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Cutting Edge |
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
| Abstract |
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production by CD8+
T cells induced either by polyclonal or Ag-specific stimuli.
CD4+CD25+ T cells inhibit the activation of
CD8+ responders by inhibiting both IL-2 production and
up-regulation of IL-2R
-chain (CD25) expression. Suppression is
mediated via a T-T interaction as activated
CD4+CD25+ T cells suppress the responses of
TCR-transgenic CD8+ T cells stimulated with soluble
peptide-MHC class I tetramers in the complete absence of APC. These
results broaden the immunoregulatory role played by
CD4+CD25+ T cells in the prevention of
autoimmune diseases, but also raise the possibility that they may
hinder the induction of effector CD8+ T cells to tumor or
foreign Ags. | Introduction |
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-chain) Ag that is present on 510% of
CD4+ T cells in normal animals
(4, 5, 6, 7, 8). The functional properties of murine
CD4+CD25+ T cells have been
extensively studied in vitro. They demonstrate profound anergy to
stimulation via their TCR, and this anergic state cannot be reversed by
costimulation with anti-CD28 (9, 10). More
importantly, when CD4+CD25+
T cells are cocultured with
CD4+CD25- T cells, they
induced profound suppression of T cell activation by down-regulating
IL-2 production in the responding
CD4+CD25- T cells
(10). The suppressive activity of the
CD4+CD25+ T cells requires
that they be activated via their TCR and is cell contact dependent but
cytokine independent (10, 11).
The mechanism by which
CD4+CD25+ T cells mediate
their suppressive effects is poorly understood. The physiologic ligand
recognized by their TCR is unknown, and considerable controversy exists
as to their cellular target. Thornton and Shevach (12)
have demonstrated that the suppressors do not modulate APC function,
whereas other laboratories have raised the possibility that they act by
suppressing APC function (13) or by competing for
APC-derived costimulatory signals (9). The potential
suppressive activity of
CD4+CD25+ T cells on
non-CD4+ responder cells has not been studied in
detail. Here, we demonstrate that
CD4+CD25+ T cells suppress
CD8+ T cell proliferation and IFN-
production
induced by polyclonal or Ag-specific stimuli. In addition, the effects
of the CD4+CD25+ T cells on
CD8+ cells are more complex than their effects on
CD4+CD25- responders,
because they suppress both IL-2 production and CD25 expression.
Finally, we made use of peptide-MHC tetramers to stimulate
CD8+ responders in a two-cell suppressor assay
system to formally demonstrate that
CD4+CD25+ cells mediate
their suppressor function via a T-T cell interaction and in the absence
of APC.
| Materials and Methods |
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Female C57BL/6 and BALB/c mice were obtained from the National Cancer Institute (Frederick, MD). OT-I (specific for OVA257264 peptide), F5 (specific for nucleoprotein (NP)366374 influenza peptide), and P14 (specific for lymphocytic choriomeningitis virus (LCMV) gp3341 peptide) CD8+ TCR-transgenic (Tg) mice were obtained from Taconic Farms (Germantown, NY). All mice used were 612 wk of age.
The mAbs
The following Abs were used for flow cytometry experiments:
biotin-anti-CD25 (7D4 clone), FITC-streptavidin, PE-anti-CD4,
PE-anti-CD8, FITC-anti-CD25, FITC-anti-CD69, and purified
anti-CD3
(2C11), all of which were purchased from BD PharMingen
(San Diego, CA).
Peptides
OT-I OVA257264, F5 NP366374, and LCMV gp3341 peptides were provided by R. Germain and J. Yewdell (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). Peptides were synthesized and purified by the Laboratory of Molecular Structure Peptide Synthesis Laboratory (National Institute of Allergy and Infectious Diseases, National Institutes of Health).
Tetramers
MHC class I H-2Kb-OVA257264 tetramer solutions were prepared at the National Institute of Allergy and Infectious Diseases tetramer facility.
Cell purification and culture
CD4+CD25+ T cells
were isolated on a FACStar cell sorter (BD Biosciences, San Jose,
CA) as described previously (10). The purity of the
final CD4+CD25+ preparation
was typically >95%. T-depleted spleen cells (10; TdS) were irradiated
at 3000 rad and pulsed for 30 min at 37°C with an appropriate
peptide. Activated
CD4+CD25+ cells were
prepared as previously described (12). Briefly,
cell-sorted CD4+CD25+ cells
were cultured with irradiated APC (1:1 ratio), anti-CD3 (0.5
µg/ml), and human IL-2 (5 ng/ml, 100 U/ml) for 72 h and
were then split and maintained in IL-2 medium for
714 days.
CD8+ T cells were purified either by negative
(depletion of B220-, CD4-, and I-Ab-positive
cells) or positive selection (using CD8
magnetic beads) on the
AutoMACS magnetic separation system (Miltenyi Biotec, Auburn,
CA). For experiments involving tetramer stimulation, OT-I
CD8+ T cells were FACS purified using Abs against
Thy1.2 and CD8
molecules, with final purities of >99%.
Proliferation assays
Proliferation assays were performed by culturing CD8+ T cells (5 x 104) in flat-bottom microtiter plates (0.2 ml) with peptide-pulsed APC (12 x 105) and resting or activated CD4+CD25+ or CD4+CD25- T cells for 72 h at 37°C in complete medium (10). Human rIL-2 was purchased from Peprotech (Rocky Hill, NJ). Cell cultures were pulsed with [3H]TdR for the last 8 h. All data represent the average cpm of triplicate determinations. All proliferation experiments were repeated at least three times.
For cytokine production, supernatants were taken at 72 h, and the
production of IFN-
was measured using an ELISA kit (R&D Systems,
Minneapolis, MN).
Flow cytometry
Cells were collected and stained with PE-CD8
and FITC-CD69 or
FITC-CD25 and analyzed with a FACScan flow cytometer (BD
Biosciences).
| Results and Discussion |
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in the absence of previous priming, we next studied
the effects of CD4+CD25+ T
cells on the capacity of CD8+ T cells to produce
IFN-
. Freshly explanted
CD4+CD25+ T cells readily
suppressed IFN-
production by CD8+ T cells
stimulated with anti-CD3 (Fig. 1
production when the CD8+ T
cells were stimulated with specific peptide (Fig. 1
secretion at a CD25+:CD8 ratio of 1:2,
and >75% suppression at a 1:1 cell ratio.
CD4+CD25+ T cells (Fig. 1
(data not shown).
CD4+CD25- T cells (Fig. 1
(data not shown).
IL-2 will reverse suppression when
CD4+CD25+ suppressors are
cocultured with CD4+CD25-
responders. It has been proposed that IL-2 can directly act on the
suppressors and reverse their anergic phenotype and consequently
disable their suppressive capability (14). Alternatively,
the addition of exogenous IL-2 may simply be circumventing the block in
IL-2 production induced in the responders by the
CD4+CD25+ suppressors. When
fresh CD4+CD25+ T cells
(Fig. 2
A) or activated
CD4+CD25+ T cells (Fig. 2
B) were cocultured with OT-I CD8+ T
cells, significant suppression of proliferation (
60%) and IFN-
production (Fig. 2
C) was observed with soluble anti-CD3
or peptide stimulation. Suppression was not reversed by the addition of
exogenous IL-2 at all suppressor:responder ratios or by enhancement of
endogenous IL-2 production by the addition of anti-CD28 (data not
shown). Increasing the amount of IL-2 added to the cocultures to 100
U/ml also had no effect. It should be noted that the proliferative
response of CD4+CD25+ T
cells to IL-2 is modest when compared with activated
CD4+CD25- T cells. Thus,
the contribution of the activated
CD4+CD25+ T cells to the
proliferative responses in the cocultures performed in the presence of
IL-2 is also minimal but may contribute some residual proliferation to
the culture. The hypothesis (9) that IL-2 abrogates
suppressor function is not supported by our studies on
CD8+ responders because suppression is clearly
maintained in the presence of IL-2.
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production
(Fig. 4
production (Fig. 4
production was seen in
the presence of CD4+CD25+ T
cells, but not CD4+CD25-
cells, even at low
CD4+CD25+:CD8+
ratios (Fig. 4
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was
also suppressed. Second, whereas
CD4+CD25+ T cells inhibit
the activation of CD4+ responders by primarily
blocking IL-2 production,
CD4+CD25+ T cells regulate
CD8+ T cell responses both by blocking IL-2
production as well as by lowering responsiveness to exogenous IL-2 and
thereby potentially disrupting CD4 help for CD8+
T cells. Finally, CD4+CD25+
T cells can inhibit T cell activation by directly acting on responder
CD8+ T cells in the absence of APC. However, this
result does not exclude the possibility that
CD4+CD25+ T cells might
also exert inhibitory/deactivating effects on APC or use the APC
surface as a platform on which the suppressor cells physically interact
with CD4+ or CD8+ effectors
in vivo.
Although immunoregulatory
CD4+CD25+ cells function
beneficially in vivo to protect the host against the development of
autoimmunity, they may simultaneously prevent the host from mounting an
immune response to autoantigens such as tumor Ags. Because IL-2
responsiveness by CD8+ T cells is a critical
factor for cytokine production (IFN-
) and cytolytic activity
(22, 23), our demonstration that
CD4+CD25+ T cells
down-regulate both IL-2 production and CD25 expression on
CD8+ T cells may represent a significant
impediment to the use of tumor or viral vaccines. Indeed, deletion of
CD4+CD25+ T cells before
the use of such vaccines may be needed for optimal immunotherapy.
| Footnotes |
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2 Abbreviations used in this paper: NP, nucleoprotein; LCMV, lymphocytic choriomeningitis virus; Tg, transgenic; TdS, T-depleted spleen cells. ![]()
Received for publication April 30, 2001. Accepted for publication June 4, 2001.
| References |
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-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
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