Studying human regulatory T cells in vivo.

In this issue of Clinical Cancer Research , Mutis et al. (1) use a model of xenogeneic graft-versus-host disease to assess the suppressive function of human regulatory T cells in vivo . This model is based on the transfer of human peripheral blood cells into RAG-2 / gc / mice that are deficient for natural killer cells as well as T and B cells. On transfer, human immune cells, especially T cells, induce a robust xenogeneic response to host antigens that kill the animals within 2 weeks. This model was first described by the same team in a previous article published in Clinical Cancer Research (2). In the present study, the authors investigate the effect of depletion or enrichment of CD4CD25 regulatory T cells in the graft inoculum on the development and severity of xenogeneic graft-versus-host disease after transplant. Regulatory T cells (Treg) have been characterized as a distinct subset of CD3CD4 T cells constitutively expressing the a chain of the high-affinity interleukin (IL)-2 receptor, CD25 (3). At first, these cells could not readily be distinguished from activated CD4 effector T cells because activated T cells transiently express a wide range of receptors also found on naturally occurring Treg. Subsequently, a unique transcription factor, forkhead box P3 (FOXP3), was found to be specifically expressed in murine and human Treg (4). Studies have also shown that forced ectopic expression of FOXP3 in CD4 T cells was sufficient to confer suppressive capabilities to CD4CD25 T cells (4). To date, no other marker has been identified that defines Treg more accurately than FOXP3. Functionally, CD3CD4FOXP3 Treg act as natural inhibitors of normal immune responses. These cells control and likely channel effector T cells responding to external and internal pathogens (5). They also restrain autoreactive immune cells from mounting destructive reactions to self-antigens (6). Likewise, Treg can also limit natural antitumor immune responses from rejecting transformed cells. A series of articles published in Clinical Cancer Research and elsewhere have reported the poor prognosis of patients when tumors are densely infiltrated with Treg (7–10). This is commonly interpreted as evidence to support an important role for Treg as mediators of resistance to effector T cells, although the role of Treg in tumor immunity is likely more complex (11). Defects in Treg have been associated with a variety of immune pathologies. Single point mutations in the FOXP3 gene are known to cause a lethal systemic autoimmune syndrome, immune dysregulation, polyendocrinopathy, enteropathy X-linked (12). Subtle alterations in Treg functions have also been reported in patients with multiple sclerosis (13), rheumatoid arthritis (14), systemic lupus erythematosus (15), type I diabetes (16), autoimmune polyglandular syndromes (17), as well as allergic reactions (18). As knowledge of Treg biology grows, it becomes apparent that these cells participate in virtually all immune responses. Artificially modulating their function could thus provide ways to enhance or reduce immune responses and lead to novel therapies. One of the contexts in which interventions to modulate Treg may first be used is after allogeneic hematopoietic stem cell transplantation (HSCT). Several retrospective clinical studies have suggested that the reconstitution of Treg after HSCT plays an important role in controlling alloimmunity and preventing graft-versushost disease (GVHD; refs. 19–21). Conversely, poor reconstitution of Treg likely leads to a persistent immune imbalance that results in chronic GVHD (22). It is also possible that Treg, by channeling effector cells, are required for any given immune response to reach its full capacity. This would explain the apparent immunodeficiency that correlates with reduced Treg pools and chronic GVHD after allogeneic HSCT. Several mouse studies have previously shown the potential use of Treg to prevent or treat GVHD across minor or major histocompatibility barriers (23–26). In most models, Treg were administered to the recipient together with the graft and were able to reduce or prevent GVHD. Interestingly, prevention of GVHD was obtained while retaining the beneficial graftversus-leukemia effect, suggesting that Treg-based therapeutic interventions could be particularly useful in patients with hematologic malignancies undergoing allogeneic HSCT (23, 24, 26). In humans, studies have shown the feasibility of purifying Treg from leukopheresis products from normal donors and expanding them in vitro for subsequent infusion in stem cell recipients (27, 28). Ex vivo –expanded Treg share a distinct phenotype with their naturally occurring counterparts and have the capacity to suppress a wide range of immune effector cells in vitro, including CD4CD25 T cells and CD8 T cells. Taken together, these preclinical studies set the stage for Treg-based therapeutic trials. Yet, without an adequate experimental model, the behavior of human Treg had not been studied in vivo until now. In this respect, the experimental model described by Mutis et al. (1) provides a unique opportunity to explore several aspects of the biological function of human Treg in vivo. Xenogeneic mouse transplantation models have previously been used by various research groups to study GVHD mediated by human effector cells. In 2003, van Rijn et al. (2) described the effects of transplanting human T cells into RAG-2 / gc / mice. Compared with the widely used nonobese diabetic-severe combined immunodeficient mice, RAG-2 / gc / doubleknockout mice that received i.v. infusions of human peripheral The Biology Behind