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. 2022 Aug 3:12:873639.
doi: 10.3389/fonc.2022.873639. eCollection 2022.

Combined treatment with epigenetic agents enhances anti-tumor activity of MAGE-D4 peptide-specific T cells by upregulating the MAGE-D4 expression in glioma

Shui-Qing Bi  1   2 , Qing-Mei Zhang  1   3 , Xia Zeng  1 , Chang Liu  4 , Wei-Xia Nong  1 , Huan Xie  1 , Feng Li  1 , Li-Na Lin  1 , Bin Luo  1   3 , Ying-Ying Ge  1 , Xiao-Xun Xie  1   3
Affiliations

Combined treatment with epigenetic agents enhances anti-tumor activity of MAGE-D4 peptide-specific T cells by upregulating the MAGE-D4 expression in glioma

Shui-Qing Bi et al. Front Oncol. .

Abstract

Objective: The study evaluated the efficacy of combined epigenetic drugs of decitabine (DAC), valproic acid (VPA), and trichostatin A (TSA) on immunotherapy against glioma.

Methods: The expression and prognosis of MAGE-D4 in glioma were analyzed online, and the expression of MAGE-D4 and HLA-A2 in glioma induced by epigenetic drugs was detected by qRT-PCR, Western blot, and flow cytometry. The methylation status of the MAGE-D4 promoter was determined by pyrosequencing. An HLA-A2 restricted MAGE-D4 peptide was predicted and synthesized. An affinity assay and a peptide/HLA complex stability assay were performed to determine the affinity between peptide and HLA. CCK8 assay, CFSE assay, ELISA and ELISPOT were performed to detect the function of MAGE-D4 peptide-specific T cells. Flow cytometry, ELISA, and cytotoxicity assays were used to detect the cytotoxicity effect of MAGE-D4 peptide-specific T cells combined with epigenetic drugs against glioma in vitro. Finally, the glioma-loaded mouse model was applied to test the inhibitory effect of specific T cells on gliomas in vivo.

Results: MAGE-D4 was highly expressed in glioma and correlated with poor prognosis. Glioma cells could be induced to express MAGE-D4 and HLA-A2 by epigenetic drugs. MAGE-D4-associated peptides were found that induce DCs to stimulate the highest T-cell activities of proliferation, IL-2 excretion, and IFN-γ secretion. MAGE-D4 peptide-specific T cells treated with TSA only or combining TSA and DAC had the most cytotoxicity effect, and its cytotoxicity effect on glioma cells decreased significantly after HLA blocking. In vivo experiments also confirmed that MAGE-D4-specific T cells inhibit TSA-treated glioma.

Conclusion: MAGE-D4 is highly expressed in glioma and correlated with the prognosis of glioma. The novel MAGE-D4 peptide identified was capable of inducing MAGE-D4-specific T cells that can effectively inhibit glioma growth, and the epigenetic drug application can enhance this inhibition.

Keywords: MAGE-D4; cytotoxic T lymphocyte; decitabine; glioma; trichostatin A; valproic acid.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Expression and survival analysis of MAGE-D4 in glioma by GEO and LinkedOmics data. (A) Expression of MAGE-D4 in glioma and normal brain tissues. (B) Kaplan–Meier survival analysis of 625 glioma patients according to MAGE-D4 expression. *P <0.05.
Figure 2
Figure 2
Expression of MAGE-D4 and HLA-A2 in glioma cells treated with epigenectic drugs. (A) Relative mRNA expression of MAGE-D4 in U251 and U87 cells treated with epigenectic drugs. (B) Relative mRNA expression of HLA-A2 in U251 and U87 cells treated with epigenectic drugs. (C) Relative protein expression of MAGE-D4 in U251 and U87 cells treated with epigenetic drugs. (D) Expression of HLA-A2 in U251 and U87 cells treated with epigenetic drugs. Con, control; D, DAC; V, VPA; T, TSA; D + V, DAC + VPA; D + T, DAC + TSA; *P <0.05.
Figure 3
Figure 3
Methylation analysis of MAGE-D4 promoter region in U251 and U87 cells treated with epigenetic drugs. (A) Overall level of MAGE-D4 promoter methylation. (B) Overall level of MAGE-D4 promoter methylation in region 1. (C) Overall level of MAGE-D4 promoter methylation in region 2. Con, control; D, DAC; V, VPA; T, TSA; D + V, DAC + VPA; D + T, DAC + TSA; *P <0.05, **P <0.01, ***P <0.001.
Figure 4
Figure 4
MAGE-D4 peptide-pulsed DCs stimulated the proliferation of T cells. (A) CCK8 assay. (B) CFSE assay. The value (inset) for the percentage of cells that divided at least once (top left corner) and the proliferation index (bottom left corner) are indicated for each sample. DC, dendritic cell; Tc, T cell; NC, negative control peptide. *P <0.05.
Figure 5
Figure 5
MAGE-D4 peptide pulsed DCs stimulated T cells secreting cytokines. (A) ELISA detected IL-2. (B) ELISA detected IFN-γ. (C, D) ELISOT detected number of T cells secreting IFN-γ. DC, dendritic cell; NC, negative control peptide; Tc,T cell. PC, positive control. *P <0.05.
Figure 6
Figure 6
Epigenetic drugs sensitize tumor cells to the cytotoxicity of MAGE-D4-activated T cells. (A, B) the ratio of CD107a-expressed T cells stimulated by epigenetic drugs treated U251 (A) and U87 (B) cells. (C–F) the cytokines expression of T cells stimulated by epigenetic drugs treated U251 (C, E) and U87 (D, F) cells. (G) The anti-tumor activity of T cells against U251 treated by DAC and TSA. (H) The anti-tumor activity of T cells against U87 treated by TSA. (I) The cytotoxicity to DAC + TSA-treated U251 by P8 induced T cells blocked by Anti-HLA antibodies. (J) The cytotoxicity to TSA-treated U87 by P8 induced T cells blocked by Anti-HLA antibodies. DC, dendritic cell; Tc, T cell; NC, negative control peptide. *P <0.05.
Figure 7
Figure 7
P8-specific T cells inhibited the in vivo growth of U87 cell-derived tumors. (A) Tumor growth was suppressed by the adoptive transfer of P8-specific T cells. (B) Tumors harvested from mice that were inoculated with different groups of T cells and glioma cells. (C) CD8+ T cells infiltrated glioma tissue in vivo. DC, dendritic cell; Tc, T cell; NC, negative control peptide. *P <0.05, **P <0.01, scale bar: 50 μm.
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