Nous remercions les Drs. Robert Bonneau et Neil Christensen pour leurs commentaires utiles. Ce travail a été soutenu par la Pennsylvania State University College de fonds de la médecine.

1. Isolement des cellules T de la rate et des ganglions lymphatiques de souris BDC2.5 <ol><li> Utilisez 6 semaines d&#39;âge pré-diabétiques féminins BDC2.5 souris en tant que donneurs de cellules CD4 + lymphocytes T diabétogéniques. Souris doivent être sans diabète telle que déterminée par la mesure de la glycosurie (voir ci-dessous). </li><li> Euthanasier chaque souris en utilisant le CO 2 asphyxie et enlever la rate, axillaires et brachial ganglions lymphatiques dans des conditions stériles. Pour enlever la rate, faire tremper la fourrure à l&#39;éthanol 70%, puis les couper et retirer la peau. La rate est visible en tant qu&#39;organe rouge foncé sur le côté gauche de la souris. Faire une incision de 1 pouce dans le péritoine à l&#39;aide des petits ciseaux et de saisir la rate doucement dans le centre d&#39;une paire de petites pinces. Couper délicatement le tissu conjonctif et la graisse attachée autant que possible et enlever la rate. </li><li> Recueillir les rates et les ganglions lymphatiques dans 10 ml moyen de Dulbecco&#39;s-Modified Eagle (DMEM) dans un tube conique de 15 ml sur la glace. </li><li> Préparer une suspension cellulaire unique en utilisant èmee fin d&#39;une seringue de 10 ml piston stérile pour appuyer doucement sur les organes lymphoïdes à travers 70 filtres cellulaires um (une rate par crépine et 4-6 ganglions lymphatiques par filtre) dans le même tube conique de 50 ml. </li></ol> Pendant le processus, rincer chaque crépine avec 1 ml de DMEM plusieurs fois afin de maximiser la récupération des cellules de la crépine. <ol start="5"><li> Transférer les cellules sont prélevées du tube conique de 50 ml dans un tube conique de 15 ml et centrifuger à 300-400 g pendant 7 min à température ambiante. </li><li> Pour lyse des globules rouges, éliminer le surnageant, remettre en suspension les cellules dans 5 ml Le chlorure de potassium-(ACK) tampon (pH 7,2), et incuber la suspension ACK-cellule à température ambiante pendant 5 min. </li><li> Ajouter 10 ml de DMEM à la suspension ACK-cellules et centrifuger le tube comme ci-dessus. Laver le culot cellulaire une fois dans 10 ml de DMEM. </li></ol> 2. Le tri cellulaire activé par fluorescence des cellules CD4 + T Cells diabétogéniques de BDC2.5 Souris <ol><li> Re-suspendre les cellules dans 5 ml FACS scontenant un tampon et de compter le nombre de cellules viables (en utilisant un microscope à contraste de phase et un hémocytomètre) par colorant bleu trypan exclusion. </li><li> En utilisant un tampon FACS, régler le volume de la suspension cellulaire à 5 x 10 7 cellules / ml. Retirer ~ 1 x 10 6 cellules par le contrôle de la coloration (1 NO, 3 taches simples taches). Colorer l&#39;échantillon de CD4 transgéniques non activés cellules T avec des anti-CD4 (APC), anti-TCR Vβ4 (FITC) et anti-CD62L (PE) anticorps monoclonaux (mAb) dans un tube de 15 ml. Effectuer coloration des cellules en utilisant les concentrations mAb suggérés par le fabricant pour 20-30 min à 4 ° C dans l&#39;obscurité. </li><li> Laver l&#39;échantillon et des contrôles simples tache avec du tampon FACS à&gt; 3X le volume de la réaction de coloration. Centrifuger le tube à 300-400 xg, éliminer le surnageant et remettre en suspension les cellules dans un tampon FACS (1-2 x 10 7 cellules / ml pour le tri des échantillons et 300 pi pour les contrôles tache unique) et magasins sur la glace jusqu&#39;à la prochaine étape. </li><li> Passer l&#39;échantillon de cellules à travers une cellule de 35 umTube cap passoire pour enlever des amas de cellules. Trier les échantillons de CD4 + TCR Vβ4 + CD62L + cellules sur un trieur de cellules avec un opérateur qualifié. Recueillir les cellules triées dans 3 ml DMEM dans un tube de 15 ml. Laisser 2,5-3 h, y compris la configuration, pour trier 1,5 x 10 8 cellules (le nombre approximatif de cellules obtenues à partir de 3 souris donneuses) à un taux de tri de 2 x 4 au total 10 événements / sec. Attendez-vous à ~ 2,5 x 10 6 cellules CD4 transgéniques non activés + T cellules par souris après tri cellulaire. </li><li> Notez le nombre absolu de cellules triées à la fin de la sorte et centrifuger les pendant 7 min à 300-400 g. Jeter le surnageant et remettre en suspension les cellules (2 x 10 6 cellules / ml) dans stérile tampon phosphate salin (PBS) (Mg 2 + / Ca 2 + libre) pour le transfert de cellules T adoptive. </li></ol> 3. Le transfert adoptif de lymphocytes CD4 + T Cells diabétogéniques de BDC2.5 Souris <ol><li> Utiliser une seringue de 1 ml et une ½ aiguille de calibre 18-1, doucement remettre en suspension les cellules CD4 + purifiées par FACSLymphocytes T et charger la seringue de 1 ml. En préparation pour injection, remplacer le 18-1 ½ aiguille de la jauge avec un 27 ½ aiguille de la jauge. </li><li> Expose 6-8 semaines, âgé de souris receveuses NOD.SCID femmes à une lampe chauffante jusqu&#39;à ce qu&#39;ils ont un comportement de toilettage. </li><li> Restrain souris receveuses dans un dispositif de retenue et essuyer leur queue avec 70% (v / v) d&#39;éthanol pour désinfecter le site d&#39;injection. </li><li> Injecter 1-2 x 10 6 FACS __gVirt_NP_NNS_NNPS&lt;__ cellules / souris triés (dans un maximum de 500 pi de PBS) dans l&#39;une des veines de la queue latérales. </li></ol> Ne forcez pas le piston. Si l&#39;aiguille se trouve de manière appropriée dans la veine, l&#39;injection aura lieu pratiquement sans résistance. 4. Suivi des souris receveuses pour Hyperglycémie et diabète de type 1 <ol><li> Commençant cinq jours après le transfert de cellules T, les souris receveuses NOD.SCID sont surveillées quotidiennement pour le glucose urinaire élevée en utilisant des bandelettes réactives (Bayer Diastix) selon les instructions du fabricant. Souris avec deux urin consécutivelectures de glucose E&gt; 250 mg / dl sont considérés comme diabétique. </li></ol>

<ol>
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L., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nonobese+diabetic+mouse+as+a+model+of+autoimmune+diabetes:+immune+dysregulation+gets+the+NOD.">The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD.</a> Immunity. 7, 727-738 (1997).</li><li>Wicker, L. S., Miller, B. J., Mullen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+autoimmune+diabetes+mellitus+with+splenocytes+from+nonobese+diabetic+(NOD)+mice.">Transfer of autoimmune diabetes mellitus with splenocytes from nonobese diabetic (NOD) mice.</a> Diabetes. 35, 855-860 (1986).</li><li>Bendelac, A., Carnaud, C., Boitard, C., Bach, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syngeneic+transfer+of+autoimmune+diabetes+from+diabetic+NOD+mice+to+healthy+neonates.+Requirement+for+both+L3T4++and+Lyt-2++T+cells.">Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates. Requirement for both L3T4+ and Lyt-2+ T cells.</a> J. Exp. Med. 166, 823-833 (1987).</li><li>Haskins, K., McDuffie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+diabetes+in+young+NOD+mice+with+a+CD4++islet-specific+T+cell+clone.">Acceleration of diabetes in young NOD mice with a CD4+ islet-specific T cell clone.</a> Science. 249, 1433-1436 (1990).</li><li>Christianson, S. W., Shultz, L. D., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+of+diabetes+into+immunodeficient+NOD-scid/scid+mice.+Relative+contributions+of+CD4++and+CD8++T-cells+from+diabetic+versus+prediabetic+NOD.NON-Thy-1a+donors.">Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors.</a> Diabetes. 42, 44-55 (1993).</li><li>Wicker, L. S., Todd, J. A., Peterson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+autoimmune+diabetes+in+the+NOD+mouse.">Genetic control of autoimmune diabetes in the NOD mouse.</a> Annual Reviews in Immunology. 13, 179-200 (1995).</li><li>Serreze, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+I-mediated+antigen+presentation+and+induction+of+CD8++cytotoxic+T-cell+responses+in+autoimmune+diabetes-prone+NOD+mice.">MHC class I-mediated antigen presentation and induction of CD8+ cytotoxic T-cell responses in autoimmune diabetes-prone NOD mice.</a> Diabetes. 45, 902-908 (1996).</li><li>Milton, M. J., Poulin, M., Mathews, C., Piganelli, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation,+maintenance,+and+adoptive+transfer+of+diabetogenic+T-cell+lines/clones+from+the+nonobese+diabetic+mouse.">Generation, maintenance, and adoptive transfer of diabetogenic T-cell lines/clones from the nonobese diabetic mouse.</a> Methods Mol. Med. 102, 213-225 (2004).</li><li>Kurrer, M. O., Pakala, S. V., Hanson, H. L., Katz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+cell+apoptosis+in+T+cell-mediated+autoimmune+diabetes.">Beta cell apoptosis in T cell-mediated autoimmune diabetes.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 213-218 (1997).</li><li>Amrani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin-independent+beta-cell+destruction+by+diabetogenic+CD8(+)+T+lymphocytes+in+transgenic+nonobese+diabetic+mice.">Perforin-independent beta-cell destruction by diabetogenic CD8(+) T lymphocytes in transgenic nonobese diabetic mice.</a> J. Clin. Invest. 103, 1201-1209 (1999).</li><li>Dobbs, C., Haskins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+a+T+cell+clone+and+of+T+cells+from+a+TCR+transgenic+mouse:+TCR+transgenic+T+cells+specific+for+self-antigen+are+atypical.">Comparison of a T cell clone and of T cells from a TCR transgenic mouse: TCR transgenic T cells specific for self-antigen are atypical.</a> J. Immunol. 166, 2495-2504 (2001).</li><li>Haskins, K., Portas, M., Bradley, B., Wegmann, D., Lafferty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-lymphocyte+clone+specific+for+pancreatic+islet+antigen.">T-lymphocyte clone specific for pancreatic islet antigen.</a> Diabetes. 37, 1444-1448 (1988).</li><li>Katz, J. D., Wang, B., Haskins, K., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+a+diabetogenic+T+cell+from+genesis+through+pathogenesis.">Following a diabetogenic T cell from genesis through pathogenesis.</a> Cell. 74, 1089-1100 (1993).</li><li>Stadinski, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromogranin+A+is+an+autoantigen+in+type+1+diabetes.">Chromogranin A is an autoantigen in type 1 diabetes.</a> Nat. Immunol. 11, 225-231 (2010).</li><li>Luhder, F., Chambers, C., Allison, J. P., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pinpointing+when+T+cell+costimulatory+receptor+CTLA-4+must+be+engaged+to+dampen+diabetogenic+T+cells.">Pinpointing when T cell costimulatory receptor CTLA-4 must be engaged to dampen diabetogenic T cells.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 12204-12209 (2000).</li><li>Tang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes.">In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.</a> J. Exp. Med. 199, 1455-1465 (2004).</li><li>Calderon, B., Suri, A., Pan, X. O., Mills, J. C., Unanue, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-gamma-dependent+regulatory+circuits+in+immune+inflammation+highlighted+in+diabetes.">IFN-gamma-dependent regulatory circuits in immune inflammation highlighted in diabetes.</a> J. Immunol. 181, 6964-6974 (2008).</li><li>Shultz, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+defects+in+innate+and+adaptive+immunologic+function+in+NOD/LtSz-scid+mice.">Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.</a> J. Immunol. 154, 180-191 (1995).</li><li>Waldner, H., Sobel, R. A., Price, N., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autoimmune+diabetes+locus+Idd9+regulates+development+of+type+1+diabetes+by+affecting+the+homing+of+islet-specific+T+cells.">The autoimmune diabetes locus Idd9 regulates development of type 1 diabetes by affecting the homing of islet-specific T cells.</a> J. Immunol. 176, 5455-5462 (2006).</li><li>Verdaguer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+autoimmune+diabetes+in+monoclonal+T+cell+nonobese+diabetic+mice.">Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.</a> J. Exp. Med. 186, 1663-1676 (1997).</li><li>Thomas, D. C., Mellanby, R. J., Phillips, J. M., Cooke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+early+age-related+increase+in+the+frequency+of+CD4++Foxp3++cells+in+BDC2.5NOD+mice.">An early age-related increase in the frequency of CD4+ Foxp3+ cells in BDC2.5NOD mice.</a> Immunology. 121, 565-576 (2007).</li><li>Prochazka, M., Gaskins, H. R., Shultz, L. D., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nonobese+diabetic+scid+mouse:+model+for+spontaneous+thymomagenesis+associated+with+immunodeficiency.">The nonobese diabetic scid mouse: model for spontaneous thymomagenesis associated with immunodeficiency.</a> Proc. Natl. Acad. Sci. U.S.A. 89, 3290-3294 (1992).</li></ol>

Toutes les souris ont été logées à la Penn State College of Medicine établissement exempt d&#39;agents pathogènes (SPF) spécifique en conformité avec les lignes directrices de la Penn State Institutional Animal Care et utiliser Comité. Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

DT1 peut être induite chez la souris bénéficiaires à des efficacités variables par transfert adoptif de cellules de rate entières ou des sous-ensembles de lymphocytes T de souris NOD diabétiques ou les souris transgéniques pour le TCR provenant de clones de lymphocytes T diabétogéniques. Nous rapportons ici une méthode reproductible pour induire le DT1 dans les souris receveuses dans les deux semaines à 100% d&#39;incidence en transférant FACS purifiés CD62L + BDC2.5 CD4 + lymphocytes T transgéniques dans...

La souris NOD développe un diabète auto-immune spontanément et a été largement utilisé comme modèle animal pour 1,2 DT1 humaine. Pathogenèse du DT1 chez les souris NOD est caractérisée par l&#39;infiltration, à partir de 3-4 semaines d&#39;âge, des îlots de Langerhans du pancréas par les cellules dendritiques et les macrophages, suivis par les cellules T et B. Cette phase de non-destructive péri-insulite conduit à une lente destruction progressive des cellules β pancréatiques productrices d&#39;insuline, entraînant un diabète patent de 4-6 mois d&#39;âge de 3 ans. Transfert de splénocytes 4,5, 6,7 ou CD4 + CD8 + 8,9 lymphocytes T de souris NOD diabétiques ont été montrés à la médiation du diabète chez la souris NOD immunodéprimés, ce qui indique que les cellules T îlots réactifs jouent un rôle central dans la pathogenèse du DT1. Selon les conditions expérimentales, le diabète développé dans les souris receveuses lentement, sur plusieurs semaines dans ces études. De même, les différents clones de lymphocytes T, dérivé par le temps et coûteuseculture de cellules T diabétogéniques, ont été signalés à la médiation diabète plusieurs semaines après transfert dans des souris receveuses 7,10. Avec la disponibilité de souris transgéniques exprimant des TCR dérivées de CD4 ou des clones de lymphocytes T CD8 diabétogéniques restriction, plusieurs laboratoires ont ensuite montré que les cellules T spléniques de ces souris ont été en mesure de transférer le diabète aux bénéficiaires 11-13. Plus précisément, BDC2.5 souris NOD sont transgéniques pour le TCR BDC2.5, qui est spécifique de la chromogranine A, une protéine dans les cellules bêta du pancréas 14-16. Le transfert de cellules en non activées rate entières ou fractionnées de diabétiques ou prédiabétiques ouvertement BDC2.5 souris transférés diabète de souris NOD néonatales ou immunodéficientes à des efficacités variables 11,17-19 vitro activé ou. Nous décrivons une méthode simple qui utilise CD4 + purifiés transgéniques cellules T de pré-diabétiques BDC2.5 souris pour induire le DT1 dans les souris receveuses à haut rendement et Consistence. Un grand nombre d&#39;îlots de Langerhans, naïfs CD4 + spécifiques d&#39;un antigène des cellules T sont isolés à partir de ces souris par tri cellulaire activé par fluorescence (FACS) pour CD4 + CD62L + des cellules T exprimant la chaîne TCR transgénique Vβ4. Les cellules T transgéniques purifiés sont ensuite transférés dans des souris sans activation NOD.SCID, qui manquent T fonctionnelles et les lymphocytes B et sont sans diabète insulite et 20. Les souris receveuses sont surveillés pour des concentrations élevées de glucose dans l&#39;urine indiquant DT1, qui se développe rapidement dans les deux semaines après le transfert de cellules T. Contrairement à d&#39;autres méthodes qui transfèrent des cellules T diabétogéniques avec des spécificités hétérogènes, notre protocole utilise triées par FACS cellules T CD4 + qui expriment presque exclusivement le diabetogenic BDC2.5 TCR. En raison de leur homogénéité, seul un petit nombre de cellules T transférées (~ 1x10 6 cellules / souris) sont nécessaires pour le développement du DT1 rapide dans les 2 semaines à 100% d&#39;incidence. Un autre avantage de notre protocole est que irradiation des souris receveuses n&#39;est pas nécessaire que ce soit pour d&#39;autres méthodes. Une limitation potentielle de cette méthode est qu&#39;elle ne permet pas à l&#39;enquête sur la contribution des deux CD4 et CD8 sous-ensembles de cellules T CD8 ou spécifiquement les lymphocytes T dans le diabète. Le protocole décrit sera utile pour étudier le développement rapide DT1, médiée par les naïfs CD4 monospécifiques cellules T, ainsi que des stratégies thérapeutiques pour intervenir dans homing des cellules Th spécifiques de l&#39;antigène îlots à l&#39;organe cible.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(Tcr&alpha;BDC 2.5, Tcr&beta;BDC 2.5)</td>
			<td>JAX</td>
			<td>004460</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NOD.SCID mice (NOD.CB17-Prkdcscid/J)</td>
			<td>JAX</td>
			<td>001303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
			<td>Themo Scientific</td>
			<td>SH30022.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bayer Diastix</td>
			<td>Fisher Scientific</td>
			<td>AM2803</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352095</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile surgical tweezers</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile small pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile large pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>70 &mu;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 &mu;m cell strainer cap tubes</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium-Chloride-Potassium (ACK) buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2 in dH2O</td>
		</tr>
		<tr>
			<td>BD FACSFlowTM sheath fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS staining buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized</td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD4 (APC) mAb</td>
			<td>Biolegend</td>
			<td>1005616</td>
			<td>clone RM4-5</td>
		</tr>
		<tr>
			<td>Anti-TCR V&beta;4 (FITC) mAb</td>
			<td>BD Biosciences</td>
			<td>553365</td>
			<td>clone KT4</td>
		</tr>
		<tr>
			<td>Anti-CD62L (PE) mAb</td>
			<td>BD Biosciences</td>
			<td>553151</td>
			<td>clone MEL-14</td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>BD Biosciences</td>
			<td>&nbsp;</td>
			<td>e.g. BD FACSAria III</td>
		</tr>
		<tr>
			<td>Heat lamp</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>18-1&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>27&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305109</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

accelerated type 1 diabetes induction in mice by adoptive transfer of diabetogenic cd4+ t cells

Les diabétiques (NOD) souris non obèses se développe spontanément diabète auto-immun après 12 semaines d&#39;âge et est le modèle le plus largement étudié animal humain de type 1 du diabète (DT1). études sur le transfert des cellules dans des souris receveuses irradiées ont établi que les lymphocytes T jouent un rôle essentiel dans la pathogenèse du DT1 dans ce modèle. Nous décrivons ici une méthode simple pour rapidement induire une DT1 par transfert adoptif de purifié, CD4 + primaires des cellules T de souris NOD pré-diabétiques transgéniques pour le récepteur des cellules T îlot spécifique (TCR) BDC2.5 dans des souris receveuses NOD.SCID. Les principaux avantages de cette technique sont que l&#39;isolement et le transfert adoptif de lymphocytes T diabétogéniques peut être complété dans le même jour, l&#39;irradiation des bénéficiaires n&#39;est pas nécessaire, et une forte incidence de diabète de type 1 est provoqué dans les 2 semaines après le transfert de cellules T. Ainsi, les études sur la pathogenèse et les interventions thérapeutiques dans le DT1 peuvent se réaliser à un rythme plus rapide que les méthodes qui s&#39;appuient sur des populations de cellules T hétérogènes ou des clonesprovenant de souris NOD diabétiques.

Nos résultats montrent l&#39;isolement des cellules transgéniques exprimant CD62L BDC2.5, ce qui est essentiel pour les cellules T à la maison vers les organes lymphoïdes secondaires comme les ganglions lymphatiques pancréatiques. Nos résultats démontrent en outre la capacité puissante de cette population de cellules T monospécifiques de transférer rapidement et efficacement à DT1 souris receveuses NOD.SCID. Isolement de CD4 diabétogéniques cellules T de souris BDC2.5 est montr?...

Watch this Scientific Journal Video about Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells at JoVE.com

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

Nous fournissons une méthode reproductible pour induire un diabète de type 1 (DT1) chez la souris dans les deux semaines par le transfert adoptif de l&#39;îlot spécifiques de l&#39;antigène, CD4 + primaires des cellules T.

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 ...

accelerated-type-1-diabetes-induction-mice-adoptive-transfer

Research

JoVE Journal

Immunology and Infection

16.6K vues.  Pennsylvania State University College of Medicine. Nous fournissons une méthode reproductible pour induire un diabète de type 1 (DT1) chez la souris dans les deux semaines par le transfert adoptif de l'îlot spécifiques de l'antigène, CD4 + primaires des cellules T.

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Préparation et utilisation de VIH-1 primaires CD4 + T-cellules comme cellules cibles dans des cellules tueuses naturelles Tests cytotoxiques

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Recherche

Immunologie et infection

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

L&#39;évaluation de l&#39;Innate détection du VIH-1 CD4 infectés<sup&gt; +</sup&gt; Cellules T par les cellules dendritiques plasmacytoïdes Utilisation d&#39;un<em&gt; Ex vivo</em&gt; Système de co-culture.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

Préparation des cellules CD4<sup&gt; +</sup&gt; Cellules T pour l&#39;analyse de GD3 et GD2 ganglioside Membrane Expression par Microscopie

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

In Vitro Differentiation of Human CD4 + FOXP3 cellules + Induced T régulatrices (de iTregs) de Naïf cellules T CD4 + en utilisant un protocole TGF-β contenant

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Induction de paralysie et de lésion du système visuel chez la souris par le particulier de cellules T pour Neuromyélite optique autoantigènes Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

Isolement et transfert adoptif de sel élevé traités des cellules dendritiques présentatrices d’antigène

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Quantification de la prolifération des cellules CD4et T spécifiques à l'antigène humain à l'aide de Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Immunothérapie adoptive des cellules iNKT dans les souris de RA induites par l'isomérase de glucose-6-Phosphate (G6PI)

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and celiac disease in the United States. With the coronavirus disease 2019 (COVID-19) pandemic, the epidemiology of COVID-19 in the general population and knowledge about the association between COVID-19 infection and T1D development are urgently needed. The currently standard screening method of the radio-binding assay (RBA) has met two great challenges: low efficiency with a single assay format and low disease specificity with a large proportion of low-affinity antibodies generated in screening. With the platform of the multiplex electrochemiluminescence (ECL) assay we established previously, a novel 6-Plex ECL assay was developed that combines, in a single well, all four islet autoantibodies (IAbs) to insulin, glutamic acid decarboxylase (GAD65), insulinoma antigen 2 (IA-2), and Zinc transporter 8 (ZnT8) for T1D, transglutaminase autoantibodies (TGA) for celiac disease, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibodies for COVID-19. The assay was validated in blind using 880 samples from the ASK study, including 325 positive samples and 555 all antibody-negative samples, and compared with the standard RBAs and a single ECL assay. With the advantages of high efficiency, low cost, and low serum volume, this assay has been accepted as the primary screening tool for the ASK study.

In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2.

Un dépistage multiplexé à haut débit pour le diabète de type 1, les maladies cœliaques et la COVID-19

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and ...