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

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 response1,2 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 immunotherapies4,5. 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 cells3,6. 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 diseases8,9. 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 research8–10. 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 circulation10,12,13. These rigorous selection processes ensure that lymphocytes obtain a broad range of reactivity to foreign antigens yet lack reactivity to self-antigens3,11. 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-β16,18. 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 Foxp32,8. 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 activity19,20. 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 diseases21,22. 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 Treg1,6. 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 periphery6,23. 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 Treg1,24 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. 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 models17,27.