Single-cell analysis of normal and FOXP3-mutant
human T cells: FOXP3 expression without
regulatory T cell development
Marc A. Gavin*†, Troy R. Torgerson*‡, Evan Houston*, Paul deRoos§, William Y. Ho¶, Asbjørg Stray-Pedersen?,
Elizabeth L. Ocheltree‡, Philip D. Greenberg*¶, Hans D. Ochs*‡, and Alexander Y. Rudensky*†§
*Department of Immunology and§Howard Hughes Medical Institute, University of Washington, Box 357370, Seattle, WA 98195;‡Children’s Hospital
Regional Medical Center, Seattle, WA 98105;¶Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and?Centre for Rare Disorders,
Rikshospitalet University Hospital, N-0027 Oslo, Norway
Edited by Richard A. Flavell, Yale University School of Medicine, New Haven, CT, and approved March 8, 2006 (received for review October 31, 2005)
Forkhead winged-helix transcription factor Foxp3 serves as the
dedicated mediator of the genetic program governing CD25?CD4?
regulatory T cell (TR) development and function in mice. In humans,
its role in mediating TR development has been controversial.
Furthermore, the fate of TRprecursors in FOXP3 deficiency has yet
FOXP3, we have addressed the relationship between FOXP3 ex-
a small subset of human CD4?and CD8?T cells transiently up-
regulated FOXP3 upon in vitro stimulation. Induced FOXP3, how-
ever, did not alter cell-surface phenotype or suppress T helper 1
cytokine expression. Furthermore, only ex vivo FOXP3?TR cells
persisted after prolonged culture, suggesting that induced FOXP3
did not activate a TR developmental program in a significant
number of cells. FOXP3 flow cytometry was also used to further
characterize several patients exhibiting symptoms of immune
dysregulation, polyendocrinopathy, enteropathy, X-linked syn-
drome (IPEX) with or without FOXP3 mutations. Most patients
lacked FOXP3-expressing cells, further solidifying the association
between FOXP3 deficiency and immune dysregulation, polyendo-
crinopathy, enteropathy, X-linked syndrome. Interestingly, one
patient bearing a FOXP3 mutation enabling expression of stable
FOXP3mutprotein exhibited FOXP3mut-expressing cells among a
subset of highly activated CD4?T cells. This observation raises the
possibility that the severe autoimmunity in FOXP3 deficiency can
be attributed, in part, to aggressive T helper cells that have
developed from TRprecursors.
CD25?CD4?regulatory T cells (TR) develop as a separate lineage
been identified in humans and have been shown to possess many of
the same phenotypic and functional properties as their murine
counterparts (4). Mutations of FOXP3 in humans lead to an
early-onset, multisystem autoimmune syndrome known as IPEX
(immune dysregulation, polyendocrinopathy, enteropathy, X-
linked) (5–7). Foxp3nulland scurfy mice exhibit an analogous auto-
by FOXP3 across phylogeny.
Although it is well established that both murine and human TR
develop as a subset of CD4 single-positive thymocytes (10, 11), the
conditions under which TR arise in peripheral organs is less
understood. In mice, no measurable role for Foxp3 has been found
in the differentiation or function of non-TRin response to T cell
receptor (TCR) agonists (9). In contrast, human CD25?CD4?and
CD8?T cells have been shown to increase FOXP3 mRNA and
protein levels upon activation, suggesting a cell-intrinsic role for
FOXP3 in the regulation of T cell responses in humans (12–14).
Furthermore, the existence of IPEX-like individuals that are phe-
notypically similar to IPEX but lack mutations within the coding
region of the FOXP3 gene calls into question the role of FOXP3 as
significant body of evidence has been derived from rodent
models demonstrating that, through Foxp3 expression,
the ‘‘master regulator’’ of human TRdevelopment and function.
Thus, two nonmutually exclusive models can be proposed for the
role of FOXP3 in regulating immune responses in humans. In the
first model, preexisting FOXP3?TRare recruited to sites of active
immune response where they suppress antigen-specific effector T
cells and expand to control the intensity of the response. In the
second model, FOXP3?CD4?T cells responding to neoantigens
expressed by target organs or to pathogens give rise to a clonal
population consisting of both effector T cells and FOXP3?TR,
the latter of which may either exist transiently or give rise to
Whether the mechanisms of TRdevelopment and function differ
in humans and mice is currently an area of significant debate.
Recent evidence suggests that the latter model of peripheral TR
development may be more operative in humans than in mice,
because some groups have found that, unlike murine cells, stimu-
lation of human CD25?CD4?T cells results in considerable
Others have not observed such conversion of naı ¨ve T cells into
FOXP3-expressing TRin vitro (16). Determining whether humans
and the mechanisms driving such TRdevelopment, is of substantial
further examine the relationship between FOXP3 deficiency and
IPEX, we investigated FOXP3 expression in ex vivo isolated and
activated T cells from normal donors and IPEX patients using our
recently developed flow cytometric methodology. Serendipitously,
the identification in one patient of activated T cells expressing a
loss-of-function mutant FOXP3 suggests the possibility that the
severity of IPEX?scurfy autoimmunity may result from an alterna-
tive proinflammatory fate of TRprecursors.
Results and Discussion
Flow Cytometric Characterization of Human FOXP3?Cells. To exam-
we developed methods for flow cytometric detection of FOXP3
polyclonal antibody. Both antibodies detect murine as well as
human FOXP3, and their utility for single-cell detection of Foxp3
expression was demonstrated by using normal and Foxp3nullmice.
Staining of mouse lymph node cells with either antibody revealed
Foxp3 expression in the majority of CD25?CD4?T cells and a
small subset of CD25?CD4?cells (Fig. 1 A and B). This Foxp3
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: Th, T helper; IPEX, immune dysregulation, polyendocrinopathy, enteropa-
thy, X-linked syndrome; TR, regulatory T cell; PBMC, peripheral blood mononuclear cell;
TCR, T cell receptor; PE, phycoerythrin; PW, Perm?Wash.
†To whom correspondence may be addressed. E-mail: email@example.com or
© 2006 by The National Academy of Sciences of the USA
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expression pattern was similar to that of Foxp3GFPknockin mice
(17). Reactivity with Foxp3 was specific, because no staining was
observed with either antibody in Foxp3nullcells (Fig. 1 A and B).
Specificity was further confirmed by mapping of the mAb 3G3
among all forkhead-family transcription factors (Fig. 5, which is
published as supporting information on the PNAS web site). No
specific staining was observed in murine CD8?cells or non-T cells
(data not shown).
FOXP3 expression profiles in human peripheral blood mono-
nuclear cells (PBMC) were very similar to those observed in
murine cells. All CD25highCD4?cells, previously shown to
exhibit potent suppressor function (4), were FOXP3?, whereas
only a minority of CD25lowCD4?and CD25?CD4?cells exhib-
ited FOXP3 expression. This finding is consistent with the
observation that CD25lowcells are not suppressive (18) (Fig. 1 C
and D). Previous estimates have proposed that the human TR
subset constitutes ?1–3% of CD4?T cells. However, the
percentage of FOXP3?cells was found to be closer to 6% in
normal donors using our FOXP3-specific rabbit polyclonal an-
tibody. This finding is in complete agreement with recently
described flow cytometric detection of human FOXP3 using
another novel mAb (14). Similar to Foxp3nullmice, patients with
FOXP3 mutations affecting mRNA splicing (IPEX-1 and
IPEX-3) have no detectable FOXP3?cells (Fig. 1 C and D and
Table 1). Interestingly, CD4?cells from IPEX patients exhibited
a similar proportion of CD25?cells as normal subjects, suggest-
ing the presence of activated effector T helper (Th) cells despite
the administration of immunosuppressants (Fig. 1 C and D and
Table 1). FOXP3?CD4?cells were also enriched in expression
of the T cell activation markers CTLA-4 and HLA-DR. In
contrast to the correlation seen between high CD25 expression
and FOXP3 positivity, however, comparably high expression
levels of CTLA-4 and HLA-DR were present on both FOXP3?
and FOXP3?CD4?T cells (Fig. 1E). In the CD8?T cell
compartment, there were negligible numbers of FOXP3?cells
(compare with the IPEX sample that lacks FOXP3 expression
altogether), showing that, in quiescent PBMC, FOXP3-
expressing CD8?cells are rare (Fig. 1 C and D). For reasons
likely due to variable epitope accessibility, our 3G3 mAb was
somewhat less efficient than the rabbit polyclonal antibody at
detecting FOXP3-expressing cells (Fig. 1). However, its utility
and specificity for staining FOXP3 in humans is demonstrated
here in normal and IPEX patient samples (Fig. 1).
FOXP3 Expression Is Induced Transiently in Some Human Non-TRCD4?
and CD8?T Cells upon Activation but Persists only in in Vivo-
Generated TR Cells. To investigate the degree to which de novo
FOXP3 expression might occur in individual human T cells, we
examined FOXP3 expression after TCR stimulation. Total or
CD25-depleted PBMC were stimulated with varying doses of
anti-CD3, and cells were analyzed by flow cytometry on days 3, 7,
and 10 of culture. This regimen relies on ‘‘presentation’’ of anti-
a situation that we feel more closely resembles TCR activation in
B) Normal or Foxp3-deficient mouse lymph node cells were stained for Foxp3
and cell-surface markers by using digoxigenin-conjugated mAb 3G3 (A) or
Foxp3-specific rabbit antibody (B). CD4?gated lymphocytes are shown. (C–E)
Normal (1792 and 1745) or FOXP3-deficient (IPEX) PBMC were stained for
FOXP3 and lymphocyte markers by using digoxigenin-conjugated mAb 3G3
(C) or digoxigenin-conjugated Foxp3-specific rabbit antibody (D and E). Both
CD4?and CD8?gated lymphocytes are shown. Additional IPEX-1 PBMC were
not available for subsequent analysis with rabbit antibody. High background
Table 1. IPEX patients
type (age at onset) Other*
Age and treatment
when PBMC drawn
IPEX-1 210-210 ? 1, GG ? AC,
c.751?753, del GAG,
c.751?753, del GAG,
Eczema IDDM (2 months)AIHA?ITP 1IgE 5 months, FK506?steroids,
6 years, intermittent
9 years, FK506
IPEX-2-P1EczemaIDDM (6 months) and
IDDM (6 months) and
IPEX-3 EczemaFood allergies 1IgE4 years, FK506
IPEX-like-1EczemaIDDM (2 years) and
thyroiditis (6 years)
IDDM (2 years)
Nephrotic syndrome 11 years, FK506
Eczema Candidiasis 1IgE
Persistent AIHA 1IgE
3 years, azathioprine
4 months, CsA
Eczema and alopecia
Mutation nomenclature is according to ref. 28. IDDM, insulin-dependent diabetes mellitus; AIHA, autoimmune hemolytic anemia; ITP, immune thrombo-
cytopenia; TPN, total parenteral nutrition; N?A, not available; 1, high concentration.
*All patients had moderate to severe enteropathy with profuse watery diarrhea.
www.pnas.org?cgi?doi?10.1073?pnas.0509484103 Gavin et al.
response to its natural ligands (i.e., peptide?MHC complexes) than
plate- or bead-immobilized antibodies. A dramatic increase in the
percentage of FOXP3?cells among both CD4?and CD8?T cells
was observed after stimulation, with up to 25% of CD4?cells and
27% of CD8?cells expressing FOXP3 on day 3 (Fig. 2 A and C).
The proportion of FOXP3?T cells diminished progressively over
time to near baseline levels by day 10. Interestingly, the relative loss
of FOXP3 expression was most dramatic for cell populations that
contained fewer FOXP3?cells before activation (CD8?cells and
CD4?cells from CD25-depleted PBMC). In contrast, CD4?cells
in cultures of total PBMC retained a FOXP3?CD25?CD4?sub-
population on day 10 of culture that was strikingly similar to freshly
isolated PBMC (Fig. 2 A and B). This pattern of transient FOXP3
and was consistent among monoclonal 3G3, rabbit polyclonal
T cell costimulation was required for FOXP3 induction, because
activation of purified T cells with plate-bound anti-CD3 and
anti-CD28, but not anti-CD3 alone, promoted similar transient
FOXP3 expression (Fig. 6, which is published as supporting infor-
mation on the PNAS web site).
The substantial size of the FOXP3?cell population after T cell
activation suggests that many of these cells may arise by transient,
activation-induced, de novo expression of FOXP3 in non-TR. How-
of FOXP3?CD25?CD4?T cells potentially capable of in vitro
expansion. Indeed, similar experiments with mouse cells revealed a
striking enrichment of Foxp3?cells because of selective outgrowth
(Fig. 7, which is published as supporting information on the PNAS
web site). To further examine human FOXP3 induction in vitro,
were evaluated for proliferative responses and FOXP3 expression
levels by flow cytometry. On day 3, when increased numbers of
FOXP3?cells were readily observed, FOXP3 expression on both
CD4?and CD8?cells was not confined to highly divided CFSElow
cells (Fig. 3D). Specifically, FOXP3 was found to be expressed in
?6% of CD8?T cells that had not yet undergone cell division (Fig.
T cells are capable of de novo FOXP3 induction in vitro. Although
FOXP3?cells on day 7 exhibited a high degree of CFSE dilution,
it is likely that most of these cells derived from the efficient
proliferation of preexisting TRbecause the depletion of CD25?
cells from starting cultures (while not dramatically affecting the
degree of FOXP3 induction on day 3) results in a paucity of
FOXP3?cells at later time points (Fig. 2 A–C). Importantly, unlike
up-regulation of CD25, only a subset of T cells induced FOXP3
expression, suggesting that FOXP3 induction is stochastic or that
some peripheral T cells are poised, i.e., precommitted, to express
Induced FOXP3 Does Not Suppress Th1 Cytokine Synthesis. Next we
sought to determine whether the induction of FOXP3 resulted in
CD25, glucocorticoid-induced TNF receptor, or CD27 expression
expressing induced FOXP3 for direct suppressor function studies.
Ectopic expression of high levels of FOXP3 in naı ¨ve human CD4?
inability of naturally developing TRto produce these cytokines.
Thus, analysis of intracellular cytokine production should serve as
an indirect way to assess acquisition of some TR properties by
activated T cells.
To determine whether the induced FOXP3 suppressed these
cytokines, cultured cells were examined for FOXP3, IL-2, and
IFN-? expression. As expected, the FOXP3?CD4?TRin freshly
isolated PBMC did not express either IL-2 or IFN-? in response to
activation on day 0 (Fig. 3). On days 3 and 7 after stimulation, both
cytokines were expressed by FOXP3?CD4?and FOXP3lowCD4?
T cells. (A–C) Total or CD25-depleted PBMC from donor 1745 were stimu-
lated with 5, 100, or 1000 ng?ml anti-CD3. FOXP3 and CD25 expression on
CD4?and CD8?cells were assessed at days 3, 7, and 10 of culture. Shown
are expression profiles for 100 ng?ml anti-CD3 (A), 5 or 1,000 ng?ml
anti-CD3 for CD4?gated cells (B), and the plotted percentage of gated cells
expressing FOXP3 (C). FOXP3 was detected with digoxigenin-conjugated
FOXP3-specific rabbit antibody. (D) Total PBMC from donor 1745 were
labeled with CFSE and stimulated with 100 ng?ml anti-CD3. FOXP3 expres-
sion was assessed at days 3 and 7 with digoxigenin-conjugated rabbit
antibody. Data are representative of four separate experiments and three
normal adult donors.
Analysis of FOXP3 expression in activated human CD4?and CD8?
Gavin et al. PNAS ?
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cells but not by a distinguishable FOXP3highpopulation observed
among CD4?but not CD8?T cells. The notable lack of Th1
cytokine expression by FOXP3highCD4?cells suggests that high
levels of FOXP3 are required to suppress cytokine synthesis or that
this population was derived from preexisting FOXP3?CD4?TR
present in the starting population (Fig. 3). The latter hypothesis is
supported by the observation that the FOXP3highCD4?population
was 20% less (day 3) and 75% less (day 7) abundant in the
CD25-depleted vs. undepleted PBMC cultures (data not shown).
FOXP3?CD8?cells demonstrated an expected lack of IL-2 pro-
duction but efficiently expressed IFN-? on both day 3 and day 7,
indicating that FOXP3 also did not promote a TR-like transcrip-
tional program in CD8?T cells. Similar results were obtained with
another FOXP3-specific monoclonal 259D using PBMC from a
second donor and costaining for TNF-? in addition to IL-2 and
IFN-? (Fig. 8, which is published as supporting information on the
PNAS web site). Cells with induced FOXP3 are likely to down-
regulate its expression rather than undergo apoptosis, because
costaining with the caspase active-site reporter substrate FITC-
VAD-FMK identified subsets of apoptotic FOXP3?CD4?but not
FOXP3?CD4?cells at days 3 and 7 (data not shown). Together,
these findings suggest that FOXP3 induced in vitro upon activation
of human non-TRdoes not promote TRphenotype development,
whereas FOXP3?TRgenerated in vivo persist and maintain their
functional characteristics after in vitro expansion.
FOXP3 Expression in IPEX Syndrome. Although FOXP3 mutations
have been characterized in more than two-thirds of IPEX patients,
we have identified a subgroup of patients exhibiting a similar
splice-site mutations. In such individuals, identified as IPEX-like
(Table 1), FOXP3 deficiency may result from uncharacterized
FOXP3 promoter mutations or from mutations in genes required
for FOXP3 expression. Alternatively, FOXP3 expression may be
intact, and the disease may result from mutations in other genes
autoimmune pathology in these IPEX-like patients, PBMC were
analyzed for FOXP3 expression with mAb 3G3. Of four IPEX-like
patients, three (IPEX-like-1, -2, and -4) lacked FOXP3 expression
in the CD25highCD4?cell population, whereas IPEX-like-3 exhib-
Thus, we have linked three of four IPEX-like patients who lack
FOXP3 coding mutations with FOXP3 deficiency. Promoter mu-
identification of which will significantly advance our understanding
of the factors and signals that promote FOXP3 transcription.
splicing and absence of protein expression. A third IPEX patient
within the leucine zipper of FOXP3 was also identified. This
mutation was of particular interest because it should allow for
expression of a full-length, mutant FOXP3 protein. Indeed, ectopic
expression of native or mutant FOXP3 in both human fibroblasts
stable and could be efficiently detected by flow cytometry (Fig. 9,
which is published as supporting information on the PNAS web
site). Furthermore, FOXP3?E251was unable to dimerize or to
suppress transcription from an IL-2 promoter–luciferase reporter
construct, confirming a lack of functional activity (T.R.T., unpub-
lished observations). The presence of the classic IPEX phenotype
in this patient strongly argues against FOXP3?E251promoting
significant TRactivity. Thus, FOXP3?E251protein should serve as
a natural reporter to examine FOXP3 expression in the apparent
absence of FOXP3 function, thereby advancing our understanding
of the requirements for persistence of TRprecursors as well as the
nature of autoimmune effector cells in IPEX. Specifically, the
presence of FOXP3?E251?cells in IPEX-2 PBMC would indicate
to survive in the absence of FOXP3 function. Such cells could
represent those that either (i) attempted TRdevelopment during
thymic maturation and migrated to the periphery or (ii) induced
FOXP3?E251expression in peripheral tissues, perhaps in response
would indicate that FOXP3 function is required for the survival of
cells committed to the TRdifferentiation pathway.
Two IPEX-2 PBMC samples (P1 and P2) were obtained 3 years
with intermittent corticosteroid therapy, before the initiation of
other potent immunosuppressants. The second (IPEX-2-P2) was
drawn after 2 years of treatment with FK506. Analysis of
differences. IPEX-2-P1 contained a population of large CD4?cells
expressing very high levels of CD25 (designated CD25??) (Fig. 4
A and B and data not shown). Thirty-three percent of these cells
expressed FOXP3?E251, but the presence of aggressive systemic
autoimmune disease in the patient at the time that the sample was
drawn argues against these cells having any significant regulatory
function. In contrast, IPEX-2-P2 lacked this population of large
incubated with PMA, ionomycin, and monensin and stained for FOXP3 (rabbit IgG-digoxigenin), IL-2, IFN-?, and surface markers as described in Materials and
Methods. (B) The percentage of cytokine-expressing cells among FOXP3high, FOXP3low, or FOXP3?cells is plotted. The distinction between high and low FOXP3
expression was not made for CD4?cells and CD8?cells on day 0. Data are representative of three separate experiments.
Induced FOXP3 does not suppress IL-2 or IFN-? synthesis. (A) Freshly isolated or stimulated (100 ng?ml anti-CD3) total PBMC from donor 1745 were
www.pnas.org?cgi?doi?10.1073?pnas.0509484103 Gavin et al.
CD25??CD4?cells and possessed a greatly reduced percentage of
the paucity of FOXP3?E251?cells in freshly isolated PBMCs,
FOXP3?E251expression was induced in 10% of CD4?IPEX-2-P2
PBMC upon stimulation with anti-CD3 for 3 days, mirroring the
kinetics of induction observed in control PBMC. We hypothesize
that the large CD25??CD4?cells found in IPEX-2-P1 are likely to
represent aggressive autoreactive effector T cells, some of which
also expressed FOXP3?E251, and that potent T cell-directed immu-
nosuppression with FK506 resulted in the loss of this population.
In the context of our findings in vitro, two nonmutually exclusive
potential mechanisms may explain the presence of FOXP3?E251-
expressing CD25??CD4?T cells in IPEX-2-P1. First, TRprecur-
sors that did not receive appropriate signals to continue down a TR
developmental pathway because of lack of functional FOXP3 may
have persisted as FOXP3?E251-expressing autoreactive effector T
cells (i.e., cells bearing TCRs that normally promote thymic TR
T cells that have induced FOXP3?E251expression in response to
activation (i.e., cells normally suppressed by TR). If the
FOXP3?E251?CD4?cells arose from non-TR precursors under
conditions similar to those that promote FOXP3 induction in vitro,
then a similar population may exist among CD8?IPEX-2-P1 cells
because we have observed FOXP3 induction with equal efficiency
in both CD4?and CD8?T cells. IPEX-2-P1 CD8?cells contained
a CD25??subset similar to their CD4?counterparts, suggesting
that some CD8?T cells were also highly reactive to self antigens
web site); however, the high degree of FOXP3?E251expression
found in CD25??CD4?cells was not observed (Figs. 4B and 10).
Thus, signals unique to CD4?cells appear to promote FOXP3
transcription in FOXP3 deficiency. If FOXP3 does not normally
rescue TRprecursors from thymic negative selection, such a signal
may derive from the increased TCR affinity TRtypically display for
self-peptide?MHC ligands (20, 21). Our recent findings of TR-
specific TCRs expressed in activated CD25?CD4?T cells from
Foxp3nullmice support this hypothesis (22).
In conclusion, we have presented the first flow cytometric
analysis of human FOXP3 expression in activated human PBMC,
demonstrating that FOXP3 induction can be uncoupled from TR
development. Although some FOXP3?T cells up-regulated
FOXP3 upon in vitro activation, Th1 cytokine synthesis was not
blocked. Furthermore, under conditions that favored the persis-
tence of in vivo-generated TR, long-lived TR were not readily
derived from activated cells. In vivo, the identification of
FOXP3?E251?CD25??CD4?T cells in IPEX-2-P1 suggests that
TR precursors contribute significantly to the severity of IPEX
symptomology. Although these two possibilities are not mutually
exclusive, the latter scenario is attractive in that it associates
self-reactive TCRs, i.e., those that promote TRdevelopment, with
T cells responsible for the multiorgan pathology observed in
FOXP3-deficient humans and mice.
Although our findings reveal a lack of functional consequences
of transiently induced FOXP3, others have reported de novo
generation of FOXP3?suppressive TRin more long-term cultures
(12, 15). Our findings support the possibility that preexisting TR,
capable of efficient expansion in vitro when in the presence of
IL-2-producing T cells, may contribute to the generated TRpop-
ulation in these experimental systems. Because we have observed a
correlation between high FOXP3 expression and repression of Th1
cytokines, sustained expression of high levels of FOXP3 may be
required to promote TRdevelopment in vitro. Indeed, our group
of murine or human FOXP3 results in the acquisition of TR
phenotype and function (J. Fontenot, personal communication)
(23). Although our methods for T cell activation did not result in
sustained, high-level expression of induced FOXP3, we cannot
exclude the possibility that some experimental conditions may
promote such expression and subsequent TRdevelopment.
In normal individuals, acute T cell stimulation by high-affinity
ligands can occur in response to various forms of neoantigen,
including infectious agents, vaccines, alloantigens presented after
organ transplantation, and self-antigens in the setting of graft-
versus-host disease. Should FOXP3 induction occur in such highly
activated T cells, as we have observed in vitro, the degree and
longevity of its expression and consequential TRdevelopment is
likely to be effected by the maturation state of antigen-presenting
dendritic cells (24–26). In mice, similar transient de novo Foxp3
expression has recently been reported for highly activated T cells
stimulated in vivo by dendritic cells presenting foreign antigen,
whereas only low levels of antigen in the absence of proinflamma-
tory signals resulted in de novo TRdevelopment (27). Our findings
support the distinct possibility that transient up-regulation of
Gated CD4?cells are shown. Histograms show FOXP3 expression (A) and side scatter (B) on CD4?cells expressing varying degrees of CD25 as delineated in the
adjacent 2D plots. Because the staining procedure results in a decrease in forward scatter, side scatter is a better indicator of cell size. Staining was performed
before the development of protocols by using digoxigenin-conjugated rabbit antibody, but additional PBMC from these patients were not available for further
study. PBMC shown in A and B were stained in separate experiments. (C) Freshly isolated or simulated (100 ng?ml anti-CD3; 3 days) normal (2020) or IPEX-2-P2
PBMC were stained for FOXP3 with mAb 259D (14).
FOXP3 expression in IPEX. (A and B) Normal (1745), IPEX-like (A), and IPEX (B) PBMC were stained for cell-surface markers and FOXP3 with mAb 3G3.
Gavin et al. PNAS ?
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vol. 103 ?
no. 17 ?
FOXP3 under proinflammatory conditions may not promote im- Download full-text
munosuppressive function in contrast to that mediated by preex-
isting TRresponding to the same antigens. In aggregate, our data
suggest that, despite the capacity for FOXP3 induction after TCR
sion for the acquisition of TRfunction. Although such conditions
may exist for TRprecursors in IPEX, they are not sufficient to elicit
suppressor function in the absence of functional FOXP3.
Materials and Methods
Antibodies. Rabbits and mice were immunized with bacterially
Fred Ramsdell; Celltech R&D, Bothell, WA) purified on Ni-NTA-
Agarose (Qiagen, Venlo, The Netherlands). Polyclonal antibodies
were produced by immunizing rabbits (R&R Rabbitry, Stanwood,
WA) every 21 days with 250 ?g of His-Foxp3. Hybridoma 3G3 was
generated by priming mice with 75 ?g of His-Foxp3 followed by
three 30-?g boosts before fusion and clone screening by ELISA.
Positive clones were subcloned and expanded in GIBCO Hybrid-
oma-SFM. Anti-Foxp3 antibodies were isolated from rabbit anti-
sera or hybridoma supernatant with protein A or protein G
Sepharose affinity chromatography (Amersham Pharmacia Bio-
sciences). Antibodies were labeled with digoxigenin-3-O-
methylcarbonyl-?-aminocaproic acid-N-hydroxysuccinimide ester
(Roche Diagnostics, Indianapolis).
donors by leukopheresis. Participants gave informed consent per
guidelines of the Institutional Review Board of the Fred Hutchin-
son Cancer Research Center. IPEX PBMC were isolated from
venous blood for the molecular diagnosis of IPEX syndrome by
sequence analysis and flow cytometry after consent of the patients.
T Cell Stimulation. Total or CD25-depleted (MACS, Miltenyi Bio-
tec) pooled mouse lymph node and spleen cells or human PBMC
were cultured at 4 ? 106cells per well (24-well plates) with titrated
anti-CD3 (2C11.145 or OKT3) in mouse cell medium (DMEM?
10% FBS?50 ?M 2-mercaptoethanol?10 mM Hepes?2 mM L-
glutamine?1 mM sodium pyruvate?penicillin–streptomycin) or hu-
2-mercaptoethanol?12.5 mM Hepes?6 mM L-glutamine?23.8 mM
Flow Cytometry. For staining with Foxp3-specific rabbit poly-
clonal IgG, cells were fixed in Cytofix?Cytoperm (BD Bio-
sciences) for 30 min on ice, washed once in DMEM?5% FBS,
and frozen at ?80°C in DMEM?20% FBS?10% DMSO. Cells
were thawed, washed twice in Perm?Wash (PW) (BD Bio-
sciences), and refixed in Cytofix?Cytoperm for 4 min on ice.
Cells were washed once with cold DMEM?5% FCS and twice
with PBS, resuspended in PBS?500 ?g/ml DNase (Roche, Indi-
anapolis)?4 mM MgCl2, and incubated at room temperature for
stained in PW supplemented with 350 mM NaCl (PW500) with
either 200 ?g?ml normal goat IgG (Jackson ImmunoResearch)
(mouse cells) or 5% normal rabbit serum (Jackson Immuno-
Research) (human cells). After 5–10 min, anti-Foxp3 rabbit IgG
or digoxigenin-labeled anti-Foxp3 rabbit IgG was added to 10
?g?ml. After three washes in PW500, cells were stained with
normal goat IgG (mouse cells) or 5 ?g?ml biotinylated mouse
anti-digoxin mAb with 5% normal mouse serum (human cells)
(all Jackson ImmunoResearch reagents). After 20–30 min at
room temperature, cells were washed three times with PW
stained in PW with allophycocyanin-conjugated streptavidin
(BD Biosciences) and other fluorophore-conjugated antibodies
specific for cell-surface antigens. After a 20-min incubation at
room temperature, cells were washed twice in PW, resuspended
in PBS, and analyzed on a FACSCalibur or FACSCanto flow
cytometer (BD Biosciences). For staining with digoxigenin-
labeled Foxp3-specific mouse mAb (3G3-dig), mouse cells were
incubated with 200 ?g?ml DNase whereas human cells were
neither refixed nor treated with DNase. Normal mouse serum
(5%) (Jackson ImmunoResearch) was included during staining
with both 3G3-dig and the secondary anti-digoxin reagent. In
later experiments, FOXP3 was detected with Alexa Fluor 488-
conjugated 259D (14) according to the manufacturer’s protocols
(BioLegend, San Diego).
For cytokine staining, cells were cultured with PMA (40 ng?ml),
ionomycin (1 ?g?ml), and monensin (3 ?M) for 5 h (day 0 PBMC)
were stained with IL-2-FITC, IFN-?-phycoerythrin (PE), 5% nor-
mal mouse serum, and cell-surface markers during incubation with
the anti-digoxigenin secondary reagent. Cells were costained with
CD4-peridinin chlorophyll protein (SK3), CD8-FITC or CD8-
allophycocyanin Cy7 (RPA-T8), CD25-PE or CD25-PECy7 (M-
A251), CTLA-4-PE (BNI3), and HLA-DR-PE (G46-6) (BD Bio-
We are grateful to all members of the A.Y.R. laboratory for provocative
discussions and to Drs. Frank Ruemmele, Andy Gennery, Lawrence
Jung, Robert Hostoffer, and Allison Jones for the referral of patient
samples for molecular diagnosis and analysis. This work was supported
by grants from Arthritis Foundation and the Lymphoma and Leukemia
Society (to M.A.G.), grants from the National Institutes of Health (to
A.Y.R., T.R.T., and H.D.O.), a Pfizer Postdoctoral Fellowship in
Rheumatology?Immunology (to T.R.T.), the Immunodeficiency Foun-
dation, and the Jeffrey Modell Foundation (H.D.O.). A.Y.R. is a
Howard Hughes Medical Institute Investigator.
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