and Foxp3+ cells. CD4 gated cells were presented. Data from three reproducible experiments are presented. Combined statistical analysis by paired t test for three experiments; P < 0.02, for PBS versus sunitinib treatment.

Figure 5

MDSC in the tumor of wild-type or ckit mutant (Wv/Wv) tumor-bearing mice with or without sunitinib treatment. Large tumor-bearing mice were treated with sunitinib or PBS for 10 d. The TILs were then isolated and stained for Gr-1 and Ly-6C to identify the MDSC. ckit Wv/Wv tumor-bearing mice had a lower percentage of MDSC in the tumor when compared with wild-type tumor-bearing mice. Sunitinib treatment did not further decrease MDSC in the tumor, suggesting that ckit is the direct target of sunitinib.

Figure 6

Sunitinib significantly improves the long-term survival rate of mice treated with Adv.mIL12 + 4-1BB activation immune modulatory therapy. Mice bearing large MCA26 tumors were divided into the following treatment groups: (a) DL312 (control vector) + PBS (solid diamond, n = 5), (b) DL312 + sunitinib (solid square, n = 8), (c) DL312 + 4-1BBL + sunitinib (solid triangle, n = 10), (d) Adv.mIL-12 + sunitinib (open square, n = 5), (e) Adv.mIL-12 + 4-1BBL + PBS (open circle, n = 25), and (f) Adv.mIL-12 + 4-1BBL + sunitinib (solid circle, n = 24). The results are combined from two reproducible experiments. The survival advantage for the Adv.mIL-12 + 4-1BBL + sunitinib–treated group was statistically significant compared with the Adv.mIL-12 + 4-1BBL + PBS group. *, P = 0.0177, by log rank.