In Vitro Functional Analysis of Regulatory T cells: Focus On Proliferation And Differentiation

Summary

This protocol provides an in vitro co-culture strategy for investigating the effects of regulatory T cells on effector T cell proliferation and differentiation.

Abstract

Regulatory T (Treg) cells play key roles in maintaining immune homeostasis. Abnormalities in Treg cell function are associated with various diseases, including autoimmune diseases, organ transplant rejection, infection, and cancer. Manipulating Treg cells is considered a potential therapeutic strategy for treating inflammatory diseases, including autoimmune disorders, solid organ transplant rejection, graft-versus-host disease, and allergic diseases. Classic in vitro Treg cell suppression assays mainly assess the inhibitory effect of Treg cells on effector T (Teff) cell proliferation. However, in addition to proliferation, T cell differentiation is important for T cells to perform their immune functions. Therefore, this protocol provides a modified in vitro Treg cell suppression assay for investigating the effects of Treg cells on the proliferation and differentiation of Teff cells. In this protocol, the preparation of mouse Treg cells and naive T cells is first introduced, followed by the co-culture strategy for studying the regulatory effects of Treg cells on the proliferation and differentiation of Teff cells, including Th1 and Th17 cells, and finally, the staining strategy for flow cytometry after co-culture is presented. This in vitro functional analysis of Treg cells can be used to investigate the effects of Treg cells on T cell proliferation and polarization.

Introduction

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells are important mediators of immune homeostasis¹. Treg cells actively suppress excessive immune responses and maintain self-immune tolerance, thereby preventing immune reactions from damaging normal tissues and organs². Manipulating Treg cells is considered a potential therapeutic strategy for treating inflammatory diseases such as autoimmune disorders, solid organ transplant rejection, graft-versus-host disease, and allergic diseases^3,4,5. Meanwhile, inhib....

Protocol

All mouse experiments were authorized by the Animal Ethics Committee at West China Hospital, Sichuan University (Approval Nos. 20250814006, 20211212A). All mice used in this experiment were on a C57BL/6 genetic background.

1. Preparation of solutions

1. Prepare fluorescence-activated cell sorting (FACS) buffer: 1× PBS containing 0.5% bovine serum albumin (BSA).
2. Prepare magnetic-activated cell sorting (MACS) buffer: 1× PBS containing 0.5% BSA and 2 mM ethylenediamine tetraacetic acid (EDTA).
3. Prepare cell culture medium: RPMI 1640 medium containing 10% fetal bovine serum (FBS), 1 mM sodi....

Results

In the representative experiment, naïve T cells were used as Tresp cells to investigate the effects of Treg-enriched cells on the proliferation and differentiation of Th1 and Th17 cells. In this experiment, Tresp cells were activated by anti-mouse CD3 and APC-enriched non-CD4⁺ cells, and then differentiated into Th1 cells under IL-12 p70 induction or into Th17 cells under TGF-β1 and IL-6 induction. Under these two induction conditions, changes in the proliferation and differentiation of Tresp cells were studie.......

Discussion

In this study, the results of the co-culture under Th1 induction conditions are consistent with previous reports that Treg cells can suppress Th1 cell proliferation and differentiation^17,18. Treg-enriched cells also inhibited the proliferation of Tresp cells under Th17 induction conditions. However, under the Th17-polarizing conditions used here, the frequency of IL-17A⁺ Tresp cells increased in the presence of Treg-enriched cells, which differs from p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL centrifuge tube	Thermo Fisher Scientific	3457IW	or equivalent
12.5 μL Filtered Pipette Tips	Thermo Fisher Scientific	94420043	or equivalent
1250 μL Filtered Pipette Tips	Thermo Fisher Scientific	94420813	or equivalent
15 mL Conical Centrifuge Tubes	Thermo Fisher Scientific	339650	or equivalent
200 μL Filtered Pipette Tips	Thermo Fisher Scientific	94420318	or equivalent
48-well plates	Corning Incorporated	3548	or equivalent
5 mL syringe piston	Shifeng Medical	SB2-061(A)	or equivalent
50 mL Conical Centrifuge Tubes	Thermo Fisher Scientific	339652	or equivalent
50 μL Filtered Pipette Tips	Thermo Fisher Scientific	94420258	or equivalent
70 μm cell strainer	Biosharp Life Sciences	BS-70-CS	or equivalent
Anti-mouse CD28 antibody	BioXcell	BE0015-1	or equivalent
Anti-mouse CD3 antibody	BioXcell	BE0001-1	or equivalent
Anti-mouse CD4 PerCP-Cy5.5 (RM4-5)	Thermo Fisher Scientific	45-0042-82	or equivalent
Anti-mouse Foxp3 PB (FJK-16s)	Thermo Fisher Scientific	48-5773-82	or equivalent
Anti-mouse IFN-gamma PE (XMG1.2)	BioLegend	505808	or equivalent
Anti-mouse IL-17A PE-Cy7 (eBio17B7)	Thermo Fisher Scientific	25-7177-82	or equivalent
Beckman CytoFLEX S Flow Cytometer	Beckman Coulter	N/A	for flow cytometric data acquisition of immune cell samples
Bovine Serum Albumin (BSA)	Sigma-Aldrich	B2064	or equivalent
C57BL/6 mice	Shanghai Model Organisms Center, Inc	SM-001	for isolation of primary lymphocytes and T cell subsets
CD25 Monoclonal Antibody (PC61.5), PE	Thermo Fisher Scientific	12-0251-83	or equivalent
CD4+CD25+ Regulatory T Cell Isolation Kit, mouse	Miltenyi Biotec	130-091-041	for cell sorting
CD4+CD62L+ T Cell Isolation Kit, mouse	Miltenyi Biotec	130-106-643	for cell sorting
CD45.1 mice	Shanghai Model Organisms Center, Inc	NM-KI-210226	for isolation of primary lymphocytes and T cell subsets
CD45.1 Monoclonal Antibody (A20), FITC	Thermo Fisher Scientific	11-0453-85	or equivalent
CD45.2 Monoclonal Antibody (104), APC-eFluor 780	Thermo Fisher Scientific	47-0454-82	or equivalent
CD62L (L-Selectin) Monoclonal Antibody (MEL-14), FITC	Thermo Fisher Scientific	11-0621-85	or equivalent
Cytofix/Cytoperm Fixation/Permeabilization Solution Kit	BD Biosciences	554714	or equivalent
eBioscience Cell Proliferation Dye eFluor 450	Thermo Fisher Scientific	65-0842-90	or equivalent
ethylenediamine tetraacetic acid (EDTA)	Solarbio Life Sciences	E1170	or equivalent
Fetal Bovine Serum	Thermo Fisher Scientific	A5669701	or equivalent
FlowJo 10.6.2	BD Biosciences	N/A	software for flow cytometry data analysis
Golgi-Plug Protein Transport Inhibitor	BD Biosciences	555029	or equivalent
HEPES (1 M)	Thermo Fisher Scientific	15630080	or equivalent
Human TGF-beta1	PEPROTECH	100-21C	or equivalent
Ionomycin calcium salt	Millipore Sigma	I3909	or equivalent
LD Columns	Miltenyi Biotec	130-042-901	for cell sorting
L-Glutamine (200 mM)	Thermo Fisher Scientific	25030081	or equivalent
LS Columns	Miltenyi Biotec	130-042-401	for cell sorting
MACS MultiStand	Miltenyi Biotec	130-042-303	for cell sorting
MS Columns	Miltenyi Biotec	130-042-201	for cell sorting
OctoMACS Separators	Miltenyi Biotec	130-042-109	for cell sorting
PBS, pH 7.4	Thermo Fisher Scientific	10010049	or equivalent
PE Rat IgG1, κ Isotype Ctrl Antibody	BioLegend	400408	or equivalent
PE/Cyanine7 Rat IgG2a kappa Isotype Control Antibody	Thermo Fisher Scientific	25-4321-82	or equivalent
Penicillin-Streptomycin	Thermo Fisher Scientific	15140122	or equivalent
PMA	Millipore Sigma	P8139	or equivalent
Purified anti-mouse CD16/32 Antibody	BioLegend	101302	or equivalent
QuadroMACS Separator	Miltenyi Biotec	130-090-976	for cell sorting
Recombinant mouse IL-12 p70	PEPROTECH	210-12	or equivalent
Recombinant Mouse IL-6	R&D Systems	406-ML	or equivalent
Red Blood Cell Lysis Buffer (ACK Lysing Buffer)	Thermo Fisher Scientific	A1049201	or equivalent
RPMI 1640 medium	Thermo Fisher Scientific	11875093	or equivalent
Sodium Pyruvate (100 mM)	Thermo Fisher Scientific	11360070	or equivalent
Trypan Blue Solution, 0.4%	Thermo Fisher Scientific	15250061	or equivalent
UltraComp eBeads Compensation Beads	Thermo Fisher Scientific	01-2222-42	or equivalent
Zombie Yellow Fixable Viability Kit	BioLegend	423104	or equivalent
β-Mercaptoethanol	Sigma-Aldrich	M3148	or equivalent