Name: rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development
Uploaded: 2018-06-19T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Siamo grati ai nostri assistenti tecnici: U. Bröder e H. Shkarlet, che ci hanno aiutato durante le procedure sperimentali. Questo lavoro è stato parzialmente finanziato dalla UE sussidi (UniVax, contratto n. 601738 e TRANSVAC2, contratto n. 730964) e una sovvenzione di Associazione Helmholtz (know-IDR). Le fonti di finanziamento non hanno influenzato la ricerca di design, generazione del manoscritto o decisione di inviarla per la pubblicazione.

Tutti i topi utilizzati in questo studio erano dallo sfondo C57BL/6. Tutti gli animali erano tenuti in condizioni esenti da patogeni. Tutti gli esperimenti sono stati eseguiti secondo la normativa della legge tedesca di protezione animale (TierSchG BGBl. I S 1105; 25.05.1998) e sono stati approvati dal Comitato Bassa Sassonia l'etica di esperimenti di animale e l'ufficio statale (Bassa Sassonia stato ufficio della protezione dei consumatori e la sicurezza alimentare), sotto il numero di permesso 33,4-42502-04-13/1281 e 162280.
1. CFSE macchiatura di OT-I T cellule e trasferimento adottivo
Nota: OT-I topi sono animali transgenically generati che esprimono un recettore delle cellule T (TCR) con fisso α e β catene che insieme riconoscono il peptide di immuno-dominante di ovuli, SIINFEKL10,11. Di conseguenza, questi topi hanno un numero considerevolmente elevato di CD8 specifici SIINFEKL+ T cellule (97%)12 rispetto ai topi normali o OVA-vaccinati (≤ 1%)13.
<ol><li>Isolamento di CD8 tracciabili+ T cellule da OT-I topi che esprimono specifici del linfocita T Thy1un (Thy1.1) allele:<ol>
<li>Eutanasia 6-9 settimane vecchio OT-I topi da inalazione di CO2 seguirono da dislocazione cervicale14. Sezionare la milza e linfonodi principali (solo per coppie di inguinale, ascellare e cervicale) tagliando la pelle con forbici chirurgiche, staccando la pelle dal corpo con l'aiuto di pinze chirurgiche e forcipe e metterli in capsule di Petri (60 x 15 mm) ognuno dei quali contiene un 100 µm poro maglia Coppa per rilascio di macinazione e delle cellule del tessuto.</li>
		<li>Mantenere di Petri (ciascuno con una mesh Coppa) in 3-5 mL di terreno RPMI completo (RPMI 1640, 10% v/v FCS, 100 U/mL penicillina, 50 µ g/mL di streptomicina) tenuta su ghiaccio.</li>
		<li>Schiacciare gli organi con l'aiuto di un stantuffo della siringa o un analogo strumento sterile (previo taglio non è necessario) e raccogliere la sospensione singola cella risultante in provette per centrifuga da 15 mL.</li>
		<li>Centrifugare la sospensione cellulare a 300 x g per 10 min a 4 ° C. Scartare il surnatante. Lavare le cellule in 10 mL di PBS freddo mediante centrifugazione e lisare gli eritrociti dalla milza sospendendo nuovamente il pellet in 1 mL/milza del cloruro di ammonio buffer (buffer di ACK, disponibili in commercio). Incubare le cellule per 1,5 min sul ghiaccio e successivamente lavare le cellule con 10 mL di PBS freddo mediante centrifugazione (come fatto in precedenza).</li>
		<li>Risospendere il pellet nello stesso tubo in 1 mL di PBS (pH 7,2) contenente 5% di siero bovino fetale per isolamento magnetico.</li>
	</ol>
</li>
	<li>Per eseguire l'isolamento magnetico, procedere alla selezione negativa di CD8+ T cellule utilizzando un kit di isolamento magnetico secondo le istruzioni del produttore o pubblicato protocolli15.</li>
	<li>Per eseguire la colorazione di CFSE, prima di contare il numero di CD8+ T cellule ottenute da OT-I topi utilizzando un contatore di cellule automatizzato (contatore di particelle). 1-5 di macchia x 107 cellule/mL in un volume di 1-5 mL con 5 µM CFSE in PBS per 7 min a 37 ° C, al riparo dalla luce in un tubo falcon di 15 ml.</li>
	<li>Placare la macchiatura CFSE aggiungendo lo stesso volume (1:1) di siero bovino fetale alle cellule e li Incubare per ulteriori 7 min a 37 ° C al riparo dalla luce. Lavare le cellule con 10 mL di PBS due volte.</li>
	<li>Contare le celle utilizzando un contatore di cellule automatizzato. Impostare il numero di cellulare da 3-5 x 107 cellule/mL di PBS al fine di iniettare 3-5 x 106 cellule/mouse in 100 µ l per via endovenosa (i.v.) tramite vena caudale.</li>
	<li>Per eseguire iniezioni della vena della coda, immobilizzare i topi in dispositivi appropriati. Scaldare la zona posteriore e la coda dei topi da iniettare utilizzando una lampada di luce rossa, posizionata tra 20 a 25 centimetri lontano i topi per 1-3 minuti consentire la vasodilatazione della vena della coda. Nota: Questo consente alla facilità della vena rilevamento e all'amministrazione della sospensione delle cellule.</li>
	<li>Garantire (con la mano appoggiata tra i topi e la lampada) che non è troppo caldo per i topi e quando le vene di coda sono chiaramente visibili, procedere all'iniezione. Iniettare le cellule nella vena caudale laterale o dorsale utilizzando una siringa da 1mL con un ago calibro 2516.</li>
</ol>2. immunizzazione (Endo-libero OVA + /-adiuvante)
<ol><li>Posizionare i topi trapiantati con OT-ho cellule (passaggio 1.7, Figura 1) in una camera di anestesia e isoflurano in ossigeno per amministrare un anestesia macchina14.</li>
	<li>Quando il mouse è completamente addormentato sotto anestesia, tirarla fuori dalla camera e radere il pelo del mouse sulla zona sopra gluteus superficialis (laterale bassa della schiena) utilizzando una macchina di taglio di capelli elettrico, al fine di eseguire un'iniezione pulito con una buona vista della zona di applicazione.</li>
	<li>Iniettare 50 µ l del vaccino, s.c. in zona rasata utilizzando un ago di calibro 25.</li>
</ol>3. isolamento dei linfociti e la macchiatura per analisi di citometria a flusso
<ol><li>Isolamento dei linfociti e splenocytes<ol>
<li>Eutanasia i topi vaccinati per inalazione di CO2 seguita da dislocazione cervicale.</li>
		<li>Estrarre i linfonodi drenanti e distanti e milza e metterli in piatti separati di Petri contenente 100 µm poro maglia tazze per rilascio di macinazione e delle cellule del tessuto. Seguire i passaggi 1.1.2 attraverso 1.1.3.</li>
		<li>Decantare il supernatante dopo centrifugazione e risospendere il pellet cellulare nello stesso volume del residuo PBS (circa 100 µ l).</li>
	</ol>
</li>
	<li>Macchiando per analisi di citometria a flusso<ol>
<li>In una provetta da 15 mL Centrifuga preparare un mix master contenenti gli anticorpi colorazione nelle concentrazioni illustrate nella tabella 1, producendo così un mix concentrato di colorazione di 2x. Preparare abbastanza volume di mix master di avere 100 µ l per campione. Mescolare le cellule e il mix master 1:1 e viene quindi incubato a 4° C per 30 min. Nota: Il volume finale di colorazione per campione dovrebbe essere 200 µ l (100 µ l di cella pellet + 100 µ l di mix master anticorpo).</li>
		<li>Lavare le cellule due volte mediante centrifugazione, come descritto in 1.1.3, aggiungere 10 mL di PBS, due volte.</li>
		<li>Risospendere le cellule marcate in 0,5-1 mL di PBS per acquisizione e trasferimento della sospensione per tubi cytometer di flusso. Sempre mantenere i campioni sul ghiaccio e al riparo dalla luce.</li>
	</ol>
</li>
</ol>4. flusso Cytometry
<ol><li>Sempre pre-filtrare i campioni di cellule utilizzando filtri di 70-100 µm.</li>
	<li>Preparare singola colorazione compensazione controlli per i fluorophores indicati nella tabella 1 (perline o celle). Nota: Compensazione dovrebbe essere eseguita nel citometro o analisi software17,18,19 e applicato a tutti i campioni.</li>
	<li>Seguire la strategia di gating rappresentata nella Figura 2. Brevemente, popolazioni di cancello in altezza di forward scatter vs zona di forward scatter (Figura 2A) e di nuovo a dispersione laterale ampia rispetto all'area di dispersione laterale (SSA, Figura 2B) al fine di escludere doppietti.</li>
	<li>Porta la popolazione da Figura 2B tracciando BV 650 (auto-fluorescenza) vs FITC (CFSE) al fine di discriminare il vero CFSE macchiato le cellule dalle cellule auto-fluorescente alte. Sono perline o cellule colorate con anticorpo coniugato a BV 650 nei controlli di compensazione. Questa trama (Figura 2) del BV 650 vs FITC è usata per cancello l'OT-ho CFSE macchiato le cellule.</li>
	<li>Al fine di distinguere le cellule derivate dal donatore vs topi destinatari del cancello la popolazione da Figura 2 C per Pe-Cy7 (Thy1.1 - CD90.1-) vs APC (CD8). Nota: Le cellule derivate da OT-I topi del donatore sono Thy1.1+ (CD90.1), mentre le cellule derivate da topi (C57BL/6, destinatario) WT sono Thy 1.2+ (CD90.2) (Figura 2D). Alcuni laboratori hanno OT-I topi in uno sfondo di Thy 1.2 e WT (C57BL/6) in un Thy1.1. In questo caso è necessario usare un anticorpo contro Thy 1.2 per OT-I cellulare gating.</li>
	<li>Ri-cancello CD8 cellule di+ da Thy1.1+ popolazione tracciando su un cancello composto da 450 BV (CD4) vs APC (CD8) (Figura 2E).</li>
	<li>Visualizzare la popolazione gated in Figura 2E utilizzando una visualizzazione dell'istogramma del CD8+ cellule che mostrano CFSE (FITC, 530/15 canale - laser blu-), cancello la popolazione proliferata includendo intensità da 102 fino al livello dove le popolazioni di controllo indivisa sono (intensità ~ 105, a seconda l'efficacia di CFSE colorazione e l'impostazione della tensione sul citofluorimetro) (Figura 2F).</li>
	<li>Fare un ulteriore cancello (non visibile in figura 2) tracciato marcatore FITC vs L/D (450/20 canale, UV laser) al fine di valutare le cellule morte in parallelo alla valutazione di proliferazione.</li>
	<li>Acquisire almeno 5000 eventi per i controlli di compensazione e 10,000 eventi (cancello E, Figura 2) dai campioni presso un citometro a flusso. Eseguire il citometro a flusso di basso o medio (non superare le portate di 20.000 eventi/s).</li>
</ol>

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Moderni vaccini sono idealmente ospita l'antigene purificato e adiuvanti, con l'eventuale aggiunta di un sistema di consegna come liposomi, particelle virus-like, nanoparticelle o vettori dal vivo. Un aspetto fondamentale durante la progettazione di un vaccino è quello di scegliere il giusto adiuvante secondo le necessità cliniche. Parte dell'ambito potrebbe coinvolgere favorendo un umorale vs risposta immunitaria cellulare (o entrambi), l'elezione di un locale vs una risposta immunitaria sistemica (o entrambi) e il ti...

Linfociti T citotossici (CTL) che inducono i vaccini sono fondamentali interventi terapeutici che sono stati sviluppati per combattere alcuni tipi di cancro1. CTL sono anche importanti per vaccini profilattici contro patogeni intracellulari2. Inoltre, CTL sono uno dei meccanismi di difesa immunitario pochi funzionalmente attivi in popolazioni a rischio come neonati3,4 quali dipendono anche CTL per combattere infezioni di vita iniziali5. A questo proposito, i vaccini contro il Virus respiratorio sinciziale (RSV) che sono stati sviluppati con un adiuvante che non suscitare le risposte CTL (allume) ha provocato un completo fallimento del vaccino che porta a gravi complicazioni su infezioni in infanti6. Questi effetti negativi della vaccinazione possono essere invertiti da un CD8+ di risposta delle cellule T7. Precedentemente abbiamo dimostrato che le principali citochine (interferoni di tipo I) suscitate da alcuni stimolatore di agonisti di geni (STING) di interferone sono essenziali per le risposte CTL generate da questi adiuvanti8, in parte misurando la proliferazione delle OT-I T cellule dopo vaccinazione e utilizzando questi risultati come una misura di CTL inducendo capacità osservata in esteso vaccinazione orari9. La misurazione della proliferazione di OT-ho CD8+ T cells in un mouse di destinatario C57BL/6 wild type (WT) di tintura di carboxyfluorescein succinimidyl ester (CFSE) diluizione è una stima robusta della capacità dell'adiuvante di un vaccino per generare cross-priming di SIINFEKL, (il peptide di immuno-dominante di ovoalbumina, uova). Variazioni di questa tecnica sono ampiamente utilizzati per la valutazione della proliferazione di OT-ho CD8+ e OT-II CD4+ T cellule. Ad esempio, esso è stato utilizzato in assenza delle citochine selezionate (topi KO) o per misurare l'efficacia del vaccino dopo il richiamo dell'antigene negli animali WT. Abbiamo messo a punto un protocollo breve (4 giorni esperimento) in cui dopo il trasferimento passivo di CFSE-macchiato OT-ho CD8+ T cells, una sottocutanea immunizzazione (s.c.) che consiste di una dose di 50 µ g di ovuli privo di endotossina completati con adiuvanti di prova viene somministrata (Figura 1). Il follow-up dei risultati 48 h dopo la vaccinazione fornisce una prova affidabile della capacità dell'adiuvante per generare risposte CTL. Da questa strategia, è possibile valutare la potenza della risposta immunitaria locale nel linfonodo drenante dopo immunizzazione, nonché l'entità della risposta misurando l'attività CTL nella milza (o linfonodi a distanza).

<table><tbody><tr><td>BD LSR Fortessa Cell Analyzer</td><td>BD</td><td>Special Order</td><td>Flow Cytometer</td></tr><tr><td>CFSE</td><td>Molecular Probes</td><td>C34554</td><td>Proliferation Dye</td></tr><tr><td>MojoSort Mouse CD8 T Cell Isolation Kit</td><td>Biolegend</td><td>480007</td><td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td></tr><tr><td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td><td>Molecular Probes</td><td>L23105</td><td>Dead Cell Marker</td></tr><tr><td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td><td>eBioscience</td><td>25-0900-82</td><td>antibody</td></tr><tr><td>APC anti-mouse CD8a Antibody</td><td>BioLegend</td><td>100712</td><td>antibody</td></tr><tr><td>BV421 Rat Anti-Mouse CD4</td><td>BD</td><td>740007</td><td>antibody</td></tr><tr><td>Z2 coulter Particle count and Size Analyzer</td><td>Beckman Coulter</td><td>9914591DA</td><td>Cell counter. Z2 Automated particle/cell counter</td></tr><tr><td>EndoGrade Ovalbumin (10 mg)</td><td>Hyglos(Germany)</td><td>321000</td><td>Ovalbumin endotoxin free tested.</td></tr><tr><td>Cell Strainer 100&amp;micro;m nylon</td><td>Corning</td><td>352360</td><td>Cell strainer (100 &amp;micro;m pore mesh cups).</td></tr><tr><td>Sample Vials</td><td>Beckman Coulter</td><td>899366014</td><td>Sample vials for Z2 automated counter</td></tr><tr><td>C57BL/6 mice (CD90.2)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Company is now Envigo</td></tr><tr><td>OT-I (C57BL/6 background, CD90.1)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Inbreed at our animal facility. Company from where adquired is now Envigo</td></tr><tr><td>FACS tubes</td><td>Fischer (Corning)</td><td>14-959-5</td><td>Corning Falcon Round-Bottom Polystyrene Tubes</td></tr><tr><td>Falcon 15 mL tubes</td><td>Fischer (Corning)</td><td>05-527-90</td><td>Falcon 15mL Conical Centrifuge Tubes</td></tr><tr><td>PBS (500 mL)</td><td>Fischer (Gibco)</td><td>20-012-027</td><td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td></tr><tr><td>Red lamp (heating lamp)</td><td>Dirk Rossmann GmbH (Germany)</td><td>405096</td><td>Heating infrred lamp (100 wats)</td></tr><tr><td>IsoFlo (Isoflurane)</td><td>Abbott Laboratories (USA)</td><td>5260.04-05.</td><td>Isoflurane anesthesic (250 mL flask).</td></tr><tr><td>&lt;strong&gt;Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber&lt;/strong&gt;</td><td>Parkland Scientific</td><td>V3000PK</td><td>Isoflurane anesthesia machine.</td></tr><tr><td>RPMI 1640 medium</td><td>Gibco (distributed by ThermoFischer)</td><td>11-875-093</td><td>Base medium with Glutamine (500 mL)</td></tr><tr><td>Pen-Strept antibiotic solution (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>15-140-148</td><td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td></tr><tr><td>Fetal Bobine Serum (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>10082147</td><td>Fetal Bovine Serum, certified, heat inactivated, US origin</td></tr><tr><td>ACK Lysing Buffer (100 ml)</td><td>Gibco (distributed by ThermoFischer)</td><td>A1049201</td><td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td></tr><tr><td>Plastic Petri Dishes</td><td>Nunc (distributed by ThermoFischer)</td><td>150340</td><td>60 x 15mm Plastic Petri Dish, Non-treated</td></tr><tr><td>Cell Clump Filter</td><td>CellTrics (Sysmex)</td><td>04-004-2317</td><td>CellTrics&amp;reg; 50 &amp;mu;m, sterile</td></tr></tbody></table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

La valutazione dei vaccini moderni sottounità rivela che la generazione di anticorpi neutralizzanti è importante ma non sufficiente per la selezione di adiuvante. Di conseguenza, adiuvanti con capacità immuno-stimolante sia umorali e cellulari che sono in grado di promuovere le risposte citotossiche (CTL) di linfociti di T sono urgentemente necessari. Quindi, monitoraggio fedele dei candidati adiuvante che inducono cross-priming e successivamente migliorano CTL generazione rappresenta un passo cruciale nello sviluppo del vaccino. Qui vi presentiamo un'applicazione per un metodo che utilizza SIINFEKL specifici (OT-ho) cellule di T per monitorare la cross-presentazione del modello antigene ovoalbumina (uova) in vivo in presenza di candidati diversi adiuvante. Questo metodo rappresenta un test rapido per selezionare adiuvanti con le migliori funzionalità di cross-priming. La proliferazione di CD8+ T cellule è l'indicazione più preziosa di cross-priming ed è anche considerato come un correlato di indotta da adiuvante cross-presentazione. Questa funzionalità può essere valutata in differenti organi immuni come i linfonodi e la milza. Nella misura della generazione CTL possa anche essere monitorata, dando intuizioni sulla natura di un locale (svuotamento linfonodale principalmente) o una risposta sistemica (linfonodi a distanza e/o milza). Ulteriormente questa tecnica permette modifiche multiple per testare i farmaci che possono inibire specifiche vie di cross-presentazione e offre anche la possibilità di essere utilizzato in diversi ceppi di topi convenzionali e geneticamente modificati. In sintesi, l'applicazione che vi presentiamo qui sarà utile per i laboratori di vaccino nell'industria o academia che sviluppano o modificare coadiuvanti chimici per vaccino ricerca e sviluppo.

Al fine di testare i trattamenti utilizzando una diversa combinazione di adiuvanti (ADJ1 e ADJ2), abbiamo valutato la capacità di generazione di CTL misurando la proliferazione di passivamente trasferiti OT-ho CD8+ T cellule mediante citometria a flusso (Figura 2). Per questo, abbiamo precedentemente macchiato le cellule isolate dal drenante nei linfonodi e milza (tabella 1). Misurando la proliferazione di CD8+ T cellul...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Qui presentiamo un'applicazione per una tecnica immunologica standard (CFSE macchiato OT-ho proliferazione) destinato a monitorare rapidamente adiuvante-mediata di linfociti T citotossici (CTL) generazione in vivo. Questa veloce stima delle capacità CTL è utile per lo sviluppo di vaccini profilattici contro patogeni intracellulari, come pure i vaccini terapeutici del cancro.

Rapida In Vivo valutazione della funzionalità di generazione i linfociti T citotossici di adiuvante per lo sviluppo di vaccini

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

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Research

JoVE Journal

Medicine

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

8.6K Views.  Helmholtz Centre for Infection Research. Qui presentiamo un'applicazione per una tecnica immunologica standard (CFSE macchiato OT-ho proliferazione) destinato a monitorare rapidamente adiuvante-mediata di linfociti T citotossici (CTL) generazione in vivo. Questa veloce stima delle capacità CTL è utile per lo sviluppo di vaccini profilattici contro patogeni intracellulari, come pure i vaccini terapeutici del cancro.

Video: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

In vitro per valutare l&#39;impatto di immunoregolatorio Pathways su HIV-specifica CD4 T effettrici delle cellule di funzione

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Ricerca

Immunologia e infezioni

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

L&#39;uso di fluorescenti target matrici per la valutazione delle cellule T risposte<em&gt; In vivo</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Imaging Whole-animale e di citometria a flusso tecniche di analisi di Antigen-specific CD8 + cellule T Risposte dopo nanoparticelle vaccinazione

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

In Vivo Test per la determinazione della funzione citolitica di cellule T antigene-specifiche utilizzando un modello di vaccinazione

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

Valutazione immunostimulatory agente: Attivazione linfoide tessuto estrazione e iniezione Route-dipendente delle cellule dendritiche

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Sartoria in vivo saggi di citotossicità per studiare l'Immunodominanza nelle risposte delle cellule CD8+ T specifiche del tumore

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Preparazione, caratteristiche, tossicità e valutazione dell'efficacia del vaccino tumorale nanoemulsione autoassemblato nasale in vitro e in vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

In vitro Valutazione dell'attività cellulare del vaccino adiuvante nanoemulsione ofiopogonina D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Determinazione dell'immunogenicità del vaccino utilizzando cellule dendritiche derivate da monociti bovini

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Source: Lirussi, D. et al., <a href="https://app.jove.com/v/57401">Rapid In Vivo&#160;Assessment of Adjuvant&#39;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.</a> J. Vis. Exp. (2018)
The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

Una tecnica immunologica per monitorare la generazione di linfociti T citotossici mediati da adiuvanti

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

Rapida In Vivo valutazione della funzionalità di generazione i linfociti T citotossici di adiuvante per lo sviluppo di vaccini

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