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*
Celltech R&D, Inc., Bothell, WA 98021; and
Virginia Mason Research Center, Seattle, WA 98101.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
TNF-
, are consistent with the range of pathological symptoms leading
to death.
In many respects, sf/Y animals are strikingly similar to
animals that lack expression of either CTLA-4 (8, 9) or
TGF- (10, 11, 12). Using adoptive transfer studies, the
effector cells in the disease have been shown to be
CD4+ T cells (6). Flow cytometric
analyses of freshly explanted sf/Y
CD4+cells reveal an increased number of cells
expressing activation-related cell surface markers, including CD44,
CD69, CD25, CD80, and CD86 (7). Previous studies have
demonstrated that purified CD4+ T cells from
mutant mice are exquisitely sensitive to stimulation through the TCR.
These cells however, retain their requirement for two activation
signals, one through the TCR and the second through a coreceptor such
as CD28, for maximal activation (7). Recent data has
demonstrated that engagement of the TCR is required for pathology, as
generation of mice that possess a single TCR specificity
(D011.10 transgene) prevents the induction of disease in
otherwise susceptible sf/Y mice (13). Thus,
like CTLA-4- or TGF--targeted mutant mice, the pathology observed in
sf/Y mice appears to result from an inability to
appropriately regulate T cell function.
The gene responsible for the defect in sf/Y mice has been identified recently using a positional cloning strategy. The gene encodes a novel member of the forkhead transcription factor family, designated Foxp3 (14). In scurfy mutant animals, a 2-bp insertion results in premature termination of translation and a potentially truncated protein that appears to be nonfunctional. The absence of disease in male mice expressing a wild-type Foxp3 transgene, in addition to the sf mutant Foxp3 gene, confirmed the direct relationship between this gene and the pathology seen in mutant animals (14). Our current studies show that in addition to preventing the disease in mutant mice, overexpression of the wild-type allele confers a dose-dependent decrease in the number and functional responsiveness of CD4+ T cells in otherwise normal animals. The effect of transgene expression appears limited to peripheral T cells, as thymic development in these mice is normal when the transgene is expressed under the control of its own regulatory elements. Overexpression of the gene using a promoter restricted to the thymus can result in altered development, but this thymic-specific expression is unable to prevent disease in sf/Y mutant animals. The results described here suggest that the amount of Foxp3 gene expression (and presumably the encoded scurfin protein) is directly related to the overall functional competence of peripheral CD4+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal studies were conducted following Public Health Service
guidelines, and all mice were maintained under specific pathogen-free
(SPF)2 conditions. The
original breeding stocks for scurfy mice were obtained from Oak Ridge
National Laboratory (Oak Ridge, TN), with mice subsequently
derived by cesarean section into SPF conditions. Transgenic mice were
generated by oocyte microinjection by DNX Transgenic Services
(Cranbury, NJ), as described previously (14). For the 2826
mouse line, a 30.8-kb cosmid construct was generated from mouse BAC K60
for injection. This cosmid contains the entire Foxp3 gene
along with 
18 kbp of 5' sequence and 4 kbp of 3' sequence.
Expression of the gene parallels that of the endogenous gene with
respect to tissue distribution (14). For construction of
the lck-Foxp3 transgene, a Foxp3 cDNA fragment
containing BglII sites at each end was generated by RT-PCR
from 10-day thymus RNA using 5'-GCAGATCTCCTGACTCTGCCTTC-3' and
5'-GCAGATCTGACAAGCTGTGTCTG-3' and cloned into the BamHI
site of p1017, which contains the lck proximal promoter
(15). Both transgenic and scurfy mice were backcrossed
onto the C57BL/6 background (Jackson) for from four to six generations
for all studies. No differences in responsiveness or phenotype were
noted during backcrossing. Northern blot analysis was performed as
described previously (14).
Flow cytometry and cell sorting
Thymus, lymph node, and splenic tissues were collected as
described previously (7) and were resuspended in staining
buffer (1% BSA, 0.1% sodium azide in PBS) at a cell
density of 20 x 106/ml. Cell aliquots were
treated with 2% normal mouse serum (Sigma, St. Louis, MO) to block
nonspecific binding and then stained by incubation on ice for 30 min
with combinations of the following fluorochrome-conjugated
anti-mouse mAbs: CD3, CD8, CD4, CD25, IgG2a control (Caltag
Laboratories, Burlingame, CA); and CD28, CD45RB, CD44, CD62L, CD69,
CD95 (BD PharMingen, San Diego, CA). The fluorescence intensity of
105 cells was examined using a MoFlo flow
cytometer (Cytomation, Fort Collins, CO) with dead cell exclusion by
addition of propidium iodide (10 µg/ml).
CD4+ T lymphocytes were sort purified from tissues for functional assays by positive selection using the MoFlo. Sort purities as determined by postsort analysis were typically greater than 95%.
Functional analyses
Thymus, lymph node, and splenic tissues were removed from appropriate animals, macerated between sterile microscope slides, filtered through a sterile 70-µm nylon mesh, and collected by centrifugation. Cells were cultured at 37°C in complete RPMI 1640 (10% FBS, 0.05 mM 2-ME, 15 mM HEPES, 100 U/ml penicillin, and 100 µg/ml each streptomycin and glutamine) in 96-well round-bottom tissue culture plates. Culture wells were prepared for T cell activation by preincubation with the indicated concentrations of purified Ab to CD3 (clone 2C11) in sterile PBS for 4 h at 37°C. Purified anti-mouse CD28 (clone 37.51) or anti-mouse KLH (control Ab) was coimmobilized at 1 µg/ml final concentration.
T cells were cultured at a cell density of 1–5 x 104 cells/well in 200 µl of complete RPMI 1640 for 72 h. Supernatant (100 µl) was removed at 48 h for analysis of cytokine production. Wells were pulsed with 1 µCi/well of [3H]thymidine (Amersham Life Science, Arlington Heights, IL) for the last 8–12 h of culture and then harvested (Tomtec, Hamden, CT). Proliferation data reported are based on mean value of triplicate wells and represent a minimum of three experiments. Cytokine levels were determined by ELISA according to the manufacturer’s directions (Biosource International, Camarillo, CA).
Histology
Tissues for histological analysis were removed from mice 8 wk
after birth and immediately fixed in buffered 10% formalin.
Paraffin-embedded sections were processed for H&E staining, and
comparative histopathology was performed on representative mice
(Applied Veterinary Pathobiology, Bainbridge, WA).
Mixed lymphocyte culture
A single-cell suspension of BALB/c spleen cells was generated to
use as stimulator cells. These cells were irradiated (3300 rad) and
incubated a 10:1 ratio (stimulator:effector) with scurfin-transgenic or
normal littermate control (NLC) spleen cells. To some cultures, IL-2
was added at 100 U/ml. For proliferation assays, cells were pulsed
after 5 days and harvested as above. For generation of CTL, splenic T
cells were stimulated in a similar manner in the presence of 100 U/ml
IL-2. After 5 days, cells were either assayed in the "just another
method" assay (16) or restimulated on a new stimulator
layer. Cells were 95% CD8+.
Cytotoxicity assay
BALB/c spleen cells were stimulated with PMA (10 ng/ml) in the presence of ionomycin (250 ng/ml) for 24 h to allow for efficient loading of cells with [3H]thymidine. After 24 h, [3H]thymidine (5 µCi/ml) was added to PMA and ionomycin-stimulated BALB/c spleen cells. Cell were incubated at 37°C for 18 h and then washed. CD8+ effector cells were plated with target BALB/c cells at increasing ratios ranging from 1.5:1 to 50:1 (E:T) in a 96-well flat-bottom plate (experimental) in a final volume of 100 µl. The cells were pelleted by centrifugation and incubated at 37°C for 4 h. A plate containing labeled BALB/c cells alone was harvested immediately and used to determine total counts (TC). A second plate containing labeled BALB/c cells alone also was incubated at 37°C for 4 h to determine spontaneous release (SR). After 4 h of incubation, cells were harvested onto glass fiber and counted in a scintillation counter. Percent lysis was determined as follows: [(TC - SR) - (experimental - SR)/(TC - SR)] x 100 = percent lysis.
Contact sensitivity response
Age-matched animals were treated on the left ear with 2% oxazalone (diluted in olive oil/acetone), using a final volume of 25 µl. After 7 days, ear thickness was measured using spring-loaded calipers, and mice were challenged on the right ear with 2% oxazalone (8 µl per ear). Ear thickness was measured at 24 h and is reported as change in ear thickness compared with prechallenge. Control mice were challenged only (no priming). Thickness of ears after initial priming (before challenge) was no different from untreated ears. Mice were subsequently treated with PMA (10 ng/ml; 8 µl/ear) on the priming ear. Ear thickness was measured at 18 h and is reported as thickness compared with pretreatment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial experiments involving the Foxp3-transgenic mice
demonstrated that in five of five lines generated from distinct founder
animals, the expression of the wild-type Foxp3 transgene
prevented disease in sf/Y mutant mice (14).
Further analysis demonstrated that the copy number of the transgene was
directly correlated to the expression of the gene at the mRNA level
(14). This is likely due to the fact that the transgene
construct consisted of a large genomic fragment including a substantial
portion of 5' sequence and much of the regulatory region. In analyzing
the various transgenic lines, it also became clear that there was a
direct relationship between the expression of the Foxp3 gene
and the number of lymph node cells (14). The relationship
between transgene copy number and cell number is shown for three of the
founder lines, with the scurfy mutant animal (sf/Y) and NLC
for comparison (Table I
). Although there
is a less dramatic, but consistent, difference in the number of splenic
cells in the transgenic mice as well, the number of thymocytes is not
significantly affected. For reasons of simplicity, except where noted,
the remainder of the studies in this report focus on the 2826
transgenic line. Animals from this line are generally healthy and
survive for greater than 1 year under SPF conditions. The line has
16 copies of the transgene and by Northern blot analysis is
expressed at 10 to 20 times the level of the endogenous gene in
lymphoid tissues (14). The transgene, like the endogenous
gene, is only poorly expressed in nonlymphoid tissues, a likely
consequence of its expression under the control of its endogenous
promoter. Lymph node cell number in mice from this line range from
15–50% of normal, with the number of cells accumulating with age.
Splenic cell number is less dramatically affected although generally
decreased, with a range of 25–90% of normal.
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The role of the Foxp3 gene in thymic selection
remains unclear. Deletion of superantigen-specific V-bearing
thymocytes appears normal in both sf/Y and 2826 transgenic
mice (F. Ramsdell, unpublished results). Consistent with this,
overexpression of the Foxp3 gene using its own endogenous
promoter (2826 line) also does not appear to result in any gross
changes in thymic development or selection. The number of thymocytes
(Table I
) and their distribution among the major phenotypic subsets
(Fig. 1) is indistinguishable from
littermate control animals. A more detailed examination of the
CD4-8- subset also
reveals a normal distribution of 
cells (not shown) and
CD25+ cells. Importantly, the fraction of
CD4+8- thymocytes
expressing the maturation markers CD69 and HSA (not shown) is identical
in 2826 and control animals, suggesting that the maturation process is
normal.
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). Two separate founder animals were
crossed to scurfy carrier females in an attempt to prevent disease. In
each case sf/Y mice carrying the lck proximal
promoter–Foxp3 transgene developed an acute
lymphoproliferative disease that was identical both in severity and
time course to that seen in nontransgenic sf/Y siblings. In
each case, expression of the transgene was seen predominantly in the
thymus with no detectable expression in peripheral organs, including
spleen (Fig. 2A). Furthermore,
thymic expression of the lck-driven transgene was
substantially greater than that of the gene in 2826 transgenic animals
or of the endogenous gene in normal littermate control mice. Hence, it
appears that the fatal lymphoproliferative disease seen in
sf/Y mice does not arise as a consequence of
scurfin-mediated developmental defects in the thymus.
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B,
Table II). T cell development still occurs in these animals, as
assessed by the generation of CD4 and CD8 single-positive cells and by
the presence of relatively normal numbers of peripheral T cells in both
lymph node and spleen (Table II). CD69 expression on
CD4+8- cells from the
thymus is similar in transgenic and wild-type littermates, suggesting
positive selection likely proceeds normally, whereas within the
double-negative compartment, the fraction of cells expressing CD25 is
diminished relative to wild-type animals (Fig. 2B). These
transgenic animals indicate that overexpression of the Foxp3 gene
within the thymic compartment specifically can alter thymic
development, but this appears to have no affect on regulating
peripheral T cell activity. Altered phenotype of peripheral T cells from scurfin-transgenic mice
In addition to a decrease in the number of peripheral T cells in
2826 mice, there are several phenotypic alterations in these transgenic
mice as well. There is a slight reduction in the percentage of
CD4+ cells in both the lymph node and spleen
relative to NLC (Fig. 3A).
Whereas the CD3 levels appear normal on peripheral T cells (data not
shown), there are a number of other surface markers with altered
expression levels. For CD4+ cells in the
transgenic mice, the most consistent changes are a small decrease in
the expression of CD62L and CD45RB as well as an increase in the
expression of CD95 (Fig. 3B, top). However,
analysis of Fas-L expression (by quantitative RT-PCR) does not show any
significant differences between sf mutant, transgenic, or
NLC (data not shown). By comparison, cells from sf mutant
animals have a very different phenotype. CD4+
cells from these mice are large and clearly activated. They are
predominantly CD44high,
CD45RBlow, CD62Llow, and
partially CD69+ (7).
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A).
This decrease is typically more dramatic (50–75%) than the decrease
in the CD4+ compartment (25–50%).
CD8+ T cells display relatively minor and
variable changes in the level of CD62L, CD45RB, and CD95 on the cell
surface in comparison to NLC. In contrast to CD4+
T cells, there is a more pronounced increase in the percentage of
CD8+ T cells that were also
CD44high. Overall, the CD8+
cells do not express surface markers at levels that characterize them
as specifically naive, activated, or memory. Histological analyses of scurfin-transgenic mice
Whereas peripheral T cells in 2826 mice are clearly
decreased in number, we also sought to determine whether the
architecture of the lymphoid organs was also perturbed. Histological
examination of the major lymphoid organs (thymus, lymph node, and
spleen) indicated that the most significant changes were found in the
mesenteric and peripheral lymph nodes (Fig. 4). As expected, the thymus appears
relatively normal, with a well-defined corticomedullary junction,
although there appears to be a slight reduction in the size of the
thymic medulla. Transgenic animals have smaller peripheral lymph nodes,
lack robust and normally distributed lymphoid follicles, lack distinct
margins between follicular and interfollicular areas, and have more
obvious sinuses than those found in the lymph nodes of the NLC mice.
Even though the spleen and Peyer’s patches appear approximately normal
in size and microarchitecture, there is a moderate decrease in total
cell number and no or minimal evidence of germinal centers in these
tissues. The changes noted here reflect a hypocellular state distinct
from a number of other targeted mutations in which the lymph nodes fail
to develop. Thus, although T cells are capable of development in an
apparently normal manner, their representation within the peripheral
lymphoid tissues, particularly the lymph nodes, is substantially
decreased.
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The phenotypic and cell number data suggest that there are
specific defects in the biology of CD4 T cells from 2826 transgenic
animals. Therefore, we evaluated the functional responses of T cells
from these animals to several stimuli, including anti-CD3 and
anti-CD28. Lymphocytes were isolated from various tissues from NLC,
2826 transgenic, or scurfy (mutant) mice, and CD4 cells were purified
by cell sorting. Proliferation (Fig. 5)
and IL-2 production (Fig. 6) are
significantly diminished in cells from the transgenic animals compared
with their littermates. This difference is maintained at later time
points after stimulation. Although transgenic animals increase their
responsiveness with increasing stimulation, they rarely reach the
levels achieved by NLC. This is particularly true for IL-2 production,
in which cells from 2826 mice consistently produce low to undetectable
amounts of this cytokine. Similar results are seen whether the cells
are derived from the spleen or the lymph nodes. As expected, cells from
scurfy animals are hyperresponsive to stimulation and produce increased
amounts of IL-2. The effect of the transgene is independent of strain
and has remained constant during the backcrossing of the animals onto
C57BL/6 through at least generation N6. T cells from transgenic mice
remain responsive to anti-CD28 in this assay, whereas stimulation
with anti-CD3 and control Ig results in generally poor responses
that were lower than but similar to NLC responses. Interestingly,
addition of high doses of IL-2 is able to partially overcome the
proliferative defect in CD4+ T cells from 2826
mice, but generally fails to restore the response to that of wild-type
animals (Fig. 5, C and D). To assess whether
reduced responsiveness of transgenic T cells was due to increased
activation induced cell death, apoptosis was measured in primary
cultures by annexin V and propidium iodide staining. No significant
differences were seen between CD4+ T cells from
transgenic or NLC (data not shown). Similar results were found with
cell cycle analysis. The CD4+ T cells from
transgenic animals were not arrested at any stages of cell cycle (data
not shown). At specific time points, there were slightly reduced
percentages of transgenic CD4+ T cells in S or
G2-M phase, but these differences were not
significant enough to account for reduced proliferative responses.
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A). However, IL-2 production
by thymic CD4+ cells is reduced substantially
from the transgenic animals (Fig. 7B). The reduction in IL-2
production by thymocytes is somewhat more variable than that seen in
lymph node or spleen and may suggest that the IL-2 produced is also
consumed during the culture. Alternatively, thymocytes may produce
other growth factors less affected by the expression of the
Foxp3 gene. Nevertheless, the data generally support the
view that a major defect in the transgenic animals is in the ability of
both thymic and peripheral T cells to produce IL-2.
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We next determined the ability of transgenic T cells to generate
and function as cytotoxic T cells in an in vitro assay. Transgenic T
cells were stimulated in a mixed-lymphocyte culture containing
increasing numbers of irradiated allogeneic stimulator cells in the
presence or absence of IL-2. We then measured the proliferative
response of either transgenic or NLC effector cells (Fig. 8A). T cells from the
transgenic animals responded poorly in the absence of exogenous IL-2,
consistent with the data for purified CD4+ cells
(above). In the presence of exogenous IL-2, transgenic T cells
displayed an increased proliferative response but still required a
higher number of stimulator cells to reach a similar level of
proliferation as control cells. The ability of mixed T cell populations
to respond to stimulation in this assay may reflect the presence of
both CD4+ and CD8+ T cells
in these cultures.
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B). However, the transgenic cells were still
effective, with 50–60% lysis at these intermediate ratios. Overall,
these data suggest that scurfin-transgenic T cells possess cytolytic
activity but are less effective than NLC. In addition, exogenous IL-2
was required to generate functional CD8+ T cells,
presumably due to the poor endogenous production of this cytokine.
As a further indicator of T cell responsiveness, we have addressed the
functional responsiveness of 2826 transgenic animals to Ag in vivo.
Contact sensitivity responses using oxazalone as the challenging agent
were conducted on 2826 mice and their littermate controls. In these
studies, transgenic animals made a consistently poor response to
oxazalone at all times examined, whereas control animals responded
normally (Fig. 9). However, the
transgenic animals responded normally to challenge with PMA, indicating
that they were capable of generating an inflammatory reaction to a
strong, non Ag-specific challenge. Further studies using animals
transgenic for both a TCR and Foxp3 will examine in vivo
responses in greater detail.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(10, 11) have profoundly
altered immune responses, which may be attributed to a failure to
control T cell function. The latter two types of mice dramatically
demonstrate the critical role for these proteins in regulation of
immune function, as these animals die from lethal immune pathology
within weeks of birth. We have focused our studies on a spontaneous
mutant, scurfy, that displays many of the characteristics of CTLA-4- or
TGF--targeted mice. The gene that is mutated in scurfy mice has recently been identified as a novel member of the forkhead/winged-helix family of transcription factors and has been designated Foxp3 (14). The protein product of this gene, scurfin, has been further shown to bind DNA (19), thus suggesting that the protein indeed regulates transcription. The identity of the target genes affected has yet to be determined. The functional activity of scurfin is largely conserved between mouse and man as evidenced by the high level of amino acid identity between these species (86%) and, more importantly, the existence of mutations in the human gene that result in a phenotype similar to the mouse mutant (20, 21). Additionally, the absolutely critical role of this protein in immune function is suggested by the fact that, to date, there are no polymorphisms in the gene other than those that result in overt disease.
In the course of confirming that Foxp3 was the gene responsible for the phenotype in scurfy mice, we generated transgenic animals that overexpress the wild-type gene from a large genomic construct. This resulted in a copy number-dependent, tissue-specific expression pattern such that the resulting animals expressed 2–70 times the amount of RNA found in normal mice, but in a tissue pattern similar to that of normal mice (14). We have studied these transgenic lines in more detail and shown that with increasing amounts of Foxp3 gene expression, peripheral T cell number is significantly reduced and that this excess expression of Foxp3 results in dramatically decreased responsiveness of CD4+ T cells.
Although Foxp3 transgenic mice appear generally healthy and remain viable for greater than 1 year, their immunological responses are significantly muted and the number of peripheral T cells is substantially reduced. This effect on cell number is more apparent in the lymph nodes than the spleen, and not evident in the thymus. Histological examination of these tissues indicates a marked hypocellularity of the peripheral lymphoid organs. This is substantially different from several other gene-targeted animals that fail to properly develop lymph nodes. The relatively minimal alterations in the thymi of animals expressing the Foxp3 transgene under the control of its own promoter suggest that under normal circumstances, the gene acts primarily on peripheral T cells. This is supported by the finding that when expression is restricted to the thymus, transgenic Foxp3 animals are unable to prevent disease in sf/Y mice. Furthermore, in these lck-driven transgenic mice, peripheral T cells appear normal in number and function. The thymic phenotype of sf/Y mutant mice (lacking scurfin) also appears normal soon after birth, with no obvious defects in selection or development (13 and our unpublished results). A more detailed analysis of selection in TCR-transgenic-Foxp3-transgenic mice will be required to address this issue more fully. However, the results to date suggest that scurfin functions primarily within peripheral T cells.
In all five Foxp3-transgenic lines generated, we observed a reduction in both proliferation and, even more strikingly, IL-2 production by purified CD4 cells from the transgenic animals. Addition of IL-2 can partially overcome the proliferative defect in cells from the 2826 line. Whether the decreases in peripheral T cell number reflect an inability to produce cytokine such as IL-2 family members or result from a distinct aspect of altered signaling is unclear at present. However, it should be noted that although endogenous scurfin expression is largely restricted to CD4 cells, there is a significant effect on CD8 cells within the transgenic mice. Analysis of scurfin expression within the CD8 cells of transgenic animals indicates an increased amount of expression relative to that seen in NLC, although substantially below that seen in 2826 transgenic CD4 cells (not shown). This may suggest that the effects on CD8 cells could be directly mediated via scurfin activity within this subset, although an indirect effect via CD4 cells cannot be ruled out. Nevertheless, the dramatic alterations in CD8 cell number, percentage, and functionality in the transgenic animals indicate that the amount of scurfin expression can significantly alter CD8 T cell biology.
The precise mechanism by which the scurfin protein controls immune responses is not yet understood. The ability of the protein to bind DNA and its sequence similarity to other transcription factors suggests that the mechanism of action involves regulation of gene expression. Scurfin lacks an obvious transcriptional activation domain and so may require binding to another factor to induce transcription. Alternatively, scurfin may act primarily by preventing the transcription of specific genes. In transient assays, transfection of the human Foxp3 gene into Jurkat cells with a luciferase reporter driven by a multimer of NFAT site in IL-2 promoter lead to decrease in activation-induced promoter activity, consistent with an inhibitory function (19). In addition to any effect(s) on gene transcription, the mechanism by which scurfin itself is regulated is unclear. Whether the scurfin protein is regulated in a manner similar to other forkhead family members, such as by phosphorylation, remains to be determined (22). However, there is no PKB/AKT consensus site within the protein for serine/threonine phosphorylation.
Because the phenotype of scurfy mice is similar to that of CTLA-4 and
TGF knock-out animals, we considered that scurfin might directly
regulate expression of these gene products. However, this does not
appear to be the case, as T cells from scurfy mutant mice express
CTLA-4 and TGF as well as the ligands/receptors for these molecules
(data not shown). There was an additional possibility that scurfin
regulates the expression of receptors involved in apoptosis. Our
analysis showed no difference in expression of Fas (FACS analysis) or
Fas-L, TNFRI, and TNFRII (RT-PCR analysis). A potential role for
scurfin in the downstream signaling from these receptors is under
study.
The present data indicate that the amount of Foxp3 message, and presumably scurfin protein, is directly correlated with the responsiveness of peripheral T cells and in their overall number. Although scurfin can have effects on T cell development, but not disease modification, when highly expressed using the proximal lck promoter, expression of the gene under its own promoter does not seem to alter thymic selection, but does prevent disease in sf/Y mice. This is consistent with the hypothesis that scurfin exerts its effects primarily within the peripheral T cell compartment. Whereas the lack of functional scurfin activity leads to a hyperactive immune state and death within 3 wk of birth, excessive scurfin activity leads to a hypoactive immune state characterized by decreased numbers and activity of peripheral T cells. This indicates that the Foxp3 gene and its protein product scurfin acts as a central regulator of T cell activity.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: SPF, specific pathogen-free; NLC, normal littermate control.
Received for publication June 26, 2001. Accepted for publication September 28, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 gene results in multifocal inflammatory disease. Nature 359:693.[Medline]
1 null mutation in mice causes excessive inflammatory response and early death. Proc. Natl. Acad. Sci. USA 90:770.1-deficient mice. J. Immunol. 153:1936.[Abstract]
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C. Chen, E. A. Rowell, R. M. Thomas, W. W. Hancock, and A. D. Wells Transcriptional Regulation by Foxp3 Is Associated with Direct Promoter Occupancy and Modulation of Histone Acetylation J. Biol. Chem., December 1, 2006; 281(48): 36828 - 36834. [Abstract] [Full Text] [PDF]
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J. E. Lopes, T. R. Torgerson, L. A. Schubert, S. D. Anover, E. L. Ocheltree, H. D. Ochs, and S. F. Ziegler Analysis of FOXP3 Reveals Multiple Domains Required for Its Function as a Transcriptional Repressor. J. Immunol., September 1, 2006; 177(5): 3133 - 3142. [Abstract] [Full Text] [PDF]
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H. W. Lim, H. E. Broxmeyer, and C. H. Kim Regulation of Trafficking Receptor Expression in Human Forkhead Box P3+ Regulatory T Cells J. Immunol., July 15, 2006; 177(2): 840 - 851. [Abstract] [Full Text] [PDF]
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P.-Y. Mantel, N. Ouaked, B. Ruckert, C. Karagiannidis, R. Welz, K. Blaser, and C. B. Schmidt-Weber Molecular Mechanisms Underlying FOXP3 Induction in Human T Cells J. Immunol., March 15, 2006; 176(6): 3593 - 3602. [Abstract] [Full Text] [PDF]
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C. Veldman, A. Pahl, S. Beissert, W. Hansen, J. Buer, D. Dieckmann, G. Schuler, and M. Hertl Inhibition of the transcription factor foxp3 converts desmoglein 3-specific type 1 regulatory T cells into th2-like cells. J. Immunol., March 1, 2006; 176(5): 3215 - 3222. [Abstract] [Full Text] [PDF]
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 M. Firan, S. Dhillon, P. Estess, and M. H. Siegelman Suppressor activity and potency among regulatory T cells is discriminated by functionally active CD44 Blood, January 15, 2006; 107(2): 619 - 627. [Abstract] [Full Text] [PDF]
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 X. Chang, J. X. Gao, Q. Jiang, J. Wen, N. Seifers, L. Su, V. L. Godfrey, T. Zuo, P. Zheng, and Y. Liu The Scurfy mutation of FoxP3 in the thymus stroma leads to defective thymopoiesis J. Exp. Med., October 17, 2005; 202(8): 1141 - 1151. [Abstract] [Full Text] [PDF]
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R. Mallone, S. A. Kochik, H. Reijonen, B. Carson, S. F. Ziegler, W. W. Kwok, and G. T. Nepom Functional avidity directs T-cell fate in autoreactive CD4+ T cells Blood, October 15, 2005; 106(8): 2798 - 2805. [Abstract] [Full Text] [PDF]
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 F Obermeier, U G Strauch, N Dunger, N Grunwald, H C Rath, H Herfarth, J Scholmerich, and W Falk In vivo CpG DNA/toll-like receptor 9 interaction induces regulatory properties in CD4+CD62L+ T cells which prevent intestinal inflammation in the SCID transfer model of colitis Gut, October 1, 2005; 54(10): 1428 - 1436. [Abstract] [Full Text] [PDF]
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 Y. Y. Wan and R. A. Flavell Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter PNAS, April 5, 2005; 102(14): 5126 - 5131. [Abstract] [Full Text] [PDF]
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E. Bettelli, M. Dastrange, and M. Oukka Foxp3 interacts with nuclear factor of activated T cells and NF-{kappa}B to repress cytokine gene expression and effector functions of T helper cells PNAS, April 5, 2005; 102(14): 5138 - 5143. [Abstract] [Full Text] [PDF]
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 C. Schramm, S. Huber, M. Protschka, P. Czochra, J. Burg, E. Schmitt, A. W. Lohse, P. R. Galle, and M. Blessing TGF{beta} regulates the CD4+CD25+ T-cell pool and the expression of Foxp3 in vivo Int. Immunol., September 1, 2004; 16(9): 1241 - 1249. [Abstract] [Full Text] [PDF]
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 T. L. Sumpter and D. S. Wilkes Role of autoimmunity in organ allograft rejection: a focus on immunity to type V collagen in the pathogenesis of lung transplant rejection Am J Physiol Lung Cell Mol Physiol, June 1, 2004; 286(6): L1129 - L1139. [Abstract] [Full Text] [PDF]
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Y. Peng, Y. Laouar, M. O. Li, E. A. Green, and R. A. Flavell TGF-{beta} regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes PNAS, March 30, 2004; 101(13): 4572 - 4577. [Abstract] [Full Text] [PDF]
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T. L. Denning, H. Qi, R. Konig, K. G. Scott, M. Naganuma, and P. B. Ernst CD4+ Th Cells Resembling Regulatory T Cells That Inhibit Chronic Colitis Differentiate in the Absence of Interactions Between CD4 and Class II MHC J. Immunol., September 1, 2003; 171(5): 2279 - 2286. [Abstract] [Full Text] [PDF]
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D. J. Kasprowicz, P. S. Smallwood, A. J. Tyznik, and S. F. Ziegler Scurfin (FoxP3) Controls T-Dependent Immune Responses In Vivo Through Regulation of CD4+ T Cell Effector Function J. Immunol., August 1, 2003; 171(3): 1216 - 1223. [Abstract] [Full Text] [PDF]
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), 2826 transgenic mice (
),
or scurfy mice (
) were plated in the absence of IL-2
(A and B). C and
D, cells from NLC or 2826 transgenic mice were cultured
in the absence (open symbols) or presence (filled symbols) of 20 U/ml
rIL-2. Similarly diminished responses are noted when cells are
stimulated with PMA and ionomycin (not shown).
), scurfy mice (
),
or 2826 mice (
) or oxazalone (