Name: transplantation of tail skin to study allogeneic cd4 t cell responses in mice
Uploaded: 2014-07-25T13:00:00
Description: Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

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.

I denne videoen publikasjonen og protokoll, ble alle prosedyrer dyre gjennomført i henhold til dyr protokollen godkjent av Cantonal Authority Basel-Stadt, Sveits. Utfør alle prosedyrer i sterile forhold der det er mulig. En. Utarbeidelse av kirurgi <ol><li> Autoklaver alle kirurgiske instrumenter og gasbind før bruk. </li><li> Varm varm pad og organisere kirurgiske instrumenter på bordet (Table of materialer / metoder). </li><li> Åpne finger stripe bandasjer. Påfør ...

<ol>
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Skin transplantasjon er en mye brukt metode for å studere avvisning og toleranse avhengig av T-celler. Siden huden transplantasjon modellen ble etablert, har flere tilpasninger og endringer blitt anvendt. I den beskrevne fremgangsmåten, er IA bm12 tail-hud transplantasjon utføres ved hjelp av anestesigass (isofluorane). Bruken av gassanestesi reduserer tiden for utførelse og mus gjenvinning, noe som reduserer belastningen på transplanterte mus. Prosedyren syssels vev lim til å feste hale hud, som har en...

Transplantasjon av faste organer som hud, hjerte og nyrer er nå en standard prosedyre i medisinsk praksis over hele verden en. Vellykket transplanterte organer kan forkastes ved aktivering av mottakerens immunsystem, som gjenkjenner de store histocompatibility antigenene til donoren. Derfor transplanterte pasienter trenger behandling med immundempende medisiner to. Allogen transplantasjon hud hos mus ble etablert av Medawar og medarbeidere i 1955, og var nyttig for å identifisere de målrettede molekyler senere beskrevet som store histocompatibility kompleks (MHC) klasse I og II. Siden den gang har hudtransplantasjon modellen vært kontinuerlig modifisert og tilpasset for å studere rollen til T celle undergrupper og relevansen av kjemiske og biologiske intervensjon i å undertrykke pode avvisning 2-4. Hud fra øret, og stammen er mer vanskelig å fremstille og er mer utsatt for hypoksi og nekrose enn hale-skin 5; Mentransplantasjon prosedyren er lik. I tillegg til overvåkning av tail-skin transplantasjoner er lett på grunn av den karakteristiske konsistens av hår på huden. Denne artikkelen gir en detaljert fremgangsmåte for MHC klasse II mismatch tail-hudtransplantasjon som gjør det mulig for studier av ulike aspekter av CD4 + T-celle-mediert allograft avvisning og toleranse hos mus. Den naturlige tre-punkt-mutasjon i MHC klasse II molekyl IA b (kalt IA bm12) 6-9 er tilstrekkelig til å indusere hud-avvisning av allografter i C57BL / 6 mus 8.. IA bm12 molekyl aktiverer CD4 + T-celler med forskjellige αβ-T-celle-reseptoren (TCR) kjeder fra C57BL / 6 mus, hvorav Vα2Vβ8-TCR-spesifikke T-celler ble identifisert for å generere en TCR-transgene mus 10. I adoptive transfer of Vα2Vβ8-TCR-spesifikke T-celler har blitt brukt for å etablere en avvisning modell i immunodeficient C57BL / 6 Rag2 - / - mus transplantert med IA bm12 hud. Genetiske forskjeller mellom giver og mottaker påvirke utfallet av transplantasjon aksept og avvisning. Det finnes forskjellige typer transplantasjoner: autografts er transplantasjoner fra mottakeren enkelte selv; syngrafts og allografts er transplantasjoner fra genetisk identiske og genetisk ubeslektede individer hhv. Aksept av forskjellige allogene organtransplantasjoner har blitt demonstrert av kjemiske og biologiske intervensjon hos pasienter og musemodeller 11,3,4. I en grunnleggende tilnærming, anti-CD3 antistoff-behandlet C57BL / 6 mus viste forlenget overlevelse av IA bm12 tail-hud (upubliserte data). Nedbryting av CD4 + og CD8 + T celler før transplantasjon i mottaker mus resulterte i aksept av MHC klasse I og II umake grafts (rev. i 12). Interessant, avvisning av skin grafts avhenger av nærværet av CD4 + </sup&gt; T-celler (rev. i 12). I denne modellen, kan målrette spesifikke interaksjoner mellom ulike immunceller ved å blokkere kostimulerende molekyler med antistoffer eller undertrykkelse med regulatoriske T-celler indusere toleranse (upubliserte data). Faktisk, blokkerer både CD40 og CD28 førte til langsiktig hud allograft toleranse 13,14. Tail-hudtransplantasjon er lett å utføre og lett å overvåke forhold til transplantering av andre organer. I tillegg, hale-skin transplantasjoner er lett å tilberede og er mindre utsatt for ischemi enn andre hudvevet. I motsetning til injiserte bedøvelsesmidler, bruk av anestesigass (isofluorane) under transplantasjon forkorter både fremgangsmåten og mottaker restitusjonstid. Curling på halen-hud transplantasjon, noe som kan føre til ufullstendig sårheling og pode avvisning, forhindres ved bruk av vev lim. Videre IA bm12 tail-hudtransplantasjon modell aktiverer utelukkende CD4 <sup&gt; + T celler i både immunkompetente og immunsvikt mus (av samme genetiske bakgrunn) å legge til rette tolkningen av resultatene. Denne protokollen beskriver en pålitelig, reproduserbar og enkelt overvåkes mus modell som gjør det mulig for kjemisk og biologisk intervensjon. Modellen er beregnet for å undersøke avvisning og toleranse induksjon av hale hud transplantasjoner.

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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.

I en første tilnærming C57BL / 6 mus ble transplantert med IA bm12 allografts og IA b syngrafts. Etter bandasje fjerning, grafts manifest tegn på sårtilheling uten nedleggelse av kontaktsonen i C57BL / 6 mus (Figur 1a). Etter bandasje fjerning, CD4 + T-celle-mediert inflammasjon førte til utseendet på nekrotiske områder (røde flekker) og avvisning av IA bm12 allografts i C57BL / 6 mus innen 13 dager etter transplantasjon (Figur 1B-C, f...

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.

Transplantasjon av Tail Skin Study Cell Responses Allogen CD4 T i Mus

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

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Isolasjon og Th17 Differensiering av naive CD4 T-lymfocytter

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

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

Induksjon av murine tarmbetennelse etter adoptiv overføring av Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Høy</sup&gt; T-celler i immundefekte mus

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

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

Mus naive CD4<sup&gt; +</sup&gt; T Cell Isolasjon og<em&gt; In vitro</em&gt; Differensiering i 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 ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

Imaging CD4 T Cell Interstitiell Migrasjon i Betente 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 ...

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

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

In Vitro og i Vivo vurdering av T, B og myeloide celler undertrykkende aktivitet og humorale svar fra transplantasjon mottakere

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

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

En suge blemme protokoll å studere Human T-celle tilbakekalling svar i 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

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

Evaluering av Zika Virus-spesifikke T-celle svar i Immunoprivileged organer av infiserte Ifnar1- / - mus

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