Regulatory T lymphocytes as a treatment method for rheumatoid arthritis – Superiority of allogeneic to autologous cells

Cellular therapies utilizing regulatory T cells (Tregs) have flourished in the autoimmunity space as a new pillar of medicine. These cells have shown a great promise in the treatment of such devastating conditions as type 1 diabetes mellitus (T1DM), systemic lupus erythematosus (SLE) and graft versus host disease (GVHD). Novel treatment protocols, which utilize Tregs-mediated suppressive mechanisms, are based on the two main strategies: administration of immunomodulatory factors affecting Tregs or adoptive cell transfer (ACT). ACT involves extraction, in vitro expansion and subsequent administration of Tregs that could be either of autologous or allogeneic origin. Rheumatoid arthritis (RA) is another autoimmune candidate where this treatment approach is being considered. RA remains an especially challenging adversary since it is one of the most frequent and debilitating conditions among all autoaggressive disorders. Noteworthy, Tregs circulating in RA patients' blood have been proven defective and unable to suppress inflammation and joint destruction. With this knowledge, adoptive transfer of compromised autologous Tregs in the fledgling clinical trials involving RA patients should be reconsidered. In this article we hypothesize that incorporation of healthy donor allogeneic Tregs may provide more lucid and beneficial results.


Introduction
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic synovial inflammation, leading to cartilage damage, bone erosion and, ultimately, severe joint destruction.Autoreactivity and dysregulation of self-tolerance are thought to be responsible for the disease onset.RA is one of the main causes of permanent work disability, associated with many social consequences [1].Between January 2005 and October 2006, the QUEST-RA (Quantitative Patient Questionnaires in Standard Monitoring of Patients with Rheumatoid Arthritis) project was conducted to achieve quantitative clinical assessment of patients with rheumatoid arthritis seen in standard rheumatology care in 15 countries.With 4363 included participants, this international multi-centre cross-sectional database provided a thorough overview of clinical status and treatments of patients with RA.Data on demographic, disease and treatment-related variables were collected and analyzed using descriptive statistics, enriching our knowledge on RA specific demographic characteristics.The QUEST-RA database has continued to grow and in June 2009 included over 8000 patients from 86 clinics in 32 countries.Based on collected data, the statistcal analysis was performed, showing that work disability rates remain high among people with RA during this millennium.More than one third (37 %) of participants reported subsequent work disability because of RA [2].
Current therapies: conventional synthetic DMARDs, targeted synthetic DMARDs (JAK inhibitors) and biological DMARDs can induce remission in approximately 70 % of the patients, however, remaining 30 % experience incurable and extremely debilitating affliction [3,4].Nowadays, all efforts are given to identify new therapeutic approaches, especially those focused on immune homeostasis [5,6].
Regulatory T lymphocytes (Tregs) are a vital part of the adaptive immune system, responsible for preventing excessive inflammatory responses and maintaining immune balance.They are a specialized subpopulation of T helper cells, essential for inhibiting pathological reactions towards self-antigen.Given their unique ability to control aberrant autoreactive responses, there has been a significant interest in targeting Tregs therapeutically [7].The pivotal role of Tregs in the maintenance of immune balance gave rise to a number of studies on their physiology and therapeutic potential [8].Currently, regulatory T cell-based therapies are way past the in vitro phase, demonstrated in murine models.They've entered the clinical phase and some promising results have been reported repeatedly.Over the years, a lot of data has been collected, showing a great efficacy of Treg-based treatment approaches, not only in autoimmune diseases but also in GVHDone of the biggest challenges to overcome in bone marrow transplantation [9,10].
As for RA, the size of its clinical data repository remains limited when compared to T1DM or GVHD, which are the diseases that have drawn researchers' attention from the very beginning of the Treg-based therapies era.Nevertheless, in several completed trials, Tregs have been proven useful in the management of RA, helping to overcome impaired functions and reduced frequencies of factors responsible for maintaining immune balance.The most promising Treg-based treatment protocol seems to be the one involving adoptive transfer of in vitro expanded Tregs [11].Adoptive T cell therapy (ACT) used in autoimmunity usually utilizes polyclonal autologous lymphocytes, extracted and isolated from the peripheral blood of the treated subject.Although peripheral blood of healthy individual usually provides a sufficient amount of Tregs, they present a terminally differentiated memory phenotype, which reduces not only the expansion capacity, but also the likelihood of successful engraftment and survival [12].This situation changes in autoimmune conditions, such as RA, in which we observe Tregs reduced frequencies and impaired functions.Moreover, different reports have shown that high plasticity of Tregs makes them particularly susceptible to inflammatory signals [13].As the result, their regulatory and anti-inflammatory profile becomes defective.This causes inability to control cytokine-activated T cell functions and results in autoimmune-mediated damage to the healthy tissue.While Tregs protect body against autoaggression in healthy individuals, under autoimmune conditions their immunosuppressive properties become insufficient and even contribute to exacerbation of the disease [14,15].This unfortunate phenomenon seems to be one of the culprit behind the inability to achieve a full therapeutic success of polyclonal autologous ACT [16,17].
We believe that changing the source of regulatory lymphocytes to a healthy donor could bring more certain and valuable conlusions and better understanding of the complex mechanisms behind the immune tolarance in RA.In this narrative review, we provide a basic background on the regulatory T lymphocytes and their role in the treatment of autoimmunity.We also hypothesize about a novel approach in the treatment of RA, based on adoptive transfer of allogeneic regulatory T cells.

The aim of this narrative review
Our aim was to give a brief overview of studies focused on harnessing regulatory T lymphocytes as a new therapeutic approach to treat autoimmune and other immunological diseases, especially RA.In this review, we highlight both the advances and the challenges in this arena, such as Tregs declined stability under inflammatory conditions.We also summarize available data (as of June 2024) on the clinical trials involving in vivo Tregs enhancement in RA patients, as well as the first implementation of allogeneic Tregs in the treatment of different diseases.

Methods
A literature search was performed in October and November 2023 and updated in January 2024 in PubMed database.Additional abbreviated update was performed in June 2024.The database was searched using a combination of subject headings and free-text terms in the title and abstract fields.Highlighted keywords for searching were: regulatory T cells, adoptive cell transfer, rheumatoid arthritis.We focused our interest on most recent papers (date of publication 2014 to present), especially extensive meta-analyses summarizing the data from the last two decades.
As for for the clinical trials research, database used was ClinicalTrials.gov.The date of our investigation was June 2024, with applied search criteria: Tregs; regulatory T lymphocytes; rheumatoid arthritis; allogeneic Tregs.
For practical purposes, the search strategies were limited to English language as no resources were available to translate papers in other languages.

Regulatory T lymphocytes
In 1995 Sakaguchi et al. reported a subset of T-helper lymphocytes (CD4 + ) that maintain immune homeostasis by suppressing proliferation of effector T-cells [18].Those lymphocytes expressed characteristic receptor for IL-2 (CD25).Over the years, more data about Tregs properties has been collected, suggesting the important role of Foxp3 transcription factor, which defines Treg identity and function.The population of CD4(+)CD25(+)Foxp3(+)CD127(low/-) T lymphocytes is currently recognized as the major immunoregulatory subset since it has been the most thoroughly studied [19].These cells are often called naturally-occurring Tregs (nTregs) and consist of two subgroups: thymus-derived Tregs (tTregs), produced by the thymus as a functionally distinct and mature population, and peripherally-derived Tregs (pTregs), generated extrathymically at peripheral sites under certain conditions.
Tregs differentiation from naïve CD4(+) cells is regulated by TCR signaling that commit precursors to a regulatory lineage.Tregs can also be induced from non-Tregs in vitro, e.g. by antigenic stimulation in the presence of TGF-β and IL-2 (induced Tregs (iTregs)) [20].Interestingly, there is an increasing evidence that some CD8(+) T cells and B cells also show suppressive activity and have major impact on the immune homeostasis [21,22].These findings confirm extremely complex nature of specific interactions between every cell in human immune system.
Naturally-occurring Treg lymphocytes present their regulatory effect via cell-cell contact or by producing anti-inflammatory cytokines [14] [Fig.1].As mentioned before, there have been some discoveries suggesting that under certain conditions, such as inflammatory environment, Tregs start to present pro-inflammatory features and fail in preventing autoaggression [23].In 2014, a ground-breaking paper by Jeffrey Bluestone's group showed, using mice fate mapping analysis, that Tregs can start to produce IL-17 due to the down-regulation of Foxp3 gene transcriptional activity, triggered by autoimmune arthritis environment [24].These IL-17 producing cells are particularly arthritogenic and capable of inducing a severe chondral damage.Tumour necrosis factor α (TNF-α), present in inflammed joints in high concentrations, is one of the cytokines that are responsible for the impaired Treg cells function [15,Table 1].

Tregs in autoimmune diseases
Treg-mediated immunosuppression provides self-tolerance and any failure in that system leads to some kind of autoimmune disorder [45].The pathological changes we observe as the result of immune imbalance may affect multiple systems and organs of the human body.Autoimmune diseases present a wide variation of symptoms: they may become chronic or acute; may present episodes of remission followed by aggressive relapses; the course of the disease may vary from mild to severe.Accordingly, their response to wide range of available treatment methods depends on many different factors and mechanisms, often not fully understood.Currently there are more than 80 types of autoimmune diseases described in the literature.They affect approximately 10 % of the global population [46].Examples of such diseases include type 1 diabetes mellitus, multiple sclerosis, psoriasis and rheumatoid arthritis.

Tregs in RA
The prevalence of RA varies globally, with generally higher occurrence in the industrialized countries [47].In the last years, RA was reported to be the most disabling disease, causing early departure from professional career path and enormous limitations in the social life area [2].Immune regulation in RA is extremely complex and may be achieved through a direct cell-cell contact or paracrine signalling.Tregs mediate the downrgulation of antigen-presenting cells (APC) and also regulate the metabolism in effector T cells [8,48] [Fig.1].In the last two decades, there has been an explosion in the research describing the role of Tregs and their relevance in different autoimmune disorders, enriching our knowledge about the etiology and pathogenesis of autoimmunity.
It has been shown that Tregs are frequently found subpopulation in the peripheral blood (PB) and synovial fluid (SF) of RA patientswith significantly higher numbers in the latter [49].In 2016, Morita et al. presented a meta-analysis of 31 studies, confirming that the proportion of Tregs, defined by both Foxp3 and CD25, was higher in SF than that in PB among RA patients [50].However, enhanced presence of regulatory cells is clearly not sufficient to prevent autoimmunity.It is believed that suppressive function of Tregs is impaired partly because of pro-inflammatory cytokines (e.g.TNF-α) produced by synovium [51,52].Unparalleled plasticity of Tregs is responsible for the loss of their anti-inflammatory phenotype in highly inflammatory environment of RA [53,54] [Table 1].As compared to healthy individuals, the quantity of Tregs in the synovial fluid of RA patients may be higher, but this increased number does not compensate for functional impairment.Tregs affected by RA simply fail to suppress auto-reactive effector lymphocytes and start to contribute to the progression of the disease via number of mechanisms, such as Treg conversion to Th17 (Bluestone et al.) [24,55,56].

Tregs in non-autoimmune diseases
Tregs function is not limited to self-tolerance and autoimmunity.Over the years, several additional properties have been suggested [57].Interestingly, emerging evidence suggest that regulatory T lymphocytes intervene in the inflammatory process that drive osteoarthritis (OA), considered for decades a simple degenerative disease.Some groups demonstrated that decreased Tregs responses may contribute in the pathogenesis of chondral damage in OA [58,59].
The role of Tregs have been also described in the regenerative process after stroke or myocardial infarction [60][61][62].If so, it may imply similar regenerative activity around the joints.
Furthermore, it appears that the adaptive immunity during severe SARS-CoV-2 infection is dysregulated and the number of Tregs significantly decreased.At the same time, their functional suppressive potential seems to be elevated with high expression of molecular markers [63].
The undeniable role of regulatory T cells in the pathophysiology of cancer should also not be forgotten.In tumor-bearing individuals, Tregs compromise the response of immune system against tumour, and therefore accelerate the progression of cancer [64][65][66].

Treg-based therapies
To date, two main strategies have been developed in the field of Treg-based therapies: the administration of immunomodulatory factors enhancing the number or function of Tregs and ACT -adoptive transfer of in vitro expanded Treg lymphocytes.The first one rely on in vivo protocols, involving specific immulomodulatory agents targeting the key molecules of Tregs maintenance, thus improving Treg-mediated immune tolerance [67].These therapies involve treatment with cytokines such as low-dose IL-2, IL-2/Anti-IL-2 Ab complex and IL-4, IL-12, IFN-γ-in murine models [10,48].However, the most promising results were provided by clinical trials focused on adoptive immunotherapy [68].
ACT refers to the ex vivo manipulation of T cells and their subsequent reinfusion into patient.While numerous in-human trials have demonstrated that Tregs reduce the severity of autoimmune reactions, several impediments still remain, including Tregs variability and the practical need for their effective in vitro expansion techniques.
Many studies on the heterogeneity of regulatory T lymphocytes population revealed distinct subsets with different functions in the control of the immune homeostasis and induction of peripheral tolerance.All these Tregs subsets and their specific properties can be exploited to develop novel treatment methods [69].
As mentioned before, the number of Tregs circulating in the peripheral blood is significantly lower than that of other lymphocytes, making them challenging to extract and multiply.There have been also some controversies on the proper identification method for Tregs, which caused a lack of clarity in the firstly emerged studies [50].Fortunately, consensus on coherent identity markers has been achieved, followed by introduction of specific protocols that allow in vitro expansion of Tregs in sufficient amount [70][71][72].
Efficient clinical-scale Treg product manufacturing involves three key stepsisolation, adequate ex vivo expansion, while maintaining regulatory properties, and quality assessment [73].Currently, the CD4(+)CD25(+)Foxp3(+)CD127(low/-) phenotype is most commonly isolated from Tregs population through flow cytometry or immunobead-separation methods.Tregs can be further distinguished from resting conventional T cells (Tconv) by hypomethylation of Foxp3, which enables to recognize regulatory profile at the genetic level.Tregs are then activated in the presence of high levels of exogenous interleukin-2 (IL-2).Next step is cell proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads [74].
Various approaches have been explored to enhance the proliferation, function and stability of Tregs, including modifying culture conditions or adding drugs, e.g.Treg + MSC (Mesenchymal Stem Cells) co-culture, rapamycin, IL-6 and TNF-α [75,76].Given the low frequency of Tregs in human peripheral blood, a feasible approach is the in vitro generation of potent Tregs (iTregs) from non-Tregs, since they are much higher in numbers and relatively easy to isolate.iTreg cells can develop from naïve CD4(+)Foxp3(− )T cells upon TGF-β stimulation.However, unlike tTregs, iTregs are highly unstable, which is a significant obstacle to their use for adoptive immunotherapy [77].Interestingly, all-trans retinoic acid (atRA), a vitamin A metabolite, has been proven essential in regulation of a wide range of biological processes, including the in vitro enhancement of Tregs function and stability.This development has become especially useful in the manufacturing of both nTregs and iTregs for adoptive transfer purposeses [78].
Most Tregs manufacturing methods have been developed and validated based on autologous polyclonal Treg products.Moreover, most clinical trials have also utilized only polyclonal Treg cells populations, acquired from the treated subject and subsequently expanded in vitro.However, evidence from pre-clinical studies has clearly shown that antigen-specific Treg-based ACT is vastly superior to polyclonal cell-based [79].The most basic method to obtain antigen-specific product includes antigen-stimulated Tregs expansion.This approach is mainly used in transplantation, to prevent graft rejection, and involves the use of APCs (Antigen Presenting Cells) from the graft donor to specifically stimulate alloreactive Tregs from the recipient.However, the implementation of this method in autoimmunity is limited due to the lack of knowledge of patient-specific TCRs (T cell receptors), which recognize disease-relevant MHC (Major Histocompatibility Complex)-peptide complexes [9].
Accessibility and safety of polyclonal Tregs have made them more frequently used in ongoing and past clinical trials.Polyclonal Tregs used in ACT may be autologous, derived from peripheral blood of the treated subject (the most popular source), or allogeneicisolated from a healthy donor.Umbilical cord blood (UCB) could also become the source of allogeneic regulatory cells [80].

Genetic engineering of Tregs
The emergence of advanced gene editing techniques has opened the door to new methods of Tregs modifications, creating therapeutics with improved specificity and function.These strategies include advanced generation of highly antigen-specific Tregs, based on viral transduction with CAR (Chimeric Antigen Receptor)-or engineered TCR-encoding vector, as well as genome editing, utilizing CRISPR technology [9,81,82].Furthermore, transgenic overexpression of Foxp3 in antigen-specific conventional CD4(+) lymphocytes has become an appealing way to produce a larger source of suppressive cells for infusion, thus overcoming the challenges of low precursor frequency [83].
Without a doubt, technological advance in genomics and cell manufacturing has made such individualized therapies possible, unfortunately, they are still extremely expensive, thus difficult to incorporate on a larger scale.Furthermore, there are several risks associated with Tregs genetic engineering, such as off-target disruption of essential genes or carcinogenesis associated with viral transduction and gene editing.There is a pressing need for careful monitoring of ongoing clinical trials utilizing genetically engineered Tregs, to further characterize the long-term risks and ensure safety of such therapies [84,85].

Treg-based therapies in RA
Currently, synthetic conventional, synthetic targeted and biological DMARDs are widely used in clinical practice and allow to achieve remission in approximately two out of three patients [3,86,87].In 2018 Chinese cross-sectional observational study, including a total of almost 2000 RA participants, showed that the proportions of patients who fulfilled remission criteria was only 1.75 %-10.90 % (depending on the method used to evaluate response to the treatment -DAS28, CDAI, ACR/EULAR).The results indicated that the rate of disease activity remission achieved by currently accepted treatment regimen is disappointing.Moreover, adverse effects of applied drugs occurred in approximately one in five patients [88].New approach is needed for those who do not respond well to standard disease-modifying drugs.To meet expectations, researchers started to develop several new therapies, especially based on the autoimmune background of RA and Tregs unique properties.
First strategyapplication of cytokines or specific antibodies that target Tregs that circulate in the body of the treated patient, provoking the increase in Tregs frequency or suppressive functionhas been repeatedly proven effective.These studies have been successfully introduced into human trials more than 10 years ago and presented highly promising results in many autoimmune diseases, such as SLE, Sjögren syndrome, dermatomyositis [89][90][91].Unfortunately, in RA population these cytokines have been used only very recently [Table 2].One of the most interesting study was TRANSREG initiative that compared the biological and clinical responses to the administration of low doses IL-2 across 14 autoimmune and inflammatory pathologies over the course of 7 years (2014-2021).Primary endpoint assessed during TRANSREG study was the change in Tregs frequency, expressed as a percentage of total CD(+) count, after the induction period, compared with baseline.Secondary biological endpoints were changes in Tregs and other immune cells (e.g.CD3(+) T cells, CD4(+) T cells, CD8(+) T cells, CD19(+) B cells, NK cells/mm3) at specific periods of time, compared with baseline, and and ratio Tregs/Teffs in induction and maintenance period.
Second strategy involves ACT, based mostly on polyclonal autologous lymphocytes (aside from GVHD patientsin this group allogeneic (donor) regulatory T cells are isolated and administered to the patient -recipient).This protocol has been efficiently implemented in number of clinical trials: completed (kidney transplant, T1DM) or ongoing (GVHD, SLE) [7,48,[101][102][103].There has been no officially registered clinical trial involving adoptive Tregs transfer in RA patients till the end of 2023.Interestingly, this situtation changed in March 2024, when Sonoma Biotherapeutics Inc. announced the recruitment of participants to Phase 1 Trial

Table 2
A brief summary of different approaches to in vivo Tregs enhancement in clinical trials involving RA patients.Database used for the research: ClinicalTrials.gov.Search criteria: Tregs; regulatory T lymphocytes; rheumatoid arthritis.The aim of TRANSREG study was to compare biological and clinical responses to the administration of low doses IL-2 across 14 selected pathologies: Rheumatoid Arthritis, Ankylosing Spondylitis, Systemic Lupus Erythematosus, Psoriasis, Behcet's Disease, Granulomatosis with Polyangiitis, Takayasu's Disease, Crohn's Disease, Ulcerative Colitis, Autoimmune Hepatitis, Sclerosing Cholangitis, Sjögren Syndrome, Systemic Sclerosis and Idiopathic Thrombocytopenic Purpura.

ClinicalTrials.gov ID Reference
"Study of Single Doses of SBT777101 in Subjects With Rheumatoid Arthritis".SBT777101 is an experimental biological treatment that consist of autologous CAR Treg lymphocytes [92].

Allogeneic Tregs ACT as a potential treatment method for RA
The great success of ACT in many autoimmune conditions (with T1DM being the leader in the field) made rheumatoid arthritis destined to become the next target.RA causes the impairment of all aspects of QOL (Quality of life) and inevitably leads to mental health, social, environmental and sexual dysfunctions [104].With its extreme negative impact on society, it is only a matter of time before we witness widespread application of Treg-based ACT in clinical trials.It is crucial to carefully choosea suitable study design and methodology, reflectng the aims of the research, and reach only for the protocols based on the latest scientific findings, to provide the most transparent and critical evidence base for evaluating the safety and efficacy of new medical product.We are no longer grasping in the dark for answers in the mysterious world of autoimmune regulation.The last couple of years were extremely productive for researchers specialized in the therapeutic potential of Tregs.It seems unwise to copy some fundamentally false assumptions that lead to vague and questionable outcomes.With our current knowledge, we should re-consider the use of autologous Tregs ACT in the first in-man studies.
It has been repeatedly proven that Tregs in RA patients present different kinds and levels of intrinsic dysfunctions [105].In these settings, expanding and adoptively transferring defective regulatory cells seems to be of limited benefit, unless we develop the way to stabilize their phenotype [106,107].It is questionable how objectively valuable is the real potential of these cells, since we deal with only the defective ones.Taking into consideration that implementation of healthy donor allogeneic Tregs has been successful in the Table 3 Clinical trials involving allogeneic Tregs ACT as a treatment method of different diseases.Our investigation was conducted at the beginning of June 2024, using data from ClinicalTrials.govregistry, with "allogeneic Tregs" as the applied search criterion.The result showed 22 clinical trials; 8 of the studies turned out to be irrelevant to the topic of allogeneic Tregs ACT.Most of the remaining trials involved adoptive transfer of donor Tregs in the treatment of GVHD.Interestingly, 3 out of 5 actively recruiting studies are not focused on hematologic conditions.3].Interestingly, allogeneic cells has already been used in the innovative therapies of RAmesenchymal stem cells (MSCs) obtained from adipose tissue or umbilical cord have become one of the most promising approaches [108,109].MSCs act as the immunosuppressive agents and, because of their important immunoprivileged properties, remain safe for allogeneic use.This results from MSCs having extremely low levels of major histocompatibility complex (MHC) class I and II molecules expression [110].Somehow similar approach has been developed in our group where autologous Tregs were expanded in the presence of allogeneic mismatched MSC, which improved significantly the regulatory potential of Tregs [111].Compared to mesenchymal, allogeneic Tregs ACT represent a challenge since administration of unmodified donor cells, unmatched in HLA complex, are likely to trigger sensitization or acute rejection by recipient immune system [83].

ClinicalTrials
The obstacle of HLA mismatch could be overcomed by application of allogeneic Tregs derived from umbilical cord blood (UCB).The successful use of partially HLA-mismatched unrelated donor UCB as the source of hematopoietic stem cells has been reported as early as 1996 [112].After the decades of studies, unrelated donor UCB has become the commonly acceptable alternative to HLA-matched bone marrow in the transplants among patients suffering from malignancies, BM failure, immunodeficiencies, etc [113].Since UCB is easier to match than any other source of potentially therapeutic allogeneic cells, it has been widely and willingly used by researchers, providing some interesting findings, even concerning the recent pandemic [114].Some newest reports show that the administration of allogeneic cord blood Tregs could be successfully used in the treatment of COVID-19 ARDS (Acute Respiratory Distress Syndrome) [115].UCB is particularly alluring for clinical trials because it is the non-controversial and absolutely ethical source of stem cells collected post-birth, which prevents any political or religious issues.Obviously, cord blood has some limitations as the source of regulatory T lymphocytes, if they were to be used as a commonly accepted option for RA treatment.Cord-blood banking system is still growing, with The World Marrow Donor Association incharge, but faces many obstacles, such as financial issues or limited implementation of UCB banking in some countries [116].

Conclusions
Though still largely an experimental procedure, Treg-based adoptive cell therapy has recently become a clinical reality.Results from in-human trials are encouraging, but many questions remain to be addressed before this approach becomes routinely applicable to RA patients.We believe that the key to achieving satisfactory results of ACT in RA is implementation of the lymphocytes of allogeneic origin, not restricted by pathologic changes triggred by ongoing inflammatory processes.
The ideal situation in the distant future would be setting up a GMP(Good Manufacturing Practice)-compatible allogeneic cell banking system to cover large populations of patientssimilar to hematopoietic stem cell transplantation (HSCT) strategies, exploited by physicians for decades [117].The extensive use of immune cells from unrelated donors, optimally matched in MHC haplotypes, would offer many advantages over autologous cells, including improved cost-effectiveness, broader availability and higher quality of the product.Furthermore, allogeneic approach has the potential to provide a ready to use "off the shelf" immunotherapeutic, such that a single manufacturing run would allow multiple administration of the product.Likewise, by increasing the scale of production and creating a specific cell banking system, average cost per patient would decrease while the availability of therapeutic agent would increase [10,118].The alternative for this 'natural source' of allogeneic Tregs might become Tregs with genetically engineered HLA that we are currently developing in our group.The idea is to create universal Treg donor lines by switching off HLA genes with either viral vectors or CRISPR technology [9,119].
Within the past decade, we have witnessed the transformative therapeutic potential of adoptive therapy for autoimmune conditions.However, at this point, we are still very far from clinical utilization of Tregs ACT on a bigger scale.At first we should focus on smaller trials, with carefully planned study design and methodology, to shed light on the exact mechanisms responsible for lymphocyte regulation in RA.This data would inevitably provide some further evidence of Tregs efficiency and safety in clinical practice.As long as we reach for defective autologous Tregs, proven abnormal or even pro-inflammatory, our results will not bring answers, only more pressing questions.

Fig. 1 .
Fig. 1.Mechanisms of Treg suppression.CTLA-4 binds to CD80/CD86 on APC (Antigen Presenting Cell), inhibits antigen presentation and increases IDO (Indoleamine 2,3-dioxygenase) expression.IDO pathway induces T cells anergy and enhances Tregs function.PD-1 (Programmed Death Receptor 1) binds to its ligand on APC resulting in suppression of antigen-reactive activity of Teff (effector T lymphocyte).TIGIT (T cell Immunoreceptor with Ig and ITIM domains) presented on Treg interacts with CD155 on dendritic cell and enhances the expression of IL-10, thus specifically suppresses proinflammatory Th1 and Th17 cells.Anti-inflammatory cytokines are also directly produced by Tregs.Exosomes (extracellular vesicles) secreted by Tregs contain many specific biological molecules, which are delivered to target cells and modulate immune responses by inhibiting T cell proliferation, inducing apoptosis and changing the cytokine expression profiles of target cells.Activated Tregs cells also utilize granzyme B and perforin to disrupt protective barrier of cell membrane and destroy integrity of the target cell.CD39/CD73 expressed on Treg degrade ATP into extracellular adenosine, which is implicated in the immunoregulatory activity by inhibiting antigen presentation by APCs.TRAIL/DR5 (TNF-Related Apoptosis-Inducing Ligand/Death Recptor 5) interaction activates caspase-8 to induce Teff apoptosis.ICOS (Inducible Tcell Co-Stimulator) signaling can mediate Tregs survival by fine-tuning the expression of multiple anti-apoptotic molecules and anti-inflammatory cytokines.CD25, also konown as IL-2 receptor (IL-2R), is expressed at high levels on the surface of Tregs.IL-2, though not produced by Tregs, is essential for their development.Tregs compete for IL-2 secreted by effector cells and prevent them from acquiring the sufficient amount for proliferation and activation.

Table 1
Impairment of Tregs suppressive functions in inflammatory conditions.
involving autoimmune and hematologic conditions, we suggest to incorporate the same protocol in RA research [Table Abbreviations: thyTregsthymic regulatory T lymphocytes; Tconconventional T lymphocytes; HSCT -Hematopoietic Stem Cell Transplantation; GVHD -Graft versus Host Disease; SIRS -Systemic Inflammatory Response Syndrome; ARDS -Acute Respiratory Distress Syndrome; ALS -Amyotrophic Lateral Sclerosis.trials