Gostaria de agradecer a Bianca Mothé, Carla Oseroff, e Marie-France DelGuercio por me apresentar ensaios imunológicos e manuseio do mouse. Trabalho no laboratório de GA Splitter é financiado pelo NIH conceder 1-RO1-AI-073558, GLRCE conceder 1-U54-AI-057153, e BARD EUA-3829-06.

1. Provocando destino-específico CTLs efetoras de Imunização (Dia 0) <ol><li> Antes de começar, decida em cima de um efetor específicos: sistema de destino. Neste exemplo de um clássico ensaio in vivo de CTL, usaremos uma imunização peptídeo purificado para provocar específicas CD8 + T células. O peptídeo mesma será usada para pulso as células-alvo singênico, criando um efetor específicos: sistema de destino. Alternativamente, você pode optar por utilizar patógeno todo ou proteínas para obter o seu efetores e metas específicas. </li><li> Descontaminar a superfície de trabalho dentro de uma capa de biossegurança para garantir a esterilidade das injeções. Prepare a imunização peptídeo primeira colocação por 50 mg de peptídeo purificado em 100 mL DMSO 10% em PBS / mouse contidos em um tubo de 2 mL. Em seguida, adicione uma quantidade igual de muito frio adjuvante de Freund incompleto e até a marca de 0,2 mL com 1 mm para contas de mistura. A partir deste ponto, mantenha imunógeno em gelo até injeção. Tampa segura com Parafilm. </li><li> Use um batedor de talão definido para RPMs máximo para misturar a emulsão por 3 minutos, colocando imediatamente a emulsão de volta no gelo após a mistura. A emulsão resultante deve ser semelhante em consistência à maionese. Permitir emulsão para sentar-se a 4 ° C durante a noite se o tempo permitir (até uma semana), a mistura de outros eventuais separação é anotado. </li><li> Use uma seringa de 1 mL e 20 ou 20,5 agulha para puxar para cima emulsão tendo o cuidado de não puxar para cima todas as pérolas (contas deve ser maior que calibre de agulha, mas ainda estar ciente). </li><li> Rato seguro para injeção subcutânea na base da cauda. Nós usamos uma versão modificada plásticos de 50 mL do tubo cônico de forma segura e suavemente imobilizar o mouse para injeção. Injetar o mouse com 0,2 mL de emulsão. </li></ol> 2. Colheita células-alvo (dia 8) <ol><li> Você vai precisar de ingênuo camundongos isogênicos para as células-alvo. Euthanize camundongos ingênuos de acordo com padrões AAALAC. </li><li> Desinfectar rato por pulverização Etanol 70% para minimizar a possibilidade de contaminação do cabelo do mouse ou de outros contaminantes externos. </li><li> Com uma tesoura de dissecção fazer snip pequenos através da pele no lado esquerdo do mouse. Puxe retalho de pele mais para descobrir saco peritoneal e órgãos internos. </li><li> Snip através do peritônio, tomando cuidado para não comprometer os órgãos subjacentes. O baço é próximo à superfície e é similar na cor para o fígado (vermelho escuro), mas menor e mais feijão rim em forma. Camundongos infectados geralmente apresentam baços notadamente maiores do que os controles não infectados. </li><li> Retire cuidadosamente o baço, tendo o cuidado de ser gentil para não quebrar o baço em várias partes, a fim de recuperar o número de células máxima. Colocar imediatamente o baço em um 15 mL cônico contendo 10 mL de meios preparados no gelo. Continue com ratos restantes. </li><li> Despeje o conteúdo do tubo de 15 mL cônica (media e do baço) em um moedor de tecido de vidro. Moer o baço usando muita força no braço. Quando todo o tecido perdeu a sua cor que você pode supor que você tenha recuperado todas as células fora do tecido conjuntivo. </li><li> Despeje o conteúdo do moinho através de um filtro de células 70 M em um tubo cônico de 50 mL. Enxágüe moedor com a mídia fresco e despeje através de um filtro de células. </li><li> Centrífuga a 4 ° C, 1300 RPM por 7 minutos. Deitar fora o sobrenadante. </li><li> Ressuspender em 10 ml de reagente de lise preparada e incubar a 37 º durante 7 minutos. </li><li> Adicionar até 50 ml com a mídia fresco e centrífuga, mesmo que o anterior. </li><li> Ressuspender em 50 ml de mídia fresco e centrífuga, mesmo que o anterior (etapa de lavagem). * Nota: Neste momento é possível realizar uma separação de células magnéticas para um tipo específico de célula (macrófagos, por exemplo), se uma população de células-alvo puro é desejado para a transferência em camundongos destinatário. Além disso, neste momento você poderá introduzir um tratamento experimental para a cultura, a fim de estudar os efeitos do que o tratamento em matar células específicas. </li><li> Contagem de células e placa de 1 x 10 7 por bem em 100μL em uma placa de 96 cultura de tecidos bem fundo redondo tratada. * Esta é uma alta concentração de células / poço, mas porque o ensaio é um rápido, temos visto nenhuma perda da viabilidade das células nesta concentração. </li></ol> 3. Tratamento e marcação das células-alvo <ol><li> Células de pulso apropriada e repetições, com peptídeo de patógenos específicos ou controle peptídeo irrelevante: Adicionar um peptídeo mcg / mL. Incubar a 4 ° C por 1,5 horas. Mover-se para 37 ° C incubadora nos últimos 30 minutos. Durante este tempo, preparar o reagente CFSE. </li><li> Prepare CFSE de acordo com as especificações do fabricante e diluir para uma concentração de trabalho entre 0,5 e 25 mM. Faça concentração &quot;baixa&quot; de 10x menor que CFSE concentração &quot;alto&quot;. Por exemplo, para [baixo] adicionar 0,5 mL CFSE a 10 mls PBS e para [alta], adicionar 5 mL CFSE a 10 mls PBS. CFSE preparado quente a 37 ° C antes de rotulagem. </li><li> Retire a placa de células de incubadora e pEllet em uma centrífuga de alta velocidade à temperatura ambiente, 1300 RPM por 7 minutos. Flick para remover o sobrenadante. </li><li> Ressuspender as células em qualquer [baixo] ou CFSE [alta], adicionando 200 mL de reagente adequado para os poços correspondentes e incubar por 15 minutos a 37 ° C. </li><li> Retire a placa de células de incubadora e de novo pellet em temperatura ambiente, 1300 RPM por 7 minutos. Flick para remover o sobrenadante. </li><li> Ressuspender as células em 200 mL / poço de mídia fresco aquecido e incubar por 30 minutos a 37 ° C. </li><li> Retire a placa de células de incubadora e pellet em temperatura ambiente, 1300 RPM por 7 minutos. Flick para remover o sobrenadante. </li><li> Ressuspender as células em 100 mL / poço de PBS. </li><li> Para a transferência adotiva, adicione um bem [baixo] células marcadas para um bem [de alta] rotulados células por mouse. Resultantes da injecção deve ser de 50% metas específicas e 50% de células irrelevante em 0,2 mls. </li><li> Não se esqueça de reservar algum células marcadas para atuar como seus controles não-transferidos durante a análise de dados e para executar no citômetro de fluxo (Figura 1). </li></ol> 4. Transferência adotiva <ol><li> Anestesiar previamente peptídeo camundongos imunizados (De parte # 1 do presente protocolo) de acordo com padrões AAALAC. </li><li> Injetar 0,2 mL de células-alvo marcado via retro-orbital rota em camundongos destinatário. </li><li> Esperar 6 horas ou tempo predeterminado outras ideal antes de eutanásia os ratos destinatário. </li></ol> 5. Colheita células marcadas * Nota: Os passos 5.1 a 5,11 são os mesmos passos 2.1 a 2,11 <ol start="12"><li> Contagem de células e placa para fora em 1 x 10 6 por poço em 100 mL em 96 cultura bem fundo redondo de tecido placa tratada. </li><li> Fix células recuperado e não transferidos pela adição de 100 mL de paraformaldeído 2% para uma concentração final de paraformaldeído 1% em todos os poços. </li></ol> 6. Citometria de Fluxo e Análise de Dados <ol><li> Executar amostras em um citômetro de fluxo e analisar os dados. </li></ol> 7. Resultados representante Um pico diminuindo epítopo específico no histograma resultante é um indicador de matar células específicas (Figura 1). Use as seguintes equações (aplicado na Tabela 1) para determinar o valor real de lise específica de células: Percentual = relação Irrelevante: Percentagem específicas Epitope (pico [baixo]: [alta] pico), Lise por cento específico = [1 - (Non-transferidos relação controle / relação Experimental)] x 100 <img src="/files/ftp_upload/2250/2250fig1.jpg" alt="Figura 1" /> Figura 1. CFSE perfil de Números recuperados esplenócitos BALB / c mouse. Indicam a porcentagem de células do fenótipo CFSE alta ou baixa. A) não transferiu o controle. Todos [alta] CFSE picos representam epítopo células-alvo específicos, enquanto os picos CFSE [baixo] representam alvos pulsadas com peptídeo irrelevante. B) As células CFSE rotulados recuperado da PBS do mouse destinatário imunizadas (Control). C) células CFSE rotulados recuperados a partir de duas diferentes peptídeo ratos destinatário específico, anote os picos diminuiu epítopo específico. <img src="/files/ftp_upload/2250/2250fig2.jpg" alt="Figura 2" /> Figura 2. Representação gráfica dos dados analisados. Três pontos de dados apresentados para cada grupo de ratos, PBS, Peptide A, e B. Peptide <table border="1"><tbody><tr><td> Amostra </td><td> CFSE Epitope% ALTA Pulsada </td><td> % CFSE LOW Irrelevante </td><td> Relação de Baixo: Alta </td><td> Matar por cento Epitope específicas </td></tr><tr><td> PBS </td><td> 42,8 </td><td> 57,2 </td><td> 1,34 </td><td> 16,97 </td></tr><tr><td> PBS </td><td> 43,1 </td><td> 56,9 </td><td> 1,32 </td><td> 15,94 </td></tr><tr><td> PBS </td><td> 44,3 </td><td> 55,7 </td><td> 1,26 </td><td> 11,74 </td></tr><tr><td> Um peptídeo </td><td> 39,7 </td><td> 60,3 </td><td> 1,52 </td><td> 32,56 </td></tr><tr><td> Um peptídeo </td><td> 38,6 </td><td> 61,4 </td><td> 1,59 </td><td> 35,61 </td></tr><tr><td> Um peptídeo </td><td> 40,6 </td><td> 59,4 </td><td> 1,46 </td><td> 29,99 </td></tr><tr><td> Peptídeo B </td><td> 38,7 </td><td> 61,3 </td><td> 1,58 </td><td> 24,68 </td></tr><tr><td> Peptídeo B </td><td> 38,5 </td><td> 61,5 </td><td> 1,60 </td><td> 25,32 </td></tr><tr><td> Peptídeo B </td><td> 39,3 </td><td> 60,7 </td><td> 1,54 </td><td> 22,76 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td> PBS NT Controle </td><td> 47,4 </td><td> 52,6 </td&gt; <td> 1,11 </td><td></td></tr><tr><td> Pep Um Controle NT </td><td> 49,4 </td><td> 50,6 </td><td> 1,02 </td><td></td></tr><tr><td> Pep B NT Controle </td><td> 45,6 </td><td> 54,4 </td><td> 1,19 </td><td></td></tr></tbody></table> Tabela 1. Análise de dados representativos. &quot;NT&quot; não é transferido.

<ol>
	<li>Blattman, J. N., Wherry, E. J., Ha, S. J., van der Most, R. G., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+epitope+escape+on+PD-1+expression+and+CD8+T-cell+exhaustion+during+chronic+infection.">Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection.</a> J Virol. 83 (9), 4386-4394 (2009).</li><li>Jenkins, M. R., Tsun, A., Stinchcombe, J. C., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+strength+of+T+cell+receptor+signal+controls+the+polarization+of+cytotoxic+machinery+to+the+immunological+synapse.">The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.</a> Immunity. 31 (4), 621-631 (2009).</li><li>Ingulli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+tolerance+and+immunity+in+vivo+by+CFSE-labeling+of+administered+cells.">Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells.</a> Methods Mol Biol. 380, 365-376 (2007).</li><li>Durward, M. A., Harms, J., Magnani, D. M., Eskra, L., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discordant+Brucella+melitensis+antigens+yield+cognate+CD8++T+cells+in+vivo.">Discordant Brucella melitensis antigens yield cognate CD8+ T cells in vivo.</a> Infect Immun. 78 (1), 168-176 (2010).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J Immunol Methods. 171 (1), 131-137 (1994).</li><li>Weston, S. A., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+dyes+for+lymphocyte+migration+studies.+Analysis+by+flow+cytometry+and+fluorescence+microscopy.">New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.</a> J Immunol Methods. 133 (1), 87-97 (1990).</li><li>Akondy, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yellow+fever+virus+vaccine+induces+a+broad+and+polyfunctional+human+memory+CD8++T+cell+response.">The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.</a> J Immunol. 183 (12), 7919-7930 (2009).</li><li>Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+antigen-driven+responses+by+flow+cytometry:+monitoring+proliferation+by+dye+dilution.">Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution.</a> Cytometry A. 73 (11), 1019-1034 (2008).</li><li>Labadi, A., Balogh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+preferences+in+serosal+homing+and+distribution+of+peritoneal+B-cell+subsets+revealed+by+in+situ+CFSE+labeling.">Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling.</a> Int Immunol. 21 (9), 1047-1056 (2009).</li><li>Suffner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+support+homeostatic+expansion+of+Foxp3++regulatory+T+cells+in+Foxp3.LuciDTR+mice.">Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice.</a> J Immunol. 184 (4), 1810-1820 .</li></ol>

Não há conflitos de interesse declarados. Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela Institutional Animal Care e do Comitê de Uso (IACUC) da Universidade de Wisconsin-Madison.

Este ensaio pode ser modificado para investigar a capacidade proliferativa das células, incluindo a expansão clonal, porque CFSE relativamente divide igualmente entre os descendentes 10,11. Também é possível usar rotulagem CFSE para investigar a migração celular 6,12, embora existam outras células rastreamento métodos que podem ser mais apropriadas dependendo da hipótese, por exemplo tetrâmeros e bioluminescência 13, que também pode ser combinado com rotulagem CFSE. <p c...

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antigen specific in vivo killing assay using cfse labeled target cells

Diacetato carboxifluoresceína succinimidyl éster (CFSE) pode ser usado para facilmente e rapidamente rótulo uma população de células de interesse para a investigação in vivo. Esta rotulagem tem sido classicamente utilizado para o estudo da proliferação e migração. No método aqui apresentado, temos reduzido o cronograma, após a transferência adotiva de olhar para a sobrevivência e morte de epítopo específico CFSE células-alvo marcado 4-6. O nível de matar específica de um clone de células T CD8 + pode indicar a qualidade da resposta, como a sua quantidade pode ser enganosa. Específicos células T CD8 + podem se tornar funcionalmente esgotado ao longo do tempo com um declínio na produção de citocinas e matando 7,8. Além disso, certos CD8 + clones de células T não pode matar, bem como outros com diferentes especificidades TCR 9. Eficazes para Imunidade mediada por células (CMI), os antígenos devem ser identificados que produzem não apenas um número adequado de células T responder, mas também funcionalmente robusta células T responder. Aqui nós avaliamos a morte de células específicas de dois por cento peptídeo clones específicos de células T em camundongos BALB / c.

Watch this Scientific Journal Video about Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells at JoVE.com

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Muitas infecções provocar uma forte resposta CTL, mas, ocasionalmente, a quantidade de células que respondem não se relaciona com o controle do patógeno<sup&gt; 1</sup&gt;. Uma medida da qualidade do CTL é a sua capacidade de matar especificamente<sup&gt; 2</sup&gt;. Rotulagem CFSE de células-alvo pode ser usado para investigar esta qualidade de resposta CTL<em&gt; In vivo</em<sup&gt; 3,4</sup&gt;.

Specific Antigen In Vivo Ensaio Killing usando células CFSE rotuladas Alvo

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

antigen-specific-in-vivo-killing-assay-using-cfse-labeled-target-cells

Research

JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

19.5K visualizações.  University of Wisconsin-Madison. Muitas infecções provocar uma forte resposta CTL, mas, ocasionalmente, a quantidade de células que respondem não se relaciona com o controle do patógeno 1. Uma medida da qualidade do CTL é a sua capacidade de matar especificamente 2. Rotulagem CFSE de células-alvo pode ser usado para investigar esta qualidade de resposta CTL In vivo

Vídeo: Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Trans-vivo Atraso Tipo Ensaio Hipersensibilidade para o Antigen Regulamento Específico

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Imunologia e Infecção

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 ...

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 ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Detecção e enriquecimento da Rare células B antígeno-específica para a análise do fenótipo e função

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Na Vivo Ensaio para detecção de células T antígeno-específicas função citolítica usando um modelo de vacinação

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Geração dos anticorpos monoclonais humanos discriminativa de rara B antígeno-específicas células circulantes no sangue

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Um ensaio de Microarray da proteína para determinação sorológica do antígeno-específicas anticorpo respostas seguintes Clostridium difficile infecção

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Ensaio de matar Opsonophagocytic para avaliar as respostas imunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

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.

Quantificação de células T antígeno-específicas humanas proliferating de CD4+ usando o éster do Succinimidyl do carboxyfluorescein

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

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Geração de alta eficiência de células T Cytotoxic do rato preliminar antígeno-específico para o teste funcional em um modelo auto-imune do diabetes

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...