T-regulatory cell-mediated immune tolerance as a potential immunotherapeutic strategy to facilitate graft survival

Introduction

T regulatory cells (Treg) are a specialised subset of T cells that engage in the maintenance of immunological self-tolerance by actively suppressing the activation, expansion of self-reactive lymphocytes, suppression of immune response^1,2 and constitute a central mechanism in immune regulation³. There is no doubt that vital organ transplantation has prolonged the survival of patients with terminal diseases and end-stage organ failure. This great advance in transplantation could not have been achieved without the rapid development of immunosuppressive agents; although the side effects of immunosuppression remain one of the major concerns in clinical transplantation. As more patients benefit from organ transplantation we also face the worldwide problem of the shortage of donor organs, and long-term graft loss. It is almost evident that conventional T cells are involved in transplant injury, and their toxic effects on the health of transplants could, therefore, be manipulated by intervening through immunotherapies^4,5.

The presence of Treg allows the human immune system to maintain self-tolerance against self-destruction while sustaining the ability to mount robust immune responses against invading microorganisms and cancerous cells^3,6. Cell-mediated suppression has been shown to play a central role in immune regulation although a suitable marker for identifying, and characterising the suppresser cells involved was not known⁷. In 1995, Sakaguchi and his colleagues identified a group of T cells from the thymus, which they showed to be responsible for protection against the development of autoimmune diseases^8,9. Reconstitution of CD4⁺CD25⁺ cells in nude mice prevented the development of autoimmune diseases associated with the transfer of the CD4⁺CD25⁻ cells⁸. These findings confirmed the contribution of CD4⁺CD25⁺ cells in maintaining self-tolerance by down-regulating the immune response of CD4⁺CD25⁻ cells, and this became a significant stepping-stone for Treg research^8–10. The development of natural Treg in the thymus was thought to occur in the Hassall’s corpuscles¹⁰. The thymus is the central organ for immunological self- and non-self-discrimination, and Treg are produced in the thymus as a mechanism for maintaining self-tolerance¹¹. Thymocytes, which originate from bone marrow, undergo two selection processes during their development in the thymus before being released into the circulation^10,12,13. These rigorous selection processes ensure that lymphocytes obtain a broad range of reactivity to foreign antigens yet lack reactivity to self-antigens^3,11. During the selection processes, most of the self-reactive lymphocytes eliminated, nevertheless, a fraction of reactive T lymphocytes do escape clonal deletion (the negative selection) in the thymus and are released into the circulation¹⁴. In vitro studies have shown that the suppression by Treg occurs in a cell-contact manner, and is likely to be cytokine-independent¹⁵. The occurrence of many autoimmune diseases is prevented by active suppression of naturally occurring Treg in the periphery⁹. These findings have also been supported by the observation that elimination of the CD4⁺CD25⁺ Treg population led to the loss of self-tolerance, and the induction of autoimmune diseases in animals. Several classes of induced Treg have been identified and shown to be capable of suppressing antigen-specific immune responses via cell-contact or of secreting soluble factors such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β)^16,17. These Treg are thought to arise from CD4⁺CD25⁻ T cells either during an immune response or after encountering a tolerogenic dendritic cell (Figure 1). Chen et al. demonstrated that, under the co-stimulation of T-cell receptors (TCR) and in the presence of TGF-β, peripheral CD4⁺CD25⁻ naive T cells were converted to CD4⁺CD25⁺ T cells, acquiring the expression of the Treg transcription factor Foxp3 and inhibiting T-cell proliferation in vitro¹⁸. Other studies also indicated that these Treg might be antigen-specific and inhibit CD4⁺ T-cell proliferation via a mechanism involving the soluble factors IL-10 and TGF-β^16,18. Following the study in 1995 by Sakaguchi et al., several additional studies investigating the function of CD4⁺CD25⁺ cells in murine models of auto-immune diseases further demonstrated a close correlation between the development and function of Treg and their expression of Foxp3^2,8. In 2001, Brunkow et al. demonstrated that the genetic cause of a fatal lymphoproliferative disorder was a defect in a gene called Foxp3 in the X chromosome¹⁹. The frame-shift mutation in this gene results in disruption of Foxp3 protein production, causing a disproportionate increase in CD4⁺CD8⁻ T lymphocyte activity^19,20. Recent studies have revealed that different genetic defects in the Foxp3 gene (single amino acid deletions in the leucine zipper domain and missense and nonsense mutations and deletions in the fork head homology domain) result in uncontrolled activation and expansion of CD4⁺ T cells, causing the human IPEX/XLAAD diseases^21,22. Studies have also shown that, in a number of allergic and autoimmune disorders, loss-of-function mutations in the Foxp3 gene cause congenital deficiency of Treg; triggering the onset of lymphoproliferation, myeloproliferation, autoimmunity, and allergic dysregulation³. Studies of Foxp3 expression in CD4⁺CD25⁺ Treg also demonstrated that Foxp3 is associated with the development and function of Treg^1,6. Using real-time quantitative polymerase chain reaction, Hori et al. showed that the expression of Foxp3 was primarily on the CD4⁺CD25⁺ T cells in mouse thymus¹. Other studies have also indicated that Foxp3 expression is predominantly restricted to the CD4⁺CD25⁺ Treg population in both mice thymus and in the periphery^6,23. Moreover, the induction of Foxp3 expression in CD4⁺CD25⁻ cells result in these cells acquiring a Treg-like phenotype and suppressive activity¹. Given the evidence that the expression of Foxp3 converts CD4⁺CD25⁻ T cells into cells that are phenotypically and functionally like Treg^1,24 and that Foxp3 expression is confined to CD4⁺CD25⁺ Treg, Foxp3 is thought to be necessary and sufficient for mouse CD4⁺CD25⁺ Treg development and function⁶. Treg can also be induced in mice via the stimulation of CD4⁺ T cells with anti-CD3/28 in the presence of TGF-β or all-trans-retinoic acid. These cells acquire Foxp3 expression and potent immunosuppressive effects²⁵. Though, the generation and expansion of Treg for clinical applications still require further investigation, these cells may be of benefit in future cellular therapy for human diseases and transplantation²⁵.

Figure 1.

A model illustrating generation of T regulatory cells from the thymus.

The CD4⁺ CD25⁺ T-cell interaction with dendritic cells facilitates expression of CD4⁺ CD25⁺ FoxP3⁺ cells. The development of Treg takes place in the thymus and in the periphery (converted from CD4⁺CD25⁻ T cells) under stimulation from activated dendritic cells.

Phenotype of T regulatory cells

Apart from CD4⁺CD25⁺Foxp3⁺ Treg, several other types of Treg have been identified in the immune system with distinct phenotypes and mechanisms of action²⁶. The immunological function and importance of each type remains speculative and under investigation. T cells with “regulatory function” can be derived from the CD4⁺CD8⁺ T cell populations or even from other minor T-cell populations such as non-polymorphic CD1d-responsive natural killer T cells²⁶. CD4⁺Tr1, CD8⁺, CD8⁺CD28⁻ and TCR⁺CD4⁻ CD8⁻ Treg have been shown in other studies to exert regulatory effects in different transplantation models^17,27.

CD25 expression

After the discovery of CD4⁺CD25⁺ Treg, CD25 was thought to be an indispensable molecule for the generation and maintenance of natural CD4⁺ Treg²⁸. Sakaguchi et al. showed that the reconstitution of BALB/c athymic nude mice with CD4⁺CD25⁺ T cells could normalise the autoimmune disease caused by the transfer of naïve BALB/c splenic cells depleted of CD4⁺CD25⁺ T cells⁸. The co-transfer of a small number of CD4⁺CD25⁺ T cells with CD4⁺CD25⁻ T cells could prevent lethal systemic autoimmunity^8,29. Furthermore, lethal autoimmunity in CD25⁻ deficient mice could be prevented by the inoculation of CD4⁺CD25⁺ Treg from normal syngeneic mice²⁹. Similar phenotypes and properties have also been observed in human CD4⁺CD25⁺ T cells as these cells have been shown to suppress the activation and proliferation of CD4⁺ and CD8⁺ T cells³⁰. Thus, the expression of CD25 seemed to be linked to the development, maintenance and function of Treg^31,32. CD25 is not expressed on resting CD4⁺ T cells; however, upon exposure to antigen and T-cell activation, CD25 can be detected on activated CD4⁺ T cells, which have no regulatory functions. Although CD25 has been very useful in identifying and enriching Treg. The reliability of CD25 as a marker for Treg has been questioned²⁷. Other studies have also suggested that the actual level of CD25 expression on CD4⁺ T cells is critical for distinguishing between regulatory and non-regulatory CD4⁺ T cells^27,33. Baecher-Allan et al. found that only CD4⁺CD25^high cells that constitute approximately 2–3% of total CD4⁺ T cells, consistently had in vitro regulatory activity closely resembling murine Treg^30,34. The CD4⁺CD25^low cells, on the other hand, contained a more heterogeneous mixture of cells with different cell surface phenotypes and did not show suppressive activity³⁰.

CD28 expression

CD28 is one of the main T-cell co-stimulatory molecules, which binds to CD80/CD86 molecules on antigen-presenting cells to deliver a positive signal for T-cell survival and proliferation^35,36. This signal promotes the secretion of cytokines, such as IL-2, and cell expansion, and prevents the induction of anergy and cell death³⁵. Recent studies have shown that CD28 interactions are essential for the development of Treg in the thymus and for their survival in the periphery³. Both CD28 and CD80/CD86 knockout mice on a non-obese diabetic background rapidly develop severe spontaneous autoimmune diabetes and have a substantial reduction in the CD4⁺CD25⁺ Treg population in both the thymus and periphery³⁷. Adoptive transfer of CD4⁺CD25⁺ Treg from a naïve animal delayed the onset of diabetes in the mice³⁷. In the transplant setting, emerging data show that CD28 signalling is required for the induction of unresponsiveness to donor allo-antigens in vivo²⁷. Soligo et al. demonstrated that unique CD28-induced NF-κB-dependent signals were sufficient to activate Foxp3 transcription in human CD4⁺CD25⁺ T cells³⁸. It has also been shown that CD28 is critical for the generation of CD4⁺CD25⁺Foxp3⁺ Treg from CD4⁺CD25⁺ T cells both in vitro and in vivo. Thus CD28 plays a critical role in Treg and the regulation of peripheral tolerance³⁸. However, the importance of CD28 signalling in the development and homeostasis of Treg has been demonstrated, CD28 is also found on other T lymphocytes³⁹ and, therefore, can not be used as a unique Treg marker. Furthermore, CD4⁺CD25⁺ Treg from CD28 knockout mice were found to have a similar phenotype and a potent suppressive effect comparable to that of the CD4⁺CD25⁺ Treg extracted from CD28⁺ animals in in vitro assays⁴⁰. In brief, the role and influence of CD28 signalling in Treg homeostasis need further defining.

CD62L is another surface molecule that is expressed constitutively at a high level by both human and mouse peripheral CD4⁺CD25⁺ Treg^30,41. Indeed, in human, more than 95% of the CD4⁺CD25^high Treg were found to be CD62L-positive^30,34. In an in vivo murine model of allogeneic bone marrow transplantation, Ermann et al. showed that CD4⁺CD25⁺CD62L⁺ T cells inhibited the expansion of alloreactive CD4⁺CD25⁻ T cells more efficiently than CD4⁺CD25⁺CD62L⁻ T cells⁴². Only the adoptive transfer of donor CD4⁺CD25⁺CD62L⁺ Treg protected the recipient mice from lethal acute GVHD induced by donor CD4⁺CD25⁻ T cells⁴². Due to the different expression of various memory/or activation markers of these two subsets of CD4⁺CD25⁺ cells, it was proposed that the CD4⁺CD25⁺CD62L⁺ cell population contained more Treg and less recently activated CD4⁺ T cells than the CD4⁺CD25⁺CD62L⁻ population⁴². The variation seen in different CD62L subsets of Treg indicated that they might differ in their ability to enter the priming sites and that CD62L expression might be important for Treg function. Furthermore, in vitro-expanded CD4⁺CD25^high T cells with suppressive function and allo-antigen-specific CD4⁺CD25⁺ Treg have also been found to express CD62L on their surface²⁷. Although CD62L has been shown to be expressed on human and mice CD4⁺CD25⁺ Treg, the expression of CD62L is not unique to Treg and can also be found on other cell types: i.e. “B cells”, naïve, memory T cells, granulocytes and thymocytes³⁰.

CTLA-4 expression

Peripheral CD4⁺CD25⁺ Treg from both humans and mice have been shown to express CTLA-4 constitutively³⁰. T cells from CTLA-4⁻/⁻ mice are resistant to gamma-irradiation-induced apoptosis, as observed in NOD mice, suggesting that CTLA-4 might contribute to the pathogenesis of autoimmune diabetes⁴³. CTLA-4⁻/⁻ mice and Foxp3⁻/⁻ mice also share similar systemic autoimmune-like syndromes, and both have a short life span due to massive lymphoproliferation. The induced expression of Foxp3 delays disease and prevents mortality in CTLA-4⁻/⁻ mice indicating the potential correlation of Foxp3 and CTLA-4 pathways²³. The link between CLTA-4 and Treg, however, remains controversial. The analysis of Treg in CTLA-4 deficient mice indicated that CLTA-4 might not be required for the development or function of peripheral Treg while other studies showed that CTLA-4 plays a vital role in controlling the homeostasis and suppressive capacity of Treg in both mice and human^44–46. Boden et al. showed that non-activating anti-CTLA-4 antibodies could block the suppressive activity of Treg in vitro⁴⁷. Like other Treg markers described above, CTLA-4 expression is not restricted to Treg and has also been reported on activated T and B cells²⁷. Foxp3 has been widely demonstrated to be expressed in mice and human CD4⁺CD25⁺ Treg, and to be critically important for the development and function of Treg¹⁰.

Foxp3 expression

Foxp3 is currently the most widely used Treg marker^{27,33,46,48,49}. It has, however, been demonstrated that Foxp3 is not necessarily an indicator of suppressive capacity in human, as has been shown for mice²³. Studies showed that stimulation via TCR in the presence of IL-2 could induce transient Foxp3 expression in human CD4⁺CD25⁻ T cells, although the level of expression was significantly lower than that in suppressive naïve Tr eg⁵⁰. The transient expression of Foxp3 in these CD4⁺CD25⁻ T cells did not induce a Treg-like phenotype or confer suppressive activity on freshly isolated CD4⁺CD25⁻ T cells⁵⁰. Furthermore, several studies have discovered different types of Treg, which do not express Foxp3, but are still able to exert inhibitory effects on the in vitro proliferation of CD4⁺CD25⁻ T cells with a similar efficiency as the CD4⁺CD25⁺ Treg²⁷. Thus, the correlation of Foxp3 with the suppressive ability in Treg in humans may not be the same as that observed in mice, in which expression of Foxp3 correlates strictly with regulatory activity⁵⁰. In contrast to the prevention of transplant rejection by Treg in animal models, clinical studies examining Foxp3 mRNA expression as a marker of Treg revealed a correlation of Treg with acute rejection of renal transplants⁵¹. A small study examining CD4⁺CD25^high cells rather than Foxp3 mRNA expression as a marker for Treg showed that the cells from recipients of rejected organs exerted greater inhibition on CD4⁺CD25⁻ cells than the cells from recipients of non-rejected organs in a small number of heart transplants⁵². The emerging information on Treg in human indicates that these cells are more divergent, less well defined, and more complex than was originally thought. Thus caution should be taken with regards to their characterisation and isolation.

Discussion

High-dose immunosuppression is used to prevent acute graft rejection and is designed according to the drug levels, graft function, and biopsy outcomes of the patient⁵³. Though, the immunosuppressive regimen is tailored for each individual patient the health risks related to the use of immunosuppression remain a major global problem. The side effects depend on the nature and amount of the immunosuppressive drugs used and also the duration of the treatment. Current immunosuppressive drugs are non-specific and damage transplanted organs³¹. In most cases, transplant recipients require lifelong treatment with immunosuppressive drugs³¹, even though, this favours the development of opportunistic infections and tumours^31,54–57. Indeed, the adverse effects of immunosuppressive treatment contribute substantially to morbidity and mortality among transplant recipients^57,31,58, becoming a burden on medical resources and lowering the frequency of transplantation^58–60. In an effort to improve outcome on the basis of individualised treatment, there is an urgent need for non-invasive assays, as well as biomarkers, to more accurately monitor allogeneic responses and predict the risk of acute and chronic allograft rejection in transplant recipients⁵³. However, the potential impact of current immunosuppression regimens on the development and function of Treg in transplant recipients is still unclear and requires further elucidation. CD4⁺CD25⁺Foxp3⁺ Treg can act as critical mediators of peripheral self-tolerance. However, Treg can avert immune reactions against tumours and pathogens. Treg were originally identified as a CD4⁺CD25⁺ T-cell population with the ability to suppress immune responses. The identification of Foxp3 as the “master-regulator” of Treg was a critical step in defining Treg as a distinct T-cell lineage.

Proliferation and cytokine production of CD4⁺CD25⁻ conventional T cells can be inhibited directly by Treg. In addition, Treg can indirectly suppress T-cell activation via inhibition of the stimulatory capacity of antigen-presenting cells (Figure 2). The immunosuppressive mechanism of Treg remains obscure, although there is increasing evidence that these cells exert their effects through a variety of mechanisms that include the secretion of immunosuppressive soluble factors such as IL-9, IL-10 and TGF-β, while cell-mediated regulation, via the high affinity TCR and other co-stimulatory molecules such as CTLA-4, GITR (glucocorticoid-induced tumour necrosis factor receptor-related protein), cytolytic activity and, therefore, a number of different immunotherapies, has been adopted to target Treg⁶¹. As illustrated in Figure 2, most Treg-directed immunotherapies use anti-CD25⁶², anti CTLA-4⁶³, anti-VEGFR2⁶⁴, anti-CD134⁶⁵ and anti-GITR monoclonal antibodies⁶⁶. Additional therapies, however, have been implemented using drugs such ascyclophosphamide⁶⁷, tacrolis mus⁶⁸, cyclosporine A⁶⁹, and other research is targeting different cell signalling pathways involved in Treg development such as the p38MAPK pathway⁷⁰ and the Notch pathway⁷¹. The impact of anti-CD25 monoclonal antibody therapy on circulating Treg in transplant recipients has been examined. In a conversion study by Demirkiran et al. a single dose of IL-2-receptor (CD25) blocking antibody (daclizumab) reduced the levels of CD4⁺CD25⁺ cells but not the levels of CD4⁺CD25⁺Foxp3⁺ cells in the circulation of liver transplant recipients⁷². These findings explain why immunosuppression has complex effects on Treg in clinical transplantation. The IL-2 signalling pathway is crucial for the development and function of Treg; thus treatments targeting or affecting this pathway might potentially influence the level or function of Treg in transplant recipients. However, the potential impact of current immunosuppression regimens on the development and function of Treg in transplant recipients is still unclear and requires further elucidation. The important role of Treg in the modulation of the immune response has ensured that these cells continue to generate significant research interest. As has been shown in numerous studies, the role of Treg in transplant recipients is diverse²⁷. Understanding the mechanisms by which Treg cells exert their effects is an area of global research with broad implications for the development of therapeutic strategies for many disease processes including pulmonary hypertension, solid organ transplants, cancer, diabetes, and other immune-mediated diseases.

Figure 2.

A model of the various cell markers and specific immunotherapeutic strategies to eliminate and inhibit T regulatory cells.

In summary, this review highlights important information about immunosuppressive treatment in transplant recipients, and provides some basis for the potential use of Treg as diagnostic and therapeutic tools in clinical transplantation.