Name: rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development
Uploaded: 2018-06-19T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Estamos em dívida com nossos assistentes técnicos: U. Bröder e H. Shkarlet, que nos ajudou durante procedimentos experimentais. Este trabalho foi parcialmente financiado por subvenções e da UE (contrato n º 601738, UniVax e TRANSVAC2, contraem n º 730964), uma subvenção de Helmholtz-gemeinschaft (HAI-IDR). As fontes de financiamento não influenciou a pesquisa concepção, geração do manuscrito ou decisão de enviá-lo para publicação.

Todos os mouses utilizados neste estudo foram do fundo C57BL/6. Todos os animais foram mantidos em condições isentos de organismos patogénicos. Todos os experimentos foram realizados de acordo com a normativa da lei alemã de proteção animal (TierSchG BGBl. EU S 1105; 25.05.1998) e foram aprovados pela Comissão Baixa Saxônia a ética de experimentos do Animal e do escritório de estado (Baixa Saxônia estado escritório de defesa do consumidor e da segurança alimentar), sob o número de autorização de 33,4-42502-04-13/1281 e 162280.
1. CFSE coloração de OT-eu T células e transferência adoptiva
Nota: OT-os ratos são animais transgenicamente gerados que expressam um receptor de células T (TCR) com α fixo e cadeias β que juntos reconhecem o peptídeo imuno-dominante de óvulos, SIINFEKL10,11. Como resultado, estes ratos têm um número consideravelmente elevado de SIINFEKL específicos CD8+ T células (97%)12 quando comparado com ratos normais ou óvulos vacinados (≤ 1%)13.
<ol><li>Isolamento de CD8 rastreável+ T células de OT-eu camundongos expressando o alelo específico dos linfócitos T Thy1um (Thy1.1):<ol>
<li>Eutanásia em OT 6-9 semanas de idade-eu ratos por inalação de CO2 , seguido por deslocamento cervical14. Dissecar o baço e linfonodos principais (pares inguinais, axilares e cervicais), cortando a pele com uma tesoura cirúrgica, destacando a pele do corpo com a ajuda do alicate cirúrgico e pinça e colocá-los em placas de Petri (15 x 60 mm) cada um contendo um 100 µm poro malha cup para libertação de moagem e células de tecidos.</li>
		<li>Manter os pratos de petri (cada um com uma malha Copa) em 3-5 mL de meio RPMI completo (RPMI 1640, 10% v/v FCS, 100 U/mL penicilina, estreptomicina 50 µ g/mL) mantido no gelo.</li>
		<li>Triture os órgãos com a ajuda de um êmbolo da seringa ou um instrumento similar de estéril (corte prévia não é necessária) e recolher a suspensão única célula resultante em tubos de centrífuga de 15 mL.</li>
		<li>Centrifugar a suspensão de eritrócitos a 300 x g durante 10 minutos a 4 ° C. Desprezar o sobrenadante. Lavar as células em 10 mL de PBS frio por centrifugação e lisar as hemácias do baço, re-suspendendo a pelota em 1 mL/baço do cloreto de amônio buffer (buffer de ACK, comercialmente disponível). Incube as celulas por 1,5 min no gelo e posteriormente lavar as células com 10 mL de PBS frio por centrifugação (como feito antes).</li>
		<li>Ressuspender o sedimento no mesmo tubo em 1 mL de PBS (pH 7,2) contendo 5% fetal soro bovino para isolamento magnético.</li>
	</ol>
</li>
	<li>Para isolamento magnético, proceder à seleção negativa de CD8+ T células usando um kit de isolamento magnético de acordo com as instruções do fabricante ou publicado protocolos15.</li>
	<li>Para executar o CFSE coloração, primeiro contar o número de CD8+ T células obtidas a partir de OT-eu ratos usando um contador de célula automatizada (contador de partículas). Mancha de 1-5 x 107 células/mL em um volume de 1-5 mL com 5 µM CFSE em PBS por 7 min a 37 ° C, protegido da luz em um tubo falcon de 15 ml.</li>
	<li>Saciar a coloração CFSE adicionando o mesmo volume (1:1) de soro fetal bovino para as células e incube-os adicional 7 min a 37 ° C, protegido da luz. As células com 10 mL de PBS lave duas vezes.</li>
	<li>Conte as células usando um contador automatizado de células. Defina o número de células com 3-5 x 107 células/mL de PBS para injetar o 3-5 x 106 células/rato em 100 µ l por via intravenosa (IV) através da veia da cauda.</li>
	<li>Para executar a cauda veia injeções, imobilize os ratos em protecções adequadas. Aquecer a área traseira e a cauda dos ratos deve ser injetado por meio de uma lâmpada de luz vermelha, colocada entre 20 a 25 cm.... os ratos para 1-3 min permitir que a cauda veia vasodilatação. Nota: Isso ajuda a deteção de veia de facilidade e administração da suspensão celular.</li>
	<li>Certifique-se (com a mão colocada entre os ratos e a lâmpada) que não é demasiado quente para os ratos e quando as veias da cauda são claramente visíveis, dirijam-se à injeção. Injete as células na veia de cauda lateral ou dorsal utilizando uma seringa de 1mL com uma agulha de calibre 2516.</li>
</ol>2. imunização (Endo-free OVA + adjuvante)
<ol><li>Coloque os ratos transplantados com OT-eu células (etapa 1.7, Figura 1) em uma câmara de anestesia e administrar isoflurano em oxigênio por uma máquina de anestesia14.</li>
	<li>Quando o mouse está dormindo sob anestesia, tirá-lo da câmara e raspar o pelo do mouse sobre a área sobre o glúteo superficial (laterais, parte inferior das costas), usando uma máquina de corte de cabelo elétrico, a fim de realizar uma injeção limpa com uma boa visão da área de aplicação.</li>
	<li>Injete 50 µ l da vacina, s.c. na área depilada, usando uma agulha de calibre 25.</li>
</ol>3. isolamento de linfócitos e mancha para análise de citometria de fluxo
<ol><li>Isolamento de linfócitos e splenocytes<ol>
<li>Abater os ratos vacinados por inalação de CO2 seguida por deslocamento cervical.</li>
		<li>Extraia o distantes e drenagem dos linfonodos e baço e colocá-los em separado placas de Petri contendo 100 µm poro malha copos para libertação de moagem e células de tecidos. Siga os passos 1.1.2 através de 1.1.3.</li>
		<li>Decante o sobrenadante após centrifugação e ressuspender as pelotas de célula no mesmo volume de remanescente PBS (aprox. 100 µ l).</li>
	</ol>
</li>
	<li>Coloração para análise de citometria de fluxo<ol>
<li>Num tubo de centrífuga de 15 mL prepare uma mistura de mestre que contém os anticorpos coloração nas concentrações descritos na tabela 1, assim, produzindo uma mistura concentrada de coloração de 2x. Prepare-se volume suficiente do mestre mix ter 100 µ l por amostra. Misture as células e a mestre mistura 1:1 e incubar é a 4° C por 30 min. Nota: O volume final de coloração por exemplo deve ser 200 µ l (100 µ l de célula pelota + 100 µ l do mix de mestre de anticorpo).</li>
		<li>Lave as células duas vezes por centrifugação, conforme descrito em 1.1.3, adicionar 10 mL de PBS, duas vezes.</li>
		<li>Ressuspender as células coradas em 0,5-1 mL de PBS para a aquisição e a suspensão de transferência para tubos de citômetro de fluxo. Sempre manter as amostras no gelo e protegido da luz.</li>
	</ol>
</li>
</ol>4. fluxo Cytometry
<ol><li>Sempre pré-filtre as amostras de células usando filtros de 70-100 µm.</li>
	<li>Prepare a coloração única compensação controles para o fluorophores detalhadas na tabela 1 (grânulos ou células). Nota: Compensação deve ser executada no citômetro ou análise software17,18,19 e aplicada a todas as amostras.</li>
	<li>Siga a estratégia associada representada na Figura 2. Brevemente, populações de portão na altura de avançar dispersão vs área de dispersão para a frente (Figura 2A) e novamente dispersão lateral ampla vs área de dispersão lateral (SSA, Figura 2B) a fim de excluir parelhas.</li>
	<li>Portão a população de Figura 2B plotando BV 650 (autofluorescência) vs FITC (CFSE) a fim de discriminar o verdadeiro CFSE manchado células de altas autofluorescente de células. Incluem grânulos ou células coradas com anticorpos conjugados a BV 650 em controles de compensação. Este enredo (Figura 2), da BV 650 vs FITC é usado para portão OT-eu CFSE manchado células.</li>
	<li>Portão da população de C da Figura 2 para a Pe-Cy7 (Thy1.1 - CD90.1-) vs APC (CD8) a fim de distinguir células derivadas de doador vs ratos destinatários. Nota: Células derivadas de OT-eu ratos doadores são Thy1.1+ (CD90.1), (WT C57BL/6, destinatários) derivado de ratos células são Thy1.2+ (CD90.2) (Figura 2D). Alguns laboratórios têm OT-eu ratos em um fundo de Thy1.2 e WT (C57BL/6) em um Thy1.1. Neste caso você tem que usar um anticorpo contra Thy1.2 para OT-eu celular gating.</li>
	<li>Re-portão CD8+ células de Thy1.1+ população plotando-a em um portão constituído por 450 BV (CD4) vs APC (CD8) (Figura 2E).</li>
	<li>Exibir a população fechada na Figura 2E usando um display de histograma do CD8+ células mostrando CFSE (FITC, canal 530/15 - laser azul-), portão a população proliferada, incluindo intensidades de 102 até o nível onde populações a indivisa de controle são (intensidades 10 ~5, dependendo a eficácia do CFSE mancha e a configuração da tensão com o citômetro de fluxo) (Figura 2F).</li>
	<li>Que uma porta adicional (não mostrado na Figura 2) plotagem FITC vs L/D marcador (450/20 canal, laser UV) a fim de avaliar as células mortas, em paralelo à avaliação de proliferação.</li>
	<li>Adquira pelo menos 5000 eventos para os controles de compensação e 10,000 eventos (portão E, Figura 2) das amostras em um citômetro de fluxo. Executar o citômetro de fluxo médio ou baixo (não excedam as taxas de fluxo de 20.000 eventos/s).</li>
</ol>

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Vacinas modernas são idealmente composedof antígeno purificado e adjuvantes, com a possível adição de um sistema de entrega como lipossomas, vírus-como partículas, nanopartículas ou vetores ao vivo. Um aspecto fundamental durante a criação de uma vacina é escolher o certo adjuvante de acordo com as necessidades clínicas. Parte do escopo poderia envolver favorecendo um humoral vs. resposta imune celular (ou ambos), a eleição de um local contra uma resposta imune sistêmica (ou ambos) e o tipo de memória que...

Linfócitos T citotóxicos (CTL) induzindo vacinas são principais intervenções terapêuticas que foram desenvolvidas para lutar contra certos tipos de câncer1. CTL são também importantes para vacinas profiláticas contra patógenos intracelulares2. Além disso, o CTL são dentre os mecanismos de defesa imune alguns funcionalmente ativos em populações de risco tais como neonatos3,4 , quem também dependem de CTL para combate de infecções início vida5. A este respeito, as vacinas contra o vírus sincicial respiratório (VSR) que foram desenvolvidas com um adjuvante que não eliciam respostas CTL (alume) resultaram em um completo fracasso da vacina levando a sérias complicações com infecções em lactentes de6. Estes efeitos negativos da vacinação podem ser revertidos por um CD8+ de resposta de células T7. Nós demonstramos anteriormente que as principais citocinas (interferons do tipo I) eliciadas algum estimulador de agonistas de genes (picada) de interferon são essenciais para as respostas CTL geradas por estes adjuvantes8, em parte, medindo-se a proliferação de OT-I T pilhas após vacinação e usando estes resultados como uma medida de CTL induzindo capacidades observadas em estendem de horários de vacinação9. A medição da proliferação de OT-eu CD8+ T em um rato de destinatário tipo selvagem (WT) C57BL/6 células por carboxyfluorescein grufo tintura de éster (CFSE) diluição é uma robusta estimativa da capacidade do adjuvante de uma vacina para gerar Cruz-ferragem de SIINFEKL, (o peptídeo imuno-dominante da ovalbumina, óvulos). Variações desta técnica são amplamente utilizadas para a avaliação da proliferação de OT-eu CD8+ e OT-II CD4+ T células. Por exemplo, ela tem sido usada na ausência de citocinas selecionadas (ratos KO) ou para medir a eficácia da vacina após retirada de antigénios em animais WT. Criámos um protocolo curto (4 dias experimento) em que após a transferência passiva de OT CFSE-manchado-eu CD8+ T cells, uma imunização (s.c.) subcutânea, consistindo de uma dose de 50 µ g de OVA livre de endotoxinas suplementado com adjuvantes de teste é administrado (Figura 1). O acompanhamento dos resultados 48 h após a vacinação fornece uma confiável prova da capacidade do adjuvante para gerar respostas CTL. Por esta estratégia, é possível avaliar a potência da resposta imune local no nó de linfa drena após a imunização, bem como a extensão da resposta, medindo-se a atividade CTL no baço (ou distante dos gânglios linfáticos).

<table><tbody><tr><td>BD LSR Fortessa Cell Analyzer</td><td>BD</td><td>Special Order</td><td>Flow Cytometer</td></tr><tr><td>CFSE</td><td>Molecular Probes</td><td>C34554</td><td>Proliferation Dye</td></tr><tr><td>MojoSort Mouse CD8 T Cell Isolation Kit</td><td>Biolegend</td><td>480007</td><td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td></tr><tr><td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td><td>Molecular Probes</td><td>L23105</td><td>Dead Cell Marker</td></tr><tr><td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td><td>eBioscience</td><td>25-0900-82</td><td>antibody</td></tr><tr><td>APC anti-mouse CD8a Antibody</td><td>BioLegend</td><td>100712</td><td>antibody</td></tr><tr><td>BV421 Rat Anti-Mouse CD4</td><td>BD</td><td>740007</td><td>antibody</td></tr><tr><td>Z2 coulter Particle count and Size Analyzer</td><td>Beckman Coulter</td><td>9914591DA</td><td>Cell counter. Z2 Automated particle/cell counter</td></tr><tr><td>EndoGrade Ovalbumin (10 mg)</td><td>Hyglos(Germany)</td><td>321000</td><td>Ovalbumin endotoxin free tested.</td></tr><tr><td>Cell Strainer 100&amp;micro;m nylon</td><td>Corning</td><td>352360</td><td>Cell strainer (100 &amp;micro;m pore mesh cups).</td></tr><tr><td>Sample Vials</td><td>Beckman Coulter</td><td>899366014</td><td>Sample vials for Z2 automated counter</td></tr><tr><td>C57BL/6 mice (CD90.2)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Company is now Envigo</td></tr><tr><td>OT-I (C57BL/6 background, CD90.1)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Inbreed at our animal facility. Company from where adquired is now Envigo</td></tr><tr><td>FACS tubes</td><td>Fischer (Corning)</td><td>14-959-5</td><td>Corning Falcon Round-Bottom Polystyrene Tubes</td></tr><tr><td>Falcon 15 mL tubes</td><td>Fischer (Corning)</td><td>05-527-90</td><td>Falcon 15mL Conical Centrifuge Tubes</td></tr><tr><td>PBS (500 mL)</td><td>Fischer (Gibco)</td><td>20-012-027</td><td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td></tr><tr><td>Red lamp (heating lamp)</td><td>Dirk Rossmann GmbH (Germany)</td><td>405096</td><td>Heating infrred lamp (100 wats)</td></tr><tr><td>IsoFlo (Isoflurane)</td><td>Abbott Laboratories (USA)</td><td>5260.04-05.</td><td>Isoflurane anesthesic (250 mL flask).</td></tr><tr><td>&lt;strong&gt;Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber&lt;/strong&gt;</td><td>Parkland Scientific</td><td>V3000PK</td><td>Isoflurane anesthesia machine.</td></tr><tr><td>RPMI 1640 medium</td><td>Gibco (distributed by ThermoFischer)</td><td>11-875-093</td><td>Base medium with Glutamine (500 mL)</td></tr><tr><td>Pen-Strept antibiotic solution (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>15-140-148</td><td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td></tr><tr><td>Fetal Bobine Serum (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>10082147</td><td>Fetal Bovine Serum, certified, heat inactivated, US origin</td></tr><tr><td>ACK Lysing Buffer (100 ml)</td><td>Gibco (distributed by ThermoFischer)</td><td>A1049201</td><td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td></tr><tr><td>Plastic Petri Dishes</td><td>Nunc (distributed by ThermoFischer)</td><td>150340</td><td>60 x 15mm Plastic Petri Dish, Non-treated</td></tr><tr><td>Cell Clump Filter</td><td>CellTrics (Sysmex)</td><td>04-004-2317</td><td>CellTrics&amp;reg; 50 &amp;mu;m, sterile</td></tr></tbody></table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

A avaliação de vacinas de subunidade moderno revela que a geração de anticorpos neutralizantes é importante mas não suficiente para a seleção de adjuvante. Portanto, adjuvantes com capacidades imuno-estimulatórios humorais e celulares que são capazes de promover respostas de linfócitos (CTL) T citotóxicas são urgentemente necessárias. Assim, fiel, monitoramento de adjuvante que induzir Cruz-ferragem e posteriormente aumentar a geração de CTL representa um passo crucial no desenvolvimento de vacinas. Aqui apresentamos um pedido de um método que usa SIINFEKL específicos (OT-eu) células T para monitorar a Cruz-apresentação do modelo do antígeno ovalbumina (OVA) na vivo na presença de candidatos de diferentes adjuvantes. Este método representa um teste rápido para selecionar adjuvantes com os melhores recursos de cruz-ferragem. A proliferação de CD8+ T células é a indicação mais valiosa da Cruz-ferragem e também é considerado como um correlato de cruz-apresentação induzida por adjuvante. Esta característica pode ser avaliada em diferentes órgãos imunes como linfonodos e baço. A extensão da geração CTL também pode ser monitorizada, conferindo-lhe insights sobre a natureza de um local (drenagem do linfonodo principalmente) ou uma resposta sistêmica (distante dos gânglios linfáticos e/ou baço). Ainda mais esta técnica permite várias modificações para testes de drogas que podem inibir a percursos específicos da Cruz-apresentação e também oferece a possibilidade de ser usado em diferentes cepas de ratos geneticamente modificados e convencionais. Em resumo, o aplicativo que apresentamos aqui será útil para laboratórios de vacina em indústria ou do meio académico que desenvolvem ou modificar adjuvantes químicos para pesquisa de vacina e desenvolvimento.

Para testar os tratamentos utilizando uma combinação diferente de adjuvantes (ADJ1 e ADJ2), avaliamos a capacidade de geração de CTL medindo-se a proliferação de OT enviaram transferido-eu CD8+ T células por citometria de fluxo (Figura 2). Por isso, nós anteriormente manchadas de células isoladas da drenagem dos gânglios linfáticos e baço (tabela 1). Medindo-se a proliferação de CD8+ T células nos linfonod...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Apresentamos aqui um pedido de uma norma técnica imunológica (CFSE manchado OT-eu proliferação) destinados a monitorar rapidamente mediada por adjuvante T linfócitos citotóxicos (CTL) geração in vivo. Esta estimativa rápida das capacidades CTL é útil para o desenvolvimento de vacinas profiláticas contra patógenos intracelulares, bem como as vacinas terapêuticas do câncer.

Rápida na Vivo avaliação dos recursos de geração de linfócitos de T citotóxicos do adjuvante para desenvolvimento de vacinas

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

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Research

JoVE Journal

Medicine

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

8.6K visualizações.  Helmholtz Centre for Infection Research. Apresentamos aqui um pedido de uma norma técnica imunológica (CFSE manchado OT-eu proliferação) destinados a monitorar rapidamente mediada por adjuvante T linfócitos citotóxicos (CTL) geração in vivo. Esta estimativa rápida das capacidades CTL é útil para o desenvolvimento de vacinas profiláticas contra patógenos intracelulares, bem como as vacinas terapêuticas do câncer.

Vídeo: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

In Vitro Ensaio para avaliar o impacto da imunorreguladora Pathways on específica para o HIV CD4 T efetoras celular Função

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Imunologia e Infecção

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

O Uso de fluorescentes alvo Matrizes para a Avaliação de respostas de células T<em&gt; In vivo</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Imaginologia todo animal e técnicas de citometria de fluxo para CD8 + respostas de células T Análise do antigénio específico da Nanoparticle após vacinação

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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 ...

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

Immunostimulatory agente avaliação: Linfoide em injeção e extração de tecido ativação rota dependente de célula dendrítica

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Adaptando os ensaios de citotoxicidade in vivo para estudar Imunodominância em respostas de células T CD8+ específicas do tumor

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Preparação, características, toxicidade e avaliação da eficácia da vacina contra tumores em nanoemulsão automontada nasal in vitro e in vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

In vitro Avaliação da Atividade Celular da Vacina de Nanoemulsão Adjuvante Ofiopogonina D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Determinação da imunogenicidade vacinal utilizando células dendríticas derivadas de monócitos bovinos

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Source: Lirussi, D. et al., <a href="https://app.jove.com/v/57401">Rapid In Vivo&#160;Assessment of Adjuvant&#39;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.</a> J. Vis. Exp. (2018)
The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

Uma técnica imunológica para monitorar a geração de linfócitos T citotóxicos mediados por adjuvantes

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

Rápida na Vivo avaliação dos recursos de geração de linfócitos de T citotóxicos do adjuvante para desenvolvimento de vacinas

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