Generation of Human Regulatory T Cell Clones

Summary

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Abstract

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as “Treg” therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4⁺CD25⁺ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

Introduction

The purpose of this protocol is to enable the in vitro propagation of ultra-high purity, clonal human Treg. Isolation of Treg-enriched populations and subsequent cloning allows for the selection of desired Treg phenotypes and their expansion for further study of the biology of these cells, exploration of their potential therapeutic usefulness, and other experimental downstream applications.

Cloning Treg will yield significantly better Treg purity than polyclonal isolation and expansion approaches. This is due to the reliable, controlled exclusion of T effector cells with similar or different phenotypes from the purified population, including FOXP3-expressing (FOXP3^intCD45RA^negCD25^int) non-Treg and CD4⁺ CD25⁺ FOXP3^– Teff¹, among others. Clonal cell lines obtained through this approach do not face the problem of overgrowth with rapidly-expanding non-Treg clones that render very long-term expansion (i.e., several months) and in vitro culture of the cells practically impossible or at least extremely challenging. Clonal Treg also allow extensive vetting of their phenotypic features postexpansion, including through methods recognized as standard for the assessment of epigenetic features indicative of human bona fide natural Treg^1,2,3,4 (e.g., stable demethylation at the TSDR).

Treg expansion has mainly been performed in the form of polyclonal cell expansion for both investigative and therapeutic purposes^5,6,7. The problems with Teff contamination are a major obstacle to the successful implementation of Treg cell-based immunotherapy approaches. Previous attempts to expand/generate monoclonal Treg in the literature are scarce and have failed to show maintenance of Treg features in the long term⁸.

This method will be of interest to anyone studying cellular, molecular, and metabolic properties of bona fide human Treg. The ultrapure Treg product generated through the use of this protocol in particular lends itself to analyses using genomic approaches. Given the relatively low expansion rates that characterize human Treg in general, this method may be of limited use to those who seek the rapid expansion of massive numbers of cells. However, given the extremely high purity of the Treg generated with this protocol, smaller numbers of Treg may have similar or even better efficacy than larger expansions of polyclonal cell lines that contain effector cells that limit the overall suppressive potential of the generated product.

Protocol

This protocol follows all institutional guidelines pertaining to the ethical conduct of research involving the use of human samples. Work with human cells and other human blood products must take place at least in a BSL-2 certified environment following BLS-2 safety guidelines at a minimum. 1. Preenrichment of human peripheral blood mononuclear cells for CD4+CD127loCD25hi cells CAUTION: Use sterile technique throughout. Discard sharps …

Representative Results

Successful implementation of this protocol will lead to the generation of stable human regulatory T cell clones and lines. Preselection/preenrichment of CD4+CD127loCD25hi cells was a straightforward method to obtain a starting population that contained most human Treg (Figure 1A–C). Not all clones displayed a Treg phenotype. Prescreening of clones by measurement of CD25hiCD127lo expressi…

Discussion

This protocol describes the propagation of ultrapure human regulatory T cells through the isolation, expansion, and careful vetting of cells obtained in a limiting dilution and feeder cell-based expansion approach from Treg-containing starting populations.

Critical steps in this approach are: 1) the choice of an appropriate starting population. Generally, the CD127^loCD25^hi compartment of CD4⁺ T cells within human PBMC contains a wide variety of Treg that suits …

Materials

0.22 µm Stericup, 500 mL	Millipore	5500	Media storage and preparation
100x Nonessential amino acids	Gibco	11140-050	Media component
15 mL conical centrifuge tubes (50/bag, case of 500)	ThermoFisher Scientific	339650
1M HEPES	Gibco	15630-080	Media component
25 ml Single Well Pipet Basin	Fischer Scientific	13-681-508
50 mL Conical Centrifuge Tube (25/sleeve)	ThermoFisher Scientific	339652
50x Penicillin Streptomycin Soln	Corning	Corning, 30-001-Cl	Media component
CryoTube Vial Int Thread Round Btm Starfoot PP Screw Stopper Sterile PP 1.8 mL	Nalge Nunc	377267
DMSO	Corning	25-950-CQC
EasySep Human CD25 positive selection kit	Stemcell Technologies	18231	Alternatives are FACS or MACS column-based sorting
EasySep Human CD4+CD127low T cell Pre-Enrichment Kit	Stemcell Technologies	19231	Alternatives are FACS or MACS column-based sorting
EasySep Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (alternative to item 12)	Stemcell Technologies	18063	Alternatives are FACS or MACS column-based sorting
Ficoll	GE Healthcare	17-5442-03	PBMC purification from peripheral blood of leukapheresis products; density gradient medium
Human AB Serum (PHS-AB)	Valley Biomedical Inc	HP1022	Media component
LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation	Thermo Fischer	L-34962	Viability dye
Phytohemagglutinin-L (PHA-L)	Millipore/Sigma	11249738001	T cell stimulation
Recombinant IL-2 (e.g., PROLEUKINâ)	Prometheus		T cell stimulation and maintenance/ Media component
RPMI 1640	Gibco	21870-076	Media component
Staining antibodies for flowcytometry (Treg phenotyping)	See "Comments"	See "Comments"	Staining antibodies are enlisted in: Nowatzky et al. (2019) PubMed PMID: 30584695; PubMed Central PMCID: PMC6497402. In case EasySep Human CD25 positive selection kit is used, stain with 2A3 or BC96 anti-CD25 antibody, e.g.: Brilliant Violet 421 anti-human CD25 Antibody (Biolegend; 302629)
TCR Vβ Repertoire Kit; IOTest Beta Mark	Beckman Coulter	PN IM3497	Vetting of expansions for monoclonality
Tissue Culture Plate, 96 Well, U-Bottom with Low Evaporation Lid	Corning	353077