Questo lavoro è stato sostenuto da un L. Dan Duncan collaborativa assegno di ricerca (CMB e EJS), il National Heart, Lung, and Blood Institute (US4HL081007), un Leukemia and Lymphoma Society Clinical Research Scholar premio (CMB), e il National Cancer Institute (RO1 CA06150816; EJS).

1. Cella di isolamento mononucleare (giorno 0) <ol><li> Inserire spike di luer femmina nella porta di uscita della frazione 20% della unità di sangue cordonale, collegare la siringa e rimuovere il sangue. Trasferimento sangue scongelati a 20 ml di RPMI riscaldato in tubo da 50 ml per centrifuga. Risciacquare sangue borsa dei cavi con 5 ml di RPMI e trasferire al tubo da centrifuga stessa. </li><li> Centrifugare le cellule per 10 minuti a 400 x g. Aspirare il surnatante. </li><li> Risospendere le cellule in 20 ml di RPMI caldo. Cellule strato su 15 ml di Lymphoprep in tubo da 50 ml per centrifuga. Centrifugare 40 minuti a 400 x g. </li><li> Raccogliere l&#39;interfaccia contenente le cellule mononucleari e lavare in 20 ml di RPMI. Centrifugare per 10 minuti a 450 x g. </li><li> Aspirare il surnatante e lavare in 20 ml di RPMI. Contare le celle e girare per 5 minuti a 400 x g. </li><li> Aspirare il surnatante e risospendere le cellule a 5 x 10 6 cellule mononucleate / mL di Cellgenix DC media (privo di siero). Togliere 1 mL per EBV-LCL generazione e mettere in 15 mL centrifuge tubo. </li></ol> 2. Cellule dendritiche Generation (a partire dal giorno 0) <ol><li> Piastra 2 ml di cellule (sono già a 5 x 10 6 cellule / ml di DC media) per pozzetto in una piastra da 6 pozzetti. Lasciare in incubatore per 1-2 ore. </li><li> Dopo 1-2 ore, lavare le cellule non aderenti mediante lavaggio 3-4 volte con PBS. Raccogliere le cellule non aderenti e congelare. Aggiungere 2 mL / pozzetto di supporti contenenti CC 1000 U / ml di IL-4 e 800 U / ml di GM-CSF. </li><li> 3-4 giorni dopo DC iniziazione, rifornire di IL-4 e GM-CSF aggiungendo 100 pl / pozzetto di supporti contenenti una concentrazione finale di 1000U/mL IL-4 e 800 U / ml di GM-CSF. </li><li> Il giorno 5-6, raccolto la DC raschiando il bene con una pipetta di trasferimento. Contare le cellule grandi e centrifugare per 5 minuti a 400 x g. Aspirare media e risospendere CC a 2x10 6 cellule / ml di DC media. Aggiungere 0,5 ml / pozzetto di una piastra a 24 pozzetti. </li><li> Trasdurre la DC con il Ad5f35pp65 vettore in una cella per MOI di 10 unità infettive. Aggiungi vector a ciascun pozzetto in una piastra da 24 pozzetti. Incubare le cellule per 1,5 ore. Dopo 1,5 ore, aggiungere 1,5 mL / pozzetto di DC supporto contenente: 1000 U / ml di IL-4, 800 U / ml di GM-CSF, 10 ng / mL TNF-alfa, 1 pg / mL PGE-1, 100 ng / mL IL-6, e 10 ng / ml di IL-1beta. Queste citochine maturare i controller di dominio. </li></ol> 3. Generazione di EBV-LCL (a partire dal giorno 0) <ol><li> Centrifugare la provetta contenente le x 5 10 6 cellule mononucleate. Aggiungere 200 ml di concentrato EBV B95.8 surnatante e 1,8 ml di mezzo completo (RPMI + 10% FBS + 2 mM L-glutammina) contenente ciclosporina (1 pg / mL). </li><li> Aliquotare 100 ml di cellule in 10 pozzetti di una piastra a 96 pozzetti e 200 pl in 5 pozzetti. Aggiungere 100 ml di mezzi completi ciclosporina + ai pozzetti contenenti solo 100 ml di cellule. Riempire i pozzetti rimanenti con acqua sterile. </li><li> Alimentare il settimanale LCL ed espandere da una piastra a 96 pozzetti a un 24-pozzetti dopo ~ 2 settimane. Dopo un&#39;altra settimana, trasferire in un matraccio T25 ed il trasferimento settimana successivauna beuta T75. In questa fase si consiglia di congelare aliquote di LCL. Si richiede in genere 1 mese a generare un numero sufficiente di LCL. </li></ol> 4. CTL Initiation - 7 giorni dopo l&#39;inizio delle DC <ol><li> DC Harvest e irradiano a 30 Gy. Lavare 4 x e il conteggio. Risospendere DC in mezzi cellule T contenenti siero umano (45% RPMI, 45% CLICK, il 10% di siero umano, 2 mM L-glutammina) @ 1 x 10 5 cellule dendritiche / mL. </li><li> Scongelare le cellule non aderenti a partire dal punto 2.2. Lavare le cellule, e contare. Risospendere a 2 x 10 6 cellule non aderenti / ml in mezzi cellule T contenenti siero umano. Aggiungere 10 ng / ml di IL-7 e IL-12, e 5 ng / ml di IL-15. Aggiungere 1 ml di cellule per pozzetto in una piastra da 24 pozzetti. Aggiungere 1 ml di DC per pozzetto. Riempire i pozzetti vuoti di 24 pozzetti con acqua sterile. </li><li> 7 giorni dopo l&#39;inizio delle cellule T, mangimi e / o dividere le celle con i media delle cellule T contenenti siero umano. </li><li> 9-12 giorni dopo l&#39;inizio delle cellule T, congelare le cellule T fino a quando il LCL sono pronti. </li><li> Once LCL si sono ampliati e sono diversi passaggi in fiasche T75, trasdurre l&#39;LCL mediante centrifugazione per 5 minuti a 400 x g. Aggiungere il vettore al pellet cellulare ad una MOI di 100 unità infettive per cellula. Incubare per 1,5 ore. Dopo 1,5 ore, risospendere in mezzi completi a 5 x 10 5 LCL / ml e aggiungere 2 ml per pozzetto in una piastra a 24 pozzetti 12. </li><li> Dopo 2 giorni, raccogliere il LCL e irradiano a 40 Gy. Lavare 4 x e il conteggio. Risospendere il LCL in mezzi cellule T contenenti siero umano a 2,5 x 10 5 cellule / ml. Aggiungere 1 ml di LCL per pozzetto di una piastra a 24 pozzetti. Inoltre, aggiungere 5 x 10 6 LCL ad un dispositivo di coltura Grex40 e 5 ng / ml di IL-15. 13 </li><li> Raccogliere le cellule T. Centrifugare per 5 minuti a 400 xg, aspirare il surnatante, risospendere in mezzi e cellule T contenenti siero umano a 1 x 10 6 cellule T / mL. Aggiungere 5 ng / ml di IL-15 e di aggiungere 1 ml / pozzetto di cellule T (totale di 1 x 10 6 cellule T) al LCL che sono già in 24-pozzetti. Nel Grex40, portareil volume totale del supporto fino a 30 mL. </li><li> Il giorno 3-4 post-stimolazione, se le cellule confluenti sono poi divisi (nel piatto) 1:1 e aggiungere mezzo fresco contenente 50 U / ml IL-2. Se non sono confluenti poi semplicemente aspirare ½ media e sostituirlo con supporti contenenti 50 U / mL di IL-2. Nel Grex40, aspirare la metà dei mezzi di comunicazione, sostituire con mezzi freschi, e aggiungere 50 U / mL di IL-2. </li><li> Il giorno 7, ripetere come nel passo 4,6 ma utilizzare IL-2 nel giorno della stimolazione non, IL-15. </li></ol> 5. Risultati rappresentativi Uno schema del GMP approvato dalla FDA protocollo di fabbricazione è rappresentato in figura 1. Il processo dura circa 50 giorni. Metodi tipici di generazione di virus-specifiche cellule T espandere preesistenti cellule T di memoria, tuttavia, il sangue del cordone non ha virus esperti cellule T, e quindi abbiamo bisogno di prime ingenuo T cellule ex vivo. Per fare ciò, usiamo cellule dendritiche e le citochine IL-7, IL-12 e IL-15,necessaria per generare specificità virale. Dopo 3 stimolazioni, il rendimento deve essere superiore a 100x10 6 celle. Se le cellule non siano disponibili sufficienti, stimolazioni supplementari possono essere eseguite fino a quando il numero desiderato di CTL sono disponibili. La maggior parte di queste cellule dovrebbe essere CD3 + con una miscela di CD4 + e CD8 + T cellule. Ci dovrebbe essere inferiore a 15% delle cellule NK (CD3-/CD56 +) e meno di 1% di cellule B CD19 + (Figura 2). Il CTL estesa dovrebbe riconoscere l&#39;antigene pp65 da CMV, hexon e Penton da adenovirus, nonché numerosi antigeni EBV che sono espressi sulla EBV-LCL. Quando la prova è in un saggio ELISPOT, CTL dovrebbe secernere più IFN-? in risposta a questi antigeni di antigeni irrilevanti (Figura 3). Il CTL dovrebbe anche lisare virali peptide-pulsati tali obiettivi blasti PHA. In un saggio di rilascio Cr 51, CTL dovrebbe lisare LCL, CMVpp65-pulsato e Adenovirus esone-e /o Penton-pulsato obiettivi, ma non obiettivi pulsate con peptidi irrilevanti (Figura 4). <img src="/files/ftp_upload/3627/3627fig1.jpg" alt="Figura 1."/> Figura 1. Generazione di multi-virus CTL specifici dal cordone ombelicale. Schematico che mostra l&#39;intero processo di fabbricazione CTL da cordone ombelicale. Prima le cellule mononucleate del sangue del cordone sono isolati dalla frazione 20% della frazionato unità di sangue del cordone ombelicale. Con l&#39;eccezione di 5x10 6 cellule salvate per generazione LCL, tutte le cellule vengono quindi piastrate in mezzi cellule dendritiche per 1-2 ore, a questo punto le cellule non aderenti sono raccolte e congelate. La DC vengono poi alimentati DC supporti contenenti IL-4 e GM-CSF. Dopo 5 giorni di coltura, la DC sono maturato e trasdotte con un vettore adenovirale contenente l&#39;antigene immunodominante CMV pp65. All&#39;iniziazione DC questi sono combinati con le cellule non aderenti e le citochine IL-7, IL-12,e IL-15. A stimolazioni successive, lo stesso vettore adenovirale viene utilizzato per trasdurre EBV-LCL, che sono utilizzati come le cellule presentanti l&#39;antigene. IL-15 viene utilizzato al secondo stimolazione e IL-2 in seguito. <img src="/files/ftp_upload/3627/3627fig2.jpg" alt="Figura 2."/> Figura 2. Fenotipo delle cellule T risultante. Mostrata è la percentuale di cellule vive inclusi nel gate linfocitario. CTL sono CD3 + e CD4 + e CD8 +, ma in gran parte CD3-/CD56- e CD19-. <img src="/files/ftp_upload/3627/3627fig3.jpg" alt="Figura 3."/> Figura 3. Funzionalità delle cellule T. Specificità delle cellule T è stato testato da IFN-γELISPOT. Le cellule T sono state pulsate con peptidi sovrapposti copre l&#39;intera proteina di hexon, Penton, pp65, e l&#39;antigene irrilevante IE-1. CTL da solo indica un mezzo solo. Autologo LCL sono stati irradiati e ha aggiunto a 1x10 5 / bene. Viene mostrato il punto medio di formazione delle cellule cim porto di pozzetti in triplicato. <img src="/files/ftp_upload/3627/3627fig4.jpg" alt="Figura 4."/> Figura 4. Attività citolitica dei CTL. La capacità del CTL risultante per lisare bersagli esprimenti antigeni virali è stato testato in un saggio di 51 Cr. 51 Cr-etichettati blasti autologhe PHA sono state pulsate con peptidi sovrapposti copre l&#39;intera o antigene marcato con 51 Cr LCL autologhe sono stati co-coltivate con CTL. Dopo 4 ore, gamma di rilascio è stato contato in un contatore gamma.

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<html> Attuali strategie volte a controllare le infezioni virali dopo CBT può essere efficace, ma sono associati a tossicità significative, sono costosi, e non conferiscono protezione a lungo termine contro l&#39;infezione in seguito. In effetti, l&#39;uso di alcuni farmaci antivirali possono limitare l&#39;espansione del virus-specifiche cellule T che altrimenti sarebbero protettivo. 14 Un&#39;altra opzione è l&#39;infusione di donatore-derivati ​​virus-cellule T specifiche. Noi e altri hanno dimostrat...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 21870-076 </td></tr><tr><td> DC supporti </td><td> CellGenix </td><td> 20801-0500 </td></tr><tr><td> EHAA (Medium Click di) </td><td> Irvine Scientific </td><td> 9195 </td></tr><tr><td> Siero umano </td><td> Gemini Bio Prodotti </td><td> 100-110 </td></tr><tr><td> Gas Permeabile Cultureware 18 </td><td> Wilson-Wolf </td><td> 80040S </td></tr><tr><td> IL-2 </td><td> Chirone (TCH Farmacia) </td><td></td></tr><tr><td> IL-12 </td><td> NCI / CTEP </td><td></td></tr><tr><td> IL-15 </td><td> CellGenix </td><td> 1013-050 </td></tr><tr><td> IL-7 </td><td> R &amp; S </td><td> AFL207 </td></tr><tr><td> IL-1beta </td><td> R &amp; S </td><td> AFL201 </td></tr><tr><td> IL-6 </td><td> CellaGenix </td><td> 1004-050 </td></tr><tr><td> GM-CSF </td><td> TCH Farmacia </td><td></td></tr><tr><td> IL-4 </td><td> R &amp; S </td><td> <a href="http://www.rndsystems.com/pdf/AFL204.pdf" target="_blank">AFL204</a> </td></tr><tr><td> TNF-alfa </td><td> R &amp; S </td><td> AFL210 </td></tr><tr><td> Ad5f35pp65 </td><td> BCM CAGT Vector Production Facility </td><td></td></tr><tr><td> Trasferimento al plasma con adattatore femmina luer </td><td> Carta Medical </td><td> 89-550-66J </td></tr><tr><td> Lymphoprep </td><td> Nycomed </td><td> 1114550 </td></tr></tbody></table>

expanding cytotoxic t lymphocytes from umbilical cord blood that target cytomegalovirus, epstein-barr virus, and adenovirus

Infezione da virus dopo il trapianto di cellule staminali sono tra le cause più comuni di morte, specialmente dopo sangue cordonale (CB) trapianto (CBT) in cui l&#39;organismo di certificazione non contiene numeri notevoli di virus-esperti cellule T in grado di proteggere il destinatario dalle infezioni 1. - 4 Noi e altri hanno dimostrato che virus specifica CTL generati da donatori sieropositivi e infuso al destinatario sono sicuri e protettivi. 5-8 Tuttavia, fino a poco tempo, virus-specifiche cellule T non può essere generato dal sangue del cordone ombelicale, probabilmente a causa del assenza di celle di memoria virus-T specifiche. Nel tentativo di imitare il meglio in condizioni di innesco in vivo di cellule T naive, abbiamo stabilito un metodo utilizzato CB derivate da cellule dendritiche (DC) trasdotte con un vettore adenovirale (Ad5f35pp65) contenente l&#39;antigene immunodominante CMV pp65, quindi guidare specificità delle cellule T verso CMV e adenovirus. 9 Alla iniziazione, usiamo queste macchineDCs strutturati come anche cellule T CB-derivato in presenza delle citochine IL-7, IL-12 e IL-15. 10 Alla stimolazione secondo abbiamo usato EBV-trasformate cellule B, o EBV-LCL, che esprimono sia latente e litiche antigeni EBV. Ad5f35pp65-trasdotte EBV-LCL sono usati per stimolare le cellule T in presenza di IL-15 alla seconda stimolazione. Stimoli successivi utilizzano Ad5f35pp65-trasdotte EBV-LCL e IL-2. Da 50x10 6 cellule mononucleate CB siamo in grado di generare l&#39;alto di 150 x 10 6 cellule T virus-specifici che lisare antigene-pulsate obiettivi e citochine rilascio in risposta alla stimolazione antigenica. 11 Queste cellule sono state prodotte in un modo conforme GMP utilizzando solo la frazione al 20% di una unità di sangue del cordone frazionato e tradotti per l&#39;uso clinico.

Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Qui si descrive la prima pratica di fabbricazione (GMP) conforme metodo per produrre virus-specifici linfociti T citotossici (CTL) da sangue del cordone ombelicale, fonte di cellule T prevalentemente ingenuo.

Espansione linfociti T citotossici da sangue del cordone ombelicale che Target Citomegalovirus, virus di Epstein-Barr virus, e Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

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Immunology and Infection

12.7K Views.  Baylor College of Medicine. Qui si descrive la prima pratica di fabbricazione (GMP) conforme metodo per produrre virus-specifici linfociti T citotossici (CTL) da sangue del cordone ombelicale, fonte di cellule T prevalentemente ingenuo.

Video: Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Preparazione e utilizzo di HIV-1 CD4 infetti primaria + T-Cells come cellule bersaglio nei test di cellule Natural Killer citotossici

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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Immunologia e infezioni

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Metodi per valutare la morte delle cellule beta Mediata da linfociti T citotossici

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Istituzione di Epstein-Barr Virus Growth-trasformata linee linfoblastoidi

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Applicazione clinica della<em&gt; Bella Addormentata</em&gt; Artificiale e cellule presentanti l&#39;antigene per modificare geneticamente le cellule T da sangue del cordone ombelicale e periferico

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Isolamento di precursori delle cellule B di sottoinsiemi di sangue del cordone ombelicale

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Isolamento e differenziazione di Th17 Naïve linfociti T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Sviluppo di un Varicella-Zoster immunità cellulo-mediata specifica-Virus-IFN γ ELISpot test per valutare seguito cordone ombelicale trapianto di sangue

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generazione di Human alloantigene-specifiche cellule T da sangue periferico

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

Radial Mobilità e citotossico Funzione retrovirale Replica vettoriale trasdotte, non aderente Alloresponsive Linfociti T

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...