EJV è sostenuto da una borsa di studio L&#39;s &amp; colite di Crohn Foundation of America (CCFA). JRM è supportato anche da finanziamenti CCFA, Cancer Research Institute (CRI), Howard H. Goodman (MGH), Massachusetts Life Science Center (MLSC) e Innovator Award nuovo direttore del NIH s.

1. Generazione ex vivo di cellule T gut-homing e di controllo (vedi Figura 1) <ol><li> Isolare splenociti da schiacciare uno milza di topi wild-type. Risospendere la sospensione le cellule in PBS e centrifugare per 5 &#39;a 400 x g. Rimuovere il supernatante e lisare i globuli rossi da risospendere il pellet in 4 ml di tampone di lisi ACK per 2-3 minuti. Dopo di che, aggiungere 5 ml di PBS. Centrifugare per 5 &#39;a 400 xg ed eliminare il surnatante. </li><li> Risospendere splenociti totale a 1 x 10 6 / ml in IMDM + 10% FBS + 50 mM di 2-mercaptoetanolo + penicillina / streptomicina (IMDM completo). Separare le cellule in due gruppi, uno dei quali è integrato con AR (o sintetiche RAR-agonisti, come Am80 o Am580) ad una concentrazione finale di 100-200 nM 3. Cellule T attivate in presenza di RA o RAR-agonisti sarà upregulate a4b7and CCR9 e diventerà gut-tropico cellule T, mentre le cellule T attivate senza RA diventeranno cellule T di controllo. </li><li> Aggiungere 1,5-2,0 mL di sospensione cellulare a ciascun pozzetto di una 24-pozzetti precedentemente rivestita con anti-CD3 (10 mg / ml) e anti-CD28 (10 mg / ml) e incubare a 37 ° C in 5% di CO 2 (per il rivestimento della piastra aggiungere 500 ml / pozzetto di anti-CD3, più anti-CD28 in PBS e incubare per 2 ore a 37 ˚ C e poi lavare due volte con 2 ml di PBS). </li><li> Al fine di evitare eccesso di stimolazione delle cellule T, dopo 2-3 giorni il trasferimento delle sospensioni di cellule in una nuova patinata 24-pozzetti. Incubare per altri 2-3 giorni. A seconda della densità delle cellule e la proliferazione dei media potrebbe diventare acido (giallo), nel qual caso potrebbe essere necessario sostituire la metà della media con fresca IMDM completa. Raccogliere le cellule dopo 4-5 giorni (contando da giorno 0). </li><li> Tipici rendimenti finali sono 1-2 x 10 6 cellule T effettrici / bene. Quindi, un minimo di 9-12 pozzi di ogni gut-tropico cellule T ed il controllo delle cellule T dovrebbe essere placcato per finire con 10-20 x 10 6 cellule T per condizione. </li></ol> 2. Analisi di gut-homing recettori su cellule T attivate Dopo 4-5 giorni di coltura si dovrebbero osservare un&#39;alta espressione di gut-homing a4b7and CCR9 recettori sulle cellule T attivate in presenza di agonisti RA o RAR, mentre il controllo delle cellule T dovrebbe esprimere livelli molto bassi di questi recettori 4. Citometria a flusso (FACS) analisi. <ol><li> Raccogliere tra 0,2-0,5 x10 6 celle e centrifugare per 5 min a 300 g a 4 ° C. </li><li> Incubare con anti-CD4-FITC, anti-a4b7-PE (BD Biosciences), anti-CCR9-APC (eBiosciences) e anti-CD8-PerCP per 15 minuti a 4 ° C al buio. </li><li> Dopo l&#39;incubazione, centrifugare per 5 min a 300 g 4 ° C. Lavare e risospendere le cellule in colorazione buffer e analizzare con il FACS. </li></ol> 3. T etichettatura cella con inseguitori cellula CFSE e CMTMR <ol><li> Tenere tutte le soluzioni a 37 ° C prima di iniziare. Lavare gut-tropico cellule T (a4b7 alta CCR9 alta) e il controllo delle cellule T (a4b7 Basso / Neg CCR9 Basso / Neg) due volte con PBS per rimuovere il siero. Regolare la concentrazione delle cellule T a 10-15 x 10 6 / ml in PBS. </li><li> Aggiungi CFSE (Carboxyfluorescein succinimidile estere) o CMTMR (clorometil-benzoil-amino-tetrametilrodamina) fluorofori a uno gut-tropico T e il controllo delle cellule T ad una concentrazione finale di 5 mm e 10 mm, rispettivamente, dolcemente vortex e incubare per 20 minuti a 37 ° C. Si raccomanda di scambiare le labelings nello stesso o in un esperimento separato per escludere potenziali effetti intrinseci di fluorofori sulla migrazione delle cellule T. </li><li> Dopo l&#39;incubazione, spegnere con 1 volume di FBS e incubare per 1-5 minuti a temperatura ambiente. Diluire 10 volte con caldo PBS e centrifugare per 5 min a 300 g. Lavare due volte con PBS e risospendere in IMDM completo. </li><li> Idealmente, 10-20 x 10 6 cellule di ogni popolazione T deve essere miscelato in rapporto 1:1 e poi centrifugato a 300 xg per 5 min. Infine, risospendere le cellule in 200-250 ml di PBS caldo e iniettare attraverso la vena della coda. </li><li> Per calcolare il rapporto esatto di ingresso (idealmente prossimo a 1) salvare 5-10 ml di sospensione cellulare iniettato e diluire con 300 ml di PBS e analizzare con il FACS. </li></ol> 4. Analisi di homing delle cellule T Anche se i topi possono essere analizzati in vari momenti (tra 1 ora e diversi giorni dopo l&#39;iniezione di cellule T), homing di gut-tropico cellule T è meglio documentato tra 12-24 ore dopo l&#39;iniezione. Punti di tempo più lungo aumentare i potenziali effetti di altre variabili che incidono sulla cella finale numeri / rapporti, come ad esempio l&#39;apoptosi delle cellule T e cellule T uscita dal tessuto. Dopo i topi sono eutanasia, sospensioni cellulari da tessuti diversi devono essere isolati senza indugio al fine di diminuire la morte delle cellule, che potrebbero influire sul rendimento finale e risultati. I tessuti di interesse sono il piccolo intestino, colon, milza, linfonodi, placche di Peyer, fegato, polmoni e sangue. Se l&#39;iniettate cellule T sono in buona fede gut-tropico cellule T, che dovrebbero a casa in media 5-10 volte migliore (o superiore) alla mucosa dell&#39;intestino tenue rispetto al controllo delle cellule T. Tuttavia, altri tessuti, come il sangue e la milza, non dovrebbe mostrare una preferenziale la migrazione delle cellule T. Isolamento dei linfociti da lamina propria intestinale e il vano intraepiteliale è stato descritto in precedenza 5. <ol><li> Colorazione FACS: I campioni sono risospesi a seconda del numero di cellule raccolte, con una densità non superiore a 3,0 x 10 6 cellule / ml. Se congenic CD45.1 + o + Thy1.1 topi sono stati usati come destinatari, le cellule sono colorate con il marcatore corrispondente congenic (ad esempio, CD45.2 o Thy1.2) in combinazione con alcuni marker lignaggio (ad esempio, TCRbchain, CD4, CD8 , CD45.1 + / CD45.2 +). </li><li> Durante l&#39;analisi FACS, le cellule sono gated sul marcatore congenic (ad esempio CD45.1) e poi sui globi lineage specifici (ad esempio CD4/CD8) e analizzati per i rapporti tra CMTMR e cellule CFSE positivo. </li><li> I dati sono di solito espressa come l&#39;Indice di Homing (HI), che è calcolato come rapporto CFSE / CMTMR (o CMTMR / CFSE) in ogni tessuto divisa per il rapporto di ingresso corrispondente (vedi sotto). Se il rapporto di ingresso è molto vicino a 1, poi i rapporti tessuto sarà equivalente al HI. </li></ol> HI = CFSE tessuto / tessuto CMTMR: CFSE ingresso / CMTMR ingresso Il sangue dovrebbe essere analizzati al fine di determinare se le due popolazioni di cellule T sono ugualmente rappresentati in circolazione o se una popolazione di cellule T è diminuito rispetto agli altri (ad esempio, intrappolando preferenziale nei polmoni / fegato o con vitalità ridotta). Ciò può accadere quando si confrontano ingenuo o di riposo delle cellule T memoria rispetto recentemente attivato le cellule T effettrici, in quanto quest&#39;ultimo potrebbe essere intrappolato in misura maggiore nei polmoni. Se il HI nel sangue è significativamente diverso da 1, si può normalizzare HI nei tessuti dalla HI nel sangue. Sangue può essere ottenuto tramite puntura cardiaca di topi anestetizzati (con l&#39;eutanasia o Avertin isofluorane precedente) e devono essere lisate due volte con tampone ACK prima di utilizzarlo per la colorazione FACS. Normalizzazione sangue = HI tessuto / HI Sangue 5. Rappresentante Risultati Topi riceventi (Thy1.1 +) sono stati dopo l&#39;iniezione euthanized18h cellulare. Sospensioni cellulari sono stati generati dalla milza, linfonodi periferici (PLN), linfonodi mesenterici (MLN) e piccola lamina propia intestino (LP). Dopo che le cellule sono state colorate per Thy1.2 e CD8 e poi analizzati da FACS di gating su valida Thy1.2 + cellule CD8 +, che sono stati infine analizzati per il rapporto tra le cellule CMTMR + (gut-tropico cellule T) e CFSE + cellule (controllo di cellule T) divisa per il rapporto di ingresso. I risultati possono essere visualizzati come mostrato nella Figura 2, in cui può essere apprezzato che gut-tropico e il controllo delle cellule T homed anche per la milza (HI vicino a 1, indicato con una linea rossa). Al contrario, gut-tropico cellule T migrate circa 10 volte più efficiente al LP rispetto al controllo delle cellule T. <img src="/files/ftp_upload/2619/2619fig1.jpg" alt="Figura 1" /> Figura 1:. Schema della generazione e l&#39;etichettatura di gut-tropico e non-gut cellule T tropico cellule T sono isolate da topi wild-type e attivato con anti-CD3/anti-CD28 e in presenza o assenza di 100-200 nM tutti - trans retinoico (RA). Dopo 4-5 giorni AR trattati con cellule T acquisire l&#39;espressione dei recettori di homing intestino CCR9 e a4b7. Poi, gut-tropico e il controllo delle cellule T sono etichettati in modo differenziale con CFSE o CMTMR, lavati, mescolati in rapporto 1:1 e iniettato in un topo destinatario attraverso la vena della coda. Alcune cellule marcate sono utilizzati per determinare il CFSE / CMTMR rapporto input (dovrebbe essere vicino a 1). <img src="/files/ftp_upload/2619/2619fig2.jpg" alt="Figura 2" /> Figura 2:. Esempio di analisi homing topi analizzati 12-18 ore dopo l&#39;iniezione di cellule T e le sospensioni singola cella sono ottenute dagli organi di interesse. Le cellule sono colorate con anti-Thy1.2 (marcatore congenic) più anti-CD8 + e poi Thy1.2 cellule T CD8 + vengono analizzati per il rapporto tra CMTMR CFSE + + e le cellule di ogni tessuto da FACS. Il CMTMR / CFSE rapporti tessuti vengono poi normalizzate dal rapporto input CMTMR / CFSE di ottenere gli indici di homing (HI). In questo esempio e + CMTMR CFSE cellule + sono gut-tropico e le cellule T effettrici controllo, rispettivamente.

<ol>
	<li>Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial+cell+recognition:+Three+(or+more)+steps+to+specificity+and+diversity.">Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity.</a> Cell. 67, 1033-1036 (1991).</li><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+for+lymphocyte+recirculation+and+leukocyte+emigration:+the+multistep+paradigm.">Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.</a> Cell. 76, 301-314 (1994).</li><li>Iwata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+imprints+gut-homing+specificity+on+T+cells.">Retinoic acid imprints gut-homing specificity on T cells.</a> Immunity. 21, 527-538 (2004).</li><li>Mora, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+and+dynamic+control+of+CD8+T+cell+homing+by+dendritic+cells+from+skin-+and+gut-associated+lymphoid+tissues.">Reciprocal and dynamic control of CD8 T cell homing by dendritic cells from skin- and gut-associated lymphoid tissues.</a> J Exp Med. 201, 303-316 (2005).</li><li>Mora, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imprinting+of+gut-homing+T+cells+by+Peyer's+patch+dendritic+cells.">Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells.</a> Nature. 424, 88-93 (2003).</li><li>Mempel, T. R., Scimone, M. L., Mora, J. R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+leukocyte+trafficking+in+blood+vessels+and+tissues.">In vivo imaging of leukocyte trafficking in blood vessels and tissues.</a> Curr Opin Immunol. 16, 406-417 (2004).</li></ol>

Nessun conflitto di interessi dichiarati.

Anche se gli esperimenti di homing fornire informazioni molto importanti sulla migrazione di popolazioni di cellule totali in qualsiasi tessuto dato, va tenuto presente che questi test non direttamente analizzare l&#39;adesione endoteliale e, pertanto, che non discriminino in cui passo (s) del adesione multistep cascata (tethering / a rotazione, l&#39;attivazione o attaccare), un recettore dato homing agisce. Il gold standard per definire il ruolo specifico dei recettori di homing in cascata l&#39;adesione è microscopi...

Animali: topi C57BL / 6 sono comunemente usati per l&#39;isolamento delle cellule T. Inoltre, CD45.1 e Thy1.1 ceppi congenic sono disponibili attraverso Jackson Laboratories (Bar Harbor, ME). Cultura dei media: IMDM (Medium Iscove di modifica Dulbecco + L-Glutamina + Hepes) più 10% di siero FBS (Fetal Bovine Serum, endotossine basse, Gibco ®, Invitrogen, Carlsbad, CA) integrata con 100 U / ml di penicillina, 100 mg / ml di streptomicina (antibiotici Hyclone, Waltham, MA), 0,5 mg / ml Fungizone / amfotericina B (Gibco), e 50 mm b-mercaptoetanolo. ACK Red Cell Lysis buffer di sangue (globuli rossi, 10 mM KHCO 3, 150 mM NH 4 Cl, 0,1 mM EDTA, pH 8,0), portato a pH 7,2-7,4 e conservare a temperatura ambiente). PBS (Phosphate Buffered Saline, Hyclone, Waltham, MA). Citometria a flusso (FACS), media (PBS o IMDM + 2% FBS + 5 mM EDTA). Quando colorazione utilizzando selectina-Fc chimere, mezzi di comunicazione con 2 mM Ca + + deve essere usato in tutte le misure (compresa l&#39;acquisizione FACS). IMDM è raccomandato in questo caso. Etichettatura delle cellule T e adottiva di trasferimento: CFSE (diacetato carboxyfluorescein, succinimidile estere), CMTMR ((5 - (e-6 )-((( 4-clorometil) benzoile) ammino) tetrametilrodamina) da Molecular Probes ®, Invitrogen, Carlsbad, CA ). 1000 x scorte dovrebbe essere effettuata in DMSO (5 CFSE mm, 20 mm CMTMR) e conservati a -20 ° C. Policlonale attivazione delle cellule T: 24-bene o piastre a 96 pozzetti (tessuto-coltura trattata, polistirolo, a fondo piatto con coperchio, BD Falcon, Franklin Lakes, NJ) vengono incubate per 2 ore a 37 ° C con 50 ml di PBS, rispettivamente , contenente anti-CD3 più anticorpi anti-CD28 (10 mg / mL). Poi, piastre di coltura vengono lavate due volte con PBS e utilizzato immediatamente per la coltura delle cellule T. In alternativa, Dynabeads rivestito con anti-CD3/anti-CD28 (Dynal, Invitrogen, Carlsbad, CA) possono essere utilizzati per l&#39;attivazione delle cellule T, invece di piatto legato anticorpi. Citometria a flusso (FACS) colorazione: attivazione policlonali: CD3 (1452C11), CD28 (37,51). Lineage mAb: CD4 (L3T4), CD8a (Ly-2), Thy1.2 (CD90.2/53-1.2), CD45.2 (104). Gut-homing recettori: purificato CCR9 (CD199/eBioCW-1.2, eBioscience, San Diego, CA), a4b7 (LPAM-1/DATK3), isotipo di controllo (IgG2a, k). Skin-homing recettori: P-selectina-Fc (depurata del mouse P-selectina - proteina di fusione IgG, BD Pharmingen, San Jose, CA), E-selectina-Fc (E-Selectin/Fc mouse ricombinante Chimera, R &amp; D Systems, Minneapolis, MN), più corrispondente secondaria F capra reagente (ab &#39;) 2 anti-IgG umane R-PE (Invitrogen, Carlsbad, CA). Acido all-trans retinoico (Sigma, St. Louis, MO) è risospeso in etanolo assoluto o DMSO utilizzando una lampadina gialla o una fonte indiretta di luce durante la preparazione. Aliquote Conservare in flaconi di vetro a -80 ° C e al riparo dalla luce in ogni momento. Sintetico RAR-agonisti: Am80 (Wako Chemicals, Richmond, VA).

competitive homing assays to study gut-tropic t cell migration

Al fine di esercitare la loro funzione di linfociti bisogno di lasciare il sangue e migrare in vari tessuti del corpo. Adesione dei linfociti alle cellule endoteliali e lo stravaso del tessuto è un processo a più fasi controllate da molecole di adesione diversi (recettori di homing) ha espresso sui linfociti e le loro rispettive ligandi (addressins) visualizzati sulle cellule endoteliali 1 2. Anche se la funzione di questi recettori di adesione può essere parzialmente studiati ex vivo, l&#39;esame finale per la loro rilevanza fisiologica è quello di valutare il loro ruolo in vivo durante l&#39;adesione e la migrazione dei linfociti. Due strategie complementari sono state utilizzate per questo scopo: microscopia intravitale (IVM) e gli esperimenti di homing. Anche se IVM è stato essenziale per definire il contributo preciso di recettori di adesione specifici durante la cascata adesione in tempo reale e in diversi tessuti, IVM richiede tempo e lavoro intensivo, spesso richiede lo sviluppo di sofisticate tecniche chirurgiche, ha bisogno di isolamento prima di omogenea popolazioni di cellule e permette l&#39;analisi di un solo tessuto / organo in un dato momento. Al contrario, competitivo esperimenti di homing consentono il confronto diretto e simultaneo nella migrazione di due (o più) sottoinsiemi cellula lo stesso mouse e permettono anche l&#39;analisi di molti tessuti e di un elevato numero di cellule nel medesimo esperimento. Qui si descrive il classico protocollo di homing competitivo utilizzato per determinare il vantaggio / svantaggio di un tipo di cellula dato casa ai tessuti specifici rispetto ad una popolazione di cellule di controllo. Abbiamo scelto di illustrare le proprietà migratorie di gut-tropico rispetto a non gut-tropico cellule T, perché la mucosa intestinale è la superficie più grande corpo a contatto con l&#39;ambiente esterno ed è anche l&#39;olio extra-tessuto linfoide con il meglio definiti i requisiti migratori . Inoltre, un recente lavoro ha stabilito che la vitamina A metabolita acido all-trans retinoico (RA) è il principale meccanismo molecolare responsabile per indurre gut-specifici recettori di adesione (integrine recettori delle chemochine a4b7and CCR9) sui linfociti. Quindi, possiamo facilmente generare un gran numero di gut-tropico e non gut-tropico linfociti ex vivo, attivando le cellule T, in presenza o assenza di RA, rispettivamente, che possono essere finalmente utilizzati negli esperimenti di homing competitivo qui descritto.

Watch this Scientific Journal Video about Competitive Homing Assays to Study Gut-tropic T Cell Migration at JoVE.com

Competitive Homing Assays to Study Gut-tropic T Cell Migration

Esperimenti di homing competitivo permettono di valutare direttamente le proprietà migratorie di due diverse popolazioni cellulari in un mouse. Qui illustrare questa procedura confrontando la migrazione di ex vivo generata gut-tropico rispetto a non-intestinali cellule T tropico.

Competitivo saggi Homing allo Studio Gut-tropico Migrazione cellulare T

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

competitive-homing-assays-to-study-gut-tropic-t-cell-migration

Research

JoVE Journal

Immunology and Infection

12.4K Views.  Massachusetts General Hospital, Harvard Medical School. Esperimenti di homing competitivo permettono di valutare direttamente le proprietà migratorie di due diverse popolazioni cellulari in un mouse. Qui illustrare questa procedura confrontando la migrazione di ex vivo generata gut-tropico rispetto a non-intestinali cellule T tropico.

Video: Competitive Homing Assays to Study Gut-tropic T Cell Migration

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento linfociti T umani, la cultura e l&#39;analisi delle migrazioni In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Ricerca

Immunologia e infezioni

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non invasiva di imaging di homing dei leucociti e la migrazione In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

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

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

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

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

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Una valutazione quantitativa della migrazione cellulare con il Test Phagokinetic motilità Traccia

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

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 ...

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.

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 ...

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 ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Un test CFSE-based per studiare la migrazione delle cellule dendritiche murini della pelle in drenanti linfonodi durante l&#39;infezione con<em&gt; Mycobacterium bovis</em&gt; Bacille Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Utilizzando cristallografia a raggi X, Biofisica, e saggi funzionali per determinare i meccanismi di amministrazione delle cellule T del recettore Riconoscimento di cancro antigeni

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

Un romanzo In Vitro ferita guarigione test per valutare la migrazione cellulare

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...