Combined Blockade of TIGIT and CD39 or A2AR Enhances NK-92 Cell-Mediated Cytotoxicity in AML.

This study aimed to characterize different natural killer (NK) cell phenotypes on bone marrow and peripheral blood cells from acute myeloid leukemia (AML) patients and healthy donors (HDs). Our data show that CD56dimCD16- and CD56brightCD16- NK cells represent the predominant NK cell subpopulations in AML, while the CD56dimCD16+ NK cells are significantly reduced compared to HDs. Moreover, TIGIT+ and PVRIG+ cells cluster on the CD56dimCD16+ subset whereas CD39+ and CD38+ cells do so on CD56brightCD16- NK cells in AML. Furthermore, functional effects of (co-)blockade of TIGIT and CD39 or A2AR on NK cell functionality were analyzed. These experiments revealed that the single blockade of the TIGIT receptor results in an increased NK-92 cell-mediated killing of AML cells in vitro. Combined targeting of CD39 or A2AR significantly augments the anti-TIGIT-mediated lysis of AML cells. Our data indicate that distinct NK cell subsets in AML exhibit different immunosuppressive patterns (via the TIGIT/PVRIG receptors and the purinergic pathway). In summary, we conclude that TIGIT, CD39, and A2AR constitute relevant inhibitory checkpoints of NK cells in AML patients. A combinatorial blockade synergistically strengthens NK-92 cell-mediated cytotoxicity. As inhibitors of TIGIT, CD39, and A2AR are clinically available, studies on their combined use could be conducted in the near future.

Radiosensitization effect by HDAC inhibition improves NKG2D-dependent natural killer cytotoxicity in hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Radiotherapy (RT) controls HCC unsatisfactorily and temporarily. Histone deacetylase inhibitor (HDACi) is a heterogeneous group of epigenetic therapeutics with promising anticancer effects and synergism in combination with RT. HDACi modulates natural killer (NK) cell ligand expression on tumor cells, and leads to immune evasion of cancer cells. Expressions of NK group 2D (NKG2D) ligands on cancer cells determine the cytotoxic effect by interacting with NKG2D receptor on NK cells. However, the role of NKG2D signaling in HCC upon combined RT and HDACi remains unclear. Method: In vitro co-culture system with NK cells was tested for human and murine HCC cell lines. Pan-HDACi (panobinostat) and specific HDAC4 knockdown (HDAC4-KD) were used for HDAC inhibition. Clonogenic assay and flow cytometry examined HCC cell survival and NKG2D ligand expression, respectively. Syngeneic mouse model was used to validate the radiosensitizing effect in vivo. Results: Combined RT and HDACi/HDAC4-KD significantly enhanced NK cell-related cytotoxicity and increased NKG2D ligands, MICA/MICB expressions in human and RAE-1/H60 expressions in murine HCC cells. Delayed tumor growth in vivo by the combinational treatment of RT and HDACi/HDAC4-KD was shown with the associated NKG2D ligand expressions. However, NKG2D receptor did not significantly change among tumors. Conclusion: Radiosensitizing effect with combined RT and HDAC inhibition increased the expression of NKG2D ligands in HCC cells and enhanced their susceptibility to NK cell-mediated cytotoxicity. These findings imply the potential use of combined RT/HDACi and NK cell-directed immunotherapy.

Assessment of NK cell-mediated cytotoxicity by flow cytometry after rapid, high-yield isolation from peripheral blood.

Natural killer (NK) cells constitute the predominant innate lymphocyte subset that mediates the anti-viral and anti-tumor immune responses. NK cells use an array of innate receptors to sense their environment and to respond to infections, cellular stress and transformation. The resulting NK cell activation, including cytotoxicity and cytokine production, is a fundamental component of the early immune response. The most recent discoveries in NK cell biology have stimulated the translational research that has led to remarkable results for the treatment of human malignancies. Therefore, the rapid isolation of NK cells from the peripheral blood or tumor microenvironment and the subsequent assessment of cytolytic function are crucial to the study of their potency and NK cell-mediated immunosurveillance. Here, we provide protocols for NK cell isolation and the assessment of NK cell cytotoxicity using flow cytometry.

Cd226-/- natural killer cells fail to establish stable contacts with cancer cells and show impaired control of tumor metastasis in vivo.

CD226 is an activating receptor expressed on natural killer (NK) cells, CD8+ T cells, and other immune cells. Upon binding to its ligands expressed on target cells, CD226 activates intracellular signaling that triggers cytokine production and degranulation in NK cells. However, the role of CD226 in contact dynamics between NK and cancer cells has remained unclear. Our time-lapse images showed that individual wild-type CD226+ NK cells contacted B16F10 melanoma cells for 23.7 min, but Cd226-/- NK cells only for 12.8 min, although both NK cell subsets showed equal contact frequency over 4 h. On the surface of B16F10 cells, CD226+ cells stayed at the same site with oscillating movement (named stable contact), while Cd226-/- NK cells moved around at a velocity of 4 µm/min (named unstable contact). Consequently, Cd226-/- NK cells did not kill B16F10 cells in vitro and did not inhibit their metastasis into the lung in vivo. Taken together, our data demonstrate that CD226 enables prolonged stable interaction between NK and cancer cells, which is needed for efficient killing of cancer cells.

Interleukin 21 - its potential role in the therapy of B-cell lymphomas.

Interleukin-21 (IL-21), a member of IL-2 cytokine family, has pleotropic biological effects on lymphoid and myeloid cells. During the past 15 years, since the discovery of IL-21, great advances have been made regarding its biological activity and the mechanisms controlling IL-21-mediated cellular responses, especially in hematological malignancies. Preclinical studies have shown that IL-21R is expressed on healthy and neoplastic B-cells and exogenous IL-21 can induce direct apoptosis of IL-21R expressing B-cell non-Hodgkin lymphomas (NHL), making it a potentially attractive anti-lymphoma therapy. However, in some hematological malignancies such as multiple myeloma, Hodgkin lymphoma and Burkitt lymphoma, IL-21 can induce proliferation of neoplastic B-cells. In NHL, the underlying mechanism of cell death was found to be different between the various subtypes, including activation of different JAK/STAT signal transduction pathways or other factors. Immunomodulatory effects of IL-21 have also been reported to contribute to its anti-tumor effects as described by earlier studies in solid tumors and B-cell associated malignancies. These effects are predominantly mediated by IL-21's ability to activate cytolytic activities by NK-cells and CD4+/CD8+ T-cells. In this review, we provide an overview of IL-21's effects in NHL, results from clinical trials utilizing IL-21, and propose how IL-21 can be therapeutically exploited for treating these lymphomas.