Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells

Summary

We describe a method for generating regulatory, memory and naïve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Abstract

The development and maintenance of immunosuppressive CD4⁺ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis¹ , type I diabetes² , rheumatoid arthritis and graft versus host disease,⁴ several fundamental differences in human versus mouse Treg biology⁵ has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion⁶.

Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4⁺ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans⁷ and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into naïve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and naïve T cells have different reported polarization requirements and plasticities⁸ , pre-sorting of the initial T cell population into CD45RA⁺ and CD45RO⁺ subsets can be used to examine these discrepancies. Consistent with others, our CD25^HiCD45RA^- iTregs express high levels of FoxP3⁹ , GITR and CTLA-4¹¹ and low levels of CD127¹² . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Protocol

1. Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) from Buffy Coat

1. Acquire one unit of buffy coat from hospital or nearby blood center location. Our blood center provides us with about 40-60 mL of buffy coat per unit obtained from normal blood donors.
2. Pour blood into an autoclaved 500 mL glass bottle containing sterile PBS to dilute buffy coat. The final volume of PBS + buffy coat should be 250 mL.
3. Fill ten 50 mL conical tubes each with 20 mL Lymphoprep solution.
4. Gently overlay the Lymphoprep solution with 25 mL of the diluted buffy coat, being careful not to disturb the Lymphoprep solution.
5. Spin tubes ....

Discussion

While Treg transfer holds enormous therapeutic promise in combating autoimmunity graft rejection and other immune or inflammatory-mediated disorders, methods for their efficient generation and stable maintenance have not yet been developed. As only 1-5% of circulating human T cells are Tregs, their controlled expansion and differentiation overcomes this paucity as a major deterrent of implementation of Tregs into the clinic. On the other hand, as we learned from the TGN1412 trial¹³ , it is scientifically and e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lymphoprep	Axis-Shield	1114547	Keep at room temperature
Refrigerated Bench-Top Centrifuge	Eppendorf	5810R
Bright-Line Hemocytometer	Hausser Scientific	3110
EasySep Human CD4+ T Cell Enrichment Kit	Stem Cell Technologies	19052
EasySep Human Memory CD4+ T Cell Enrichment Kit	Stem Cell Technologies	19157
EasySep Human Naïve CD4+ T Cell Enrichment Kit	Stem Cell Technologies	19155
The Big Easy EasySep Magnet	Stem Cell Technologies	18001	Silver
5 mL Round Bottom Polystyrene Tubes	BD Biosciences	352008
15 mL Polypropylene Centrifuge Tubes	VWR international	89004-368
14 mL Polypropylene Round-Bottom Tubes	BD Biosciences	352059
50 mL Polypropylene Centrifuge Tubes	VWR international	89004-364
Human anti-CD3 Antibody	Bio X Cell	BE0001-2	Clone: OKT3
24 Well Cell Polystyrene Culture Plate	BD Biosciences	353047
96 Well Round Bottom Tissue Culture Plate	Greiner Bio-One	650180
RPMI 1640 with Glutamax-I and HEPES Buffer	GIBCO, by Life Technologies	72400
Fetal Bovine Serum (FBS)	GIBCO, by Life Technologies	16000
β-Mercapt–thanol	Sigma-Aldrich	M7522
Recombinant Human TGF-β1	eBioscience	14-8348-62
Recombinant Human IL-2	eBioscience	14-8029-63
Bovine Serum Albumin (BSA)	MP Biomedicals	810531
0.5 M EDTA	Amresco	E177
Mouse Anti-Human CD25-PE	Miltenyi Biotec	130-091-024	Clone: 4E3
Mouse Anti-Human CD45RA-PE-Cy5	eBioscience	15-0458-42	Clone: HI100
Mouse Anti-Human CD127-APC	Miltenyi Biotec	130-094-890	Clone: MB15-18C9
DNase II	MP Biomedicals	190370
MoFlo Flow Cytometer	Beckman Coulter Inc.
FlowJo Software	Tree Star, Inc.
Cell Quest Pro Software	BD Biosciences
Newborn Calf Serum	GIBCO, by Life Technologies	16010
L-glutamine	GIBCO, by Life Technologies	25030
AIM-V	GIBCO, by Life Technologies	0870112
CellTrace CFSE Cell Proliferation Kit	Invitrogen	C34554
Treg Suppression Inspector Beads	Miltenyi Biotec	130-092-909
Penicillin-Streptomycin	GIBCO, by Life Technologies	15140
40 μM Nylon Cell Strainer	BD Biosciences	352340