Questo lavoro è supportato da una produzione di assistenza per le terapie cellulari (contratto NIH-NHLBI (HB-10-03) HHSN26820100000C) (CMR), un centri specializzati per la terapia cellulare di Grant NIH-NHLBI 1 U54 HL081007 (CMR), una ASBMT Investigator Award giovani (UG e JV), una società Leukemia and Lymphoma Fellow Award speciale in Ricerca Clinica (UG) e un Amy Strelzer Scholar Award Manasevit (AML).

1. DC nucleofection <ol><li> Harvest DC derivate da monociti, che sono stati arricchiti con l&#39;adesione di plastica, colta per 5 giorni utilizzando cellule Genix multimediale integrato con IL4 (1000U/ml), GMCSF (800IU/ml) e ulteriormente stagionato per 24 ore utilizzando le citochine maturazione DC IL4 (1000U / ml), GMCSF (800IU/ml), IL6100ng/ml, TNF-α 10ng/ml, IL1-β 10ng/ml e PGE2 (1μg/ml) 1, dalla risospensione dolce con una pipetta di trasferimento da 3 ml. </li><li> Conte DC praticabile con trypan blu, trasferimento in 3x tubi 15ml con non meno di 0.5x10 6 e non più di 2x10 6 cellule / tubo. </li><li> Centrifugare per 10 minuti DC @ 200g. Durante questo periodo di pre-riscaldare cella Genix multimediali integrate con citochine maturazione della DC (media maturazione DC) - 2ml/well in tre pozzi di un 12-e colture di tessuti trattati piastra a 37 ° C / 5% CO 2 incubatore. </li><li> Una volta che le cellule hanno finito di girare, aspirare il supernatante e aggiungere i plasmidi DNA relativi a ciascuno dei tubi in una concentrazione finale di DNA 5μg / tubo. In questo caso aggiungere la codifica plasmide IE1-pp65 al tubo # 1, Hexon-Penton al tubo # 2, e EBNA1-LMP2-BZLF1 per tubo # 3. </li><li> Risospendere DC e del DNA con 100μl di soluzione nucloefection Amaxa, mescolare bene e trasferimento in cuvette nucleofection. </li><li> Cuvette posto nella nucleofector 4D, scegliete programma CB150 (Amaxa / Lonza), e premere start. </li><li> Immediatamente dopo nucleofection aggiungere 500μl di 2 ml preriscaldata cella Genix dei media maturazione DC alla cuvetta, mescolare delicatamente pipettando su e giù per 2-3 volte, e DC trasferimento nucleofected al preparato 12-pozzetti contenenti il ​​restante 1,5 ml di preriscaldata DC maturazione media. Trasferimento ai 37 ° C / 5% CO 2 incubatore per un ulteriore 12-18hrs. </li></ol> 2. Stimolazione delle cellule T <ol><li> Raccolta e il conteggio DC nucleofected, e irradiare a 30Gy. Lavare una volta con 10 ml di medium CTL (45% RPMI, 45% clic EHAA, 10% FBS, 2mM Glutamax) e risospendere @ 3 x 10 5 DC per ml di mezzi di CTL. </li><li> Piscina un minimo di 7.5x10 5 (2,5 ml) e un massimo di 15x10 5 (5 ml) di DC contenenti ognuno dei plasmidi e trasferire la DC in pool per la G-Rex dispositivo che sarà posto in incubatrice. </li><li> Per la preparazione delle cellule responder utilizzare PBMC precedentemente congelate o cellule mononucleari non aderente che rimangono dopo la selezione DC (aderenza o CD14 selezione). Scongelare le cellule, trasferimento al terreno di coltura preriscaldata, lavare una volta con i media CTL. Risospendere le cellule in Media CTL, contare le cellule e portarli a una concentrazione di 2x10 6 cellule per ml. Prendere 15x10 6 celle o 7.5ml e integrare con 30000U IL4 (1000U/ml -. Conc finale) e 300ng IL7 (10ng/ml - conc finale.). </li><li> Trasferimento 7.5ml di PBMC (15x10 6 celle) al G-Rex e rabboccare il bioreattore con i media CTL per un volume totale di 30ml. </li><li> Cultura del G-Rex per 6-7 giorni a 37 ° C / 5% CO 2 incubatore umidificato. </li></ol> 3. L&#39;espansione delle cellule T <ol><li> Il giorno di 6-7, 10ml aspirato di media, poi mescolare le cellule del restante 20 ml di media con una pipetta da 10 ml e conta delle cellule vitali con trypan blu. Se ci sono &lt;50x10 6 ricostituire con mezzi freschi + citochine. Se ci sono&gt; 50x10 6 celle rimuovere 10 ml di sospensione cellulare, trasferimento in un nuovo G-Rex, e quindi alimentazione sia G-Rexs fresca mezzi CTL + citochine. </li><li> Cultura per altri 4-6 giorni. Una volta che le cellule sufficiente sono stati ampliati, eseguire la caratterizzazione fenotipica e funzionale del CTL in eccesso e crioconservare per un uso futuro. </li></ol> 4. Rappresentante dei risultati: Uno schema del nostro approvato dall&#39;FDA multivirus specifico processo di generazione CTL è illustrato nella figura 1. A differenza di convenzione multivirus CTL protocolli che utilizzano adenovectors e EBV-LCL per stimolare virus cellule T reattive 2 abbiamo sostituito con materiale infetto da virus plasmidi DNA che codificano antigeni multipli derivati ​​da ciascuno dei virus 3. Per stimolare CTL trivirus abbiamo progettato tre multicistronic plasmidi codifica Hexon e Penton di adenovirus, IE1 e pp65 del CMV, e EBNA1, LMP2 e BZLF1 di EBV. Questi antigeni sono stati scelti sulla base di incoraggianti risultati clinici del nostro e di altri gruppi che mostrano che le cellule T diretta contro Adv-hexon e Penton 2,4-6, e di CMV-1E1 e per CMV pp65 sono protettivi in vivo 7. Per EBV, EBNA1 è un immunodominanti CD4 + antigene delle cellule T obiettivo espresso in tutte le neoplasie EBV-associati e in normali cellule infettate da EBV-B 8,9, LMP2 è immunogenico per tutti i tipi HLA più ed espresse nella maggior parte dei tumori EBV, 10,11 mentre BZLF1 codifica un immunodominanti, immediato antigene precoce ciclo litico che stimola sia CD4 + e CD8 + T dalla maggior parte degli individui ed è probabile che importante per la control di cellule replicazione del virus 12. Per ottimizzare ulteriormente i nostri metodi di produzione abbiamo collaborato con la tecnologia che la Natura ha generato minimizzato, antibiotico-free (FDA-compliant) plasmidi per la stimolazione CTL 13,14. Utilizzando questa strategia abbiamo sempre raggiungere efficienze nucleofection del&gt; 35%, pur mantenendo alta la vitalità delle cellule (dati non riportati) 3. La figura 2 mostra che la frequenza del virus-specifiche cellule T in risposta a plasmidi DNA ottimizzato come misurato da IFNγ ELISPOT, era maggiore che in risposta a pShuttle tradizionali a base di plasmidi di espressione che esprimono gli stessi antigeni (n = 8 Adenovirus, n = 4 CMV e EBV n = 2). Il rapporto ottimale tra DC: PBMC è stata importante per la potente stimolazione delle cellule T, come mostrato nella Figura 3, dove un rapporto di 1:50 prodotta sub-ottimali di attivazione rispetto a S 1:20 a: rapporto R (n = 2 donatori). Produzione di sufficiente numero di CTL con specificità antigenica ampio è un pre-requisito per l&#39;efficacia clinica nei confronti di tutti e tre i virus. Questo risultato è ottenuto dalla cultura CTL nel G-Rex, che supporta l&#39;espansione delle cellule T superiore rispetto ai tradizionali 24 pozzetti (Figura 4A) 15, mentre l&#39;aggiunta di IL4 e IL7 ad aumenti delle culture del repertorio e la specificità come mostrato nella Figura 4B dove la frequenza di cellule T reattive contro il CMV pp65-derivati ​​HLA-A2 resticted NLV peptide è stata valutata nelle culture generate in presenza o assenza di IL4 e / o IL7 16,17. Per valutare la capacità fenotipo e funzionale delle cellule espanse che effettuare analisi citometria a flusso, intracellulare di citochine colorazione / IFNγ ELISPOT, e Cr 51 saggi di rilascio del prodotto finale per la crioconservazione / infusione. Tipicamente le cellule generate sono policlonali con una popolazione mista di cellule CD4 + e CD8 + T con l&#39;antigene-specificità rilevabili in entrambi i comparti delle cellule T. Il CTL sono in grado di uccidere che esprimono l&#39;antigene virale cellule bersaglio del virus, ma non negativo parzialmente HLA obiettivi abbinati, ad indicare che non devono indurre graft-versus-host disease (GvHD) in vivo (Figura 5). <img src="/files/ftp_upload/2736/2736fig1.jpg" alt="Figura 1"/> Figura 1. Protocollo generazione RCtl. In primo luogo, DC sono nucleofected con l&#39;antigene virale codifica plasmidi e poi mescolato con PBMC autologo a R: S di 10 o 20:1. Le cellule vengono espanse nel G-Rex per 10-14 giorni in presenza di IL4 e IL7, poi raccolte, contate, testato per la funzione, l&#39;identità e la sterilità, e poi crioconservati per uso clinico. <img src="/files/ftp_upload/2736/2736fig2.jpg" alt="Figura 2"/> Figura 2. Plasmidi DNA Ottimizzato indurre superiore attivazione delle cellule T in vitro. DC sono stati ottimizzati con nucleofected, FDA-compliant plasmidi codifica Hexon e Penton (Adv), IE1 e pp65 (CMV), e EBNA1, LMP2, e BZLF1 (EBV) o plasmidi pShuttle convenzionale codifica gli stessi antigeni. Questi sono stati utilizzati per stimolare le cellule T e la specificità è stata analizzata da ELISPOT IFNγ 10 giorni post-stimolazione. <img src="/files/ftp_upload/2736/2736fig3.jpg" alt="Figura 3"/> Figura 3 CC ottimale:. Ratio per l&#39;attivazione delle cellule T CTL. DC da 2 donatori erano nucleofected con tutti e tre i plasmidi ottimizzato e poi utilizzati per stimolare PBMC autologhi a 1:20 o 1:50 DC: rapporto di PBMC. La frequenza di cellule T è stata valutata riattivato il giorno 10 da IFNγ ELISPOT. <img src="/files/ftp_upload/2736/2736fig4.jpg" alt="Figura 4"/> Figura 4. Espansione delle cellule T nel G-Rex con citochine migliorare. Un pannello mostra la G-Rex dispositivo, nonché l&#39;aspetto CTL sulla membrana permeabile ai gas, valutata al microscopio. Un confronto tra la produzione delle cellule ottenuti in colture di tessuti trattati convenzione piatti vs G-Rex è anche mostrato. Pannello B mostra la frequenza del CTL CMV pentamero positivi ottenuti in colture espanse in presenza di nessuna delle citochine, IL4 solo, IL7 solo e IL4 + IL7. <img src="/files/ftp_upload/2736/2736fig5.jpg" alt="Figura 5"/> Figura 5. Fenotipo e la funzione del CTL ampliato. Panel A presenta un esempio rappresentativo del fenotipo del CTL ampliato multivirus, che sono policlonali con una miscela di CD4 + (45% - helper) e CD8 + (42% - citotossici) cellule T, di cui la maggioranza (95%) ha espresso il memoria marcatore CD45RO + / CD62L +. pannello B mostra che queste cellule sono specifici per tutti gli antigeni stimolante e sono polifunzionali come valutato dalla colorazione delle citochine intracellulari di rilevare la produzione di IFNΓ e TNFa dopo la stimolazione antigene. Panel C mostra che il CTL espanse sono funzionali misurate da Cr 51 test. Autologo LCL, da solo o trasdotte con un vettore nullo o un vettore adenovirale che esprime CMV pp65 sono state usate come catrameottiene. Alloreattività è stata valutata utilizzando allogenico esplosioni PHA come bersaglio.

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N. Engl. J. Med. 333, 1038-1044 (1995).</li></ol>

Juan F. Vera è un consulente per la produzione di Wilson Wolf. Gli altri autori non hanno informazioni rilevanti.

Le infezioni virali rappresentano morbilità e mortalità nei pazienti immunocompromessi a causa della malattia o il suo trattamento. Dopo trapianto, per esempio, infezioni causate da herpes virus persistente come EBV e CMV, oltre che da virus respiratori quali virus respiratorio sinciziale (RSV), sono ben noti, mentre l&#39;importanza delle infezioni causate da Adv, virus BK e umano herpesvirus (HHV) -6 hanno più recentemente apprezzato. Mentre gli agenti farmacologici sono la terapia standard per alcune infezioni, hanno tossicità sostanziale, generare varianti resistenti, e sono spesso inefficaci. Al contrario, i virus-cellule T specifiche derivate da donatori di cellule staminali hanno dimostrato sicuro ed efficace per la prevenzione e il trattamento di infezioni virali o malattie il trapianto di cellule staminali emopoietiche (HSCT) impostazione 2,5,6,18-21. Tuttavia, l&#39;applicazione più ampio di immunoterapia delle cellule T è in ultima analisi, limitate dalla complessità dei costi, e il tempo necessario per la produzione di CTL. Il nostro approccio nuovo e rapido per generare multivirus CTL, descritti nel manoscritto attuale, dovrebbe aumentare notevolmente la fattibilità della terapia delle cellule T citotossiche per le malattie virali, che permette la strategia per diventare uno standard di cura per l&#39;ospite immunocompromessi. L&#39;uso del plasmide DC nucleofected come APC consente la presentazione dell&#39;antigene da entrambe le MHC di classe I e II, senza la concorrenza dei vettori virali o addirittura provenienti da molteplici antigeni virali viene espressa all&#39;interno di una singola cella in quanto diverse popolazioni di DC sono utilizzati per ogni plasmide 3. L&#39;impiego di IL-4 / 7 aumenta la sopravvivenza e la proliferazione delle cellule T, che aiuta di conseguenza aumentare la frequenza e repertorio di risposta antigene-specifica delle cellule T 16,17. Infine, la cultura del G-Rex riduce drasticamente l&#39;apoptosi delle cellule T in coltura. Scambio di gas (O 2 e CO 2 out) si verifica attraverso una membrana permeabile al silicio gas alla base del pallone, impedendo l&#39;ipossia, consentendo una maggiore profondità di media sopra le cellule, fornendo più sostanze nutritive e la diluizione di prodotti di scarto. Questa piattaforma può essere esteso anche ai virus aggiuntivi come quando antigeni protettivi vengono identificati.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> CellGenix </td><td> CellGenix </td><td> 2005 </td></tr><tr><td> IL4 </td><td> R &amp; D Systems </td><td> 204-IL/CF </td></tr><tr><td> IL7 </td><td> Peprotech </td><td> 200-15 </td></tr><tr><td> Hyclone RPMI 1640 </td><td> Thermo Scientific </td><td> SH30096.01 </td></tr><tr><td> Siero umano AB </td><td> Valle Biomedical Inc. </td><td> HP1022 </td></tr><tr><td> Nucleofector </td><td> Amaxa / Lonza </td><td> AAF-1001B e AAF-1001X </td></tr><tr><td> Nucleofection Kit </td><td> Amaxa / Lonza </td><td> V4XP-3012 </td></tr><tr><td> Plasmidi </td><td> NTC </td><td> n / a </td></tr><tr><td> GM-CSF </td><td> R &amp; S </td><td> 215-GM/CF </td></tr><tr><td> IL1 </td><td> R &amp; S </td><td> 201-LB-025 </td></tr><tr><td> IL6 </td><td> R &amp; S </td><td> 206-IL-CF </td></tr><tr><td> TNFa </td><td> R &amp; S </td><td> 210-TA-010 </td></tr><tr><td> PGE2 </td><td> Sigma </td><td> P6532-1MG </td></tr><tr><td> G-Rex </td><td> Wilson Lupo Produzione </td><td> AY11-00027 </td></tr></tbody></table>

generation of multivirus-specific t cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant

Le infezioni virali causare morbilità e mortalità nei trapianto allogenico di cellule staminali ematopoietiche (HSCT). Noi e gli altri sono riusciti a generare e infuse cellule T specifiche per il virus di Epstein Barr (EBV), citomegalovirus (CMV) e Adenovirus (Adv) utilizzando monociti e EBV-trasformate linfoblastoidi (EBV-LCL) gene modificato con un vettore adenovirus come cellule presentanti l&#39;antigene (APC). Da un minimo di 2x10 5 / kg trivirus specifici linfociti T citotossici (CTL) proliferavano in base ai registri diversi dopo l&#39;infusione e apparve per prevenire e curare anche una grave malattia virale resistente ad altre terapie disponibili. L&#39;applicazione più ampio di questo approccio incoraggiante è limitata da alti costi di produzione, la complessità della produzione e del tempo prolungato (4-6 settimane per EBV-LCL generazione, e 4-8 settimane per la produzione di CTL - totale 10-14 settimane) per la preparazione. Per superare questi limiti, abbiamo sviluppato un nuovo protocollo di GMP CTL di produzione. In primo luogo, al posto di adenovectors per stimolare le cellule T si usa cellule dendritiche (DC) nucleofected con DNA plasmidi codifica LMP2, EBNA1 e BZLF1 (EBV), Hexon e Penton (Adv), e pp65 e IE1 (CMV) che presentano l&#39;antigene cellule. Queste APC riattivare le cellule T specifiche per tutti gli antigeni stimolante. In secondo luogo, la cultura di cellule T attivate in presenza di IL-4 (1.000 U / ml) e IL-7 (10ng/ml) aumenta e sostiene il repertorio e la frequenza di cellule T specifiche nelle nostre linee. In terzo luogo, abbiamo utilizzato un nuovo dispositivo di cultura gas-permeabili (G-Rex) che promuove l&#39;espansione e la sopravvivenza dei numeri a grandi cellule dopo un singolo stimolo, eliminando così la necessità di EBV-LCLs e riducendo l&#39;intervento tecnico. Mediante l&#39;attuazione di questi cambiamenti possiamo produrre CTL multispecifico targeting EBV, CMV, e Adv ad un costo per 10 6 cellule che si riduce&gt; 90%, e in soli 10 giorni invece di 10 settimane con un approccio che può essere esteso a ulteriori protezione antigeni virali. Il nostro FDA ha approvato l&#39;approccio deve essere di valore per le applicazioni di profilassi e di trattamento per i destinatari alto rischio HSCT allogenico.

Watch this Scientific Journal Video about Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant at JoVE.com

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Un rapido, protocollo semplice e conveniente per la generazione di donatore-derivati ​​multivirus specifici CTL (RCtl) per infusione a trapianto allogenico di cellule staminali emopoietiche (HSCT) a rischio di sviluppare infezioni da CMV, EBV o Adv. Questo processo di fabbricazione è GMP e deve assicurare l&#39;applicazione più ampia di cellule T immunoterapia al di là di centri specializzati.

Generazione di Multivirus-cellule T specifiche per prevenire / curare infezioni virali dopo trapianto allogenico di cellule staminali ematopoietiche

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

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JoVE Journal

Immunology and Infection

17.1K Views.  Baylor College of Medicine. Un rapido, protocollo semplice e conveniente per la generazione di donatore-derivati ​​multivirus specifici CTL (RCtl) per infusione a trapianto allogenico di cellule staminali emopoietiche (HSCT) a rischio di sviluppare infezioni da CMV, EBV o Adv. Questo processo di fabbricazione è GMP e deve assicurare l'applicazione più ampia di cellule T immunoterapia al di là di centri specializzati.

Video: Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

A seguito di Cell-destino<em&gt; E. coli</em&gt; Dopo l&#39;infezione da fago lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

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Immunologia e infezioni

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generazione di cellule T regolatrici indotte da cellule primarie umane T naive e memoria

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

L&#39;identificazione del DNA Mutazioni in depurata ematopoietiche cellule staminali / progenitrici

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Il trapianto di coda della pelle per studiare allogenico di cellule T CD4 risposte in topi

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Arricchimento cellulare automatizzata di Citomegalovirus specifici linfociti T per le applicazioni cliniche che utilizzano il sistema di citochine cattura

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation e Multi-fenotipica Screening-Alto contenuto di<em&gt; Coxiella burnetii</em&gt; Mutants transposon

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Lo sviluppo di cellule staminali derivate antigene-specifiche cellule T regolatorie contro autoimmunità

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

Misura della T cellulare alloreattività Utilizzando Imaging Citometria a Flusso

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Valutazione delle risposte a cellula T Zika Virus-specifici in organi di nuova di Ifnar1 infetti- / - Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...