Regulatory T cells (Tregs) are now among the most intensively studied of T cell subsets and among the major targets of immune drug development because of their fundamental role in immune tolerance. The three papers featured as Pillars of Immunology in this issue of The Journal of Immunology were published in 2003 and describe the discovery of Foxp3 as the determinant of Treg cell fate and function (1–3). These papers were essential in validating the existence of Tregs as a specialized cell lineage and provided the molecular underpinnings for the revival of the suppressor T cell field that was under siege for decades. Moreover, the demonstration that the devastating autoimmune syndrome, immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX), was due to a deficiency in Tregs established direct evidence for Tregs as critical regulators of immune homeostasis (4).
Ever since Medawar won the Nobel Prize for his work in neonatal tolerance, immunologists have thought that the induction and maintenance of immune tolerance would be the key to treating diseases ranging from autoimmunity, allergy, and organ transplant rejection (by inducing tolerance) to cancer and infectious disease (by breaking tolerance). The description of a specialized subset of T suppressor cells, proposed by Gershon and Kondo (5), was heralded as a fundamental discovery for the basis of immune tolerance. This compelling concept motivated dozens of passionate young researchers (myself included) and led to hundreds of studies designed to dissect the mechanisms of action and functions of these T suppressor cells. Unfortunately, many of the follow-on studies suffered from an absence of molecular precision, as they were done long before the development of the molecular tools at our disposal today. Failure to find reliable markers to uniquely identify T suppressor cells, ambiguity in the molecular basis of suppression, and inability to isolate pure populations of Ag-specific T suppressor cells for detailed analyses plagued the field. The core principles of a suppressor cell network were discredited and the T suppressor cell field was soon derided by most card-carrying immunologists (6, 7).
The underpinnings of the T suppressor cell renaissance were established in the 1990s as investigators in the field of neonatal tolerance, and later autoimmunity, transplantation, and cancer, established a cellular basis for immune regulation (7–10). Concepts such as bystander suppression and infectious tolerance were verified in adoptive transfer experiments using small numbers of what turned out to be CD4+ lymphocytes marked by various cell surface markers, including CD62L and CD45RB (10). Most importantly, Sakaguchi et al. (11) identified constitutive high CD25 expression on a subset of CD4+ T cells that could transfer immune tolerance in a previously described model of neonatal thymectomy-induced autoimmunity (8). These in vivo observations were complemented by the development of an in vitro assay to assess suppressor cell activity (12). As a result, the field of T suppressor cells, rebranded as Tregs, was reborn. However, because expression of CD25 as well as other molecules implicated in suppressor activity (e.g., CTLA-4, GITR, TGF-β, and IL-10) was not limited to Tregs, skepticism prevailed among many immunologists, as the existence of a dedicated subset of T cells specialized for immune suppression continued to be challenged.
The groundwork for the results published in these three highlighted papers was laid back in 1949 when a mouse strain was identified at the Oak Ridge National Laboratory in Tennessee that had developed a spontaneous mutation resulting in severely scaly and ruffled skin, enlarged spleen and lymph nodes, and ultimately death at just a few weeks of age. By the early 1990s, it became clear that these so-called “scurfy” mice suffered from a systemic, T cell–dependent, autoimmune disease that could be rescued in trans by mixed bone marrow reconstitution. Importantly, the pathology of the scurfy mouse resembled a human disease, IPEX (13). Then, in late 2000 and early 2001, Ramsdell and colleagues (14) systematically mapped the gene responsible for scurfy disease, Foxp3, an essentially undefined transcription factor. Contemporaneously, similar efforts by Chatila et al. (15) and others (16) demonstrated that FOXP3 was responsible for the aggressive and rapid lethal lymphoproliferative syndrome characterized by multisystem autoimmune sequelae in patients with IPEX. Interestingly, a notable function of FOXP3 was the repression of IL-2 expression in T cells. Thus, the prevailing view was that FOXP3 functioned in a cell-intrinsic manner to restrain T cell activation directly in activated effector T cells.
The seminal finding in the three Pillars in Immunology papers was the demonstration that Foxp3 was uniquely expressed in the CD4+CD25+ Treg subset and responsible for dominant suppression mediated by Tregs. Foxp3 was not expressed in effector T cells, although CD25 was upregulated by T cell activation. The Ramsdell and colleagues (1) paper showed that “functional” CD4+CD25+ Tregs were missing in scurfy mice, whereas Rudensky and colleagues (3) demonstrated that targeted deletion of Foxp3 in mice recapitulated the systemic autoimmunity observed in the scurfin mutant (17). All three papers showed that forced expression of Foxp3 conferred a regulatory phenotype that included the constitutive expression of CD25, CTLA-4, and GITR, inhibition of cytokine production (namely IL-2), and the ability to suppress conventional T cell activation in vitro and in vivo. Furthermore, Fontenot et al. (3) showed that neonatal transfer of CD4+CD25+ Tregs into Foxp3 mutant mice rescued recipients from the lymphoproliferative autoimmune syndrome, whereas Hori et al. (2) showed that the adoptive transfer of Foxp3-transduced T cells prevented the development of autoimmune colitis and gastritis in a mouse model. Interestingly, overexpression of Foxp3 in CD8+ T cells resulted in suppressive cell activity, suggesting that Foxp3 was sufficient to induce a Treg phenotype in cell types that do not normally have Treg activity (1). Finally, forced expression of Foxp3 delayed, but did not prevent, lymphoproliferation in CTLA-4−/− mice, providing a hint of CTLA-4–independent function(s) of Tregs (1).
The discovery of FOXP3 as a master regulator of Treg lineage development and function by these three groups marked an inflection point of research in Treg biology. Expression of the gene encoding FOXP3 both directly and indirectly controls the expression of hundreds of proteins that are involved in many differentiation and effector functions of other T cell subsets, including Th1, Th2, and Th17 cells. Disruption of FOXP3, even in mature Tregs, can lead to Treg instability and exacerbation of autoimmunity or reversal of immune tolerance in the cancer and organ transplant setting (18–20). It has become increasingly clear that under certain conditions in the gut and other mucosal tissues, FOXP3 can be induced in conventional T cells, which alters the function of these cells and, in some instances, turns these potential effector T cells into Tregs (21). Thus, it is not surprising that efforts are underway to develop drugs that can modulate FOXP3 expression both directly and indirectly, leading to enhancement or repression of FOXP3 and FOXP3-dependent gene expression. In cancer immunotherapy studies, Abs that affect Treg homing are being tested as a means to enhance local cancer immunity, whereas in autoimmune disease, organ transplantation, and graft-versus-host disease, drugs that expand Tregs in vivo, such as low-dose IL-2, as well as direct Treg adoptive immunotherapy, are being pioneered as potential tolerogenic therapies (22, 23).
In summary, the discovery of FOXP3 and its role in Treg development changed the field of immunology. These three papers and the elegant continued contributions of these and other investigators to the field have altered our basic understanding of immune tolerance and led to new drugs that will transform the treatment of immunological diseases and many other diseases in which altered immunity and inflammation may play a role.
J. A. B. serves as a consultant for and receives a research grant and stipend from Juno Therapeutics on genetically engineered Tregs and also works with Caladrius Biosciences on clinical trials relating to Tregs in T1D. J.A.B. has intellectual property for which patents are pending licensed to these companies.
Abbreviations used in this article:
immunodysregulation, polyendocrinopathy, enteropathy, X-linked
regulatory T cell.