<em>In Situ</em> Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

Chandirasegaran Massilamany; Arunakumar Gangaplara; Ting Jia; Christian Elowsky; Qingsheng Li; You Zhou; Jay Reddy

doi:10.3791/51679

A detecção in situ de células T CD4 autoreactive em Cérebro e Coração Usando pr...

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Immunology and Infection

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JoVE Journal Immunology and Infection

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

A detecção in situ de células T CD4 autoreactive em Cérebro e Coração Usando principal de histocompatibilidade Dextramers classe II

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13:10 min

August 1, 2014

DOI: 10.3791/51679-v

Chandirasegaran Massilamany¹, Arunakumar Gangaplara¹, Ting Jia¹, Christian Elowsky², Qingsheng Li³, You Zhou², Jay Reddy¹

¹School of Veterinary Medicine and Biomedical Sciences,University of Nebraska, Lincoln, ²Center for Biotechnology,University of Nebraska, Lincoln, ³Nebraska Center for Virology and School of Biological Sciences,University of Nebraska, Lincoln

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Overview

This article describes a protocol for detecting self-reactive CD4 T cells in the brain and heart using MHC Class II dextramers. The method allows for a comprehensive analysis of antigen-specific CD4 + T cell frequencies in situ.

Key Study Components

Area of Science

Neuroscience
Immunology
Autoimmunity

Background

Self-reactive T cells play a critical role in autoimmune diseases.
MHC Class II dextramers are tools for identifying specific T cell populations.
Accurate enumeration of T cell frequencies is essential for understanding immune responses.
This study focuses on the brain and heart, key areas affected in autoimmune conditions.

Purpose of Study

To develop a reliable method for detecting self-reactive CD4 T cells.
To enumerate antigen-specific T cell frequencies in situ.
To enhance understanding of autoimmune mechanisms in the brain and heart.

Methods Used

Harvesting brain and heart tissues from experimental autoimmune encephalomyelitis mice.
Preparing tissue sections using a microtome.
Staining and fixing the tissue sections for analysis.
Mounting sections on microscopic slides for visualization.

Main Results

Successful identification of self-reactive CD4 T cells in brain and heart tissues.
Demonstrated the effectiveness of MHC Class II dextramers in T cell detection.
Provided a reliable method for enumerating T cell frequencies.
Contributed to the understanding of autoimmune responses in specific tissues.

Conclusions

The protocol offers a valuable tool for studying self-reactive T cells.
Findings may inform future research on autoimmune diseases.
Further studies are needed to explore the implications of these T cell populations.

Frequently Asked Questions

What are MHC Class II dextramers?

MHC Class II dextramers are specialized reagents used to identify and analyze specific T cell populations based on their antigen specificity.

Why is it important to detect self-reactive T cells?

Detecting self-reactive T cells is crucial for understanding the mechanisms underlying autoimmune diseases and developing targeted therapies.

What type of tissues were used in this study?

The study utilized brain and heart tissues harvested from experimental autoimmune encephalomyelitis mice.

How are the tissue sections prepared for analysis?

Tissue sections are prepared by embedding in paraffin, cutting into thin slices, and then staining and fixing for microscopic examination.

What is the significance of enumerating T cell frequencies?

Enumerating T cell frequencies helps researchers understand the immune response and the role of specific T cell populations in disease processes.

Can this method be applied to other tissues?

While this study focuses on the brain and heart, the method may be adaptable to other tissues affected by autoimmune conditions.

O protocolo para detectar células auto-reactivas T CD4 no cérebro e no coração por coloração direta com complexo de histocompatibilidade principal dextramers classe II foi descrito neste relatório. Para análise detalhada, um método confiável para enumerar as freqüências de células T CD4 ⁺ antígeno-específicas in situ também é concebido.

O objetivo geral deste procedimento é detectar células T auto-reativas com alto grau de especificidade usando aparadores MHC Classe dois dex. Neste vídeo, o cérebro é colhido do cérebro de um camundongo com encefalomielite autoimune experimental induzida por proteína lipídica protio de mielina. Isso é feito primeiro colhendo cuidadosamente os tecidos e preparando os blocos de aros.

O segundo passo é cortar o tecido embebido em aros em seções de 200 micrômetros de espessura com um viome. As seções são então transferidas para câmaras de tecido em uma placa de 24 poços, onde são coradas e fixadas. A etapa final é montar as seções de tecido em lâminas microscópicas.

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