Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses

Abstract

Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+), but not CD4(+) T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Impacts of DAC treatment on EL4 tumorigenicity and tumor growth.

(A) EL4 cells were treated with DAC (0.25 µM in culture medium) or Cytidine or PBS for 72 h. Cells were then injected into each mouse s.c. at a dose of 1×104 cells/mouse. Tumor size was measured in two directions every two or three days. Tumor volume was calculated using a formula: volume = (L x W2)/2 where L = length; W = width. *P<0.05 when DAC-treatment is compared to PBS or Cytidine treatment. Five to six mice were used in each group and data were pooled from two experiments. (B) EL4 cells treated with DAC were injected into C57BL/6 mice s.c. at a dose of 1×104 cells/mouse (G1), 8×104 cells/mouse (G2) or 16×104 cells/mouse (G3). PBS-treated EL4 cells (1×104 cells/mouse) served as control. Mouse survival data is shown. Five mice were included in each group and data shown are representative of three independent experiments. (C) DAC-treated or PBS-treated EL4 cells were injected into each mouse i.v. at a dose of 1×104 cells/mouse. Mouse survival was monitored up to 100 days after tumor cell injection. Ten mice per group were used, and data shown are representative of three independent experiments. (D) Mice with established EL4 tumors were treated with DAC or PBS i.p. for 5 consecutive days starting on day 10. Data shown are representative of three experiments. Asterisks indicate statistical significance of P<0.05.

Figure 2. DAC treatment induces T cell infiltration into tumors.

(A–B) C57BL/6 mice with established EL4 tumors were treated with DAC (1 mg/kg body weight) or PBS once daily for 5 consecutive days. 7–10 days after treatment, mice were sacrificed, tumors were harvested, and disassociated tumor cells were stained for the expression of different cell surface markers, followed by flow cytometry quantification for CD8+, CD4+ and NK1.1+CD3− cells. (C) Flow cytometric analysis and quantification of intracellular IFN-γ production by tumor infiltrating CD8+ T cells. Bars represent mean+SD; n = 3–7 mice per group. (D) Flow cytometric analysis of intracellular IFN-γ production by tumor infiltrating CD4+ T cells. Bars represent mean+SD; n = 3–7 mice per group. Student’s t test was used for the statistical analysis. Numbers in flow cytometric figures indicate % positive cells corresponding to each gate.

Figure 3. DAC-treatment leads to CD8+ T cell dependent tumor rejection.

Four doses of anti-CD8 or anti-CD4 antibody (400 µg/per mouse, i.p.) were injected at 4 day intervals beginning on day 1 after DAC treatment. Three mice per group were used for the experiment shown. (A) CD8+ T cells in spleens and tumors were analyzed by flow cytometry after anti-CD8 antibody or an isotype-matched control antibody treatment. (B) Tumor growth in mice treated with anti-CD8 or an isotype-matched control mAb following DAC administration. Data is representative of two independent experiments with similar results. Asterisks indicate statistical significance of P<0.05. (C) CD4+ T cells in spleens and tumors were analyzed by flow cytometry after anti-CD4 antibody or an isotype-matched control antibody treatment. (D) Tumor growth of mice treated with anti-CD4 or an isotype-matched control mAb following DAC administration. Data is representative of two independent experiments with similar results. Numbers in flow cytometric figures indicate % positive cells corresponding to each gate.

Figure 4. Up-regulated gene expression in DAC treated EL4 cells.

The mouse SmartArray chips were hybridized with RNA derived from DAC or PBS treated EL4 cells. (A) Heatmap of some upregulated genes by DAC treatment. Columns represent microarray data obtained from 3 independent biological replicates. (B) RT-PCR was used to validate the up-regulated genes. (C) qRT-PCR was used to quantify up-regulated genes in DAC and PBS treated EL4 cells.

Figure 5. Characterization of DAC-induced CD80 gene expression in cancer cells.

(A) EL4 cells were treated with DAC (0.25 µM) or PBS for 72 h. RT-PCR was used to detect CD80 gene expression in DAC and PBS treated EL4 cells. The GAPDH gene was simultaneously amplified as a loading control. (B) Flow cytometric analysis of CD80 expression in DAC-treated and PBS- treated EL4 cells. (C) EL4 cells were treated with DAC and PBS for 3 consecutive days and were maintained in the culture. Cells were harvested every two days and stained with anti-CD80 followed by flow cytometric analysis. (D) EL4 cells (CD45.2) were injected s.c. into C57BL/6 mice or i.v. into CD45.1 congenic C57BL/6 mice. DAC was administrated i.p. 2 weeks later for 5 consecutive days. EL4 cells from disassociated tumors (left panel) and bone marrows (BM) (right panel) were analyzed for the CD80 expression by flow cytometry. (E) Induction of CD80 expression in human cancer cell lines. Cancer cells were treated with DAC (0.25 µM) or PBS for 72 h. RT-PCR was used to determine CD80 gene expression. Numbers in flow cytometric figures indicate % positive cells corresponding to each gate.

Figure 6. DNA methylation of CD80 promoter region in EL4 and its variant cells.

(A) The distribution of CpG dinucleotides in a region 1 kb upstream of Exon 1 of CD80 gene. Numbers refer to position relative to the 5′ end of CD80. Two CpG enriched regions (F1 and F2) were selected for Bisulfite sequencing analysis. (B) Flow cytometric analysis of CD80 expression on two EL4 variants (EL4C3 and EL4C45). Numbers in flow cytometric figures indicate % positive cells corresponding to each gate. (C) Bisulfite sequencing of the CD80 promoter region in EL4C45 (upper panel) and EL4C3 (lower panel). Open and filled circles represent demethylated and methylated CpG sites, respectively. The methylation frequency at CpG sites of each cell line is indicated. (D) Bisulfite sequencing analysis of CD80 promoter region in DAC- and PBS-treated EL4 cells. (E) EL4 cells were first treated with DAC or PBS, CD80+ and CD80− EL4 cells were then sorted by flow cytometry-based sorting. Bisulfite sequencing analysis was performed on DAC-treated, CD80+ and CD80− EL4 cells.

Figure 7. DAC educated CD80 positive, but not CD80 negative EL4 cells induce T cell responses.

(A) EL4 cells were treated with DAC. CD80+ and CD80− EL4 cells were separated by flow cytometry-based sorting. Flow cytometric analysis of CD80 expression in EL4 cells before and after sorting is shown. (B) Tumor growth kinetics of DAC-treated CD80+ and CD80− EL4 cells. 2×104 CD80+ or CD80− DAC-treated EL4 cells were injected s.c. into each mouse. Tumor growth in individual mouse is shown. (C) Flow cytometry analysis of T cell and NK cell infiltration in CD80+ and CD80− tumors. Single cell suspensions of tumors were stained for CD4, CD8, CD3, NK1.1 followed by flow cytometry analysis. Percentage of CD8+ (D), CD4+ (E) and NK1.1+CD3− (F) in CD80+ tumors (n = 6) and CD80− tumors (n = 6) were quantified. Data shown are mean+SEM and are representative of four independent experiments with similar results. Numbers in flow cytometric figures indicate % positive cells corresponding to each gate.

Figure 8. Role of DAC induced CD80 expression in stimulation of T cells.

Six million irradiated EL4 cells were injected i.p. into each BALB/c mouse twice with a week interval between injections. Spleen and lymph node cells from EL4 immunized BALB/C mice were co-cultured with DAC-treated or PBS-treated EL4 cells for 6 days. (A) T cell proliferation was assessed using Cell Counting Kit-8 (CCK8, Dojindo, Kumomoto, Japan) on day 6. IL-2 (B) and IFN-γ (C) production in the culture supernatants were detected by ELISA. For CD80 blocking, spleen and lymph node cells from EL4-immunized mice were co-cultured with DAC-treated EL4 cells in the presence of 5 µg/ml anti-CD80 antibody or an isotype matched control antibody. (D) T cell proliferation was assessed using the CCK8 assay. Supernatants of co-culture were harvested 48 hours later for detection of IL-2 (E) and IFN-γ (F) by ELISA. Student’s t-test was used for statistical analysis. Data shown are representative of three experiments with similar results.

Figure 9. The model of DAC action in T cell response.

DNA hypermethylation in the promoter and exon 1 region of CD80 gene repress the expression of CD80 in leukemic/cancer cells. Lack of co-stimulatory signal leads to T cell tolerance. Low dose decitabine treatment can restore cancer cell expression of CD80 by demethylation of the CD80 promoter region in leukemic/cancer cells. The expression of CD80 on leukemic/cancer cells can overcome T cell tolerance and lead to activation of T cells. MHC = Major histocompatibility complex; TCR = T cell receptor.