Immune cells and platelets maintain plasma membrane phospholipid asymmetry. Upon activation, this asymmetry is disrupted by phospholipid scrambling (PS), which is a major step during activation of immune cells, hemostasis and apoptosis. Anoctamin 6 (Ano6; TMEM16F) causes chloride (Cl−) and cation currents and is required for Ca2+-dependent PS. It is defective in blood cells from patients with Scott syndrome, a rare bleeding disorder. We examined if Cl− currents and PS are related, whether both processes are Ca2+ dependent, and whether Ca2+-independent scrambling during intrinsic and extrinsic apoptosis is controlled by Ano6. Ca2+ increase by ionomycin activated Ano6 Cl− currents and PS in normal lymphocytes, but not in B-lymphocytes from two different patients with Scott syndrome. Fas ligand (FasL) did not increase intracellular Ca2+, but activated Cl− currents in normal but not in Scott lymphocytes. Whole-cell currents were inhibited by Cl− channel blockers and by siRNA knockdown of Ano6. In contrast, intrinsic mitochondrial apoptosis by ABT-737 did not induce Cl− currents in lymphocytes. PS was not inhibited by blockers of Ano6 or removal of Cl− ions. Remarkably, Ca2+-independent scrambling due to extrinsic (FasL) or intrinsic (ABT-737) apoptosis was unchanged in Scott cells. We conclude that: (i) Ano6 Cl− currents are activated by increase in cytosolic Ca2+, or Ca2+ independent by stimulation of Fas receptors; (ii) Ca2+-dependent PS induced by Ano6 does not require Cl− currents; (iii) Ca2+-independent PS does not require Ano6; (iv) Ano6 is necessary for Ca2+-dependent PS, but not by increasing intracellular Ca2+.

The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular, T cell regeneration is generally delayed, resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled, feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect, indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore, the addition of AA to feeder cocultures resulted in development of DP and SP T cells, whereas without AA, a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy.

Besides their role in destruction of altered self-cells, NK cells have been shown to potentiate T-cell responses by interacting with DC. To take advantage of NK-DC crosstalk in therapeutic DC-based vaccination for infectious diseases and cancer, it is essential to understand the biology of this crosstalk. We aimed to elucidate the in vitro mechanisms responsible for NK-cell recruitment and activation by DC during infection. To mimic bacterial infection, DC were exposed to a membrane fraction of Klebsiella pneumoniae, which triggers TLR2/4. DC matured with these bacterial fragments can actively recruit NK cells in a CCR5-dependent manner. An additional mechanism of DC-induced NK-cell recruitment is characterized by the induction of CCR7 expression on CD56 dim CD16 1 NK cells after physical contact with membrane fraction of K. pneumoniae-matured DC, resulting in an enhanced migratory responsiveness to the lymph node-associated chemokine CCL19. Bacterial fragment-matured DC do not only mediate NK-cell migration but also meet the prerequisites needed for augmentation of NK-cell cytotoxicity and IFN-c production, the latter of which contributes to Th1 polarization.Key words: CCR5 . CCR7 . NK-DC interaction . Th1 polarization Supporting Information available online Introduction NK cells are important effector cells in the innate immune response against virally infected or malignantly transformed cells and their cytotoxicity is regulated by a delicate balance of inhibitory and activating signals [1]. Recent studies suggest that the interplay between NK cells and DC, the specialized antigenpresenting cell of the innate immune system [2], is critical in shaping the adaptive immune response [3]. This concept originates from several lines of evidence including: the discovery of NK cells colocalizing with DC in the T-cell areas of lymph nodes [4,5], the coupling of NK-cell recruitment to lymph nodes à These authors contributed equally to this work. 3138with the induction of more potent Th1 skewing [3], and the identification of NK-cell subpopulations with helper properties [6]. Although the exact mechanisms of NK-DC interaction remain to be elucidated, increasing evidence supports the importance of bidirectional NK-DC crosstalk [7,8].On the one hand, NK-DC crosstalk is characterized by the capacity of activated NK cells to induce DC maturation with elevated IL-12p70 production and subsequently an increased capacity to induce Th1 and CTL responses [9]. This NK-induced DC maturation depends at least in part on soluble factors such as and as well as on engagement of the natural cytotoxicity triggering receptor 30 [12]. Moreover, NK cells control the quality of the adaptive immune response by natural cytotoxicity triggering receptor 30-mediated lysis of immature or inadequately matured DC [13], enabling only fully mature DC to migrate into lymph nodes and subsequently prime T cells. On the other hand, DC are able to induce NK-cell proliferation, augmentation of cytotoxicity and cytokine secretion [8]. The DC-induced modulati...

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Among prostaglandins (PGs), PGE2 is abundantly expressed in various malignancies and is probably one of many factors promoting tumor growth by inhibiting tumor immune surveillance . In the current study, we report on a novel mechanism by which PGE2 inhibits in vitro natural killer-dendritic cell (NK-DC) crosstalk and thereby innate and adaptive immune responses via its effect on NK-DC crosstalk. The presence of PGE2 during IFN-␥/membrane fraction of Klebsiella pneumoniae DC maturation inhibits the production of chemokines (CCL5, CCL19, and CXCL10) and cytokines (IL-12 and IL-18), which is cAMP-dependent and imprinted during DC maturation. As a consequence, these DCs fail to attract NK cells and show a decreased capacity to trigger NK cell IFN-␥ production, which in turn leads to reduced T-helper 1 polarization. In addition, the presence of PGE2 during DC maturation impairs DC-mediated augmentation of NK-cell cytotoxicity. Op- IntroductionProstaglandins (PGs) are potent immune modulators that are produced during inflammation after the conversion of arachidonic acid by cyclooxygenase (COX). 1 Furthermore, PGs are also abundantly produced by various types of tumors. 2 COX2 expression, which is correlated with a poor prognosis, is induced in a variety of human premalignant and malignant tumors, including solid tumors as well as hematologic malignancies. [3][4][5][6] Several lines of evidence demonstrate that COX2-derived PGs are involved in the promotion of tumor growth by regulation of cancer cell proliferation, apoptosis, migration, and invasion. [7][8][9][10][11] PGs are also produced by tumor-surrounding cells, creating a tumor-supporting environment by enhancing angiogenesis and inhibiting tumor immune surveillance. 2,[11][12][13][14] Of all prostaglandins, PGE2 has a pivotal role in tumor immunosuppression. It has been hypothesized that this effect is caused by induction of a permanent state of inflammation, 2 resulting in phenotypic and functional changes of T-helper (T H ) cells, cytotoxic T-lymphocyte (CTL) cells, dendritic cells (DCs), natural killer (NK) cells, and myeloid-derived suppressor cells. 12 PGE2 has been shown to deviate T H cell skewing from an antitumor T H 1 response toward a T H 2/T H 17 response by direct binding to these cells. [15][16][17] In addition, PGE2 is responsible for shifting the balance of IL-12/IL-23 production by DCs toward IL-23, which is a very potent cytokine responsible for T H 17 expansion and survival. 18,19 As a consequence, less IL-12 and other proinflammatory cytokines are produced, thereby inhibiting T H 1 polarization. 17,20 PGE2 also decreases the cytotoxic capacity of CTLs directly by inducing the expression of inhibitory receptors on CTLs 21 and indirectly by inhibiting DC maturation and antigen presentation. 22,23 Moreover, tumor-associated PGE2 has been reported to be responsible for the preferential attraction and induction of regulatory T cells, creating an immune-regulatory microenvironment. 24,25 Next to the modulating effects of PGE2 on the effector mech...