Hot on Tolerance's Trail

A UNIQUE FACTOR

CD4⁺CD25⁺ T_regs express a unique transcription factor called Foxp3 (also called scurfin). Loss-of-function mutations in Foxp3 produce fatal T cell-mediated autoimmune diseases in mice and humans. This and several other lines of evidence led researchers to speculate that Foxp3 might control CD4⁺CD25⁺ T_regs. For example, Shimon Sakaguchi and colleagues from Kyoto University in Japan became interested in the transcription factor because a recessive human genetic disease called IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome), which arises from mutations in the human version of FOXP3, is clinically and pathologically similar to the autoimmune disease induced when researchers deplete T_regs in experimental animals. "This prompted us to ask whether Foxp3 plays a key role in the development and/or function of natural regulatory T cells," Sakaguchi says.

In the first Hot Paper,¹ published in February 2003, Sakaguchi and colleagues showed that FOXP3 expression is confined predominately to CD4⁺CD25⁺ T_regs in the thymus and periphery. Furthermore, retroviral transfer of FOXP3 converts naïve nonregulatory T lymphocytes into a phenotype similar to T_regs. For example, transfected cells suppress autoreactive T cells and express another antigenic hallmark of T_regs, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Buckner explains that the observation that introducing Foxp3 into mature T cells confers a regulatory phenotype shows that the transcription factor is vital not only to the development of T_regs, but also to their function.

The scurfy mouse strain underscores Foxp3's functional importance. Scurfy males, which express mutated Foxp3, die of a lymphoproliferative disease mediated by CD4⁺ T cells that is characterized by wasting and infiltration of lymphocytes into numerous organs. In April 2003, Howard Hughes Medical Institute investigator Alexander Rudensky and colleagues at the University of Washington, Seattle, reported that scurfy males show low levels of CD4⁺CD25⁺ T_regs. Further research showed that Foxp3 is essential for the normal development of CD4⁺CD25⁺ T_regs in the thymus. Finally, the Seattle team used a retrovirus to introduce FOXP3 into peripheral lymphocytes that express CD4 but not CD25. After the ectopic expression of Foxp3 in these CD4⁺CD25^- cells, lymphocytes gain the ability to suppress autoreactive cells and counter the scurfy phenotype.²

In the third paper,³ Fred Ramsdell and collaborators at Bothell, Wash.-based Celltech determined that mice overexpressing Foxp3 show high levels of CD4⁺CD25⁺ T_regs. Conversely, mice deficient in Foxp3 lack CD4⁺CD25⁺ T_regs. Moreover, CD4⁺CD25^- and CD4^-CD8⁺ Tregs do not normally suppress autoreactive cells. Foxp3 over-expression, however, confers suppressive activity on both lines. Further, mice that lack CTLA-4 show similar symptoms to those that lack Foxp3. High levels of Foxp3 activity delay the manifestations of CTLA-4 related disease, suggesting that the pathways are linked. Taken together, Steven Ziegler, director of the immunology program at the Benaroya Research Institute, notes that the papers, along with other research, show that in mice at least, Foxp3 expression is "necessary and sufficient" for the development and function of CD4⁺CD25⁺ T_regs.

PROFOUND IMPLICATIONS

The papers gave extra impetus to studies into the immunology of self/nonself. Indeed, Foxp3 is the only marker so far identified that appears to be specific for CD4⁺CD25⁺ T_regs. "Unfortunately, it is a nuclear protein, so detecting it means killing the cell," Ziegler says.

"There is an urgent need to find a specific cell-surface marker than can differentiate T_reg from other activated T cells," Sakaguchi adds. For example, numerous reports now link CD25⁺ T_regs with various conditions, including inflammatory bowel disease and rheumatoid arthritis. But CD25 is a general activation marker, and Sakaguchi argues that these reports are not reliable until the analyses can be repeated using more specific markers. "Once such a cell-surface molecule becomes available, we can expect sound progress in this area," Sakaguchi says.

T_reg cells expressing Foxp3 are produced in the thymus, but CD4⁺cells in the periphery may also acquire regulatory function. Dendritic cell (DC) triggering overcomes this suppression to allow effector response to pathogens. Neonatal thymectomy or loss-of-function mutations in FOXP3 leads to wasting disease, autoimmunity and lymphoproliferation. FOXP3 is also mutated in the recessive genetic disorder IPEX. (A. O'Garra, P. Vieira, Nat Immunol, 4:304–6, 2003.)

The hunt for specific markers is especially pressing, given that recent research suggests that Foxp3 may be important to other subsets of T cells. "Human T-cell clones and lines may express specific splice variants of Foxp3 that are not unique to regulatory T cells," says Mary Morgan, at the Nijmegen Center for Molecular Life Sciences, the Netherlands. "Research on splice variants of Foxp3 may be crucial to truly understanding the functions of this transcription factor," says Morgan.

Indeed, Ziegler says, the papers suggest that CD4⁺ T cells can be defined by the transcription factors they express. For example, Th1 cells express T-Bet, Th2 cells express GATA3, and T_regs express Foxp3. So changing the expression of a single gene potentially alters the functional fate of a CD4⁺ T cell. According to Ziegler, this raises the possibility of using ectopic Foxp3 expression to covert pathogenic T cells to a regulatory phenotype. "A possible scenario would be the isolation of self-reactive T cells from a patient with an autoimmune disease, such as type 1 diabetes, followed by introduction of Foxp3, for example with a lentivirus vector, and reinfusion," he suggests. Indeed, therapeutic vaccination with CD4⁺CD25⁺ T_regs might induce and maintain tolerance in patients with autoimmune diabetes.⁴

The research also leads to numerous other therapeutic speculations. Ziegler suggests that turning expression down or off may increase tumor immunogenicity. Sakaguchi adds that modulating CD4⁺CD25⁺ T_regs might offer novel approaches for reducing the risk of organ-transplant rejection, enhancing the efficacy of vaccinations, alleviating allergic responses, and preventing miscarriage. Morgan notes, however, that Foxp3 is a transcription factor and needs to be in the nucleus, which raises delivery issues for the protein. She suggests, however, that researchers could devise pharmaceuticals that either stimulate or inhibit Foxp3 transcription or translation.

FOX ON THE RUN

Although researchers are bullish about the prospects, several issues remain unresolved. For example, identifying the genes controlled by Foxp3, Ziegler says, would bring researchers closer to understanding how the transcription factor converts effector T cells into T_regs. "Several groups are pursuing these genes, and they should be identified shortly," he says. In particular, researchers want to determine those cell-surface molecules involved in T_regs suppressive actions. "Pre-sumably Foxp3 is somehow involved in their expression, and it is these proteins that will be a major target for the manipulation of T_regs," he predicts.

Similarly, Sakaguchi says that the mechanism by which CD4⁺CD25⁺ T6.5 suppress the autoimmune response remains unclear. The literature contains at least 10 possible mechanisms. "A key question is how the FOXP3 gene is activated and how it controls T_reg suppressive activity," he says. "To know this, we must first know the mechanism of suppression."

Finally, Ziegler says that the meaning of the differences between human and animal Foxp3 expression and function remain unresolved. For example, in mice, Foxp3 expression appears to be limited to T_regs. Activation mediated by T-cell receptors, which bind tightly to viral and other potentially pathogenic proteins, does not seem to influence Foxp3 expression. However, in human T cells, FOXP3 acts more like an activation-induced gene, says Ziegler. Moreover, Morgan says that further research will find FOXP3 splice variants specific for human T_regs.

Like its vulpine namesake, human Foxp3 remains elusive. Researchers are hot on its trail, but as yet have caught only a glimpse of its detailed function and potential therapeutic applications.