Vorremmo ringraziare Aaron Selya di discussione utile. Questo lavoro è stato sostenuto da NIH concedere AI29575, CA108835, AI077097 a JS, una borsa di studio pilota della Johns Hopkins Malaria Research Institute e il Dipartimento della Difesa concedere PC di MO 040972

1. Fare, HLA-A2-Ig base aAPC <ol><li> Preparare un tampone sterile borato <ol><li> Sciogliere l&#39;acido borico in acqua per fare 0.1M. Regolare il pH a 7,0. </li><li> Filtrare attraverso un filtro 0.22μm sterile e conservare a 4 ° C. </li></ol></li><li> Preparare tampone sterile Bead Lavare <ol><li> Prendere 956ml PBS w / o magnesio o di calcio. </li><li> Aggiungere 30 ml di siero umano AB. </li><li> Aggiungi EDTA concentrazione finale 2mm. </li><li> Aggiungi 0.1gsodium azide. </li><li> Filtrare attraverso un filtro di 0,22 micron sterile e conservare a 4 ° C. </li></ol></li><li> Prendere 1 ml di Invitrogen M-450 perle di resina epossidica a magazzino, circa 400 milioni di perline (sfere possono essere contati da emocitometro), e messo in una sterile, flaconcino di vetro tappo a vite. </li><li> Mettete la fiala contro una calamita Dynal MPC-1, mentre le perline aderire al lato del flaconcino, togliere il surnatante tramite aspirazione. Lavare una volta con perle di 1 ml di tampone borato. </li><li> Perline risospendere in una miscela di tampone borato da 1 ml e 20 mg di HLA-A2-Ig dimero e 20 mg di mAb anti-CD28 umano (clone 9,3). </li><li> Proteine ​​per coniugazione tallone: ​​mettere il flacone di vetro sul rotore e ruotare a 4 ° C per 24 ore. </li><li> Porre il tubo in MPC-1 magnete e rimuovere tutti tampone borato. </li><li> Lavare le perle due volte con 1 ml di tampone di lavaggio Bead. </li><li> Incubare le perline in 1 ml tampone di lavaggio perlina, ruotare a 4 ° C per 24 ore. Perché tampone di lavaggio contiene microsfere di siero umano AB, bloccherà tutto il sito residuo legame con le proteine ​​sulla perline. </li><li> Rimuovere il surnatante dal flacone di vetro, sostituire con 1 ml di tampone fresco Lavare Bead. </li></ol> 2. Controllo della qualità dei aAPC e peptide carico, stoccaggio <ol><li> Aggiungere ~ 5 × 10 5 perline di FACS 100μl tampone di lavaggio in tubi di FACS, e la macchia con 1ml di anti-mouse IgG1-PE (riconoscere la parte Fc di HLA-A2-Ig) e 1ml di anti-topo IgG2a-FITC (riconoscere la parte Fc di mAb anti-CD28). Dopo la colorazione per 20 minuti in FACS lavaggio tampone, lavare di nuovo e leggere il risultato colorazione immediatamente citofluorimetro (Figura 4). </li><li> Peptide carico sul perle: lavare le perle due volte in flaconcino di vetro con 1 ml di PBS sterile. Risospendere con 1 ml di PBS sterile quindi aggiungere 10μl di CMV peptide (1mg/ml) </li><li> Contare le perline da emocitometro, e l&#39;etichetta del flaconcino con data e concentrazione. </li><li> aAPC sono pronti per l&#39;uso dopo almeno 3 giorni di incubazione peptide a 4 ° C, per consentire un tempo sufficiente di legame peptidico sul HLA-A2-Ig dimero. Le sfere possono essere conservato a 4 ° C e rimangono funzionali per almeno 6 mesi. </li></ol> 3. CTL isolamento umano <ol><li> Raccogliere ~ 100 ml di sangue fresco periferico di sano HLA-A2 donatore positivo in 10 tubi di eparina sodica Vacutainer BD. Utilizzare una 21-gauge o più grande per evitare l&#39;emolisi. </li><li> Centrifugare a 300xg per 10 minuti a temperatura ambiente </li><li> Rimuovere con cura lo strato superiore al plasma per aspirazione. Il plasma può essere usato come supplemento per il terreno di coltura. </li><li> Sostituire il plasma raccolto con PBS sterile e trasferire il sangue in una sterile T75 fiasco cultura o provette coniche da 50 ml. Mescolare il sangue con PBS ben pipettando su e giù. </li><li> Una volta che tutto il sangue viene raccolto, preparare altri quattro tubi conici da 50 ml e aggiungere 15 ml di Ficoll-Paque Plus. </li><li> Lentamente overlay 30-35ml di cellule del sangue sulla parte superiore del Ficoll di ciascun tubo. Mantenere l&#39;interfaccia distinte tra Ficoll e le cellule del sangue. </li><li> Centrifugare a 500xg per 20 minuti a temperatura ambiente. Girare il freno &quot;off&quot; e l&#39;accelerazione più basso possibile di mantenere una chiara interfaccia tra gli strati. </li><li> Usando una pipetta seriological, aspirare con cura lo strato PBMC e raccogliere il PBMC in una fresca 50ml tubo conico. Quando tutti i PBMC vengono raccolti aggiungere 30 ml di PBS e far girare a 400xg per 10 minuti. Scartare il surnatante e lavare una volta con 30 ml di PBS per rimuovere tutti i residui di Ficoll. </li><li> Procedere alla isolamento delle cellule CD8 + T utilizzando Miltenyi umane CD8 + kit di isolamento delle cellule T secondo il protocollo del produttore. Questo kit arricchisce molto per CD8 + (di solito&gt; 95%) mediante deplezione CD8 - cellule. </li><li> Contare le cellule T CD8 +. La purezza attesi dovrebbero essere superiore al 95%. A conferma di ciò 2x10 uso 5 celle ed eseguire una CD4/3/8 analisi FACS. Il CTL rimanente può essere utilizzata subito per l&#39;antigene specifico per la stimolazione aAPC oppure possono essere congelati per un uso futuro. </li></ol> 4. Nel sistema di coltura in vitro aAPC base <ol><li> Preparare un terreno di coltura <ol><li> Per TF (fattore di crescita delle cellule T, realizzato in laboratorio 4) 2X terreno di coltura: medium RPMI completo più il 5% del donatore autologo plasma e l&#39;8% fattore di crescita delle cellule T. </li><liDonatori&gt; plasma autologo può essere sostituito da inattivato al calore umano siero AB. </li></ol></li><li> Risospendere 1 milioni CTL a 8ml di media cultura TF 2X più 8ml di completo media RPMI, aggiungere 1 × 10 6 aAPC, mescolare bene. </li><li> Utilizzare un multi-canale pipetter alle cellule sulla piastra da 96 pozzetti U fondo piastre di coltura dei tessuti. (160 microlitri per pozzetto) </li><li> Cellule in coltura a 37 ° C, 5% CO 2 incubater per 7 giorni. Nutrire le cellule il giorno 4 con 80 microlitri / e medio TF 2X. </li><li> Le cellule sono pronte per essere raccolte il giorno 7. Dopo la raccolta, posto le cellule contro il magnete e rimuovere il vecchio aAPC. </li><li> Specificità antigenica può essere determinata colorazione tetramero secondo il protocollo del produttore. Fenotipo colorazione e colorazione delle citochine intracellulari sono eseguite in conformità alle nostre 3 precedente studio. </li><li> Cellule raccolte possono essere saltuariamente con aAPC di nuovo nelle stesse condizioni. Numero di cellulare e specificità antigenica è previsto un aumento dopo stimolazione ripetuta. </li></ol> 5. Rappresentante dei risultati: Un esempio di aAPC dopo coniugazione HLA-A2 Ig e anti-CD28 è mostrata in Figura 4. Coniugazione di proteine ​​di successo è evidente da un chiaro spostamento di colorazione degli anticorpi corrispondenti. Mentre la frequenza di specifici CTL CMV nel sangue periferico è tipicamente 0,5-1%, dopo una sola settimana di aAPC-mediata stimolazione, la specificità può raggiungere il 55 - 93% (Figura 2 e 3). L&#39;espansione del CTL antigene specifico può essere molto variabile tra i diversi donatori, ma i risultati sono riproducibili all&#39;interno dello stesso donatore. Per estrapolazione, l&#39;espansione delle cellule CMV specifiche possono essere migliaia di volte rispetto ai livelli precursore direttamente ex vivo (dati non riportati). Colorazione intracellulare delle citochine (Figura 3) mostra che questi CTL espanso sono polifunzionali, piuttosto che esausto, dopo la coltura cellulare e la proliferazione prolungato significativo. <img src="/files/ftp_upload/2801/2801fig1.jpg" alt="Figura 1"/> Figura 1. Diagramma di flusso rappresentante di aAPC espansione basata ex vivo di CTL umane per l&#39;immunoterapia adottiva in HSCT allogenico <img src="/files/ftp_upload/2801/2801fig2.jpg" alt="Figura 2"/> Figura 2. Rappresentante tetramero risultato colorazione dei CTL CMV specifiche generate da aAPC dopo una settimana di cultura <img src="/files/ftp_upload/2801/2801fig3.jpg" alt="Figura 3"/> Figura 3. Rappresentante intracellulare risultato colorazione citochine di CTL CMV specifiche generate da aAPC (CMV è stata specificità 61%) <img src="/files/ftp_upload/2801/2801fig4.jpg" alt="Figura 4"/> Figura 4. Risultato colorazione rappresentante di M-450 perle di resina epossidica dopo coniugazione di proteine ​​colorate con anticorpi anti-mouse IgG1-PE e anti-mouse IgG2-FITC

<ol>
	<li>Walter, E. A. <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, 1038-1038 (1995).</li><li>Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer:+a+clinical+path+to+effective+cancer+immunotherapy.">Adoptive cell transfer: a clinical path to effective cancer immunotherapy.</a> Nat Rev Cancer. 8, 3669-3669 (2008).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nat Med. 9, 619-619 (2003).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+antigen-presenting+cells:+artificial+solution+for+real+diseases.">Artificial antigen-presenting cells: artificial solution for real diseases.</a> Trends Mol Med. 11, 412-412 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

Il sistema aAPC che descriviamo qui è un sistema efficiente per l&#39;espansione ex vivo di CTL umana contro una varietà di antigeni. Particolare cura deve essere presa per quanto riguarda la qualità della coniugazione di proteine ​​e la distribuzione anche di aAPC e CTL nella cultura piastra da 96 pozzetti. Utilizzando questo approccio siamo stati in grado di espandere CTL per più di 8 settimane, durante i quali abbiamo ampliato antigene-specifici CTL fino a un milione di volte 4. Ci sono stati vari sistemi APC artificiali che utilizzano linee cellulari o altre piattaforme acellulare 5, tuttavia, secondo i dati pubblicati ogni sistema ha il suo profilo unico per quanto riguarda l&#39;espansione e la specificità supportare le applicazioni differenti. Importante, poiché la qualità del CTL è importante quanto la quantità, la polifunzionalità del CMV-specifici CTL generati dal nostro sistema sono tenuti a conferire superiore anti-virale efficienza.

<table border="1"><tbody><tr><th> Reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Vacutainer tubo (contiene eparina) </td><td> Becton Dickinson </td><td> 367874 </td></tr><tr><td> Umane CD8 + kit di isolamento delle cellule T </td><td> Miltenyi </td><td> 130-094-156 </td></tr><tr><td> Dynabeads M-450 epossidica </td><td> Invitrogen </td><td> 140,11 </td></tr><tr><td> Dynal MPC-1 Magnete </td><td> Invitrogen </td><td> 120-01D </td></tr><tr><td> Ficoll-Paque Plus. </td><td> GE Healthcare </td><td> 17-1440-03 </td></tr><tr><td> RPMI 1640 medium </td><td> Gibco </td><td> 11875 </td></tr><tr><td> HLA-A2-Ig dimero X </td><td> Becton Dickinson </td><td> 551263 </td></tr><tr><td> iTAgMHC tetramero (HLA-A2-CMV)-PE </td><td> Beckman Coulter </td><td> T20099 </td></tr><tr><td> Falcon 96-ben chiaro piastrina </td><td> Becton Dickinson </td><td> 353077 </td></tr><tr><td> Ratto anti-topo IgG2a-FITC </td><td> Becton Dickinson </td><td> 553390 </td></tr><tr><td> Capra anti-topo IgG1-PE </td><td> Invitrogen </td><td> P21129 </td></tr><tr><td> Siero umano di tipo AB </td><td> Atlanta biologici </td><td> S40110 </td></tr><tr><td> Topo anti-umano CD8a-FITC </td><td> Sigma-Aldrich </td><td> F0772 </td></tr><tr><td> Topo anti-umano CD8a-APC </td><td> Becton Dickinson </td><td> 340684 </td></tr><tr><td> Topo anti-umano IFNγ-FITC </td><td> Becton Dickinson </td><td> 340449 </td></tr><tr><td> Topo anti-umana TNF-PE </td><td> Becton Dickinson </td><td> 340512 </td></tr></tbody></table>

hla-ig based artificial antigen presenting cells for efficient ex vivo expansion of human ctl

CTL con ottima funzione effettrice giocano un ruolo critico nel mediare la protezione contro varie infezioni intracellulari e cancro. Tuttavia, gli individui possono presentare soppressivi microambiente immunitario e, in contrasto con l&#39;attivazione di CTL, le loro cellule autologhe che presentano l&#39;antigene possono tendere a tolerize o anergize CTL antigene specifico. Come risultato, anche se ancora in fase sperimentale, CTL-immunoterapia adottiva si è evoluto fino a diventare un promettente trattamento per varie malattie come il cancro e le infezioni da virus. In esperimenti iniziali ex vivo ampliato CMV (citomegalovirus) CTL specifici sono stati utilizzati per il trattamento delle infezioni da CMV nei pazienti immunocompromessi trapianto allogenico di midollo osseo. Mentre è comune avere pericolose per la vita viremia CMV in questi pazienti, nessuno dei pazienti trattati ampliato CTL sviluppare malattie legate CMV, che implica l&#39;anti-CMV immunità è stabilito dal adoptively trasferiti CTL 1. Risultati promettenti sono stati osservati anche per il melanoma e può essere estesa ad altri tipi di cancro 2. Mentre ci sono molti modi per ex vivo stimolare e ampliare CTL umane, di approcci attuali sono limitati dai limiti di costo e tecnico. Ad esempio, il gold standard attuale si basa sull&#39;uso di DC autologhe. Questo richiede che ogni paziente di donare un significativo numero di leucociti ed è anche molto costoso e laborioso. Inoltre, dettagliata caratterizzazione in vitro di DC ampliato CTL ha rivelato che questi hanno solo non ottimale funzione effettrice 3. Qui vi presentiamo una efficiente sistema di aAPC base per l&#39;espansione ex vivo di CMV umano CTL specifici per l&#39;immunoterapia adottiva (Figura 1). Il aAPC sono state fatte da accoppiamento cellula magnetica con perline di dimensioni umane HLA-A2-Ig dimero e anti-CD28mAb 4. Una volta aAPC sono fatti, possono essere caricati con peptidi di interesse, e rimangono funzionali per mesi. In questo rapporto, aAPC sono stati caricati con un peptide dominante da CMV, pp65 (NLVPMVATV). Dopo coltura purificata umano CD8 + CTL da un donatore sano con aAPC per una settimana, aspirazione specifici CTL può essere aumentata drammaticamente nella specificità fino al 98% (Figura 2) e amplificato più di 10.000 volte. Se più CMV-specifici CTL sono richiesti, l&#39;ulteriore espansione può essere facilmente ottenuto con stimolazione ripetitiva aAPC. Caratterizzazione fenotipica e funzionale mostra queste cellule espanse hanno fenotipo un effettore-memoria e una notevole quantità di entrambi TNFa e IFNγ (Figura 3).

Watch this Scientific Journal Video about HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL at JoVE.com

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Un nuovo metodo DC indipendente per l&#39;induzione e l&#39;espansione di antigene-specifica delle cellule T è descritta. HLA-A2 Ig Cellule artificiali basati Antigen Presenting (aAPC) vengono caricati con HLA-A2 peptidi limitato ad ampliare in modo efficace CTL di specificità antigenica diversi. Questa tecnologia ha un grande potenziale per CTL-immunoterapia adottiva.

HLA-Ig celle in base Antigen Presenting artificiale per Efficiente Ex vivo Estensione dei diritti dell&#39;uomo e CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

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Research

JoVE Journal

Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

15.3K Views.  Johns Hopkins University. Un nuovo metodo DC indipendente per l'induzione e l'espansione di antigene-specifica delle cellule T è descritta. HLA-A2 Ig Cellule artificiali basati Antigen Presenting (aAPC) vengono caricati con HLA-A2 peptidi limitato ad ampliare in modo efficace CTL di specificità antigenica diversi. Questa tecnologia ha un grande potenziale per CTL-immunoterapia adottiva.

Video: HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Antigene specifico In Vivo Analisi Uccidere utilizzando cellule bersaglio CFSE etichetta

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

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Immunologia e infezioni

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Espansione ex vivo di tumore cellule T reattive per mezzo di 1/Ionomycin Bryostatin e il Comune catena gamma Citochine Formulazione

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Espansione, di purificazione, e la valutazione funzionale delle cellule del sangue umano periferico NK

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Espansione del sangue periferico umano γδ cellule T con Zoledronato

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Antigen Presenting artificiale cellulare (AAPC) Attivazione mediata ed espansione delle cellule Natural Killer T

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Killer artificiale cellule presentanti l&#39;antigene (KaAPC) per una efficiente<em&gt; In Vitro</em&gt; L&#39;esaurimento delle Umana antigene-specifiche cellule T

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Linfociti Isolamento da Pelle umana per l&#39;analisi fenotipica e<em&gt; Ex Vivo</em&gt; Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Sviluppo di un modello colite Antigen-driven per studiare presentazione di antigeni da cellule presentanti l&#39;antigene alle cellule T

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...