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The TNF receptor family member OX40 promotes activation and proliferation of T cells, which fuels efforts to modulate this immune checkpoint to reinforce antitumor immunity. Besides T cells, NK cells are a second cytotoxic lymphocyte subset that contributes to antitumor immunity, particularly in leukemia. Accordingly, these cells are being clinically evaluated for cancer treatment through multiple approaches, such as adoptive transfer of ex vivo expanded polyclonal NK cells (pNKC). Here, we analyzed whether and how OX40 and its ligand (OX40L) influence NK-cell function and antileukemia reactivity. We report that OX40 is expressed on leukemic blasts in a substantial percentage of patients with acute myeloid leukemia (AML) and that OX40 can, after stimulation with agonistic OX40 antibodies, mediate proliferation and release of cytokines that act as growth and survival factors for the leukemic cells. We also demonstrate that pNKC differentially express OX40L, depending on the protocol used for their generation. OX40L signaling promoted NK-cell activation, cytokine production, and cytotoxicity, and disruption of OX40-OX40L interaction impaired pNKC reactivity against primary AML cells. Together, our data implicate OX40/OX40L in disease pathophysiology of AML and in NK-cell immunosurveillance. Our findings indicate that effects of the OX40-OX40L receptor-ligand system in other immune cell subsets and also malignant cells should be taken into account when developing OX40-targeted approaches for cancer immunotherapy. Cancer Immunol Res; 6(2); 209-21. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

The reciprocal interaction of tumor cells with the immune system is influenced by various members of the tumor necrosis factor (TNF)/TNF receptor (TNFR) family, and recently, glucocorticoid-induced TNFR-related protein (GITR) was shown to stimulate antitumor immunity in mice. However, GITR may mediate different effects in mice and men and impairs the reactivity of human natural killer (NK) cells. Here, we studied the role of GITR and its ligand (GITRL) in human acute myeloid leukemia (AML). Surface expression of GITRL was observed on AML cells in six of seven investigated cell lines, and 34 of 60 investigated AML patients whereas healthy CD34(+) cells did not express GITRL. Furthermore, soluble GITRL (sGITRL) was detectable in AML patient sera in 18 of 55 investigated cases. While the presence of GITRL was not restricted to a specific AML subtype, surface expression was significantly associated with monocytic differentiation. Signaling via GITRL into patient AML cells induced the release of TNF and interleukin-10 (IL-10), and this was blocked by the inhibition of mitogen-activated protein kinases extracellular signal-regulated kinase 1/2. Furthermore, triggering GITR by surface-expressed and sGITRL impaired NK cell cytotoxicity and IFN-gamma production in cocultures with leukemia cells, and NK cell reactivity could be restored by blocking GITR and neutralization of sGITRL and IL-10. Thus, whereas a stimulatory role of the GITR-GITRL system in mouse antitumor immunity has been reported, our data show that in humans GITRL expression subverts NK cell immunosurveillance of AML. Our results provide useful information for therapeutic approaches in AML, which, like haploidentical stem cell transplantation, rely on a sufficient NK cell response.