Cytotoxic Granule Endocytosis in Primary Mouse Cytotoxic T Lymphocytes

Cytotoxic T lymphocytes (CTLs) fight against infection by eliminating virus-infected and malignant cells from the body. CTLs recognize its target upon specific T cell receptor engagement and consequently triggers cytotoxic granules (CGs) release to the cell-cell contact site, termed immunological synapse (IS) to induce target cell death. CTLs are efficient killers as multiple routes of fast lethal deliveries suggest, however whether the sustained cytotoxic supply from retrieved granules after fusion contributes to multiple target cell killing remains unknown. By using primary CTLs isolated from Synaptobrevin2-mRFP knock-in mice, we demonstrate endocytosis of the specific CG membrane protein Syb2 at the IS upon target cell contact by confocal live cell imaging. CG endocytosis is Clathrin- and Dynamin-dependent, and later on endocytosed CGs became re-filled with toxic substances like Granzyme B to again enable cytotoxicity. These endocytic granules polarized to respective synapses upon sequential target cell contacts. Importantly, blocking CG endocytosis reduces the serial killing efficiency by about 50%, indicating endocytic CGs contribute significantly to the overall cytotoxic capacity. These data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process. We further investigate the involvement of the vesicular membrane protein Flower in the calcium-dependence of CG endocytosis. It was previously suggested to function as a calcium channel to regulate synaptic endocytosis in drosophila. Flower is proposed to transfer from vesicle to plasma membrane through vesicle fusion and thus regulates calcium influx. We showed that the Flower protein, which contains four transmembrane domains, is expressed in mouse CTLs. Flower-deficiency does not affect degranulation or CG exocytosis, however, it leads to a block of CG endocytosis at an early stage. This defect in endocytosis can be rescued by re-introducing Flower or by increasing extracellular calcium, suggesting that the phenotype is specifically due to a lack of Flower and that other calcium sources could compensate its function. Additionally, Flower vesicles polarize early to the IS and localize around exocytic and endocytic granules. Intriguingly, a Flower mutant with tyrosin motifs (YxxF) substituted to alanine shows a re-distribution from vesicles to the plasma membrane, but fails to rescue the endocytosis defect. This result implies that either Flower functions on vesicles rather than at the plasma membrane or that active Flower requires interaction with other molecules via its tyrosin motifs. Taken together, these data identify Flower as an essential molecule initiating granule endocytosis. However, further studies are required to answer the question whether Flower plays a role in calcium-dependence of endocytosis.Zytotoxische T Lymphozyten (CTLs) bekämpfen Infektionen, indem sie virusinfizierte oder entartete Zellen eliminieren. CTLs identifizieren ihre Zielzellen mit Hilfe spezifischer T-Zell Rezeptoren, was zur Ausbildung eines Zell-Zell-Kontaktes, der sogenannten Immunologischen Synapse, führt. An dieser IS erfolgt letztlich die Fusion zytotoxischer Granula (CGs), die den Zelltod der Zielzelle induzieren. CTLs sind effektive Tötungsmaschinen, da sie hintereinander Tausende an Zielzellen in kurzer Zeit eliminieren können. Allerdings ist unklar, inwieweit an dieser seriellen Zytotoxizität rezyklierte Granula beteiligt sind. Durch die Nutzung von CTLs, die aus Synaptobrevin2-mRFP Knock-in Mäusen gewonnen wurden, konnten wir mit Hilfe konfokaler Mikroskopie zeigen, dass Syb2 an der IS nach Zielzellkontakt endozytiert wird. Die Endozytose von CGs erfolgt über Clathrin- und Dynamin-abhängige Wege. Später werden endozytierte CGs mit toxischen Substanzen wie GranzymeB aufgefüllt, um die Zytotoxizität erneut herzustellen. Diese endozytotischen Granula polarisieren dann zu den jeweiligen Synapsen neuer Zielzellkontakte. Wichtiger Weise führt die Blockierung der Endozytose zu einer etwa um 50% reduzierten Tötungseffizienz, was ein Hinweis darauf ist, dass Endozytose zur zytotoxischen Gesamtkapazität signifikant beiträgt. Diese Daten zeigen, dass die Endozytose von CG Membranproteinen eine Voraussetzung für effizientes serielles Abtöten von Zielzellen durch CTLs ist und identifizieren Schlüsselereignisse dieses Prozesses. Weiterhin untersuchten wir ein Kandidatenmolekül für die Kalziumabhängigkeit der CG Endozytose. Dieses Flower genannte, vesikuläre Protein könnte in Drosophila melanogaster ein endozytoseregulierender Kalziumkanal sein. Es wird angenommen, dass Flower durch Fusion vom Vesikeln zur Plasmamembran transferiert wird und dort den Kalziumeinstrom reguliert. Wir konnten die Expression des Flower-Proteins, welches vier Transmembrandomänen enthält, in CTLs der Maus nachweisen. Das Fehlen des Flower-Proteins beeinflusste weder die Degranulation noch die Exozytose der CGs, führte aber zu einem vollständigen Block der CG Endozytose in einem frühen Stadium. Dieser Endozytose-Defekt konnte durch erneutes Hinzufügen von Flower oder durch eine Erhöhung der extrazellulären Kalziumkonzentration behoben werden. Es kann also angenommen werden, dass der Defekt spezifisch für das Fehlen von Flower war und andere Kalziumquellen das Fehlen von Flower kompensieren können. Hinzu kommt, dass Flower Vesikel früh zur IS polarisieren und in der Nähe exo- und endozytotischer Vesikel zu finden sind. Interessanter Weise zeigen Flower Mutanten, bei denen bestimmte Tyrosine zu Alanin ausgetauscht wurden, eine Neuverteilung von Flower von Vesikeln hin zur Plasmamembran, ohne jedoch den Endozytose-Defekt zu beheben. Diese Ergebnisse können in zweierlei Weise interpretiert werden: der Ort des Flower- Effekts ist eher das Vesikel als die Plasmamembran oder aktives Flower benötigt Interaktionen mit anderen Molekülen über seine Tyrosin Motive. Diese Daten legen nahe, dass Flower ein essentielles Molekül für die Auslösung der Endozytose ist. Allerdings sind für die Klärung der Frage, ob Flower eine Rolle in der Kalziumabhängigkeit spielt, weitere Experimente in zukünftigen Untersuchungen notwendig

Local Activities of the Membranes Associated with Glycosaminoglycan-Chitosan Complexes in Bone Cells

Chitosan is a cationic polysaccharide derived from the partial deacetylation of chitin. Hyaluronic acid (HA), chondroitin sulfate (CS) and heparin (HP) are anionic glycosaminoglycans (GCGs) which can regulate osteogenic activity. In this study, chitosan membranes were prepared by glutaraldehyde crosslinking reaction and then complexed with three different types of GCGs. 7F2 osteoblasts-like cells and macrophages Raw264.7 were used as models to study the influence of chitosan membranes on osteometabolism. Although chitosan membranes are highly hydrophilic, the membranes associated with GCG-chitosan complexes showed about 60-70% cell attachment. Furthermore, the membranes associated with HP-chitosan complexes could increase ALP activity in comparison with chitosan films only. Three types of the membranes associated with GCG-chitosan complexes could significantly inhibit LPS induced-nitric oxide expression. In addition, chitosan membranes associated with HP and HA can down-regulate tartrate-resistant acid phosphatase (TRAP) activity but not CS-chitosan complexes. Based on these results, we conclude that chitosan membranes associated with HP can increase ALP activity in osteoblasts and chitosan membranes associated with HP and HA reduce TRAP activity in osteoclasts

Study of Effector CD8+ T Cell Interactions with Cortical Neurons in Response to Inflammation in Mouse Brain Slices and Neuronal Cultures

Cytotoxic CD8+ T cells contribute to neuronal damage in inflammatory and degenerative CNS disorders, such as multiple sclerosis (MS). The mechanism of cortical damage associated with CD8+ T cells is not well understood. We developed in vitro cell culture and ex vivo brain slice co-culture models of brain inflammation to study CD8+ T cell–neuron interactions. To induce inflammation, we applied T cell conditioned media, which contains a variety of cytokines, during CD8+ T cell polyclonal activation. Release of IFNγ and TNFα from co-cultures was verified by ELISA, confirming an inflammatory response. We also visualized the physical interactions between CD8+ T cells and cortical neurons using live-cell confocal imaging. The imaging revealed that T cells reduced their migration velocity and changed their migratory patterns under inflammatory conditions. CD8+ T cells increased their dwell time at neuronal soma and dendrites in response to added cytokines. These changes were seen in both the in vitro and ex vivo models. The results confirm that these in vitro and ex vivo models provide promising platforms for the study of the molecular details of neuron–immune cell interactions under inflammatory conditions, which allow high-resolution live microscopy and are readily amenable to experimental manipulation

Investigation of Cytotoxic T Lymphocyte Function during Allorejection in the Anterior Chamber of the Eye

Cytotoxic T lymphocytes (CTL) are an essential part of our immune system by killing infected and malignant cells. To fully understand this process, it is necessary to study CTL function in the physiological setting of a living organism to account for their interplay with other immune cells like CD4+ T helper cells and macrophages. The anterior chamber of the eye (ACE), originally developed for diabetes research, is ideally suited for non-invasive and longitudinal in vivo imaging. We take advantage of the ACE window to observe immune responses, particularly allorejection of islets of Langerhans cells by CTLs. We follow the onset of the rejection after vascularization on islets until the end of the rejection process for about a month by repetitive two-photon microscopy. We find that CTLs show reduced migration on allogeneic islets in vivo compared to in vitro data, indicating CTL activation. Interestingly, the temporal infiltration pattern of T cells during rejection is precisely regulated, showing enrichment of CD4+ T helper cells on the islets before arrival of CD8+ CTLs. The adaptation of the ACE to immune responses enables the examination of the mechanism and regulation of CTL-mediated killing in vivo and to further investigate the killing in gene-deficient mice that resemble severe human immune diseases

Lytic granule exocytosis at immune synapses: lessons from neuronal synapses

Regulated exocytosis is a central mechanism of cellular communication. It is not only the basis for neurotransmission and hormone release, but also plays an important role in the immune system for the release of cytokines and cytotoxic molecules. In cytotoxic T lymphocytes (CTLs), the formation of the immunological synapse is required for the delivery of the cytotoxic substances such as granzymes and perforin, which are stored in lytic granules and released via exocytosis. The molecular mechanisms of their fusion with the plasma membrane are only partially understood. In this review, we discuss the molecular players involved in the regulated exocytosis of CTL, highlighting the parallels and differences to neuronal synaptic transmission. Additionally, we examine the strengths and weaknesses of both systems to study exocytosis

Heterozygous OT-I mice reveal that antigen-specific CD8+ T cells shift from apoptotic to necrotic killers in the elderly

Numerous alterations in CD8+ T cells contribute to impaired immune responses in elderly individuals. However, the discrimination between cell-intrinsic dysfunctions and microenvironmental changes is challenging. TCR transgenic OT-I mice are utilized to investigate CD8+ T-cell immunity, but their immunodeficient phenotype hampers their use especially in aging. Here, we demonstrate that using a heterozygous OT-I model minimizes the current limitations and provides a valuable tool to assess antigen-specific T-cell responses even at old age. We analyzed phenotypic and functional characteristics of CD8+ T cells from OT-I +/+ and OT-I +/− mice to prove the applicability of the heterozygous system. Our data reveal that OVA-activated CD8+ T cells from adult OT-I +/− mice proliferate, differentiate, and exert cytolytic activity equally to their homozygous counterparts. Moreover, common age-related alterations in CD8+ T cells, including naive T-cell deterioration and decreased proliferative capacity, also occur in elderly OT-I +/− mice, indicating the wide range of applications for in vivo and in vitro aging studies. We used the OT-I +/− model to investigate cell-intrinsic alterations affecting the cytotoxic behavior of aged CD8+ T cells after antigen-specific in vitro activation. Time-resolved analysis of antigen-directed target cell lysis confirmed previous observations that the cytotoxic capacity of CD8+ T cells increases with age. Surprisingly, detailed single cell analysis revealed that transcriptional upregulation of perforin in aged CD8+ T cells shifts the mode of target cell death from granzymemediated apoptosis to rapid induction of necrosis. This unexpected capability might be beneficial or detrimental for the aging host and requires detailed evaluation

Pseudomonas aeruginosa sepsis with ecthyma gangrenosum and pseudomembranous pharyngolaryngitis in a 5-month-old boy

Pseudomonas aeruginosa infection that induced pseudomembranous laryngopharyngitis and ecthyma gangrenosum simultaneously in a healthy infant is rare. We reported on a previously healthy 5-month-old boy with initial presentation of fever and diarrhea followed by stridor and progressive respiratory distress. P. aeruginosa sepsis was suspected because ecthyma gangrenosum over the right leg was found at the emergency department, and the diagnosis was confirmed by the blood culture. Fiberscope revealed bacterial pharyngolaryngitis without involvement of the trachea. Because of early recognition and adequate treatment, including antimicrobial therapy, noninvasive ventilation, incision, and drainage, he recovered completely without any complications

Cocaine Hydrolase-Fc Fusion Proteins for Cocaine and Methods for Utilizing the Same

The presently-disclosed subject matter includes isolated polypeptides that comprise a butyrylcholinestrase (BChE) polypeptide and a second polypeptide. The BChE polypeptide as well as the second polypeptide can be variants and/or fragments thereof. The presently-disclosed subject matter also includes a pharmaceutical composition that comprises the present isolated polypeptide and a suitable pharmaceutical carrier. Further still, methods are provided for treating cocaine-induced conditions, and comprise administering the isolated polypeptide and/or pharmaceutical compositions thereof to an individual