In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Hongzheng Bi; Samiksha Wasnik; David  J. Baylink; Chenfan Liu; Xiaolei Tang

doi:10.3791/60585

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

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08:02 min

January 22, 2020

DOI: 10.3791/60585-v

Hongzheng Bi^1,2, Samiksha Wasnik¹, David J. Baylink¹, Chenfan Liu^1,3,4, Xiaolei Tang^1,3

¹Division of Regenerative Medicine, Department of Medicine,Loma Linda University, ²Zhengzhou University, ³Department of Veterinary Biomedical Sciences, College of Veterinary Medicine,Long Island University, ⁴Jinan Infectious Disease Hospital,Shandong University

Overview

This protocol describes a novel therapeutic strategy for enhancing gut-homing regulatory T cell induction through engineered dendritic cells. It can be adapted for human applications and is effective in pro-inflammatory environments.

Key Study Components

Area of Science

Immunology
Cell Biology
Therapeutic Strategies

Background

Regulatory T cells play a crucial role in maintaining immune homeostasis.
Active vitamin D and vitamin A are important for immune system function.
Engineering dendritic cells can enhance T cell responses.
This protocol aims to improve the understanding of vitamin roles in immunity.

Purpose of Study

To develop a protocol for the induction of gut-homing regulatory T cells.
To explore the effects of active vitamin D and A on the immune system.
To provide a method that can be used in highly inflammatory conditions.

Methods Used

Seeding 293T cells in cell culture medium.
Using concentrated HBS solution and plasmids for dendritic cell engineering.
Incubation at controlled temperature and CO2 levels.
Step-by-step visual demonstration of the protocol.

Main Results

Successful generation of engineered dendritic cells.
Enhanced induction of gut-homing regulatory T cells observed.
Protocol applicable in various inflammatory contexts.
Insights into the role of vitamins in immune modulation.

Conclusions

The protocol offers a promising approach for T cell induction.
Engineering dendritic cells can be a valuable tool in immunotherapy.
Further research is needed to explore clinical applications.

Frequently Asked Questions

What are gut-homing regulatory T cells?

Gut-homing regulatory T cells are specialized immune cells that help maintain tolerance and prevent inflammation in the gut.

How does vitamin D influence the immune system?

Vitamin D plays a role in modulating immune responses and enhancing the function of regulatory T cells.

Can this protocol be used in human applications?

Yes, the protocol is designed to be adaptable for potential human therapeutic applications.

What is the significance of dendritic cells in immunity?

Dendritic cells are key antigen-presenting cells that initiate and regulate immune responses.

What are the conditions for cell culture in this protocol?

Cells are cultured at 37 degrees Celsius with 5% CO2 for optimal growth.

What role does retinoic acid play in T cell induction?

Retinoic acid is involved in the differentiation and migration of T cells to the gut.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]₂D) and the active vitamin A (retinoic acid or RA) de novo.

This protocol describes a potentially novel therapeutic strategy that can be readily modified for human applications. This technique directly enhances the induction of gut-homing regulatory T cells in peripheral lymphoid tissues and can be stably implemented even in highly pro-inflammatory environments. These techniques can also be used to investigate the role of active vitamin D and vitamin A within the peripheral immune system.

This visual demonstration will provide step by step instructions for the generation of high quality engineered dendritic cells that are critical for the success of this technique. Begin by seeding one times 10 to the seven 293T cells in 20 milliliters of CM-10-D cell culture medium in 150 by 25 millimeter culture plates for a 24 hour incubation at 37 degrees Celsius and 5%carbon dioxide. The next day, add 1.62 milliliters of freshly prepared two-fold concentrated HBS solution, 9.5 micrograms of envelope plasmid, 17.5 micrograms of packaging plasmid, and 27 micrograms of the LENTI-CYP-ALDH plasmid to a 50 milliliter conical tube.

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