ObjectivesAnemia is a common complication of malignancy, which could result from either compromised erythropoiesis or decreased lifespan of circulating erythrocytes. Premature suicidal erythrocyte death, characterized by cell shrinkage and phosphatidylserine (PS) externalization, decreases erythrocyte lifespan and could thus cause anemia. Here, we explored whether accelerated eryptosis participates in the pathophysiology of anemia associated with lung cancer (LC) and its treatment.MethodsErythrocytes were drawn from healthy volunteers and LC patients with and without cytostatic treatment. PS exposure (annexin V-binding), cell volume (forward scatter), cytosolic Ca2+ (Fluo3 fluorescence), reactive oxygen species (ROS) production (DCFDA fluorescence) and ceramide formation (anti-ceramide antibody) were determined by flow cytometry.ResultsHemoglobin concentration and hematocrit were significantly lower in LC patients as compared to healthy controls, even though reticulocyte number was higher in LC (3.0±0.6%) than in controls (1.4±0.2%). The percentage of PS-exposing erythrocytes was significantly higher in LC patients with (1.4±0.1%) and without (1.2±0.3%) cytostatic treatment as compared to healthy controls (0.6±0.1%). Erythrocyte ROS production and ceramide abundance, but not Fluo3 fluorescence, were significantly higher in freshly drawn erythrocytes from LC patients than in freshly drawn erythrocytes from healthy controls. PS exposure of erythrocytes drawn from healthy volunteers was significantly more pronounced following incubation in plasma from LC patients than following incubation in plasma from healthy controls.ConclusionAnemia in LC patients with and without cytostatic treatment is paralleled by increased eryptosis, which is triggered, at least in part, by increased oxidative stress and ceramide formation.

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 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..

NK cells play an important role in tumor immunosurveillance and largely contribute to the therapeutic success of anti-tumor antibodies like Rituximab. Here, we studied the role of the TNF family member 4-1BB ligand (4-1BBL) during the interaction of NK cells with chronic lymphocytic leukemia (CLL) cells. 4-1BBL was highly expressed on patient B-CLL cells in all 56 investigated cases. Signaling via 4-1BBL following interaction with 4-1BB, which was detected on NK cells of CLL patients but not healthy individuals, led to the release of immunoregulatory cytokines including TNF by CLL cells. CLL patient sera contained elevated levels of TNF and induced 4-1BB upregulation on NK cells, which in turn impaired direct and Rituximab-induced NK-cell reactivity against 4-1BBL-expressing targets. NK-cell reactivity was not only enhanced by blocking the interaction of NK cell-expressed 4-1BB with 4-1BBL expressed by CLL cells, but also by preventing 4-1BB upregulation on NK cells via neutralization of TNF in patient serum with Infliximab. Our data indicate that 4-1BBL mediates NK-cell immunosubversion in CLL, and thus might contribute to the reportedly compromised efficacy of Rituximab to induce NK-cell reactivity in the disease, and that TNF neutralization may serve to enhance the efficacy of Rituximab treatment in CLL.

3548 The TNF/TNF receptor (TNFR) family comprises various molecules that substantially influence cellular functions of both tumor and immune effector cells. The TNFR family member OX40 has been shown to influence proliferation and differentiation of T cells in autoimmune diseases. Here we studied the yet unknown role of OX40 in acute myeloid leukemia (AML). Substantial surface expression of OX40 was detected on malignant cells of AML patients in 24 of 60 (40%) investigated cases. Expression of OX40 mRNA and protein by leukemic cells was confirmed by analysis of AML cells lines, which displayed substantial surface levels in 6 of 7 investigated cases. Induction of OX40 signaling into AML cells by recombinant OX40L or agonistic antibodies lead to the release of cytokines like IL-10 and TNF which contribute to AML pathophysiology and stimulated metabolic activity (WST test) of the leukemia cells. Moreover, we found that NK cells, which play an important role in anti-tumor immunity and largely contribute to the clinical success of allogeneic stem cell transplantation (SCT) in AML, express OX40 ligand (OX40L) following activation, and OX40L triggering stimulated NK cell reactivity. Functional analyses with OX40 transfectants and OX40-negative controls confirmed that OX40L signaling promotes NK cell cytotoxicity and IFN-γ production. Furthermore, disruption of OX40-OX40L interaction by blocking OX40 F(ab)2 fragments resulted in reduced cytotoxicity and cytokine production of allogenic NK cells, thereby further confirming the stimulatory effect of OX40L on NK cell anti-leukemia reactivity when interacting with its AML-expressed counterpart. Our data suggest that OX40 is involved in disease pathophysiology of AML and identify OX40-OX40L interaction as previously unknown modulator of NK cell immunosurveillance in AML. Disclosures: No relevant conflicts of interest to declare.

<p>Individual data of cytokine release and metabolic activity upon OX40 stimulation of AML cells.</p>

<p>Correlative analysis of basal cytokine release, proliferation and apoptosis with OX40 expression on AML cells.</p>

<p>Correlation of OX40 expression on AML cells and NK cell count and activation.</p>

<p>Individual data of cytokine release and metabolic activity upon OX40 stimulation of AML cells.</p>