The role of FOXP3⁺ regulatory T cells in human autoimmune and inflammatory diseases

Summary

CD4⁺ regulatory T cells (T_reg) expressing the forkhead box protein 3 (FOXP3) transcription factor (T_regs) are instrumental for the prevention of autoimmune diseases. There is increasing evidence that the human T regulatory population is highly heterogeneous in phenotype and function. Numerous studies conducted in human autoimmune diseases have shown that T_reg cells are impaired either in their suppressive function, in number, or both. However, the contribution of the FOXP3⁺ T_reg subpopulations to the development of autoimmunity has not been delineated in detail. Rare genetic disorders that involve deficits in T_reg function can be studied to develop a global idea of the impact of partial or complete deficiency in a specific molecular mechanism involved in T_reg function. In patients with reduced T_reg numbers (but no functional deficiency), the expansion of autologous T_reg cells could be a suitable therapeutic approach: either infusion of in‐vitro autologous expanded cells, infusion of interleukin (IL)‐2/anti‐IL‐2 complex, or both. T_reg biology‐based therapies may not be suitable in patients with deficits of T_reg function, unless their deficit can be corrected in vivo/in vitro. Finally, it is critical to consider the appropriate stage of autoimmune diseases at which administration of T_reg cellular therapy can be most effective. We discuss conflicting data regarding whether T_reg cells are more effectual at preventing the initiation of autoimmunity, ameliorating disease progression or curing autoimmunity itself.

Introduction

CD4⁺ T regulatory cells (T_reg) expressing the forkhead box protein 3 (FOXP3) transcription factor (T_reg cells) are capable of suppressing immune responses, especially their initiation, by preventing the activation and proliferation of T and B cells 1. The importance of T_reg cells has been demonstrated in animal models whereby their depletion is associated with loss of self‐tolerance and development of severe autoimmunity [e.g. immunodysregulation, polyendocrinopathy enteropathy X‐linked (IPEX) syndrome] 2, 3, 4, 5. Further data in murine models have also demonstrated their ability to prevent progression of and even cure established autoimmune/inflammatory disease 6, 7. Overall, these cells are also considered to play a role in allergy prevention 8, gestational tolerance 9, the promotion of graft tolerance post‐transplantation 10 and the prevention of tumour immune responses 11.

Human T_reg cells were initially characterized as CD4⁺ T cells co‐expressing the interleukin (IL)‐2 receptor alpha chain 12, 13, 14, 15, 16, 17, 18. This was based on murine data demonstrating that depletion of CD4⁺CD25⁺ T cells led to the development of severe autoimmunity 2. Since then, T_reg cells have been more precisely described as CD4⁺ T cells expressing the FOXP3 transcription factor in mice 3, 4, 5 and in humans 19. However, while the CD25⁺FOXP3⁺ phenotype defines T_reg cells in mice, both CD25 and FOXP3 can also be induced upon activation in naive CD4⁺ T cells (of mice and humans) 20. Intriguingly, CD25 and FOXP3 can also be induced in conventional T cells, although this does not equate to them adopting ‘regulatory’ function 21, 22. All this indicates that the mere combination of CD25 and FOXP3 expression is insufficient to define human T_reg cells phenotypically in health and diseases 23.

In recent years, we have progressed to further categorize human CD4⁺FOXP3⁺ T cells into three distinct subpopulations based upon their phenotypical and functional differences. These subpopulations are (a) Fraction I: CD45RA⁺FOXP3^lo naive T_reg cells, considered equivalent to natural T_reg cells arising from the thymus (tT_reg cells) and demonstrating immunosuppressive properties in vitro; (b) Fraction II: CD45RA^–FOXP3^hi activated effector T_reg cells, also immunosuppressive in vitro; and (c) Fraction III: CD45RA^–FOXP3^lo cytokine‐secreting but not immunosuppressive cells 23. We were also able to classify these populations similarly using CD25 (instead of FOXP3) and CD45RO (instead of CD45RA). Since then, further elegant work has demonstrated that the Fraction III population could be further subdivided on the basis of CD127 to identify two subpopulations. The proteomic analyses performed identified these subpopulations to closely resemble memory conventional T cells or effector T_reg cells, respectively 24. Importantly, these aforementioned subpopulations can be distinctly identified in healthy and diseased states. For example, CD45RA⁺FOXP3^lo T_reg cells are the main T_reg population identified in cord blood 23, whereas effector T_reg cells are highly prevalent in tumours or in peripheral blood of patients with sarcoidosis 25, 26 or mycosis fungoides 27. Interestingly, a small proportion of CD45RA^–FOXP3^lo cells have been identified in the peripheral blood of patients with active systemic lupus erythematosus 27 or in some tumours 28. The impact of these cell subpopulations on their respective pathologies is not yet known.

Numerous studies conducted in human autoimmune diseases have shown that T_reg cells were impaired either in their suppressive function, in number, or both. A further mechanism involves resistance of conventional T cells to T_reg‐mediated suppression via the presence of certain cytokines [tumour necrosis factor (TNF) and IL‐6] in the microenvironment and over‐activated phosphatidylinositol 3‐kinase/protein kinase B (PI3K/Akt) signalling 29. Of note, these findings have been demonstrated in a range of autoimmune diseases 29, 30, 31. The conclusions of those studies have been drawn following different phenotypical definitions of human T_reg cells, mainly based on the assumption the either that FOXP3‐expressing and/or CD25^high CD4⁺T cells constitute a single homogeneous population of T_regs 19. Hence, the contribution of the heterogeneous FOXP3⁺ T_reg subpopulations to the development of autoimmunity has not been delineated in detail. In this review, we discuss several unresolved questions 32, 33 and emerging issues regarding the role of T_reg cells in human autoimmune and inflammatory diseases.

Heterogeneity of human FOXP3‐expressing CD4⁺ T cell subsets

There is increasing evidence that the human T regulatory population is highly heterogeneous in phenotype and function 34. While FOXP3‐expressing cells can be roughly separated into three subsets (naive T_reg cells, effector T_reg cells and FOXP3^lo‐activated T cells) 23, there are novel data indicating that these subpopulations can be subdivided phenotypically even further (Fig. 1).

Figure 1.

Human regulatory T cell (T_reg) subsets. Thymus produces CD45RA⁺forkhead box protein 3 (FOXP3)^lo naive T_reg (nT_reg) cells as well as naive CD45RA⁺ non‐T_reg cells. nT_reg cells can differentiate into CD45RA^–FOXP3^hi effector T+ (eT_reg) cells, which are potently suppressive, but they can also maintain low levels of FOXP3 and become non‐T_reg cells. Effector T_reg cells can also convert into non‐T_reg cells, while non‐T_reg cells can up‐regulate FOXP3 transiently to become induced effector T_reg cells. As shown, all FOXP3‐expressing subsets can be divided into subsets by differential expression of surface markers or transcription factors.

CD45RA⁺ naive T_reg cells can be separated into the CD31⁺ recent thymic emigrant (RTE) and the CD31^– naive T_reg cell population 35 (Fig. 1). Recent data obtained via cytometry by time of flight (CyTOF) has demonstrated that naive T_reg cells can be subdivided based on their expression of CD49b, CD62L and certain chemokine receptors. This has led to their subcategorization as CD49b⁺, CD49b⁺CXCR3⁺CCR4⁺CCR6⁺ and CXCR3⁺RORC2⁺CD62L⁺ subsets 36. It is currently unknown how CD31⁺ RTE T_reg cells differentiate into each of these subsets; the prevalence of each subset in human health and disease and how each of those subsets differentiates into other FOXP33⁺ T cells have also to be determined.

Interestingly, CCR4 can also delineate six different subsets among the Fraction III FOXP3^lo non‐T_reg cell population when combined with CD127 and CD49d. (Fig. 1). Among these subsets, the CD127⁺CD49d⁺CCR4^– population contains most of the cytokine‐producing cells (IL‐2, IL‐17 and IFN‐γ), while the CD127^–CD49d^–CCR4⁺ contains the lowest number of cytokine‐producing cells. This latter subpopulation is, therefore, functionally and phenotypically most similar to the effector T_reg subpopulation, as most effector T_reg cells are CD127^–CD49d^–CCR4⁺ 24. The origin of Fraction III FOXP3^lo cells has not yet been completely elucidated: these cells can be derived from naive T_reg cells that fail to up‐regulate high levels of FOXP3 upon activation, for instance, because of weak signal transducer and activator of transcription (STAT)‐5 signalling; they may also be derived from some FOXP3^hi effector T_reg cells through reduced expression of FOXP3, and they can also be derived from conventional CD4⁺ T cells that transiently up‐regulate FOXP3 upon activation. Of note, all of these phenotypical changes have been observed in vitro, generally in the presence of low‐dose IL‐2 37.

Effector T_reg cells constitute a functionally homogeneous suppressive subset that is highly proliferative in vivo but poorly proliferative in vitro, and prone to apoptosis in the absence of IL‐2 1, 19. However, from a phenotypical perspective, these cells can be subcategorized based on their co‐expression of effector T cell transcription factors such as T‐bet [T helper type 1 (Th1), GATA binding protein 3 (GATA‐3) (Th2) and retinoid‐related orphan receptor γt (RORC)] (Th17) 32. This is supported by murine data indicating that Th1‐, Th2‐ and Th17‐like T_reg cells suppress their related effector Th cell counterparts, respectively 38, 39, 40. These T_reg subsets also express their effector Th cell counterparts’ chemokine receptors. Hence, Th1‐like T_reg cells are CXCR3⁺, Th2‐like T_reg cells are CCR6^–CCR4⁺ and Th17‐like T_reg cells are CCR6⁺CCR4⁺ 41. A further T_reg subset comprise T follicular regulatory (Tfr) cells, which are found in germinal centres and can directly influence B cells. 42 These cells interfere with the interaction between T follicular helper (Tfh) cells and B cells to alter subsequent B cell differentiation towards antibody‐producing plasma cells or a memory phenotype. This interaction requires further study, particularly to improve our understanding of antibody‐mediated autoimmune disease [e.g. thyroiditis, type 1 diabetes (T1D)].

Additionally, recent studies have demonstrated phenotypical heterogeneity between T_reg cells in the peripheral circulation and those present in the healthy/diseased tissue 43. For instance, IL‐1 receptor type II (IL‐1R2), a decoy receptor for IL‐1, is highly expressed on breast and colonic T_reg cells but not on their peripheral circulation counterparts 44, 45. Similarly, CD15s (sialyl Lewis X) is present on peripherally circulating effector T_reg cells but is absent on their pulmonary T_reg counterparts 46.

Overall, it is clear that historical studies in human autoimmune and/or inflammatory diseases have been conducted on the basis that circulating FOXP3⁺ T cells were a homogeneous population 30. On the basis of the data discussed above and access to novel technologies, we anticipate the future determination of the distinct contributions of FOXP3⁺ T_reg subsets to human health and disease (Fig. 1).

The significance of FOXP3⁺ T_reg cells abnormalities in autoimmune diseases: from pernicious anaemia to IPEX

In their seminal publication demonstrating that murine T_reg cells displayed the CD4⁺CD25⁺ phenotype, Sakaguchi et al. showed that the canonical autoimmune abnormality observed in sick mice depleted of T_reg cells was the occurrence of autoimmune gastritis with circulating anti‐parietal autoantibodies 2, a condition that is reminiscent of pernicious anaemia in humans 47. However, the role of T_reg cells in pernicious anaemia in humans has not yet been delineated. This knowledge gap can also be extrapolated to other autoimmune diseases whereby the role of T_reg cells in their development has been studied using animal or human culture systems that are not necessarily reflective of true human pathology 33.

On one hand, upon review of published literature into human autoimmunity, one may be tempted to conclude that all autoimmune diseases could be characterized by either a deficit in T_reg number and/or function or resistance of conventional T cells to T_reg‐mediated suppression 29, 30, 31, 48, 49. On the other hand, the only known condition with clear evidence for total depletion of T_reg cells is IPEX 3, 4, 5. This condition is provoked by different genetic defects in the FOXP3 gene and is characterized by the occurrence of enteropathy, eczema, T1D, thyroiditis, cytopenia, hepatitis, nephritis and gastritis 50, 51, 52. Indeed, the scurfy mouse model is widely utilized for the study of T_reg cells, as equivalent defects in the FOXP3 gene lead to a pathologically similar autoimmune disease. Scurfy mice die within a few weeks after birth, while untreated newborns with IPEX die rapidly, both of severe inflammation, allergy and autoimmunity 3, 4, 5. Hence, it is clear that a complete defect in T_reg cells leads to the development of this lethal systemic autoimmune and inflammatory disease.

Due to the rapid progression of IPEX in murine and human newborns (fortunately, a rare condition), the detailed study of T_reg cell deficiency in adults with autoimmune disease has remained a challenge. Rudensky et al. and Sparwasser et al. attempted to address this by developing mice with T_reg cells bearing the diphtheria toxin receptor 53. Thus, the injection of diphtheria toxin would provoke the complete depletion of T_reg cells within days, and thereby lead to a rapidly lethal systemic autoimmune disease that is similar to that observed in IPEX 53, 54.

Overall, it is clear from the experimental observations discussed above that a complete deficiency of T_reg cells at any stage of life rapidly leads to a fatal systemic disease. However, it is also clear from the clinical literature that human autoimmune diseases tend not to evolve towards an IPEX‐like condition. Indeed, we would reconcile these findings by hypothesizing that T_reg number deficiency and dysfunction in human autoimmune diseases is incomplete at any stage of life and is probably mild or minimal. For example, pernicious anaemia may be associated with some organ‐specific endocrine autoimmune diseases such as Graves’ disease, but rarely to other systemic autoimmune diseases 55. Importantly, neither of these conditions evolve towards an IPEX‐like condition. Although not yet proven, if there is indeed a T_reg deficit in pernicious anaemia it is likely to be mild or minimal.

FOXP3⁺ T_reg cell function deficiencies in human autoimmune diseases: lessons from genetic diseases

The lack of validated experimental assays reflective of in‐vivo human T_reg biology remains a major limitation in the field. To what extent in‐vitro T_reg suppressive activity correlates with in‐vivo T_reg function has not yet been established in humans 48. This limitation is important to overcome, as T_reg cells have numerous mechanisms of action which require different experimental design and reagents to reliably elicit 56. It is indeed plausible that observed in‐vitro T_reg functional deficiency in human autoimmune diseases may be explained by the partial deficiency of one or several mechanisms of suppression. One must also not discount the potential for effector T cells to be resistant to T_reg‐mediated suppression mechanisms 29. While the specific roles of these mechanisms can be studied in mice (via different conditional knock‐out models), their corresponding contributions in humans have been mainly elicited using in‐vitro models instead 57. For this reason, patients with rare genetic disorders that involve deficits in T_reg function can be studied to develop a global idea of the impact of partial or complete deficiency in a specific molecular mechanism involved in T_reg function.

One of the most well‐established mechanisms of action of T_reg cells is through their cytotoxic T lymphocyte‐associated protein 4 (CTLA‐4) receptor. Indeed, constitutive expression of CTLA‐4 by T_reg cells is instrumental for their in‐vivo suppressive capacity 58. Interestingly, CTLA‐4 haploinsufficiency has been described (albeit rarely) in certain families 59, 60. It is therefore noteworthy that patients with heterozygous non‐sense mutations of CTLA‐4 genes develop a systemic autoimmune disease manifesting as diarrhoea, granulomatous interstitial lung disease, autoimmune cytopaenia, thyroiditis, arthritis and skin disease—all of which are reminiscent of IPEX (but with less severity). Of note, none of these patients studied developed autoimmunity in early infancy, but a significant proportion had their first autoimmune abnormality diagnosed in adulthood. From a cellular perspective, although this mutation could have impacted on the CTLA4‐induction properties and function of all activated T cells, the impact on T_reg cells specifically is important. This is because normal T_reg cells express disproportionally higher surface and intracellular CTLA4 61. Interestingly, in patients with CTLA‐4 haploinsuffiency, they had higher numbers of T_reg cells but their individual expression of CTLA‐4 was reduced, especially after activation 59, 60. Hence, CTLA‐4 haploinsufficiency could be considered as a partial CTLA‐4‐related T_reg functional deficiency. Additionally, the unintended manifestations of blocking CTLA‐4 have recently been demonstrated in humans with cancer who are receiving anti‐CTLA‐4 checkpoint blockade therapy 62. These therapies work by boosting effector T cell activity and inhibiting T_reg cells; however, pharmacovigilance data suggest that some patients develop enteropathy and colitis similar to that of inflammatory bowel disease. It is important to understand why these patients specifically have effector T cells targeting and infiltrating the gut, as the anti‐CTLA‐4 antibody itself is systemically administered.

There is also another rare genetic disorder, which leads instead to a complete CTLA‐4‐related functional deficiency. This involves a deficiency in lipopolysaccharide‐responsive and beige‐like anchor protein (LRBA), which is an intracellular protein involved in the membrane expression of CTLA‐4 63. This condition is clinically characterized by a systemic autoimmune syndrome that resembles CTLA‐4 haploinsufficiency as well as IPEX (as some patients develop T1D). However, LRBA deficiency onsets in early infancy and, indeed, in a few patients shortly after birth 64.

Similarly, mild reduction in CTLA‐4 expression has also been observed in T_reg cells isolated from patients with rheumatoid arthritis (RA). While the CTLA‐4 gene is highly demethylated in normal T_reg cells (thus indicating stable CTLA‐4 expression) 65, the CTLA‐4 promoter was instead methylated in T_reg cells from RA patients 66.

All the above findings are also important from an age‐related perspective; in comparison to LRBA deficiency, which onsets in early infancy, the onset of RA is usually in adults (older than 45–50 years) 67. Hence, by comparing patients with RA, CTLA‐4 haploinsufficiency, LRBA insufficiency and IPEX we can study the relationship between the intensity of CTLA‐4‐related functional deficiency on T_reg cells, the effect on T_reg biology and the extent of any clinical presentation (including its severity and time of onset) (Fig. 2).

Figure 2.

Relationship between the intensity of regulatory T cell (T_reg) functional deficiency and the severity and time to onset of the disease. By comparing patients with rheumatoid arthritis (RA), cytotoxic T lymphocyte antigen 4 (CTLA‐4) haploinsufficiency, lipopolysaccharide‐responsive and beige‐like anchor protein (LRBA) insufficiency and immunodysregulation polyendocrinopathy enteropathy X‐linked (IPEX), we can study the relationship between the intensity of CTLA‐4 related functional deficiency on T_reg cells, the effect on T_reg biology and the extent of any clinical presentation (including its severity and time of onset).

Finally, although T_reg functional deficiencies have been described in numerous other autoimmune diseases, the detailed molecular mechanism(s) responsible for these deficiencies is/are currently unknown 68. For example, impaired T_reg suppression has been described in multiple sclerosis (MS) 69. It is indeed noteworthy that a key mechanism of suppression by T_reg cells is related to their high expression of CD25, as T_reg cells act as a sink for IL‐2 2, 70, 71. Interestingly, a polymorphism in CD25 has been associated with a high risk for developing MS through a genome‐wide association study (GWAS) 72. Therefore, it is plausible that a CD25‐related mechanism of suppression may be altered in the T_reg cells of MS patients. These T_reg cells may also be unstable, as loss of CD25 on T_reg cells is known to alter FOXP3 expression, T_reg cell function and precipitate Th17 effector cell differentiation 73. In parallel with RA patients who have a mild deficiency in CTLA‐4, the extent of the defect in T_reg suppression is probably mild or moderate. This is further supported by clinical observations that both RA and MS never evolve toward an IPEX‐like syndrome and rarely occur in early infancy.

Targeting FOXP3⁺ T_reg cells for the control of autoimmune responses

As deficiencies in T_reg number/function or resistance of T conventional cells from T_reg‐mediated suppression are observed in most human autoimmune diseases, it seems logical to propose stable and functionally superior T_reg‐based immunotherapies as a new therapeutic strategy in order to reinstate immune homeostasis. The two T_reg‐based therapeutic strategies currently being clinically evaluated in autoimmunity are in‐vivo T_reg expansion and infusion of in‐vitro expanded T_reg cells 19, 74, 75.

From the perspective of in‐vivo expansion, low‐dose IL‐2 has been evaluated in Phases I and II trials as a therapeutic targeting human T_reg cells in order to expand them in vivo within the context of autoimmunity (e.g. T1D, alopecia areata and systemic lupus) or inflammatory conditions (hepatitis C‐related cryoglobulinaemic vasculitis) 76. However, although injection of IL‐2 expands the circulating T_reg cell population, it also expands effector cells such as natural killer (NK) cells or eosinophils—thus indicating the lack of T_reg specificity 77, 78. This has been overcome through the development of an IL‐2/anti‐IL‐2 complex that can specifically promote the binding of IL‐2 to the high‐affinity receptor of IL‐2 that is expressed by activated T_reg cells and promote T_reg cell expansion in vivo without modifying other effector cells 79, 80, 81.

A second approach is the autologous expansion of T_reg cells in vitro in order to reinfuse a large number of T_reg cells into patients. Due to the recognized potential for reduced FOXP3 expression or reduced immunosuppressive capacity upon in‐vitro expansion, it is important to culture the right cell subpopulation in conditions that favour maintenance of T_reg phenotype and function 82. This will help to optimize any expansion protocols and more reliably predict the phenotype of the end product 83. The addition of rapamycin to the culture conditions is important to eliminate contaminating conventional effector cells. Finally, we also consider it important to utilize molecules capable of modifying T_reg epigenetics (e.g. DNA methyltransferase inhibitors or vitamin C) 37, 84, 85. These molecules work to maintain FOXP3 expression as well as various other T_reg‐specific demethylation patterns, which consequently lead to more stable and functional T_reg cells 37, 65, 85.

Strategies aiming at increasing the number of autologous T_reg cells are suitable for diseases with reduced but fully functional T_reg cells. However, it is currently unknown in diseases with deficiencies in T_reg function whether the deficiency is present within the entire T_reg population (i.e. all T_reg cells are impaired) or specific to a distinct subset (indicating that some T_reg cells would be functionally impaired while others would be fully functional). In the first case, all expanded T_reg cells would be functionally impaired with no beneficial therapeutic effect. In the second case the expansion, either in vitro or in vivo, of the global pool of T_reg cells would lead to the expansion of deficient T_reg cells and of fully functional T_reg cells. It could, therefore, be speculated that the number of expanded fully functional T_reg cells would be sufficient to overcome and compensate the functional deficiency of the expanded deficient subset. However, it is important not to overlook the possibility that expanded dysfunctional T_reg cells could convert into pathogenic cells when in a proinflammatory environment and also exacerbate disease 86. There are data demonstrating that FOXP3⁺ human T_reg cells can start secreting IL‐17 when exposed to cytokines such as IL‐1β, ‐2, ‐6, ‐15, ‐21 and ‐23 87, 88. These IL‐17‐secreting T_reg cells can subsequently lose their anti‐inflammatory function despite continuous FOXP3 expression 88.

A third approach is to utilize peripherally induced T_reg cells (pT_reg cells or iT_reg cells if in vitro), which differentiate from naive CD4⁺ T cells in the presence of transforming growth factor (TGF)‐β and IL‐2 89. One advantage of these cells is the potential to generate antigen‐specific subsets corresponding to the antigens key to the immunopathogenesis of different autoimmune diseases. However, the partially demethylated nature of FOXP3 gives rise to the instability of FOXP3 expression and subsequent loss of suppressive function 89. The in‐vivo stability of human iT_reg cells within a proinflammatory microenvironment needs to be optimized if they are to be considered as a safe and non‐pathogenic clinical product.

In diseases with functional T_reg deficiencies, infusion of autologous T_reg cells would be feasible if the impaired molecular mechanism of suppression is identified and corrected by the use of small molecules in vitro or via genetic modifications. An alternative strategy could be the infusion of allogeneic expanded T_reg cells sourced from cord blood or other healthy donors 90. Another strategy is to give those patients therapeutics that can compensate for the impaired T_reg mechanism; e.g. in patients with CTLA‐4 deficiency, the use of CTLA‐4‐Ig has proved effective in preventing autoimmune events 63.

As discussed above, T_reg‐based strategies are already being clinically evaluated in some human autoimmune diseases with reported deficiencies in T_reg cell numbers 78, 91 (and even in T_reg functions) 92, with the assumption that increasing their number would ameliorate or even cure the diseases. The first in‐human trial evaluating T_reg cells in autoimmunity was conducted in the setting of T1D 92. Fourteen patients received expanded autologous polyclonal T_reg cells (CD25⁺CD127^lo) in a dose‐escalation study (from 0·5 to 26 × 10⁸ cells). The reliability of the expansion process was demonstrated by the purity of the final product, 76–96·9% FOXP3⁺. Although two patients had serious adverse events of severe hypoglycaemia and ketoacidosis, no directly T_reg‐related adverse events were reported. This study was not powered for disease‐specific outcomes, as it was a Phase 1 study—hence, results of future Phase 2 studies are awaited. Further novel work using T_reg cells is also ongoing in the contexts of graft‐versus‐host disease and solid organ transplantation 93, 94, 95, 96.

T_reg cells are capable of inhibiting the initiation of immune responses, although the evidence regarding their ability to control active autoimmune/inflammatory disease is more controversial 2, 6, 7, 74. There are in‐vitro data demonstrating that T_reg cells cannot inhibit the proliferation of preactivated effector T cells 97, and when transferred into mice after pathogenic cells the T_reg cells are incapable of preventing the onset of autoimmunity 2 (Fig. 3). Indeed, this resistance of effector T cells to T_reg‐mediated suppression is another key mechanism in autoimmunity. The effector T cells are supported by a signalling pleiotrophic microenvironment consisting of TNF, IL‐6, IL‐1β as well as over‐activated intracellular signalling via the PI3K/Akt pathways 29. Additionally, in another murine model of severe colitis, the progression of this disease was ameliorated and reversed when the mice underwent adoptive transfer of T_reg cells 7. This effect was inhibited when mice were administered antibodies to IL‐10, CTLA‐4 or TGF‐β. Together, these data suggest that it is not only T_reg cells but also the presence of particular cytokines in the microenvironment that can modulate disease progression.

Figure 3.

Timing of regulatory T cell (T_reg)‐mediated suppression of immune responses. T_reg cells inhibit the initiation of immune responses by preventing the activation of effector cells. One important mechanism of suppression is their ability to adsorb interleukin (IL)‐2 produced by effector cells (top). When effector cells are preactivated, T_reg cells are not capable of suppressing the ongoing immune responses. In this case, IL‐2 is massively produced and amplifies the activated effector cell pool and also the T_reg cells. They can still suppress some non‐activated cells present in the vicinity by bystander suppression (bottom). Mild beneficial therapeutic effects observed in active diseases treated with T_reg biology‐based therapies can be explained by the absence of suppression of activated immune cells and the prevention of the activation of dormant pathogenic cells.

Hence, there was little surprise that only modest benefits are observed in trials evaluating T_reg‐based therapies for ongoing autoimmune or inflammatory diseases 78, 98. However, while they are inefficient at controlling activated pathogenic cells, the expanded T_reg cells could theoretically prevent the activation of resting pathogenic cells 74, 75. Therefore, some modest beneficial effect can be expected, as T_reg cells would inhibit the activation of dormant pathogenic cells. This would indeed be a viable therapeutic approach, as clinical observations have identified the role of steroids and/or immunosuppressants in controlling active autoimmune diseases as they target activated effector cells first. Secondly, when the disease is considered in remission, another line of immunosuppressants and/or of immunomodulatory drugs are given to patients in order to prevent relapses or disease flare‐up 75. As T_reg cells are professionally involved in the prevention of the initiation of pathogenic autoimmune responses, we believe that T_reg cell‐based treatments should only be considered as a maintenance therapy for the prevention of flares or relapses after the elimination of pathogenic cells. Such strategies would, therefore, be suitable for remitting and relapsing diseases such as MS, RA or anti‐neutrophil cytoplasmic antibodies (ANCA)‐associated vasculitis only during the remission period to prevent relapses.

These trials have also not addressed concerning whether or not it is necessary for infused T_reg cells in humans to migrate to the diseased tissue in order to prevent further flares/relapses. This is important in the context of autoimmunity, as although patients have a focal site of inflammation (e.g. joints in RA, gut in inflammatory bowel disease), they also have pathology elsewhere (e.g. extra‐articular/intestinal manifestations) 99, 100. Thus, in order to optimize the T_reg therapeutic effect and dose, it may be necessary to culture T_reg cells that pre‐emptively express tissue‐specific homing markers (if not, induce this phenotype genetically or pharmacologically). This is also important to minimize any off‐target effects such as the risk of malignancy from interference with anti‐tumour immunity.

Interestingly, the T_reg‐based therapeutic strategies available in humans aim at expanding polyclonal T_reg cells, without considering their antigen specificity 74, 75. Studies in mice indicate that antigen‐specific T_reg cells are more efficient that polyclonal T_reg cells in preventing autoimmune diseases. However, if the activation of T_reg cells is dependent on the target antigen of their T cell receptors (TCRs), the suppressive function itself is not only antigen‐specific. Once activated, T_reg cells can suppress effector T cells with the same or any other antigen specificity (via bystander suppression) 48. However, as T_reg cells expanded in vitro for cell therapy are highly activated, it may not be necessary to take into account antigen‐specificity of T_reg cells to obtain beneficial results in the settings of human autoimmune diseases.

Conclusions

From the data discussed so far, it is clear that abnormalities in the quantity or function of T_reg cells are observed in most, if not all, human autoimmune and/or inflammatory diseases. Although numerous T_reg subsets have been defined, their individual contributions to human autoimmune and/or inflammatory diseases are largely unknown. The clinical observations of genetic diseases involving deficiencies in T_reg function indicate that the severity and the age of disease onset correlate to the depth of T_reg functional impairment.

T_reg biology‐based therapies may not be suitable in patients with deficits of T_reg function, unless their deficit can be corrected in vivo/in vitro. It is also critical to consider the appropriate stage of autoimmune diseases whereby administration of T_reg cellular therapy can be effective. As highlighted, there are conflicting data regarding whether T_reg cells are more effectual at preventing the initiation of autoimmunity, ameliorating disease progression or curing autoimmunity itself. This is because, although T_reg cells can prevent the initiation of autoimmune responses, they cannot terminate ongoing responses.

We therefore propose here a global sequential therapeutic strategy for autoimmune diseases that includes (1) induction treatments for diseases flares or chronic disease with chronic activity and (2) maintenance treatments that are suitable for diseases during remission phases by utilizing T_reg cell biology‐derived therapies to prevent relapses or subsequent flares (Fig. 4).

Figure 4.

Therapeutic strategies for autoimmune diseases with regulatory T cell (T_reg) biology‐based treatments. The current gold standard treatment regimen for autoimmune diseases includes an induction phase, which aims to eliminate pathogenic cells. Usually, high‐dose steroids are given intravenously during the first days, followed by oral steroids with a tapering scheme. Immunosuppressants such as cyclophosphamide, mycophenolate or rituximab are also necessary to induce remission. When remission is obtained, it is necessary to maintain mild immunosuppressants such as azathioprine or mycophenolate to prevent the occurrence of subsequent flares or relapses of the disease. In diseases with number deficiency of functional T_reg cells, autologous T_reg cells infusion or interleukin (IL)‐2/anti‐IL‐2 complexes can be used as a maintenance treatment when the disease is controlled by standard induction treatments. In diseases with dysfunctional T_reg cells, if the dysfunctional mechanism of suppression is identified molecules mimicking the T_reg function, genetically corrected autologous T_reg cells or allogeneic or cord blood T_reg cells can be infused as maintenance treatment.

In patients with reduced T_reg numbers (but no functional deficiency), the expansion of autologous T_reg cells could be a suitable therapeutic approach (either infusion of in‐vitro‐expanded autologous cells, infusion of IL‐2/anti‐IL‐2 complex, or both). In contrast, patients with diseases involving deficiencies of T_reg function would benefit from a detailed understanding of the impaired mechanisms of action of their T_reg cells. We anticipate the development of suitable therapeutics to correct/reduce the severity of their T_reg deficiencies and thereby reduce their disease burden. Another feasible therapeutic option would be to administer functional allogeneic expanded T_reg cells.

Disclosures

None declared.