Este trabalho foi apoiado por subsídios do projeto # 1010395 (BQ e PB) e # 525431 (CR), e um Programa de Grant # 455395 (CP) do Conselho Nacional de Saúde e Pesquisa Médica da Austrália, um Centro Australiano para Hepatite e HIV Virologia EOI 2012 bolsa (CR e RJJ) e uma bolsa da Fundação Bootes Gordon and Gretel (BQ e CR). Queremos agradecer Harpreet Vohra e Michael Devoy por sua excelente manutenção do laboratório JCSMR FACS, a Facilidade Australian Cancer Research Foundation Biomolecular Resource, JCSMR, ANU, para síntese de peptídeos, e Dr. David Boyle, CSIRO Animais Laboratórios de Saúde, Geelong, Austrália por fornecer os estoques de vacinas HIV pais.

<ol>
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Os autores declaram que não têm interesses financeiros concorrentes.

A vantagem de ensaios baseados em TLC é que eles permitem a discriminação de&gt; 250 populações de células alvo viável e completamente funcional a partir de um único animal hospedeiro por citometria de fluxo. Isso proporciona um nível de complexidade para in vivo fluem ensaios baseados citometria de que não foi possível antes. Isto é realçado nas duas experiências com animais mostradas acima, em que as respostas aos epítopos virais 7 distintas em 6 concentrações poderiam ser monitorizadas em re...

As células T desempenham um papel central na resposta imune adaptativa e são frequentemente alvo de manipulação em imunoterapia. As células CD4 + T efectoras responder ao antigénio estranho pela secreção de citocinas que regulam muitos aspectos da imunidade e também podem ajudar directamente as células B para fabricar anticorpos. As células T citotóxicas CD8 + (CTLs) podem também responder ao antigénio estranho pela secreção de citocinas, bem como desempenha um papel central em matar directamente as células que expressam um antigénio estranho. A interacção fundamental que inicia estas funções efectoras de células T envolve a interacção do receptor de células T (TCR) com peptídeos estranhos apresentados em moléculas de MHC na superfície das células. As células CD4 + T reconhecem péptidos apresentados em moléculas de MHC de classe II nas células que apresentam os antigénios e células T CD8 + reconhecem péptidos apresentados em moléculas de MHC de classe I que são normalmente apresentadas em células microbianas infectado. Em ordempara avaliar o papel de células T desempenham na resposta imune, é essencial que as suas funções efectoras são medidos por técnicas fiáveis ​​e sensíveis. Os métodos comuns de avaliação de resposta das células T incluem; MHC class-I/II/peptide tetramer reatividade; produção de citocinas por ELISPOT e coloração de citocinas intracelulares; e matando a capacidade em 51 ensaios de Cr-lançamento. Estes ensaios, no entanto, são tipicamente realizados ex vivo com estimulação in vitro, ou fornecer uma visão limitada em função das células T. Idealmente, ao medir as respostas das células T, seria benéfico para os avaliar in situ, in vivo à medida que ocorrem, sem a manipulação de células T, de modo a evitar alterações nos parâmetros funcionais que podem ocorrer através de estimulação in vitro. Alguns dos mais vulgarmente usados ​​em células T ensaios funcionais in vivo são baseados na medição de morte por CTL mediada de células alvo pulsadas com péptidos do MHC de classe I de ligação, que são enumerados in vivovia sua detecção através de marcação fluorescente com corantes vitais como CFSE. Embora estes tipos de ensaios podem monitorar morte mediada por CTL de metas quando acontecem in vivo, eles já tinham uma capacidade relativamente limitada para avaliar a morte de múltiplos alvos que apresentam diferentes concentrações e tipos diferentes de epítopos peptídicos, que é necessário para permitir que os parâmetros qualitativos, tais avidez tão funcional e variante epítopo reatividade cruzada a ser avaliado. Estes ensaios também não fornece nenhuma informação sobre as respostas mediadas por células T CD4 +. Para superar muitas das limitações com os métodos atuais usados ​​para avaliar as respostas das células T, que recentemente desenvolveu um ensaio multiplex com base em matrizes fluorescentes alvo (ACL), que permite o monitoramento de respostas de células T contra&gt; 250 células alvo simultaneamente em um animal por citometria de fluxo 1, 2. FTAs são compostas de linfócitos marcados com SEVeral concentrações e combinações de corantes vitais como CFSE, CTV e CPD permitindo&gt; 250 grupos de células de fluorescência único a ser gerado. Uma vez que estas células permanecem viáveis ​​e completamente funcional, que pode ser injectado no animal para permitir a monitorização da sua interacção com células T efectoras in vivo 3. Por exemplo, os agregados de células FTA podem ser pulsadas com péptidos do MHC-I de ligação à classe para permitir a avaliação de morte por CTL específica de antigénio mediada por células-alvo 1. Além disso, os agregados de células FTA também podem ser pulsadas com péptidos de MHC de classe II de ligação, permitindo a avaliação do antigénio específico para a célula T auxiliar (T H), avaliando a actividade de activação (por avaliação de marcadores de activação, como CD69, CD44 e / ou CD62L) de células B dentro do FTA rolamento peptídeo cognato 2. Uma vez que mais de 250 alvos pode ser detectado em simultâneo, é possível medir as respostas de CTL e de T H contra muitos grupos de células alvo pulsadas com npeptídeos umerous em diferentes concentrações ea inclusão de muitas repetições. Por isso, o ensaio FTA fornece um nível sem precedentes de avaliação da resposta das células T efetoras in vivo. Aqui, descrevemos em pormenor a construção de um FTA e mostram como podem ser aplicados para avaliar as respostas de células T in vivo. O processo descreve a construção de um FTA composta de 252 agrupamentos de células discerníveis por meio da utilização de três corantes vitais, composta de seis repetições de 42 conjuntos de células pulsadas com o MHC de classe I e II, os péptidos de ligação. A rotulagem de 42 conjuntos de células ocorre em 10 ml de fundo cónico de tubos e que é útil para colocar estes em um suporte para tubos, conforme ilustrado na Tabela 1. Esse método pode ser ajustado para um número menor de aglomerados discerníveis como requerido ao reduzir a quantidade de rotulagem de cada corante realizada 1. Destaca-se a utilidade do ensaio, mostrando como ele pode medir responses geradas pela vacinação pox-vírus recombinante contra múltiplos epitopos em um pequeno grupo de ratos. Isto mostra como o ensaio de TLC pode ser usado para medir as respostas cumulativas e avidez funcional através de, respectivamente, o uso da área sob a curva (AUC) de avaliações e medições da concentração de péptido eficaz necessária para produzir respostas máximas metade (EC 50).

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			<td height="22" style="height:22px;width:216px;">RPMI</td>
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			<td style="width:119px;">R8758</td>
			<td style="width:167px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Fetal calf serum</td>
			<td style="width:71px;">Serana</td>
			<td style="width:119px;">FCS-500</td>
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			<td height="43" style="height:43px;width:216px;">CTV</td>
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			<td height="43" style="height:43px;width:216px;">CFDA, SE</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C1157</td>
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			<td height="43" style="height:43px;width:216px;">CPD</td>
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			<td style="width:119px;">65-0840-90</td>
			<td style="width:167px;"></td>
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			<td height="43" style="height:43px;width:216px;">anti-B220 PerCp-Cy5.5</td>
			<td style="width:71px;">eBioscience</td>
			<td style="width:119px;">110730</td>
			<td style="width:167px;"></td>
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			<td height="43" style="height:43px;width:216px;">anti-CD69 brilliant violet 605</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">104529</td>
			<td style="width:167px;"></td>
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			<td height="22" style="height:22px;width:216px;">PKH-26</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
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			<td height="22" style="height:22px;">EQUIPMENT:</td>
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			<td height="43" style="height:43px;width:216px;">Material Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td style="width:167px;">Comments (optional)</td>
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			<td height="64" style="height:64px;width:216px;">Vortex</td>
			<td style="width:71px;">Scientific Industries Inc&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Centrifuge</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="64" style="height:64px;width:216px;">Flow cytometer (Fortessa or equivalent with blue, red and violet laser source)</td>
			<td style="width:71px;">BD Bioscience</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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the use of fluorescent target arrays for assessment of t cell responses in vivo

A capacidade de monitorar as respostas das células T in vivo é importante para o desenvolvimento da nossa compreensão da resposta imune e da criação de imunoterapias. Aqui nós descrevemos o uso de tecnologia de matriz alvo fluorescente (FTA), que utiliza corantes vitais, tais como éster carboxifluoresceína succinimidyl (CFSE), corantes excitáveis ​​a laser violeta (violeta CellTrace: CTV) e corantes excitáveis ​​de laser vermelho (celular Proliferação Dye eFluor 670: CPD ) para combinatorialmente linfócitos etiqueta rato em&gt; 250 agrupamentos de células fluorescentes discerníveis. Aglomerados de células dentro dessas ACL pode ser pulsada com histocompatibilidade principal (MHC) de péptidos de ligação de classe I e MHC de classe II e assim actuar como células-alvo para as células CD8 + e células T CD4 +, respectivamente. Estas células FTA permanecer viável e totalmente funcional, e pode, portanto, ser administrado em ratos para permitir a avaliação de CD8 + T mediada por células morte de células alvo FTA e CD4 + T hel meditado-cellp de TLC células B alvo de células em tempo real in vivo, por citometria de fluxo. Desde&gt; 250 células alvo pode ser avaliada de uma só vez, a técnica permite o monitoramento de respostas de células T contra vários epítopos de antígeno em várias concentrações e em múltiplas repetições. Como tal, a técnica pode medir as respostas das células T, tanto a um (por exemplo, a magnitude da resposta cumulativa) quantitativa e um (por exemplo, avidez funcional. Eo epitopo de reactividade cruzada da resposta) qualitativa nível. Aqui, nós descrevemos como esses TLCs são construídos e dar um exemplo de como eles podem ser aplicados para avaliar as respostas de células T induzidas por uma vacina de vírus da varíola recombinante.

Como um exemplo da utilização do ensaio de TLC, um ratinho BALB / c foi imunizado com vírus vaccinia recombinante (VV) expressando epítopos de HIV-I (VV-HIV) e as respostas aos epítopos de CTL de VIH-I, Gag, mut Gag Env e Pol, o VV epitopos CTL F2L e F2L mut, e o epitopo de célula T de HIV-I H, Gag Th (tal como descrito em 2) foram avaliados utilizando um ensaio de TLC parâmetro 252 (Figura 1B). Variantes de epitopos de mordaça (mut Gag) e F2L (F2L mut) não são expressos ...

Watch this Scientific Journal Video about The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo at JoVE.com

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

A capacidade de monitorar as respostas das células T em detalhe, in vivo é importante para o desenvolvimento da nossa compreensão da resposta imune. Aqui nós descrevemos o uso de matrizes alvo fluorescentes (ACL) em um ensaio in vivo de células T em que avalia&gt; 250 parâmetros simultaneamente por citometria de fluxo.

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 ...

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Immunology and Infection

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

14.3K visualizações.  Australian National University.  A capacidade de monitorar as respostas das células T em detalhe, in vivo é importante para o desenvolvimento da nossa compreensão da resposta imune. Aqui nós descrevemos o uso de matrizes alvo fluorescentes (ACL) em um ensaio in vivo de células T em que avalia> 250 parâmetros simultaneamente por citometria de fluxo. 

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transdução retroviral de receptor de células T no rato células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Imunologia e Infecção

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

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

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

Preparação e Uso de HIV-1 infectados CD4 + T-Primária células como as células-alvo em Ensaios Natural Killer Celular citotóxicos

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

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Indução de enxerto-versus-hospedeiro de Doenças e<em&gt; Em Vivo</em&gt; Monitoramento celular T Usando um modelo murino de MHC de correspondência

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

In Vitro e In Vivo de avaliação da atividade supressiva, T, B e células mieloides e respostas Humoral de pacientes transplantados

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

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 ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento de Salmonella typhimurium-contendo Phagosomes de macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Um protocolo de aspiração da bolha para estudar células T humanas Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Avaliação das respostas de células T Zika vírus específicos em órgãos Immunoprivileged de infectados Ifnar1- / - ratos

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

Uso da tecnologia de MHC única cadeia para investigar Co-Genealogia na ativação de células T humanas CD8 +

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...