Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells
Takahiro Kamiya, … , Murray Robinson, Dario Campana
Takahiro Kamiya, … , Murray Robinson, Dario Campana
Published March 12, 2019
Citation Information: J Clin Invest. 2019;129(5):2094-2106. https://doi.org/10.1172/JCI123955.
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Research Article Immunology Oncology

Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells

Abstract

A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA class I molecule (HLA-E) in tumor cells, which suppresses NK cell activity via ligation of the NK inhibitory receptor CD94/NK group 2 member A (NKG2A). Gene expression data from approximately 10,000 tumor samples showed widespread HLAE expression, with levels correlating with those of KLRC1 (NKG2A) and KLRD1 (CD94). To bypass HLA-E inhibition, we developed a way to generate highly functional NK cells lacking NKG2A. Constructs containing a single-chain variable fragment derived from an anti-NKG2A antibody were linked to endoplasmic reticulum–retention domains. After retroviral transduction in human peripheral blood NK cells, these NKG2A protein expression blockers (PEBLs) abrogated NKG2A expression. The resulting NKG2Anull NK cells had higher cytotoxicity against HLA-E–expressing tumor cells. Transduction of anti-NKG2A PEBL produced more potent cytotoxicity than interference with an anti-NKG2A antibody and prevented de novo NKG2A expression without affecting NK cell proliferation. In immunodeficient mice, NKG2Anull NK cells were substantially more powerful than NKG2A+ NK cells against HLA-E–expressing tumors. Thus, NKG2A downregulation evades the HLA-E cancer immune checkpoint and increases the antitumor activity of NK cell infusions. Because this strategy is easily adaptable to current protocols for clinical-grade immune cell processing, its clinical testing is feasible and warranted.

Authors

Takahiro Kamiya, See Voon Seow, Desmond Wong, Murray Robinson, Dario Campana

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Figure 6

Cytotoxicity of NKG2Anull NK cells against tumor cells with endogenous HLA-E expression.

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Cytotoxicity of NKG2Anull NK cells against tumor cells with endogenous H...
(A) Four-hour cytotoxicity of NK cells transduced with anti-NKG2A PEBL or GFP only (Control) against cell lines expressing endogenous HLA-E (see Supplemental Figure 10). EW8 and PLC/PRF/5 were transduced with luciferase. BrightGlo was added after 4 hours of coculture, and luminescence was measured using a Flx 800 plate reader. Cytotoxicity of U937 and OP-1 was measured by flow cytometry. Box (25th–75th percentile, median) and whiskers (minimum-maximum) plots from 3 experiments with cells from 3 donors (EW8, PLC/PRF/5), and 6 experiments with cells from 2 donors (U937, OP-1) in triplicate, at 2:1, 1:1, or 1:2 E/T. (B) Spheroid tumors of U2OS-mCherry were cocultured with NK cells at 1:2 E/T in triplicate and analyzed with IncuCyte Zoom System. Data are shown as mean (± SD) red calibrated unit (RCU)/μM2. Representative images at end of culture are shown. Scale bars: 300 μm. (C) Four-hour cytotoxicity against cell lines exposed to IFN-γ (300 ng/ml; 12 hours). Plots are from 3 experiments with NK cells from 3 donors (EW8, PLC/PRF/5), and 6 with NK cells from 2 donors (U937) in triplicate at 2:1, 1:1, or 1:2 E/T. (D) Similar experiments targeting cells exposed for 12 hours to conditioned medium (C.M.) from 24-hour cocultures of NK cells with the respective cell lines. Four-hour cytotoxicity was compared with that against cells not exposed to conditioned medium. (E) Four-hour cytotoxicity against primary AML cells from 4 patients, exposed to IFN-γ (300 ng/ml; 12 hours). Data are from 4 experiments with NK cells from 2 donors in triplicate at 2:1 and 1:1 E/T. (F) NKG2A-negative NK cells from 3 donors were stimulated with K562-mb15-41BBL for 7 days, transduced with anti-NKG2A PEBL or GFP alone, and then exposed to IL-12 (20 ng/ml) for 5 days. Percentage of NKG2A+ cells at each stage is shown. (G) PEBL-transduced and control NK cells were exposed to IL-12 and tested in 4-hour cytotoxicity assays against K562-GpHLA-E cells. Data are shown as mean (± SD) of triplicate measurements at each E/T. **P < 0.01; ***P < 0.001; ****P < 0.0001, t test.