Phospholipid Phosphatidylserine Exposure Research Articles

EXTH-15. A NOVEL IMMUNE-BASED TREATMENT STRATEGY FOR COMBATTING PROTEIN TUMOR HETEROGENEITY

Abstract Cell-based immunotherapy has revolutionized the treatment of many hematological malignancies but has mostly fail against solid tumors, including glioblastoma (GBM). The reason for this failure is multi-fold, but one of the most significant barriers is the extensive intratumoral heterogeneity that characterizes these tumors. Unfortunately, this problem has not been eliminated by personalized medicine strategies aimed at sequencing individual tumors and targeting the uncovered neoantigens. Thinking “outside the box” led us to examine other types of tumor-specific alterations that might more ubiquitously characterize tumor cells, while still being targetable by an immune-based platform. Our investigations led us to the phospholipid phosphatidylserine (PS). PS is typically present on the cytoplasm-facing side of the plasma membrane, where it remains out of view to the immune system. Cellular stresses can lead to dysregulation of processes that keep PS facing internally and instead promote PS exposure on the cell surface. We show here that tumor cells frequently lose the capacity to regulate their plasma membrane and exhibit detectable levels of PS on their surface. Moreover, inducing cell stress through radiation and chemotherapy of tumor cells further enhanced PS exposure. In this study, we aimed to target PS with an immune-based therapy. To this end, we engineered a bi-specific T cell engager (BiTE) consisting of the natural high-affinity PS receptor TIM4 linked to an anti-CD3scFv. This TIM4 BiTE is hypothesized to redirect all T cells to PS, resulting in T cell-mediated killing of tumor cells with exposed PS. Indeed, TIM4 BiTE facilitated potent killing of multiple human tumor cell lines in vitro, including in models of protein heterogeneous tumors. When TIM4 BiTE was delivered intratumorally to a heterogeneous glioma-bearing mouse, survival was significantly extended. These data provide evidence that targeting PS with an immune-based platform represents a potential novel treatment strategy for combatting tumor heterogeneity.

Scramblases as Regulators of Proteolytic ADAM Function.

Proteolytic ectodomain release is a key mechanism for regulating the function of many cell surface proteins. The sheddases ADAM10 and ADAM17 are the best-characterized members of the family of transmembrane disintegrin-like metalloproteinase. Constitutive proteolytic activities are low but can be abruptly upregulated via inside-out signaling triggered by diverse activating events. Emerging evidence indicates that the plasma membrane itself must be assigned a dominant role in upregulation of sheddase function. Data are discussed that tentatively identify phospholipid scramblases as central players during these events. We propose that scramblase-dependent externalization of the negatively charged phospholipid phosphatidylserine (PS) plays an important role in the final activation step of ADAM10 and ADAM17. In this manuscript, we summarize the current knowledge on the interplay of cell membrane changes, PS exposure, and proteolytic activity of transmembrane proteases as well as the potential consequences in the context of immune response, infection, and cancer. The novel concept that scramblases regulate the action of ADAM-proteases may be extendable to other functional proteins that act at the cell surface.

Delivery of IL-2 to the T Cell Surface Through Phosphatidylserine Permits Robust Expansion of CD8 T Cells.

The phospholipid phosphatidylserine (PS) is naturally maintained on the cytoplasmic side of the plasma membrane. Independent of apoptosis, PS is redistributed to the surface of CD8 T cells in response to TCR-mediated activation. Annexin V (AnnV) is a protein known to bind PS with high affinity and has been effectively utilized to anchor antigen to the surface of CD8 T cells. To expand these studies, we aimed to exploit TCR activation driven PS exposure as a target to deliver cytokine, namely interleukin-2 (IL-2), to the surface of CD8 T cells. This was accomplished using a novel chimeric fusion protein of annexin V and interleukin 2 (AnnV-IL2). In vitro analysis revealed that AnnV-IL2 is able to specifically bind PS on the T cell surface following TCR stimulation. Consequently, AnnV-IL2 proved to be significantly more effective at enhancing T cell activation compared to recombinant IL-2. In vivo, AnnV-IL2 promotes robust expansion of antigen-specific cells capable of interferon gamma (IFNγ) production when administered following peptide vaccination. Importantly, upon antigen rechallenge, AnnV-IL2 treatment mice demonstrated a stronger secondary expansion, indicating durability of AnnV-IL2 mediated responses. Our data supports the use of AnnV-IL2 to modulate antigen-specific T cell immunity and demonstrates that the PS-AnnV axis is a feasible mechanism to target diverse cargo to CD8 T cells.

Phosphatidylserine exposure promotes increased adhesion in Dictyostelium Copine A mutants.

The phospholipid phosphatidylserine (PS) is a key signaling molecule and binding partner for many intracellular proteins. PS is normally found on the inner surface of the cell membrane, but PS can be flipped to the outer surface in a process called PS exposure. PS exposure is important in many cell functions, yet the mechanisms that control PS exposure have not been extensively studied. Copines (Cpn), found in most eukaryotic organisms, make up a family of calcium-dependent phospholipid binding proteins. In Dictyostelium, which has six copine genes, CpnA strongly binds to PS and translocates from the cytosol to the plasma membrane in response to a rise in calcium. Cells lacking the cpnA gene (cpnA-) have defects in adhesion, chemotaxis, membrane trafficking, and cytokinesis. In this study we used both flow cytometry and fluorescent microscopy to show that cpnA- cells have increased adhesion to beads and bacteria and that the increased adhesion was not due to changes in the actin cytoskeleton or cell surface proteins. We found that cpnA- cells bound higher amounts of Annexin V, a PS binding protein, than parental cells and showed that unlabeled Annexin V reduced the increased cell adhesion property of cpnA- cells. We also found that cpnA- cells were more sensitive to Polybia-MP1, which binds to external PS and induces cell lysis. Overall, this suggests that cpnA- cells have increased PS exposure and this property contributes to the increased cell adhesion of cpnA- cells. We conclude that CpnA has a role in the regulation of plasma membrane lipid composition and may act as a negative regulator of PS exposure.

Cofilin-1 Activation in Stored Platelets

BackgroundPlatelets, harvested for transfusion, have a relatively short “shelf life” of 5 days. With increasing storage, platelets develop structural and functional changes called “platelet storage lesions”. These changes result not only in reduced hemostatic response to agonists but also in an accelerated clearance from the circulation in vivo. The precise mechanisms of clearance of platelets aged in vitro (or vivo) are not known. There is an increased exposure of the anionic phospholipid phosphatidylserine (PS) on the cell surface of stored platelets. PS is a well-known tag for clearance of cells from the circulation by the macrophage PS receptors. PS-expressing stored platelets are also rapidly cleared from the circulation, presumably through the similar mechanism. Targeting this pathway could improve the recovery of stored platelets following transfusion by inhibiting storage-induced PS exposureResults and DiscussionActin is a major PS binding proteins in platelets. In resting platelets, actin filaments are at the periphery and in close proximity to the membrane. We hypothesize that the interaction with actin filaments constrains the mobility of PS in the inner leaflet. We generated artificial bilamellar liposomes mimicking the intra (PS) or extra (PC) cellular phospholipid composition of the membrane bilayer. When these liposomes were incubated with platelet lysate, the most striking enrichment was seen in actin, which was increased 5-fold in PS-containing liposome compared to control PC liposomes. These data suggest that actin and actin-associated proteins may, in fact, be the major inner leaflet PS-binding proteins in resting platelets.Cortical actin retracts from the membrane with PS exposure in senescent platelets: We examined the morphological changes in resting fresh platelets, five-day-old platelets and ABT-737 (10 μM) treated platelets by immunofluorescence microscopy. Platelets immobilized on polylysine-coated plates were immunostained with FITC-lactadherin and Alexa- 647-phalloidin. As shown in Figure 1, resting platelets do not express PS (no or minimal FITC-lactadherin staining). Actin filaments (Alexa-647-phalloidin) are distributed in the subcortical region as a thin submembranous network. In 6-day old stored platelets, there is significant increase in platelet surface FITC-lactadherin staining. In addition, in most platelets actin filaments have retracted from the subcortical region towards the center of the platelet (compare figure 1 panels A and BBad phosphorylation is significantly diminished in stored platelets. Phosphorylated Bad is sequestered in the cytosol by binding to the scaffolding protein 14-3-3. Furthermore, phosphorylation of S118 inhibits apoptosis by preventing its association with Bcl-xL. Dephosphorylation of Bad may initiate intrinsic apoptosis by associating with Bcl-xL. We determined the phosphorylation status of Bad in stored platelets by Phosphoproteomic and WB analyses of stored and fresh human platelets ( Figure-2) Both methods showed a drastic decrease in Bad is phosphorylation at serine residues 99 and 118 in stored platelets.Activation of cofilin-1 by dephosphorylation in stored platelets. Like Bad, phosphorylation of cofilin-1 is also markedly decrease in stored platelets as shown by WB analyses (Figure-3). It is unclear how the apoptotic pathways lead to cofilin-1 activation (dephosphorylation). Caspases are known to activate slingshot phosphatase which could then activate cofilina1 by its dephosphorylation. LIM- kinases phosphorylate and, thus, inactivate cofilin-1 whereas slingshot phosphatases dephosphorylate and activate cofilin-1. Our data show that the Lim kinase inhibitor, as expected increased ABT-737-induced PS exposure on platelets (Figure 4A), whereas slingshot phosphatase inhibitor inhibited ABT-737-induced PS exposure at a 1-μM concentration (Figure 4B)ConclusionOur data show that activation of apoptotic pathways is responsible for activation of cofilin and subsequent PS exposure by actin reorganization in stored platelets. Targeting this pathway could improve the recovery of stored platelets following transfusion by inhibiting storage-induced PS exposure. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tyrosine Kinase Inhibitor Pazopanib Inhibits Platelet Procoagulant Activity in Renal Cell Carcinoma Patients.

Pazopanib is an angiostatic tyrosine kinase inhibitor (TKI) presently used for cancer treatment, particularly in patients with renal cell carcinoma (RCC). This treatment can be accompanied by mild bleeding as an adverse effect. Given the role of protein tyrosine kinases in platelet activation processes, we investigated whether and how pazopanib can affect platelet functions in purified systems and during treatment of advanced RCC patients. In isolated platelets from healthy volunteers, pazopanib dose-dependently reduced collagen-induced integrin activation and secretion, as well as platelet aggregation. Pazopanib addition diminished glycoprotein (GP) VI-dependent tyrosine phosphorylation of multiple platelet proteins, including the tyrosine kinase Syk. Furthermore, pazopanib inhibited GPVI-induced Ca2+ elevation, resulting in reduced exposure of the procoagulant phospholipid phosphatidylserine (PS). Upon perfusion of control blood over a collagen surface, pazopanib inhibited thrombus size as well as PS exposure. Blood samples from 10 RCC patients were also analyzed before and after 14 days of pazopanib treatment as monotherapy. This treatment caused an overall lowering in platelet count, with 3 out of 10 patients experiencing mild bleeding. Platelets isolated from pazopanib-treated patients showed a significant lowering of PS exposure upon activation. In addition, platelet procoagulant activity was inhibited in thrombi formed under flow conditions. Control experiments indicated that higher pazopanib concentrations were required to inhibit GPVI-mediated PS exposure in the presence of plasma. Together, these results indicated that pazopanib suppresses GPVI-induced platelet activation responses in a way partly antagonized by the presence of plasma. In treated cancer patients, pazopanib effects were confined to a reduction in GPVI-dependent PS exposure. Together with the reduced platelet count, this may explain the mild bleeding tendency observed in pazopanib-treated patients.

Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation

AbstractIt is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in β3−/− platelets or by integrin antagonists. The impaired MV release and PS exposure in β3−/− platelets were rescued by expression of wild-type β3 but not a Gα13 binding–deficient β3 mutant (E733EE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα13 or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα13-integrin interaction, suggesting that Gα13-dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα13 delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα13 diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, β3 integrins serve as a shear sensor activating the Gα13-dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα13-integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation.

Regulation of Platelet Procoagulant Activity

Platelets play prominent and unique roles in the kinetics of the coagulation process. Modulated by the flow conditions, they control coagulation in various ways: (1) by integrin activation and ensuing clot retraction; (2) via exposure of the procoagulant phospholipid phosphatidylserine (PS), which causes binding of a specific set of coagulation factors at the platelet surface with as a result tenase activation and thrombin generation; (3) by initiating fibrin fiber formation at the platelet surface, a process specifically catalyzed by coated platelets. Recent evidence points to fibrin as an active structural network, which provides binding sides for plasma factors and platelets and, thereby, provides an important feedback mechanism for the stimulation of platelet activation. This overview will focus on novel insights into the signaling and activation processes in platelets that regulate these distinct procoagulant functions. One key mechanism is the activation and secondary closure of integrin alphaIIb-beta3. Another key mechanism concerns the calcium-dependent exposure of PS, which is accompanied by swelling of the platelets. Together, these processes control not only platelet adhesion, but also the accessibility for coagulation factors. Major distortions in these procoagulant functions are seen in platelets from a patient with the Scott syndrome, a rare bleeding disorder associated with mutations in the calcium-dependent ion channel protein anoctamin-6 (gene ANO6, previously TMEM16A). These platelets are deficient in calcium-induced PS exposure, swelling, integrin inactivation and in the formation of thrombin and fibrin at the platelet surface. These dysfunctions are precisely phenocopied in mice lacking anoctamin-6 expression. Advanced platelet proteomics analysis indicated that the deficiency in anoctamin-6 was accompanied by decreased calpain-dependent cleavage of a whole spectrum of intracellular proteins, and an increased phosphorylation state of many signaling and cytoskeletal proteins. However, calcium signaling in the Scott platelets was unchanged. In contrast, reports on patients with a gain in platelet procoagulant function, the Stormorken syndrome, indicate a link with genetic mutation in the calcium signaling protein STIM1. The possible consequences of these novel insights for our understanding of haemorrhagic and thrombotic deseases will be discussed. DisclosuresNo relevant conflicts of interest to declare.

Plant Food Anthocyanins Induced Platelet Apoptosis Via BCL-2/BCL-XL Pathway

Background: Platelets are versatile cells and play important roles in hemostasis/thrombosis, inflammation, and atherosclerosis. The pathogenesis of cardiovascular diseases (CVDs) is linked to platelet hyperactivity which is considered an independent risk factor for CVDs. Platelets are critical for promoting the progression of CVDs, and platelet apoptosis have been reported to be involved in platelet activation. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies demonstrated that anthocyanins from plant food significantly inhibited platelet activation, adhesion, aggregation and granule secretion, as well as attenuated thrombus growth at both arterial and venous shear stresses in vitro and in vivo, however, the effects of anthocyanin on platelet apoptosis and its mechanisms have not been explored. In the present study, we examined whether anthocyanin Cyanidin-3-glucoside (Cy-3-g) affect platelet apoptosis and the BCL-2/BCL-XL intrinsic apoptotic pathway.Methods: Cy-3-g, the predominant bioactive compound of anthocyanin preparations, was obtained from Polyphenol AS Company in Norway.Purified gel-filtered platelets from healthy volunteers were incubated at 37oC for 40 minutes with different concentrations of Cy-3-g (0.5、5、50μM) or PBS buffer as a control. the activated platelets were triggered with 0.5U thrombin for 15min to induce apoptosis. Mitochondria membrane potential (Δψm) and membrane phospholipid phosphatidylserine (PS) exposure in both activated and resting platelets were assessed by flow cytometry. Cytochrome C release, activation of caspase-3, caspase-8, caspase-9, cleavage of gelsolin, the levels of anti-apoptotic BCL-2 family proteins such as BCL-2, BCL-XL and proapoptotic BCL-2 family proteins Bax, Bak, Bad, Bid and tBid in both activated and resting platelets were measured by western blotting.Results: Cy-3-g at 5μM and 50μM directly induced significant ΔΨm dissipation in activated platelets dose dependently. Correspondingly, 50μM Cy-3-g increased cytochrome C release compared to control. The expression of pro-caspase-8 and pro-caspase-9 decreased, activation of caspase-3, caspase-8 and caspase-9 was induced in activated platelets in both 5μM and 50μM Cy-3-g groups. Both PS exposure and the cleavage of gelsolin increased in activated platelets, however these effects were only observed at Cy-3-g doses as high as 50μM. Cy-3-g did not induce the above changes in resting platelets. The intrinsic apoptotic pathway was initiated by Cy-3-g treatment in activated platelets; Cy-3-g significantly inhibited the expression of BCL-2, BCL-XL and increased the levels of Bax, Bak, Bad and Bid in activated platelets dose dependently. No significant difference was observed in resting platelets.Conclusions: Our data demonstrate for the first time that purified anthocyanin Cy-3-g directly accelerated apoptosis in activated platelets via the BCL-2/BCL-XL pathway. Anthocyanins may possess therapeutic potential for patients suffering from thrombotic conditions. DisclosuresNo relevant conflicts of interest to declare.

Diurnal, localized exposure of phosphatidylserine by rod outer segment tips in wild-type but notItgb5−/−orMfge8−/−mouse retina

In the mammalian retina, life-long renewal of light-sensitive photoreceptor outer segments (POS) involves circadian shedding of distal rod POS tips and their subsequent phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning after light onset. Molecular mechanisms that promote or synchronize POS tip shedding have thus far remained unknown. Here we examined plasma membrane asymmetry of living POS by quantifying surface exposure of the membrane phospholipid phosphatidylserine (PS) using antibodies, annexin V, and pSIVA (polarity-sensitive indicator of viability and apoptosis), an annexin-based biosensor with switchable states of fluorescence. We found that isolated POS particles possess externalized PS, whose blockade or removal reduces their binding and engulfment by RPE in culture. Imaging of live photoreceptors in freshly dissected mouse retina detected PS externalization restricted to POS tips with discrete boundaries. In wild-type mice, frequency of rod tips exposing PS and length of tips with exposed PS peak shortly after light onset. In contrast, PS-marked POS tips do not vary in mice lacking the diurnal phagocytic rhythm of the RPE due to loss of either the phagocytosis receptor αvβ5 integrin, expressed by the RPE but not by photoreceptors, or its extracellular ligand milk fat globule-EGF factor 8 (MFG-E8). These data identify a molecular distinction, localized PS exposure, that is specific to the surface of rod POS tips. Enhanced PS exposure preceding rod shedding and phagocytosis suggests that surface PS promotes these processes. Moreover, our results demonstrate that the diurnal rhythm of PS demarcation of POS tips is not intrinsic to rod photoreceptors but requires activities of the RPE as well.

In search of a novel target — Phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy

This study was performed in the aim to identify potential targets for the development of novel therapy to treat cancer with poor outcome or treatment efficacy. We show that the negatively charged phospholipid phosphatidylserine (PS) is exposed in the outer leaflet of their plasma membrane not only in tumor cell lines, but also in metastases and primary cultures thereof, which contrasts with a lack of PS exposure by differentiated non-tumorigenic counterparts. Studied tumor cell lines were derived from non-tumorigenic and malignant melanomas, prostate- and renal cancer, glioblastoma and a rhabdomyosarcoma. Importantly, also metastases of melanoma expose PS and there is a correlation between malignancy of melanoma cell lines from different stages of tumor progression and PS exposure. The PS exposure we found was neither of apoptotic nor of experimental artificial origin. Finally potentially malignant and non-malignant cells could be differentiated by sorting of a primary cell culture derived from a glioblastoma based on PS exposure, which has so far not been possible within one culture due to lack of a specific marker. Our data provide clear evidence that PS could serve as uniform marker of tumor cells and metastases as well as a target for novel therapeutic approaches based on e.g. PS-specific host defense derived peptides.

An Important Role for Rac1 and RhoA In Regulating Platelet Microparticle Formation and Phosphatidylserine Exposure

AbstractAbstract 2023Platelets activated by physiological agonists such as thrombin and collagen shed procoagulant microparticles (MPs) and externalize the procoagulant phospholipid phosphatidylserine (PS), both of which are critical to hemostasis and play an important role in inflammation. To date, the signaling mechanisms that regulate agonist-induced MP formation and PS exposure in platelets remain unclear. In this study, we demonstrate that the small GTPases Rac1 and RhoA play important roles in regulating the procoagulant activity of platelets. Rac1 null (-/-) mouse platelets or human platelets treated with the Rac1 inhibitor NSC23766 (NSC) displayed a significant defect in MP formation and PS exposure induced by various agonists. Furthermore, Rac1-/- platelets and NSC-treated human platelets displayed a defect in procoagulant activity as demonstrated by a prolonged coagulation time following recalcification of citrated PRP. The stimulatory role of Rac1 in platelet MP formation and PS exposure is distinct from the known function of Rac1 in facilitating platelet granule secretion and secretion-dependent amplification of platelet aggregation, because supplementation of the granule content ADP rescued the defect in platelet aggregation caused by Rac1 inhibition, but failed to rescue the defect in MP formation caused by Rac1 inhibition. In contrast to Rac1, RhoA plays an inhibitory role in regulating platelet procoagulant activity, because treatment of platelets with the Rho inhibitor C3-toxin (C3) significantly enhanced agonist-induced MP formation, PS exposure, and procoagulant activity. The enhancing effect of C3 on platelet procoagulant activity is not caused by an overall enhancement of platelet activation because C3 significantly inhibited platelet secretion and aggregation. Thus, our data demonstrates that while Rac1 and RhoA both play important stimulatory roles in platelet granule secretion and aggregation, they play opposing roles in MP formation and PS exposure in platelets. Rac1 is important for stimulating platelet MP formation, PS exposure, and procoagulant activity, which is antagonized by RhoA. Disclosures:No relevant conflicts of interest to declare.

Antigen recognition induces phosphatidylserine exposure on the cell surface of human CD8+ T cells

AbstractIn 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 inward-directed 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.

Antigen Recognition Induces Phosphatidylserine Exposure on the Cell Surface of Human CD8+ T Cells.

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 (CTL) expose PS upon T-cell receptor (TCR)-mediated antigen recognition: antigen-specific CTL, recognizing the HLA-A2 binding Melan-A peptide, demonstrated a marked exposure of PS as determined by annV-FITC staining, a highly specific PS-binding protein, after 4 h of stimulation with Melan-A-loaded antigen presenting cells (APC). PS exposure was Ag-specific (>80% annV-positive T cells) but control peptide (gp100)-pulsed and unpulsed APC also induced a slight background PS exposure on the CTL. To follow more precisely the fate of annV-positive CTL after antigen-specific stimulation, we labeled Melan-A-specific CTL with the membrane dye PKH-26 and isolated all annV+ PKH-26+ CTL 4 h after stimulation with peptide-pulsed APC cells by cell sorting. Results demonstrate that the annV-positive T-cell population is heterogenous: while the annV-high T-cell population retained the annV-high phenotype and consisted of apoptotic T cells, the annV-intermediate(int) T-cell population revealed a constant decrease of annV binding and became annV-negative at 54 to 72 h after stimulation. Using three independent assays for apoptosis we found that annV-int T cells are propidium iodide negative, do not exhibit DNA strand brakes and contain no active caspase 3. In contrast, annV-int CTL revealed a strongly activated phenotype indicated by an upregulation of CD69 expression and downregulation of the TCRαβ chain expression. Fluorescence microscopic analysis demonstrated that PS is distributed inhomogenously over the plasma membrane and concentrated in membrane lipid raft domains at the immunological synapse. By studying the activity of PS transport proteins using a fluorescence-labeled PS analogue, we determined a constitutive outward transfer of PS molecules in Melan-A-specific CTL. The constitutive PS outward transport was not further accelerated after antigen re-stimulation. In sharp contrast, the inward transporting flippase was strongly inhibited in stimulated CTL resulting in an accumulation of PS molecules on the cell surface. Shielding of exposed PS by annexinV protein during antigen recognition diminished cytokine secretion, activation and cell-cell clustering of antigen-specific CTL. In summary, our data demonstrate for the first time that externalized PS on antigen-stimulated CTL is linked to T-cell activation and probably involved in cell-cell contact formation at the immunological synapse.