Questo lavoro è stato supportato anche da finanziamenti del NIH (5U01CA137070) e l&#39;American Cancer Society (RSG-09-070-01-LIB), una ricerca sul cancro Investigator Award Institute (MK), un American Heart Association pre-dottorato (KM) e un Ministero spagnolo dell&#39;Educazione e della Scienza Fellowship (APG). Vorremmo ringraziare il Dr. Nicholas Restifo e il Dr. Zhiya Yu (NIH / NCI) per suggerimenti utili al protocollo.

1. Preparare Costruire retrovirali <ol><li> Sub-clone del recettore delle cellule T (TCR) gene di interesse in un vettore retrovirale (Figura 1, vettori esempio PMSG 7, pMIGII 5,8, pMXs da Cellbiolabs). Il TCR α e β gene della catena dovrebbe essere lo stesso vettore sotto il controllo del promotore stesso per garantire la parità di espressione. Se il TCR di interesse è umano, i domini umano costante bisogno di essere sostituito da domini del mouse costante 9. La nostra osservazione è che TCR lunghezza umani non esprimono stabilmente il topo le cellule T. </li><li> Preparare il DNA plasmidico di alta qualità utilizzando Qiagen Maxi Prep Kit o prodotto analogo. La concentrazione del plasmide deve essere di circa 1 mg / mL. </li></ol> 2. Trasfezione e T-cella di isolamento Day -1 (cellule imballaggio Plate) <ol><li> Sloggiare Platinum-E (Plat-E, Cellbiolabs) cellule imballaggio retrovirale con tripsina-EDTA e neutralizzare con DMEM media. </li><li> Centrifugare le cellule a 1000 xg per 5 min. Aspirare il surnatante e risospendere il pellet cellulare in DMEM media. </li><li> Determinare il numero delle cellule e diluire le cellule a 0.6x10 6 / mL. Tavola 10 ml di sospensione cellulare in un 10mm poli-lisina piatto rivestito coltura tissutale e crescere durante la notte in una cella incubatore a 37 ° C, 5% di CO 2. </li></ol> Giorno 0 (Transfection) <ol start="4"><li> La mattina dopo, esaminare le cellule al microscopio ottico. Le cellule dovrebbero essere circa l&#39;80% confluenti. </li><li> Rimuovere delicatamente il supporto dal piatto. Lavare le cellule una volta con PBS 1x e aggiungere 10 ml di pre-riscaldato, fresco DMEM media senza penicillina-streptomicina (Pen / Strep). (Nota: Pen / Strep possono interferire con la trasfezione) </li><li> Preparare complesso trasfezione. Preparare mix A e B come segue: <table border="0"><tbody><tr><td> Mix A: DNA plasmidi </td><td> 9 mcg </td></tr><tr><td> PCL-Eco plasmide helper </td><td> 6,3 mcg </td></tr><tr><td> OptiMEM </td><td> 1,5 ml </td></tr><tr><td colspan="2"></td></tr><tr><td> Mix B: Lipopfectime 2000 </td><td> 60 microlitri </td></tr><tr><td> OptiMEM </td><td> 1,5 ml </td></tr></tbody></table> Incubare A e B separatamente per 5 minuti a temperatura ambiente Mix A e B insieme delicatamente e incubare per 20 minuti a temperatura ambiente per formare complessi trasfezione. </li><li> Lentamente gocciolare la miscela (totale 3 ml) in Plat-E cellule. Agitare delicatamente la piastra avanti e indietro per distribuire uniformemente la miscela di trasfezione. </li><li> Incubare le cellule a 37 ° C / 5% di CO 2 in un incubatore cellulare. </li><li> Dopo 6-8 ore, sostituire media con 10 ml di DMEM medio fresco (con il Pen / Strep) per la produzione virale. </li></ol> Piastra cappotto con anticorpi <ol start="10"><li> Topo le cellule T devono essere attivati ​​prima di trasduzione virale. Ci sono diversi modi per attivare le cellule T. Qui usiamo piatto anticorpi legati anti-CD3e e anti-CD28. Preparare una miscela di anticorpi di 1 mg / mL di anti-CD3e e 2 mg / mL di anti-CD28 in PBS sterile. </li><li> Dispensare 250 microlitri della miscela di anticorpi a ciascun pozzetto di una 24-pozzetti di coltura tissutale. La piastra può essere coperti per una notte a 4 ° C o 2 ore a 37 ° C. </li></ol> Preparazione di singole cellule sospensione dalla milza del mouse <ol start="12"><li> Vendemmia milza del mouse e trasferimento in media RPMI. </li><li> Posizionare la milza in un colino cellula. Schiacciate la milza con un stantuffo della siringa in una piastra di coltura tissutale 5 cm. Lavare le cellule fuori dal filtro cella con PBS sterile. </li><li> Centrifuga 1000 xg, 5 min. </li><li> Gettare il surnatante. Risospendere il pellet di cellule in tampone di lisi ACK (2 mL per milza). Incubare 2 a 3 min a temperatura ambiente. Aggiungere mezzo RPMI fino a 20 ml e centrifugare 1000 xg per 5 minuti. </li><li> Gettare il surnatante. Risospendere il pellet di cellule in PBS sterile. Determinare numero di cellulare e centrifugare 1000 xg per 5 minuti a 4 ° C. Procedere all&#39;isolamento delle cellule T. </li></ol> L&#39;isolamento e l&#39;attivazione di topo le cellule T <ol start="17"><li> Magneticamente etichetta le cellule in base al manuale del prodotto (CD8 + T-cellule kit di isolamento dal Miltenyi Biotec). </li><li> Inserire una colonna LS (Miltenyi Biotec) nel magnetic campo. Applicare sospensione cellulare etichettati sulla colonna. Raccogliere flow-through (arricchito topo CD8 + T-cellule). Lavare la colonna tre volte con 3 mL di tampone in base al manuale del prodotto e si combinano con precedenti flow-through. </li><li> Macchia le cellule con anticorpi anti-CD3e e anti-CD8a e analizzare in citometria a flusso (Figura 2a). Per una separazione tipica ~ 8-10% di cellule totali può essere isolato. ~ 90% delle cellule isolate sono CD8 + T-cellule. </li><li> Centrifugare le cellule a 1000 xg, 5 min. Scartare il surnatante. Risospendere il pellet di cellule in medium RPMI con umana ricombinante IL-2 (rhIL2, 20 ng / mL) a 1x10 6 / ml. </li><li> Poco prima di aggiungere le cellule, rimuovere soluzione di anticorpi dalla piastra (preparato precedentemente in 2.11). Lavare ogni pozzetto con PBS sterile e rimuovere PBS. </li><li> Aggiungere 1 ml di sospensione cellulare in ciascun pozzetto. Mettere il piatto a 37 ° C / 5% di CO 2 in una cella incubatrice. </li></ol> 3. (Day 2 e Day 3) L&#39;infezione di cellule T attivate <ol><li> Dopo 2 giorni di produzione del virus, il mezzo in Plat-E piastra cellulare dovrebbe diventare gialla. Trasferire il supernatante virale ad un tubo da 15 ml. Aggiungere 10 ml di DMEM medio fresco alla piastra per l&#39;ulteriore produzione virale (opzionale). </li><li> Centrifugare surnatante virus a 1000 xg per 5 minuti per rimuovere i detriti delle cellule. Con attenzione trasferimento sopranatante virus in una nuova provetta. Lasciare un po &#39;di liquido sul fondo del tubo e non disturbare i detriti cellule. </li><li> Raccogliere cellule T attivate dalla piastra in un tubo da 50 ml. Determinare il numero delle cellule. Risparmiare un po &#39;le cellule in una provetta a parte per essere usato come negativo (un-trasdotte) di controllo. Centrifugare a 1000 xg per 5 minuti e scartare il surnatante. </li><li> Risospendere il pellet cellulare in virus supernatante a 10 6 cellule per 1 ml a 20 ng / mL rhIL2 e 10 mg / mL di solfato di protamina. Aggiungere 1 ml di sospensione cellulare in ciascun pozzetto di una piastra ben 24. Risospendere le cellule in provetta di controllo nel medio RPMI alla densità stessa cella con la stessa quantità di solfato di rhIL2 e protamina. Aggiungi le cellule alla piastra stessa. </li><li> Avvolgere il piatto con film plastico. Centrifugare 90 minuti a 2000 xg, a 32 ° C senza freno. </li><li> Dopo la centrifugazione, rimuovere la pellicola di plastica. Aggiungere 1 ml di fresca media RPMI con 20 ng / mL rhIL2 a ciascun pozzetto. Mettere la piastra posteriore per l&#39;incubatrice. </li><li> (Opzionale) livello di espressione Superiore di TCR può essere raggiunto attraverso la raccolta del surnatante virus e ripetendo la procedura di infezione al giorno 3. </li></ol> 4. Espansione di cellule e di valutazione del rendimento di trasduzione <ol><li> Esaminare il trasdotte cellule T quotidiano. Dividere le celle con un rapporto 01:03, quando necessario, in RPMI medio rhIL2. Non lasciate che il proliferare delle cellule (medium diventa giallo). </li><li> Al giorno 6 o 7 ° giorno, macchia le cellule con l&#39;anticorpo e tetramero MHC specifici per il TCR per valutare l&#39;espressione del TCR sulle cellule T di superficie. Utilizzare il non-trasdotte cellule T come controllo (Figura 2c). </li><li> Il trasdotte cellule T sono pronti per ulteriori applicazioni a valle. </li></ol> 5. Rappresentante Risultati Spesso è vantaggioso per purificare cellule T sottoinsiemi prima di trasduzione splenociti anche se possono essere trasdotte senza purificazione. Elevata purezza delle cellule T sottoinsiemi può essere ottenuto utilizzando commerciale sfere magnetiche (Figura 2a). Per valutare il livello di espressione del TCR sulle cellule T, anticorpi specifici per le catene di TCR alfa o beta può essere utilizzato. Tipicamente 30% al 80% delle cellule può esprimere trasdotte TCR (Figura 2b), a seconda dei geni TCR e titoli virus. MHC tetramero specifici per il TCR può essere utilizzato anche per verificare ulteriormente espressione funzionale di TCR (Figura 2c). <img src="/files/ftp_upload/2307/2307fig1.jpg" alt="Figura 1" /> Figura 1. Esempio di cellule T del gene del recettore costruire sub-clonato in un vettore retrovirale. Alfa a tutta lunghezza e geni catena beta sono collegati da un auto-cleavable linker peptide 2A 8. <img src="/files/ftp_upload/2307/2307fig2.jpg" alt="Figura 2" /> Figura 2. Esempio di isolamento di successo e di trasduzione del mouse cellule-T. (A) CD8 T-cellule sono state isolate da splenociti utilizzando CD8a + T cella di isolamento Kit (Miltenyi Biotec) e colorati con gli anticorpi anti-CD3e e anti-CD8a. Isolato CD8 T-cellule sono state trasdotte con R6C12 TCR specifico per l&#39;uomo gp100: 209 -217 quattro peptidi e colorati con anti-umano Vbeta 8 anticorpi (b) e HLA-A2kb gp100 tetramero (c).

<ol>
	<li>Krogsgaard, M., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+T+cells+'see'+antigen.">How T cells 'see' antigen.</a> Nat. Immunol. 6, 239-245 (2005).</li><li>Berg, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen/MHC-specific+T+cells+are+preferentially+exported+from+the+thymus+in+the+presence+of+their+MHC+ligand.">Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.</a> Cell. 58, 1035-1046 (1989).</li><li>Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Yu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+immunogenicity+of+a+self/tumor+antigen+derives+from+peptide-MHC-I+instability+and+is+independent+of+tolerance.">Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.</a> J. Clin. Invest. 114, 551-559 (2004).</li><li>Holst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li><li>Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+regression+in+patients+after+transfer+of+genetically+engineered+lymphocytes.">Cancer regression in patients after transfer of genetically engineered lymphocytes.</a> Science. 314, 126-129 (2006).</li><li>Hawley, R. G., Lieu, F. H., Fong, A. Z., Hawley, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+retroviral+vectors+for+potential+use+in+gene+therapy.">Versatile retroviral vectors for potential use in gene therapy.</a> Gene Ther. 1, 136-138 (1994).</li><li>Szymczak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat. Biotechnol. 22, 589-594 (2004).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66, 8878-8886 (2006).</li><li>Letourneur, F., Malissen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+T+cell+hybridoma+variant+deprived+of+functional+T+cell+receptor+alpha+and+beta+chain+transcripts+reveals+a+nonfunctional+alpha-mRNA+of+BW5147+origin.">Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin.</a> Eur. J. Immunol. 19, 2269-2274 (1989).</li><li>Bendle, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+graft-versus-host+disease+in+mouse+models+of+T+cell+receptor+gene+therapy.">Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.</a> Nat. Med. 16, 565-570 (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

Diversi passaggi sono fondamentali per ottenere risultati ottimali. Plat-E linea cellulare di packaging deve essere conservato in condizioni di buona salute. Se necessario, le cellule possono essere diversi passaggi un paio di volte in medium DMEM con 1 mg / ml puromicina, 10 mg / ml blasticidin per prevenire la perdita di espressione delle proteine ​​retrovirali struttura. Il giorno della transfezione, le cellule dovrebbero essere confluenti ~ 80%. Celle troppo pochi o troppi possono ridurre il titolo virus. La qualità virus può essere controllata mediante la prova su linee cellulari che possono essere facilmente trasdotte. Dal TCR richiedono CD3 complessi per essere espressi sulla superficie cellulare, si usano abitualmente 58α-/β- ibridomi 10 (una linea cellulare che non esprime endogeno TCR α o β catena, ma non esprime complesso CD3). Si ottiene quasi il 100% di efficienza di trasduzione ibridomi quando trasduzione cellule con 1 ml di sopranatante virus. Infine, le cellule T devono essere attivati ​​e proliferano perché retrovirus possono infettare solo attivamente divisione delle cellule. Il metodo presentato qui per la produzione di antigene-specifiche cellule T ha diversi limiti. In primo luogo, è difficile raggiungere l&#39;efficienza di trasduzione del 100% per le primarie del mouse T-cellule. Una parte delle cellule non esprimono il TCR di interesse. In secondo luogo, il trasdotte TCR α e β catene possono mispair con TCR endogena 9, che possono ridurre il livello di espressione del TCR di interesse. Inoltre, il TCR mispaired può causare l&#39;autoimmunità in vivo 11. Infine, vi è solo un periodo di tempo limitato la trasdotte cellule T possono essere utilizzati. Dopo circa una settimana, le cellule subiscono apoptosi salvo per essere nuovamente stimolata. Tuttavia, le cellule si esaurisca e perdere con funzioni ripetitive ri-stimolazioni.

Media: DMEM medio (calore DMEM +10% FBS inattivato + Pen / Strep + L-Glu + + Piruvato di sodio non-aminoacidi essenziali). DMEM mezzo senza Pen / Strep (Stesso medium DMEM, ma senza aggiungere Pen / Strep). RPMI medio (calore RPMI +10% FBS inattivato + L-Glu + non-aminoacidi essenziali + β-Mercaphoethanol + Pen / Strep + Piruvato di sodio).

retroviral transduction of t-cell receptors in mouse t-cells

Recettori delle cellule T (TCR) svolgono un ruolo centrale nel sistema immunitario. TCR sulle cellule T superfici possono riconoscere specificamente antigeni peptidici presentati da cellule presentanti l&#39;antigene (APC) 1. Questo riconoscimento comporta l&#39;attivazione delle cellule T e una serie di risultati funzionali (ad esempio la produzione di citochine, l&#39;uccisione delle cellule bersaglio). Comprendere il ruolo funzionale di TCR è fondamentale per sfruttare la potenza del sistema immunitario per trattare una varietà di malattie immunologia correlate (ad esempio il cancro o autoimmunità). E &#39;conveniente per studiare TCR in modelli murini, che può essere realizzato in diversi modi. Fare TCR transgenico modelli murini è costoso e richiede molto tempo e attualmente ci sono solo un numero limitato di metterli a disposizione 2-4. In alternativa, i topi con l&#39;antigene-specifiche cellule T possono essere generati dal midollo osseo chimera. Questo metodo richiede anche diverse settimane e richiede competenze 5. Trasduzione retrovirale di TCR in in vitro attivato topo le cellule T è un metodo rapido e relativamente facile da ottenere cellule T del desiderato peptide-MHC specificità. Antigene-specifiche cellule T possono essere generati in una settimana e utilizzato in tutte le applicazioni a valle. Studiare trasdotte cellule T ha anche applicazione diretta di immunoterapia umano, come il trasferimento adottivo di cellule T umane trasdotte con antigene-specifica TCR è una strategia emergente per il trattamento del cancro 6. Qui vi presentiamo un protocollo per trasdurre retrovirally TCR in in vitro attivato topo le cellule T. Sia umane che il mouse geni TCR può essere utilizzato. Retrovirus trasporto specifici geni TCR sono formati ed utilizzati per infettare topo le cellule T attivate con anticorpi anti-CD3 e anti-CD28. Dopo l&#39;espansione in vitro, le cellule T trasdotte vengono analizzati mediante citometria di flusso.

Watch this Scientific Journal Video about Retroviral Transduction of T-cell Receptors in Mouse T-cells at JoVE.com

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Vi presentiamo un protocollo per la produzione antigene-specifica topo le cellule T mediante trasduzione retrovirale

Trasduzione retrovirale di recettori delle cellule T nel topo le cellule T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

retroviral-transduction-of-t-cell-receptors-in-mouse-t-cells

Research

JoVE Journal

Immunology and Infection

34.7K Views.  New York University School of Medicine. Vi presentiamo un protocollo per la produzione antigene-specifica topo le cellule T mediante trasduzione retrovirale

Video: Retroviral Transduction of T-cell Receptors in Mouse T-cells

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac Transposon modifica del sistema di cellule primarie umane T

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Ricerca

Immunologia e infezioni

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Trasduzione Adenoviral di CD4 T naive Cells allo Studio Differenziazione Treg

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivazione di cellule T<em&gt; In Vitro</em&gt; Da cellule staminali embrionali di topo

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Mouse Naïve CD4<sup&gt; +</sup&gt; Isolamento T cellulare e<em&gt; In vitro</em&gt; Differenziazione in T sottopopolazioni cellulari

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Trasduzione retrovirale di cellule progenitrici del midollo osseo per generare cellule T del recettore Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Generazione<em&gt; De Novo</em&gt; Antigene-specifici T umana recettori cellulari di trasduzione retrovirale di Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Trasduzione retrovirale di cellule T helper come un approccio genetico per studiare meccanismi che controllano la loro differenziazione e funzione

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Isolamento delle cellule CD8 + T residenti neireni del topo per l'analisi della citometria a flusso

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Purificazione ed espansione di cellule T Natural Killer invarianti di topo per studi in vitro e in vivo

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

Analisi dell'afflusso di calcio indotto dal recettore delle cellule T nelle cellule T murine primarie mediante citometria a flusso a spettro completo

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...