Human NK cells activated by EBV⁺lymphoblastoid cells overcome anti-apoptotic mechanisms of drug resistance in haematological cancer cells

Abstract

Natural killer (NK) cells recognize and eliminate transformed or infected cells that have downregulated MHC class-I and express specific activating ligands. Recent evidence indicates that allogeneic NK cells are useful to eliminate haematological cancer cells independently of MHC-I expression. However, it is unclear if transformed cells expressing mutations that confer anti-apoptotic properties and chemoresistance will be susceptible to NK cells. Allogeneic primary human NK cells were activated using different protocols and prospectively tested for their ability to eliminate diverse mutant haematological and apoptotic-resistant cancer cell lines as well as patient-derived B-cell chronic lymphocytic leukemia cells with chemotherapy multiresistance. Here, we show that human NK cells from healthy donors activated with Epstein Barr virus positive (EBV)-lymphoblastoid cells display an enhanced cytotoxic and proliferative potential in comparison to other protocols of activation such a K562 cells plus interleukin (IL)2. This enhancement enables them to kill more efficiently a variety of haematological cancer cell lines, including a panel of transfectants that mimic natural mutations leading to oncogenic transformation and chemoresistance (e.g., overexpression of Bcl-2, Bcl-X and Mcl-1 or downregulation of p53, Bak/Bax or caspase activity). The effect was also observed against blasts from B-cell chronic lymphocytic leukemia patients showing multi-resistance to chemotherapy. Our findings demonstrate that particular activated NK cells may overcome anti-apoptotic mechanisms and oncogenic alterations frequently occurring in transformed cells, pointing toward the use of EBV-lymphoblastoid cells as a desirable strategy to activate NK cells for the purpose of treating haematological neoplasia with poor prognosis.

Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells

“…Cell death was almost completely inhibited by EGTA indicating the granule exocytosis was the main mechanism employed by NK cells to kill B-CLL cells (data not shown). As previously found in NK cells activated with R69 LCLs (51), the NK cell receptors NKG2A, NKp30, NKp44, and DNAM and the cytotoxic protease granzyme B were upregulated in activated NK cells (data not shown).…”

“…Our group has previously described that NK cells activated with EBV-transformed R69 or 721.211 LCLs are able to eliminate cells from B-CLL patients without requiring supplementation with exogenous cytokines (51). Importantly, activated NK cells did not kill healthy non-transformed PBMCs.…”

“…NK cells were labeled with 1 μM of CellTracker™ Green CMFDA (Life Technologies) and incubated with target cells at 9:1 e:t ratio for 4 h. Subsequently, phosphatidylserine (PS) translocation and membrane damage were analyzed in the green or violet fluorescence negative target cell population by flow cytometry using annexin V and 7AAD as previously described (51). …”

“…We have previously shown that the selection of a proper activating stimulus is critical to generate activated NK cells able to kill chemoresistant hematological cancer cell lines as well as cells from B-CLL patients (51, 52). Allogeneic NK cells activated in the presence of EBV-transformed B-cell lymphoblastoid cell lines (LCL) presented significantly higher cytotoxicity than those generated with K562 cells and IL-2/IL-15.…”

How Do Cytotoxic Lymphocytes Kill Cancer Cells?

“…It has been found that Tc cells use PRF1 and GZMB to kill cells in which both the intrinsic mitochondrial pathway and caspases are blocked (93), highlighting the potential benefits of immunotherapy to treat cancer cells that do not respond to conventional therapy (3,4). We have recently confirmed in humans that allogeneic activated NK cells expressing GZMB eliminate hematologic cancer cells in which apoptosis is blocked by p53 mutation and overexpression of Bcl-x L or downregulation of Bak and Bax even in the presence of caspase inhibitors (99). However, under these circumstances, the phenotype of dying cells is not apoptotic, and PS translocation did not preceded membrane permeability.…”