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 4

Downregulation of NKG2A increases NK cell cytotoxicity against tumor cells expressing HLA-E with HLA-G signal peptide (GpHLA-E).

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Downregulation of NKG2A increases NK cell cytotoxicity against tumor cel...
(A) Four-hour cytotoxicity assays with NKG2A+ NK cells transduced with anti-NKG2A PEBL or GFP only (Control). Target cell lines were transduced with GpHLA-E (see Supplemental Figure 7) and luciferase. BrightGlo was added after 4 hours of coculture, and luminescence was measured using a Flx 800 plate reader. Data are shown as mean (± SD) of cell killing using target cells cultured without NK cells as reference. NK cells from 11 donors were tested with K562, 6 with U2OS, 8 with ES8, and 5 with EW8, all in triplicate. (B) Expression of CD107a among NK cell subsets after 4-hour coculture with K562-GpHLA-E cells. Percentages are shown as mean of triplicate measurements with NK cells from 1 donor. (C) Four-hour cytotoxicity (measured as in A) of GFP-transduced NK cells against GpHLA-E–transduced target cells in the presence of the anti-NKG2A antibody Z199 compared with that of anti-NKG2A PEBL-transduced NK cells. An isotype-matched (mIgG2b) nonreactive immunoglobulin served as a control. Data are shown as mean (± SD) of triplicate measurements with NK cells from 2 donors (K562, ES8) or 1 donor (U2OS). (D) Long-term cytotoxicity of PEBL-transduced and control NK cells against GpHLA-E+ target cells. Experiments were performed at E/T 1:8 for K562, 1:2 for U2OS, and 1:4 for ES8. Tumor cell growth was measured with IncuCyte Zoom System. Data are shown as mean (± SD) of triplicate measurements with NK cells from 1 representative donor (out of 3 tested) compared with growth of the cell line without NK cells. (E) Spheroid tumors formed with U2OS-GpHLA-E cells transduced with mCherry were cocultured with PEBL-transduced or control NK cells at a 2:1 E/T. Images were collected with the IncuCyte Zoom System. Scale bars: 300 μm. Numerical data are shown in Supplemental Figure 9. *P < 0.05; **P < 0.01; ****P < 0.0001, t test.