Soutenu par des subventions du National Institutes of Health (CA133085 à KL) et l&#39;American Cancer Society (RSG-09-209-01-TBG à KL).

1. Modèle expérimental de la souris Métastases <ol><li> Le jour avant les injections de cellules tumorales, une graine flacon T75 avec un maximum de 1 x 10 7 CMS4-Met cellules dans 10 ml de milieu RPMI contenant 10% de sérum pour obtenir croissance rapide des cellules tumorales. Incuber une nuit à 37 ° C. </li><li> Le jour de l&#39;injection, éliminer le milieu et rincer les cellules une fois avec du PBS, puis la récolte des cellules tumorales avec 0,05% de trypsine-EDTA à 37 ° C pendant 5 minutes. Arrêter la réaction avec 10 ml de milieu RPMI contenant 10% de sérum. Transfert des cellules à un tube conique. </li><li> Isoler les cellules à 1300 rpm dans une centrifugeuse Sorvall Legend RT pendant 3 minutes à température ambiante. Enlever le surnageant. Resuspendre les cellules dans 10 mL frais 1X HBSS à laver, puis à nouveau spin-down et remettre de la même manière. </li><li> Compter les cellules tumorales sur un hématimètre. Diluer les cellules dans HBSS, de sorte que les cellules nécessaires à chaque injection sont remis en suspension dans un volume total de 100 ul. </li><li> Chauffer le 6-8 semaines vieilles souris Balb / c dans un bécher immergé dans l&#39;eau chaude pour dilater la veine de la queue. Placez la souris sur une veine de la queue de contention sur le banc. Utilisez une microseringue à injecter 100 ml de cellules tumorales dans la veine latérale de la queue. Utiliser une technique aseptique pour éviter l&#39;infection. Après l&#39;injection, appliquez une légère pression au site d&#39;injection jusqu&#39;au saignement s&#39;est arrêté. </li><li> Retour de la souris dans la cage, et permettre à la tumeur de croître sur la scène désirée. </li></ol> 2. Lymphocytes T cytotoxiques (CTL) Immunothérapie transfert adoptif <ol><li> Le jour du transfert, une pipette de haut en bas pour resuspendre cytotoxique des lymphocytes T purifiés, ou CTL, préparé comme décrit dans le texte. Transfert de toutes les cellules d&#39;une plaque à un tube de 15 ml coniques, ne laissant pas le volume dépasse de plus de 2 / 3 du tube. </li><li> Insérer une pipette Pasteur stérile dans le tube conique et laïcs milieu de séparation des lymphocytes, ou de LSM, sous les cellules jusqu&#39;à ce que le volume total se rapproche de 14 ml. </li><li> Soyez prudent de ne pas déranger les couches. Centrifuger à 2000 rpm pendant 20 minutes à température ambiante. Ne pas utiliser le frein pour ralentir le rotor après le filage. </li><li> Transfert des CTL à un nouveau 15 ml tube conique. Ajouter HBSS à un volume d&#39;environ 10 ml à laver. </li><li> Compter les cellules. Isoler à 1300 rpm pendant 5 minutes à température ambiante. Remettre en suspension dans HBSS à la densité de cellules nécessaires pour les injections, en gardant le volume total par injection à 100 pi. </li><li> Utilisez la technique d&#39;injection queue mêmes comme vu plus tôt dans cette vidéo pour injecter le CTL en souris porteuses de tumeurs. </li><li> Retour de la souris dans la cage, et permettre au CTL à interagir avec la tumeur. Les souris sont souvent sacrifiés pour l&#39;analyse de 14-21 jours après le traitement de CTL. </li></ol> 3. Visualisation des métastases pulmonaires <ol><li> Pour visualiser les métastases pulmonaires, placer une souris sacrifiées sur son dos sur une planche en mousse de polystyrène. Pin des jambes pour assurer un libre accès à la trachée. Vaporisez la face ventrale avec de l&#39;éthanol à 70%. </li><li> A partir du milieu de l&#39;abdomen, utilisez des ciseaux pour couper le long de la ligne médiane, à travers la cage thoracique, et jusqu&#39;à vers les glandes salivaires. Exposer la trachée. Utilisez une pince pour retirer les tissus entourant la trachée. </li><li> Enfilez une pointe de pipette 200 uL sous la trachée. Tenir la pointe d&#39;une main, soulevez doucement la trachée et loin du corps. </li><li> Tournez la plate-forme de 180 °. Utiliser une seringue de 50 ml à injecter l&#39;encre de Chine dans les poumons par la trachée. Complètement gonfler les poumons avec de l&#39;encre jusqu&#39;à ce que vous sentez une forte résistance. </li><li> Utilisez des ciseaux pour couper la trachée. Faites glisser une paire de pinces dans les poumons et les sortir de la souris. Rincez les poumons brièvement dans un bécher de 1 litre d&#39;eau. </li><li> Dans une hotte, le transfert des poumons à un flacon à scintillation en verre contenant 5 ml de solution de Fekete. Le tissu tumoral va s&#39;imposer comme des nodules blancs sur les poumons noirs après quelques minutes. Les tumeurs peuvent maintenant être comptés et enregistrés dans une solution de Fekete indéfiniment. </li></ol> 4. Métastases Représentant en Poumons visualisé <ol><li> Ici, les poumons des souris qui ont été injectés avec 4T1 carcinome mammaire lignée cellulaire montrent des taches blanches indiquant les tumeurs. Les souris qui ont été injectées avec des cellules 4T1 transfectées avec un IRF8 dominante-négative K79E mutant montrer le potentiel améliorée métastatique des cellules tumorales. </li><li> Ces images montrent une analyse histologique de l&#39;efficacité de l&#39;immunothérapie adoptive de transfert de CTL. Souris porteuses de tumeurs injectées avec une solution saline a montré de multiples tumeurs 6 jours plus tard, indiquées par des flèches jaunes (A). Souris injectées avec des cellules T spécifiques de la tumeur, d&#39;autre part, a montré une réduction de tumeurs (B). </li><li> Dans la même expérience, le traitement encre de Chine donne un moyen plus simple pour mesurer l&#39;efficacité du transfert adoptif de CTL. Ici, les nodules tumoraux blanc sur porteurs de tumeurs des poumons clairement les distinguer des poumons qui ont subi avec succès le transfert adoptif de CTL, et en comptant les nodules permet un degréde quantification n&#39;est pas possible avec l&#39;histologie. </li></ol> 5. Les résultats représentatifs <img src="/files/ftp_upload/2077/2077fig1.jpg" alt="Figure 1" /> Figure 1. Dispositif expérimental pour la métastase des tumeurs expérimentales et de transfert de CTL adaptative immunothérapie modèle de souris. Les points rouges indiquent les cellules tumorales et des points verts indiquent les CTL. <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="Figure 2" /> Figure 2. Perturbation des fonctions améliorées IRF8 le potentiel métastatique des cellules tumorales. Mammaire de souris 4T1 carcinome ligne a été transfectées de façon stable avec le vecteur (4T1.Vector) ou vecteur exprimant une IRF8 mutant dominant négatif K79E (4T1.IRF8K79E) (15, 16). Les cellules tumorales ont été injectées dans les veines de souris iv latérale de la queue. Porteurs de tumeurs des poumons étaient gonflés à l&#39;encre de Chine de visualiser des nodules tumoraux. Nodules tumoraux sont facilement considérées comme des taches blanches sur le fond noir du tissu pulmonaire. <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="Figure 3" /> Figure 3. L&#39;analyse histologique de l&#39;efficacité de l&#39;immunothérapie adoptive de transfert de CTL. Lignée cellulaire de souris sarcome de CMS4-Met a été injecté dans les veines de souris iv latérale de la queue. Trois jours plus tard, une solution saline (A) ou spécifiques d&#39;une tumeur des cellules T (B) ont été injectées dans des souris porteuses de tumeur. Poumons ont analysé six jours après le traitement de CTL par coloration H &amp; E conventionnels histologique. Nodules tumoraux sont indiquées par des flèches. <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="Figure 4" /> Figure 4. Visualisation des nodules tumoraux par l&#39;inflation encre de Chine. Lignée cellulaire de souris sarcome de CMS4-Met a été injecté dans les veines de souris iv latérale de la queue. Trois jours plus tard, une solution saline (contrôle) ou une tumeur des cellules T spécifiques (+ CTL) ont été injectées dans des souris porteuses de tumeur. Poumons ont été analysés quatorze jours après le traitement de CTL. Les taches blanches, qui sont des nodules tumoraux, permettant la quantification aisée de l&#39;efficacité du traitement de CTL.

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	<li>Ryan, M. H., Bristol, J. A., McDuffie, E., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regression+of+extensive+pulmonary+metastases+in+mice+by+adoptive+transfer+of+antigen-specific+CD8(+)+CTL+reactive+against+tumor+cells+expressing+a+naturally+occurring+rejection+epitope.">Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope.</a> J Immunol. 167 (8), 4286-4292 (2001).</li><li>Caldwell, S. A., Ryan, M. H., McDuffie, E., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fas/Fas+ligand+pathway+is+important+for+optimal+tumor+regression+in+a+mouse+model+of+CTL+adoptive+immunotherapy+of+experimental+CMS4+lung+metastases.">The Fas/Fas ligand pathway is important for optimal tumor regression in a mouse model of CTL adoptive immunotherapy of experimental CMS4 lung metastases.</a> J Immunol. 171 (5), 2402-2412 (2003).</li><li>Liu, K., Caldwell, S. A., Greeneltch, K. M., Yang, D., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTL+Adoptive+Immunotherapy+Concurrently+Mediates+Tumor+Regression+and+Tumor+Escape.">CTL Adoptive Immunotherapy Concurrently Mediates Tumor Regression and Tumor Escape.</a> J Immunol. 176 (6), 3374-3382 (2006).</li><li>Yang, D., Stewart, T. J., Smith, K. K., Georgi, D., Abrams, S. I., Liu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+IFN-gammaR+in+association+with+loss+of+Fas+function+is+linked+to+tumor+progression.">Downregulation of IFN-gammaR in association with loss of Fas function is linked to tumor progression.</a> International journal of cancer. 122 (2), 350-362 (2008).</li><li>Pages, F., Berger, A., Camus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+memory+T+cells,+early+metastasis,+and+survival+in+colorectal+cancer.">Effector memory T cells, early metastasis, and survival in colorectal cancer.</a> N Engl J Med. 353 (25), 2654-2666 (2005).</li><li>Galon, J., Costes, A., Sanchez-Cabo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type,+density,+and+location+of+immune+cells+within+human+colorectal+tumors+predict+clinical+outcome.">Type, density, and location of immune cells within human colorectal tumors predict clinical outcome.</a> Science. 313 (5795), 1960-194 (2006).</li><li>Strater, J., Hinz, U., Hasel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+CD95+expression+predisposes+for+recurrence+in+curatively+resected+colon+carcinoma:+clinical+evidence+for+immunoselection+and+CD95L+mediated+control+of+minimal+residual+disease.">Impaired CD95 expression predisposes for recurrence in curatively resected colon carcinoma: clinical evidence for immunoselection and CD95L mediated control of minimal residual disease.</a> Gut. 54 (5), 661-665 (2005).</li><li>Camus, M., Tosolini, M., Mlecnik, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+intratumoral+immune+reaction+and+human+colorectal+cancer+recurrence.">Coordination of intratumoral immune reaction and human colorectal cancer recurrence.</a> Cancer research. 69 (6), 2685-2693 (2009).</li><li>Dudley, M. E., Wunderlich, J. R., Yang, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer+therapy+following+non-myeloablative+but+lymphodepleting+chemotherapy+for+the+treatment+of+patients+with+refractory+metastatic+melanoma.">Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma.</a> J Clin Oncol. 23 (10), 2346-2357 (2005).</li><li>Srivastava, M. K., Sinha, P., Clements, V. K., Rodriguez, P., Ostrand-Rosenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid-derived+suppressor+cells+inhibit+T-cell+activation+by+depleting+cystine+and+cysteine.+Cancer+research.">Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine. 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W., Kranenburg, O., Rinkes, B. o. r. e. l., H, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+colorectal+cancer+and+liver+metastases.">Mouse models of colorectal cancer and liver metastases.</a> Digestive surgery. 22 (1-2), 1-2 (2005).</li><li>Yang, D., Ud Din, N., Browning, D. D., Abrams, S. I., Liu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+lymphotoxin+beta+receptor+with+tumor-specific+T+lymphocytes+for+tumor+regression.">Targeting lymphotoxin beta receptor with tumor-specific T lymphocytes for tumor regression.</a> Clin Cancer Res. 13 (17), 5202-5210 (2007).</li><li>Yang, D., Thangaraju, M., Browning, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN+Regulatory+Factor+8+Mediates+Apoptosis+in+Nonhemopoietic+Tumor+Cells+via+Regulation+of+Fas+Expression.">IFN Regulatory Factor 8 Mediates Apoptosis in Nonhemopoietic Tumor Cells via Regulation of Fas Expression.</a> J Immunol. 179 (7), 4775-4782 (2007).</li><li>Yang, D., Thangaraju, M., Greeneltch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+IFN+regulatory+factor+8+by+DNA+methylation+is+a+molecular+determinant+of+apoptotic+resistance+and+metastatic+phenotype+in+metastatic+tumor+cells.">Repression of IFN regulatory factor 8 by DNA methylation is a molecular determinant of apoptotic resistance and metastatic phenotype in metastatic tumor cells.</a> Cancer research. 67 (7), 3301-3309 (2007).</li><li>Wexler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+identification+of+experimental+pulmonary+metastases.">Accurate identification of experimental pulmonary metastases.</a> Journal of the National Cancer Institute. 36 (4), 641-645 (1966).</li></ol>

Les souris ont été achetés auprès de l&#39;Institut national du cancer (Friderick, MD) et logé dans le Medical College of Georgia animalerie. Les expériences et les soins / bien-être sont en accord avec les règlements fédéraux et d&#39;un protocole approuvé par le Medical College of Georgia IACUC comité.

Solutions: Solution d&#39;encre en Inde (17): <ol><li> Verser 150 ml d&#39;eau distillée dans un flacon de 250 ml. </li><li> Ajouter 4 gouttes d&#39;hydroxyde d&#39;ammonium à l&#39;eau distillée. </li><li> Ajouter 30 ml d&#39;encre en Inde stocks (Sanford Black Magic dessin à l&#39;encre imperméable 4465 Item 44011) au mélange d&#39;ammoniaque et d&#39;eau. </li><li> Terminez avec de l&#39;eau distillée pour un volume de 200 ml. La solution est prête pour l&#39;injection. </li></ol> La solution Fekete (17): Solution de Fekete est utilisé pour blanchir l&#39;encre de Chine-gonflés porteurs de tumeurs des poumons pour distinguer les nodules tumoraux blanc du fond noir des tissus normaux. <ol><li> Ajouter 350ml EtOH 95% à bouteille en verre 1L. </li><li> Ajouter 150ml d&#39;eau distillée </li><li> Ajouter 50 ml de formaldéhyde </li><li> Ajouter 25 ml d&#39;acide glaciaire acides </li></ol>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

Experimental modèle de souris est un modèle de métastases métastases simple et pourtant physiologiquement pertinents. Les cellules tumorales sont injectées par voie intraveineuse (iv) dans les veines queue de souris et de coloniser dans les poumons, ce qui, ressemblant à la dernière étape de la métastase des cellules tumorales spontanées: la survie dans la circulation, l&#39;extravasation et la colonisation dans les organes distaux. D&#39;un point de vue thérapeutique, le modèle expérimental de métastases est le modèle le plus simple et idéale puisque la cible de thérapies est souvent le point final de la métastase: tumeur métastatique établie dans l&#39;organe distal. Dans ce modèle, les cellules tumorales sont injectées dans les veines iv queue de souris et a permis de coloniser et à croître dans les poumons. Spécifiques de la tumeur CTL sont ensuite injectés iv dans la souris porteuses de métastases. Le nombre et la taille des métastases pulmonaires peuvent être contrôlés par le nombre de cellules tumorales doit être injecté et le temps de la croissance tumorale. Par conséquent, les différentes étapes de la métastase, de métastases minimale de métastases étendues, peuvent être modélisés. Métastases pulmonaires sont analysés par l&#39;inflation avec de l&#39;encre, permettant ainsi plus facile l&#39;observation visuelle et de quantification.

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Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Une métastase pulmonaire expérimentale et CTL modèle de souris d&#39;immunothérapie pour l&#39;analyse de l&#39;interaction des cellules tumorales des cellules-T<em&gt; In vivo</em&gt;.

Métastases expérimentales et CTL Modèle adoptifs souris Transfert immunothérapie

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

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Immunology and Infection

19.3K vues.  Medical College of Georgia. Une métastase pulmonaire expérimentale et CTL modèle de souris d'immunothérapie pour l'analyse de l'interaction des cellules tumorales des cellules-T In vivo.

Vidéo: Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

Protocoles pour l&#39;inoculation vaginale et prélèvement d&#39;échantillons dans le modèle expérimental chez la souris des Candida Vaginite

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

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Immunologie et infection

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

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

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

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Surmonter Absence de réaction dans encéphalomyélite allergique expérimentale (EAE) souches de souris résistantes par transfert adoptif et une stimulation antigénique

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

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

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

Accélération de diabète de type 1 chez la souris par induction transfert adoptif de lymphocytes CD4 + T Cells diabétogéniques

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

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Isolement, la purification et l&#39;étiquetage des neutrophiles osseuse de souris de moelle pour les études fonctionnelles et expériences de transfert adoptif

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

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

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Évaluer le développement de cellules murines dendritiques plasmacytoïdes dans les plaques de Peyer Utiliser le transfert adoptif de progéniteurs hématopoïétiques

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

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Génération de la RCA cellules T pour la thérapie adoptive dans le contexte de glioblastome norme de diligence

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

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

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

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

Induction de murin inflammation intestinale par transfert adoptif de effecteur CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Haute</sup&gt; T cellules dans des souris immunodéficientes

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

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

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Stéréotaxique transfert adoptif de cellules immunitaires cytotoxiques dans des modèles murins de xénogreffes de glioblastome Multiforme Orthotopic homme

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

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Un modèle de souris contrôlée pour infection polymicrobienne néonatale

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Création du modèle de souris TK-NOG humanisé double pour la pathogénie du foie associée au VIH

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...