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PVRIG is a novel natural killer cell immune checkpoint receptor in acute myeloid leukemia

This study explored the novel immune checkpoint poliovirus receptor- related immunoglobulin domain-containing (PVRIG) in acute myeloid leukemia (AML). We showed that AML patient blasts consistently expressed the PVRIG ligand (poliovirus receptor-related 2, PVRL2). Furthermore, PVRIG blockade significantly enhanced naural killer (NK)-cell killing of PVRL2+, poliovirus receptor (PVR)lo AML cell lines, and significantly increased NK-cell activation and degranulation in the context of patient primary AML blasts. However, in AML patient bone marrow, NK-cell PVRIG expression levels were not increased. In order to understand how PVRIG blockade might potentially be exploited therapeutically, we investigated the biology of PVRIG and revealed that NK-cell activation resulted in reduced PVRIG expression on the cell surface. This occurred whether NK cells were activated by tumor cell recognition, cytokines (interleukin 2 [IL-2] and IL-12) or activating receptor stimulation (CD16 and NKp46). PVRIG was present at higher levels in the cytoplasm than on the cell surface, particularly on CD56bright NK cells, which further increased cytoplasmic PVRIG levels following IL-2 and IL-12 activation. PVRIG was continually transported to the cell surface via the endoplasmic reticulum and Golgi in both unstimulated and activated NK cells. Taken together, our findings suggest that anti-PVRIG blocking antibody functions by binding to surface-bound PVRIG, which undergoes rapid turnover in both unstimulated and activated NK cells. We conclude that the PVRIG-PVRL2 immune checkpoint axis can feasibly be targeted with PVRIG blocking antibody for NK-mediated immunotherapy of PVRL2+ AML.


Flow cytometry staining
In all experiments, cells were first labelled with LIVE/DEAD Fixable Yellow (ThermoFisher) in phosphate buffered saline (PBS) for 20 min at 4ºC and washed. For AML patient samples, cells were pre-incubated with human Fc block (BD Biosciences) for 10 min at 4ºC prior to surface staining. Cells were then stained with antibodies against surface markers in FACS buffer (PBS with 2% FCS) for 30 min at 4ºC. Cells were then washed and fixed in 2% paraformaldehyde (PFA, ThermoFisher) for 15 min at 4ºC, then resuspended in FACS buffer before acquisition on a FACSymphony (BD). For total (intracellular + surface) PVRIG staining, after staining for surface markers (CD56, CD16 and CD69) cells were washed and fixed using Fixation Buffer (eBioscience) for 20 min at 4ºC, then washed and stained with anti-PVRIG-PE in Permeabilization Buffer (eBioscience) for 30 min at 4ºC. Where indicated, DMFI of each marker was calculated as geometric mean of test -iso.

Chromium release assay
Chromium release assay was performed as described previously. 1 Briefly, target cells were labelled with 100 µCi Chromium-51 ( 51 Cr, PerkinElmer) for 1 hr at 37ºC, washed, then cocultured with PBMCs in triplicate wells at effector:target ratios from 32:1 to 2:1. Blocking or isotype control antibodies were each added at a final concentration of 10 µg/ml. EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,Nʹ,Nʹ-tetraacetic acid, Sigma) was added at a final concentration of 4 mM. Wells with targets alone (spontaneous release) and targets with 10% Triton X 100 (Sigma, maximum release) were included as controls. After 4 hr, supernatants were collected and the amount of 51 Cr released detected using a gamma counter (Wallac Wizard). The % specific lysis was calculated by [(experimental releasespontaneous release)/ (maximum release -spontaneous release)] * 100. NK:target ratio was calculated from the percentage of NK cells found in PBMCs, determined by flow cytometry.

Degranulation assay
Target cells were labelled with Cell Trace Violet (ThermoFisher) in PBS for 10 min at 37ºC, washed, then co-cultured with PBMCs in triplicate wells at the specified effector:target ratios.
Blocking or isotype control antibodies were each added at a final concentration of 10 µg/ml, and anti-CD107a AF488 was included during the co-culture period. Wells with targets alone or PBMCs alone were included as controls. After 4 hr, cells were washed and stained with LIVE/DEAD Fixable Yellow followed by antibodies against CD56, CD16, CD3 and CD69. Due to variation in baseline CD69 levels between donors, CD69 MFI was normalised as a percentage of the isotype control treated group. Supplementary Table S1. Antibodies used for flow cytometry.

Marker
Fluorochrome  antibodies, measured by 51 Cr release assay. Each graph shows mean ± SD of triplicates for an individual healthy donor (donor ID labelled above each graph), with NK:target ratios calculated using % of NK cells in PBMC as determined by flow cytometry. Significance determined by Student's t-test, * p < 0.05, ** p < 0.01, *** p < 0.01; red = anti-PVRIG vs iso, green = anti-TIGIT vs iso. Figure S3. Gating strategy for AML bone marrow. AML blasts were identified as CD45 lo SSC int , along with various combinations of the markers CD33, CD34, CD117, depending on the patient. Mature myeloid cells (CD45 hi SSC hi ) could be further subdivided into CD14 + CD11b + or CD14 -CD11b + cells. The CD14 -CD11b + subset was not present in all patients, and was absent in all healthy donors, therefore was not included in further analysis. Within the lymphoid population (CD45 hi SSC lo ), NK cells (CD56 + CD3 -), NKT cells (CD56 + CD3 + ), CD8 + T cells (CD3 + CD56 -CD8 + ) and CD8 -T cells (CD3 + CD56 -CD8 -) could be identified. For healthy donor BM, the same gating strategy was employed, with CD45 lo SSC int immature myeloid cells used as an analogous population to compare with AML blasts.