Analysis of Simian Immunodeficiency Virus-specific CD8<sup>+</sup> T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Lucas Gonzalez-Nieto; Aline Domingues; Michael Ricciardi; Martin J. Gutman; Helen S. Maxwell; Nuria Pedre&#241;o-Lopez; Varian Bailey; Diogo M. Magnani; Mauricio A. Martins

doi:10.3791/54881

유인원 면역 결핍 바이러스 특정 CD8 분석 + 펩티드 - MHC-I 테트라 머 염색에 의해 붉은 털...

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Analysis of Simian Immunodeficiency Virus-specific CD8⁺ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

유인원 면역 결핍 바이러스 특정 CD8 분석 + 펩티드 - MHC-I 테트라 머 염색에 의해 붉은 털 원숭이의 T 세포

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December 23, 2016

DOI: 10.3791/54881-v

Lucas Gonzalez-Nieto¹, Aline Domingues¹, Michael Ricciardi¹, Martin J. Gutman¹, Helen S. Maxwell¹, Nuria Pedreño-Lopez¹, Varian Bailey¹, Diogo M. Magnani¹, Mauricio A. Martins¹

¹Department of Pathology,University of Miami

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Overview

This article presents an optimized protocol for enumerating and characterizing CD8 + T cells specific to the AIDS virus in rhesus macaques. The method allows for the quantification of antigen-specific CD8 T-cells in biological samples obtained ex vivo, providing insights into T-cell responses in various disease contexts.

Key Study Components

Area of Science

HIV immunology
T-cell immunology
Biomedical research

Background

CD8 + T cells play a crucial role in immune responses against viral infections.
Understanding the frequency of antigen-specific CD8 T-cells is vital for vaccine development.
Challenges exist due to incomplete knowledge of rhesus macaque MHC genetics.
Flow cytometric staining requires precise antibody application.

Purpose of Study

To enumerate and characterize CD8 positive T-cell populations against the Simian Immunodeficiency Virus.
To evaluate T-cell responses induced by vaccination or primary infection.
To provide a reliable method for studying CD8 T-cell responses in various disease settings.

Methods Used

Re-suspension of rhesus PBMC in R10 medium.
Flow cytometry for quantifying antigen-specific CD8 T-cells.
Direct analysis of biological samples without in vitro re-stimulation.
Characterization of T-cell populations generated by vaccination or infection.

Main Results

The method successfully quantifies antigen-specific CD8 T-cells in ex vivo samples.
It provides insights into the frequency of T-cell responses to vaccination and infection.
Facilitates the study of CD8 T-cell responses in multiple disease contexts.
Addresses challenges related to MHC genetics and antibody application.

Conclusions

This optimized protocol is a valuable tool for HIV immunology and broader biomedical research.
It enhances understanding of CD8 T-cell dynamics in response to viral infections.
The technique can be adapted for various research applications involving T-cell responses.

Frequently Asked Questions

What is the significance of CD8 + T cells in HIV research?

CD8 + T cells are crucial for controlling viral infections, including HIV, and understanding their responses can inform vaccine development.

How does this method differ from traditional techniques?

This method allows for the quantification of T-cells directly from biological samples without the need for in vitro re-stimulation, providing more accurate results.

What challenges do researchers face when using this protocol?

Researchers may struggle with the complexities of rhesus macaque MHC genetics and the need for precise antibody application during flow cytometry.

Can this technique be applied to other diseases?

Yes, the technique can be adapted to study CD8 T-cell responses in various disease settings beyond HIV.

What are the implications of this research?

The findings can enhance our understanding of immune responses and improve strategies for vaccine development against viral infections.

Is prior experience required to use this method?

While prior experience can be beneficial, detailed protocols and guidance can help new researchers successfully implement the technique.

여기에서는 AIDS 바이러스를 붉은 털 원숭이 CD8 ⁺ T 세포를 열거하고 특성화하기위한 최적의 프로토콜을 제시한다. 이 문서에서는 HIV의 면역학 분야에뿐만 아니라, CD8 ⁺ T 세포 반응이 질병의 결과에 영향을 미치는 것으로 알려져있다 생물 의학 연구의 다른 영역뿐만 아니라 유용합니다.

이 분석의 전반적인 목표는 백신 접종 또는 1차 감염에 의해 생성된 Simian Immunodeficiency Virus-specific CD8 양성 T 세포 집단을 열거하고 특성화하는 것입니다. 이 방법은 백신 접종 또는 감염에 의해 유도된 항원 특이적 CD8 T 세포의 빈도와 같은 T 세포 면역학 분야의 주요 질문에 답하는 데 도움이 될 수 있습니다. 이 기술의 주요 장점은 시험관 내 재자극 없이 생체 외에서 직접 얻은 생물학적 샘플에서 항원 특이적 CD8 T 세포를 정량화할 수 있다는 것입니다.

이 기술의 의미는 붉은털 원숭이의 절개 특이적 CD8 T 세포를 평가하고 여러 질병 환경에서 CDB T 세포 반응을 연구하는 데까지 확장됩니다. 일반적으로 이 방법을 처음 접하는 개인은 붉은털 원숭이 MHC 유전학에 대한 지식이 불완전하고 유세포 분석 염색에 정확한 항체 적용이 필요하기 때문에 어려움을 겪을 것입니다. R10 배지에서 rhesus PBMC를 다시 현탁하여 시작합니다.

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