Scurfy, the Foxp3 locus, and the molecular basis of peripheral tolerance.

The ability to rapidly and efficiently recognize and eliminate pathogens while sparing normal self tissue is a hallmark of the mammalian immune system. When it fails, however, autoimmune disease results. The genetic and environmental factors that control the process of making such distinctions, not to mention the specific targeted tissues, are extraordinarily complex in the human population; only now are we characterizing the candidate genes responsible for these responses to pathogens. The examination of specific traits in murine models of disease has led to the identification of many of the candidate genes for human disease. The study of mouse mutations (both induced and spontaneous) has also greatly advanced our understanding of the immune responses and autoimmune disease. Here, we describe the use of classical mouse genetics to identify one gene centrally involved in the control of immune responses. Furthermore, although mutations in the orthologous human gene result in a virtually identical phenotype to that seen in the mouse, it is unlikely that studying the human disease populations alone would have successfully identified this gene. Thus, despite the complete sequencing of the human and mouse genomes, the examination of murine mutations remains a powerful and unbiased tool to connect genotype and phenotype.

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-beta 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.

A rare polyadenylation signal mutation of the FOXP3 gene (AAUAAA-->AAUGAA) leads to the IPEX syndrome.

The mouse scurfy gene, Foxp3, and its human orthologue, FOXP3, which maps to Xp11.23-Xq13.3, were recently identified by positional cloning. Point mutations and microdeletions of the FOXP3 gene were found in the affected members of eight of nine families with IPEX (immune dysfunction, polyendocrinopathy, enteropathy, X-linked; OMIM 304930). We evaluated a pedigree with clinically typical IPEX in which mutations of the coding exons of FOXP3 were not detected. Our reevaluation of this pedigree identified an A-->G transition within the first polyadenylation signal (AAUAAA-->AAUGAA) after the stop codon. The next polyadenylation signal is not encountered for a further 5.1 kb. This transition was not detected in over 212 normal individuals (approximately 318 X chromosomes), excluding the possibility of a rare polymorphism. We suggest that this mutation is causal of IPEX in this family by a mechanism of nonspecific degradation of the FOXP3 gene message.

Scurfin (FOXP3) acts as a repressor of transcription and regulates T cell activation.

We have recently identified and cloned Foxp3, the gene defective in mice with the scurfy mutation. The immune dysregulation documented in these mice and in humans with mutations in the orthologous gene indicates that the foxp3 gene product, scurfin, is involved in the regulation of T cell activation and differentiation. The autoimmune state observed in these patients with the immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome, or X-linked autoimmunity-allergic dysregulation syndrome also points to a critical role for scurfin in the regulation of T cell homeostasis. FOXP3 encodes a novel member of the forkhead family of transcription factors. Here we demonstrate that this structural domain is required for nuclear localization and DNA binding. Scurfin, transiently expressed in heterologous cells, represses transcription of a reporter containing a multimeric forkhead binding site. Upon overexpression in CD4 T cells, scurfin attenuates activation-induced cytokine production and proliferation. We have identified FKH binding sequences adjacent to critical NFAT regulatory sites in the promoters of several cytokine genes whose expression is sensitive to changes in SFN abundance. Our findings indicate that the ability of scurfin to bind DNA, and presumably repress transcription, plays a paramount role in determining the amplitude of the response of CD4 T cells to activation.

X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy.

To determine whether human X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome (IPEX; MIM 304930) is the genetic equivalent of the scurfy (sf) mouse, we sequenced the human ortholog (FOXP3) of the gene mutated in scurfy mice (Foxp3), in IPEX patients. We found four non-polymorphic mutations. Each mutation affects the forkhead/winged-helix domain of the scurfin protein, indicating that the mutations may disrupt critical DNA interactions.

The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3.

IPEX is a fatal disorder characterized by immune dysregulation, polyendocrinopathy, enteropathy and X-linked inheritance (MIM 304930). We present genetic evidence that different mutations of the human gene FOXP3, the ortholog of the gene mutated in scurfy mice (Foxp3), causes IPEX syndrome. Recent linkage analysis studies mapped the gene mutated in IPEX to an interval of 17-20-cM at Xp11. 23-Xq13.3.

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 or Tgf-beta, 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.

Cellular and molecular characterization of the scurfy mouse mutant.

Mice hemizygous (Xsf/Y) for the X-linked mutation scurfy (sf) develop a severe and rapidly fatal lymphoproliferative disease mediated by CD4+CD8- T lymphocytes. We have undertaken phenotypic and functional studies to more accurately identify the immunologic pathway(s) affected by this important mutation. Flow cytometric analyses of lymphoid cell populations reveal that scurfy syndrome is characterized by changes in several phenotypic parameters, including an increase in Mac-1+ cells and a decrease in B220+ cells, changes that may result from the production of extremely high levels of the cytokine granulocyte-macrophage CSF by scurfy T cells. Scurfy T cells also exhibit strong up-regulation of cell surface Ags indicative of in vivo activation, including CD69, CD25, CD80, and CD86. Both scurfy and normal T cells are responsive to two distinct signals provided by the TCR and by ligation of CD28; scurfy cells, however, are hyperresponsive to TCR ligation and exhibit a decreased requirement for costimulation through CD28 relative to normal controls. This hypersensitivity may result, in part, from increased costimulation through B7-1 and B7-2, whose expression is up-regulated on scurfy T cells. Although the specific defect leading to this hyperactivation has not been identified, we also demonstrate that scurfy T cells are less sensitive than normal controls to inhibitors of tyrosine kinases such as genistein and herbimycin A, and the immunosuppressant cyclosporin A. One interpretation of our data would suggest that the scurfy mutation results in a defect, which interferes with the normal down-regulation of T cell activation.

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.

Fas ligand mediates activation-induced cell death in human T lymphocytes.

A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T cells resulted in the expression of Fas-L mRNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T cells is mediated by Fas/Fas-L interactions.

Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice.

Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.

Differential ability of Th1 and Th2 T cells to express Fas ligand and to undergo activation-induced cell death.

Stimulation of previously activated T cells through the antigen receptor can result in the apoptotic death of the responding cell, a process referred to as activation-induced cell death (AICD). This process appears to involve Fas (CD95) and its ligand (Fas-L). The distribution of Fas and Fas-L on various T cell subsets has not been extensively characterized. We have therefore analyzed cells committed to a Th1- or Th2-type differentiation pattern for the expression and function of Fas-L. Using both a sensitive bioassay and flow cytometry, we demonstrate that cloned Th1 cells express high levels of Fas-L, whereas cloned Th2 cells express only low levels. The expression of Fas-L by Th1 and Th2 cells correlates with the relative abilities of these two cell types to undergo AICD. Whereas AICD is readily observed in cultures of cloned Th1, but not Th2 cells, Th2 cells are capable of undergoing apoptosis in the presence of Th1 cells expressing Fas-L. The ability of T cells to undergo AICD appears to be unrelated to the presence of various cytokines. Thus, the Fas/Fas-L pathway appears to be critical for the induction of AICD and this pathway is differentially regulated in cells committed to either Th1 or Th2 differentiation.

Molecular characterization of murine and human OX40/OX40 ligand systems: identification of a human OX40 ligand as the HTLV-1-regulated protein gp34.

A ligand was cloned for murine OX40, a member of the TNF receptor family, using a T cell lymphoma cDNA library. The ligand (muOX40L) is a type II membrane protein with significant identity to human gp34 (gp34), a protein whose expression on HTLV-1-infected human leukemic T cells is regulated by the tax gene. The predicted structures of muOX40L and gp34 are similar to, but more compact than, those of other ligands of the TNF family. Mapping of the muOX40L gene revealed tight linkage to gld, the FasL gene, on chromosome 1. gp34 maps to a homologous region in the human genome, 1q25. cDNAs for human OX40 receptor were cloned by cross-hybridization with muOX40, and gp34 was found to bind the expressed human receptor. Lymphoid expression of muOX40L was detected on activated T cells, with higher levels found on CD4+ rather than CD8+ cells. The cell-bound recombinant ligands are biologically active, co-stimulating T cell proliferation and cytokine production. Strong induction of IL-4 secretion by muOX40L suggests that this ligand may play a role in regulating immune responses. In addition, the HTLV-1 regulation of gp34 suggests a possible connection between virally induced pathogenesis and the OX40 system.

The activation antigen CD69.

One of the earliest cell surface antigens expressed by T cells following activation is CD69, which is detectable within one h of ligation of the T cell receptor/CD3 complex. Once expressed, CD69 acts as a costimulatory molecule for T cell activation and proliferation. In addition to mature T cells, CD69 is inducibly expressed by immature thymocytes, B cells, natural killer (NK) cells, monocytes, neutrophils and eosinophils, and is constitutively expressed by mature thymocytes and platelets. Recently, cDNA clones encoding human and mouse CD69 were isolated and showed CD69 to be a member of the C-type lectin superfamily. Gene mapping studies have placed CD69 on distal mouse chromosome 6 and human chromosome 12p13, close to, if not in, the NK gene complex. The structure, chromosomal localization, expression and function of CD69 suggest that it is likely a pleiotropic immune regulator, potentially important not only in NK cell function but also in the activation and differentiation of a wide variety of hematopoietic cells.

Recombinant CD40 ligand exerts potent biologic effects on T cells.

Murine and human CD40 ligand (CD40L) were recently cloned, expressed, and shown to possess potent activity on human and murine B cells, including stimulation of proliferation and Ig secretion in the presence of cytokines. In addition to its action on B lymphocytes, this report demonstrates that CD40L induced both CD4+ and CD8+ T cells isolated from murine lymphoid tissues to proliferate in the presence of submitogenic dosages of Con A, PHA, CD3 mAb, and TCR-alpha beta mAb. The presence of CD40L during suboptimal TCR stimulation resulted in increased expression of the activation Ags IL-2R alpha and CD69 and increased IL-2 production. Taken together, these results show that CD40L is a potent activator of murine T cells and suggest that CD40L is involved in the regulation of T cell function mediated through T:T cell interaction.

gld/gld mice are unable to express a functional ligand for Fas.

Mice homozygous for either the lpr or gld genes develop phenotypically identical autoimmune disorders. The gene responsible for the pathology in lpr/lpr mice encodes the Fas antigen, a protein associated with the induction of programmed cell death. To determine if the defect associated with gld represents a mutation in the ligand for Fas, we have assessed the ability of lymphoid cells from homozygous gld/gld mice to lyse target cells in a Fas-dependent manner. Using an antagonistic antibody to Fas, we demonstrate that activated T cells from normal and lpr mice are capable of inducing Fas-mediated lysis of tumor target cells. In contrast, activated T cells from gld/gld mice fail to induce lysis of tumor targets, although cells from gld mice are able to lyse specific allogeneic targets following mixed lymphocyte culture. In addition, activated T cells from gld/gld homozygous animals are not capable of binding to a Fas.Fc fusion protein at high levels, whereas activated T cells from normal and lpr/lpr animals bind Fas.Fc efficiently. These data indicate that mice homozygous for gld are unable to express a functional ligand for Fas.

CD40 ligand acts as a costimulatory signal for neonatal thymic gamma delta T cells.

The stimulatory requirements for T cells bearing gamma delta T cell receptors are distinct from those of alpha beta T cells. We have analyzed the ability of the CD40 ligand (CD40L) to activate neonatal thymic gamma delta T cells. CD40L is expressed on activated T cells and has been shown to induce B cell proliferation and Ig secretion as well as monocyte activation. We now demonstrate that, in the presence of an anti-TCR-gamma delta Ab, CD40L is able to induce the proliferation of neonatal thymic gamma delta cells. The presence of CD40L also leads to enhanced expression of a variety of activation-associated Ag including CD25, CD69, CD44, and Ly6C. In addition to proliferation, CD40L induces lectin-mediated cytolytic activity in thymic gamma delta T cells as well as the production of IFN-gamma and TNF-alpha. We were unable to detect IL-2 or IL-4 production in response to CD40L, and Ab-blocking studies indicate that the mechanism of activation appears to involve IL-1 but is independent of IL-2, IL-4, and IL-7. These results suggest that, in addition to its effects on B cells and monocytes, CD40L can costimulate the activation of thymic gamma delta T cells.

The mouse CD69 gene. Structure, expression, and mapping to the NK gene complex.

CD69 is a rapidly induced T cell activation Ag that is also expressed in an inducible fashion on cells of most, if not all, hematopoietic lineages. Molecular cloning has shown that CD69 is a type II membrane glycoprotein that is a member of the C-type lectin family. In this report we have shown that induction of CD69 mRNA in activated murine thymocytes and T cells is very rapid, peaking between 30 and 60 min poststimulation, and transient, dropping to nearly resting levels by 8 h. An analysis of the mouse CD69 gene structure showed the gene to consist of 5 exons and have a phorbol ester-inducible promoter element within the first 700 bp upstream of the start of transcription. Chromosomal mapping placed the mouse CD69 gene on the long arm of chromosome 6 near the NK gene complex that contains the related NKR-P1 and Ly-49 gene families. The human CD69 gene mapped to chromosome 12p13 near the related NKG2 gene cluster and in a region associated with rearrangements in approximately 10% of cases of childhood acute lymphocytic leukemia.