Vorrei ringraziare Bianca Mothe, Carla Oseroff, e Marie-France DelGuercio per avermi fatto conoscere saggi immunologici e gestione del mouse. Il lavoro nel laboratorio di GA Splitter è finanziato dal NIH concedere 1-RO1-AI-073558, GLRCE concedere 1-U54-AI-057153, e BARD USA-3829-06.

1. Suscitando CTL effettori target specifici di Immunizzazione (giorno 0) <ol><li> Prima di iniziare, decidere un effettore specifico: sistema di destinazione. In questo esempio di un classico test in vivo CTL, useremo una immunizzazione peptide purificato per suscitare specifiche cellule T CD8 +. Il peptide stesso sarà utilizzato per impulso delle cellule bersaglio singenici, creando un effettore specifico: sistema di destinazione. In alternativa, si può scegliere di utilizzare patogeno intero o proteine ​​per ottenere il effettori e gli obiettivi specifici. </li><li> Decontaminare il piano di lavoro all&#39;interno di una cappa di biosicurezza per garantire la sterilità delle iniezioni. Preparare l&#39;immunizzazione peptide di prima immissione 50 mg di peptide purificato in 100 microlitri di DMSO 10% in PBS / mouse contenuta in una provetta da 2 ml. Successivamente, aggiungere una quantità uguale di molto freddo adiuvante incompleto di Freund e fino alla tacca 0,2 ml con 1 perle mm per la miscelazione. Da questo punto, tenere immunogeno in ghiaccio fino ad iniezione. Coperchio sicuro con Parafilm. </li><li> Utilizzare un battitore tallone impostato RPM massimo di mescolare l&#39;emulsione per 3 minuti, prontamente ponendo l&#39;emulsione di nuovo sul ghiaccio dopo la miscelazione. L&#39;emulsione risultante dovrebbe essere simile nella consistenza alla maionese. Lasciare emulsione per sedersi a 4 ° C per una notte, se il tempo lo permette (fino ad una settimana), mescolando altri eventuali separazione è notato. </li><li> Utilizzare una siringa da 1 ml e 20 o 20,5 gauge a tirare su emulsione facendo attenzione a non tirare a qualsiasi perline (perline dovrebbe essere maggiore di calibro di ago, ma ancora essere a conoscenza). </li><li> Del mouse sicuro per iniezione sottocutanea alla base della coda. Usiamo un modificata plastica tubo da 50 ml conica in modo sicuro e delicatamente immobilizzare il mouse per l&#39;iniezione. Iniettare il mouse con 0,2 ml di emulsione. </li></ol> 2. Cellule bersaglio raccolta (giorno 8) <ol><li> Avrete bisogno ingenuo singenici topi per le vostre cellule bersaglio. Euthanize topi ingenuo secondo gli standard AAALAC. </li><li> Disinfettare il mouse a spruzzo 70% etanolo per ridurre al minimo il rischio di contaminazione da capelli mouse o altri contaminanti esterni. </li><li> Dissezione con forbici fare snip piccoli attraverso la pelle sul lato sinistro del mouse. Tirare lembo cutaneo oltre a scoprire sacco peritoneale e gli organi interni. </li><li> Snip attraverso il peritoneo, facendo attenzione a non compromettere uno degli organi sottostanti. La milza è vicino alla superficie ed è simile a colori per il fegato (rosso scuro), ma più piccoli e fagiolo più in forma. Topi infettati mostrano generalmente milza in particolare maggiore rispetto ai controlli non infetti. </li><li> Estrarre delicatamente la milza, avendo cura di essere gentile per non rompere la milza in più parti al fine di recuperare il massimo numero di cellule. Collocare immediatamente la milza in un 15 ml conica contenente 10 ml di supporti preparati sul ghiaccio. Continuare con i topi rimanenti. </li><li> Versare il contenuto della provetta 15 ml conica (media e milza) in un macinino fibra di vetro. Macinare la milza con un sacco di potenza superiore del braccio. Quando tutto il tessuto ha perso il suo colore che si può supporre che avete recuperato tutte le cellule dal tessuto connettivo. </li><li> Versare il contenuto della smerigliatrice attraverso un filtro a 70 micron di cellule in una provetta da 50 ml. Sciacquare smerigliatrice con mezzi freschi e versare attraverso un colino cellula. </li><li> Centrifugare a 4 ° C, a 1300 RPM per 7 minuti. Eliminare il surnatante. </li><li> Risospendere in 10 ml di reagente di lisi preparata e incubare a 37 ° per 7 minuti. </li><li> Aggiungere fino a 50 ml con mezzi freschi e centrifuga, come sopra. </li><li> Risospendere in 50 ml mezzi freschi e centrifuga, come prima (fase di lavaggio). * Nota: In questo momento è possibile eseguire una separazione magnetica cella per un tipo specifico di cellule (macrofagi esempio) se una pura popolazione di cellule bersaglio è desiderato per il trasferimento in topi riceventi. Inoltre, in questo momento si potrebbe introdurre un trattamento sperimentale nella cultura al fine di studiare gli effetti di tale trattamento sulla morte delle cellule specifiche. </li><li> Contare le celle e la piastra di 1 x 10 7 per bene in 100μL in 96 pozzetti rotonda coltura tissutale piastra inferiore trattati. * Si tratta di un&#39;alta concentrazione di cellule / pozzetto, ma perché il saggio è una veloce, abbiamo visto alcuna perdita di vitalità delle cellule a questa concentrazione. </li></ol> 3. Trattamento e etichettatura delle cellule bersaglio <ol><li> Cellule impulsi appropriati e replica con peptide specifico agente patogeno o di controllo peptide irrilevante: Aggiungere 1 mg / ml peptide. Incubare a 4 ° C per 1,5 ore. Passare a 37 ° C incubatore negli ultimi 30 minuti. Durante questo tempo preparare il reagente CFSE. </li><li> Preparare CFSE secondo le specifiche del produttore e diluire ad una concentrazione di lavoro tra 0,5 e 25 micron. Fai la concentrazione &quot;basso&quot; 10 volte inferiore a quello CFSE &quot;alta&quot; concentrazione. Ad esempio, per [basso] CFSE aggiungere 0,5 microlitri a 10 ml PBS e per [alto], aggiungere 5 CFSE microlitri a 10 ml PBS. Caldo CFSE preparati a 37 ° C prima di etichettatura. </li><li> Rimuovere la piastra di cellule da incubatore e pEllet in una centrifuga ad alta velocità a temperatura ambiente, 1300 RPM per 7 minuti. Flick per rimuovere supernatante. </li><li> Risospendere le cellule in entrambi [basso] o [alto] CFSE con l&#39;aggiunta di 200 ml di reagente appropriato ai pozzetti corrispondenti e incubare per 15 minuti a 37 ° C. </li><li> Rimuovere la piastra di cellule da incubatore e di nuovo pellet a temperatura ambiente, 1300 RPM per 7 minuti. Flick per rimuovere supernatante. </li><li> Risospendere le cellule in 200 l / pozzetto di scaldato mezzi freschi e incubare per 30 minuti a 37 ° C. </li><li> Rimuovere la piastra di cellule da incubatore e pellet a temperatura ambiente, 1300 RPM per 7 minuti. Flick per rimuovere supernatante. </li><li> Risospendere le cellule in 100 l / pozzetto PBS. </li><li> Per il trasferimento adottivo, aggiungere un bene [basso] cellule marcate ad un pozzetto [alto] etichettati cellule per topo. Conseguente iniezione deve essere del 50% obiettivi specifici e il 50% di cellule irrilevante in 0,2 ml. </li><li> Non dimenticare di mettere da parte alcune cellule etichettati in modo da agire come i controlli non trasferiti durante l&#39;analisi dei dati e per l&#39;esecuzione del citofluorimetro (vedi figura 1). </li></ol> 4. Trasferimento adottivo <ol><li> Anestetizzare topi immunizzati precedentemente peptide (Da parte # 1 di questo protocollo) secondo gli standard AAALAC. </li><li> Iniettare 0,2 ml di cellule bersaglio marcato via retro-orbitale percorso in topi riceventi. </li><li> Attendere 6 ore o altro tempo predeterminato ottimale prima di eutanasia i topi destinatario. </li></ol> 5. La raccolta cellule marcate * Nota: I punti 5.1 e 5,11 sono gli stessi passi da 2.1 a 2,11 <ol start="12"><li> Contare le celle e la piastra presso 1 x 10 6 per bene in 100 ul in 96 cultura fondo piatto e tondo tessuti trattati. </li><li> Fissare le cellule recuperate e non trasferiti aggiungendo 100 microlitri paraformaldeide al 2% ad una concentrazione finale di 1% paraformaldeide a tutti i pozzetti. </li></ol> 6. Citometria a flusso e analisi dei dati <ol><li> Esegui campioni su un citofluorimetro e analizzare i dati. </li></ol> 7. Rappresentante Risultati Un picco epitopo diminuendo specifiche sul istogramma risultante è un indicatore di uccidere cellule specifiche (Figura 1). Usare le seguenti equazioni (applicato nella tabella 1) per determinare il valore reale di lisi delle cellule specifiche: = Rapporto Irrilevante Percentuale: percentuale epitopi specifici (di picco [basso]: [alto] di picco), Percentuale Lysis specifica = [1 - (no trasferiti rapporto di controllo / rapporto Sperimentale)] x 100 <img src="/files/ftp_upload/2250/2250fig1.jpg" alt="Figura 1" /> Figura 1. CFSE profilo dei numeri recuperato BALB / c splenociti mouse. Indicare la percentuale di cellule del fenotipo CFSE alto o basso. A) non ha trasferito il controllo. Tutti [alto] CFSE picchi rappresentano epitopo cellule bersaglio specifico, mentre [basso] picchi CFSE costituiscono un obiettivo di impulsi con peptide irrilevante. B) le cellule CFSE etichettati recuperato dal destinatario del mouse immunizzati PBS (controllo). C) le cellule CFSE etichettati recuperato da due differenti topi riceventi peptide specifico, notare i picchi epitopo diminuita specifici. <img src="/files/ftp_upload/2250/2250fig2.jpg" alt="Figura 2" /> Figura 2. Rappresentazione grafica dei dati analizzati. Tre punti dati mostrati per ogni gruppo di topi, PBS, peptide A e B. Peptide <table border="1"><tbody><tr><td> Campione </td><td> Epitopo% CFSE pulsata ad alta </td><td> CFSE% BASSO Irrilevante </td><td> Rapporto di Basso: Alta </td><td> Uccidere per cento epitopi specifici </td></tr><tr><td> PBS </td><td> 42,8 </td><td> 57,2 </td><td> 1,34 </td><td> 16,97 </td></tr><tr><td> PBS </td><td> 43,1 </td><td> 56,9 </td><td> 1,32 </td><td> 15,94 </td></tr><tr><td> PBS </td><td> 44,3 </td><td> 55,7 </td><td> 1,26 </td><td> 11,74 </td></tr><tr><td> Un peptide </td><td> 39,7 </td><td> 60,3 </td><td> 1,52 </td><td> 32,56 </td></tr><tr><td> Un peptide </td><td> 38,6 </td><td> 61,4 </td><td> 1,59 </td><td> 35,61 </td></tr><tr><td> Un peptide </td><td> 40,6 </td><td> 59,4 </td><td> 1,46 </td><td> 29,99 </td></tr><tr><td> Peptide B </td><td> 38,7 </td><td> 61,3 </td><td> 1,58 </td><td> 24,68 </td></tr><tr><td> Peptide B </td><td> 38,5 </td><td> 61,5 </td><td> 1,60 </td><td> 25,32 </td></tr><tr><td> Peptide B </td><td> 39,3 </td><td> 60,7 </td><td> 1,54 </td><td> 22,76 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td> PBS NT controllo </td><td> 47,4 </td><td> 52,6 </td&gt; <td> 1,11 </td><td></td></tr><tr><td> Pep Un Controllo NT </td><td> 49,4 </td><td> 50,6 </td><td> 1,02 </td><td></td></tr><tr><td> Pep B NT controllo </td><td> 45,6 </td><td> 54,4 </td><td> 1,19 </td><td></td></tr></tbody></table> Tabella 1. Analisi dei dati rappresentativi. &quot;NT&quot; non è trasferito.

<ol>
	<li>Blattman, J. N., Wherry, E. J., Ha, S. J., van der Most, R. G., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+epitope+escape+on+PD-1+expression+and+CD8+T-cell+exhaustion+during+chronic+infection.">Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection.</a> J Virol. 83 (9), 4386-4394 (2009).</li><li>Jenkins, M. R., Tsun, A., Stinchcombe, J. C., Griffiths, 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=The+strength+of+T+cell+receptor+signal+controls+the+polarization+of+cytotoxic+machinery+to+the+immunological+synapse.">The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.</a> Immunity. 31 (4), 621-631 (2009).</li><li>Ingulli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+tolerance+and+immunity+in+vivo+by+CFSE-labeling+of+administered+cells.">Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells.</a> Methods Mol Biol. 380, 365-376 (2007).</li><li>Durward, M. A., Harms, J., Magnani, D. M., Eskra, L., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discordant+Brucella+melitensis+antigens+yield+cognate+CD8++T+cells+in+vivo.">Discordant Brucella melitensis antigens yield cognate CD8+ T cells in vivo.</a> Infect Immun. 78 (1), 168-176 (2010).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J Immunol Methods. 171 (1), 131-137 (1994).</li><li>Weston, S. A., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+dyes+for+lymphocyte+migration+studies.+Analysis+by+flow+cytometry+and+fluorescence+microscopy.">New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.</a> J Immunol Methods. 133 (1), 87-97 (1990).</li><li>Akondy, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yellow+fever+virus+vaccine+induces+a+broad+and+polyfunctional+human+memory+CD8++T+cell+response.">The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.</a> J Immunol. 183 (12), 7919-7930 (2009).</li><li>Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+antigen-driven+responses+by+flow+cytometry:+monitoring+proliferation+by+dye+dilution.">Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution.</a> Cytometry A. 73 (11), 1019-1034 (2008).</li><li>Labadi, A., Balogh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+preferences+in+serosal+homing+and+distribution+of+peritoneal+B-cell+subsets+revealed+by+in+situ+CFSE+labeling.">Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling.</a> Int Immunol. 21 (9), 1047-1056 (2009).</li><li>Suffner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+support+homeostatic+expansion+of+Foxp3++regulatory+T+cells+in+Foxp3.LuciDTR+mice.">Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice.</a> J Immunol. 184 (4), 1810-1820 .</li></ol>

Nessun conflitto di interessi dichiarati. Gli esperimenti sugli animali sono stati eseguiti secondo le linee guida e regolamenti stabiliti dalla cura degli animali e del Comitato Istituzionale uso (IACUC) presso l&#39;Università del Wisconsin-Madison.

Questo test può essere modificato per indagare la capacità proliferativa delle cellule, tra cui l&#39;espansione clonale, perché CFSE divide equamente tra progenie relativamente 10,11. E &#39;anche possibile utilizzare l&#39;etichettatura CFSE per studiare la migrazione delle cellule 6,12, anche se ci sono altre cellule tracciamento metodi che possono essere più appropriato a seconda delle ipotesi, per esempio tetrameri e bioluminescenza 13, che può anche essere combinato con l&#39;...

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<td>1 mm glass beads</td>
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<td>Biospec Products</td>
<td>11079110</td>
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<td>BD Falcon</td>
<td>352350</td>
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<td>96-well plate</td>
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<td>Adjulite Incomplete Freund's Adjuvant</td>
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<td>Invitrogen (Molecular Probes)</td>
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antigen specific in vivo killing assay using cfse labeled target cells

Carboxyfluorescein diacetato succinimidile estere (CFSE) può essere utilizzato per etichettare facilmente e rapidamente una popolazione di cellule per un esame più vivo. L&#39;etichettatura è stata classicamente utilizzato per studiare la proliferazione e la migrazione. Nel metodo presentato qui, abbiamo accorciato la timeline dopo il trasferimento adottivo di guardare la sopravvivenza e l&#39;uccisione di epitopo specifiche cellule bersaglio CFSE etichettato 4-6. Il livello di uccidere specifiche di una T CD8 + clone di cellule in grado di indicare la qualità della risposta, in quanto la loro quantità può essere fuorviante. Specifiche cellule T CD8 + può diventare funzionale esaurito nel tempo con un calo della produzione di citochine e uccidendo 7,8. Inoltre, alcuni cloni di cellule CD8 + T non può uccidere così come gli altri con diverse specificità TCR 9. Efficace per l&#39;immunità cellulo-mediata (CMI), devono essere identificati antigeni che producono non solo un numero adeguato di risposta delle cellule T, ma anche funzionale, robusto cellule T risponde. Qui valutare l&#39;uccisione delle cellule specifiche per cento dei due cloni T peptide specifico delle cellule in topi BALB / c.

Watch this Scientific Journal Video about Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells at JoVE.com

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Molte infezioni provocano una forte risposta CTL, ma a volte, la quantità di cellule rispondono non è correlata al controllo del patogeno<sup&gt; 1</sup&gt;. Una misura della qualità CTL è la loro capacità di uccidere specificamente<sup&gt; 2</sup&gt;. Etichettatura CFSE di cellule bersaglio può essere utilizzato per indagare questa qualità CTL risposta<em&gt; In vivo</em<sup&gt; 3,4</sup&gt;.

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.  ...

antigen-specific-in-vivo-killing-assay-using-cfse-labeled-target-cells

Research

JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

19.5K Views.  University of Wisconsin-Madison. Molte infezioni provocano una forte risposta CTL, ma a volte, la quantità di cellule rispondono non è correlata al controllo del patogeno 1. Una misura della qualità CTL è la loro capacità di uccidere specificamente 2. Etichettatura CFSE di cellule bersaglio può essere utilizzato per indagare questa qualità CTL risposta In vivo

Video: Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Trans-vivo Delayed Type Ipersensibilità Assay per Antigen regolamento specifico

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

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Immunologia e infezioni

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 ...

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

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

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

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

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

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Rilevamento e l&#39;arricchimento di Rare antigene-specifiche cellule B per l&#39;analisi del fenotipo e funzione

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

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 ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Generazione di anticorpi monoclonali umani discriminante da rari B antigene-specifiche cellule circolanti nel sangue

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Un'analisi di Microarray della proteina per determinazione sierologica dell'infezione di Clostridium difficile anticorpo seguenti risposte antigene-specifiche

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Anticorpli uccisione test per valutare le risposte immunologiche contro batteri patogeni

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Quantificazione delle cellule CD4 specifiche dell'antigene umano che proliferano utilizzando Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Generazione ad alta efficienza di cellule T Topo primario specifico dell'antigene per test funzionali in un modello di diabete autoimmune

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...