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. 2013 Mar;24(3):295-305.
doi: 10.1089/hum.2012.143. Epub 2013 Mar 1.

Chimeric NKG2D CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by histone deacetylase inhibition

De-Gang Song  1 , Qunrui Ye, Stephen Santoro, Chongyun Fang, Andrew Best, Daniel J Powell Jr
Affiliations

Chimeric NKG2D CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by histone deacetylase inhibition

De-Gang Song et al. Hum Gene Ther. 2013 Mar.

Abstract

NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested. To redirect human T cells against NKG2DLs, an NKG2DL-specific CAR was generated by fusing the extracellular domain of the NKG2D receptor to the 4-1BB costimulatory and CD3-ζ chain signaling domains. In vitro expansion of chimeric NKG2D CAR T cells was delayed compared with untransduced T cells and control CAR T cells; the likely result of fratricide among activated T cells expressing NKG2DLs. However, NKG2D CAR T cells did expand and were selectively enriched during prolonged culture. In coculture, CD4(+) and CD8(+) NKG2D CAR T cells specifically recognized and killed NKG2DL-expressing ovarian cancer cell lines but not NKG2DL-negative cells. Notably, pretreatment of ovarian cancer cells expressing moderate to low levels of NKG2DLs with the histone deacetylase inhibitor sodium valproate (VPA) upregulated NKG2DL cell surface expression and consequently enhanced their immune recognition by chimeric NKG2D CAR T cells. Our results demonstrate that VPA-induced upregulation of NKG2DL expression enhances the immune recognition of ovarian cancer cells by engineered NKG2D CAR T cells, and rationalizes the use of VPA in combination with NKG2DL-targeted immunotherapy in ovarian cancer.

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Figures

FIG. 1.
FIG. 1.
Surface expression of NKG2D ligands in ovarian cancer cells. A panel of human ovarian cancer cell lines was stained with specific antibodies that recognize MICA; MICB; ULBP-1, -2, -3, or -4; or with a soluble human NKG2D–human IgG1 fusion protein (open histogram) or matched isotype controls (shaded histogram) and analyzed by flow cytometry. A murine mesothelioma cell line, AE17, was used as negative control.
FIG. 2.
FIG. 2.
Construction, expression of an NKG2D ligand (NKG2DL)-specific chimeric antigen receptor (CAR), and expansion of CAR T cells in vitro. (A) Schematic illustration of the lentiviral construct for the NKG2DL- and folate receptor-α (FR)-specific CARs (left) and domain architecture of endogenous NKG2D and engineered NKG2DL CAR (right). The chimeric NKG2D (chNKG2D) CAR contains the extracellular portion of the human NKG2D receptor, which linked to a CD8α hinge and transmembrane region, followed by an intracellular CD137 (4-1BB) costimulatory domain and a CD3ζ signaling motif. A similar FR-specific CAR using an anti-FR single-chain variable fragment (scFv) was constructed as an antigen-specific control CAR for assays. (B) NKG2DL-specific CAR and GFP coexpressed by both transduced human CD4+ and CD8+ T cells 14 days after transduction. (C) Comparison of expansion of chNKG2D CAR T cells, untransduced and FR-CAR T cells. (D) Temporal induction of apoptosis in untransduced (UNT), chNKG2D CAR, and FR-transduced CAR T cells after stimulation and transduction. (E) NKG2DLs are expressed on activated CD4+ and CD8+ T cells after activation in vitro. Shown is the mean±SD NKG2D+ T cell frequency from three independent assays. (F) NKG2DL-specific CARpos (GFP-expressing) T cells are enriched over time. Results for three independent donors are shown. 7-AAD, 7-aminoactinomycin D; ECD, extracellular domain; ICD, intracellular domain. Color images available online at www.liebertpub.com/hum
FIG. 3.
FIG. 3.
Recognition of human ovarian cancer cells by NKG2DL-specific CAR T cells. (A) Chimeric NKG2D (chNKG2D) CAR-modified T cells secrete IFN-γ during overnight culture with all NKG2DL-expressing ovarian cancer cells, but not NKG2D-negative AE17 mesothelioma cells. Mean IFN-γ concentration±SEM (pg/ml) from triplicate cultures is shown. (B) To show NKG2D dependence, NKG2DL-specific CAR T cells were incubated with anti-NKG2D antibodies or with isotype control IgG antibodies before incubation with tumor cells for 6 hr. Blocking NKG2D markedly reduced the amount of IFN-γ released by NKG2DL-specific CAR T cells against tumor cells at all ratios compared with control. (C) Lysis of NKG2DL-expressing tumor cells (A1847 and OVCAR5) by CAR CD4+ and CD8+ T cells in an 18-hr bioluminescence assay at the indicated effector-to-target (E/T) ratios. Untransduced CD4+ and CD8+ human T cells and AE17 mesothelioma cells served as negative effector and target cell controls, respectively.
FIG. 4.
FIG. 4.
Increased NKG2DL surface expression by human ovarian cancer cells after sodium valproate (VPA) treatment. (A) Effect of VPA on cell viability. The viability of ovarian cancer cells was analyzed via 7-aminoactinomycin D (7-AAD) staining 48 and 96 hr after VPA (final concentration, 0–32 mM) treatment. (B) Histograms show the surface expression of the respective NKG2DLs on OVCAR5, A2780, and PEO-1 ovarian cancer cells treated with VPA (dashed histograms) or without treatment (open histograms). Shaded histograms represent matched isotype controls. (C) Histograms show the surface expression of the respective NKG2DLs on normal primary ovarian cells from donors #1744 and #1804 with VPA (dashed histograms) or without VPA (open histograms). Ovarian cancer cells from patient PT#1801 are shown for comparison. Shaded histograms represent matched isotype controls.
FIG. 5.
FIG. 5.
Upregulation of NKG2DL expression after VPA treatment enhances the susceptibility of ovarian cancer cells to NKG2DL-specific CAR T cell-mediated attack. The antitumor effect of NKG2DL-specific CAR T cells against untreated or VPA-pretreated ovarian cancer cells was assessed by IFN-γ release after overnight coculture, using lymphocytes from three different donors. Mean values of triplicate assays are shown. p<0.05 for all VPA versus untreated conditions for all cancer cell lines and donor lymphocytes tested.
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