Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Sebastian C. Warth; Vigo Heissmeyer

doi:10.3791/50455

Trasduzione Adenoviral di CD4 T naive Cells allo Studio Differenziazione Treg

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Trasduzione Adenoviral di CD4 T naive Cells allo Studio Differenziazione Treg

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15:33 min

August 13, 2013

DOI: 10.3791/50455-v

Sebastian C. Warth¹, Vigo Heissmeyer¹

¹Institute for Molecular Immunology,Helmholtz Zentrum München

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Overview

This protocol outlines a method for producing infectious adenoviral particles to transduce genes into naive CD4 T cells. The transduced cells can then differentiate into regulatory T cells (Tregs) under specific conditions.

Key Study Components

Area of Science

Gene transfer techniques
Immunology
Cell differentiation

Background

Adenoviral vectors are effective tools for gene delivery.
Understanding Treg differentiation is crucial for immunological research.
This method allows for the study of gene function in T cell biology.
Naive CD4 T cells can be manipulated to study various immune responses.

Purpose of Study

To develop a protocol for adenoviral gene transfer into T cells.
To analyze the differentiation of naive T cells into Tregs.
To facilitate molecular studies of T cell regulation.

Methods Used

Cloning of the gene of interest into an adenoviral vector.
Transfection of the vector into adenovirus producer cell line HC 2 93 A.
Production of infectious adenoviral particles.
Infection of naive T cells from mice expressing the adenovirus receptor.

Main Results

Successful production of adenoviral particles for gene delivery.
Transduced naive T cells can differentiate into Tregs under polarizing conditions.
Expression of the gene of interest is confirmed post-infection.
This method provides insights into T cell differentiation mechanisms.

Conclusions

The protocol enables effective gene transfer into T cells.
It allows for the study of regulatory T cell differentiation.
This approach can advance understanding of immune regulation.

Frequently Asked Questions

What is the significance of using adenoviral vectors?

Adenoviral vectors are efficient for gene delivery and can infect dividing and non-dividing cells.

How are Tregs differentiated from naive T cells?

Tregs are induced under specific polarizing conditions after activation of naive T cells.

What are the applications of this protocol?

This protocol can be used to study T cell biology and immune responses in various contexts.

Can this method be applied to other cell types?

While this protocol focuses on T cells, adenoviral vectors can be adapted for other cell types.

What precautions should be taken during the protocol?

Ensure proper biosafety measures are in place when handling viral vectors and infected cells.

Adenovirale trasferimento genico nelle cellule T CD4 naive con transgenici espressione del recettore adenovirus Coxsackie permette l'analisi molecolare della differenziazione delle cellule T regolatorie

Questo protocollo fornisce un flusso di lavoro per la produzione di particelle adenovirali infettive che vengono utilizzate per trasdurre un gene di interesse in cellule T naive, che dopo l'espressione atopica del transgene o differenziate in cellule T regolatorie o treg. In primo luogo, il gene di interesse viene clonato nel vettore di destinazione adenovirale. Il vettore viene quindi trasferito nella linea cellulare produttrice dell'adenovirus, HC 2 93 A e vengono generate particelle adenovirali infettive.

I virus risultanti vengono utilizzati per infettare le cellule T naive dei topi con un transgene del recettore dell'adenovirus dopo l'infezione. Le cellule vengono quindi fatte riposare per garantire l'espressione del gene di interesse. Nelle cellule T naive, le cellule T vengono attivate in condizioni di polarizzazione per indurre la differenziazione delle treg.

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