The Journal of Experimental Medicine
JEM © The Rockefeller University Press
Vol. 201, No. 1, January 3, 2005 127–137
Conversion of CD4
CD25regulatory T cells in vivo requires
B7 costimulation, but not the thymus
cells into CD4
Shuang Liang, Pascale Alard, Yuan Zhao, Sarah Parnell, Sherry L. Clark,
and Michele M. Kosiewicz
Department of Microbiology and Immunology, University of Louisville Health Science Center, Louisville, KY 40202
is known about their development and maintenance. In this study, we investigated whether
CD4CD25cells can convert to CD4 CD25
conditions. CD4CD25cells from CD45.1mice were sorted and transferred into congenic
CD45.2mice. Converted CD4CD25cells could be detected in lymphoid organs as early as
1 wk after transfer and by 6 wk after transfer, 5–12% of transferred CD4
CD25. Converted CD4CD25cells themselves failed to proliferate after stimulation, but
could suppress proliferation of responder cells in vitro, and also expressed high levels of
Foxp3 mRNA. In addition, CD4CD25cells transferred into thymectomized congenic mice
converted to CD4CD25cells that also suppressed responder cell proliferation in vitro, and
expressed high levels of Foxp3 mRNA. Finally, CD4
failed to convert into CD4 CD25cells that exhibit the regulatory phenotype. These data
indicate that CD4CD25cells convert into CD4
vivo and suggest that this conversion process could contribute significantly to the
maintenance of the peripheral CD4CD25regulatory T cell population.
regulatory T cells play a critical role in controlling autoimmunity, but little
? ? ? ?
regulatory T cells in vivo under natural
? ? ?
? ? ?
cells transferred into B7
? ? ?
regulatory T cells spontaneously in
Regulating potentially autoreactive cells that
have escaped negative selection in the thymus
is an important function of peripheral toler-
ance. A growing body of evidence suggests
that a population of regulatory T cells, CD4
CD25T cells, first identified by Asano et al.
(1) and Sakaguchi et al. (2), is critical for con-
trolling a wide variety of immune responses in-
cluding those that cause many types of autoim-
mune disease. Depletion of this population of
cells results in multi-organ autoimmune diseases
in a variety of strains of mice (1, 2). It is still
unclear to date whether the CD4
ulatory T cell population represents a distinct
lineage of T cells. Several lines of evidence
suggest that these cells develop in the thymus.
For example, CD4
can be found in the thymus and exhibit phe-
notypic and functional characteristics that are
identical to those found in peripheral CD4
CD25 T cells. Adoptive transfer of this popu-
lation of thymocytes prevents development of a
variety of autoimmune and inflammatory diseases
(3). Furthermore, CD4
T cells develop
directly in fetal thymic organ cultures (3).
These cells appear to be positively selected on
thymic epithelium because mice that do not
express MHC class II on their thymic cortical
epithelium fail to develop CD4
latory T cells, and transgenic mice that express
specific peptide on their thymic stromal cells
produce extremely high percentages of CD4
CD25regulatory T cells (4, 5). Although it
appears very likely that most CD4
regulatory T cell development occurs in the
thymus, accumulating evidence suggests that
these cells may also develop in the periphery
(i.e., extrathymically). For example, although
under normal circumstances all CD4
RAGTCR transgenic mice are CD25
studies have shown that a percentage of
TCR transgenic T cells adoptively
transferred into antigen-expressing transgenic
mice or mice that have received a tolerizing
dose of peptide antigen administered either i.v.
or orally can convert to a CD4
latory T cell phenotype (6, 7). It is unclear,
however, whether CD4
do convert to a CD4CD25
phenotype under natural conditions, i.e., with
T cells can or
regulatory T cell
The online version of this article contains supplemental material.
Michele M. Kosiewicz:
Abbreviations used: CFSE, car-
boxyfluorescein diacetate succin-
imidyl ester; CTLA-4, cytotoxic
T lymphocyte–associated anti-
gen 4; GITR, glucocorticoid-
induced TNF receptor gene.
CELLS CONVERT INTO CD4
REGULATORY T CELLS IN VIVO | Liang et al.
expression of the natural TCR repertoire and exposure to the
natural endogenous antigen load. The purpose of this study
was to address this issue and identify the requirements for this
conversion process. We have found that mature peripheral
CD4 CD25T cells can indeed convert to a CD4
regulatory T cell phenotype and do so in a thymus-indepen-
dent but B7-dependent manner.
? ? ?
vivo in both sublethally irradiated and nonirradiated mice
It is not clear whether the CD4
distinct lineage of cells that develops exclusively in the thy-
mus or whether these cells can be induced in the periphery.
The following experiments were designed to determine
whether CD4 CD25T cells from wild-type mice are capa-
ble of converting to a CD4
type in vivo. LN and spleen cells were harvested from con-
genic CD45.1mice and CD4
99.7% purity (Fig. 1 A). 10
T cells can convert into CD4
T cells in
T cells represent a
regulatory T cell pheno-
T cells were sorted
and thymus were harvested either 1 or 6 wk after injection of
cells. Donor CD45.1
CD4cells were gated (Fig. 1 B, left)
and analyzed for the presence of CD45.1
cells. The CD45.1
detected in blood (not depicted), LN (Fig. 1 B, middle), and
spleen (see Fig. S1, middle, which is available at http://
not thymus (not depicted), as early as 1 wk after i.v. injection
of CD45.1CD4 CD25T cells. By 6 wk after injection,
5–12% of the transferred CD45.1
LN (Fig. 1 B, right) as well as in the thymus (not depicted).
The percentage of CD25 cells in the spleen was somewhat
less and averaged
5–7% (Fig. S1, right).
Sublethal levels of irradiation create significant “space” in
the immune compartment and induce T cells to undergo dra-
matic homeostatic proliferation to fill this space. There is,
therefore, a possibility that the CD4
to this environment may up-regulate CD25 as a consequence
T cells were injected i.v. into sublethally irradiated
mice. Blood was collected weekly and LN, spleen,
? ? ?
T cells (2–4%) could be
? ? ?
cells expressed CD25 in
hibit regulatory function in sublethally irradiated mice. (A) Purity of
CD4? CD25? cells for transfer. CD4? CD25? cells from LN and spleen cells
of CD45.1? mice were purified using T and CD4 cell affinity columns, mag-
netic beads, and high speed sorting. CD4? CD25? cells account for ?10%
of CD4? cells before sorting (left). CD4? CD25? cells were purified to
?99.7% (?0.3% contamination with CD4? CD25? cells; right). (B) 10 ? 106
million purified CD45.1? CD4? CD25? cells were transferred into sub-
lethally irradiated CD45.2? recipients. (B) LN cells or (C and D) LN and spleen
cells were collected and labeled for CD45.1, CD4, and CD25. CD45.1? CD4?
cells (B) were gated and analyzed for the presence of converted CD4?
CD25? cells 1 (middle) and 6 wk (right) after transfer or 6 wk after transfer
and were (C) tested for regulatory function or (D) Foxp3 expression. (C) To
CD4? CD25? cells convert into CD4? CD25? cells that ex-
test for regulatory function, 25,000 freshly harvested CD4? CD25? cells
(Responder cells) were purified and cocultured at a 1:1 (regulatory/responder)
ratio with either freshly harvested CD4? CD25? cells (Control) or sorted
CD45.1? CD4? CD25? cells (Converted) in the presence of irradiated spleen
cells (APCs) and anti-CD3 for 3 d. (D) For quantitative analysis of Foxp3
expression, freshly harvested CD4? CD25? (Control) or CD4? CD25? (Con-
trol) cells, or CD45.1? CD4? CD25? (Transferred) or CD45.1? CD4? CD25?
(Converted) cells were sorted and Foxp3 mRNA levels were quantified by
real-time PCR. Data are presented as normalized Foxp3 mRNA expression
levels in the samples relative to normalized Foxp3 mRNA expression levels
in the control CD4? CD25? cells (fold change ? 1). *, a significant differ-
ence from the positive control (P ? 0.01).
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