Gli autori desiderano ringraziare il Dr. Boris Reizis del Dipartimento di Microbiologia e Immunologia presso la Columbia University per le discussioni utili. Desideriamo inoltre ringraziare il Dr. Margaret Bynoe, il Dr. Rodman Getchell e Deequa Mahamed del Dipartimento di Microbiologia e Immunologia presso il College di Medicina Veterinaria, Cornell University di assistenza tecnica con la citometria a flusso e Huang Lu per l&#39;assistenza con l&#39;editing video. Gli autori desiderano ringraziare i membri del Duhamel e Weiss laboratori per la loro revisione critica del manoscritto e la produzione video, e Stephanie Yazinski dal laboratorio Weiss per l&#39;allevamento e la genotipizzazione p53 - / - topi. Gli esperimenti sugli animali sono stati eseguiti secondo le linee guida e regolamenti stabiliti dalla cura degli animali Cornell Università Istituzionale e del Comitato Usa. Questa ricerca è stata sostenuta in parte dai fondi messi a disposizione dal Dipartimento dell&#39;Agricoltura degli Stati Uniti, la ricerca sullo stato Cooperativa, Educazione e Extension Service, la salute degli animali e della Ricerca Programma di Disease (a GED e RSW) e le sovvenzioni NIH R03 HD058220 e R01CA108773 (a RSW). Il video è stato prodotto utilizzando strutture in-house dal personale dei laboratori Duhamel &amp; Weiss.

1. Dissezione di tumore <ol><li> Per la dissezione del tumore, un tovagliolo di carta pulito o teli chirurgici monouso viene posto sulla rampa di dissezione e spruzzato con il 70% di etanolo. </li><li> I topi di questo protocollo sono regolarmente eutanasia con CO 2. Eutanasia Posizionare il mouse sul tappetino dissezione e spruzzare su entrambi i lati dell&#39;animale con etanolo. Il mouse è tenuto in posizione sul pad dissezione utilizzando quattro o più push-pin inseriti attraverso le gambe e spruzzato abbondantemente con etanolo al 70%. </li><li> La pelle sopra la linea mediana è tagliato dal pube fino alla base della testa usando forbici sterili, evitando i muscoli sottostanti e penetrando nelle cavità del corpo. </li><li> Aprire la cavità addominale con un nuovo paio di forbici sterili e pinze facendo attenzione a non danneggiare gli organi interni. </li><li> Aprire la cavità toracica da sezionare e deviando la gabbia toracica per rivelare il tumore, senza danneggiare i vasi sanguigni. </li><li> Separare il tumore da altri organi, e trasferire un pezzo di circa il 40-50% del tumore totale (circa fino a 1,5 X 0,8 X 0,8 cm) in PBS freddo tenuta in ghiaccio. </li><li> La parte restante del tumore possono essere salvate per altri test e una necroscopia completa può essere eseguita in questo momento. </li></ol> 2. Coltura Cellule Linfoma <ol><li> Spruzzare il tubo contenente il tumore sezionato con il 70% di etanolo e trasferirli in un armadio biosicurezza. </li><li> Aggiungere 10 ml di terreno di coltura ad una sterile 150 mm 2 piastra di Petri. </li><li> Trasferire il pezzo di tumore nella capsula di Petri di aspirazione delicata con una pipetta. </li><li> Piegare due sterili 18 gauge rivolto verso il lato smussato fuori. </li><li> Dissociare il tumore con gli aghi piegati. </li><li> Agitare il piatto, toccare leggermente e porre in un angolo per raccogliere la sospensione cellulare ad un lato, evitando grossi pezzi di tumore. </li><li> Aspirare la sospensione cellulare e trasferire in una provetta da 50 ml conica, evitando grossi pezzi di tumore. </li><li> Lavare la piastra con 10 ml di medio e di trasferimento all&#39;interno del tubo stesso, evitando grossi pezzi di tumore. </li><li> Mescolare la sospensione cellulare composto da linfociti e cellule stromali delicatamente e trasferire 5 ml in un T-25 fiasco di coltura tissutale e 10 ml in un T-75 fiasco di coltura tissutale. Mescolare delicatamente i fiaschi. </li><li> Utilizzare la sospensione di cellule rimanenti per fare diluizioni 2, 4, 10100 e 1000 volte in 2,5 ml di volume finale (la coltura delle cellule in diverse densità utilizzando diversi tipi di recipienti di coltura massimizza la probabilità di stabilire una linea di cellule T). </li><li> Piastra di 2,0 ml di ciascuna diluizione in singoli pozzetti di un 6-pozzetti. Agitare delicatamente la piastra. </li><li> Incubare le cellule per 48 ore in atmosfera umidificata CO 2 in aria. </li></ol> 3. Alimentazione cellule del linfoma <ol><li> Dopo 48 ore di incubazione, aggiungere 5 ml di terreno al T-25 pallone e 10 ml di medio-T-75 fiasco lungo la parete con il minimo disturbo delle cellule. </li><li> Aggiungere 2 ml di medium in ciascun pozzetto della piastra ben 6 lungo la parete con il minimo disturbo delle cellule. </li><li> Incubare le cellule per ulteriori 72 ore </li></ol> 4. Passaging cellule del linfoma <ol><li> Passaggio iniziale <ol><li> Alla fine del quinto giorno di incubazione, le cellule sono diversi passaggi con un rapporto 1:1, pur mantenendo i palloni originali e piatti (mantenendo la densità delle cellule ad una concentrazione di circa 3x106 / ml durante i passaggi prima istituzione promuove il successo di un linea cellulare continua). <ol class="lalpha"><li> Per il T-25 borraccia, trasferire da 5 ml di sospensione cellulare in un pallone nuovo contenente 5 ml di terreno. Aggiungere 5 ml di terreno nel pallone originale. </li><li> Per il T-75 fiasco, il trasferimento di 10 ml di sospensione cellulare in un pallone nuovo che contiene 10 ml di mezzo. Aggiungere 10 ml di terreno nel pallone originale. </li><li> Per il 6-pozzetti, trasferimento 2 ml di sospensione cellulare da ogni pozzetto nei pozzetti corrispondenti di una nuova piastra contenente 2 ml di mezzo in ogni bene. Aggiungere 2 ml di mezzo in ciascun pozzetto della pozzi originale. </li></ol></li><li> Incubare tutti i palloni e le piastre per altre 72 ore Nella nostra esperienza più linee cellulari sono stabiliti da questi passaggio uno culture (la presenza di aggregati sospensione liberamente aderente cellule nel corso del cellule stromali allegato è una prima indicazione di una linea di cellule prestabilite). </li><li> Per le culture con meno o nessun vagamente aderente aggregati di cellule sospensione, nutrire i palloni o piastre con attenzione la sostituzione di circa il 50% della media ogni 72 h. Per evitare di rimuovere le cellule dalla cultura d&#39;origine, recipienti di coltura sono rimasto in piedi e indisturbati per almeno 5 minuti per consentire il sedimento cellule in basso dalla forza di gravità prima di aspirazione dolce del mezzo sopranatante. </li></ol></li><li> Il mantenimento di linee cellulari stabilizzate <ol><li> Linee cellulari stabilizzate sono diversi passaggi in fiaschi e nuovi piatti. Per i T-25 e T-75 boccette, aggiungere 5 ml dell&#39;originale opassaggio una sospensione in 5 ml di terreno. Per 6-pozzetti, aggiungere 3 ml di originale o di passaggio una sospensione di cellule in 7 ml di mezzo in un T-25 pallone. Mangimi e conservare l&#39;originale o passaggio una cultura sostituendo il volume rimosso con terreno di coltura fresco. </li><li> Passaggio stabilito linee cellulari in nuovi T-25 fiaschi. Le linee di cellule hanno un tempo di raddoppio di 18-24 h. Le culture sono regolarmente mantenuti ad una densità di 1-3x106 / ml e diversi passaggi ogni 3 giorni. Aggiungere il volume richiesto di cultura passaggio precedente al nuovo mezzo per rendere fino a 10 ml. Mangimi e mantenere la cultura d&#39;origine. </li><li> Dopo alcuni passaggi, il non-aderenti cellule si auto senza sostenere le cellule stromali aderenti e un minor numero di cellule aderenti sono riportati ad ogni passaggio. Al di là di questo punto, le culture possono essere mantenuti in non tessuto coltura trattata T-25 fiaschi. </li></ol></li></ol> 5. Congelamento e Recupero linee cellulari linfoma <ol><li> Le linee cellulari sono preparati al momento congelati in RPMI 1640 medium contenente 20% FBS e il 10% di DMSO. </li><li> Cellule di una coltura di 48 h vecchio T-25 pallone (circa 1.5x10 6 cellule / ml) sono centrifugate a 1500 rpm per 5 minuti a temperatura ambiente. Dopo aver scartato il surnatante, le cellule sono risospese in 2 ml di medium freddo gelo. Due volumi di 1,0 ml di sospensione cellulare da ciascuno dei palloni sono collocati in fiale congelamento, immediatamente congelati a -80 ° C, e trasferito ad azoto liquido il giorno successivo. </li><li> Le cellule sono recuperate dallo stoccaggio di azoto liquido da scongelamento rapido, e risospendere il contenuto in 10 ml di terreno di coltura fresco. In alternativa, le cellule vengono lavate appena scongelato una volta terreno di coltura caldo per rimuovere il DMSO prima di risospendere in 10 ml di terreno di coltura fresco. Le cellule sono coltivate per almeno 72 ore prima del passaggio successivo. </li></ol> 6. Rappresentante Risultati Un Atm - / - T-linea cellulare sviluppato usando questo protocollo e designato dwj-Atm-1 è stato caratterizzato da colorazione con anti-CD3-FITC, anti-CD4-APC e anti-CD8-PE e analisi di citometria a flusso (Figura 1). Più del 90% delle cellule T sono state CD3, CD4 e CD8 triple-positivi. <img src="/files/ftp_upload/2598/2598fig1.jpg" alt="Figura 1" /> Figura 1. Caratterizzazione di una linea di cellule T sviluppato usando questo protocollo. I marcatori di superficie cellulare di un Atm stabilito - / - linea cellulare designato dwj-Atm-1 sono stati caratterizzati da colorazione con anti-CD3-FITC, anti-CD4-APC, anti-CD8-PE e analisi di citometria a flusso. In breve, 1x10 6 cellule fresche sono stati lavati una volta in PBS freddo (1500 giri / 5 min), e incubate a 4 ° C per 10 minuti con l di cocktail di anticorpi (ogni anticorpo a diluizione 1:200 in PBS con 1% BSA 200. Le cellule possono essere esaminate immediatamente mentre le cellule fisso freschi o fisso per la successiva analisi. vengono preparati con l&#39;aggiunta di formalina tamponata neutra ad una concentrazione del 4% (vol / vol) finale e incubate a 4 ° C per 30 minuti, raccolte mediante centrifugazione (1500 rpm / 5 min) e risospeso in 200 ml di PBS con BSA 1%. Un totale di 1 x 10 4 cellule sono state analizzate utilizzando un BD FACS Calibur analizzatore e CellQuest software (BD Biosciences, San Jose, CA).

<ol>
	<li>Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm-deficient+mice:+a+paradigm+of+ataxia+telangiectasia.">Atm-deficient mice: a paradigm of ataxia telangiectasia.</a> Cell. 86, 159-171 (1996).</li><li>Elson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+defects+in+ataxia-telangiectasia+protein-deficient+mice.">Pleiotropic defects in ataxia-telangiectasia protein-deficient mice.</a> Proc Natl Acad Sci U S A. 93, 13084-13089 (1996).</li><li>Kuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Atm-/-+thymic+lymphoma+cell+proliferation+in+vitro+and+in+vivo+by+dexamethasone.">Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.</a> Cancer Chemother Pharmacol. 55, 203-212 (2005).</li><li>Browne, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+a+catalytic+antioxidant+corrects+the+neurobehavioral+defect+in+ataxia-telangiectasia+mice.">Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.</a> Free Radic Biol Med. 36, 938-942 (2004).</li><li>Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+spectrum+analysis+in+p53-mutant+mice.">Tumor spectrum analysis in p53-mutant mice.</a> Curr Biol. 4, 1-7 (1994).</li><li>Chervinsky, D. S., Lam, D. H., Zhao, X. F., Melman, M. P., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+T+cell+leukemia+cell+lines+established+from+SCL/LMO1+double+transgenic+mice.">Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.</a> Leukemia. 15, 141-147 (2001).</li><li>Sharma, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+contributes+to+mouse+T-cell+leukemia+by+directly+inducing+the+expression+of+c-myc.">Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.</a> Mol Cell Biol. 26, 8022-8031 (2006).</li><li>Maser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers.">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447, 966-971 (2007).</li></ol>

Nessun conflitto di interessi dichiarati.

In questo protocollo, forniamo le procedure particolareggiate per stabilire murino linee di cellule T da due genotipi differenti del mouse; Atm - / - [FVB/N- Atm tm1Led / J] e p53 - / - [129/S6- Trp53 tm1Tyj / J] . Usando questo protocollo, per un totale di 6 (su 7 tentativi) Atm - / - e tre (su 5 tentativi) p53 - / - murini linee di cellule T sono state istituite nel nostro laboratorio. Dati rappresen...

<ul><li> Secchiello </li><li> Etanolo al 70% </li><li> Dissezione pad composto da blocco di schiuma ricoperto di un foglio di alluminio e perni </li><li> Due serie di piccole forbici e pinze sterili </li><li> Sterile 150 mm 2 piastra di Petri (Thermo Fisher Scientific, Waltham, MA;. Cat # 08-757-13) </li><li> Calibro 18 aghi sterili </li><li> 50 ml provette coniche (Corning Inc., Corning NY; cat # 430829) </li><li> Tamponata al 10% formalina </li><li> Tessuto cultura fiaschi <ul><li> T-25 (TPP, Trasadingen, Svizzera; cat # 90025) </li><li> T-75 (TPP, Trasadingen, Svizzera; cat # 90075) </li></ul></li><li> 6 pozzetti (Corning Inc., Corning NY; cat # 3506) </li><li> Fosfato soluzione salina sterile tamponata pH 7,2 (PBS) </li><li> Terreno di coltura <ul><li> RPMI 1640 con 25 HEPES mm, 200 mm L-glutamina (Lonza, Walkersville, MD; gatto # 12-115F), integrato con il seguente: <ul><li> 10% inattivato con il calore (56 ° C, 30 min) di siero fetale bovino (FBS; Hyclone, Logan, UT; cat # SH30088.03) </li><li> 1% di penicillina e streptomicina (Mediatech, Manassas, VA; gatto # 30-002-CI) </li><li> 1% amino acidi essenziali (Mediatech, cat # 25-025-CI) </li><li> 55 mm 2β-mercaptoetanolo (Sigma, St Louis, MO; cat # M752) </li></ul></li></ul></li><li> Dimetil solfossido (DMSO; Calbiochem, La Jolla, CA; cat # 317275) </li><li> Anticorpi (eBioscience, San Diego, CA) <ul><li> anti-CD3-FITC (cat. # 11-0031-85) </li><li> anti-CD4-APC (cat. # 17-0041-83) </li><li> anti-CD8-PE (cat. # 12-081-85) </li></ul></li><li> Albumina di siero bovino (BSA; Life Technologies, Grand Island, NY;. Cat # 11018-017) </li><li> Concanavalina A (gatto # Sigma C5275) </li><li> 1,8 ml fiale congelamento (Nunc, Roskilde, Danimarca; cat # 368632) </li></ul>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

Linee cellulari stabilizzate sono uno strumento di ricerca critica che può ridurre l&#39;uso di animali da laboratorio nella ricerca. Alcuni ceppi di topi geneticamente modificati, come Atm - / - e p53 - / - costantemente sviluppare linfoma timico primi anni di vita 1,2, e quindi, può servire come una fonte affidabile per la derivazione di cellule T murino linee. Qui vi presentiamo un protocollo dettagliato per lo sviluppo di linfoma timico stabilito murino linee di cellule T, senza la necessità di aggiungere interleuchine, come descritto in precedenti protocolli 1,3. I tumori sono stati raccolti dai topi di età compresa tra tre a sei mesi, alla prima indicazione di tumori visibili basa sull&#39;osservazione della postura ingobbita, respiro affannoso, governare poveri e sprecare in un 1,4 ceppo sensibile. Abbiamo stabilito con successo diverse linee di cellule T usando questo protocollo e ceppi inbred di Atm - / - [FVB/N- Atm tm1Led / J] 2 e p53 - / - [129/S6- Trp53 tm1Tyj / J] 5 topi. Abbiamo inoltre dimostrato che oltre il 90% della stabilito cellule T popolazione esprime CD3, CD4 e CD8. Coerentemente con le linee cellulari in modo stabile, le T-cellule generate utilizzando il protocollo attuale sono stati diversi passaggi per oltre un anno.

Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In questo video si dimostra un protocollo per stabilire le linee del mouse linfoma delle cellule del timo. Seguendo questo protocollo, abbiamo stabilito con successo diverse linee di cellule T da Atm-/ - e p53-/ - i topi con linfoma del timo.

Derivazione di timica linfoma a cellule T Linee da Atm - / -</sup E P53 - / -</sup Mouse

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

derivation-of-thymic-lymphoma-t-cell-lines-from-atm-and-p53-mice

Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

12.1K Views.  Cornell University. In questo video si dimostra un protocollo per stabilire le linee del mouse linfoma delle cellule del timo. Seguendo questo protocollo, abbiamo stabilito con successo diverse linee di cellule T da Atm-/ - e p53-/ - i topi con linfoma del timo.

Video: Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

I topi con Transnuclear predefiniti Specificità cellule T contro il recettore Toxoplasma gondii Ottenuto Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Ricerca

Immunologia e infezioni

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Esame di Selezione timica positivi e negativi per Citometria a Flusso

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivazione di cellule T<em&gt; In Vitro</em&gt; Da cellule staminali embrionali di topo

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

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 ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Trasduzione retrovirale di cellule progenitrici del midollo osseo per generare cellule T del recettore Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Un metodo semplice ed efficace per stabilire NK e linee a cellula T da pazienti con infezione attiva cronica del Virus di Epstein - Barr

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

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 ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Gene Knock-in di CRISPR/Cas9 e smistamento cellulare in macrofagi e linee cellulari T

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

Derivazione di timica linfoma a cellule T Linee da Atm - / - E P53 - / - Mouse

Derivazione di timica linfoma a cellule T Linee da Atm^{- / - E P53^{- / - Mouse}}