Mouse Naïve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Summary

Naïve CD4⁺ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of naïve CD4⁺ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Abstract

Antigen inexperienced (naïve) CD4⁺ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a naïve CD4⁺ T cell. Although the cytokine environment during naïve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4⁺ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4⁺ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of naïve T cells from mouse and the subsequent steps necessary for polarizing naïve cells to various T helper effector lineages in vitro.

Introduction

The concept of distinct lineages or subsets of CD4⁺ T helper (Th) cells has been around since the latter part of the 20^th century¹. Recognition of cognate antigen in the presence of costimulatory signals results in several rounds of cellular proliferation and the eventual differentiation into effector Th cells. The type of Th cell generated during this process is dependent on the cytokine environment present during activation². Initially, naïve Th cells were thought to polarize into 2 distinct lineages following T cell receptor (TCR) activation, costimulatory CD28 ligation, and cytokine signaling. Type 1 helper cells (T....

Protocol

All experimental procedures are performed using protocols approved by the office of Environmental Health and Safety at the Rosalind Franklin University of Medicine and Science. C57BL/6 mice (purchased from NCI) used for this protocol were housed under specific pathogen-free conditions, and all animal experiments were performed using protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Rosalind Franklin University of Medicine and Science.

1. Preparation of Instruments, Supplies, and Reagents

1. Coat 48-well or 24-well plates with anti-CD3 and anti-CD28 (Table 1) in sterile PBS, cover ....

Results

The time point for the analysis of differentiation can vary depending on the Th condition being tested as well as the strength of T cell receptor activation. After 2-3 days of differentiation, cells can be visualized by light microscopy to determine the extent of T cell proliferation. Wells exhibiting extensive proliferation and clumping of cells will most likely be ready for analysis at day 4. Differentiation conditions relying on the addition of exogenous IL-2, such as Th1 and Th2, will likely exhaust the media after 4.......

Discussion

While the spleen contains naïve Th cells, the proportion of this population in lymph nodes is much higher. Failure to properly identify and remove lymph nodes in this protocol will result in a poor yield of naïve cells. This can be especially difficult in older mice or male mice that have more fat tissue. As shown in Figure 1, proper fixing and pinning of the animal limbs and skin will allow for easier visualization of the accessible exterior lymph nodes. Once lymph nodes and spleens are proces.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Complete RPMI:			Warm in a 37 oC water bath before use
RPMI 1640 Media	Life Technologies	11875119
10 % FBS	Life Technologies	26140-079
1000X 2-mercaptoethanol	Life Technologies	21985023
100X Pen/Strep	Life Technologies	15140122
100X L-glutamine	Life Technologies	25030081
120 micron nylon mesh	Amazon	CMN-0120-10YD	Cut into 2 cm2 squares and autoclave
Alternative: 100 micron cell strainers	Fisher	08-771-19	Alternative to cutting nylon mesh
autoMACS running buffer	Miltenyi	130-091-221	Warm in a 37 oC water bath before use
autoMACS rinsing solution	Miltenyi	130-091-222	Warm in a 37 oC water bath before use
CD4 beads	Miltenyi	130-049-201
ACK lysis buffer	Life Technologies	A10492-01
Cytokines:
Human (h) IL-2	Peprotech	200-02
Recombinant mouse (rm) IL-4	Peprotech	214-14
rmIL-6	R & D Systems	406-ML-025
rmIL-12	Peprotech	210-12
hTGFb	R & D Systems	240-B-010
Antibodies:
2C11 (anti-CD3)	BioXcell	BE0001-1
37.51 (anti-CD28)	BioXcell	BE0015-1
11B11 (anti-IL-4)	BioXcell	BE0045
XMG1.2 (anti-IFNg)	BioXcell	BE0055
anti-CD62L-FITC	BioLegend	104406	Use at 1:100
anti-CD25-PE	BioLegend	102008	Use at 1:400
anti-CD4-PerCP	BioLegend	100434	Use at 1:1000
anti-CD44-APC	BioLegend	103012	Use at 1:500
Phorbol  12-myristate 13 acetate (PMA)	Sigma-Aldrich	P-8139	Prepare a stock at 0.1 mg/ml in DMSO and freeze aliquots at -20 oC
Ionomycin	Sigma-Aldrich	I-0634	Prepare a stock at 0.5 mg/ml in DMSO and freeze aliquots at -20 oC
Brefeldin A	eBioscience	00-4506-51	Use at 1:1000