Treg Cells

TYPE Review
PUBLISHED 19 April 2023

DOI 10.3389/fimmu.2023.1166135
Opportunities for Treg cell

OPEN ACCESS therapy for the treatment of
EDITED BY
Lesley Ann Smyth,
University of East London, United Kingdom
human disease
REVIEWED BY
Carolina Isabel Rojas, Jeffrey A. Bluestone*, Brent S. McKenzie, Joshua Beilke
University of Chile, Chile
Oksana Kehoe, and Fred Ramsdell
Keele University, United Kingdom
Sonoma Biotherapeutics, South San Francisco, CA, United States
*CORRESPONDENCE
Jeffrey A. Bluestone
[email protected]
SPECIALTY SECTION
This article was submitted to
Regulatory T (Treg) cells are essential for maintaining peripheral tolerance,
Autoimmune and Autoinflammatory
Disorders : Autoimmune Disorders, preventing autoimmunity, and limiting chronic inflammatory diseases. This
a section of the journal small CD4+ T cell population can develop in the thymus and in the peripheral
Frontiers in Immunology
tissues of the immune system through the expression of an epigenetically
RECEIVED 14 February 2023 stabilized transcription factor, FOXP3. Treg cells mediate their tolerogenic
ACCEPTED 22 March 2023
PUBLISHED 19 April 2023
effects using multiple modes of action, including the production of inhibitory
cytokines, cytokine starvation of T effector (e.g., IL-2), Teff suppression by
CITATION
Bluestone JA, McKenzie BS, Beilke J and metabolic disruption, and modulation of antigen-presenting cell maturation or
Ramsdell F (2023) Opportunities for function. These activities together result in the broad control of various immune
Treg cell therapy for the treatment
of human disease.
cell subsets, leading to the suppression of cell activation/expansion and effector
Front. Immunol. 14:1166135. functions. Moreover, these cells can facilitate tissue repair to complement their
doi: 10.3389/fimmu.2023.1166135 suppressive effects. In recent years, there has been an effort to harness Treg cells
COPYRIGHT as a new therapeutic approach to treat autoimmune and other immunological
© 2023 Bluestone, McKenzie, Beilke and
Ramsdell. This is an open-access article
diseases and, importantly, to re-establish tolerance. Recent synthetic biological
distributed under the terms of the Creative advances have enabled the cells to be genetically engineered to achieve
Commons Attribution License (CC BY). The tolerance and antigen-specific immune suppression by increasing their
use, distribution or reproduction in other
forums is permitted, provided the original specific activity, stability, and efficacy. These cells are now being tested in
author(s) and the copyright owner(s) are clinical trials. In this review, we highlight both the advances and the challenges
credited and that the original publication in
this journal is cited, in accordance with
in this arena, focusing on the efforts to develop this new pillar of medicine to treat
accepted academic practice. No use, and cure a variety of diseases.
distribution or reproduction is permitted
which does not comply with these terms.
KEYWORDS
Treg, immune tolerance, Foxp3, autoimmunity, immune regulation
1 History and biology of T regulatory cells

The demonstration of immune tolerance dates to Owens, Burnet, Medawar, and others
in the 1950s, including the awarding of a Nobel Prize in 1960 (reviewed in (1). Importantly,
over the next two decades, tolerance was shown to be transferable with leukocytes from
tolerant animals, suggesting that a specialized cell population was responsible. Although
the concept of suppressive T cells was around for many decades starting with the work of
Kondo and Gershon (2), the field was littered with phenomenology, lack of reliable
Frontiers in Immunology 01 frontiersin.org

Bluestone et al. 10.3389/fimmu.2023.1166135
molecular markers of the suppressor cell population, and proteins of the cell surface of antigen-presenting cells (24). In
conflicting data; yet, studies by Nishizuka and colleagues showed addition, Tregs function indirectly and mediate bystander
that mice thymectomized at 3 days after birth achieved systemic suppression through the production of suppressive cytokines
organ-specific autoimmunity (3). Critically, these diseases could be (including IL-10, IL-35, and TGFb), consumption of T cell
prevented or even reversed with the adoptive transfer of growth factors (including IL-2), and altering the local metabolic
CD4+CD25+ T cells (4). This was reminiscent of the Medawar environment to limit Teff cell activity (11). It is important to note
work demonstrating the importance of tolerance induction early in that Treg activation is dependent on activation through the T cell
life and implicated a small subset of T cells in the biology. receptor and CD28 engagement as a co-stimulatory signal. In fact,
In the late 1990s, studies of a mouse strain with multi-system the ability to respond to IL-2 is also TCR/CD28 dependent,
autoimmunity (scurfy mice) led to the identification of the supporting a critical interplay of these signaling pathways in
transcription factor Foxp3 (5). The scurfy mouse, which was determining the ability of these cells to mediate bystander
originally developed at Oak Ridge National Labs during the suppression (25–27). Finally, Tregs have also been shown to
Manhattan (atomic bomb) project, displayed many of the same mediate what is called infectious tolerance (28–31). Gershon and
attributes of the disease seen in neonatally thymectomized mice (6). Kondo were the first to coin the phrase “infectious tolerance” in the
Furthermore, a disease in male children—immune-mediated, 1970s (2, 28). The term was based on the observation, by this group
polyendocrinopathy, X-linked—resembled the pathology found in and others (29–31), that long-term tolerance to allogeneic skin
scurfy mice, and it was shown that these patients also possessed grafts and other immune settings was induced by adoptively
mutations in FOXP3 (7). Further studies by multiple groups transferred Tregs but, once established, remained durable by the
demonstrated that FOXP3 is expressed primarily in the recruitment and development of other immunosuppressive cell
CD4+CD25+ subset of T cells that were shown to be capable of populations. This ability of Tregs to promote other regulatory
controlling autoimmunity in mice (8–10). Thus, by the mid-2000s, cells is likely to provide more robust and durable tolerance. In
T regulatory (Treg) cells could be defined at the molecular level in addition to inhibition of inflammation, Treg cells also promote the
both mice and humans, and their critical role in maintaining repair of damage by releasing tissue and stem cell factors (32, 33).
immunological tolerance was unequivocally established. The multifaceted activity of Treg cells provides a means to reduce
Over the last 20 years, immunological tolerance has been linked inflammation with one cell type but via many different
to multiple mechanisms (1, 11). However, thymically derived Treg mechanisms, and thus Treg and Treg-friendly immune therapies
cells represent the dominant mechanism for maintaining normal have therapeutic potential in a variety of autoimmune,
immune homeostasis and preventing fatal multi-organ inflammatory, and transplant-related diseases.
autoimmunity. These cells possess a broad repertoire of self- One major concern with any potential tolerogenic therapy is the
reactivity and are found in every tissue of the body. Preclinical durability of the treatment. For most therapies, patients must take
studies have demonstrated that Treg cells from the periphery can their medications on a daily, weekly, or monthly basis. In many
traffic to many tissues and can treat many different models of instances, the inflammatory mechanisms can become resistant to
autoimmune disease, and their persistence within the tissue the pathway modulators, leading to a continued reduction in
depends on the presence of self-antigens and commensal bacteria original efficacy. Living drugs, such as Treg cell therapies, hold
(12–14). Importantly, there is strong evidence that the local tissue the prospect of a one-and-done therapy due to the potential of the
and the infiltrating Tregs have a selective activity for proteins drug to be long-lived—for instance, in the case of CAR-T cell
present in the specific inflamed tissue and, in some instances, the therapies in cancer, June and colleagues have shown that the cells
local microbiota (15–17). While Treg cells from the blood are can survive for more than 10 years (34). However, it is yet to be
primarily naïve-like cells, tissue-resident Tregs are “tuned” to the shown that Tregs have the same longevity, that is, where infectious
specific environment within a given tissue. They can display tolerance may be a distinct advantage. Several pre-clinical studies
different chemokine receptors, transcription factors, and have shown that one consequence of the multifunctional activity of
metabolic properties. Tregs is their ability to recruit and influence other immune cells
In addition to the dominant population of thymically derived (such as myeloid and naive T cells) to differentiate into regulatory
FOXP3+ Treg cells, in the periphery, factors such as TGFb can cell populations (MDSCs, Tr1 cells, and additional Tregs), leading
convert conventional T cells to FOXP3+ Tregs, especially in the gut to an amplification of the immunoregulation such that the long-
(18). Such peripherally “induced” treg cells are not as clearly defined term effects of Treg therapy can be durable. In the end, the depth
at the molecular level as the thymically derived FOXP3+ Treg cells, and the breadth of the therapeutic effect of Tregs will be crucial as
and in pre-clinical models, they do not appear to be as stable (19, new modalities of treatments for people with chronic and
20). In this regard, Dijke et al. performed an examination using debilitating diseases. These therapies must, by necessity, lead
discarded human thymuses as a source of therapeutic Tregs. They ultimately to more than a modest step-wise improvement to the
concluded that thymic Tregs are superior to Tregs derived from standard of care. In addition, a combination of precision medicine
peripheral or cord blood (21). using biomarkers to identify specific patient segments that are most
There are multiple mechanisms by which Treg cells have been likely to respond to the therapy, combined with an ability to take
shown to inhibit immune function (22, 23). Tregs directly suppress advantage of the multiple immune regulatory activities or even
antigen presentation directly through the engagement of checkpoint manipulate the cells to maximize the therapeutic impact, will be
receptors, CTLA-4, and stripping major histocompatibility complex required to fulfil the promise of these new therapies.

2 The tolerogenic potential of Treg inflamed tissues. In contrast, TCR-engineered Treg cells engage
HLA peptide-expressing cells which can be expressed in both the
therapies draining lymph node and the inflammatory sites. Although there
are concerns that broad immunosuppression might occur in the
Treg cells provide unique opportunities for meaningful therapeutic
presence of an acute infection or even in the context of cancer,
differentiation that can be envisioned to tackle the complex
healthy individuals routinely combat pathogens and cancer in the
pathobiologies that underlie most autoimmune and inflammatory
presence of antigen-specific Treg activity. There are examples where
diseases. In contrast to other approaches, Treg therapies have been
the deletion of Tregs in pre-clinical models can improve effector
shown to induce immune tolerance, which means the ability to treat for
immunity, but, in general, Tregs are part of the homeostatic balance
a short period of time resulting in long-term disease-free existence.
of a healthy immune response (48, 49). Data in the NOD mouse
Moreover, Tregs can exert their activities in both lymphoid and non-
model of T1D, in which Treg therapy prevents the autoimmune
lymphoid tissues due to the multiple mechanisms that the cells use to
destruction of pancreatic beta cells, demonstrate that viral
control the immunological responses (35). Indeed, in a variety of
immunity is preserved. In adoptive Treg therapy for clinical bone
preclinical studies, Treg adoptive immunotherapy has been shown to
marrow transplant, immunity toward the cancer is likewise
effectively ameliorate systemic inflammation and specific organ injury.
maintained. In fact, studies have shown that Treg activity at sites
These model systems include a wide variety of autoimmune models,
of pathogenic infection can limit the overall tissue damage, and
including inflammatory bowel diseases (36), type 1 diabetes (T1D)
uncontrolled chronic inflammation can be a driver for cancer in
(37), experimental autoimmune encephalomyelitis (EAE) (38),
several settings (50). Finally, in recent years, low-dose IL-2 and IL-2
collagen-induced arthritis (39), as well as organ transplant rejection
muteins have been tested as therapeutics in a variety of clinical
(40) and non-immune diseases, such as stroke (41) and amyotrophic
settings given their ability to expand Tregs in vivo (51). In many of
lateral sclerosis (ALS) (42), where inflammation plays a role in disease
these studies, there has been two- to fivefold increases in Treg
pathogenesis. Thus, Treg therapies have the potential to significantly
numbers in the circulation, even over a long-term treatment
improve a variety of autoimmune and inflammatory disease activities
regimen, yet there have been no reported increases in pathogen
given the broad applicability of the therapeutic approach.
infections or cancer, suggesting that Tregs are not overly
However, given the bystander properties of Tregs and the
immunosuppressive in the context of infections. Finally, as
recognition that organ-specific Tregs play a critical role in
discussed above, Tregs need to constantly engage antigens to
controlling autoimmunity in the local tissue environment, it was
survive—that is, without ongoing TCR and CD28 engagement,
hypothesized that Tregs with single-antigen specificities might have
Tregs fail to persist. Thus, the current preclinical and human data
an increased specific activity and a greater safety profile. In fact, data
suggest that Treg therapy does not inhibit the natural immunity to
in multiple autoimmune (43) and transplant settings (44) showed
pathogens or cancer.
that Treg cells expressing a single T cell receptor (TCR) suppressed
However, despite the early results suggesting that Tregs will be a
autoreactive, multi-specific Teff cells and led to long-term tolerance.
safe therapy, the potential liability of a broad anti-inflammatory
In fact, preliminary data in humans suggest that alloantigen-specific
activity is also being addressed in a variety of ways. First, several of
Treg cells can be effective in the transplantation setting (45). More
the companies are incorporating tags and kill switches in
recent studies have utilized new synthetic biology approaches to
therapeutic Treg products that might allow the patient to be
provide singular antigen specificity to Tregs, including the
treated with clinically approved monoclonal antibodies and small
introduction of specific TCR (46) and chimeric antigen receptors
molecules to eliminate the adoptively transferred Treg.
(CAR) (47) to target specific inflamed tissues. These studies have
Additionally, efforts are underway to create regulated CARs and
shown not only efficacy in multiple pre-clinical models but also 10–
TCRs that allow the specificity to be modulated in case of adverse
25-fold increased activity when compared with polyclonal Treg
effects of the treatment. In addition, as in the CAR-T space in
populations. Thus, given the polypharmacy nature of Tregs, their
cancer, the use of mRNA or short-lived DNA to deliver the
activity in mechanistically different diseases, and their ability to be
specificity will lead to a transient expression of the CAR or TCR.
engineered, Treg therapeutics represent a new approach to creating
This might be especially useful in conditions in which a severe
deep and durable treatments.
pathology is driven in a large part by inflammation and, once
resolved, does not require ongoing Treg persistence. Examples
include acute respiratory distress syndrome (ARDS) or stroke,
3 Challenges and key features to be among others. Such settings would benefit from or perhaps
addressed in developing Treg require the development of an off-the-shelf Treg cell product that
therapeutics might be short-lived.
3.1 Will Treg therapy promote broad

immunosuppression? 3.2 What will be the potential adverse
events following Treg therapies?
The advantage of CAR-engineered Treg therapy is that it is
directed to a single antigen that is not dependent on HLA Assuming that the immunosuppressive activity of Tregs can be
presentation and thus can be focused on antigens expressed in harnessed effectively to inhibit a disease-specific inflammation,

several other concerns must be addressed in considering a robust in expansion capabilities and potential phenotype changes, such as
cell therapy. Most importantly, the Tregs must maintain their increased interferon production, when compared with Treg derived
phenotype and function stably over time, even in the harshest from healthy individuals. Thus, investigators employed similar tools
inflammatory settings. There are some studies of mouse Tregs as those used for T conventional cell expansion, namely, the use of
showing that, under certain inflammatory conditions, a subset of anti-CD3/anti-CD28-coated beads in combination with IL-2 to
Treg cells can lose Foxp3 expression and produce proinflammatory expand Tregs. In previously published studies, the cells are
cytokines (52, 53). However, other studies suggest that Tregs are routinely expanded 300- to 500-fold in 14 days. The purity of the
quite stable (54). Similar conflicting results have been seen in expanded Treg populations is usually over 90% FOXP3+, and high
patients with autoimmune diseases, usually at the site of levels of multiple phenotypic and functional markers are expressed,
inflammation. This has raised concerns related to cytokine release including CTLA-4, GITR, LAP, and CD25 (55, 80).
syndrome (CRS) as seen in some cancer cell therapy settings using Most importantly, the expanded Tregs have equal or better
CAR-T cells. However, in these settings, CRS primarily occurs in phenotypic and functional activity when compared with freshly
the context of hematological malignancies that carry large antigen isolated Tregs (55). This is especially evident in Tregs isolated from
loads within the blood. Moreover, in many settings (such as patients with autoimmune diseases, where genetic and/or
transplantation and autoimmunity), the patients are on other environmental influences often result in Tregs that are less
immunosuppressive drugs known to moderate the effector effective in multiple suppressive activities and, in many cases, less
cytokine production. In fact, the human clinical data suggests that stable as read out by reduced FOXP3, CD25, and CTLA-4
Treg therapy does not promote CRS in a fashion like that of CAR expression as well as less ability to respond to growth factors such
Teff cells. The stability of Tregs is maintained, and Treg function as IL-2. However, the process of expansion generally leads to a
has been shown to reduce inflammation and have less infection population expressing higher levels of key functional markers and
events compared with immunosuppression. That said, as “repair” of functional defects. In fact, in early studies of IL-2
highlighted below, there are multiple academic and company therapies, the repair of endogenous Treg’s inability to respond
efforts to ensure the stability of these cells (Table 1). effectively to IL-2 was not only reversed but found to be stable
even a year after the discontinuation of cytokine therapy (81). Most
importantly, the expansion of the Tregs derived from peripheral
4 Lessons learned from clinical trials blood as well as cord blood was highly stable when examined over
time after Treg transfer into patients—for instance, an analysis of
Over the last 15 years, there have been more than 25 early-stage circulating adoptively transferred Treg cells demonstrated the
clinical trials conducted, mostly in an academic setting, to continued expression of key functional and phenotypic markers
investigate the safety and biological activity of Tregs in diseases for at least a year post-transfer (55). The results of an extensive bulk
ranging from autoimmunity, organ transplantation, graft versus and single-cell T cell receptor analysis suggest that the expanded
host, and other inflammatory diseases. An overall summary of a population of Tregs remains broad after expansion. Thus, it is not
small “subset” of clinical trials is found in Table 2. These trials have surprising that the current efforts to develop Treg populations for
led to clear evidence that the therapy is feasible, can be administered therapy have begun to employ novel genetic approaches to
safely, and is tolerated for a long term. To date, there have been no homogenize and maximize Treg phenotype and functional
life-threatening adverse events, including no observed increase in activity. This includes the use of antigen-specific receptors,
infections or evidence of cancer development (either the including CARs or TCRs, to focus the tissue specificity of the
administered cells or in the patients). In fact, as confidence in the Treg product.
therapy has grown, more than 10 companies have initiated Treg
programs using both conventional and engineered Treg cells. The
first industry-sponsored trials are underway, with efficacy studies on 4.2 Experiences in Treg stability in vitro and
the horizon, to assess the therapeutic potential of these novel cell in vivo
therapies. That said, there are key lessons that have been learned
from past studies that inform future efforts and have created Despite the previously described limited animal studies, to date,
potential opportunities to improve the therapies. there is no evidence of Treg instability in any human clinical
studies. This result may be due to multiple reasons. First, the
expansion process is much more robust in human versus mouse,
4.1 Experiences in Treg expansion and the likely preferential expansion of thymus-derived CD45RA
naïve Tregs using current approaches may selectively result in the
Fundamental research on Tregs demonstrated that the cells expansion of the most stable Treg populations. In fact, the literature
signal in a similar fashion as conventional T cells, requiring both a suggests that the majority of “unstable” Tregs are likely derived
TCR and CD28 co-stimulatory signals for maximal expansion. In from induced peripheral Tregs, generated in the periphery in vivo
addition, Tregs have been shown to be exquisitely dependent on the upon encounter with TGFb and other Treg-promoting cytokines
IL-2 growth factor for both expansion and survival. Moreover, (19, 22). Moreover, in vitro studies suggest that human Tregs
Tregs coming from individuals with autoimmune or other isolated and expanded under the conditions noted above are
inflammatory diseases can be dysfunctional, leading to differences “resistant” to certain inflammatory cytokines suspected of

TABLE 1 Companies involved in regulatory cell adoptive immunotherapy.
Company Cell type Cell source Modification Lead indication

Sonoma Biotherapeutics FOXP3+ Treg cells Autologous peripheral blood Citp-CAR RA
PolTREG FOXP3+ Treg cells Autologous peripheral blood MOG-CAR T1D
Quell Therapeutics FOXP3+ Treg cells Autologous peripheral blood FOXP3 and HLA-A2-CAR Liver transplant
Abata Therapeutics FOXP3+ Treg cells Autologous peripheral blood MBP-TCR MS
Sangamo Therapeutics FOXP3+ Treg cells Autologous peripheral blood HLA-A2-CAR Kidney transplant
Coya Therapeutics FOXP3+ Treg cells Autologous peripheral blood NONE ALS
TeraImmune FOXP3+ Treg cells Autologous peripheral blood FVIII-TCR Hemophilia
Tract Therapeutics FOXP3+ Treg cells Autologous peripheral blood NONE Kidney transplant
Cellenkos FOXP3+ Treg cells Allogeneic cord blood NONE ARDS
GentiBio CD4+ T cells Autologous peripheral blood FOXP3 and islet-specific TCR T1D
Tr1x Tr1 cells Adult peripheral blood Undisclosed Undisclosed
Cabaletta Peripheral blood T cells Autologous and Allogeneic T cells CD19-CAR Pemphigus vulgaris
Kyverna Peripheral blood T cells Autologous and Allogeneic peripheral regulated CD19-CAR B cell autoimmunity
blood T cells
Atara Peripheral blood T cells Adult peripheral blood EBV-reactive T cells PPMS
ORCA Bio FOXP3+ Treg cells Allogeneic peripheral blood NONE GvHD
promoting instability in rodent Tregs—for instance, culturing was seemingly apparent in the first cord blood-derived Treg study
human Tregs with IL-6 or TNFa (both present in highly in GvHD, where the cell product had a shorter duration in vivo
inflamed tissues) routinely results in increased expansion and when compared with autologous cells in other settings (71, 72).
function, which are likely due to the upregulation of the TNFR2 However, there are circumstances where an off-the-shelf allogeneic
receptor on the expanded Tregs (52, 82, 83). Finally, it cannot be product may be required, such as in the treatment of acute ARDS,
ruled out that unstable Tregs are only present in tissues subject to such as COVID, where the cells need to be administered
extreme inflammatory milieu; however, as will be noted below, in immediately (79). As will be highlighted elsewhere, new
several clinical settings, biopsy analysis post-transfer showed that approaches to both genetically engineered Tregs as well as
Tregs in target tissues expressed higher levels of FOXP3 and CTLA- potential stem cell-derived Treg products may enable the
4 at 3 months post-transfer of Treg. Thus, it is likely that current disruption of core allogeneic proteins, such as MHC antigens, and
expansion technologies result in the production of highly purified avoid rejection. Interestingly, the inherent alloreactivity of Tregs has
Treg populations that are stable in disease settings (including been exploited in the organ and islet transplant setting, where
autoimmune lesions and inflamed transplanted organs). investigators have either relied on the resident alloreactive Tregs in
At present, the majority of Treg products generated for clinical the polyclonal Treg population or developed approaches to
application are autologous. For autoimmune diseases, this means selectively expand the alloreactive cells prior to transfer. This
that the expansion protocol needs to consider the possibility of approach has great potential but may be superseded by the
contaminated activated Teff cells, which express high levels of CD25 genetic engineering approaches described below.
like Tregs, as well as genetically and environmentally induced Treg It should be noted that, in some settings, the challenge of high
dysfunction. One approach to avoid these potential issues has been purity and stability has required additional precautions in the
to use allogeneic Tregs derived from healthy individuals or development of Treg products—for instance, Teff cell
umbilical cord blood (UCB) (72, 79). UCB Tregs are contamination has been an issue due to inadequate separation
overwhelmingly naïve and have a very broad repertoire (84). In technology and/or patient cell surface phenotype variation. As
some cases, such as in the suppression of graft versus host disease, noted previously, preclinical studies suggest that peripherally
this has the added advantage of 5%–15% of the Tregs having derived Tregs, especially Helios- Tregs, are less stable than their
alloreactivity and thus recognize the class II MHC of the host. thymus-derived counterparts. Thus, depending on the source of
However, there are multiple risks associated with this approach. Tregs cells, the potential for Teff or Teff-like activity is possible. To
First, should the Treg cells become unstable in patients, there is an avoid some of the challenges to isolating and expanding highly
expanded repertoire that can target the allogeneic tissue, be it host stable and pure Tregs, investigators have taken advantage of the
or transplanted tissues. Secondly, the host immune system can selective ability of rapamycin to suppress effector T cell growth
recognize and destroy the adoptively transferred Treg product. This while preserving Treg expansion. The differential mechanistic role

TABLE 2 Summary of a subset of clinical trials with Tregs in multiple diseases.
Study ID Study title Study Reference

Phase Product
(www.clinicaltrials.com) (sponsor) status number
Autoimmune Diseases
T1DM immunotherapy using

Expanded autologous CD4+CD127lo/
NCT01210664 CD4+CD127lo/−CD25+ polyclonal Tregs 1 − Completed (55)
CD25+ polyclonal Treg
(University of California San Francisco)
Autologous polyclonal Tregs for lupus Expanded autologous CD4+CD127lo/

NCT02428309 1 − Completed (56)
(University of California San Francisco) CD25+ polyclonal Treg
Safety and Efficacy of CLBS03 in

adolescents with recent onset type 1
NCT02691247 diabetes 2 − Completed (57)
(The Sanford Project T-Rex Study - Lisata
Therapeutics, Inc.)
T1DM immunotherapy using polyclonal

NCT02772679 Tregs + IL-2 (TILT) 1 − Completed (58)
Safety study and therapeutic effects of

umbilical cord blood Treg on autoimmune
Expanded umbilical cord Tregs CD25+
NCT02932826 diabetes 1/2 Active NA
CD4+ T cells
(Second Xiangya Hospital of Central South
University)
Safety and efficacy of antigen-specific

regulatory T cell therapy for patients with Expanded autologous CD4+CD127lo/
NCT03185000 1/2 − Completed (59, 60)
refractory Crohn’s disease CD45RA+/CD25+ polyclonal Treg
(King’s College London)
Polyclonal regulatory T cells (PolyTregs)

NCT03239470 for pemphigus 1 − Completed NA
Autologous ex vivo expanded regulatory T

Expanded autologous CD25+ CD4+ plus
NCT04691232 (University of Erlangen-Nürnberg Medical 1 Active (61)
rapamycin
School)
Cellular therapy of type 1 diabetes with T

Expanded autologous
ISRCTN06128462 regulatory cells 1 Completed (62)
CD4+CD127lo/−CD25+ polyclonal Treg
(Medical University of Gdansk)
Islet transplantation for type 1 diabetes
PolyTreg immunotherapy in islet

NCT03444064 transplantation 1 − Completed NA*
(University of Alberta)
Infusion of autologous T regulatory cells

(Treg) at the time of treatment of allogenic Enriched autologous, CD4+CD127lo/
−
NCT04820270 islets NA CD25+ polyclonal Treg Active NA
(The Nordic Network For Clinical Islet (No expansion)
Transplantation)
Cryopreserved polyclonal regulatory T cell

Cryopreserved expanded autologous
NCT05349591 immunotherapy in islet transplantation 1 Active NA
CD4+CD127lo/−CD25+ polyclonal Treg
(University of Alberta)
Kidney transplantation
Treatment of children with kidney

transplants by Injection of
CD4+CD25+FoxP3+ T cells to prevent −
NCT01446484 1/2 CD25+ Polyclonal Treg Active NA
organ rejection
with/without Campath
(Pirogov Russian National Research
Medical University)
Treg adoptive therapy for Subclinical Expanded autologous CD4+CD127lo/

Inflammation in Kidney Transplantation CD25+ FOXP3+ Tregs
(Continued)

TABLE 2 Continued

Phase Product
(TASK)
Infusion of T-regulatory cells in kidney

NCT02091232 transplant recipients (The ONE Study) 1 − Completed (64)
CD25+ FOXP3+ Tregs
(Massachusetts General Hospital)
The ONE Study (UK Treg Trial)

Expanded autologous regulatory T cell
NCT02129881 (Guy’s and St Thomas’ NHS Foundation 1 Completed (64)
product
Trust)
Trial of Adoptive Immunotherapy with

TRACT to Prevent Rejection in Living Expanded autologous CD4+CD127lo/
Donor Kidney Transplant Recipients CD25+ FOXP3+ Tregs
(TRACT) (Northwestern University)
Donor-alloantigen-reactive regulatory T cell

(darTreg) therapy in renal transplantation Expanded autologous alloantigen-specific
NCT02244801 1 Completed (64)
(The ONE Study) CD4+CD25+FOXP3+ Tregs
The ONE Study trial Expanded autologous polyclonal

NCT02371434 1/2 Completed (64, 66)
(Charité University) CD4+CD25+FOXP3+ polyclonal Tregs
Treg Therapy in Subclinical Inflammation

NCT02711826 in Kidney Transplantation (TASK) 1/2 − Completed NA
CD25+ FOXP3+ polyclonal Tregs
A pilot study using autologous regulatory T

Cell Infusion Zortress (everolimus) in renal Expanded autologous CD4+CD127lo/
NCT03284242 1 − Active NA
transplant recipients CD25+ FOXP3+ Tregs + Everolimus
(University of Kentucky)

therapy and tocilizumab together with Expanded autologous regulatory T cell
NCT03867617 donor BM infusion in HLA-mismatched 1/2 therapy, tocilizumab, and donor bone Active NA
living donor kidney transplant recipients marrow
(Medical University of Vienna)
TLI, TBI, ATG, and hematopoietic stem

cell transplantation and recipient Tregs
NCT03943238 therapy in living donor kidney 1 Expanded autologous Tregs Active NA
transplantation
(Stanford University)
Safety and tolerability study of chimeric

antigen receptor T-reg cell therapy in living
HLA-A2 CAR CD4+/CD45RA+/CD25+/
NCT04817774 donor renal transplant recipients 1/2 Active NA
CD127lo/-
(STeadfast)
(Sangamo Therapeutics)
TWO study: cell therapy trial in renal

ISRCTN Expanded autologous.
transplantation 2 Active (67)
11038572 polyclonal Tregs
(University of Oxford)
Liver transplantation and other treatments
Safety study of using regulatory T cells

induce liver transplantation tolerance Autologous Treg cells from thymic tissue
NCT01624077 1 Unknown NA
(Treg) (thyTreg), CD25+ Foxp3+ cells
(Nanjing Medical University)
Safety and Efficacy Study of Regulatory T

Cell Therapy in Liver Transplant Patients
NCT02166177 (ThRIL) 1 Completed (68)
product
(Guy’s and St Thomas’ NHS Foundation
Trust)
(Continued)

TABLE 2 Continued

Phase Product
Donor-alloantigen- reactive regulatory T
Cell (darTregs) in Liver Transplantation Expanded autologous donor-alloantigen-
NCT02188719 (deLTa) 1/2 reactive Tregs Terminated NA
(NIAID/University of California San (darTregs)
Francisco)
Donor alloantigen reactive Tregs (darTregs)

for calcineurin inhibitor (CNI) reduction Alloantigen-expanded autologous
NCT02474199 (ARTEMIS) 1/2 CD4+CD127lo/−CD25+ Completed (69)
(NIAID/University of California San Treg cells
Francisco)
Expanded autologous
Liver transplantation with Tregs
donor alloantigen-reactive
NCT03577431 (LITTMUS MGH) 1/2 Terminated NA
CD4+CD127lo/−CD25+
(NIAID/Mass Gen Hosp)
Treg cells (arTregs)
Liver transplantation with Tregs Expanded autologous

(LITTMUS-UCSF) donor alloantigen-reactive
NCT03654040 1/2 Terminated NA
(NIAID/University of California San CD4+CD127lo/−CD25+
Francisco) Treg cells (arTregs)
Cell Therapy With Treg Cells Obtained

From Thymic Tissue (thyTreg) to Prevent
Thymic tissue (thyTreg)-derived CD25+
NCT04924491 Rejection in Heart Transplant in Children 1 Active (70)
Foxp3+ cells
(Hospital General Universitario Gregorio
Marañon)
Safety and clinical activity of QEL-001 in

A2-mismatch liver transplant patients
NCT05234190 1/2 HLA-A2 CAR-Treg Active NA
(LIBERATE)
(Quell Therapeutics Limited)
Tolerance induction by a regulatory T cell-

based cell therapy in living donor liver
UMIN-000015789 transplantation 1/2 Donor-reactive Treg-enriched cell product Active NA
(Masonic Cancer Center, University of
Minnesota)
Graft versus host disease
T-Regulatory Cell Infusion Post Umbilical

Cord Blood Transplant in Patients With
Umbilical cord blood expanded
NCT00602693 Advanced Hematologic Cancer 1 Completed (71)
CD4+CD25+ Tregs
Minnesota)
First-in-human phase 1 trial of induced

Human CD4+25- T cells cultured in IL-2,
Treg cells for graft-versus-host disease
rapamycin, and transforming growth factor
NCT01634217 prophylaxis in HLA-matched siblings 1 Completed (72)
b (TGFb) along with anti-CD3-loaded
artificial antigen-presenting cells
Minnesota)
Phase 1-2 MAHCT w/ T Cell Depleted

Graft w/ Simultaneous Infusion
NCT01660607 1 Donor regulatory T-cells Completed (73, 74)
Conventional and Regulatory T Cell
(Stanford University)
Ex-vivo Expanded Donor Regulatory T

Cells for Prevention of Acute Graft-Versus-
NCT01795573 Host Disease 1 Expanded donor regulatory T-cells Completed (73)
(H. Lee Moffitt Cancer Center and
Research Institute)
Donor regulatory T cell infusion in patients

with chronic graft-versus-host disease
NCT01903473 1 CD4+CD25+FoxP3+ T cells + IL-2 + Rapa Terminated NA
(GVHD)
(University of Liege)
(Continued)

TABLE 2 Continued

Phase Product
Trial of regulatory T-cells plus low-dose
interleukin-2 for steroid-refractory chronic CD4+CD127lo/−CD25+ Treg + low dose IL-
NCT01937468 1 Active NA
graft-versus-host disease 2
(Dana-Farber Cancer Institute)
Donor regulatory T cells in treating

patients with visceral acute graft-versus-host
NCT02526329 1 CD4+CD127lo/−CD25+ Treg cells Suspended NA
disease after stem cell transplant
(Dana-Farber Cancer Institute)
Multiple donor Treg DLI for severe

Non-expanded purified Tregs (CD8 and
NCT02749084 refractory chronic GVHD (TREG2015001) 1 Completed NA
CD29 negative, CD25hi cells)
(Universitaria di Bologna)
Donor regulatory T-cells for steroid-

refractory chronic graft-versus-host disease
NCT03683498 (GVHD-TReG) 1 Expanded donor regulatory T-cells Completed (75)
(Fundació n Pú blica Andaluza para la
gestió n de la Investigació n en Sevilla)
A Study of Engineered Donor Grafts

(TregGraft/Orca-T) in Recipients
Non-expanded purified Tregs
NCT04013685 Undergoing Allogeneic Transplantation for 1 Active (76)
CD4+CD127lo/−CD25+
Hematologic Malignancies
(Orca Biosystems, Inc.)
Other diseases
Pilot study for cell-based therapies in

NCT01087515 patients with asthma 1 Fresh Treg Completed NA
(Hannover Medical School)
T-Regulatory Cells in Amyotrophic Lateral

NCT03241784 CD4+CD25+FOXP3+ Tregs with low-dose
Sclerosis 1 Active (77, 78)
NCT04055623 IL-2 in ALS
(The Methodist Hospital Research Institute)
Regulatory T Cell infuSion fOr Lung Injury

NCT04468971 Due to COVID-19 PnEumonia (RESOLVE) 1 Umbilical cord blood-derived Treg Completed (79)
(Cellenkos, Inc.)
Regulatory T cells for amyotrophic lateral

NCT05695521 sclerosis (REGALS) 1 Umbilical cord blood-derived Treg Active NA
(Cellenkos, Inc.)
Tregs for the treatment of acute respiratory

distress syndrome (ARDS) associated with Cryopreserved ex vivo expanded polyclonal
NCT05027815 1 Terminated NA
COVID-19 (regARDS) CD4+CD127lo/-CD25+ T regulatory cells
NA, not available.
of mTORC1 and mTORC2 in Treg versus Teff cells results in the there have been only a limited number of presentations and no
rapamycin-induced increased stability of Tregs, selectively published phase 2 studies using polyclonal or antigen-specific
inhibiting Teff cell expansion (85, 86). In the future, new genetic engineered Tregs. In the case of the T1D study, NCT02691247,
engineering approaches that are now being developed will both the company, Caladrius, released a press release saying that the trial
increase the ability to isolate highly pure populations of Tregs as did not meet its primary endpoint. In the case of the ALS study,
well as provide safety switches and a more stable Treg population in recruitment was impacted by COVID-19, and no meaningful
settings where instability remains a risk. evaluation could be made. The early Treg studies were performed
in patients experiencing acute or chronic GvHD, using either cord
blood allogeneic or adult-derived autologous Tregs, based on
4.3 Experiences in early phase 1/2 trials supportive pre-clinical mouse data. In the first study reported out
of Tregs of Poland, ex vivo-expanded CD4+CD127lo/-CD25+ Treg cells were
infused into patients with active GvHD (87). A similar study was
As noted in Table 2, there have been over 25 phase 1/2 clinical performed with cord blood-derived Tregs by Wagner and
trials to test the efficacy of autologous and allogeneic polyclonal colleagues (71, 72). The studies showed that pure Tregs could be
adult and UCB-derived Tregs in a variety of diseases. However, isolated, expanded, and transferred into patients with the disease. It

is also worth noting that one of the impetuses for using cord blood- liver, and, most recently, islet transplantation (Table 2). Once again,
derived Tregs was the greater ease in purifying the population due the therapies have been deemed safe, and in several instances, there
to the distinctly high levels of CD25 on these cells. However, has been biomarker evidence (such as reduced effector cytokine
although the typical expansion time—14 days—of adult-derived production in the urine of patients treated at the time of protocol
autologous Tregs is sufficient to generate target doses, in the case of biopsy proven inflammation) suggesting biologic activity. One
cord blood, the small number of Treg at the start of culture required biomarker study, conducted in the liver transplant setting,
more extensive expansion, often three to four cycles (71, 72). This showed that the therapy was safe, and there was an altered
can lead to increased instability of the product and, in some cases, effector cytokine profile in the blood. Another example of a small
diversion to a Th2 (IL-4)-producing Treg functional state. In the safety trial was a study conducted by Appel and colleagues looking
cord blood-derived allogeneic Treg study, there was an early at the effect of Treg therapy in patients with ALS. The pre-clinical
suggestion of efficacy. This approach in GvHD is now being results had suggested that, even in this non-immune-mediated
examined, with or without low-dose IL-2, in a formal phase II disease, the control of the associated inflammation could impact
study to determine the efficacy and biomarker activity. disease progression. In the study of Appel et al., three patients with
highly advanced ALS were treated with multiple courses of
autologous Tregs along with low-dose IL-2 and monitored for
4.4 Autologous Tregs have been tested in a disease progression (77, 78). There were some suggestions that
variety of clinical settings the disease progression slowed during immunotherapy; however, it
was not clear what role the IL-2 was playing in the
The first clinical studies of autologous Tregs in autoimmunity therapeutic effects.
were conducted in patients with T1D. These studies included One of the challenges in the early Treg interventional studies in
children and adults recently diagnosed with the disease and were autoimmunity has been the small number of patients enrolled in
based on extensive preclinical data that suggested that Tregs were any given trial and the limited biomarker analyses. Although
able to reverse diabetes in a spontaneous animal model of T1D, the biopsies were possible in the transplant studies, any effects were
NOD mouse, if given soon after the diagnosis. The protocol for Treg difficult to interpret given the concomitant treatment of patients
therapy in T1D patients required the transfer of ex vivo-expanded with multiple immune-suppressive drugs. Furthermore, in many
autologous polyclonal CD4+CD127lo/-CD25+ Treg cells within 6 settings, biopsies were not obtained due to logistical and safety
weeks of diagnosis (55, 58, 62). In these studies, the c-peptide levels, reasons. However, several phase 1 studies in autoimmune settings
as a readout of insulin production, were monitored for up to 2 years. have been performed where serial biopsies could be performed. In
While there was a suggestion of efficacy, the studies were small one published study, a patient with cutaneous lupus erythematosus
and uncontrolled. In the study conducted in Poland, the treated was treated with 1 × 108 ex vivo-expanded CD4+CD127lo/-CD25+
children had higher c-peptide levels and lower insulin requirements Treg cells. The affected skin was biopsied both on day 0 (prior to
than the historical data in untreated children (62). In the first phase Treg injection) and at 12 weeks. The biopsy was assessed for
I clinical trial conducted in the United States, ex vivo-expanded changes in Tregs and Teff cells. As noted above, there was a
polyclonal Treg cells were infused into 14 patients with new-onset significant increase in the number of Tregs when the 12-week
T1D in doses ranging from 5 × 106 to 2.6 × 109 cells. The infusions biopsy was compared with that of day 0. More importantly, the
were well tolerated, and the c-peptide levels in most patients level of FOXP3 was significantly increased in the cells isolated from
remained stable for 1 year, although efficacy could not be the biopsy at week 12. In contrast, at the same time point, there was
conclusively shown (55). Notably, the cell pharmacokinetic an approximately 75% reduction in interferon-producing CD4+
analysis of Treg cells labeled with deuterium during ex vivo Teff cells when compared with the day 0 biopsy. Interestingly, in
expansion showed that a subset of the infused Treg cells persisted some patients, IL-17-producing T cells were preserved. Thus, a shift
in peripheral blood for at least 1 year and that the Treg cells from IFNg to IL-17 may lead to reduced inflammation and
remained phenotypically stable after the infusion. A phase II clinical increased tissue repair. Similar results were observed in a phase 1
trial performed by Caladrius Biosciences, in which 113 newly trial in patients with pemphigus (unpublished results/manuscript in
diagnosed (less than 100 days post-T1D diagnosis) adolescents preparation). In one patient with paired samples, the percentage of
with T1D were either untreated or infused with autologous ex Tregs cells increased in the skin biopsy at week 12, similar to that
vivo polyclonally expanded Treg cells (56). The treated group failed observed in the lupus patient (56). There was a significant decrease
to show a clinical effect of the therapy, i.e., preservation of c-peptide in both the number of Teff cells and their ability to produce
production 1 year after the infusion. While the highly enrolled study interferon at 12 weeks. These results together suggest that the
was found to be safe, with no major serious adverse events reported, Treg therapy had an impact on the microenvironment of the
the study highlighted the need to consider a more targeted antigen- pathogenic lesion, resulting in diminished inflammatory processes.
specific Treg population to gain true efficacy as seen in the pre- There have been several studies looking at alloantigen-reactive
clinical studies. Tregs based on preclinical efficacy studies. Overall, Treg cell
Recent years have seen an increased number of polyclonal Tregs infusions have been safe and well tolerated. A challenge in these
trials in multiple different clinical settings. There have been multiple studies, however, has been in manufacturing the cells, potentially
studies of autologous Treg therapy in patients receiving kidney, because these patients are chronically immunosuppressed, which

alters the Tregs both quantitatively and qualitatively. In addition, it 5.2 Enhancing efficacy
appears that the alloreactive Tregs can be sequestered in the target
tissue—in the cited instance, the liver—resulting in a reduced Most new anti-inflammatory drugs fail because of two main
precursor frequency in the blood (69). reasons: lack of clinical safety and lack of clinical efficacy. If the early
clinical work with polyclonal Tregs continues to be a guide, Treg
therapies are likely to be safe. As such, the lack of clinical efficacy
5 Opportunities to enhance Treg cell remains the biggest unknown in the field today. Harnessing the
natural homing and tissue-specific regulatory mechanisms of Tregs
therapy as well as engineering new mechanisms of immune regulation may
be the key to achieving a meaningful and durable clinical effect.
5.1 Enhanced antigen-specific activation
Here are some key areas of focus for the field.
Controlling the local activation and proliferation of Tregs is key

to maximize their therapeutic potential. Because Treg cells require 5.2.1 Cytokines
engagement through the TCR/CD28 for Treg cell survival and As potent immune regulators, cytokines are particularly
regulatory function, harnessing these physiological signals is an important controllers of Treg-mediated effects and provide many
attractive option for controlling Treg activation. TCR or CAR opportunities for engineered products. Enhancing or stabilizing the
engineering has the potential to direct Treg activation in an secretion of anti-inflammatory cytokines (such as IL-10 and TGFb)
antigen- or tissue-restricted way, thus leading to the control of may, in certain indications, enhance the anti-inflammatory actions
Treg activation, and only in the tissues of particular interest. For and better control the magnitude of tissue damage. Tregs could also
TCR-Treg development, the identification of tissue-specific be engineered to express dominant negative or decoy receptors (cell
antigen/peptide targets is critical. With CD4+ restriction, class II surface or secreted) negating the effects of local and systemic pro-
peptides are the most obvious choice, but the tools to identify and inflammatory cytokines. The remarkable ability of Tregs to home to
characterize class II-restricted peptides fall far behind those of class sites of inflammation could also be harnessed to deliver locally
I. For class I-restricted TCRs, it is possible to isolate receptors that acting anti-inflammatory molecules to the right cell at the right
are independent of CD8 co-receptor activity or the Tregs can be time. This may give new life to dozens of therapies whose off-tissue
genetically modified to express CD8 which would allow MHC class pharmacology precluded further clinical development. Tregs could
I-restricted antigens to become viable targets for TCR-engineered also be engineered to respond to certain cytokine stimuli in new
Tregs. From an engineering perspective, endogenous TCRs can be ways—for instance, there may be an opportunity to control Treg
left intact if engineered TCRs are still able to preferentially pair and activation, persistence, and responsiveness to local inflammatory
out compete endogenous receptor complexes for TCR signaling signals. Logic gated systems have gained popularity in the CAR-T
components needed to transduce the signals required. Forcing a effector space in the past few years by harnessing tissue-specific
very high level of expression of the engineered TCRs on the cell signals (including cytokines) to turn on (or shut down) proliferation
surface, utilizing known mutations to induce preferential chain or effector functions (89, 90).
pairing, capturing, or tethering them to the cell surface, and
enhancing recycling are all viable methods being explored in 5.2.2 Tissue-protective and repair factors
the field. In addition to the well-documented anti-inflammatory effects of
While using TCRs for Treg recognition is appropriate in many Tregs, it is becoming increasingly clear just how important these
settings—particularly those with a strong HLA association, another cells can be in maintaining tissue homeostasis (32, 33)—for
approach is the use of chimeric antigen receptors (CARs) which can example, amphiregulin (shown to be secreted by Tregs) can halt
avoid HLA restriction completely. One advantage is that the CAR tissue damage and encourage tissue repair and the reestablishment
affinities can be finely tuned to change both the level of antigen- of normal tissue function. Creating Tregs capable of producing or
specific Treg activation as well as the amount of off-target “tonic” enhancing the natural tissue-protective and repair factors within a
signaling. Moreover, the CARs can be engineered to alter the site specific tissue will increase the potential that Treg therapies will lead
and timing of activation. Whereas TCR-restricted specificities to transformative, durable treatments—for example, logic gated
would need cell–cell contact, CAR activation can also be driven systems could be used to “switch” Treg cells from an anti-
by soluble factors, tissue matrix, or exogenous ligands. Advances in inflammatory function to a repair function once inflammation
high throughput binder generation with exquisite specificity and has subsided.
selectivity have enabled the generation of novel CARs with
confirmational epitopes, overcoming some of the challenges to 5.2.2.1 Homing
the identification of tissues and disease-specific reactivities. CARs As mentioned above, Treg cells have the capacity to enter most
can be re-engineered to have TCR-like properties and even tissues of the body, but they most efficiently enter sites of
recognize specific peptide/MHC complexes, bypassing the need inflammation. In some instances, this may lead to unwanted off-
for TCR engineering while capturing the natural site of Treg disease effects and/or reduce the number of transferred cells homing
activation at the cell surface of antigen-presenting cells (88). to important sites of action. To induce tissue homing bias, it is

possible to select for unique tissue homing subsets or to engineer a destabilized Treg product. Nonetheless, due to the lack of relevant
homing capabilities to bulk populations of Tregs to send them to long-term model systems for using human Treg cells, there is
specific locations—for example, in treating inflammatory bowel considerable interest in maximizing Treg product stability. There
diseases, it may be advantageous to select for a4b7-expressing Tregs are several approaches to further enhance Treg stability, ranging from
capable of homing to MAdCAM-1-expressing endothelium in sites overt over-expression of FOXP3 to modification of endogenous
of tissue inflammation and in Peyer’s patches and mesenteric lymph genomic loci. One such approach is epigenetic modification of the
nodes (91). This may enhance the total tissue load of Treg and FOXP3 locus to stabilize Treg phenotype and functional activity (93).
enhance both the anti-inflammatory and the pro-repair By targeting the CAR or TCR specificity to the FOXP3 locus, the
mechanisms. Alternatively, one could select against or modify stability of the Tregs would be linked to the expression of an
homing receptors that aid the transit of Tregs to other tissues and introduced TCR or CAR. Other pathways (including IL-2
sites of cell clearance—for example, in the liver (a major site of T cell modulation) may also be effective at promoting FOXP3 expression
clearance). Homing receptors are interesting targets for logic gated and lineage stability. In all cases, stability will need to be assessed in
systems as described above. A homing receptor-mediated logic gate the context of a pro-inflammatory environment.
could be used to selectively kill cells homing to unwanted sites of
action (for example, the CNS) and avoid any consequences of 5.3.3 Allogenic Treg products
tissue-specific immunosuppression in sites of ongoing infection or There is an increasing interest in the use of allogenic cell
increased tumor risk. In this regard, Hoeppli et al. demonstrated products, including gene-modified mature Tregs and iPSC-
that the migration capacity of human Tregs can be tailored by derived off-the-shelf cell platforms. Beyond the advantages from a
adding cytokines (IL-12 and IFNg) and or/metabolites (retinoic cost-of-goods and deliverability perspective, a key advantage is the
acid) to culture conditions during in vitro expansion (92). ability to ensure product consistency and limit patient-to-patient
variability. Additionally, the ability to use healthy donor cells would
eliminate any patient segment considerations on Treg quality or
5.3 Durability and stability quantity. Finally, as noted earlier, an off-the-shelf product would be
more amenable to rapid dosing for acute indications such as ARDS
5.3.1 Enhanced growth factor independence or stroke in which it would be impossible to make autologous Tregs
As noted above, Treg cells are highly dependent on growth factors quickly enough to impact pathology.
for survival, activation, and proliferation. Although many cytokines Another significant advantage to an allogeneic product,
and growth factors can influence Tregs, IL-2 is a critical target for particularly based on iPSC technology, would be the ability to make
advanced genetic engineering. As discussed above, in proinflammatory several (maybe even dozens) of genetic modifications in a serial
conditions, particularly those mediated by T cells, IL-2 is in abundance, manner. It is conceivable to generate “panels” of engineered cells
and Treg are readily able to harness IL-2 through their high-affinity IL- with, for example, differing specificities and then match a particular
2 receptor complexes. This has a twofold effect: it stimulates Treg antigen-specific Treg with a patient segment. Each modification can be
expansion (and function) while capturing IL-2 and thus prevent Teff thoroughly characterized in isolation or in conjunction with other
cells in the environment from receiving a needed growth factor, leading modifications before clinical studies. Allogenic products may be more
to activation-induced cell death. In those indications where there is amenable to repeat dosing as well as exploring combination therapies.
insufficient IL-2—for example, in non-T cell driven inflammation—it Despite the promise of allogenic products, CAR-T effector clinical
may be necessary to provide IL-2 signals directly. As noted previously, development is just beginning. Both engineering and validation have
in the ALS clinical trials, exogenous IL-2 was provided as support for proven more difficult than expected.
expanded Treg cells following infusion. Tregs may be engineered to
provide a cell-intrinsic IL-2 signal in several ways, including IL-2
secretion, IL-2 tethering, and modulation of intracellular signaling 6 Concluding remarks
pathways. Given the central importance of IL-2 signaling in Tregs,
coupled with the inconsistent presence of IL-2 in inflammatory disease Autoimmune disorders arise from defects in immune tolerance
of interest, it is likely that IL-2 will remain a key focus for Treg growth and affect more than 50 million people in the United States and
and persistence. more than 4% of the world’s population. Autoimmune disorders
have a high impact on an individual’s morbidity and mortality as
5.3.2 Enhancing stability well as their quality of life, given that their chronicity, various organ
Although data surrounding the plasticity of adoptively manifestations, and association with co-morbidities are common
transferred human Tregs is lacking, in certain disease settings, and devastating for many sufferers. Despite several transformative
Tregs can become destabilized (as shown by changes in FOXP3 medicines that have improved many autoimmune diseases, most
expression and genome methylation status). As Treg therapies utilize patients do not adequately respond to existing therapies. Long-term
TCRs and CARs to confer disease and/or tissue specificity, ensuring drug therapy is required to maintain long-term efficacy; thus, there
the stability of any Treg product becomes paramount. As noted remains a major unmet medical need. Importantly, the goal of
previously, existing clinical trials, even those using alloantigen- achieving true immune tolerance by re-establishing immune
specific expanded Treg cells, have proven safe without evidence of homeostasis remains unrealized.

As summarized in this review, Tregs, which constitute only a Treg stability and durability, and the development of novel
small percentage of circulating T cells, play a pivotal role in techniques to build new and enhanced activities. Although the
establishing and maintaining peripheral tolerance, preventing field of Treg cell therapies is only at the “end of the beginning”,
autoimmune diseases, and limiting chronic inflammatory diseases many have suggested that this living drug may finally enable
(94–97). In fact, Treg cells are now widely regarded as the primary achieving immune tolerance—the holy grail of immunotherapy.
cells involved in the persistence of peripheral tolerance. Their
essential role is based on the cells’ multiple functionalities ranging
from the production of key immunosuppressive cytokines and Author contributions
metabolites to the cell surface expression of key checkpoint
molecules to control antigen presentation and metabolism. JAB, BM, JB and FR contributed to the writing, revision,
Importantly, the cells exhibit broad bystander suppression and reading, and approval of the submitted manuscript. All authors
can mediate infectious tolerance amplifying the impact of the contributed to the article and approved the submitted version.
cells, thus resulting in robust and durable efficacy.
As cell therapies have flourished in the cancer space as a new pillar
of medicine, it is not surprising that Treg therapies, which have the Acknowledgments
capacity to control inflammation and autoimmunity, have become the
latest approach to treat these devastating diseases. A wealth of pre- The authors wish to acknowledge the contributions of many
clinical data has shown that adoptive Treg cell therapy can be effective colleagues who have pioneered the work summarized in this review.
in the treatment of diseases ranging from autoimmunity and
prevention of organ transplant rejection to cancer-related GvHD and
neurological diseases such as ALS and stroke. Efforts are underway to Conflict of interest
exploit these cells as immunotherapies to treat and potentially prevent
these diseases (97). Early clinical studies suggest that Treg adoptive All the authors are employed by Sonoma Biotherapeutics.
immunotherapy is safe and can lead to biological and molecular
changes that alter disease biology. In the oncology setting, however,
it is likely that modifications to Treg cells may be required to fully Publisher’s note
achieve durable and long-lasting effects. Making use of synthetic
biology, efforts are underway to genetically manipulate these cells to All claims expressed in this article are solely those of the authors
incorporate novel antigen specificities, altered cell characteristics and do not necessarily represent those of their affiliated
including the reliance of certain growth factors and a carrier of organizations, or those of the publisher, the editors and the
payloads that can modify local inflamed tissues. reviewers. Any product that may be evaluated in this article, or
The future for exploiting Treg therapies will depend on solving claim that may be made by its manufacturer, is not guaranteed or
key questions regarding process development, the assessment of endorsed by the publisher.
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Treg Cells

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Treg Cells

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TYPE Review

PUBLISHED 19 April 2023

Opportunities for Treg cell

Treg, immune tolerance, Foxp3, autoimmunity, immune regulation

1 History and biology of T regulatory cells

Frontiers in Immunology 01 frontiersin.org

Frontiers in Immunology 02 frontiersin.org

3.1 Will Treg therapy promote broad

Frontiers in Immunology 03 frontiersin.org

Frontiers in Immunology 04 frontiersin.org

TABLE 1 Companies involved in regulatory cell adoptive immunotherapy.

Company Cell type Cell source Modiﬁcation Lead indication

PolTREG FOXP3+ Treg cells Autologous peripheral blood MOG-CAR T1D

Abata Therapeutics FOXP3+ Treg cells Autologous peripheral blood MBP-TCR MS

TeraImmune FOXP3+ Treg cells Autologous peripheral blood FVIII-TCR Hemophilia

Cellenkos FOXP3+ Treg cells Allogeneic cord blood NONE ARDS

Tr1x Tr1 cells Adult peripheral blood Undisclosed Undisclosed

Frontiers in Immunology 05 frontiersin.org

TABLE 2 Summary of a subset of clinical trials with Tregs in multiple diseases.

Study ID Study title Study Reference

T1DM immunotherapy using

Autologous polyclonal Tregs for lupus Expanded autologous CD4+CD127lo/

Safety and Efﬁcacy of CLBS03 in

T1DM immunotherapy using polyclonal

Safety study and therapeutic effects of

Safety and efﬁcacy of antigen-speciﬁc

Polyclonal regulatory T cells (PolyTregs)

Autologous ex vivo expanded regulatory T

Cellular therapy of type 1 diabetes with T

Islet transplantation for type 1 diabetes

PolyTreg immunotherapy in islet

Infusion of autologous T regulatory cells

Cryopreserved polyclonal regulatory T cell

Treatment of children with kidney

Treg adoptive therapy for Subclinical Expanded autologous CD4+CD127lo/

Frontiers in Immunology 06 frontiersin.org

Study ID Study title Study Reference

Infusion of T-regulatory cells in kidney

The ONE Study (UK Treg Trial)

Trial of Adoptive Immunotherapy with

Donor-alloantigen-reactive regulatory T cell

The ONE Study trial Expanded autologous polyclonal

Treg Therapy in Subclinical Inﬂammation

A pilot study using autologous regulatory T

Expanded autologous regulatory T cell

TLI, TBI, ATG, and hematopoietic stem

Safety and tolerability study of chimeric

TWO study: cell therapy trial in renal

Liver transplantation and other treatments

Safety study of using regulatory T cells

Safety and Efﬁcacy Study of Regulatory T

Frontiers in Immunology 07 frontiersin.org

Study ID Study title Study Reference

Donor alloantigen reactive Tregs (darTregs)

Liver transplantation with Tregs Expanded autologous

Cell Therapy With Treg Cells Obtained

Safety and clinical activity of QEL-001 in

Tolerance induction by a regulatory T cell-

Graft versus host disease

T-Regulatory Cell Infusion Post Umbilical

First-in-human phase 1 trial of induced

Phase 1-2 MAHCT w/ T Cell Depleted

Ex-vivo Expanded Donor Regulatory T

Donor regulatory T cell infusion in patients

Frontiers in Immunology 08 frontiersin.org

Study ID Study title Study Reference

Donor regulatory T cells in treating

Multiple donor Treg DLI for severe

Donor regulatory T-cells for steroid-

A Study of Engineered Donor Grafts

Pilot study for cell-based therapies in

T-Regulatory Cells in Amyotrophic Lateral

Regulatory T Cell infuSion fOr Lung Injury

Regulatory T cells for amyotrophic lateral

Tregs for the treatment of acute respiratory

NA, not available.