Histone deacetylase inhibitors induce TAP, LMP, Tapasin genes and MHC class I antigen presentation by melanoma cells

Abstract

Histone deacetylase inhibitors (HDACi), including trichostatin A (TSA) and valproic acid, can alter the acetylation of histones in chromatin and enhance gene transcription. Previously we demonstrated that HDACi-treated tumor cells are capable of presenting antigen via the MHC class II pathway. In this study, we show that treatment with HDACi enhances the expression of molecules (TAP1, TAP2, LMP2, LMP7, Tapasin and MHC class I) involved in antigen processing and presentation via the MHC class I pathway in melanoma cells. HDACi treatment of B16F10 cells also enhanced cell surface expression of class I and costimulatory molecules CD40 and CD86. Enhanced transcription of these genes is associated with a significant increase in direct presentation of whole protein antigen and MHC class I-restricted peptides by TSA-treated B16F10 cells. Our data indicate that epigenetic modification can convert a tumor cell to an antigen presenting cell capable of activating IFN-gamma secreting T cells via the class I pathway. These findings suggest that the abnormalities, observed in some tumors in the expression of MHC class I antigen processing and presentation molecules, may result from epigenetic repression.

Fig. 1

HDACi treatments enhance the genes involved in antigen processing and presentation via MHC class I pathway in tumor cells. a B16F10, b B16F0 and c Colon 26 cells were treated with TSA (250 nM) or IFN-γ (100 U/ml) and mRNA for H-2D, TAP1, TAP2, LMP2, LMP7 and Tapasin was amplified by RT-PCR at 24 h. The data presented represent more than three independent experiments

Fig. 2

Quantitative analysis of MHC class I antigen processing gene expression in HDACi-treated melanoma cells. TSA treatment (24 h) induced approximately 5–20-fold increase in mRNA for TAP1, TAP2, Tapasin and H-2D in B16F10 cells. LMP2 and LMP7 mRNA was also increased approximately 75 and 30-fold respectively in B16F10 cells after TSA treatment compared to untreated control. These real-time PCR experiments were repeated three times with similar results

Fig. 3

HDACi treatments enhance expression of MHC class I, CD40 and CD86 on melanoma cells. B16F10 cells were stained with specific mAb and isotype controls after treatment with TSA (500 nM for 48 h), VA (500 μM for 48 h) or IFN-γ (100 U/ml for 24 h) and analyzed by flow cytometry to assay expression of the indicated surface markers. Isotype controls are shown as shaded peaks and heavy lines represent expression determined by specific antibody staining. Values indicated in the histograms are the percent of cells positive for the respective mAb relative to the isotype staining. The data presented represent more than three independent experiments

Fig. 4

MHC class I restricted antigen presentation by epigenetically altered melanoma cells. CD8⁺ T cells, isolated from ova-primed OT-I mice and purified magnetically with microbead-labeled antibodies, were cultured in triplicate with TSA treated (500 nM for 48 h) or untreated B16F10 cells for 24 h. Splenocytes isolated from OT-I mice were used as positive control. Splenocytes and B16F10 cells (TSA-treated and untreated) were pulsed with ova, ova–peptide_257–266 or the control peptide mgp100 and irradiated before use in the assay. Standard ELISpot assays were performed to measure IFN-γ secreting CD8⁺ T cells. Asterisks indicate number of IFN-γ spots detected with ova and ova–peptide_257–266-pulsed TSA-treated B16F10 is statistically significant compared with untreated cells. Similar results were obtained from three independent experiments