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⁺CD127^loCD25^hi cells

CAUTION: Use sterile technique throughout. Discard sharps immediately in an appropriate sharps container. Bleach anything that has come into contact with blood and/or blood products prior to disposal. Work in a biosafety cabinet.

1. Obtain human peripheral blood or blood products preenriched for human leukocytes (i.e., “leukopaks”) from peripheral blood draws or leukapheresis. Process cells immediately.
   NOTE: If overnight storage cannot be avoided, store and transport cells at room temperature (RT). Avoid exposure to cold.
2. Isolate peripheral blood mononuclear cells (PBMCs) by gradient centrifugation over density gradient medium as previously described⁹.
3. Carefully count PBMCs using a hemocytometer or cell counter. If possible, use at least 300 x 10⁶ PBMC to proceed with Treg isolation.
4. Resuspend PBMCs in isolation buffer (2% pooled human AB serum [PHS-AB] with 1.5 mM EDTA in phosphate buffered saline [PBS]) at a concentration of 50 x 10⁶ cells/mL and proceed with magnetic sorting according to the manufacturer's instructions of the sorting kit used. For example, use magnetic cell sorting (Table of Materials) for negative isolation of a CD4⁺CD127^lo T cell population, followed by positive selection sorting for CD25⁺ cells.
   NOTE: A variety of products can be used for the magnetic purification of Treg, including column-based and column-free approaches. Alternatively, fluorescence-activated cell sorting (FACS) with gating on CD127^loCD25^high CD4⁺ T cells may be performed. Complete sterility is not possible with standard FACS equipment, however, posing a significant risk of contamination. In addition, cellular stress and damage conferred by the fluidics system cannot be excluded, and the instrument lasers may impact results.
5. Check the resulting Treg-enriched population for purity through staining for CD4, CD3, CD127, and CD25 (Figure 1). Choose an anti-human CD25 antibody that recognizes a CD25 binding domain different from the one used in the sorting kit, such as the one specified in Table of Materials, to obtain an accurate result. Stain and fix cells using a standard surface staining protocol such as that described in Nowatzky et al.¹⁰.

2. Cloning of Treg from a CD127^loCD25^hi preenriched human CD4⁺ T cell suspension

1. Resuspend the Treg-enriched (CD4⁺CD127^loCD25^hi) cells obtained in step 1.4 in T cell media ([TCM]; RPMI 1640, 5% PHS-AB, 1% streptomycin/penicillin, 1% HEPES, 1% nonessential amino acids, and 1% glutamine) with 300 IU/mL of human recombinant interleukin-2 (IL-2) (Table of Materials) aiming for a concentration of ~1−3 x 10⁶ cells/mL and count. Obtain at least three separate cell counts and calculate the average cell numbers before proceeding because accurate counts are absolutely crucial.
2. Prepare a single cell suspension of cells at two concentrations: (1) 3 cells/mL, and (2) 6 cells/mL. Load five round-bottom 96 well plates with 100 µL/well of suspension 1, and five plates with suspension 2. Take great care to keep cells in suspension when loading the plates to ensure a distribution of 0.3 and 0.6 cells/well, respectively.
   NOTE: Do not increase the cell concentration to 1 cell/well, because this increases the risk of obtaining oligoclonal cell lines and not true clones.
3. Prepare feeder cells from freshly isolated human allogeneic PBMCs obtained through density gradient centrifugation as in step 1.2.
4. Prepare at least 10 x 10⁶ feeder cells per cloning plate for irradiation by resuspending human allogeneic PBMCs in TCM without IL-2 in a 50 mL polypropylene tube at a concentration of ~10 x 10⁶ PBMC/mL. Tightly close the tube and irradiate with 35 Gy in either a gamma irradiator or an X-ray based irradiation device.
   NOTE: Irradiation triggers the secretion of large amounts of cytokines from the feeders that facilitate the proliferation of Teff, but may adversely affect Treg expansion. These will next be removed by washing.
5. Centrifuge cells at 450 x g and RT for 5 min after irradiation, and aspirate the supernatant. Wash cells by resuspending them in TCM without IL-2 using at least 10x the pelleted feeder cell volume of media/PBS.
   NOTE: It is recommended to determine a human leukocyte antigen (HLA) expression profile of the donor cells to be cloned. This can be done through simple staining for one or several HLA types that are common in the population a respective donor is derived from (i.e., HLA-A2 or HLA-A24). PBMC used as feeder cells should not express this HLA to enable reidentification/isolation of target cells from expansion cultures prior to irradiation-induced apoptosis of the feeders.
6. Resuspend the irradiated and washed feeder cells in TCM with 300 IU/mL IL-2 at a concentration of 1 x 10⁶ cells/mL.
7. Add phytohemagglutinin-L (PHA-L) (Table of Materials) at a concentration of 4 µg/mL (2x the required concentration in culture) and quickly proceed to step 2.8.
8. Add 100,000 irradiated feeder cells in 100 µL of TCM with 4 µg/mL PHA-L and 300 IU/mL IL-2 to the plated Treg-enriched cells, resulting in a total volume of 200 µL and a PHA-L concentration of 2 µg/mL in each well. Mix well by pipetting up and down 5x. Limit exposure time of the feeder cells to PHA-L before their addition to the Treg to the absolute minimum necessary, and keep in suspension until plated.
   NOTE: Ensure a PHA-L concentration of 2 µg/mL in the culture at the initial seeding step, but use a lower, 1 µg/mL concentration upon subsequent expansion of Treg clones. Use 300 IU/mL of IL-2 throughout.
9. Incubate at 37 °C. Change 50% of the media using TCM with 300 IU/mL IL-2 but without PHA-L on days 5−7.

3. Expansion of Treg and maintenance of clones in culture

1. Beginning on day 12, check cultures for the presence of pellets of proliferating cells.
   NOTE: There will be pellets formed by feeder cells, but on microscopic examination those will appear as small, round, dying or dead cells, whereas proliferating T cells will be larger in size, with bright contrast and healthy appearance, often forming clusters of adjacent cells that appear brown on macroscopic examination.
2. Continue to examine cultures every 1−2 days. Isolate proliferating tentative clones by transferring them onto single wells. Place each tentative clone on a single 96 well plate to avoid cross-contamination of any given tentative or established clone though inadvertent cell transfer from other wells.
3. Maintain cells through 50% media changes every 2−3 days and split as necessary. Monitor cells closely.
   NOTE: Delaying media changes and splitting can harm cells and “over splitting” can arrest their proliferation and result in cell death.
4. Restimulate cells with irradiated allogeneic feeders as described in steps 2.3−2.9, but use lower PHA-L concentrations (i.e., 1 µg/mL in TCM with 300 IU/mL IL-2). Restimulate when cell size decreases and cells become round as opposed to elongated oval which is typically after 2−3 weeks.
   NOTE: Maintain the original cloning culture for at least 6−8 weeks. Many of the ‘early’ clones that become visible at or shortly after 2 weeks tend to not be true Treg, but fast proliferating non-Treg/Teff cells and are more likely to fail vetting than ‘late’ cells, some of which will take more than 1 month to visibly appear in culture.

4. Vetting of tentative Treg clones

1. Begin vetting of tentative clones once they have proliferated to ~1 x 10⁶ cells for monoclonality and TSDR methylation status.
2. Optionally, prescreen cell expansions by staining for CD3, CD4, CD127, and CD25 in order to identify those of interest (i.e., CD3⁺CD4⁺CD127^loCD25^hi) for further assessment (Figure 2).
3. Establish monoclonality through identification of the presence of a single Vβ chain in the cellular product by Vβ staining using sets of commercially available staining antibodies (Table of Materials; Figure 3) following manufacturer's instructions. Ensure to acquire at least 1 x 10⁶ events when running samples on a flow cytometer for a reliable analysis, so that minor contaminating populations can be detected. Use a viability dye.
4. Have TSDR methylation status at the FOXP3 locus assessed by commercial providers or in-house^11,12. Obtain at least 1 x 10⁵ cells for adequate results.
5. Once Treg identity and clonality have been confirmed in steps 4.3 and 4.4, cryopreserve the cells or use for downstream applications.
   NOTE: Alternative, but far less reliable vetting approaches, are determining Treg phenotype by FACS stainings^1,10 and in vitro or in vivo suppression assays^13,14. Avoid overreliance on Treg suppression assays¹³. The assays are typically, but not exclusively, based on the coculture of Treg in graded numbers with responder T cells (i.e., PBMC or purified T cells, sometimes Treg-depleted) in the presence of third party antigen-presenting cells or CD3/CD28 with dye-dilution representing proliferation as the read-out. Many of these assays have severe limitations and may either over- or underestimate the actual degree of suppression mediated by Treg. TSDR methylation status remains the most definite/reliable measure of the Treg phenotype or “identity”^3,15,16. Note that FOXP3 is located on the X chromosome and, in females, one X chromosome is inactivated by DNA methylation, which affects the results of TSDR methylation analysis.

5. Cryopreservation of Treg

NOTE: Treg can be stored long-term after successful cryopreservation with DMSO and PHS-AB.

1. Determine the number of cryovials required to cryopreserve the cells in. This is equal to the total final volume of cell suspension (in mL) to be cryopreserved, because 1 mL is frozen per vial. Add a 10% safety margin to this volume in order to account for losses due to pipetting/surface tension.
   NOTE: Cells can be cryopreserved at a wide range of concentrations, typically in between 0.1−100 x 10⁶ cells/mL.
2. Label the cryovials.
3. Generate solution A (SA): mix 50% RPMI and 50% PHS-AB or human plasma. If plasma is used, spin at 2,000 x g for 20 min at 4 °C.
   NOTE: The volume of SA should be 75% of the total final volume of cell suspension to be cryopreserved as determined in step 5.1.
4. Generate solution B (SB): mix 60% (v/v) PBS or RPMI and 40% (v/v) dimethyl sulfoxide (DMSO).
   NOTE: The volume of SB should be 25% of the total final volume of cell suspension to be cryopreserved as determined in step 5.1.
5. Chill both solutions to 4 °C.
6. Prepare the freezing medium by mixing all of SB with equal parts of SA and keep on ice.
7. Spin Treg from step 4.5 at 450 x g for 5 min at 4 °C. Aspirate and discard media, and resuspend cells in the remaining ice-cold SA and keep on ice.
8. Slowly add the chilled freezing medium 1:1 to the cells resuspended in SA in a large tube on ice while shaking the tube.
9. Quickly aliquot into cryovials at 1 mL/vial. Immediately place the cryovials into a cupboard box at RT and transfer to a -80 °C freezer without delay. Transfer to liquid N₂ after 24 h.

Results

Successful implementation of this protocol will lead to the generation of stable human regulatory T cell clones and lines.

Preselection/preenrichment of CD4⁺CD127^loCD25^hi 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 CD25^hiCD127^lo 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

Name	Company	Catalog Number	Comments
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