Name: stereotactic adoptive transfer of cytotoxic immune cells in murine models of orthotopic human glioblastoma multiforme xenografts
Uploaded: 2018-09-01T13:00:00
Description: Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

Forfatterne takke personalet i Universitetshospital animalske faciliteten (UTE) af Nantes for dyrehold og pleje, cellulære og tissular imaging core facilitet af Nantes Universitet (MicroPICell) til billedbehandling, og funktionen flowcytometri (Cytocell) fra Nantes for deres ekspert teknisk bistand. Dette arbejde blev finansieret af INSERM, CNRS, Université de Nantes, Institut National du kræft (Inka #PLBio2014-155), Ligue Nationale contre le Cancer (AO interregionale 2017) og det europæiske konsortium ERA-Net Transcan2 (Immunoglio). Holdet er finansieret af Fondation pour la Recherche Médicale (DEQ20170839118). Dette arbejde blev realiseret i forbindelse med LabEX IGO og IHU-Cesti programmer, støttet af National forskning agenturet Investissements d'Avenir via programmer ANR-11-LABX-0016-01 og ANR-10-IBHU-005, henholdsvis. Projektets IHU-Cesti understøttes også af Nantes Metropole og Pays de la Loire-regionen. Forfatterne takke Chirine Rafia for at yde hjælp i rette håndskriftet.

De følgende procedure der involverer dyrs emner blev udført efter institutionelle retningslinjer (aftale #00186.02, regionale etiske komité i Pays de la Loire [Frankrig]). Menneskelige PBMC blev isoleret fra blod indsamlet fra informeret raske donorer (Etablissement Français du Sang Nantes, Frankrig). Alle trin er udført under sterile forhold.
1. ikke-specifikke udvidelse af cytotoksiske effektor T lymfocytter
<ol><li>Forberede og bestråle feeder celler på 35 Gy. Stimulering af 2 x 10...

<ol>
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En adoptiv overførsel af valgt naturlig eller manipuleret immun effektor celler udgør en lovende tilgang til effektiv behandling af tumorer, såsom infiltrativ hjernen kræft, pasning af begrænse reactivities mod ikke-transformeret celler15, 16,17,18. Det centrale nervesystem, som omfatter hjernen, har imidlertid en særlig immunstatus, navnlig på grund af eksistensen af blod - hjerne barr...

GBM (der klasse IV astrocytoma), er den mest hyppige og aggressive primære hjerne kræft hos voksne. På trods af aggressive behandlinger, der kombinerer kirurgi og radio-kemoterapi, forbliver GBM forbundet med en meget dårlig prognose (median overlevelse på 14,6 måneder og en 2-årsmortalitet > 73%)1. Dette er beviset interstatslig handelspraksis at få effektive terapeutiske fremskridt er blevet valideret over de sidste ti år2. Blandt kandidaterne til design af mere effektive terapeutiske strategier3,4,5udforskes immunoterapi6 i øjeblikket for at spore og fjerne meget invasiv og radio/kemo-resistente tumor celler, mistænkt for deres vigtige bidrag til hurtig og fatal tumor tilbagefald7. Forskellige potentielle immunologiske mål blev identificeret og foreslåede for immunoterapi, der involverer fysiske eller modificerede αβ eller ϒδ T lymfocytter GBM-specifikke tumor antigener eller stress-induceret molekyler8,9, 10. Mulighed for at administrere valgte GBM-reaktiv immun celle effektorer repræsenterer en enestående mulighed for at levere forhøjede mængder af effektor lymfocytter direkte i stedet for resterende malignitet. For at støtte denne strategi, har vi for nylig vist, at modeller baseret på immundefekte mus transporterer orthotopic primære menneskelige GBM xenografts trofast sammenfatte udviklingen af hjernetumorer i GBM patienter9,11. Desuden, disse modeller blev brugt til at vise den stærke antitumor effektiviteten af adoptively overførte allogene menneskelige Vϒ9Vδ2T lymfocytter.
Denne protokol beskriver de kritisk eksperimenterende trin for at opnå stereotactic immunoterapi af hjernetumorer, såsom GBM, baseret på adoptiv overførsel af allogene T-lymfocytter. Artikel viser: (i) forstærkning af terapeutiske allogene immun effektor T-lymfocytter, såsom menneskelige Vϒ9Vδ2T lymfocytter; (ii) udarbejdelse af disse effektor T lymfocytter til injektion; (iii) proceduren for stereotactic administration i hjernen, i nærheden af svulsten. Denne artikel giver også indsigt i funktionen af disse cellulære effektorer efter stereotactic injektion.
Den terapeutiske tilgang præsenteres her er baseret på indsprøjtning af 20 x 106 effektor-celler pr. dosis for hver hjerne tumor-bærende immundefekte mus. En første i vitro ekspansion skridt er forpligtet til at producere store mængder af immunceller. Derfor, ikke-specifik celle udvidelser udføres ved hjælp af phytohemagglutinin (PHA-L) og bestrålet allogene feeder celler: perifert blod mononukleære celler (PBMC) fra raske donorer og Epstein Barr Virus EBV-transformeret B-lymphoblastoid celle linjer (BLCLs), stammer fra PBMC ved in vitro- infektion med EBV-holdige kultur supernatanten fra linjen silkeabe B95-8 celle, i nærværelse af 1 µg/mL ciclosporin-A.
GBM-reaktiv effektor immunceller sammenholdes og valgt fra in vitro- assays9. Disse effektor celler er aktiveret og forstærkes ved hjælp af standardprotokoller, ifølge deres natur (f.eks., human Vγ9Vδ2 T lymfocytter9 eller menneskelige anti-herpes virus αβ T lymfocytter12) med en Minimumsrenhed på > 80%, som rutinemæssigt kontrolleret af cytometric analyse. Celle ekspansion proceduren beskrevet nedenfor gælder for forskellige menneskelige lymfocyt delmængder.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PBMCs</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:232px;">from 3 different healthy donors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">BLCLs</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td>from 3 different donors</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">Roswell Park Memorial Institute medium (RPMI)</td>
			<td style="width:136px;">Gibco</td>
			<td>31870-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">FCS</td>
			<td style="width:136px;">Dutscher</td>
			<td style="width:119px;">S1810-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">L-glutamine</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:119px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">penicillin/streptomycin</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">IL-2</td>
			<td style="width:136px;">Novartis</td>
			<td style="width:119px;">proleukin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PHA-L</td>
			<td style="width:136px;">Sigma</td>
			<td>L4144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Stereotaxic frame</td>
			<td>Stoelting Co.</td>
			<td align="right">51600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Mouse adaptator for stereotaxic frame&#160;&#160;</td>
			<td>Stoelting Co.</td>
			<td align="right">51624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microsyringe pump injector&#160;</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">UMP3-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoFil Syringe</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">NF34BV-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">NSG mice</td>
			<td style="width:136px;">Charles River</td>
			<td style="width:119px;">NSGSSFE07S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Ketamine</td>
			<td style="width:136px;">Merial</td>
			<td style="width:119px;">Imalg&#232;ne 1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Xylazine</td>
			<td style="width:136px;">Bayer</td>
			<td style="width:119px;">Rompur 2%</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Scissors</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">201758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Forceps</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">501215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">OmniDrill 115/230V</td>
			<td style="width:136px;">WPI</td>
			<td align="right">503598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Vicryl 4-0</td>
			<td style="width:136px;">Ethicon</td>
			<td>VCP397H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Xylocaine</td>
			<td style="width:136px;">Astrazeneca</td>
			<td>3634461</td>
			<td></td>
		</tr>
	</tbody>
</table>

stereotactic adoptive transfer of cytotoxic immune cells in murine models of orthotopic human glioblastoma multiforme xenografts

Glioblastom multiforme (GBM), den mest hyppige og aggressive primære hjerne kræft hos voksne, er generelt forbundet med en dårlig prognose, og knappe effektive behandlingsformer har været foreslået i det sidste årti. Blandt de lovende kandidater til at designe nye terapeutiske strategier, cellulære immunoterapi har været målrettet for at fjerne meget invasiv og kemo-radioresistant tumorceller, sandsynligvis involveret i en hurtig og fatal tilbagefald af denne kræft. Således administrationer af allogene GBM-reaktiv immun celle effektorer, såsom menneskelige Vϒ9Vδ2 T-lymfocytter, i nærheden af tumor vil repræsenterer en enestående mulighed for at levere effektiv og koncentreret terapeutiske agenter direkte ind i den site af hjernen maligniteter. Vi præsenterer her, en protokol for forberedelse og stereotaxisk administration af allogene humane lymfocytter i immundefekte mus transporterer orthotopic menneskelige primær hjernetumorer. Denne undersøgelse giver en prækliniske proof-of-concept for både gennemførligheden og den antitumor effekt af disse cellulære immunoterapi, der er afhængige af stereotactic injektioner af allogene humane lymfocytter i intrabrain tumor senge.

Denne undersøgelse beskriver strategi med adoptiv overførsel af cellulære immun effektor celler i hjernen tumor-bærende mus, baseret på stereotactic injektioner udføres direkte i tumor seng.
For at minimere enhver risiko for hjerneskade er forbundet med en stor Injektionsvolumen, skal effektor cellesuspension koncentreres (20 x 106 celler i 15-20 µL af PBS). For at kontrollere, om denne celle koncentration trin...

Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Stereotactic adoptiv overførsel af cytotoksiske immunceller i Murine modeller af Orthotopic menneskelige glioblastom Multiforme Xenografts

Vi præsenterer her, en protokol for forberedelse og stereotaxisk administration af allogene humane lymfocytter i immundefekte mus transporterer orthotopic menneskelige primær hjernetumorer. Denne undersøgelse giver et proof-of-concept for både gennemførligheden og den antitumor effekt af intrabrain-leveret cellulære immunoterapi.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Research

JoVE Journal

Immunology and Infection

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Overvinde unresponsiveness i eksperimentel autoimmun encephalomyelitis (EAE) Resistente musestammer ved adoptiv overførsel og antigenudfordring

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

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

Accelerated Type 1 Diabetes Induktion i mus ved adoptiv overførsel af diabetogene CD4 + T-celler

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

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

Vurdere udviklingen i murine Plasmacytoid dendritiske celler i Peyer&#39;ske Patches Brug adoptiv overførsel af Hematopoietic Progenitorer

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Generation af CAR T-celler til adoptiv terapi i forbindelse med glioblastom standard for pleje

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

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

Induktion af murine tarmbetændelse ved adoptiv overførsel af effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Høj</sup&gt; T celler i immunsvækkede mus

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