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J. Li et al.
1, suggesting that the inhibitory signal is dominant.1,11 PVRIG inhibitory function was shown using anti- PVRIG blocking antibodies. Xu et al. demonstrated that PVRIG blocking antibodies significantly increased NK-cell cytotoxicity against breast cancer cell lines in vitro, an effect that was further enhanced when used in combina- tion with TIGIT blocking antibodies.2 An independent group using a different anti-PVRIG antibody similarly showed that PVRIG blockade enhanced T-cell cytotoxicity against melanoma and pancreatic cancer cell lines, which was also augmented by combination with TIGIT block- ade.3 Notably, Whelan et al. demonstrated that T cells iso- lated from patient tumors and activated via CD3 increased interferon g production in response to combination PVRIG/TIGIT blockade.3 PVRIG blockade also reduced tumour burden in a mouse model when combined with anti-PDL1.14 On the basis of these data, a human IgG4 anti-PVRIG blocking antibody is currently undergoing phase I clinical trials in patients with advanced solid
tumors.15
As PVRIG is present on both T cells and NK cells, block-
ing PVRIG provides the opportunity to augment both major cytotoxic effector cell types. Although many studies focused on the capacity for immune checkpoint blockade to enhance T-cell responses, the contribution of NK cells should not be overlooked. For instance, tumors often downregulate human leukocyte antigen (HLA) class I to evade CD8+ T-cell recognition.16 However, this simultane- ously removes the ligand for killer cell immunoglobulin- like receptors (KIRs) on NK cells, rendering tumors more sensitive to NK cell-mediated killing.17 Reducing the inhibitory signal from KIR has also been shown to be effec- tive in controlling acute myeloid leukemia (AML). AML is an aggressive disease in which myeloid progenitor cells proliferate uncontrollably, and which is frequently treated with allogeneic hematopoietic stem cell transplant (allo- HSCT) when patients relapse after front-line chemothera- py. In a seminal study of allo-HSCT patients, mismatches between KIR on donor NK cells and recipient HLA was a key predictor of survival.18,19 Recipients lacking HLA lig- ands for one or more of the KIR expressed by the donor experienced graft-versus-host NK alloreactivity, which was significantly associated with a lower relapse rate.19,20
Given the pivotal role of NK cells in AML, strategies to enhance NK-cell activity could provide significant benefit for patients with AML, who have a 5-year survival rate of less than 30% with current treatments.21 This study aimed to determine whether PVRIG blockade could be used to enhance NK-cell responses against AML. Using healthy donor and AML patient blood or bone marrow samples, we evaluated the expression of PVRIG and PVRL2 on NK cells and AML blasts respectively. We also investigated whether PVRIG blockade could enhance NK-cell-mediat- ed killing of AML blasts, and the kinetics of PVRIG surface expression to reveal when the target is expressed follow- ing AML target cell activation of NK cells.
Methods
Reagents and cell lines
Anti-PVRIG and anti-TIGIT blocking antibodies were provided by Compugen, USA, Inc. Anti-DNAM-1 (11A8), anti-CD16 (3G8), anti-NKp46 (9E2), anti-2B4 (eBioC1.7) and anti-NKG2D (1D11) purified antibodies were purchased from Biolegend.
Recombinant human IL-2, IL-12, IL-15 and IL-18 were purchased from Peprotech. Monensin (GolgiStop, BD Biosciences) and brefeldin A (eBioscience) were both used at 1:1,000. Antibodies used for flow cytometry staining are listed in the Online Supplementary Table S1. SKBR3, KG1a, K562, ML-2, THP-1 and Kasumi-1 cell lines were maintained in RPMI 1640 (Gibco) supple- mented with Glutamax, penicillin, streptomycin and 10% (or 20% for Kasumi-1) fetal calf serum (FCS). AML-193 cell line was maintained in Iscove's Modified Dulbecco's Media supplemented with 5% FCS, 5 mg/mL transferrin, 5 mg/mL insulin and 2 ng/mL granulocyte-macrophage colony-stimulating factor. All cell lines tested negative for mycoplasma.
Acute myeloid leukemia patient and healthy donor bone marrow samples
All patient and healthy donor samples were obtained under ethics approval from the Peter MacCallum Cancer Center human ethics committee (HREC approval numbers 01/14 and 10-61). Cryopreserved AML patient diagnostic bone marrow samples were obtained from the Cancer Collaborative Biobank (Metro South Health, Queensland, Australia). Patient clinical characteris- tics are summarised in the Online Supplementary Table S2. Healthy donor bone marrow samples were obtained from Royal Melbourne Hospital (Melbourne, Australia) or purchased from AllCells (Alameda, California). All bone marrow samples were used for flow cytometry staining immediately after thawing.
Healthy donor peripheral blood mononuclear cells and natural killer cells
Peripheral blood mononuclear cells (PBMC) were isolated from healthy donor buffy coats (Australian Red Cross Blood Service) by density gradient (Ficoll-Paque, GE Healthcare Life Science) and cryopreserved. One day prior to experiments, PBMC were thawed and treated with DNase I (Merck) for 15 minutes at 37oC. Where required, NK cells were isolated by negative selection using a human NK Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions (except antibodies and beads were used at half the recommended concentration). The purity of NK cells as determined by flow cytometry was > 95%. Bulk PBMC or isolated NK cells were incubated in media containing 25 U/mL IL-2 overnight at 37oC before use in assays.
Natural killer cell stimulation
Isolated NK cells were incubated at 37oC alone, with the spec- ified combination of cytokines, with target cells at a 1:1 ratio, or in wells precoated (overnight 4oC) with agonistic antibodies against CD16, NKp46, 2B4 or NKG2D. After 24 hr, cells were washed and stained with LIVE/DEAD Fixable Yellow (ThermoFisher) followed by antibodies against CD56, CD16, CD69, PVRIG, TIGIT and DNAM-1. For analysis of short term kinetics, cells were incubated at 37oC with the indicated stimuli, and at the specified time points were transferred to 4oC. Cells for the 0 time point were kept at 4oC. Upon completion of all time points, cells were stained with LIVE/DEAD Fixable Yellow fol- lowed by antibodies against CD56, CD16, CD69 and PVRIG.
Other methods
Details of experimental procedures (flow cytometry, Chromium release assay, degranulation assay) are provided in the Online Supplementary Methods.
Data analysis
Flow cytometry data were analysed using FlowJo software (BD), and statistical analysis was performed in Prism (GraphPad). Figure 4G was created with BioRender.com.
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haematologica | 2021; 106(12)