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Individual data of cytokine release and metabolic activity upon OX40 stimulation of AML cells.

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.

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-γ 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. [Cancer Res 2009;69(3):1037–45]

PDF file - 264K, (A) The ability of MM cells to release sRANKL does not influence NK reactivity induced by RANK-Fc-ADCC. The increase in cytotoxicity (left) and cytokine production (right) of allogeneic NK cells using RANKL surface-positive MM cells that do (+sRANKL) or do not (-sRANKL) release sRANKL as targets upon treatment with RANK-Fc-ADCC was calculated. To this end, specific lysis (E:T ratio 40:1) or cytokine release (E:T ratio 1:1) of NK cells in the presence of isotyp control-treated target cells was set to 1 in each individual data set. No statistically significant difference (p=0.39 and p=1, respectively; Mann-Whitney U-Test) was observed upon analysis of results from 8 (cytotoxicity) and 6 (cytokine release) independent experiments. (B) Surface levels of RANKL do not correlate with induction of NK reactivity by RANK-Fc-ADCC. Expression of RANKL on patient MM (circles) and CLL cells (triangles) was correlated with the increase in cytotoxicity (left) and cytokine production (right) of allogeneic NK cells upon treatment with RANK-Fc-ADCC calculated by as described in A. Each symbol represents the result of one independent experiment. The number of independent experiments, correlation coefficients (Pearson correlation) and p values (T-Test) are indicated. (C) NK reactivity against RANKL surface-negative MM cells is not affected by the fusion proteins. RPMI 8226 MM cells (left) and primary MM cells (right) that lack RANKL surface expression were incubated with allogeneic NK cells in the presence or absence of 10mug/ml of the indicated RANK fusion proteins or isotype control. Cytotoxicity was determined by 2h Europium release assays (upper panels) and IFN-gamma levels in supernatants were analyzed after 24h by ELISA (lower panels). (D) RANKL transfectants were cultured with allogeneic NK cells in the presence or absence of RANK-Fc-ADCC or isotype control (10�g/ml each) and the indicated concentrations of rRANKL. Cytotoxicity was determined by 2h Europium release assays (left) and IFN-� levels in supernatants were analyzed after 24h by ELISA (right). One repesentative experiment each of a total of three with similar results is shown in C and D.

PDF file - 184K, (A) The RANKL antibodies MIH23 and MIH24 specifically bind to RANKL. Binding of MIH23 and MIH24 to surface expressed RANKL was determined by FACS using RANKL transfectants (L-RANKL) and control cells (L cells). Shaded peaks, specific mAb (MIH23 and MIH24); open peaks, isotype controls (mouse IgM and mouse IgG2b, respectively). (B) MIH23 and MIH24 block RANK-RANKL interaction. RANKL transfectants and the parental controls were stained with RANK-Ig (shaded peaks) or human IgG1 (open peaks) followed by anti-human-PE and investigated by FACS. Dotted line: staining after preincubation with the indicated RANKL mAb; dashed line: after preincubation with mouse IgM and mouse IgG2b as respective isotype controls. (C) Sensitivity and specificity of the sandwich ELISA for detection of sRANKL. The indicated serial dilutions of rRANKL in culture medium were analyzed as described in the methods section. X represents 100ng/ml rGITRL as control. The dotted line represents the chosen detection limit of 0.05ng/ml. Means of triplicates with standard deviations are shown. (D, E) MM cell lines do not display RANKL protein on the surface despite expression of mRANKL mRNA. The indicated MM cell lines were analyzed for RANKL surface and mRNA expression by FACS (D) and RT-PCR (E) as described in the methods section. Results obtained with RANKL transfectants and parental cells are shown as controls.

Supplementary Figure 1 from Glucocorticoid-Induced Tumor Necrosis Factor Receptor–Related Protein Ligand Subverts Immunosurveillance of Acute Myeloid Leukemia in Humans

The Chapter was inadvertently published without Acknowledgement. We have now added the acknowledgement in the chapter. Please find the same below.

Transcriptomic profiling by RNA sequencing (RNA-Seq) represents the preferred approach to measure genome-wide gene expression for understanding cellular function, tissue development, disease pathogenesis, as well as to identify potential biomarkers and therapeutic targets. For samples with small cell numbers, multiple methods have been described to increase the efficiency of library preparation and to reduce hands-on time and costs. This chapter reviews our approach, which combines flow cytometry and the most recent high-resolution techniques to perform RNA-Seq for samples with low cell numbers as well as for single-cell samples. Our approach reduces technical variability while increasing sensitivity and efficiency. Thus, it is well-suited for large-scale gene expression profiling studies with limited samples for basic and clinical studies.

Recruitment of Fc-receptor-bearing effector cells, such as natural killer (NK) cells, is a feature critical for the therapeutic success of antitumor antibodies and can be improved by the modifications of an antibody's Fc part. The various ligands of the activating immunoreceptor NKG2D, NKG2DL) are selectively expressed on malignant cells including leukemia. We here took advantage of the tumor-associated expression of NKG2DL for targeting leukemic cells by NKG2D-immunoglobulin G (IgG)1 fusion proteins containing modified Fc parts. Compared to NKG2D-Fc containing a wild-type Fc part (NKG2D-Fc-WT), our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell Fc receptor, respectively. Functional analyses with allogenic as well as autologous NK cells and primary malignant cells of leukemia patients revealed that NKG2D-Fc-KO significantly reduced NK reactivity by blocking immunostimulatory NKG2D-NKG2DL interaction. NKG2D-Fc-WT already enhanced antileukemia reactivity by inducing antibody-dependent cellular cytotoxicity (ADCC) with NKG2D-Fc-ADCC mediating significantly stronger effects. Parallel application of NKG2D-Fc-ADCC with Rituximab caused additive effects in lymphoid leukemia. In line with the tumor-associated expression of NKG2DL, no NK cell ADCC against resting healthy blood cells was induced. Thus, NKG2D-Fc-ADCC potently enhances NK antileukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL and may constitute an attractive means for immunotherapy of leukemia.