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IMMUNOBIOLOGY
From the Department of Internal Medicine II, the Core
Research for Evolutional Science and Technology Project, and the
Department of Molecular Immunology, Graduate School of Medicine, Chiba
University, Japan; and the Department of Immunology, Tokyo Metropolitan
Institute of Medical Science, Tokyo Metropolitan Organization for
Medical Research, Japan.
It has recently been shown that
CD4+CD25+ T cells are immunoregulatory T cells
that prevent CD4+ T-cell-mediated organ-specific
autoimmune diseases. In this study, the regulatory mechanism of
CD4+CD25+ T-cell development were investigated
using T-cell receptor (TCR) transgenic mice. It was found that
CD4+CD25+ T cells preferentially expressed the
endogenous TCR Immunologic tolerance is a feature of the immune
system essential for discrimination between self and nonself. Recent
data suggest that in addition to clonal deletion and anergy, regulatory T cells play a significant role in the generation and maintenance of
tolerance.1-4 The term regulatory T cells is used for a
variety of immunoregulatory cells that can be subdivided into a number of subsets based on expression of cell surface proteins and pattern of
cytokine production.1-4 Because these subsets of
regulatory T cells have been characterized in experimental models using
different assays, the interrelationship between the subsets is
difficult to understand.1-4
One of the best-characterized subsets of CD4+ regulatory T
cells is defined by its constitutive expression of interleukin-2 receptor (IL-2R)- CD4+CD25+ T cells can be subdivided by the
expression pattern of CD45RBhigh or CD45RBlow,
CD38+ or CD38 Precise signals that promote the development of
CD4+CD25+ T cells remain elusive, but
considerable evidence suggests that costimulatory molecules and
cytokines play important roles. It has been shown that
CD4+CD25+ T cell levels are severely decreased
in mice lacking CD288 or CD40L,15 indicating
that signaling by CD28 and by CD40L regulates the number of
CD4+CD25+ T cells in periphery. In addition,
CD4+CD25+ T-cell levels are decreased in mice
lacking IL-216 or IL-2R component,17
indicating that IL-2 signaling also regulates the number of
CD4+CD25+ T cells.
The CD25+ population constitutes approximately 5% of
CD4+CD8 Recently, Itoh et al18 have shown that
CD4+CD25+ T cells can develop in TCR transgenic
mice of wild-type background but not of RAG-2-deficient background.
Thus, it is possible that B cells, In the current study, to determine whether positive or negative
selection in thymus regulates the development of
CD4+CD25+ T cells, we investigated
CD4+CD25+ T-cell development in mice expressing
TCR transgene in positively selecting and negatively selecting MHC
backgrounds. We also investigated the requirement of B cells and NK T
cells for the development of CD4+CD25+ T cells.
Our results indicate that the size of the
CD4+CD25+ T-cell pool in periphery is regulated
at least 2 distinct levels. First, in the thymus, where
CD4+CD25+ T cells are produced, a certain range
of TCR-MHC affinity is required for their development. In addition, B
cells, but not NK T cells, regulate the expansion and/or survival of
CD4+CD25+ T cells in periphery.
Mice
Flow cytometric analysis
Annexin V staining After cells were stained with anti-CD4 APC and anti-CD25 PE and washed twice with phosphate-buffered saline-1% bovine serum albumin, cells were stained with annexin V-FITC (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Cells were then analyzed on a FACScalibur.Data analysis Data are summarized as mean ± SD. Statistical analysis of the results was performed by the unpaired t test. P < .05 was considered significant.
CD4+CD25+ T cells preferentially express
the second TCR-  chain
is almost perfect, whereas that of the TCR- chain is
incomplete.25 Thus, a considerable fraction of T cells
express 2 different TCR- pairs.26 Because it has
been shown that CD4+CD25+ T cells can develop
in TCR transgenic mice of wild-type background but not of
RAG-2-deficient background,18 it is possible that CD4+CD25+ T cells preferentially express the
endogenous TCR- chain and that signaling through the endogenous
TCR- chain coupled with the transgenic TCR- chain is involved in
the development of CD4+CD25+ T cells in TCR
transgenic mice. To examine this possibility, we first investigated the
frequency of CD4+ T cells that express both transgenic TCR
(recognized by anti-idiotypic monoclonal antibody, KJ1-26) and one of
the endogenous TCR V- chains, V-2, in the CD25+ or
CD25 population in ovalbumin-specific TCR transgenic
(DO10+) mice. As shown in Figure
1A, splenic CD4+ T cells that
express transgenic TCR and TCR V-2 chain were found at a higher
frequency in CD4+CD25+ T cells than in
CD4+CD25 T cells
(CD4+CD25+ T cells 12.0% ± 1.6% versus
CD4+CD25 T cells 2.5% ± 1.0%, mean ± SD; n = 5; P < .001). Moreover, CD4+ T
cells that express both transgenic TCR and TCR V-11 were also found
at a higher frequency in CD4+CD25+ T cells
(data not shown). These results suggest that
CD4+CD25+ T cells preferentially express the
second TCR- chain in DO10+ mice. For controls, we
performed the same analysis for splenocytes in DO10+
RAG-2-deficient mice. We found that, consistent with the previous report by Itoh et al,18 CD4+CD25+
T cells were almost absent in these mice (Figure 1B) and that no
CD4+CD25 T cell expressed TCR V-2 (Figure
1B) or TCR V-11 (data not shown) in these mice.
Dual TCR- chains in DO10+ mice (Figure 1A). To determine
whether dual TCR- expression is required for the development of
CD4+CD25+ T cells, we investigated the
development of CD4+CD25+ T cells in TCR-
heterozygous mice, in which only one allele of TCR- was available
for the expression. As shown in Figure 2,
the number of CD4+CD25+ T cells in thymus and
in spleen was normal in TCR- heterozygous mice, suggesting that
though CD4+CD25+ T cells preferentially
expressed the second TCR- chain in TCR transgenic mice, dual TCR-
expression was not essential for the development of
CD4+CD25+ T cells.
CD4+CD25+ thymocytes are severely decreased
in DO10+ TCR- / mice (Figure 1B) and because
CD4+CD25+ T cells preferentially express the
second TCR- chain in DO10+ mice (Figure 1A), the
expression of endogenous TCR- chain and subsequent interaction with
self-MHC at an appropriate affinity may be required for the development
of CD4+CD25+ T cells. To test this possibility,
we investigated the development of CD4+CD25+ T
cells in DO10+ TCR--deficient (TCR-/
) mice (Figure 3). Indeed, the frequency
of the CD25+ population in
CD4+CD8 mature thymocytes was severely
decreased in DO10+ TCR-/ mice with
positively selecting H-2d background compared with that in
DO10+ TCR-+/+ mice (Figure 3B). In contrast,
no significant difference was observed in the number of conventional
CD4+CD25 mature thymocytes between
DO10+ TCR-/ mice and
DO10+ TCR-+/+ mice (Figure 3B).
These results indicate that expression of the endogenous TCR- chain
is required for the development of CD4+CD25+ T
cells in DO10+ mice. In addition, they indicate that the
appropriate affinity of TCR to self-MHC for the development of
CD4+CD25+ thymocytes is different from that of
CD4+CD25 thymocytes and that the interaction
of TCR with self-MHC at a certain affinity is essential for the
development of CD4+CD25+ thymocytes.
We next analyzed CD4+CD25+ T-cell development
by transgenic TCR in DO10+ mice in a negatively selecting
H-2b background.27,28 For this purpose, we
investigated DO10+ TCR- Interestingly, though the number of CD4+CD25+
thymocytes was severely decreased in DO10+
TCR- B cells but not natural killer T cells regulate the peripheral pool of CD4+CD25+ T cells CD4+CD25+ T cells exist in the spleens of DO10+ TCR-/ H-2d mice
(Figure 3C) but not in spleens of DO10+
RAG-2/ mice (Figure 1B). Thus, it is possible that a
certain population of cells absent in RAG-2-deficient mice regulates
the size of the CD4+CD25+ T-cell pool in
periphery. The possibility that B cells might affect
CD4+CD25+ T-cell development was examined by
flow cytometric analyses of CD4+CD25+ T cells
in µ-chain-deficient mice, which lack mature B cells.19 Interestingly, a profound decrease of splenic
CD4+CD25+ T cells was observed in
µ-chain-deficient mice (Figure 4A).
Less than 7% of CD4+ T cells in µ-chain-deficient mice
expressed CD25 compared with 13% in wild-type mice (n = 5 each)
(Figure 4A). Because the absolute number of splenic T cells was
decreased in µ-chain-deficient mice, the number of splenic
CD4+CD25+ T cells was significantly decreased
in µ-chain-deficient mice (approximately 20% of wild-type levels;
P < .001; Figure 4B). Interestingly, in contrast to
splenic CD4+CD25+ T cells, the number of
CD25+ population in CD4+ CD8
mature thymocytes was normal in µ-chain-deficient mice (Figure 5). Taken together, these results
indicate that though the development of
CD4+CD25+ T cells in thymus does not require
help from B cells, the size of CD4+CD25+ T-cell
pool in periphery is regulated by B cells. In contrast, the number of
splenic CD4+CD25+ T cells (Figure 4) and
CD4+CD25+ thymocytes (Figure 5) was normal in
NK T cell-deficient mice, indicating that NK T cells are not essential
for the development of CD4+CD25+ T
cells.
In this study, we show that the size of the peripheral
CD4+CD25+ T-cell pool is regulated by at least
2 distinct mechanisms. First, in the thymus, where
CD4+CD25+ T cells are produced, a certain
TCR-MHC affinity is required for the development of
CD4+CD25+ T cells. We found that
CD4+CD25+ T cells preferentially expressed the
second TCR- Although it is accepted that CD4+CD25+ T cells
need activation by TCR for regulatory function, the antigen specificity
of CD4+CD25+ T cells is unknown. We found that
CD4+CD25+ thymocytes were severely decreased in
DO10+ TCR- Although the expression of dual TCR- We found that B cells played a significant role in the regulation of
the CD4+CD25+ T-cell pool in the spleen (Figure
4) but not in the development of CD4+CD25+ T
cells in the thymus (Figure 5). Because it has been shown that B7/CD28
interaction8 and CD40/CD40L interaction15
regulate the CD4+CD25+ T-cell pool in
periphery, B cells may regulate the CD4+CD25+
T-cell pool through B7/CD28 interaction or CD40/CD40L interaction. Recently, it has been shown that the number of dividing cells is
modestly increased in CD4+CD25+ T cells
compared with that in CD4+CD25 Another subset of CD4+ regulatory T cells, isolated after T
cells were activated with alloantigens in the presence of IL-10, was
termed type 1 T regulatory (Tr1) cells.30 Tr1 cells are distinct from classical Th1 or Th2 cells in that they produce large
amounts of IL-10 and moderate amounts of transforming growth factor- In the past 10 years, a great deal has been learned about the
regulation of the CD25 gene. It is known that the CD25 gene has at
least 3 important elements for regulation, denoted positively regulatory regions (PRR) I, II, and III.33 NF- In summary, we have shown that the peripheral pool of CD4+CD25+ T cells is regulated at least 2 distinct levels. First, a certain TCR-MHC affinity is required for the development of CD4+CD25+ T cells in thymus. Second, B cells are required for the expansion of CD4+CD25+ T cells in periphery. The identification of TCR-MHC affinity that preferentially induces the development of CD4+CD25+ T cells will extend our understanding of the nature of CD4+CD25+ T cells.
After our submission of this paper to Blood, Jordan et al reported that selection of CD4+CD25+ thymocytes required a TCR with high affinity for a self-peptide.35
We thank Dr T. Saito for BALB/c RAG-2
Submitted March 27, 2001; accepted July 9, 2001.
Supported in part by grants from the Ministry of Education, Science and Culture, Japan and from Uehara Memorial Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hiroshi Nakajima, Dept of Internal Medicine II, Chiba University School of Medicine, 1-8-1 Inohana, Chiba 260-8670, Japan; e-mail: nakajimh{at}intmed02.m.chiba-u.ac.jp.
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Top
Abstract
Introduction
T cells, or natural killer
(NK) T cells, all of which are absent in TCR transgenic
RAG-2-deficient mice but are present in TCR transgenic mice, are
required for the development of CD4+CD25+ T
cells. It is also possible that the expression of endogenous TCR and
the subsequent interaction with self-major histocompatibility complex
(MHC) at a certain affinity is required for the development of these
cells. However, a great deal of uncertainty remains about differentiation factors and antigen specificity of
CD4+CD25+ T cells.
B and SRF
bind PRRI, Elf-1 and HMG-I (Y) bind PRRII, and Stat5 and Elf-1 bind PRRIII.