Figure 7: HDAC3 inhibition induces antigen-dependent immune responses.
A) A schematic of the generation of antigen-specific T-cells and epigenetic priming of DLBCL cells. A human DLBCL cell line (OCI-Ly18) was engrafted into immunodeficient mice and allowed to establish. Human T-cells were then engrafted, exposing them to tumor antigens prior to harvesting of the tumor-infiltrating T-cell (TIL) fraction. These TILs were cultured with fresh DLBCL cells that had been epigenetically primed with different concentrations of BRD3308, and the cell viability of the DLBCL cells measured after 72h. B) TIL and DLBCL co-culture resulted in activation of the CD4 T-cells in a dose-dependent manner, as measured by flow cytometry for the CD69 activation marker. Data represent the fold change in CD69 expression compared to vehicle treated DLBCL cells (T-test vs DMSO control, *P<0.05). C) The cell viability of DLBCL cells in TIL co-culture experiments was measured by CellTiterBlue assay. Treatment with BRD3308 resulted in some cell killing through cell-intrinsic mechanisms in the absence of TILs (black). The addition of TILs at a 1:1 ratio led to a significant increase in cell death of the DLBCL cells. This was partially reduced by blocking of either MHC class I or MHC class II using neutralizing antibodies. Blocking of MHC class I and class II together completely eliminated the TIL-associated increase in cell death, suggesting that killing was mediated through MHC:TCR interactions. (T-test, *P<0.05, ***P<0.001) D) The production of IFN-γ was measured by ELISPOT and found to increase in cultures with epigenetically-primed DLBCL cells. (T-test vs DMSO control, **P<0.01, ***P<0.001) E) A syngeneic BCL6-dependent lymphoma model for in vivo testing of BRD3308 and PD-L1 blocking antibodies. Splenocytes were taken from Ezh2Y641 x IμBcl6 mice and injected into irradiated wild-type recipients that were treated upon the onset of lymphoma. F) Serum IFN-γ levels measured in mice following treatment. G-N) Representative immunofluorescence images of mouse spleens following treatment and quantification of mean fluorescence intensities from multiple mice for CD8 (G, H), CD4 (I, J), PD-L1 (K, L) and B220 (M, N), showing increased T-cell infiltration following treatment with BRD3308 and cooperation with αPD-L1 in eliminating B220+ tumor cells within the spleen. (T-test; *P<0.05, **P<0.01, ***P<0.001)