This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R. We thank E. Palmer and B.T.H. Hausmann for mice and technical expertise.

In questa pubblicazione video e protocollo, sono state condotte tutte le procedure sugli animali conformemente al protocollo animali approvato dall&#39;Autorità cantonale di Basilea-Città, Svizzera. Eseguire tutte le procedure in condizioni sterili, ove possibile. 1. Preparazione di Chirurgia <ol><li> Sterilizzare in autoclave tutti gli strumenti chirurgici e garza prima dell&#39;uso. </li><li> Riscaldare il pad calda e organizzare strumenti chirurgici sulla tabella (tabella dei materiali / metodi). </li><li> Aprire le bende striscia dito. Applicare vaselina sul pad ferita con un batuffolo di cotone (assicurarsi che la garza è completamente coperto). </li><li> Pesare i topi e somministrare analgesia Buprenorfina 0,05 mg / kg di peso corporeo. Nota: questo fornirà analgesia subito dopo il trapianto. </li></ol> 2. Preparazione della coda-pelle per il trapianto <ol><li> Euthanize topi donatori (ad esempio, IA BM12) per CO 2 asfissia e disinfect le code con una soluzione di disinfezione prima escissione. </li><li> Posizionare la coda con la striscia nera verso il basso su una tavola di plastica pulito e tagliato longitudinalmente tutta la linea mediana, facendo una incisione superficiale coinvolge solo lo strato di pelle. </li><li> Estrarre la pelle dalla coda con pinze di presa. </li><li> Mettere la coda pelle in una coltura di cellule piatto di 10 centimetri riempito con 10 ml HBSS. </li></ol> 3. Procedura chirurgica <ol><li> Anestetizzare topi riceventi (ad esempio, C57BL / 6) per inalazione di una soluzione isofluorano 3% in una scatola trasparente induzione. Nota: Ci vogliono 3-5 minuti per il mouse per raggiungere l&#39;anestesia piano chirurgico. Utilizzare veterinario unguento sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Posizionare il mouse sul tappetino tiepida e applicare 1,5% isofluorane tramite una maschera bocca e asini ogni riflesso animale punta pizzicare. </li><li> Shave il sito dorsale dei topi riceventi (ad esempio, C57BL / 6) e rimuovere i peli contaminanti utilizzando una garza sw asciuttoab. </li><li> Disinfettare sito trapianto rasata con una soluzione asettica. </li><li> Pizzicare la pelle con una pinza nel sito centrale / destra del dorso e usare le forbici curve per fare un giro incisione di circa 1 x 0,6 centimetri. </li><li> Se l&#39;emorragia si verifica, quindi pulire la ferita con un tampone di cotone sterile. </li><li> Valutare la dimensione della incisione e tagliare un trapianto della dimensione stimata dalla coda-pelle con il bisturi. </li><li> Tagliare gli angoli per arrotondare il trapianto. </li><li> Caricare il trapianto sul bisturi, eccesso secco di HBSS su un batuffolo di cotone sterile e posizionarlo nel letto innesto del mouse destinatario. </li><li> Evitare la sovrapposizione di donatore e la pelle host e applicare l&#39;adesivo di tessuto sulla zona di contatto in esclusiva. </li><li> Applicare un cerotto, senza pieghe intorno alla vita del mouse. </li><li> Applicare l&#39;adesivo bendaggio non elastico per fissare l&#39;intonaco al mouse (3 mm spostato). </li><li> Incidere con le forbici circa 3 mm nel sito ventrale superiore del bendaggio. Nota: Questo per evitare trANALITICI dei denti del mouse durante la fase di recupero. </li><li> Rimuovere l&#39;anestetico-bocca-maschera e mantenere i topi trapiantati sulla rampa di riscaldamento fino sveglio. </li><li> Ritorno topi trapiantati nelle loro gabbie solo quando completamente recuperato e valutare la loro mobilità per garantire che la fasciatura non sia troppo stretto. </li><li> Sterilizzare tutti gli strumenti chirurgici con etanolo al 70% prima di procedere con un intervento chirurgico successivo. </li></ol> 4. Cure post-operatorie <ol><li> Aggiungere sciroppo contenente paracetamolo per l&#39;acqua potabile (4 mg / ml) e somministrare per 7 giorni. </li><li> Ritorno mouse per stanza degli animali una volta che ha recuperato. </li><li> Monitorare attentamente i topi per segni clinici, tra cui la depressione o altri cambiamenti comportamentali (alimentari o di immobilità), aspetto anomalo (mancanza di grooming) o la postura (pilo-erezione o postura ricurva) per il tempo la benda è a posto (1 settimana). </li></ol> 5. Fasciatura Removal (6-7 giorni post-trapianto) <ol><li> Anesthetize topi per inalazione di una soluzione isofluorano 3% in una scatola trasparente induzione (vedi sopra). </li><li> Tagliare benda e gesso con le forbici arterie e cura rimuoverle. Nota: evitare di allungamento del sito trapianto. </li><li> Monitorare i topi trapiantati di segni clinici (v. 4.3) e il punteggio per il rifiuto di innesti cutanei (Tabella 1). Nota: Il sistema di punteggio innesto sfrutta le diverse texture dei capelli sulla coda-pelle rispetto alla dorsale pelle. L&#39;aspetto del trapianto e il suo rifiuto è classificato in 3 gradi. Pelle allogenico (MHC II non corrispondenti, ad esempio, IA BM12) innesti di C57BL / 6 topi immunocompetenti di solito sono rifiutati entro 12-15 giorni dopo il trapianto (dati propri e 15-20). </li></ol>

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The authors declare that they have no competing financial interests.

Trapianto di pelle è un metodo ampiamente usato per studiare rifiuto e tolleranza dipende cellule T. Poiché il modello di trapianto di pelle è stato istituito, sono stati applicati diversi adattamenti e modifiche. Nella procedura descritta, IA BM12 trapianto coda-pelle viene eseguita utilizzando il gas anestetico (isofluorane). L&#39;uso di anestesia gas diminuisce il tempo di esecuzione e topi di recupero, che diminuisce lo stress sui topi trapiantati. La procedura impiega tessuto adesivo per fissare la c...

Il trapianto di organi solidi come la pelle, cuore e reni è ormai una procedura standard nella pratica medica in tutto il mondo 1. Organi trapiantati con successo possono essere rifiutati dalla attivazione del sistema immunitario ricevente, che riconosce i principali antigeni di istocompatibilità del donatore. Pertanto, i pazienti trapiantati necessitano di un trattamento con farmaci immunosoppressori 2. Il trapianto allogenico pelle nei topi è stata fondata da Medawar e colleghi nel 1955 ed è stato utile per identificare le molecole mirate successivamente descritto come complesso maggiore di istocompatibilità (MHC) di classe I e II. Da allora, il modello di trapianto di pelle è stato costantemente modificato e adattato per studiare il ruolo di sottoinsiemi di cellule T e la rilevanza di prodotti chimici e di intervento biologico nel sopprimere rigetto 2-4. Pelle dall&#39;orecchio e il tronco sono più difficili da preparare e sono più suscettibili di ipossia e necrosi di coda pelle 5; tuttavia,la procedura di trapianto è simile. Inoltre il monitoraggio dei trapianti coda-pelle è facile a causa della caratteristica struttura dei capelli della pelle. Questo articolo fornisce una procedura dettagliata per MHC di classe II trapianto coda della pelle disadattamento che permette lo studio dei diversi aspetti del CD4 + T cellulo-mediata rigetto e la tolleranza nei topi. La mutazione naturale tre punti nella molecola MHC di classe II IA b (chiamato IA BM12) 6-9 è sufficiente per indurre il rigetto di alloinnesti cutanee in topi C57BL / 6 8. Il BM12 molecola IA attiva le cellule T CD4 + con vari recettore delle cellule T αβ-(TCR) catene da C57BL / 6 topi, tra cui cellule T Vα2Vβ8-TcR-specifici sono stati identificati al fine di generare un topo transgenico TcR-10. Il trasferimento adottivo di cellule Vα2Vβ8-TcR specifiche T è stato utilizzato per creare un modello di rigetto nei C57B immunodeficientiL / 6 Rag2 - / - topi trapiantati con la pelle IA BM12. Differenze genetiche tra donatore e ricevente impatto l&#39;esito di trapianto di accettazione e di rifiuto. Ci sono diversi tipi di trapianti: autologhi sono trapianti dal singolo destinatario stesso; syngrafts e allotrapianti sono trapianti da individui geneticamente identici e geneticamente imparentati, rispettivamente. L&#39;accettazione di diversi trapianti di organi allogenici è stata dimostrata dagli interventi chimici e biologici nei pazienti e nei modelli murini 11,3,4. In un approccio di base, anti-CD3 anticorpo trattati C57BL / 6 topi hanno mostrato una sopravvivenza prolungata di IA BM12 tail-pelle (dati non pubblicati). L&#39;esaurimento delle CD4 + e CD8 + T cellule prima del trapianto in topi riceventi provocato l&#39;accettazione delle MHC di classe I e II innesti non corrispondenti (Rev. in 12). È interessante notare che il rigetto di innesti di pelle dipende dalla presenza di CD4 + </sup&gt; Cellule T (Rev. in 12). In questo modello, il targeting interazioni specifiche tra le diverse cellule immunitarie, bloccando le molecole di costimolazione con anticorpi o soppressione di cellule T regolatorie potrebbe indurre tolleranza (dati non pubblicati). Infatti, il blocco sia CD40 e CD28 ha portato a lungo termine di tolleranza allogenico pelle 13,14. Trapianto Tail-pelle è facile da eseguire e facile da controllare rispetto al trapianto di altri organi. Inoltre, trapianti coda-pelle sono facili da preparare e sono meno sensibili a ischemia di altri tessuti cutanei. In contrasto con anestetici iniettati, l&#39;uso di gas anestetico (isofluorano) durante il trapianto accorcia sia la procedura e tempi di recupero destinatario. Arricciamenti del trapianto coda-pelle, che può portare alla guarigione della ferita incompleta e rigetto del trapianto, è impedita dalla applicazione di adesivo tissutale. Inoltre, il modello di trapianto di coda-pelle IA BM12 attiva esclusivamente CD4 <sup&gt; + Cellule T sia immunocompetenti e topi immunodeficienti (dello stesso background genetico) facilitare l&#39;interpretazione dei risultati. Questo protocollo descrive un modello di topo affidabile, riproducibile e facilmente controllato che permette intervento chimico e biologico. Il modello è stato progettato per indagare il rifiuto e la tolleranza induzione di trapianti di coda della pelle.

<table border="0" cellpadding="0" cellspacing="0" width="831">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:231px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine Standard solution</td>
			<td>Mundipharma</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton swab</td>
			<td style="width:183px;">Carl Roth GmbH</td>
			<td style="width:119px;">31025060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dafalgan , UPSA</td>
			<td>Bristol Myers Squibb SA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hansaplast Finger Strips</td>
			<td>Beierdorf AG</td>
			<td style="width:119px;">REF.76861</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histoacryl Tissue adhesive</td>
			<td style="width:183px;">Braun</td>
			<td style="width:119px;">REF.1050052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leukotape classic , 2cm x 10 m</td>
			<td>BSN Medical SAS</td>
			<td>REF.02204-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS, Phosphate-Buffered Saline, pH 7.4</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:119px;">10010015</td>
			<td>GIBCO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile gauze, 5 x 5cm, 8ply</td>
			<td>MaiMed GmbH</td>
			<td>21010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical instruments&#160;</td>
			<td style="width:183px;">FST</td>
			<td style="width:119px;">11003-12</td>
			<td>Narrow pattern forceps,&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14095-11</td>
			<td>Fine iris scissor curved,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14094-11</td>
			<td>Fine iris scissor&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14010-15</td>
			<td>Mayo scissors,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14080-11</td>
			<td>Artery scissors ball tip 11.5 cm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">11021-14</td>
			<td>Tissue forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Surgical Blade No.20</td>
			<td style="width:183px;">Swann-Morton LTD</td>
			<td style="width:119px;">3006</td>
			<td>Carbon Steel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Surgical blade Handles</td>
			<td style="width:183px;">Swann-Morton LTD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe, 1ml</td>
			<td style="width:183px;">ARTSANA</td>
			<td style="width:119px;"></td>
			<td>disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temgesic , Buprenorphine</td>
			<td style="width:183px;">ESSEX Chemie AG</td>
			<td style="width:119px;"></td>
			<td>0.3mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Tissue Culture dishes 10 cm, 60.1 cm2</td>
			<td style="width:183px;">TPP</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Vaseline</td>
			<td style="width:183px;">Vifor SA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Warm pad</td>
			<td style="width:183px;">Solis</td>
			<td style="width:119px;"></td>
			<td>Type 223</td>
		</tr>
	</tbody>
</table>

transplantation of tail skin to study allogeneic cd4 t cell responses in mice

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

In un primo approccio C57BL / 6 topi sono stati trapiantati con IA allotrapianti BM12 e IA b syngrafts. Dopo la rimozione della fasciatura, innesti segni manifesti di guarigione della ferita senza chiusura della zona di contatto in topi C57BL / 6 (Figura 1A). Dopo la rimozione della fasciatura, CD4 + T infiammazione cellulo-mediata portato alla comparsa di zone necrotiche (macchie rosse) e rigetto di alloinnesti IA BM12 in C57BL / 6 topi entro 13 giorni dopo i...

Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

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

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

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

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

transplantation-tail-skin-to-study-allogeneic-cd4-t-cell-responses

Research

JoVE Journal

Immunology and Infection

12.2K Views.  University of Basel and University Hospital Basel. Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

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

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

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

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

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

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Immunologia e infezioni

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Accelerated diabete di tipo 1 nei topi induzione trasferimento adottivo di cellule CD4 + T Cells diabetogenici

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

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 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Isolamento e differenziazione di Th17 Naïve linfociti T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alto</sup&gt; T Cells in topi immunodeficienti

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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 ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Imaging CD4 T cellule interstiziali migrazione nel derma infiammate

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

In Vitro e In Vivo valutazione della attività soppressiva T, B e cellule mieloidi e risposte umorali dai destinatari del trapianto

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Un protocollo di aspirazione Blister per studiare T-cellula umana richiamo Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

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

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

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

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

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

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