Download Counterparts − CD25 + Repertoires with CD4 Share Equally

Human CD4+CD25+ Regulatory T Cells
Share Equally Complex and Comparable
Repertoires with CD4+CD25− Counterparts
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Kimberly A. Kasow, Xiaohua Chen, James Knowles, David
Wichlan, Rupert Handgretinger and Janice M. Riberdy
J Immunol 2004; 172:6123-6128; ;
doi: 10.4049/jimmunol.172.10.6123
http://www.jimmunol.org/content/172/10/6123
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References
The Journal of Immunology
Human CD4ⴙCD25ⴙ Regulatory T Cells Share Equally
Complex and Comparable Repertoires with CD4ⴙCD25ⴚ
Counterparts1
Kimberly A. Kasow, Xiaohua Chen, James Knowles, David Wichlan, Rupert Handgretinger,
and Janice M. Riberdy2
D4⫹CD25⫹ T cells are an essential component of peripheral immune tolerance (1). Murine studies characterizing this population have become an area of intense investigation and have demonstrated a therapeutic potential for these
cells in preventing the onset of autoimmune disease and graft-vshost disease (2– 4). However, the precise mechanisms that control
their development and functional activity have remained elusive.
Studies of human CD4⫹CD25⫹ regulatory T cells have been even
more limited (5–9).
Despite the many unresolved questions regarding regulatory T
cells, the general consensus is that both murine and human
CD4⫹CD25⫹ T cells require activation through their TCR and
cell-cell contact to mediate their activity (10 –12). An important
feature of these cells is that once activated, they are able to downmodulate immune responses in an Ag-nonspecific manner (13). In
terms of the development of CD4⫹CD25⫹ T cells, murine studies
have shown that this subset can be generated in the thymus and
requires signals through the IL-2R (14, 15). Additional murine
studies have also demonstrated that the expression of a member of
the Forkhead transcription factor family, Foxp3, is necessary for
the development of CD4⫹CD25⫹ T cells, and animals deficient in
C
Division of Stem Cell Transplantation, St. Jude Children’s Research Hospital, Memphis, TN 38105
Received for publication October 30, 2003. Accepted for publication March 16, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Training Grant T32CA70089 from the National Cancer
Institute (to K.A.K.) and the American Lebanese and Syrian Associated Charities.
2
Address correspondence and reprint requests to Dr. Janice M. Riberdy, Division of Stem
Cell Transplantation, Mail Stop 321, St. Jude Children’s Research Hospital, 332 North
Lauderdale Street, Memphis, TN 38105. E-mail address: [email protected]
Copyright © 2004 by The American Association of Immunologists, Inc.
this molecule develop severe autoimmune and inflammatory dysfunctions (16 –18). The human allele, Foxp3, is likely to be a critical factor in the development of human CD4⫹CD25⫹ T cells as
well. Individuals with the fatal syndrome of immune dysfunction,
polyendocrinopathy, enteropathy, and X-linked inheritance have
mutations at this locus (19).
It is clear that the development of a functional CD4⫹CD25⫹ T
cell population is critical for the maintenance of a healthy immune
system. However, as activated CD4⫹CD25⫹ T cells can suppress
an immune response in an Ag-nonspecific manner, it is equally
important to understand what mechanisms regulate the activation
and functional capability of these cells (13). One feature that is
likely to contribute to the regulation of these cells is the overall
avidity of the TCR for its cognate ligand. Studies have suggested
that thymocytes with high affinity TCRs can be directed toward a
regulatory T cell lineage (20). Consistent with this idea is the observation that reducing the threshold of activation in thymocytes
preferentially selects a population of CD4⫹CD25⫹ T cells with
phenotypic and functional characteristics of regulatory T cells
(21). Yet, the high affinity nature of CD4⫹CD25⫹ T cells does not
appear to skew the repertoire, as examination of murine T cell
repertoires by V␤ staining has shown a diverse usage of V␤ gene
segments, with a similar distribution of the CD4⫹CD25⫺ T cell
population (22–24). Limited analysis of V␤ gene usage in human
CD4⫹CD25⫹ regulatory T cells from peripheral blood suggests
that these cells comprise a diverse repertoire. However, a comprehensive study examining the repertoire of human CD4⫹CD25⫹
regulatory T cells from multiple anatomical niches has been lacking (25). Interestingly, emerging data suggest that humans may
also expand CD4⫹CD25⫹ T cells in the periphery to compensate
for an increased life span accompanied by decreased thymic output
(26, 27). Temporal studies to examine whether such peripheral
mechanisms alter the repertoire as an individual ages may shed
0022-1767/04/$02.00
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CD4ⴙCD25ⴙ T cells are critical mediators of peripheral immune tolerance. However, many developmental and functional characteristics of these cells are unknown, and knowledge of human regulatory T cells is particularly limited. To better understand
how human CD4ⴙCD25ⴙ T cells develop and function, we examined the diversity of CD4ⴙCD25ⴙ and CD4ⴙCD25ⴚ T cell
repertoires in both thymus and peripheral blood. Levels of T receptor excision circles (TREC) were comparable in purified
CD4ⴙCD25ⴙ and CD4ⴙCD25ⴚ thymic populations, but were significantly higher than those in samples derived from peripheral
blood, consistent with murine studies demonstrating thymic development of CD4ⴙCD25ⴙ regulatory T cells. Surprisingly,
CD4ⴙCD25ⴚ T cells isolated from peripheral blood had greater TREC quantities than their CD4ⴙCD25ⴙ counterparts, supporting the possibility of extrathymic expansion as well. CD4ⴙCD25ⴙ and CD4ⴙCD25ⴚ T cells from a given individual showed
overlapping profiles with respect to diversity by V␤ staining and spectratyping. Interestingly, CD4ⴙCD25ⴙ T cells have lower
quantities of CD3 than CD4ⴙCD25ⴚ T cells. Collectively, these data suggest that human CD4ⴙCD25ⴙ T cells recognize a similar
array of Ags as CD4ⴙCD25ⴚ T cells. However, reduced levels of TCR on regulatory T cells suggest different requirements for
activation and may contribute to how the immune system regulates whether a particular response is suppressed or
augmented. The Journal of Immunology, 2004, 172: 6123– 6128.
6124
CD4⫹CD25⫹ REGULATORY T CELL REPERTOIRE
Materials and Methods
Thymic and peripheral blood cells
Peripheral blood cells from normal healthy donors were collected at St.
Jude Children’s Research Hospital (Memphis, TN) with permission from
the institutional review board. Thymic tissues were obtained from children
undergoing cardiac procedures at LeBonheur Children’s Medical Center
(Memphis, TN). The University of Tennessee Memphis institutional review board and the LeBonheur executive committee granted approval for
use of these specimens.
Purification of T cell subsets
Single-cell suspensions were prepared from thymic tissue. Both thymic and
peripheral blood cells were centrifuged over a gradient of Ficoll-Paque
Plus (Pharmacia Biotech, Uppsala, Sweden). Cells were washed with Dulbecco’s PBS (Cambrex, Walkersville, MD) and resuspended in MACS
buffer (PBS, 2 mM EDTA, and 0.5% human serum albumin or BSA).
CD4⫹CD25⫹ and CD4⫹CD25⫺ T cells were purified by magnetic bead
separation using an autoMACS cell separator (Miltenyi Biotec, Bergisch
Gladbach, Germany). Thymocytes were first depleted of CD8⫹ cells using
CD8 microbeads (Miltenyi Biotec). Untouched CD4⫹ T cells were purified
from both peripheral blood cells and CD8-depleted thymocytes by magnetic depletion using a CD4⫹ T cell isolation kit (Miltenyi Biotec). CD4⫹
T cell populations were fractionated into CD25⫹ and CD25⫺ subsets by
either a single-step positive selection using anti-CD25 microbeads (Miltenyi Biotec) or a two-step isolation using anti-CD25 PE (ACT-1; DAKO
Cytomation, Glostrup, Denmark), followed by anti-PE microbeads (Miltenyi Biotec). All microbead isolations followed the manufacturer’s instructions (Miltenyi Biotec). For the purified PBL and thymus products, the
CD4⫹ purity range was 82–98% (see Fig. 1, inset, for representative
CD4⫹CD25⫹ purification).
Flow cytometry
Thymocytes and PBL were analyzed using a BD LSR or BD LSRII flow
cytometer and Cell Quest Pro, BD FACSDiVa (BD Biosciences, San Jose,
CA), or FlowJo software (Tree Star, Ashland, OR). The following mAbs
were used to check the purity of the positive and negative selected cells:
anti-CD3 and anti-CD4 (SK7 and SK3, respectively; BD Biosciences),
anti-CD8 and anti-CD25 (DK25 and ACT1, respectively; DAKO Cytomation), and anti-CD25 (M-A251; BD PharMingen San Diego, CA). The
anti-V␤ family Abs were purchased from Immunotech (Marseilles,
France).
DNA and RNA isolation
DNA was isolated from purified CD4⫹CD25⫹ and CD4⫹CD25⫺ T cells
using the QIAamp DNA Mini kit (Qiagen, Valencia, CA) following the
manufacturer’s instructions. RNA was isolated from purified CD4⫹CD25⫹
3
Abbreviations used in this paper: TREC, T receptor excision circle; ⌬Ct, change in
Ct value; sj, signal joint.
FIGURE 1. Relative TREC levels. A, CD4⫹CD25⫹ and CD4⫹CD25⫺
T cells from human thymus and PBL were purified. The inset shows CD25
expression on the isolated populations after a typical purification. Relative
TREC levels were determined by real-time PCR, and the mean of duplicate
reactions is shown in arbitrary units. Bars with the same color scheme
represent purified CD4⫹CD25⫹ and CD4⫹CD25⫺ T cells from the same
individual, as indicated. Note that thymic and PBL samples are not from
the same individual. All reactions had slopes between ⫺3.1 and ⫺3.4. B,
Amplification curves for CD4⫹CD25⫹ (f) and CD4⫹CD25⫺ (E) T cell
populations. The y-axis represents the values of normalized target-specific
fluorescence signals (change in reaction), and the x-axis shows the number
of PCR cycles. The Ct values are defined as the PCR cycle at which the
change in reaction passes a threshold of 10 SD above the baseline fluorescence (threshold shown as solid line). ⌬Ct in these figures represents the
difference in Ct value between the CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell
populations. The amplification reactions for each sample were performed
in duplicate.
and CD4⫹CD25⫺ T cells using the Rneasy Mini kit (Qiagen) following the
manufacturer’s instructions.
Detection and quantitation of signal-joint (sj) TREC
Relative TREC levels were determined as previously described (28). In
brief, DNA was isolated from purified CD4⫹CD25⫹ and CD4⫹CD25⫺ T
cell populations, and real-time PCR was performed on 200 ng of DNA
using an ABI 7700 system (Applied Biosystems, Foster City, CA). All
samples were studied in duplicate reactions, using primers and conditions
described by Hazenberg et al. (28). The C␣ constant region was used as an
internal control to normalize for input DNA. A standard curve was generated by cloning the sj TREC fragment in pCR 2.1-TOPO vector using
TOPO TA Cloning kit (Invitrogen, Carlsbad, CA). The standard curve and
TREC values were analyzed by Sequence Detector software. The number
of TREC molecules in the sample was calculated as number of copies per
105 cells.
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light on the role of CD4⫹CD25⫹ T cells in immunological defects
associated with the elderly.
To better understand how human CD4⫹CD25⫹ T cells develop
and function, we have extensively analyzed the diversity of the
CD4⫹CD25⫹ T cell repertoire in both thymic and peripheral blood
compartments by V␤ staining and spectratyping. We have also
compared the regulatory T cell subset with the CD4⫹CD25⫺ counterpart for T receptor excision circle (TREC)3 content and CD3
levels. Together, our data demonstrate that human CD4⫹CD25⫹ T
cells are probably generated in the thymus with a repertoire of
comparable diversity to that of CD4⫹CD25⫺ T cells. Interestingly,
TREC data are consistent with the hypothesis that peripheral expansion could contribute to the maintenance of this population (26,
27). Finally, lower levels of CD3 in both thymic and peripheral
blood populations of naturally selected human CD4⫹CD25⫹ T
cells support the idea that these cells are a high affinity population
with different requirements for activation. Such alterations in the
threshold of activation provide the immune system with key mechanisms to control the level of suppressor activity associated with
CD4⫹CD25⫹ regulatory T cells.
The Journal of Immunology
TCR V␤ CDR3 size spectratyping
The CDR3 size distribution of 27 distinct TCR V␤ families was determined by RT-PCR as previously described (29). In brief, RNA was extracted and cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen). PCR was performed with a forward primer specific for
one of the TCR V␤ families along with a constant C␤ reverse primer
labeled with fluorescent FAM, using the conditions described by Gorski et
al. (29). RT-PCR products were analyzed on an ABI PRISM 310 Genetic
Analyzer (Applied Biosystems), using GeneScan software. The normal
TCR V␤ CDR3 size is characterized by a Gaussian distribution, containing
8 –10 peaks for each V␤ subfamily. The overall complexity of TCR V␤
subfamilies was determined by spectratype complexity scoring as previously described (30).
Quantitation of CD3-⑀ by real-time RT-PCR
Relative levels of CD3-⑀ were determined by real-time RT-PCR as previously described (31). Briefly, RNA was extracted, cDNA was synthesized,
and PCR was performed using the conditions and CD3-⑀ forward and reverse primers described by Pennington et al. (31). Experimental samples
and data points for the standard curve were assayed in duplicate. Human
GAPDH mRNA was used as the endogenous control to normalize for
input RNA.
CD4⫹CD25⫹ T cells are potent mediators of tolerance and can
down-modulate immune responses in the settings of both autoimmunity and viral infection (1–3, 32). Once activated through the
TCR, these cells can regulate a variety of effector cell functions in
an Ag-nonspecific manner (13). The lack of Ag specificity suggests a promising therapeutic potential for these cells for human
disease, because one does not have to identify the precise antigenic
epitope in each MHC haplotype. However, delineating the mechanisms that control the development and function of CD4⫹CD25⫹
T cells has been a challenge, and many questions remain unanswered. In particular, studies addressing how human CD4⫹CD25⫹
T cells develop, maintain homeostasis, and regulate activity have
been both difficult and limited (5–11, 27). Here, we assay for evidence of thymic development and compare the repertoires of
CD4⫹CD25⫹ T cells with the CD4⫹CD25⫺ counterparts to determine whether the same spectrum of Ags capable of eliciting a
robust effector cell response might also stimulate potent regulatory
T cell activity.
for the purified PBL cells ranged from 0.5 to 4.3 between the
CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell subsets (Fig. 1).
TCR V␤ staining
To address whether human CD4⫹CD25⫹ T cells exhibit a diverse
repertoire, CD4⫹CD25⫹ and CD4⫹CD25⫺ T cells from both thymus (n ⫽ 2) and peripheral blood (n ⫽ 4) were isolated. Purified
cells were doubly stained with anti-CD25 and a specific V␤ Ab to
analyze a panel of 21 subfamilies by flow cytometry. Although the
precise fingerprint varied between individuals, the profiles of TCR
V␤ usage from CD4⫹CD25⫹ T cells were strikingly similar to
those of the CD4⫹CD25⫺ counterparts (Fig. 2). This was true for
purified thymic subsets as well as isolated PBL populations.
Spectratyping
Although TCR V␤ staining examines the prevalence of specific
V␤ families within the repertoire, spectratyping dissects the diversity within a particular family (29). Although we did not observe
any repertoire skewing in the CD4⫹CD25⫹ subset at the level of
TCR V␤ gene usage, the high avidity of CD4⫹CD25⫹ T cells
could alter the distribution within a given V␤ family. Thus, we
compared the spectratyping profiles of purified human
CD4⫹CD25⫹ T cells with the CD4⫹CD25⫺ counterparts for five
thymii and six peripheral blood samples. Fig. 3 shows the profiles
of representative thymocyte and PBL samples. In general, the profiles for both thymic and PBL CD4⫹CD25⫹ T cell populations
were as broadly complex as those from the CD4⫹CD25⫺ T cell
subset. To validate that the CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell
subsets were equally complex, each isolated sample was given a
spectratype complexity score, as described by Wu et al. (30). In
brief, the number of oligoclonal peaks for each V␤ family was
counted. For CD4⫹CD25⫹ T cells isolated from thymus and PBL,
the ranges of complexity scores were 195–210 and 187–210, respectively. Similarly, the score ranges of CD4⫹CD25⫺ T cells
from thymus and PBL were 206 –214 and 188 –215, respectively.
T receptor excision circle analysis
One marker that can be used as a rough estimate of developmental
proximity to the thymus is the level of T receptor excision circles.
To ascertain whether the level of TREC was different between the
CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell subsets, specific populations were isolated from human thymus (n ⫽ 3) and PBL (n ⫽ 5).
Note that thymic and PBL samples were not from the same individual and could not be directly compared, but ranges of TREC
levels for purified thymic and PBL cells could be compared to
determine relative trends. TREC values for the various purified
subsets were assayed by real-time PCR. Fig. 1A illustrates the
range of TREC content observed in different individuals (5,175–
200,410 copies/105 cells for thymus and 23–2,386 copies/105 cells
for PBL). Despite variation in TREC values between different thymii, CD4⫹CD25⫹ and CD4⫹CD25⫺ thymocytes isolated from the
same thymus did not have significantly different levels of TREC.
Relative TREC values were independent of the actual number of
cells or the quantity of DNA isolated (data not shown). Importantly, the range of TREC values from thymocytes was significantly higher than that found in the PBL samples (Fig. 1A; p ⫽
0.001). In contrast to purified thymocytes, four of five individuals
had decreased levels of TREC in the CD4⫹CD25⫹ PBL compared
with the CD4⫹CD25⫺ counterpart. The change in Ct values (⌬Ct)
FIGURE 2. V␤ analysis. Purified CD4⫹CD25⫹ and CD4⫹CD25⫺ thymocyte and lymphocyte populations were stained for CD25 and TCR V␤
before analysis by flow cytometry. f, CD25⫹ T cells; E, CD25⫺ T cells.
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Results
6125
6126
CD4⫹CD25⫹ REGULATORY T CELL REPERTOIRE
In all comparisons of CD4⫹CD25⫹ T cells with CD4⫹CD25⫺ T
cells the ranges overlapped, and no significant differences were
observed in the overall breadth of the repertoires. Occasionally, an
oligoclonal expansion was observed in a particular V␤ family that
was unique to either the CD4⫹CD25⫹ or CD4⫹CD25⫺ T cell
subset (see V␤ 25 for thymus and V␤ 23 for PBL; Fig. 3), but, in
general, the repertoires were overlapping.
CD3 expression
The striking similarity between the repertoires suggested that the
spectrum of Ags capable of either selecting CD4⫹CD25⫹ T cells
in the thymus or activating CD4⫹CD25⫹ T cells in the periphery
could similarly stimulate the CD4⫹CD25⫺ T cell counterparts.
Although many parameters contribute to both thymic selection and
immune responses in the periphery, one factor that could affect
whether the CD4⫹CD25⫹ T cell subset was preferentially activated would be the density of TCR expression (21). We have examined CD3 expression by real-time RT-PCR for CD4⫹CD25⫹
and CD4⫹CD25⫺ T cell populations isolated from both thymus
(n ⫽ 2) and PBL (n ⫽ 3). In all cases, the CD4⫹CD25⫹ T cell
population had dramatically lower levels of CD3 RNA (Fig. 4A).
Flow cytometric analysis of mean fluorescence intensity also demonstrates an inverse correlation between CD25 and CD3 expression, with the CD4⫹CD25bright population having the lowest levels
of CD3 (Fig. 4B). This correlation was observed for both thymus
and PBL samples. In addition, purified CD4⫹CD25⫹ T cells exhibited decreased surface expression of CD3 compared with the
CD4⫹CD25⫺ counterpart whether they were isolated from thymus
(n ⫽ 2) or PBL (n ⫽ 4). Preliminary studies showed similar results
with anti-TCR staining (data not shown).
Discussion
Ideally, one would compare thymic and peripheral blood samples
from the same individual. However, thymic samples originated
from children undergoing cardiac surgery, and obtaining peripheral blood was not feasible. Peripheral blood samples were obtained from adult donors. Thus, our strategy was to compare the
CD4⫹CD25⫹ T cell population with the CD4⫹CD25⫺ counterpart. This approach allowed us to determine which trends were
associated with either a particular subset of cells or an anatomical
niche, but did not allow for a direct comparison between thymic
and peripheral blood repertoires.
At first, we sought evidence that human CD4⫹CD25⫹ T cells
were generated in the thymus, as many murine studies have elegantly shown (14, 15, 33). As a thymocyte rearranges its TCR
genes, TREC is generated, and high levels of these excision circles
are generally thought to reflect recent developments in the thymus.
However, as TREC is not replicated during mitosis, proliferation
of a particular T cell subset will affect the overall level of TREC
(28). Higher relative TREC values in both CD4⫹CD25⫹ and
CD4⫹CD25⫺ thymocytes compared with the PBL samples
strongly suggests that human CD4⫹CD25⫹ thymocytes are not
simply peripheral lymphocytes relocated in the thymus. Rather,
these cells are most likely generated in the thymus. Higher TREC
in purified thymocytes was not simply due to the fact that thymic
samples were from children and PBL were from adults. Average
TREC levels from the PBL of children posthemopoietic stem cell
transplant with documented immune reconstitution were still noticeably lower than the range seen for thymic samples (X. Chen,
data not shown). Comparable TREC values in both CD4⫹CD25⫹
and CD4⫹CD25⫺ thymocytes also suggest that any proliferation is
likely to be limited and equivalent in both populations.
Comparative studies of patients with thymic hypoplasia to
healthy age-matched controls have shown that the functional capacity of a thymus is important in maintaining normal numbers of
CD4⫹CD25⫹ T cells. One striking observation from this study is
a decline in the absolute number of CD4⫹CD25⫹ T cells from
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FIGURE 3. Spectratyping. Purified CD4⫹CD25⫹ and CD4⫹CD25⫺ thymocytes and lymphocytes were assayed by RT-PCR for CDR3 length to
determine the spectratyping profiles of each V␤ subfamily. The Gaussian curve represents expression of the V␤ subfamilies within each TCR V␤ family.
The Journal of Immunology
birth to 3 years of age in the control group. Importantly, the absolute number of CD4⫹CD25⫹ T cells remained relatively constant in older children and adults (34). It has been also been suggested that human CD4⫹CD25⫹ T cells may expand in the
periphery to compensate for the increased life span and decreased
thymic output seen in humans (27). A decreased amount of TREC
in the CD4⫹CD25⫹ T cells isolated from PBL is consistent with
the idea that human regulatory T cells may also expand in the
periphery and thus provide a mechanism to maintain absolute
numbers of this population (26, 27, 34). The extent to which such
putative mechanisms exist remains unknown and warrants further
investigation. Alternatively, one must consider the possibility that
some of the CD4⫹ T cells expressing CD25 are recently activated
T cells responding to Ag. Proliferation in response to stimulation
would artificially dilute the overall TREC content of the
CD4⫹CD25⫹ T cell population. It is unlikely, however, that four
of the five individuals were acutely infected at the time of sam-
pling. Furthermore, routine functional analysis of CD4⫹CD25⫹ T
cells isolated with the CD25 microbeads demonstrated functional
suppressor activity (D. Wichlan, data not shown). Interestingly,
purification methods that isolate both CD25bright and CD25int cells
do not yield functionally active suppressor cells. In contrast,
CD25bright cells alone demonstrate potent activity (D. Wichlan,
manuscript in preparation). Experiments to address whether the
CD25 intermediate cells are actively inhibiting the function of the
CD25bright cells are underway (D. Wichlan, data not shown). Thus,
understanding the functional composition of purified
CD4⫹CD25⫹ T cells remains a challenge for future clinical
strategies.
It is clear that CD4⫹CD25⫹ T cells must be stimulated through
the TCR to become functionally active (10 –12). However, once
activated, this population is able to down-modulate immune responses in an Ag-nonspecific manner (13). Thus, a critical question is whether the same spectrum of Ags capable of activating a
conventional CD4⫹ T cell response is able to stimulate
CD4⫹CD25⫹ T cells. Previous murine studies have compared the
CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell repertoires with respect to
V␤ gene usage and demonstrated that the regulatory T cell repertoire is indeed diverse and remarkably similar to the CD4⫹CD25⫺
T cell subset (22–24). However, murine studies have also suggested that CD4⫹CD25⫹ T cells are a high avidity population (20,
35). In support of this idea, experimental manipulation to reduce
the threshold of activation demonstrated that thymocytes can be
diverted into the CD4⫹CD25⫹ T cell lineage (21). Thus, it was
conceivable that certain high avidity clones could be preferentially
selected in the thymus, and the clonal distribution could vary between the CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell subsets. Likewise, exposure to Ag in the periphery could alter the clonal
makeup of particular V␤ families. Our spectratyping has shown
that despite the high avidity nature of CD4⫹CD25⫹ T cells, the
distribution of clones within the various V␤ subfamilies largely
overlaps with that of the CD4⫹CD25⫺ counterparts. Also worth
considering is the possibility that although the repertoires are very
similar with respect to V␤ gene usage and complexity, important
differences may still exist at the level of Ag specificity.
In a simplified view, having equally complex repertoires between subsets would imply that the same Ags capable of eliciting
a robust immune response from the CD4⫹CD25⫺ T cells could
activate the regulatory T cell component. In response to autoimmune reactions it would be highly desirable to activate
CD4⫹CD25⫹ regulatory cells as efficiently as effector cell responses. In contrast, during an infection it would be advantageous
to preferentially activate the effector cells before stimulating a population capable of initiating a dampening effect. One characteristic
that may contribute to whether the CD4⫹CD25⫹ T cells are optimally stimulated is the density of TCR expression (21). The realtime RT-PCR of CD3 levels demonstrates a dramatically lower
level of expression in the CD4⫹CD25⫹ T cell population in both
thymocytes and PBL. Altering the threshold of activation by modulating the density of TCR provides a mechanism to differentially
activate the CD4⫹CD25⫹ and CD4⫹CD25⫺ T cell subsets, despite the overall similarity in repertoires.
In summary, our TREC data with human thymocytes and PBL
are consistent with murine studies demonstrating that
CD4⫹CD25⫹ T cells are generated in the thymus before migrating
to the periphery (14, 15, 33). Interestingly, the decreased levels of
TREC in the CD4⫹CD25⫹ T cells isolated from peripheral blood
support the idea that peripheral mechanisms of homeostasis may
be important in maintaining the levels of regulatory T cells as
thymic output decreases (26, 27, 34). Nonetheless, it is clear that
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FIGURE 4. CD3⑀ expression. A, Amplification curves for CD3⑀ expression in CD4⫹CD25⫹ and CD4⫹CD25⫺ T cells purified from thymus and
PBL (f, CD4⫹CD25⫹ T cells; E, CD4⫹CD25⫺ T cells). The change in
reaction and ⌬Ct defined are described in Fig. 1B. B, Flow cytometry of
CD3 expression on unpurified CD8⫺CD4⫹ thymocytes and CD4⫹ lymphocytes with the indicated level of CD25 (CD25⫺, CD25int, and
CD25bright). The insets show the actual CD25 gates for cells depicted in the
CD3 histograms.
6127
6128
the repertoire of human CD4⫹CD25⫹ T cells is as broad and complex as the CD4⫹CD25⫺ T cell population. Finally, the precise
mechanisms that determine whether an immune response is preferentially ablated by regulatory T cells remain unclear, but differential TCR expression may be one of the many parameters contributing to the overall outcome.
Acknowledgments
We thank Drs. Ray Barfield, Thasia Leimig, and Mario Otto for their assistance in procuring and processing the thymic specimens, and
Jim Houston and Marti Holladay for expert help with flow cytometry. We
also thank Dr. Wing Leung for critical reading of the manuscript and help
with statistical analysis.
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CD4⫹CD25⫹ REGULATORY T CELL REPERTOIRE