Este trabajo fue apoyado por becas del proyecto # 1010395 (BQ y CP) y # 525431 (CR), y un Programa de Subsidios # 455395 (CP) de la Salud y medicina Consejo de Investigación Nacional de Australia, el Centro Australiano para la hepatitis y el VIH Virología EOI 2012 de subvención (CR y JJR) y una beca de la Fundación Gordon Bootes y Gretel (BQ y CR). Queremos agradecer Harpreet Vohra y Michael Devoy por su excelente mantenimiento del laboratorio JCSMR FACS, el Fondo para la Investigación del Cáncer de Australia Fundación de Recursos Biomolecular, JCSMR, ANU, para la síntesis de péptidos, y el Dr. David Boyle, CSIRO animales Laboratorios de Salud, Geelong, Australia para proporcionar a las poblaciones parentales vacunas contra el VIH.

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+VITAL+assay:+a+versatile+fluorometric+technique+for+assessing+CTL-+and+NKT-mediated+cytotoxicity+against+multiple+targets+in+vitro+and+in+vivo.">The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo.</a> J Immunol Methods. 285, 25-40 (2004).</li><li>Busch, D. H., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+affinity+maturation+by+selective+expansion+during+infection.">T cell affinity maturation by selective expansion during infection.</a> J Exp Med. 189, 701-710 (1999).</li><li>Savage, P. A., Boniface, J. J., Davis, M. 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R., Kongsbak, M., Geisler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+behind+functional+avidity+maturation+in+T+cells.">Mechanisms behind functional avidity maturation in T cells.</a> Clin Dev Immunol. , (2012).</li><li>Prussin, C., Metcalfe, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intracytoplasmic+cytokine+using+flow+cytometry+and+directly+conjugated+anti-cytokine+antibodies.">Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies.</a> J Immunol Methods. 188, 117-128 (1995).</li><li>Foster, B., Prussin, C., Liu, F., Whitmire, J. K., Whitton, J. 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Los autores declaran que no tienen intereses financieros en competencia.

La ventaja de ensayos basados ​​en TLC es que permiten la discriminación de&gt; 250 poblaciones de células objetivo viable y completamente funcional de un solo animal huésped por citometría de flujo. Esto proporciona un nivel de complejidad a fluir ensayos in vivo basados ​​en citometría de que no ha sido posible antes. Esto se pone de relieve en los 2 experimentos con animales muestran arriba, donde las respuestas a 7 epítopos virales distintas a 6 concentraciones podrían ser monitoread...

Células T juegan un papel central en la respuesta inmune adaptativa y, a menudo son objeto de manipulación en la inmunoterapia. Las células T efectoras CD4 + responden al antígeno extraño secretando citoquinas que regulan muchos aspectos de la inmunidad y también puede ayudar directamente a las células B para la fabricación de anticuerpos. Células T citotóxicas CD8 + (CTL) también pueden responder a antígeno extraño mediante la secreción de citoquinas así como jugando un papel central en matar directamente las células que expresan un antígeno extraño. La interacción fundamental que inicia estas funciones efectoras de células T implica la interacción del receptor de células T (TCR) con péptidos extraños que aparecen en las moléculas MHC sobre la superficie de las células. Células T CD4 + reconocen péptidos presentados en las moléculas de MHC de clase II en las células presentadoras de antígeno y células T CD8 + reconocen péptidos presentados en las moléculas de MHC de clase I que típicamente se muestran en las células infectadas por microbios. En ordenpara evaluar el papel de las células T juegan en una respuesta inmune, es esencial que sus funciones efectoras se miden por técnicas fiables y sensibles. Los métodos comunes para la evaluación de la respuesta de células T incluyen; MHC class-I/II/peptide reactividad tetrámero; producción de citocinas por ELISPOT y tinción intracelular de citoquinas; y matando a la capacidad por 51 ensayos de Cr-liberación. Estos ensayos, sin embargo, se llevan a cabo típicamente ex vivo con la estimulación in vitro, o proporcionan información limitada en función de células T. Idealmente, cuando la medición de respuestas de células T sería beneficioso para evaluar in situ, in vivo como se producen, sin la manipulación de las células T a fin de evitar cambios en los parámetros funcionales que se pueden producir a través de la estimulación in vitro. Algunas de las más utilizadas en vivo T ensayos funcionales de células se basan en la medición de la matanza CTL mediada por células diana pulsadas con péptidos MHC de clase I-vinculantes, que se enumeran en vivoa través de su detección mediante marcaje fluorescente con colorantes vitales como la CFSE. Si bien estos tipos de ensayos pueden supervisar la matanza CTL mediada de objetivos cuando ocurren in vivo, han tenido previamente una capacidad relativamente limitada para evaluar la matanza de blancos múltiples que presentan diferentes concentraciones y diferentes tipos de epítopos peptídicos, que se requiere para permitir a los parámetros cualitativos, avidez como funcional y variante epítopo reactividad cruzada que deben evaluarse. Estos ensayos también no proporcionan ninguna información sobre las respuestas mediadas por células T CD4 +. Para superar muchas de las limitaciones con los métodos actuales utilizados para evaluar las respuestas de células T, hemos desarrollado recientemente un ensayo múltiple basado en matrices fluorescentes objetivo (TLC), lo que permite el seguimiento de las respuestas de células T contra&gt; 250 células diana simultáneamente en un animal por la citometría de flujo 1, 2. TLC se componen de linfocitos marcados con sevrales concentraciones y combinaciones de colorantes vitales como la CFSE, CTV y CPD permitiendo&gt; 250 grupos de células de fluorescencia único que se generen. Puesto que estas células permanecen viables y completamente funcional, que pueden ser inyectados en los animales para permitir la monitorización de su interacción con células T efectoras in vivo 3. Por ejemplo, los grupos de células del TLC pueden ser pulsadas con péptidos I-vinculante-MHC de clase para permitir la evaluación de matar CTL mediada por antígeno específico de células diana 1. Además, los grupos de células TLC también pueden ser pulsadas con péptidos del MHC de clase II-de unión, lo que permite la evaluación de antígeno específico de células T auxiliares (T H) mediante la evaluación de la actividad de activación (por evaluación de marcadores de activación tales como CD69, CD44 y / o CD62L) de las células B en el TLC que llevan péptido afín 2. Desde hace más de 250 objetivos se pueden detectar de forma simultánea, es posible medir las respuestas de CTL y T H contra muchos grupos de células diana pulsadas con npéptidos umerosos a diferentes concentraciones y la inclusión de muchas repeticiones. Por tanto, el ensayo de TLC proporciona un nivel sin precedentes de la evaluación de la respuesta T efectoras de células in vivo. A continuación se describe en detalle la construcción de un acuerdo de libre comercio y mostrar la forma en que se pueden aplicar a la evaluación de las respuestas de células T in vivo. El procedimiento describe la construcción de un TLC compuesta de 252 grupos de células discernibles a través de la utilización de tres colorantes vitales, compuestos de 6 repeticiones de 42 grupos de células pulsadas con MHC de clase I y II de unión a péptidos. El etiquetado de los 42 grupos de células se produce en 10 ml de fondo cónico de tubos y que es útil para poner éstos hacia fuera en un bastidor de tubo como se muestra en la Tabla 1. Este método se puede ajustar para un número menor de grupos discernibles como es requerido por la reducción de la cantidad de etiquetado de cada colorante a cabo 1. Se destaca la utilidad del ensayo, mostrando cómo se puede medir la responsES generados por la vacunación de la viruela de virus recombinante contra múltiples epítopos en una pequeña cohorte de ratones. Esto muestra cómo el ensayo de TLC se puede utilizar para medir las respuestas acumuladas y avidez funcional a través de, respectivamente, el uso de área bajo la curva (AUC) y las evaluaciones de medición de la concentración de péptido eficaz requerida para generar respuestas máximas media (CE 50).

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			<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>
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			<td height="43" style="height:43px;width:216px;">Centrifuge</td>
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the use of fluorescent target arrays for assessment of t cell responses in vivo

La capacidad de monitorear las respuestas de células T in vivo es importante para el desarrollo de nuestra comprensión de la respuesta inmune y el diseño de inmunoterapias. Aquí se describe el uso de la tecnología fluorescente matriz de destino (TLC), que utiliza colorantes vitales como succinimidiléster carboxifluoresceína (CFSE), colorantes excitables láser violeta (violeta CellTrace: CTV) y colorantes excitables láser rojo (proliferación de células tinte eFluor 670: CPD ) para combinatoriamente linfocitos de ratón de etiquetas en&gt; 250 grupos de células fluorescentes discernibles. Grupos de células dentro de estos TLC pueden ser pulsados ​​con mayor de histocompatibilidad (MHC) de péptidos de unión de clase I y MHC de clase II y por lo tanto actúan como células diana para CD8 + y células T CD4 +, respectivamente. Estas células TLC siguen siendo viables y completamente funcional, y por lo tanto se pueden administrar en ratones para permitir la evaluación de CD8 + T muerte celular mediada de células diana TLC y CD4 + T de células HEL-meditadop de las células diana de células B TLC en tiempo real in vivo por citometría de flujo. Desde&gt; 250 células diana pueden evaluarse a la vez, la técnica permite el seguimiento de las respuestas de células T contra varios epítopos de antígeno a varias concentraciones y en múltiples repeticiones. Como tal, la técnica puede medir las respuestas de células T a una cuantitativa (por ejemplo, la magnitud de la respuesta acumulativa) ambos y un (por ejemplo. Avidez funcional y-epítopo Reactividad cruzada de la respuesta) cualitativa nivel. Aquí se describe cómo se construyen estos TLC y dan un ejemplo de cómo se pueden aplicar para evaluar las respuestas de células T inducida por una vacuna de virus de la viruela recombinante.

Como un ejemplo de la utilización del ensayo de TLC, un ratón BALB / c fue inmunizado con el virus vaccinia recombinante (VV) que expresan epítopos del VIH-I (VV-VIH) y las respuestas a los epítopos de CTL VIH-I, de la mordaza, de la mordaza de mut, Env y Pol, el VV CTL epítopos F2L y mut F2L, y el epítopo de células VIH-I T H, Gag Th (como se describe en 2) se evaluó a través de un parámetro de 252 TLC ensayo (Figura 1B). Epítopo variantes de la mordaza (mut GAG) y F2L ...

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

La capacidad de controlar las respuestas de células T en detalle en vivo es importante para el desarrollo de nuestra comprensión de la respuesta inmune. Aquí se describe el uso de conjuntos de blancos fluorescentes (TLC) en un ensayo in vivo de células T en el que evalúa&gt; 250 parámetros simultáneamente por citometría de flujo.

El uso de fluorescentes Arrays objetivo para la evaluación de las respuestas de células T<em&gt; En 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 vistas.  Australian National University.  La capacidad de controlar las respuestas de células T en detalle en vivo es importante para el desarrollo de nuestra comprensión de la respuesta inmune. Aquí se describe el uso de conjuntos de blancos fluorescentes (TLC) en un ensayo in vivo de células T en el que evalúa> 250 parámetros simultáneamente por citometría de flujo. 

Video: 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

Transducción retroviral de receptores de células T en ratones las 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 ...

Investigación

Inmunología e Infección

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.

Preparación y uso de VIH-1 en células CD4 infectadas Primaria + T-células como células diana en las células asesinas naturales ensayos 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.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión 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.

La inducción de la enfermedad de injerto contra huésped y<em&gt; En Vivo</em&gt; T Monitoreo Celular Utilizando un modelo murino MHC de concordancia

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 la evaluación de la actividad supresora T, B y las células mieloides y las respuestas humorales de los receptores de trasplante

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.

En Vivo Ensayo para la detección de la función citolítica de células T específicas de antígeno utilizando un modelo de vacunación

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.

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los 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.

Un protocolo de aspiración de la ampolla para estudiar células de T humanas recordar respuestas en 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.

Evaluación de respuestas de células T Zika Virus-específica en Immunoprivileged órganos de ratones- / - Ifnar1 infectados

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 de la única cadena MHC tecnología investigar Co-agonismo humano CD8 + activación de células T

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