Key Points Monocytic IDOhi MDSCs are increased in CLL patients, suppress T cells, and promote TReg induction. CLL cells induce conversion of monocytes into MDSCs suggesting bidirectional crosstalk between CLL cells, MDSCs, and TRegs.

Our data indicate that the adoptive transfer of antigen-specific T cells in melanoma patients can induce clinical tumor-specific immune responses without major adverse effects.

Key Points• Increased mitochondrial ROS production, adaptation to intrinsic oxidative stress, and mitochondrial biogenesis are interconnected in CLL.• Targeting the respiratory chain and promoting mitochondrial ROS lead to selective cytotoxicity in CLL cells.Alterations of cellular metabolism represent a hallmark of cancer. Numerous metabolic changes are required for malignant transformation, and they render malignant cells more prone to disturbances in the metabolic framework. Despite the high incidence of chronic lymphocytic leukemia (CLL), metabolism of CLL cells remains a relatively unexplored area. The examined untreated CLL patients displayed a metabolic condition known as oxidative stress, which was linked to alterations in their lymphoid compartment. Our studies identified mitochondrial metabolism as the key source for abundant reactive oxygen species (ROS). Unlike in other malignant cells, we found increased oxidative phosphorylation in CLL cells but not increased aerobic glycolysis. Furthermore, CLL cells adapted to intrinsic oxidative stress by upregulating the stress-responsive hemeoxygenase-1 (HO-1). Our data implicate that HO-1 was, beyond its function as an antioxidant, involved in promoting mitochondrial biogenesis. Thus ROS, adaptation to ROS, and mitochondrial biogenesis appear to form a self-amplifying feedback loop in CLL cells. Taking advantage of the altered metabolic profile, we were able to selectively target CLL cells by PK11195. This benzodiazepine derivate blocks the mitochondrial F1F0-ATPase, leads to a surplus production of mitochondrial superoxide, and thereby induces cell death in CLL cells. Taken together, our findings depict how bioenergetics and redox characteristics could be therapeutically exploited in

In eukaryotic cells the phospholipid phosphatidylserine (PS) is restricted to the inner plasma-membrane leaflet. This lipid asymmetry, which is maintained by the concerted action of phospholipid transport proteins, is mainly lost during apoptosis. Here, we demonstrate that primary human CD8 ؉ cytotoxic T lymphocytes (CTLs) expose PS on T-cell receptor (TCR)-mediated antigen (Ag) recognition. In contrast to PS externalization on apoptotic cells, activation-induced PS exposure is less pronounced and reversible. Fluorescence microscopic analysis revealed that PS is distributed nonhomogenously over the plasma membrane and concentrated in membrane lipid raft domains at the immunologic synapse. By studying the activity of PS transport proteins using a fluorescence-labeled PS analogue, we found that activation of CTLs inhibited the flippase-mediated inwarddirected PS transport without affecting the outward transport. Shielding of exposed PS by annexin V protein during Ag recognition diminished cytokine secretion, activation, and cell-to-cell clustering of Ag-specific CTLs. In summary, our data demonstrate for the first time that externalized PS on Ag-stimulated CTLs is linked to T-cell activation and probably involved in cell-to-cell contact formation at the immunologic synapse. IntroductionMost eukaryotic cells exhibit an asymmetric distribution of phospholipids (PLs) in their plasma membrane with phosphatidylcholine and sphingomyelin concentrated in the outer leaflet and phosphatidylethanolamine and phosphatidylserine (PS) predominantly located in the cytosolic leaflet. 1 PS is the only PL thereof that is completely restricted to the inner membrane layer.This PL asymmetry is maintained by an energy-dependent flippase, known as the aminophospholipid translocase (APTL), that uses ATP hydrolysis to catalyze a fast, inward-directed transport of aminophospholipids PS and phosphatidylethanolamine across the plasma membrane. 2 A second class of ATP-dependent lipid transporters, the floppases, mediates outward-directed PL transfer. In contrast to these energydependent lipid transporters the activation of a Ca 2ϩ -dependent PL scramblase, a putative membrane protein facilitating a rapid equilibration of PLs between the 2 plasma-membrane leaflets, causes the disruption of lipid asymmetry. The appearance of PS at the cell surface as a consequence of the loss of lipid asymmetry is described predominantly during early apoptosis and is reported to be a phylogenetically conserved process. [3][4][5][6] For apoptotic lymphocytes PS exposure has been ascribed to an inhibition of the APTL activity in parallel to an activation of the PL scramblase. Both processes rapidly lead to a randomization of the trans-bilayer lipid distribution. [7][8][9] Annexin V (annV) is a member of a large family of Ca 2ϩ -and PL-binding proteins. 10 In the presence of physiologic concentrations of Ca 2ϩ annV has a high affinity for PS. Fluorescence-labeled annV is widely used to detect early apoptotic cells by flow cytometry. 11,12 However, an externalizati...

Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the “classical” pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine rather than putrescine may be the main purpose of the alternative pathway of polyamine synthesis, while the classical pathway supplies putrescine and spermidine/spermine in these neurons.