Fred Ramsdell

An essential role for Scurfin in CD4+CD25+ T regulatory cells.

The molecular properties that characterize CD4+CD25+ regulatory T cells (TR cells) remain elusive. Absence of the transcription factor Scurfin (also known as forkhead box P3 and encoded by Foxp3) causes a rapidly fatal lymphoproliferative disease, similar to that seen in mice lacking cytolytic T lymphocyte–associated antigen 4 (CTLA-4). Here we show that Foxp3 is highly expressed by TR cells and is associated with TR cell activity and phenotype. Scurfin-deficient mice lack TR cells, whereas mice that overexpress Foxp3 possess more TR cells. In Foxp3-overexpressing mice, both CD4+CD25− and CD4−CD8+ T cells show suppressive activity and CD4+CD25− cells express glucocorticoid-induced tumor-necrosis factor receptor–related (GITR) protein. The forced expression of Foxp3 also delays disease in CTLA-4−/− mice, indicating that the Scurfin and CTLA-4 pathways may intersect and providing further insight into the TR cell lineage.

Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse

Scurfy (sf) is an X-linked recessive mouse mutant resulting in lethality in hemizygous males 16–25 days after birth, and is characterized by overproliferation of CD4+CD8– T lymphocytes, extensive multiorgan infiltration and elevation of numerous cytokines. Similar to animals that lack expression of either Ctla-4 (refs. 5,6) or Tgf-β (refs. 7,8), the pathology observed in sf mice seems to result from an inability to properly regulate CD4+CD8– T-cell activity. Here we identify the gene defective in sf mice by combining high-resolution genetic and physical mapping with large-scale sequence analysis. The protein encoded by this gene (designated Foxp3) is a new member of the forkhead/winged-helix family of transcriptional regulators and is highly conserved in humans. In sf mice, a frameshift mutation results in a product lacking the forkhead domain. Genetic complementation demonstrates that the protein product of Foxp3, scurfin, is essential for normal immune homeostasis.

Fas and FasL in the homeostatic regulation of immune responses

Studies of the biological effects of Fas signaling, using transformed cell lines as targets, indicate that ligation of the Fas receptor induces an apoptotic death signal. Chronically activated normal human T cells are also susceptible to Fas-mediated apoptosis. However, interactions between Fas and Fas ligand can also yield a costimulatory signal. Here, David Lynch, Fred Ramsdell and Mark Alderson present a model for the role of As and FasL in the homeostatic regulation of normal immune responses. They discuss how dysregulation of the Fas apoptotic pathway may contribute to certain disease states, including autoimmune disease and human immunodeficiency virus (HIV)-induced depletion of CD4+ T cells.

Maintenance of in Vivo Tolerance by Persistence of Antigen

T cells of the immune system respond only to foreign antigens because those cells with reactivity for self proteins are either deleted during their development or rendered non-responsive (anergic). The maintenance of the nonresponsive state was found to require the continual exposure of the anergic T cells to antigen. When anergic T cells were removed from the self antigen by adoptive transfer to a mouse strain lacking the antigen or by in vitro culture, nonresponsiveness was reversed and the anergic cells returned to normal functional status.

Thymic selection in CD8 transgenic mice supports an instructive model for commitment to a CD4 or CD8 lineage

Immature thymocytes, which coexpress CD4 and CD8, give rise to mature CD4+CD8- and CD4-CD8+ T cells. Only those T cells that recognize self-MHC are selected to mature, a process known as positive selection. The specificity of the T cell antigen receptor (TCR) for class I or class II MHC influences the commitment to a CD4 or CD8 lineage. This may occur by a directed mechanism or by stochastic commitment followed by a selection step that allows only CD8+, class I-specific and CD4+, class II-specific cells to survive. We have generated a mouse line expressing a CD8 transgene under the control of the T cell-specific CD2 regulatory sequences. Although constitutive CD8 expression does not affect thymic selection of CD4+ cells, selection of a class I-specific TCR in the CD8 subset is substantially improved. This outcome is consistent with a model for positive selection in which selection occurs at a developmental stage in which both CD4 and CD8 are expressed, and positive selection by class I MHC generates an instructive signal that directs differentiation to a CD8 lineage.

The Amount of Scurfin Protein Determines Peripheral T Cell Number and Responsiveness

In the absence of the recently identified putative transcription factor scurfin, mice develop a lymphoproliferative disorder resulting in death by 3 wk of age from a pathology that resembles TGF-β or CTLA-4 knockout mice. In this report, we characterize mice that overexpress the scurfin protein and demonstrate that these animals have a dramatically depressed immune system. Mice transgenic for the Foxp3 gene (which encodes the scurfin protein) have fewer T cells than their littermate controls, and those T cells that remain have poor proliferative and cytolytic responses and make little IL-2 after stimulation through the TCR. Although thymic development appears normal in these mice, peripheral lymphoid organs, particularly lymph nodes, are relatively acellular. In a separate transgenic line, forced expression of the gene specifically in the thymus can alter thymic development; however, this does not appear to affect peripheral T cells and is unable to prevent disease in mice lacking a functional Foxp3 gene, indicating that the scurfin protein acts on peripheral T cells. The data indicate a critical role for the Foxp3 gene product in the function of the immune system, with both the number and functionality of peripheral T cells under the aegis of the scurfin protein.

The Level of CD8 Expression Can Determine the Outcome of Thymic Selection

During thymic development, thymocytes that can recognize major histocompatability complex (MHC) molecules on thymic epithelial cells are selected to survive and mature (positive selection), whereas thymocytes that recognize MHC on hematopoietic cells are destroyed (negative selection). It is not known how MHC recognition can mediate both death and survival. One model to explain this paradox proposes that thymocytes whose T cell antigen receptors (TCRs) recognize MHC with high affinity are eliminated by negative selection, whereas low affinity TCR-MHC interactions are sufficient to mediate positive selection. Here we report that, while the expression of a 2C TCR transgene leads to positive selection of thymocytes in H-2b mice, expression of both a CD8 transgene and a 2C TCR transgene causes negative selection. This observation indicates that quantitative differences in TCR-MHC recognition are a critical determinant of T cell fate, a finding predicted by the affinity model for thymic selection.

FOXP3 and scurfy: how it all began

It has been 65 years since the scurfy mutation arose spontaneously in mice at the Oak Ridge National Laboratory in the United States, and it is 13 years since the molecular cloning of the forkhead box P3 (FOXP3) gene was reported. In this Timeline article, we review the events that have occurred during and since this time. This is not meant as an exhaustive review of the biology of FOXP3 or of regulatory T cells, rather it is an attempt to highlight the landmark events that have demonstrated the importance of FOXP3 in immune function. These events have driven, and continue to drive, the extensive research effort to fully understand the role of regulatory T cells in the immune system.

A Novel Mutation in CD83 Results in the Development of a Unique Population of CD4+ T Cells

Using a mouse mutagenesis screen, we have identified CD83 as being critical for the development of CD4+ T cells and for their function postactivation. CD11c+ dendritic cells develop and function normally in mice with a mutated CD83 gene but CD4+ T cell development is substantially reduced. Additionally, we now show that those CD4+ cells that develop in a CD83 mutant animal fail to respond normally following allogeneic stimulation. This is at least in part due to an altered cytokine expression pattern characterized by an increased production of IL-4 and IL-10 and diminished IL-2 production. Thus, in addition to its role in selection of CD4+ T cells, absence of CD83 results in the generation of cells with an altered activation and cytokine profile.

Dynamic regulation of FoxP3 expression controls the balance between CD4+ T cell activation and cell death.

The forkhead‐family transcription factor FoxP3 is important for the development and function of CD4+CD25+ regulatory T cells. While the overall phenotypic effects of FoxP3 expression are evident, the mechanism by which FoxP3 regulates T cell activation is not well understood. CD4+ T cells from mice that express a FoxP3 Tg are refractory to TCR‐mediated stimulation, failing to proliferate or produce cytokines, but possess suppressive activity towards normal T cells. In this report we show that these T cells express elevated levels of mRNA for pro‐apoptotic genes and undergo rapid apoptosis following stimulation. These T cells also display slower cell cycle transit following activation, suggesting that FoxP3 is capable of regulating the ability of T cells to respond to TCR‐mediated activation. Lastly, we show that contrary to expected results, under Th1 or Th2 driving conditions, CD4+ T cells from FoxP3 Tg mice differentiate into effector cells. Concomitant with differentiation is a loss of FoxP3 mRNA and protein. These data demonstrate that FoxP3 levels regulate T cell function, and that FoxP3 itself is dynamically regulated during effector T cell differentiation.