Commentary

IPEX and the role of Foxp3 in the development and function of human Tregs

Séverine Le Bras and Raif S. Geha

Division of Immunology, Children’s Hospital, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

Address correspondence to: Raif Geha, Karp Building, Division of Immunology, Children’s Hospital, 1 Blackfan Circle, Boston, Massachusetts 02115, USA. Phone: (617) 919-2482; Fax: (617) 730-0528; E-mail: raif.geha@childrens.harvard.edu .

Published June 1, 2006

In the past decade, naturally arising CD4⁺CD25⁺ Tregs have emerged as being critical for the maintenance of peripheral immunological tolerance (1). Neonatal thymectomy in mice and thymic hypoplasia in humans (DiGeorge syndrome) result in impaired Treg generation and in the development of organ-specific autoimmune diseases (2, 3). Treg generation in the thymus requires interaction with MHC class II molecules expressed by cortical epithelial cells (4). Other signals have also been implicated, including those delivered by CD28, CD40 ligand, and the cytokines TNF-α and TGF-β (5). However, IL-2, which is necessary for Treg survival in the periphery, is dispensable for their development (6, 7).

Tregs represent a small subset of CD4⁺ T cells (5–10%). They express several surface markers including CD25 (the IL-2 receptor α-chain), CTL-associated antigen 4 (CTLA4), and glucocorticoid-induced TNF receptor (GITR). However, these molecules are upregulated in naive CD4⁺CD25^– T cells following TCR stimulation and cannot therefore serve as absolute Treg markers. Recently, several groups identified the forkhead family transcription factor forkhead box protein P3 (Foxp3) as a specific molecular marker of Tregs (8, 9). Foxp3 expression seems to be largely restricted to a small subset of TCRαβ T cells and defines 2 pools of Tregs with suppressive activity: CD4⁺CD25^high cells and a minor population of CD4⁺CD25l^ow/– T cells. Thus, Foxp3 expression correlates better with suppressor activity than CD25 expression (10). Ectopic expression of Foxp3 in vitro and in vivo is sufficient to convert naive murine CD4⁺ T cells to Tregs (11). In contrast, overexpression of FOXP3 in human CD4⁺CD25^– T cells in vitro is not sufficient to generate potent suppressor activity, suggesting that additional factors are required for bona fide Treg activity (12). Foxp3 expression cannot be readily induced in murine T cells by stimulation in vitro. In humans, cross-linking of the TCR and CD28 stimulation, or antigen-specific stimulation, can induce CD4⁺CD25^– Foxp3^– cells to become Tregs and express FOXP3 and exert suppressor function (11, 13). These results suggest that de novo generation of Tregs may be a natural consequence of the immune response in humans.

Tregs are anergic in vitro. They fail to proliferate and secrete IL-2 in response to TCR ligation, and they depend on IL-2 generated following activation of CD4⁺CD25^– T cells to survive and exert their function. An in vitro assay that measures the ability of CD4⁺CD25⁺ T cells to suppress CD4⁺CD25^– T cell proliferation is commonly used to assess Treg function (14). The mechanisms mediating the suppression of immune responses in vivo by Tregs are still unclear. Several studies support the involvement of contact-dependent inhibition, while others suggest that Tregs can exert their function by secreting immunosuppressive cytokines such as IL-10 or TGF-β or by directly killing their target in a perforin-dependent manner (15).

Role of Foxp3 in the generation and function of Tregs

Several lines of evidence demonstrate that Foxp3 is both necessary and sufficient for the development and function of CD4⁺CD25⁺ Tregs in mice. Mutation of Foxp3 was identified as being responsible for an X-linked recessive inflammatory disease in Scurfy mutant mice (16). Male mice hemizygous for the mutation succumb to a CD4⁺ T cell–mediated lymphoproliferative disease characterized by wasting and multiorgan lymphocytic infiltration (17, 18). Tregs are absent in Scurfy mice and in mice with disruption of the Foxp3 gene. Moreover, T cell–specific ablation of Foxp3 was sufficient to induce the full lymphoproliferative autoimmune syndrome observed in Foxp3-knockout mice. Finally, the phenotype of Scurfy mice can be rescued by introduction of a Foxp3 transgene or by bone marrow reconstitution (10, 16), demonstrating the causative role of Foxp3 in disease pathogenesis. Thus, in mice, the lack of Foxp3-expressing Tregs is sufficient to break down self tolerance and induce autoimmune disease.

As in mice, human CD4⁺CD25^high cells, which express FOXP3, act as suppressors. FOXP3 mutations underlie an X-linked autoimmune lymphoproliferative disorder in humans termed immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX), similar to that seen in Scurfy and Foxp3-knockout mice (19–21). To date, about 20 different mutations in FOXP3 have been described in patients suffering from IPEX. However, the correlation between FOXP3 mutations and the generation and function of Tregs has not been studied. In this issue of the JCI, Bacchetta et al. (22) analyzed Treg numbers and function in 4 IPEX patients with a variety of phenotypes that ranged from no detectable symptoms to severe disease requiring bone marrow transplantation. They concluded that CD4⁺CD25⁺ T cells were present in all 4 patients, including one with a mutation in the first codon (ATG) that resulted in no detectable FOXP3 protein, but that the capacity of these cells to suppress the proliferation of CD4⁺CD25^– T cells was differentially impaired depending on the type of mutation, the strength of TCR stimuli, and the genotype of effector T cells. This is in sharp contrast to Scurfy mice and Foxp3-knockout mice, which lack both detectable Foxp3 and Tregs. Bacchetta et al. defined Tregs as CD25^high cells that express CTLA4 and GITR but did not analyze the presence of CD4⁺FOXP3⁺ cells. However, the mean fluorescence intensity of CD25 and GITR in one patient was respectively 4- and 6-fold less than that in the control, and his sorted CD4⁺CD25⁺ cells totally lacked suppressive activity. These findings support a critical role for FOXP3 in the generation and function of human Tregs.

Correlation between FOXP3 mutations and clinical phenotype

Analysis of the other 3 patients reported by Bacchetta et al. (22) provides potential insight into the function of discrete domains of FOXP3. A missense mutation (F373A) in the forkhead (FKH) domain of FOXP3, which did not interfere with protein expression, did not impair Treg development yet resulted in severe disease. Since this mutation interferes with the transcriptional activity of FOXP3, it suggests that the DNA-binding FKH domain is critical for Treg function but not development. Data from mice suggest that the FKH domain is essential for Treg development (16, 23). However, the studied mutation resulted in the absence of the FKH domain. It is possible that the missense mutant in the patient retained residual transcriptional activity sufficient for Treg development but not function. Another missense mutation (F324L) in the region between the leucine zipper and FKH domains of FOXP3, which did not impede protein expression or transcriptional activity, was associated with either mild or no disease and a partial defect in the suppressive activity of freshly isolated CD4⁺CD25⁺ cells. The differences in the clinical presentation of 2 brothers with this mutation illustrate the well-known influence of other genes on the clinical manifestations of monogenic primary immunodeficiency diseases. The observation that T cell lines derived from CD4⁺CD25⁺ cells of all 3 patients with missense mutations suppress the proliferation of CD4⁺CD25^– T cells normally must be interpreted with caution and may not reflect the suppressive potential of Tregs in vivo in the patients. In fact, the finding that infusion of Tregs cures autoimmune disease in Foxp3-knockout mice suggests that defective Treg function is critical for disease development.

Despite their disparate clinical phenotypes, in all 4 IPEX patients, a defect in cytokine production was detected after TCR/CD28-mediated activation of PBMCs. This finding is surprising given the fact that FOXP3 downregulates cytokine production (12, 24). Furthermore, serum levels of Th1, Th2, and inflammatory cytokines are elevated in Foxp3-deficient mice (23), suggesting ongoing cytokine production in vivo by activated T cells. It is possible that the decreased cytokine production by the patients’ T cells following in vitro stimulation with anti-CD3/anti-CD28 may have resulted from exhaustion of their in vivo–activated T cells.

The current study by Bacchetta et al. (22) should spur investigators to analyze in detail the number and function of Tregs in IPEX patients. In addition, introduction of human FOXP3 mutations in mice that correspond to mutations that cause IPEX provides a useful strategy to investigate the relationship between FOXP3 expression and Treg development and function and to define the function of FOXP3 domains. It will also be important to search for proteins that interact with FOXP3, as mutations in the genes that encode for FOXP3 partners may underlie the IPEX-like phenotype in patients who lack detectable mutations in the FOXP3 gene.

Footnotes

Nonstandard abbreviations used: FKH, forkhead; Foxp3, forkhead box protein P3; GITR, glucocorticoid-induced TNF receptor; IPEX, immune dysregulation, polyendocrinopathy, enteropathy, X-linked.

Conflict of interest: The authors have declared that no conflict of interest exists.

Citation for this article:J. Clin. Invest.116:1473–1475 (2006). doi:10.1172/JCI28880.

See the related article beginning on page 1713.

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