JHN is supported by the Swiss National Foundation (SNSF 310030_146290).

The authors have nothing to disclose.

Tal como acontece com todos os outros modelos, o modelo de colite-driven antigénio descrito acima pode apresentar alguns problemas que o investigador realização da técnica deve estar ciente. Ao injetar o OT-II / Vermelho + CD4 + T CD62L + células nos hospedeiros, o investigador deve ser muito gentil e cuidadosa para inserir a agulha para dentro da cavidade peritoneal. Não fazer isso pode resultar na ruptura do intestino do rato que pode levar à morte ou a administração subcutân...

O intestino é a maior superfície do corpo que é exposta ao ambiente externo. Vastas matrizes de micróbios residentes colonizar o intestino humano para formar a microbiota intestinal (ou microflora). Este é estimado para consistir de até 100 trilhões de células microbianas e constitui um dos habitats bacterianas mais densamente povoadas conhecidos na biologia 1-3. No GIT bactérias colonizar um nicho intestinal onde eles sobreviver e se multiplicar 4. Em troca, a microbiota dota o anfitrião com características funcionais adicionais não codificados em seu genoma 1. Por exemplo a microbiota estimula a proliferação de células epiteliais, produz vitaminas que hospeda não pode produzir por si mesmas, regula o metabolismo e protege contra agentes patogénicos 4-6. Dada esta relação benéfica, alguns autores têm sugerido que os seres humanos são &quot;super-organismos&quot; ou &quot;holobionts&quot;, que são uma mistura de genes de bactérias e humanos 7,8. Dado o impacto benéfico da microbiota do hospedeiro (humano), o sistema imunitário do intestino necessita de tolerar micróbios comensais para permitir a sua existência no lúmen, mas também matar os patogénios que invadem a partir do lado luminal 9-11. O sistema imunológico intestinal tem desenvolvido mecanismos para distinguir entre os micróbios luminais inofensivos e potencialmente prejudiciais; No entanto, estes mecanismos ainda não são bem compreendidos 12. Manter a integridade intestinal requer uma homeostase imune fortemente regulada para manter o equilíbrio entre tolerância e imunidade 13. Um desequilíbrio na homeostase imune contribui para a indução de doenças intestinais, tais como doença inflamatória do intestino (IBD) 3,14. Existem dois principais tipos de DII: doença de Crohn (CD) e colite ulcerativa (UC). Os pacientes com estas doenças geralmente sofrem de sangramento retal, diarréia severa e dor abdominal 15,16. A única causa da DII ainda édesconhecida, mas uma combinação de fatores genéticos, influências ambientais e respostas imunes desreguladas pode ser o evento chave para o desenvolvimento da doença 15. Modelos animais para IBD têm sido utilizados por mais de 50 anos. Nas últimas décadas foram desenvolvidos novos sistemas modelo IBD para testar as várias hipóteses relativas à patogénese da DII 17,18. O modelo melhor caracterizado de colite crónica é o modelo de transferência de células T que induz perturbações da homeostase de células T 19,20. Este modelo envolve a transferência de células T naive a partir de ratinhos imunocompetentes em hospedeiros que não têm T e as células B (tais como RAG - / - e ratinhos SCID) 16,21. O desenvolvimento da doença neste modelo é monitorizada durante 3-10 semanas por avaliação da presença de diarreia, a redução da actividade física, e perda de peso corporal. Este é assim chamado a síndrome de caquexia 16. Em comparação com os ratos saudáveis ​​do tecido do cólon dos exércitos transplantados é thickeR, mais curtos e mais pesado 16. Utilizando o modelo de transferência de células T, é possível compreender como as diferentes populações de células T pode contribuir para a patogénese do IBD 22. O modelo de transferência de células T não analisar as interacções entre as APCs e as células T no processo da doença de uma forma específica para o antigénio. Demonstrou-se que uma interacção entre as células mielóides e células linfóides pode ser responsável pelo desenvolvimento de inflamação intestinal 23. Embora muitos aspectos de DII foram clarificados, os eventos iniciais que conduzem ao desenvolvimento da doença têm ainda de ser claramente entendido. Foi demonstrado que na ausência da colite transferência microbiota não pode ser estabelecida 24. Recentemente, várias teorias que sugerem IBD pode ser um resultado de uma resposta imunitária contra bactérias comensais 25. Os autores propuseram também que as bactérias comensais são essenciais para induzir a inflamação no intestino distal26. Em livre de germes (GF) os animais o sistema imunitário do intestino é geralmente deficientes 27,28, mas uma colonização destes ratos com uma mistura de-específica isentos de agentes patogénicos bactérias resulta no desenvolvimento do sistema imunitário intestinal totalmente competente 29. Assim, a microbiota parece ser um elemento chave na patogénese do IBD, ou como um mecanismo que predispõe para ou protege contra o desenvolvimento de inflamação intestinal 30,31. As teorias atuais sugerem que IBD é um resultado de desequilíbrio microbiano, chamado disbiose, em pacientes geneticamente predispostos 32, mas ainda não está claro se a disbiose é a causa ou a consequência da doença 12. Considerando o papel de microorganismos no desenvolvimento de DII, em experiências in vitro mostraram que as células T CD4 + podem ser activadas por APCs pulsadas com bactérias intestinais 33,34. Além disso, demonstrou-se que os antigénios deespécies bacterianas diferentes comensais, tais como E. coli, Bacteroides, Eubacterium e Proteus, é capaz de activar células T CD4 + 35. Isto indica que a apresentação de antigénios bacterianos de células T é de importância para o desenvolvimento de IBD. Para reduzir a complexidade de múltiplos antigénios derivados pela microflora no processo da doença, de uma estirpe de E. coli foi criada que produz o antigénio OVA. Transferência colite foi induzida por injecção de células T específicas de OVA em RAG - / - de animais colonizado com E. expressam OVA coli. Este modelo baseia-se em evidências recentes sugerindo que CX 3 CR1 + MPs, um grande subconjunto de células na lâmina do cólon (CLP) 36, estão interagindo com células T CD4 + durante a transferência de colite 37. MPs provar o lúmen intestinal para antígeno particulado, tais como bactérias, usando suas dendrites 36, 38,39. Estudos anterioresdemonstrou que MPs também pode levar até antigénios solúveis, tais como óvulos, introduzidos na intestinal lúmen 40,41. Dada a abundância de CX 3 CR1 + MPs no CLP, é possível que essas células podem provar bactérias luminais e interagir com células T CD4. De imagem confocal de murganhos transplantados com células específicas de OVA T CD4 + colonizadas com E. coli PCP-OVA, mostram que CX 3 CR1 + MPs estão em contacto com células AT-II T CD4 + durante o desenvolvimento de colite conduzido pelo antigénio. Este modelo permite o estudo do processo de apresentação de antigénio entre as APCs e as células intestinais T específicas apenas para determinadas bactérias que expressam os antigénios no lúmen do intestino.

<table border="0" cellpadding="0" cellspacing="0" width="801">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">LB Broth, Miller (Luria-Bertani)</td>
			<td style="width:200px;">Difco</td>
			<td style="width:219px;">244620</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">2 mm gap couvettes&#160;</td>
			<td style="width:200px;">Peqlab Biotechnologie GmbH</td>
			<td style="width:219px;">71-2020</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">G5516-100ML</td>
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			<td height="21" style="height:21px;width:216px;">Gene Pulser Xcell system&#160;</td>
			<td style="width:200px;">BioRad Laboratories GmbH</td>
			<td style="width:219px;">1652660</td>
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			<td height="21" style="height:21px;width:216px;">LB Agar, Miller (Luria-Bertani)</td>
			<td style="width:200px;">Difco</td>
			<td style="width:219px;">244510</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">A9393-5G</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">SOC Medium</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td>S1797-100ML</td>
			<td></td>
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			<td height="16" style="height:16px;width:216px;">High Pure Plasmid Isolation Kit</td>
			<td style="width:200px;">Roche</td>
			<td style="width:219px;">11754777001</td>
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			<td height="21" style="height:21px;width:216px;">Agarose</td>
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			<td style="width:219px;">3810.1</td>
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			<td height="23" style="height:23px;width:216px;">EDTA</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">E9884-100G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris-HCl</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">T5941</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Glacial acetic acid</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">537020&#160;</td>
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			<td height="26" style="height:26px;">Gel chamber&#160;</td>
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			<td style="width:219px;">40-0708</td>
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			<td height="21" style="height:21px;width:216px;">Loading Dye</td>
			<td style="width:200px;">Thermo Fisher</td>
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			<td height="21" style="height:21px;width:216px;">GeneRuler 1 kb DNA Ladder&#160;</td>
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			<td style="width:219px;">SM0312</td>
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			<td height="21" style="height:21px;width:216px;">Ethidium bromide solution</td>
			<td style="width:200px;">Carl Roth GmbH &#38; Co. KG</td>
			<td style="width:219px;">2218.3</td>
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			<td height="21" style="height:21px;width:216px;">Photo-documentation system&#160;</td>
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			<td style="width:219px;">DeVision G&#160;</td>
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			<td height="21" style="height:21px;width:216px;">DNA sequencing&#160;</td>
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			<td style="width:219px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Phosphate buffered saline (PBS)</td>
			<td style="width:200px;">Biochrom</td>
			<td>L182-50</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescent microscope&#160;</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:219px;">HBO 100</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mini-PROTEAN Tetra System</td>
			<td style="width:200px;">Bio-Rad Laboratories GmbH</td>
			<td style="width:219px;">1658005</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">PageRuler Prestained Protein Ladder&#160;</td>
			<td style="width:200px;">Fermentas, St. Leon-Rot, Germany</td>
			<td style="width:219px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">IstanBlue Solution</td>
			<td style="width:200px;">Expedeon, Cambridgeshire, United Kingdom</td>
			<td style="width:219px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Nitrocellulose membrane&#160;</td>
			<td style="width:200px;">Macherey-Nagel GmbH &#38; Co. KG</td>
			<td>741280</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Electro blotter&#160;</td>
			<td style="width:200px;">Biometra GmbH</td>
			<td style="width:219px;">846-015-600</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumins (BSA)</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">A6003-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Ovalbumin antibody&#160;</td>
			<td style="width:200px;">Abcam</td>
			<td style="width:219px;">ab181688</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-rabbit IgG&#160; HRP</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">A0545&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce ECL Plus Western Blotting Substrate</td>
			<td style="width:200px;">Pierce Biotechnology, Thermo Fischer Scientific Inc</td>
			<td style="width:219px;">32132</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forene</td>
			<td>Abbott</td>
			<td style="width:219px;">2594.00.00</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:219px;">10500-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Falcon Cell Strainers</td>
			<td style="width:200px;">Fischer Scientific&#160;</td>
			<td style="width:219px;">08-771-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium chloride</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">254134-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris Base</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">10708976001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">CD4+ CD62 L+ T isolation kit&#160;</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:219px;">130-093-227&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS LS Columns&#160;</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:219px;">130-042-401</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS MS Columns&#160;</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MidiMACS Separator</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:219px;">130-042-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MiniMACS Separator</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:219px;">130-042-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS MultiStand</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:219px;">130-042-303</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Feeding Needle 20G</td>
			<td style="width:200px;">SouthPointe Surgical Supply, Inc</td>
			<td style="width:219px;">FN-7903</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">HT501128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraffin</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">1496904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hematoxylin</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">H9627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eosin Y</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">230251&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dithiothreitol</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">D9779&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase type VIII</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">C-2139</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Roswell Park Memorial Institute (RPMI) medium</td>
			<td style="width:200px;">AppliChem</td>
			<td style="width:219px;">A2044, 9050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Percoll (density 1.124 g/ml)</td>
			<td style="width:200px;">Biochrome</td>
			<td style="width:219px;">L-6145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium azide</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">438456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse BD Fc Block</td>
			<td style="width:200px;">BD Pharmingen</td>
			<td style="width:219px;">553141</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FITC-conjugated mAb binding V&#223; 5.1, 5.2&#160;</td>
			<td style="width:200px;">BD Pharmingen</td>
			<td style="width:219px;">553189</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">APC-conjugated mAb binding CD4 GK1.5&#160;</td>
			<td style="width:200px;">eBioscience</td>
			<td style="width:219px;">17-0041-83</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS Calibur&#160;</td>
			<td style="width:200px;">BD Biosciences</td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FCS Express V3 software</td>
			<td style="width:200px;">DeNovo</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Meta scanning confocal microscope&#160;</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:219px;">LSM 710&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss Workstation</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:219px;">LSM 7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss ZEM software&#160;</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:219px;">v4.2.0.121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Maxisorp immuno plates&#160;</td>
			<td style="width:200px;">NUNC, Roskilde</td>
			<td style="width:219px;">442404</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Streptavidin conjugated alkaline phosphatase</td>
			<td style="width:200px;">Jackson Immuno Research</td>
			<td style="width:219px;">016-050-084</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Alkaline phosphatase substrate 4-Nitrophenyl phosphate disodium salt hexahydrate</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">71768-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mAb R4-6A2</td>
			<td style="width:200px;">BD Biosciences</td>
			<td style="width:219px;">551216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mAb XMG1.2&#160;</td>
			<td style="width:200px;">BD Biosciences</td>
			<td>554410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TECAN microplate-ELISA reader</td>
			<td style="width:200px;">Tecan</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EasyWin software</td>
			<td style="width:200px;">Tecan</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to t cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

Para estabelecer um modelo de colite-driven antígeno um E. coli Foi construído um plasmídeo que contém na qual o gene para a PCP é fundido à sequência de codificação para a proteína de ovalbumina de galinha e a construção de fusão é expressa sob o controlo do promotor forte constitutivo hiper P (Figura 1A). Microscopia de fluorescência demonstra que a E. recombinante coli pCFP-OVA, mas não a de...

Watch this Scientific Journal Video about Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells at JoVE.com

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Desenvolvimento de uma colite Modelo Antigen-driven para o Estudo de apresentação de antígenos por células apresentadoras de antígenos às células T

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

development-an-antigen-driven-colitis-model-to-study-presentation

Research

JoVE Journal

Immunology and Infection

8.9K visualizações.  University College Cork. In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Vídeo: Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA-Ig Baseado Artificial células apresentadoras de antígenos para a eficiente Ex vivo Ampliação da CTL Humanos

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Imunologia e Infecção

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Antígeno Artificial célula apresentadora de ativação mediada (AAPC) e expansão de células T Natural Killer

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Aplicação Clínica de<em&gt; A Bela Adormecida</em&gt; Artificial e Antigen Presenting Cells para modificar geneticamente células T de Sangue do Cordão Umbilical e periférica

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Transdução de adenovírus Naive CD4 células T para estudar a diferenciação Treg

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Uma<em&gt; In vitro</em&gt; Modelo para estudar respostas imunes de sangue periférico humano células mononucleares para Vírus Respiratório Sincicial Humano Infecção

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Assassino Artificial células apresentadoras de antígenos (KaAPC) para a eficiente<em&gt; In Vitro</em&gt; Depleção de células T humanas antígeno específico da

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

As células T Capturar Bactérias por Transinfection das dendríticas

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Desenvolvimento de Células-Tronco de derivados de células T regulatórias antígeno específico da Contra Auto-imunidade

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Desenvolvimento e validação de uma única molécula ultra-sensível de matriz Digital ensaio imunossorvente ligado à enzima para humana Interferon-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...