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PVRIG blockade in acute myeloid leukemia
be a means to increase the net activation signal, which, in conjunction with decreased PVRIG expression, could serve to lower the activation threshold of the NK cell.
Intracellular PVRIG does not decrease upon activation
We next investigated whether PVRIG levels are regulat- ed differently in the two principal peripheral blood NK-cell subsets, CD56dimCD16+ and CD56brightCD16–. After IL-2 and IL-12 stimulation, the CD56dim subset showed decreased PVRIG surface expression, as was seen previ- ously for unfractionated NK cells (Figure 5A and B). This was unsurprising given that CD56dim comprise 90-95% of all circulating NK cells. By contrast, CD56bright NK cells showed no loss of PVRIG (Figure 5A and B). This was not due to failed activation, as both subsets upregulated CD69 equally (data not shown). Interestingly, this distinct pattern of regulation of PVRIG occurred only with cytokine stim- ulation (Figure 5B), but not following interaction with tar- get cells, which caused both subsets to downregulate PVRIG (Figure 5C). We next determined whether an intra- cellular pool of PVRIG exists, or if it is solely expressed on the cell surface. Using permeabilized cells, we detected total (surface plus intracellular) PVRIG levels. For both CD56dim and CD56bright NK-cell subsets, total PVRIG stain- ing was far greater than surface staining, indicating that a pool of PVRIG is present in the cytosol (Figure 5A). Interestingly, while CD56dim NK cells lost their surface PVRIG upon activation with IL-2 and IL-12, the total amount of PVRIG was unchanged compared with untreat- ed NK cells (Figure 5D). This suggested that PVRIG lost from the cell surface upon activation was internalized. Alternatively, the total PVRIG level could be maintained despite decreased surface expression by synthesis of new molecules, which then egress to the cell surface. The latter appeared to be the case for CD56bright NK cells, which maintained PVRIG surface expression and increased total PVRIG levels upon activation by IL-2 and IL-12 (Figure 5E).
Natural killer cell surface PVRIG levels are maintained by continuous trafficking to the cell surface
In order to further explore the mechanisms by which NK cells regulate surface PVRIG levels, we first assessed the kinetics of PVRIG loss from the cell surface under dif- ferent stimulation conditions using a short term time course. NK cells co-cultured with K562 cells showed loss of PVRIG expression within 1-2 hours, at which time CD69 began to be upregulated (Figure 6A-B). Stimulation of NK cells with anti-CD16 caused a similar level of PVRIG loss and activation, although K562 appeared to be the stronger stimulus at early time points (Figure 6A and B). Within the 4 hours assessed, IL-2 and IL-12 stimulation had no appreciable effect on PVRIG expression, and min- imal effect on activation (Online Supplementary Figure S5), indicating that cytokine stimulation influences PVRIG lev- els more slowly. Next, we used monensin and brefeldin A to determine whether intracellular trafficking via the endoplasmic reticulum (ER) and Golgi network was important for maintaining NK-cell surface PVRIG. Both monensin and brefeldin A inhibit the trafficking of mole- cules to the cell surface, by disrupting the Golgi apparatus, trans-Golgi network or endosomal network.23-26 Untreated NK cells maintained constant PVRIG surface levels over 4 hours. However, the addition of either monensin or brefeldin A resulted in significant loss of surface PVRIG levels (Figure 6C). Interestingly, the presence of monensin
or brefeldin A also caused greater loss of PVRIG in NK cells undergoing activation via anti-CD16 stimulation (Figure 6D). These results suggest there is trafficking of PVRIG molecules to the cell surface, via the ER and Golgi, in both untreated and activated NK cells (Figure 6E).
In summary, PVRIG is downregulated on the NK-cell surface following activation by tumor targets, anti-CD16 or cytokines. Furthermore, a pool of PVRIG is present in the NK-cell cytoplasm, and cell surface PVRIG is main- tained by trafficking to the surface. Taken together, our findings suggest that anti-PVRIG blocking antibodies enhanced NK-cell killing of AML target cells by blocking PVRIG present on the NK-cell surface. This resulted in decreased PVRL2-PVRIG mediated inhibition, and a decreased threshold for NK-cell activation and increased AML blast killing.
Discussion
Enhancing the activity of NK cells following HSCT may be beneficial for AML patients. NK cells are the first lym- phoid cells to be reconstituted after HSCT, reaching nor- mal levels within 1 month after transplant, much earlier than T cells.27,28 However, their capacity to kill residual leukemic blasts can be limited by the interaction of NK- inhibitory receptors with ligands in the tumor microenvi- ronment.27,29,30 Thus, blocking inhibitory receptors such as PVRIG could potentially be useful after HSCT to enhance NK-cell activity to delay or prevent relapse.
In this study, we showed PVRIG blockade enhanced human NK-cell activity against PVRL2hiPVRlo AML target cells. AML blasts in patient bone marrow were PVRL2hiPVRlo, suggesting PVRIG blockade may increase NK-mediated killing of AML blasts. The AML blast PVRL2hiPVRlo phenotype is consistent with previous stud- ies in AML patients.31 Our study is the first to report NK cell PVRIG expression in AML patient bone marrow. NK-cell PVRIG expression was not upregulated in AML patients. Our subsequent analysis suggested PVRIG upregulation is not required for PVRIG blockade to be effective. Even though interaction with AML cells caused loss of PVRIG from the NK-cell surface, PVRIG molecules trafficked via the ER-Golgi network and were then expressed on the cell surface. This suggests that, over time, a far greater amount of PVRIG is available on the cell surface to be blocked by anti-PVRIG antibodies than is detected at a single time point.
In contrast to PVRIG, other NK-cell immune checkpoint receptors, such as TIGIT, are upregulated with activa- tion.2,11 Despite sharing the same ligand as TIGIT, the modulation of PVRIG does not follow this model, suggest- ing it could have a distinct biological function. It is possi- ble that PVRIG acts as a regulator to keep NK cells in check in the steady state. The downregulation of PVRIG in response to cytokines or target recognition would then allow greater activation of NK cells in response to inflam- matory stimuli. Our data showed NK cell PVRIG was present at higher levels in the cytoplasm than on the cell surface; this intracellular PVRIG was not decreased by activation. This cytoplasmic pool of PVRIG could repre- sent either newly synthesized or recycled protein. A recent study by Whelan et al.3 examined PVRIG expres- sion on isolated human T cells, and observed a similar trend for loss of PVRIG expression immediately after acti-
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