Regulatory T Cells: A Review of Manufacturing and Clinical Utility

Introduction

Regulatory T cells (Tregs) are a population of T cells that are specialized for suppressing the activation and expansion of aberrant or overreactive lymphocytes¹. Tregs comprise 5 - 10% of the CD4⁺ T cell population² and are characterized mainly by the transcription factor (and lineage marker) forkhead box protein P3 (FOXP3)^3,4. Other important markers include high surface expression of CD25 (the IL-2 receptor α-chain) and low expression of CD127 (the IL-7 receptor α-chain)⁵. Therefore, phenotypically, Tregs can be defined as CD4⁺CD25⁺CD127^low T cells and this combination of markers can be used to identify them from other cells⁶.

The role of Tregs in immune regulation and autoimmune diseases became clear after the discovery of intracellular FOXP3 and certain Treg surface markers (e.g., CD25, or IL-2 receptor α). The importance of FOXP3, in particular, was better understood after it was discovered that mutations in the gene encoding this transcription factor led to a severe autoimmune polyendrocrine syndrome⁷. Mutations in the gene encoding FOXP3 lead to immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) - a syndrome in which patients manifest features of autoimmunity such as enteropathy, endocrinopathy, and dermatitis as well as other noninfectious, autoimmune features such as autoimmune hepatitis⁸. This discovery established the importance of Tregs in immune tolerance and additionally opened the door for the idea of adoptive transfer of Tregs as therapy for various autoimmune diseases.

In vivo, Tregs originate either through direct differentiation in the thymus or they develop from conventional CD4⁺ T cells in the periphery⁹. In the thymus, immature CD4 single-positive thymocytes that receive T cell antigen receptor (TCR) signals and have high affinity for self-peptides differentiate into FOXP3⁺ Treg cells (thymic Tregs or tTregs, also known as natural Tregs or nTregs)¹⁰. In the periphery, conventional CD4⁺ T cells encounter antigens (mostly non-self antigens such as allergens, food, and the commensal microbiota) in the presence of specific factors such as TGF-β, which leads to development of peripheral Treg cells (pTregs)^9,10. Despite the difference in origin, tTregs and pTregs are similar in function and there is currently no protein marker to differentiate them in humans⁹.

Tregs suppress immune function through both cell-contact mechanisms and the secretion of inhibitory cytokines⁹. They target T cells directly or indirectly by modulating antigen presenting cells (APCs)¹¹. Direct cell-contact mechanisms involve the expression of negative regulatory cell surface receptors such as cytotoxic T lymphocyte antigen 4 (CTLA-4)¹¹. Another mechanism of action for Tregs is through the release of anti-inflammatory soluble mediators released such as IL-10, TGFβ and IL-35 as well as through consumption of IL-2 (acting as an IL-2 sink)^9–11. Tregs also act by promoting the emergence of other immunosuppressive cell populations such as Tregs with different specificities (such as type 1 T helper cell (T_H1 cell)-like Tregs) and T regulatory 1 (Tr1) cells¹¹. For a detailed overview of how Tregs function and act, we direct the reader to an excellent review by Josefowicz et al¹⁰. Understanding these mechanisms of action has inspired several approaches to harness the potential of Tregs for adoptive cell therapy.

Given their immune suppressive functions and therapeutic potential, there has been an increasing interest in adoptive transfer of Tregs for treatment of autoimmune diseases and prevention of graft-versus-host disease (GvHD) and transplant rejection^12–16. Key factors to consider for successful use of Tregs as a cell therapy include their ability to cause immunosuppression and their survival, stability, and specificity once administered to the patient⁶. These are all essential factors to consider during the pre-clinical studies and scale-up and optimization of clinical manufacturing of Tregs, as well as while testing them in clinical trials. While progress has been made in manufacturing Tregs over the past decade with promising clinical trial results, a successful Treg cell therapy product has yet to undergo biologics license application and reach the market. In this review, we discuss the clinical-grade manufacturing strategies that have been explored, highlight the key clinical trials that have taken place and provide a brief outlook of future technologies.

Clinical-Grade Manufacturing of Tregs

Many studies have demonstrated the potential for Tregs in clinical practice while using current good manufacturing processes (cGMP) and clinical-grade reagents when possible (Table 1). These various manufacturing techniques have primarily used peripheral blood mononuclear cells (PBMC) or umbilical cord blood (UCB) as the source of Tregs. The technologies to isolate, purify and expand Tregs have advanced over the past decade and newer approaches continue to be explored. Here, we will review the key cGMP methods employed thus far by discussing the details of the starting materials and the various tools employed in isolating, purifying, and expanding the Tregs.

Table 1:

Summary of Treg cell therapy manufacturing methods

Source of cells; type of Tregs	Summary of protocol	Product specification/ Release criteria	Product purity (method: flow cytometry)	Reference
PBMC ➔ iTregs	Apheresis product was used to obtain CD4+25− T cells with immunomagnetic selection; cells were stimulated with anti-CD3 mAb-loaded, irradiated transduced K562 (KT64/86) in IL-2, rapamycin and TGFß. Tregs were restimulated with KT64/86 cells (t = 7 days). Cultures were harvested and infused (t = 14 days).	Viability ≥70%, CD4+CD25+ ≥60%, CD4−/CD8+ cells ≤10%, Gram stain with “no organisms seen,” and endotoxin <5 EU/kg.	CD4+CD25+: 95% [86-99%] Foxp3+ ~ 60%	MacMillan et al.18
PBMC ➔ tTregs	Cryopreserved leukapheresis products were used to isolate and expand Tregs using immunomagnetic beads and IL-2, TGFβ, and rapamycin (t = 0 and 7 days). Cultures were evaluated (t = 14 days) and final testing completed (t = 21 days). The cellular product was harvested (t = 21 days) and expansion beads were removed before infusion.	Negative aerobic, anaerobic and fungal sterility culture, negative mycoplasma and negative gram stain; <5.0 EU/kg endotoxin; >70% viable; >70% CD4+ CD25+; <10% CD8+ and CD19+; <3000 Exp-Act beads/10E8 cells.	CD4+CD25+: ≥ 99 % Foxp3+ > 80%	Mathew et al.20
PBMC ➔ tTregs	Apheresis product was used to isolate and purify Tregs using FACS. Purified Tregs stimulated with anti-CD3 and anti-CD28 beads (t = 0 and 9 days) in IL-2 and deuterated glucose. Cells were harvested (t = 14 days) after culture initiation and stimulating beads were removed.	FOXP3% >60%; CD4% >95%; CD8% <5%; Viability >85%.	Foxp3+ > 93%	Chandran et al.22
PBMC ➔ iTregs	Leukapheresis was used to obtain donor and recipient lymphocytes. Recipient lymphocytes were co-cultured with irradiated donor lymphocytes in the presence of anti-CD80/86 antibodies (no IL-2) to obtain inducible donor antigen-specific monoclonal Tregs.	Cell viability >80% and CD4+CD25+Foxp3+ cell count >1 x 106/kg. Absence of bacteria, fungi, mycoplasma, and endotoxins.	CD4+CD25+Foxp3+ cell count: 3.39 ± 2.12 x 106/kg (range: 0.23 to 6.37 x 106/kg)	Todo et al.26
PBMC ➔ tTregs	Apheresis products were used to isolate Tregs with CD8+ reduction and CD25+ enrichment with immunomagnetic separation. Enriched cells were activated with anti-CD3/CD28-coated beads in rapamycin and IL-2 (t = 4-6 days; replenished every 2-3 days). At final harvest, beads were removed from pooled cells.	Positive for CD4, CD25 and FOXP3; ≥ 60% CD4+CD25+ FOXP3; ≤ 10% CD8+; ≤100 beads per 3x106 cells; ≥ 70% viability; ≥ 70% recovery; ≥ 60% suppression; Sterility-no growth; Endotoxin ≤175IU/mL; Mycoplasma - not detected.	CD4+CD25+: 91.3% ± 2.33 CD4+CD25+Foxp3+: 90.7% ± 2.19	Safinia et al.21
PBMC ➔ tTegs	Leukapheresis product was used to isolate Tregs using immunomagnetic separation; stimulated with aniti-CD3/anti-CD28-coated beads. Cells were expanded (t = 7 or 12 days) with IL-2 and rapamycin (t = 0 or 2 days after isolation; replenished every 2-3 days). Beads removed at the end.	CD4+CD25highCD127low FOXP3+	CD4+CD25highCD127low FOXP3+: 84.1% [77.7-91.8]	Thiel et al.24
PBMC ➔ tTregs	Apheresis products were used to isolate Tregs using FACS. Tregs were activated with anti-CD3/anti-CD28–coated beads in the presence of IL-2 and rapamycin (t = 7 days). Cells were resuspended and fresh media and IL-2 were added every 2-3 days. Cells were restimulated (t = 9 days) with fresh anti-CD3/anti-CD28–coated beads.	Viability ≥ 85%; ≥ 60% FOXP3+ and ≥ 95% CD4+ cells; ≤ 5% CD8+ cells, <100 beads per 3 x 106 cells, and endotoxin ≤3.5 EU/ml; negative for mycoplasma, anaerobic and aerobic bacteria, gram stain, fungal culture and KOH exam.	CD4+ CD127low/−CD25+ > 95% Foxp3+: ≥ 76 %	Bluestone et al.23
PBMC ➔ tTregs	Apheresis products were used to isolate Tregs using combined immunomagnetic/FACS technique. Isolated cells were activated and expanded in the presence of IL-2, and anti-CD3/anti-CD28 beads. Tregs were cultured until the required number was achieved, but for no longer than 2 weeks (median t =10 days; range t = 7-14 days).	FoxP3 expression > 90%, passed interferon (IFN)-g suppression assay, and negative microbiological tests (no genetic material of hepatitis B virus, hepatitis C virus, or HIV and no microbial contamination in the culture supernatants)	Foxp3+: 93% [90-97]	Marek-Trzonkowska et al.28
UCB ➔ tTregs	HLA matched UCB units were thawed, processed, and enriched for CD25+ using immunomagnetic selection. CD25+ cells were co-cultured with CD3/28 co-expressing beads in the presence of IL-2 before culture was harvested (t = 14 days).	Viability ≥70%, Endotoxin <5EU/kg, Gram Stain: No organism seen, Mycoplasma: Negative, Sterility: No organism at time of infusion, CD4−CD8+ cells: <10 %, CD4+CD25+ cells > 60% and <100 beads per 3x106 cells	CD4+CD25+CD127lo: 90% [86-93%]	Kellner et al.27
UCB ➔ tTregs	HLA matched UCB units were enriched for CD25+ cells with immunomagnetic selection; then stimulated with anti-CD3 mAb loaded KT64/86 artificial APCs (aAPCs). Cultures were supplemented with IL-2 (t = 3 days). Cultures were washed and restimulated with frozen/thawed, anti-CD3 mAb-loaded KT64/86 aAPCs (t = 12 days).	Viability ≥ 70%, CD4+CD25+ purity ≥ 60%, < 10% CD4−/CD8+ cells, negative gram stain, and low endotoxin (≤5 EU/kg)	CD4+CD25+: 86% [62-97] CD4+FoxP3+CD127−: 64% [31%−96]	Brunstein et al.25
UCB ➔ tTregs	HLA matched UCB units were enriched for CD25+ cells with immunomagnetic selection; isolated cells were cultured with anti-CD3/anti-CD28 monoclonal antibody-coated beads (t = 18 days) and cultures were supplemented with IL-2 (t = 3 days).	Viability ≥ 70%, CD4+CD25+ purity ≥ 60%, < 10% CD4−/CD8+ cells, anti-CD3/anti-CD28 mAB bead count < 100 per 3 x 106 cells, gram stain with “no organisms,” and endotoxin < 5 EU/kg	CD4+CD25+: 97% [88-99] CD4+FoxP3+CD127−: 87% [78-95]	Brunstein et al.17

The major source of Treg products generated under cGMP is PBMCs collected by apheresis (Table 1). PBMCs are particularly important in manufacturing autologous products as they are easily accessible from patients. Apheresis-derived PBMCs are collected aseptically using a closed system and are easy to further manipulate in downstream processing. As Tregs comprise only 5 - 10% of the CD4⁺ T cell population^3,4, obtaining a pure population of Tregs from PBMCs requires multiple steps of isolation and expansion. To achieve this, various techniques have been developed and improved over the years – some of these will be discussed below.

UCB is the other major source of Tregs manufactured under cGMP for clinical trials (Table 1). While relatively fewer clinical trials have used UCB as the source of Tregs, the potential of third-party UCB units to be used as an allogeneic source makes them very attractive. In addition, UCB is an attractive source because of the relatively high proportion of circulating tTregs and paucity of T memory cells in the term fetus compared with adult peripheral blood¹⁷. Regardless, UCB also requires advanced techniques for isolating and expanding Tregs to achieve high enough dose for clinical applications. Our group and others have shown that Tregs must undergo multiple rounds of expansion in vitro to generate enough cells for a clinically relevant dose. These techniques of isolation and expansion will be discussed below.

The majority of Tregs derived from PBMCs and UCBs that have entered clinical trial have been tTregs. However, the need for high Treg doses for reliable disease control coupled with the low tTreg frequency and relatively poor proliferation of tTregs compared to T-conventional cells (Tcons) in ex vivo culture, have inspired exploring other options. CD4⁺ Tcons derived from PBMCs can be driven to a Treg phenotype and function in vitro in the context of TGFβ, IL-2 and rapamycin, leading to the development of iTregs. Our group has recently published the results of the first-in-human phase 1 trial of iTregs for GvHD prophylaxis in HLA-matched siblings¹⁸. The unique methods employed in inducing and expanding these cGMP grade iTregs will also be discussed below.

The initial important step in manufacturing Tregs from both PBMCs and UCB is cell selection or sorting. While various methods have been explored in the past¹⁹, currently the most common method of isolating Tregs is using immunomagnetic selection performed in a closed system. CD4⁺CD25⁺ Tregs are isolated using CD25hi as the main surface marker for positive selection²⁰. This involves incubation of the starting cell product with a cGMP-grade anti-CD25 conjugated to a magnetic nanoparticle followed by exposure to a strong magnetic field, allowing retention of CD25 rich cells that will be separated from the rest of the product. Some groups also perform CD8⁺ reduction through negative selection²¹. In addition to immunomagnetic selection, cGMP-grade FACS has also been used to isolate Tregs²². Cell surface markers such as CD4 and CD25 are used for positive selection of CD4⁺CD25⁺ Tregs; some groups also use CD127 for negative selection^22,23. While direct comparison of immunomagnetic selection versus FACS based sorting in clinical trials have not been performed yet, cGMP-grade FACS sorting is still in its early stages compared to established immunomagnetic selection.

Immunomagnetic selection is also used for iTreg manufacturing from PBMCs, but the process is modified from the above steps to enrich CD4⁺CD25⁻ cells (the starting cells that are induced into iTregs). The method involves depleting CD25⁺ cells (including tTregs) from PBMCs with anti-CD25 magnetic beads followed by purifying CD4⁺ cells from CD25⁻ PBMCs using anti-CD4 magnetic microbeads¹⁸. The CD4⁺CD25⁻ cells are then stimulated/activated before they go through the process that converts them into iTregs.

Once tTregs or CD4⁺CD25⁻ cells are isolated from the starting material, they are expanded in culture by stimulating through TCR and co-stimulatory signals. The two most common approaches for stimulating/activating these cells have been antibodies bound to beads or cell-based artificial antigen-presenting cells (aAPCs). Anti-CD3/anti-CD28–coated microbeads are widely used despite the requirement of removing the magnetic beads prior to infusion^21–24. The most widely used aAPCs to activate Tregs are K562 cells expressing CD86 and CD64 (KT86/64) that are loaded with anti-CD3 mAb^18,25. These cells are irradiated prior to use and disappear from culture over time, hence do not require a removal step prior to infusion.

Expansion culture is critical to attain a higher number of cells, as the clinical dose required is much higher than the number of cells derived through standard isolation procedures. As in the expansion of other cell types used in cell therapy, IL-2 is one of the important cytokines used to expand Tregs^20,24. The timing and frequency of IL-2 addition is variable among various groups, but in most cases, it is added in the first few days (0-3 days) and replenished as needed either every 2-3 days or on day 7 (Table 1). The immunosuppressive drug rapamycin is the other commonly used agent in expanding Tregs and is added to the media along with IL-2²³. Rapamycin, which is an inhibitor of cytotoxic T cells, works by suppressing proliferation of effector T cells while sparing Treg cells, resulting in the relative selective expansion of Tregs. For iTregs, the process of inducing the cells from CD4⁺25⁻ T cells and then expanding them happens essentially simultaneously. The CD4⁺25⁻ T cells are induced into Tregs (iTregs) through incubation with IL-2, rapamycin and TGFβ, as mentioned above¹⁸. While there are other cytokines and small molecules that may induce Treg phenotype, the combination of IL-2/rapamycin/TGFβ is the unique cocktail commonly used in both inducing and expanding iTregs.

In addition to manufacturing tTregs and iTregs through the process mentioned above, there is another unique approach to manufacturing Tregs. In this process, leukapheresis is used to obtain donor and recipient lymphocytes from PBMCs, and then the recipient lymphocytes are co-cultured with irradiated donor lymphocytes in the presence of anti-CD80/86 monoclonal antibodies without IL-2 or rapamycin²⁶. Todo et al call these types of Tregs inducible donor antigen-specific monoclonal Tregs (different than iTregs mentioned above). In addition to the difficulty of obtaining sufficient number of recipient cells and possible contamination with leukemia cells, the final product has very low purity (the mean number of CD4⁺CD25⁺Foxp3⁺ T cells was only 24.8% of the CD4⁺ cells infused).

The cell culture media used in expanding Tregs have also been variable among groups, but X-VIVO 15 has been the most consistently used^22–25,27. The extensive experience of using X-VIVO 15 in other cell therapy applications in a cGMP-compliant setting makes it a safe and reliable option. Other commonly used cGMP-compliant media include the TexMACS GMP media and the CellGro media^21,24,28. While serum free X-VIVO 15 exists and is used in some cases, the supplementation of these various media with serum is one additional factor to consider while ensuring cGMP-compliance. Supplementation of the media with 2.5 to 10% human AB serum results in enhanced Treg function. The culture conditions for expanding Tregs in these media have consistently been the standard, 37°C and 5% CO₂. Major limitations of current Treg manufacturing procedures using magnetic bead based approaches include less than 100% purity of the cellular therapy product and potential toxicities that might occur due to contaminating effector T cells. As shown in Table 1, the purity of the products is determined by measuring the percentage of CD4⁺CD25⁺ and/or Foxp3⁺ cells. While the percentage of CD4⁺CD25⁺ cells is mostly above 90%, the percentage of Foxp3⁺ cells could be as low as 60%. Another limitation of current manufacturing process is difficulty to meet target cell dose. While manufacturing Tregs with a dose ranging from 0.5-10 x 10⁶/kg and as high as 3 x 10⁸/kg have been reported, some studies report failure to meet the target dose^17,23,25.

In comparing the manufacturing approaches employed, one of the big differences is the source material used to make the Tregs. As discussed above, using PBMCs and UCBs as source material have their own advantages and disadvantages. Tregs from PBMCs requires multiple depletion steps, although they have the advantage of being easily accessible from donors while the relatively high proportion of circulating Tregs in UCBs make them an attractive source. Additionally, the potential of third-party UCB units as an allogeneic source of Tregs is another advantage. Another difference in manufacturing steps is the type of agents used in the activation and expansion stage. While IL-2 is used in almost all approaches (except in the approach used to make inducible Tregs²⁶), the use of rapamycin is not consistent across all approaches. Although rapamycin is known to suppress proliferation of contaminating effector T cells and hence improve purity of the product, it is not yet clear whether the use of rapamycin leads to a clinically significant improvement.

In addition to IL-2 and rapamycin, the process of manufacturing iTregs adds TGFβ which induces a regulatory phenotype in CD4⁺CD25⁻ T cells. iTregs manufactured through this process had an average CD4⁺CD25⁺ expression of 95%, but the average Foxp3⁺ expression was close to 60%¹⁸. Manufacturing tTregs through the activation/expansion of CD4⁺CD25⁺ has been reported to produce Tregs with Foxp3⁺ expression as high as 93% (Table 1). While the iTreg manufacturing approach produces Tregs with high dose, this lower Foxp3⁺ expression will need improvement. Whether there will be significant differences in the clinical effectiveness of iTregs versus tTregs remains to be seen.

Several of the methods involved in manufacturing Tregs are similar to those involved with other immune cell therapies. However, the need to activate/stimulate Tregs through TCR and co-stimulatory signals such as anti-CD3/anti-CD28 microbeads or aAPCs adds to the complexity of the process. While the basic aspects of methods for expanding Tregs have been delineated, the ideal culture/cytokine cocktail and the frequency/timing of when these are added continues to be optimized. With further studies and clinical trials, the important variables that will lead to a highly potent, pure, and safe Treg product will continue to be identified.

Clinical Trials

There are many ongoing clinical trials that are trying to utilize the benefits of Tregs both in adoptive cellular therapy as well as through in vivo expansion strategies with low dose IL-2. According to clinicaltrials.gov, there are currently about 10 active clinical trials utilizing Tregs in adoptive cell therapy for various autoimmune diseases. A summary of the major clinical trials involving Tregs for adoptive cell therapy over the past decade is provided in Table 2. These clinical trials have focused on using Treg cell therapy in patients with a range of conditions including GvHD, transplant rejection, and autoimmune disorders. Most of these trials have been early stage trials (mainly phase 1) recruiting limited number of patients (<20). As noted with the variations in the approach to manufacturing, the clinical protocols for Treg cell therapy have also varied with differences, for example, in dosing and the end points investigated (Table 2). Despite these variations, the one common focus of these early clinical trials has been demonstrating the safety and tolerability of Treg adoptive immunotherapy. As shown in Table 2, most clinical trials included product specifications designed to ensure a safe product, including sterility testing, viability, Gram stain, Mycoplasma testing and cell phenotype determined by flow cytometry.

Table 2:

Summary of Clinical Trials using cGMP-grade Treg cells for therapy

Patient characteristics	Treg cell product	Outcome	Reference (Listed by year)
14 adults with high risk malignancy treated with reduced intensity conditioning and mobilized peripheral blood stem cells from HLA-identical sibling donors	Induced regulatory T cells (iTregs) (PBMC derived, prepared from human CD4+25− T cells cultured in IL-2, rapamycin and TGFß along with anti-CD3 mAb-loaded artificial antigen-presenting cells); dose of 3.0 x 106/kg, 3.0 x 107/kg, and 3.0 x 108 cells/kg.	No severe infusion toxicities with all patients achieving neutrophil recovery (median day 13). Circulating iTregs were detectable through day 14. iTregs can be delivered safely at doses as high as 3 x 108 cells/kg.	MacMillan et al.18 (2021)
14 patients treated with relapsing-remitting multiple sclerosis	Autologous Tregs (PBMC derived, activated, and stimulated with anti-CD3/anti-CD28 beads and IL-2), either delivered intravenously (IV), dose 40 x 106 cells/kg or intrathecally (IT), dose 1.0 x 106 cells.	No severe adverse events were observed. Relapses were noted in five IV treated patients, while no patients in the IT group experienced a relapse.	Chwojnicki et al.32 (2021)
104 adult patients who received living-donor kidney transplants; 38 of them received Treg cell therapy	Four out of the six clinical trials pooled in this study used Tregs manufactured at four different sites using various methods.	Treg cell therapy is achievable and safe in living-donor kidney transplant recipients; associated with fewer infectious complications, but similar rejection rates in the first year.	Sawitzki et al.29 (2020)
11 recipients of living donor kidney transplant (compared with nine control patients from a corresponding reference group trial)	Autologous Tregs (PBMC derived, activated, and stimulated with anti-CD3/anti-CD28 beads, rapamycin, and IL-2); given seven days after kidney transplant, dose 0.5, 1.0, or 2.5-3.0 x 106 cells/kg.	No dose limiting toxicity observed. Stable monotherapy immunosuppression was achieved in 73% of patients, while reference group remained on standard dual or triple therapy.	Roemhild et al.33 (2020)
Nine adult patients who were enrolled while awaiting liver transplantation or 6-12 months posttransplant	Autologous Tregs (PBMC derived, activated, and stimulated with anti-CD3/CD28 beads, IL-2, and rapamycin); administered intravenously at either 0.5-1 x 106 cells/kg or 3-4.5 x 106 cells/kg.	Treg transfer was safe, transiently increased the pool of circulating Tregs and reduced anti-donor T cell responses.	Sanchez-Fueyo et al.30 (2020)
Nine adult recipients of living donor renal allografts that would not require hemodialysis during the first week following renal transplantation	Autologous Tregs (PBMC derived, activated, and stimulated with anti-CD3/CD28 beads, IL-2, TGFβ, and rapamycin); infused at doses of 0.5 x 109, 1.0 x 109, and 5 x 109 cells/recipient.	All doses of Treg therapy tested were safe with no adverse infusion related side effects, infections, or rejection events up to two years post-transplant.	Matthew et al.20 (2018)
Three kidney transplant recipients with inflammation on their 6-month surveillance kidney transplant biopsy	Autologous Tregs (PBMC derived, stimulated with anti-CD3 and anti-CD28 beads in media containing IL-2), delivered at doses of ~320 x 106 cells/recipient.	There were no infusion reactions or serious therapy related adverse events. The infused cells demonstrated persistence and stability.	Chandran et al.22 (2017)
11 patients with advanced or high-risk lympho-hematopoietic malignancy: 22 identically treated controls	Allogeneic Tregs (derived from HLA matched UCB, stimulated with anti-CD3 mAb loaded KT64/86 artificial APCs in the presence of IL-2), at doses of 3-100 x 106 cells/kg.	No adverse events observed. Incidence of grade II-IV acute GVHD at 100 days was significantly lower (9% vs 45%, P = 0.05). Chronic GVHD at 1 year was zero in Tregs and 14% in controls.	Brunstein et al.25 (2016)
10 adult patients early post-liver transplantation	Inducible donor antigen-specific monoclonal Tregs (PBMC derived, co-cultured with irradiated donor lymphocytes in the presence of anti-CD80/86 monoclonal antibodies) infused at a dose of 3.39 ± 2.12 x 106/kg, ranging from 0.23-6.37 x 106 cells/kg.	No significant adverse events were observed. All 10 recipients maintained stable graft function and seven patients successfully achieved weaning and completed cessation of immunosuppressive agents.	Todo et al.26 (2016)
14 adult subjects with Type 1 Diabetes	Autologous Tregs (PBMC derived using FACS, activated with anti-CD3/anti-CD28–coated microbeads and cultured in the presence of IL-2 and rapamycin); dose of 0.05 x 108 to 26 x 108 cells.	No infusion reactions or cell therapy–related high-grade adverse events. A subset of the Tregs was long-lived, with up to 25% of the peak level remaining in the circulation at 1 year after transfer.	Bluestone et al.23 (2015)
Five adult patients with treatment-refractory cGvHD	Allogeneic Tregs (derived from PBMC, expanded in the presence of aniti-CD3/anti-CD28 beads, IL-2, and rapamycin); delivered at a mean dose of 2.4 x 106 cells/kg.	Two patients showed clinical response with improvement of symptoms, the others showed stable symptoms for up to 21 months. In four patients, increased counts of Treg were detectable and immunosuppressionwas reduced.	Thiel et al.24 (2015)
10 children (aged 8-16 years) with recent-onset Type 1 diabetes (2 months since diagnosis)	Autologous Tregs (derived from PBMC, activated and stimulated in the presence of IL-2 and anti-CD3/anti-CD28 beads); delivered at a dose of 20 x 106 cells/kg.	Administration of Tregs was safe and tolerable. No toxicity was noted. A significant increase in percentage of Tregs in peripheral blood was observed.	Marek-Trzonkowska et al.28 (2012)
The first two patients with GvHD who received the treatment with ex vivo expanded Tregs (first-in-man clinical trial for Tregs)	Allogeneic Tregs (derived from PBMC, sorted with immunomagnetic separation and FACS, activated with anti-CD3/anti-CD28 beads and IL-2); delivered at a dose of 1 x 105 cells/kg.	Tregs led to significant alleviation of the symptoms and reduction of pharmacologic immunosuppression in the case of chronic GvHD; in the case of grade IV acute GvHD, the improvement was transient.	Trzonkowski et al.34 (2009)

In general, the studies so far show that Treg adoptive cell therapy is safe and well-tolerated among the multiple types of patient groups studied. These studies have shown safe delivery of Tregs with doses ranging from 0.5-10 x 10⁶/kg and as high as 3 x 10⁸/kg, with no serious infusion related adverse events documented (Table 2). While the optimal Treg dose for clinical application is yet to be determined, the early safety studies with the high dose of Tregs pave the way for future efficacy studies. In addition, infused cell therapy products were shown to be persistent in circulation up to 1 year retaining their Foxp3⁺CD25⁺ Treg phenotype²³. However, this study also showed that there was a rapid decline in the percentage of infused Tregs in circulation, with the majority of the Tregs being undetectable within 90 days. Moreover, long-term persistence has not been observed in many studies and this has been a limitation of other clinical trials which mostly report in vivo persistence of 14-30 days^25,30. A possible solution for this could be combining Tregs with treatments that promote Treg expansion and persistence in vivo. An example of this is combining low-dose IL-2 with Tregs, which has shown promise in early clinical studies³⁵.

While safety has been the main focus of these early trials, some of the results have indicated promising efficacy as Treg therapy resulted in low risk of GvHD²⁵ and fewer infectious complications or transplant rejections post-kidney transplant^20,29. The fact that some of these early trials showed efficacy is encouraging but phase 2 trials with larger number of patients will be required before broader conclusions can be made regarding the wider clinical applications of Treg immunotherapy.

In addition to the use of Tregs as stand-alone cell therapy agents, the role that Treg cell therapy could play in combination with other immunosuppressive agents is an exciting possibility. This is particularly important in patients receiving immunosuppressive drugs after organ transplantation. Current standard of care in organ transplantation such as liver or kidney requires putting patients on immunosuppressive drugs for many years and in some cases for lifetime. Tregs have the potential to be combined with existing immunosuppressive drugs to enhance immunosuppression with a goal to one day either reduce or completely avoid the need for immunosuppressive drugs post organ transplantation. One recent clinical trial has shown that patients receiving Treg cell therapy, and minimization of immunosuppression, can be successfully weaned within the first-year post-transplantation to monotherapy²⁹. The results from other studies^26,30 also indicate that there is enough evidence to encourage future trials that investigate the use of Tregs to facilitate the reduction or even the complete discontinuation of immunosuppressive medications following organ transplantation.

Overall, the clinical studies so far show that Treg cellular therapy products have a favorable safety profile along with early signs of therapeutic benefit. With early focus on GvHD and transplant rejection, these areas may be where the first successful implementation of Treg cellular therapy occurs. Factors influencing the long-term efficacy of Treg therapy are still under investigation³¹. Turning advances in the field into clinical reality in the later phases of clinical trials will require overcoming some remaining challenges. These challenges include further understanding Treg biology to ensure efficient and targeted approaches are used to isolate, induce, and expand Tregs, as well as optimizing the variables involved in manufacturing and administering the Tregs. In addition to optimizing the manufacturing methods, well-designed clinical trials will be crucial. Finally, given the complexity of the patients and the diseases targeted by Tregs, the timing and frequency of administration of the Treg cell therapy products may play a role in enhancing efficacy. Therefore, the success of Treg cellular immunotherapy will depend on the effective collaboration of various teams to design and implement future clinical trials.

Future Directions

As the manufacturing methods and clinical trials discussed above are moving forward, there are also exciting preclinical studies with novel ideas that are ongoing and promise even more advanced ways of using Tregs in clinical applications. Most of these novel ideas involve improvements in manufacturing Tregs, including utilizing new sources as well as engineering Tregs to make them more specific and target-focused. While new Treg sources such as discarded human thymus tissue, routinely removed during pediatric cardiac surgery^36,37, provide additional options for large-scale Treg manufacturing, it is the engineering of Tregs using chimeric antigen receptors (CARs) and genome editing to enhance their specificity and functionality that may provide significant contributions³⁸. These advances make the manufacturing process more complex but have the potential to make Tregs more versatile and applicable.

The success and approval of the CD19 CAR T cell therapy have inspired the use of the CAR T technology for a wide range of applications including the engineering of CAR Tregs³⁹. The main advantage of using CAR Tregs is to achieve antigen specificity by redirecting Tregs toward a target antigen. Preclinical studies have shown that CAR can be used to redirect Treg specificity⁴⁰ and to generate alloantigen specific Tregs for induction of transplant tolerance⁴¹. The generation of CAR specific Tregs in autoimmune diseases has been achieved⁴² and MHC molecule specific-CAR-Tregs have also been shown to have the capacity to prevent transplant rejection^43,44. Despite these exciting preclinical studies and the extensive experience with manufacturing CAR T cells, we are still awaiting the first CAR Treg clinical trial.

The next decade promises to lead to significant advances in Treg clinical applications as more phase II and possibly phase III clinical trials based on existing and advancing technology take place. Moreover, current preclinical studies, particularly with CAR Tregs, may also enter phase I trials paving the way for more potentially dramatic advances in treatment of autoimmune disease and transplant rejection. These clinical trials and additional possible clinical applications will depend on continued progress in both the basic science and manufacturing technologies.

Figure 1:

An overview of the regulatory T (Treg) cell manufacturing process. Tregs are isolated from peripheral blood mononuclear cells (PBMC) or umbilical cord blood (UCB) by immunomagnetic selection or fluorescence activated cell sorting (FACS). They are then activated/stimulated by incubation with anti-CD3/CD28 coated beads or artificial antigen-presenting cells (aAPCs) consisting of K562 which express CD86 and CD64 (FcR1). This is followed by the expansion of the Tregs using interleukin (IL)-2 and in some cases rapamycin. In the case of induced Tregs (iTregs), TGFβ is also added. After expansion, Tregs are purified (by removing the beads and other agents) followed by flow cytometric analysis to ensure their purity. The final Treg product is then infused to the patient.