time-lapse imaging to study activated murine cd8+ t-cell migration dynamics

Elucidando a cinética das células T: métodos para análise de migração em tempo real

T cells are the major effectors of the adaptive, antigen-specific immune response. On a population level, T cells are heterogeneous, comprised of cellular subsets with distinct specialized functions. Importantly, CD8+ T cells are the main cytolytic effectors of the immune system, which directly eliminate infected or dysfunctional cells1.
Mature CD8+ T cells reside in tissue and circulate through blood and lymphatics in search of antigens. During infection, T cells are presented with antigens in blood or tissue and quickly drain to the spleen or nearest draining lymph node to begin a productive immune response. In either case, T cells become activated, undergo clonal expansion, and leave the lymphatic system to enter the blood, if not already there. During this process, intracellular signaling confers the downregulation of lymphatic homing receptors and the upregulation of numerous integrin and chemokine receptors essential for tissue-specific migration2. Ultimately, the directed migration of T cells to sites of infection is driven by converging environmental signals that include integrin and chemokine signaling.
Chemokines can be broadly categorized into two main classes: (1) homeostatic signals, which are essential for differentiation, survival, and basal function, and (2) inflammatory signals, such as CXCL9, CXCL10, and CCL3, which are required for chemotaxis. Generally, chemokines create a signal gradient that drives directional migration, known as chemotaxis, in addition to activating integrin expression1. Chemotaxis is finely regulated and highly sensitive, with T cells capable of responding to tiny changes in gradient that can lead them toward a specific direction or location.
In addition to these T cell-related factors, migration is also affected by the extracellular matrix (ECM) composition and density. The ECM is made up of a dense network of proteins, including collagen and proteoglycans, which provide the scaffold for adhesive integrin receptors on T cells. Integrins are a diverse family of transmembrane proteins, each with highly specialized binding domains and downstream signaling effects. Dynamic expression of integrin receptors on the surface of a T cell enables quick adaptation to their changing environments3. Importantly, integrins connect the ECM and intracellular cytoskeletal actin networks that work together to generate the propelling force required for T cell movement.
In summary, migration patterns vary based on the immune cell phenotype or environmental signals. These complex biological processes are tightly regulated by the expression of cytokines, chemokines, and integrins on the surface of the T cell, surrounding cells, and the local, infected tissue. In vivo, these migratory mechanisms can be&#160;complex and may result from several additive signals4. Due to this complexity, it can be impossible to establish a causal relationship between seemingly interlocked variables. To overcome this, there are several in vitro approaches to study specific aspects of T cell migration such as response to specific chemokine signals and the interaction between T cell integrins and ECM binding proteins. This protocol addresses methods to isolate and activate murine CD8+ T cells, with in vitro migration assays in two-dimensional space and computational analysis tools for analyzing specified T cell migration. These methods are advantageous to the user because they do not require sophisticated materials or devices, as with some other cell migration assays described in the literature. Cell migration data generated with these methods can&#160;provide evidence of immune responses in a simplistic manner that enables further, informed investigation in vivo.

Autor em destaque: Compreendendo os mecanismos da doença por meio da análise em tempo real da migração de células T

Ensaio de migração in vitro em tempo real para células T CD8+ murinas primárias

The goal of our research is to understand T cell migration patterns in highly defined environmental conditions to enable informed in vitro experimentation and decipher molecular interactions impacting migration. Live imaging, such as intravital multiphoton microscopy, is used for in vivo understanding of immune cell kinetics. But microfluidic devices are a common in vitro tool that offer precise control over the microenvironment that cannot be afforded in a live specimen.Our protocol allows us to analyze intricate signaling pathways and provides better understanding for targeted in vitro experiments to further address the biological roles of individual signals in a cell type specific manner. Our method is simple, reproducible and easy to access in research labs that have a brightfield microscope featuring an attached digital camera. Therefore, our approach ensures a reliable set of methods to study dynamic cell migration events.Our lab will focus on investigating the migration of immune cells, including T cells and neutrophils in a variety of disease settings, such as tumors and infection, thereby comprehending the mechanisms that are important to regulate immune cell migration. This will help in developing the therapeutic methods to help treat and prevent disease.

Imagem de lapso de tempo para estudar a dinâmica de migração de células T CD8+ murinas ativadas

Para começar, isole os linfócitos vivos dos tecidos linfóides secundários de camundongos e visualize as células sob um microscópio óptico com uma objetiva de 4X ou 10X. Usando uma pipeta de um mililitro, interrompa as células ativadas e transfira-as para um novo tubo cônico de 15 mililitros. Retire as células e adicione meios de separação de linfócitos para separar as células vivas das mortas.Para microscopia de lapso de tempo, coloque uma placa de dez elevado às quintas células T CD8 + no meio em uma câmara de 37 graus Celsius. Depois de realizar o bloqueio da integrina, adquira imagens de campo claro e fluorescentes a cada 10 a 60 segundos por 10 a 60 minutos. Na imagem de lapso de tempo, as células T CD8 + ativadas aumentaram a velocidade média, o deslocamento e o índice de meandros em comparação com as células virgens.

time-lapse-imaging-to-study-activated-murine-cd8-t-cell-migration

Research

JoVE Journal

Immunology and Infection

Time-Lapse Imaging to Study Activated Murine CD8+ T-Cell Migration Dynamics

87 visualizações.  University of Rochester. Para começar, isole os linfócitos vivos dos tecidos linfóides secundários de camundongos e visualize as células sob um microscópio óptico com uma objetiva de 4X ou 10X. Usando uma pipeta de um mililitro, interrompa as células ativadas e transfira-as para um novo tubo cônico de 15 mililitros. Retire as células e adicione meios de separação de linfócitos para separar as células vivas das mortas.Para microscopia de lapso de tempo, coloque uma placa de dez elevado às quintas...

Vídeo: Imagem de lapso de tempo para estudar a dinâmica de migração de células T CD8+ murinas ativadas

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...