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Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to data from experimental studies, there is also evidence that T cells with immune regulatory activities exist in patients with long-term surviving allografts (15, 16). However, reliable strategies for identifying such cells using functional assays in vitro have been difficult to develop (17). For the most part, when T cells from tolerant recipients are cultured in vitro, they exhibit a Th1 phenotype and proliferate normally in response to donor alloantigens, responses that are clearly not an accurate reflection of their functional activity in vivo (18, 19). The majority of these studies analyzed the response of unseparated T cells; thus, the effects of regulatory T cells may have been masked by the presence of naive T cells responding to alloantigens. Further characterization of putative regulatory T cells may be facilitated by using markers that distinguish naive from Ag-experienced CD4+ T cells.
Abs reactive with different isoforms of the CD45 molecule have revealed phenotypic and functional heterogeneity in CD4+ T cells (20, 21, 22). In mice, the majority of naive CD4+ T cells express high levels of the CD45RB molecule (CD45RBhigh CD4+), whereas CD4+ T cells that have previously encountered Ag express lower levels of CD45RB (CD45RBlow CD4+) (23). Polyclonal activation of the CD45RBhigh and CD45RBlow subsets in vitro showed that the CD45RBhigh CD4+ T cells synthesized predominantly IL-2, whereas CD45RBlow CD4+ T cells synthesized IL-4 (24).
Functional analysis of CD45RBhigh and
CD45RBlow CD4+ T cells
responding to self and alloantigens has demonstrated that important
regulatory interactions occur between these subsets in vivo (25, 26). Transfer of CD45RBhigh
CD4+ T cells to scid mice led to the development
of a Th1-mediated colitis and wasting disease that could be inhibited
by cotransfer of the reciprocal CD45RBlow
CD4+ population (27, 28). Immune
suppression in this system by the CD45RBlow
subset was shown to be dependent on IL-10 and TGF-
, but independent
of IL-4 (29, 30). Further subdivision of the
CD45RBlow subset revealed that the regulatory T
cells were enriched within the CD25+ population
(31). CD25 has also been shown to be a marker of
regulatory T cells in other models of autoimmune disease (32, 33) and neonatal tolerance (34).
Previous studies from this laboratory have shown that donor-specific blood transfusion combined with depleting or nondepleting anti-CD4 Ab pretreatment can induce operational tolerance to B10 cardiac allografts in H-2k recipients (35). CD4+ T cells were shown to be responsible for both the induction (10) and maintenance of tolerance (M.N. and K.J.W., unpublished data). In this study, we have used Abs reactive with CD45RB and CD25 to subdivide the CD4+ T cell subset from tolerant mice to further investigate the phenotype and function of these cells. Our results indicate that CD4+ T cells capable of inhibiting allogeneic responses in vitro and in vivo were contained within the CD45RBlow and CD25+ populations. Importantly, neutralization of IL-10, but not IL-4, abrogated the adoptive transfer of tolerance to alloantigens by CD45RBlow CD4+ T cells, indicating that IL-10 plays a key role in this mechanism of immune suppression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBA.Ca (CBA, H-2k), C57BL/10 (B10, H-2b), BALB/c (H-2d), (CBA x B10)F1, and (CBA x BALB/c)F1 were obtained from Harlan (Bicester, U.K.) and bred in the specific pathogen-free facility at Biomedical Services Unit, John Radcliffe Hospital (Oxford, U.K.). Sex-matched mice between 8 and 12 wk of age at the time of first experimental procedure were used in all experiments.
Reagents and mAbs
The following mAbs were used for flow cytometry and cell
isolation: CD4 (anti-CD4)-Tricolor (Caltag, South San Francisco,
CA); TIB120 (anti-class II; American Type Culture Collection
(ATCC), Manassas, VA). YTA3.1.2 (anti-CD4), YTS169.4.2
(anti-CD8) (36) hybridomas were kindly provided by H.
Waldmann (Oxford, U.K.). RM4-5 (anti-CD4) PerCP or PE, 53-6.7
(anti-CD8a) APC, 16A (anti-CD45RB) FITC or PE, IM7
(anti-CD44) PE, MEL-14 (anti-CD62L) PE, H1.2F3 (anti-CD69)
PE, 7D4 (anti-CD25) biotin, and mouse rIL-2 were purchased from
PharMingen (San Diego, CA). The following mAbs were used for in vivo
assays: 11B11 (rat IgG1), a neutralizing anti-mouse IL-4 mAb (ATCC
HB188); JES5-2A5 (rat IgG1), a neutralizing anti-mouse IL-10 mAb;
and GL113 (rat IgG1), an isotype control mAb reactive with
-galactosidase (37).
Tolerance induction protocol
The protocol for the induction of operational tolerance was as described previously (10). Briefly, adult CBA mice were treated with 50 µg of the depleting anti-CD4 mAb, YTA3.1.2, 28 and 27 days before transplantation of a vascularized B10 heart. Two hundred fifty microliters of donor blood or 1 x 107 donor splenocytes (B10) were also injected on the second day of mAb treatment. The spleen and lymph nodes were harvested from mice with functioning heart grafts, confirmed by ECG and histology, over 100 days after heart transplantation, and used for cell sorting.
Skin transplantation
Full thickness tail skin grafts were transplanted to beds prepared on the flanks of recipient mice. Graft survival was defined by complete destruction of the skin graft, as assessed by visual inspection and confirmed by a third independent blinded observer. Allograft survival between two groups was compared using the log-rank test.
T cell-depleted mice
CBA mice were thymectomized and rested for 2 wk before being treated with depleting anti-CD4 (YTA3.1.2; 200 µg) and anti-CD8 (YTS169.4.2; 200 µg) mAbs. To allow time for the depletion of the majority of T cells and for clearance of the mAbs, the mice were rested for an additional 10 days. The mice were then either reconstituted i.v. with fractionated T cells or not treated. The day after reconstitution, mice received either a B10 or BALB/c skin graft.
Flow cytometry analysis
Spleen cells were resuspended in PBS supplemented with 0.1% BSA and 0.02% sodium azide (Sigma, St. Louis, MO) and incubated with CD4 PerCP, CD8 APC, and an Ab specific for an activation marker directly conjugated to PE for 30 min at 4°C. After washing the cells twice, they were resuspended in PBS containing 2% v/v formaldehyde and stored in the dark at 4°C until acquisition. The data were acquired by FACS and analyzed using the CellQuest software package (Becton Dickinson, Oxford, U.K.).
Cell purification
Lymphocyte cell suspensions were prepared from pooled spleen and lymph nodes (cervical, axillary, inguinal, mesenteric, and paraaortic) harvested from tolerant or naive mice in PBS containing 0.1% BSA (PBS/BSA). Erythrocytes were removed by hypotonic lysis. Cells were then incubated with PBS/BSA containing mAbs (anti-class II, anti-CD8) for 30 min at 4°C. After washing, the mAb-labeled cell suspension was incubated on a rotating wheel for 30 min with sheep anti-rat-coated Dynabeads at a ratio of one bead/cell (Dynabeads; Dynal, Wirral, U.K.). Negative cells were isolated by magnetic separation. The enriched population of CD4+ T cells (80% pure) was then labeled with mAbs specific for CD45RB or CD25 and CD4 for 30 min and fractionated into CD45RBhigh CD4+ and CD45RBlow CD4+ or CD25+CD4+ and CD25-CD4+ fractions by cell sorting using a FACSVantage (Becton Dickinson). The CD45RBhigh and CD45RBlow populations were defined as the brightest staining 40% and the dullest staining 15% of CD4+ T cells, respectively. All populations were >98% pure on reanalysis.
Proliferation assay
Various doses of fractionated CD45RBhigh
CD4+ or CD45RBlow
CD4+ T cells were cultured with irradiated T
cell-depleted spleen cells (5 x 105/well,
2000 rad) in 200 µl of RPMI 1640 containing 10% FCS (Myoclone; Life
Technologies, Paisley, U.K.), 2 mM L-glutamine, 0.05 mM
2-ME, and 100 U/ml each of penicillin and streptomycin. Supernatants
were harvested after 72 h for analysis of IL-2 production and
96 h for analysis of IFN-
, IL-4, and IL-10 production. The
cultures were pulsed with 0.5 µCi
[3H]thymidine (Amersham International,
Amersham, U.K.) at 96 h and harvested onto glass fiber filters
18 h later. Proliferation was measured as
[3H]thymidine incorporation using liquid
scintillation counting.
Detection of cytokines
Cytokine levels in supernatants were detected by two-site
sandwich ELISA essentially as described for IL-2, IFN-, IL-4, and
IL-10 (37). The lower limits of detection were IL-2, 125
pg/ml; IFN-, 188 pg/ml; IL-4, 7.8 pg/ml; and IL-10, 31.2 pg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBA mice treated with depleting anti-CD4 mAb in combination with donor alloantigen (B10) 28 days before transplantation accept donor (B10), but not third-party (BALB) heart grafts indefinitely (10). These mice are operationally tolerant to donor alloantigens, as assessed by their ability to accept second heart or skin grafts from the same, but not third-party donors 100 days after transplantation of the first graft. Moreover, the adoptive transfer of leukocytes from the tolerant mice to naive syngeneic recipients has been shown to result in the prolonged survival of heart grafts from the original donor, but not third-party strains (38). Despite the clear evidence for the existence of the tolerant state in vivo, unfractionated peripheral lymphocytes from tolerant mice proliferate and produce Th1 cytokines normally when they are cultured with donor APC in vitro (data not shown).
To address the conflicting behavior of leukocytes from tolerant mice in
vivo and in vitro, CD4+ T cells were isolated
from CBA mice operationally tolerant of a B10 cardiac allograft (>100
days after transplantation) and further subdivided into two fractions
based on the level of expression of the CD45RB Ag. FACS-sorted
CD45RBhigh and CD45RBlow
CD4+ T cells were then stimulated in vitro with T
cell-depleted allogeneic spleen APCs. B10 and (CBA x
B10)F1 APCs were used to evaluate the
contributions of the direct pathway and the direct plus indirect
pathway of alloantigen presentation, respectively. T cell proliferation
was evaluated by [3H]thymidine incorporation;
supernatants were harvested from the cultures after 72 h for
determination of IL-2 content, or 96 h for analysis of IFN-,
IL-4, and IL-10 production. The results from a representative
experiment are shown in Fig. 1
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in
response to B10, (CBA x B10)F1, and
(CBA x BALB/c)F1 APCs (Fig. 1
, A
and C; cytokine production in response to B10 and (CBA
x BALB/c)F1 stimulators not shown). IL-4 and
IL-10 production were below the limit of detection of the ELISA. Like
the naive population, CD45RBhigh
CD4+ T cells isolated from CBA mice tolerant of
B10 cardiac allografts also mounted a proliferative response and
produced IL-2 and IFN-, but not IL-4 or IL-10, in response to each
of the APC populations (Fig. 1, B and D; cytokine
production in response to B10 and (CBA x
BALB/c)F1 stimulators not shown). Cytokine
production was Ag specific, as cytokine levels were below the limit of
detection in cultures that did not contain APC (data not shown). In
contrast, CD45RBlow CD4+ T
cells isolated from either naive or tolerant mice failed to proliferate
or produce Th1 (IL-2, IFN-) or Th2 (IL-4, IL-10) cytokines in
response to B10, (CBA x B10)F1, or
(CBA x BALB/c)F1 (Fig. 1 and data not
shown). The lack of responsiveness of the
CD45RBlow CD4+ population
to stimulation with alloantigens was not due to differences in the
kinetics of the response, as similar results were obtained at 24, 48,
and 72 h after stimulation (data not shown). The finding that the
CD45RBlow CD4+ population
from naive mice failed to mount a significant response against
alloantigens in vitro is in accord with previous studies
(39). Adoptive transfer of fractionated CD4+ cells to T cell-depleted CBA mice
Phenotypic and functional characterization of T cell-depleted mice. To identify and characterize putative regulatory T cells present in long-term tolerant mice, an adoptive transfer system was established using T cell-depleted mice. These T cell-depleted hosts were restored with T cell subsets from naive or tolerant mice and transplanted with B10 or third-party allografts.
Initially, the phenotype of T cell-depleted mice was characterized
(Fig. 2). After thymectomy and depletion
of peripheral CD4+ and CD8+
T cells by administration of anti-CD4 and anti-CD8 mAbs, the
number of T cells present in the spleen was significantly reduced (Fig. 2B: CD4+, 37.5 ± 9.9% vs
1.6 ± 1.4%; CD8+, 11.7% vs 0.5 ±
0.4% in naive vs T cell-depleted mice, respectively; n
= 4). The small number of CD4+ T cells remaining
exhibited an activated phenotype (Fig. 2C);
CD45RBint
low, 34.5
± 1.4% vs 72.3 ± 7.0%; CD44+, 16.5
± 4.5% vs 89.3 ± 3.5%; CD62L-,
17.7 ± 4.5% vs 67.1 ± 4.9% in naive and T cell-depleted
mice, respectively. Most of the remaining CD8+
cells were also CD44+ (17.7 ± 2.2% naive
vs 49.3 ± 14.3% T cell depleted; data not shown). Despite the
presence of these residual T cells, T cell-depleted mice were unable to
reject allogeneic skin grafts (Table I).
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To determine whether CD45RBhigh
CD4+ T cells from naive CBA mice could induce
rejection of B10 skin grafts, T cell-depleted CBA mice were
reconstituted with varying numbers of purified
CD45RBhigh CD4+ T cells,
and their effect on graft survival was monitored (Table I). Mice
reconstituted with less than 1 x 105
CD45RBhigh CD4+ T cells
were unable to reject B10 skin grafts consistently. When 1 x
105 of CD45RBhigh
CD4+ T cells were used for reconstitution, 88%
of mice rejected allogeneic skin grafts. Reconstitution with 1 x
105 CD45RBhigh
CD4+ T cells did not induce wasting disease, and
all mice remained healthy throughout the study (data not shown). Mice
reconstituted with 1 x 105
CD45RBhigh CD4+ naive T
cells will be referred to as minimally reconstituted
(MR)3 mice.
CD45RBlow CD4+ and CD25+ CD4 T cells from tolerant mice contain cells with regulatory activity in vivo
The adoptive transfer of spleen cells or purified CD4+ T cells from CBA mice operationally tolerant to B10 alloantigens to naive secondary CBA recipients results in the acceptance of B10 heart grafts, indicating that tolerance in this system is an active process mediated by a subset of CD4+ T cells (38) (M.N. and K.J.W., unpublished data). To address which subpopulation of CD4+ T cells was responsible for immunoregulation in vivo, CD4+ T cells were fractionated based upon expression of CD45RB or CD25.
FACS-sorted CD45RBhigh or
CD45RBlow CD4+ T cells from
tolerant mice were transferred into MR mice (i.e., T cell-depleted mice
reconstituted with 1 x 105 naive
CD45RBhigh CD4+ T cells—MR
mice). The following day, mice were transplanted with B10 skin grafts,
and graft survival was monitored. When 5 x
105 CD45RBlow
CD4+ T cells from tolerant mice were
cotransferred, B10 skin graft rejection was abrogated and indefinite
survival of the skin grafts was observed in five of six MR recipients
(Fig. 3A; p <
0.001 vs MR mice; p < 0.05 vs MR mice treated with
5 x 105 tolerant
CD45RBhigh CD4+ T cells).
In contrast, cotransfer of 5 x 105
CD45RBhigh CD4+ T cells
from tolerant mice resulted in the rejection of B10 skin grafts that
was not statistically different from that observed in T cell-depleted
mice reconstituted with 1 x 105 naive
CD45RBhigh CD4+ cells alone
(Fig. 3A; p = NS MR mice treated with 5
x 105 tolerant CD45RBhigh
CD4+ T cells vs MR mice).
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; p < 0.01 vs
B10 graft). Immunoregulation by CD45RBlow
CD4+ T cells from tolerant mice was less
effective at lower cell doses (data not shown). Moreover, cotransfer of
5 x 105 CD45RBlow
CD4+ cells from naive mice into MR mice did not
prevent the rejection of B10 skin grafts (Fig. 3). These latter
findings contrast with those reported by Davies and colleagues, in
which CD45RBlow CD4+ cells
from naive mice were found to be capable of preventing
CD45RBhigh CD4+ cells from
causing rejection of neonatal islet grafts, as assessed by histology
(40). This may reflect differences between the mechanisms
of rejection of islet and skin allografts or the level of alloantigen
expression between neonatal and adult tissue. Our results indicate that
the CD45RBlow CD4+
population in tolerant mice contains a population of regulatory T cells
capable of adoptively transferring tolerance, and that this functional
activity is dependent on previous exposure to donor alloantigens on the
primary graft.
As CD25+CD4+ T cells have
been shown to contain the regulatory population in a number of systems,
we investigated whether
CD25+CD4+ T cells from
tolerant mice could prevent rejection in MR mice (31, 32, 33, 34).
When 5 x 105
CD25+CD4+ T cells from
tolerant mice were cotransferred, B10 skin graft rejection was
abrogated and indefinite survival of the skin grafts was observed in
four of six MR recipients (Fig. 3B; p <
0.001 vs MR mice). The activity of the
CD25+CD4+ T cells from
tolerant mice was donor alloantigen specific; BALB/c skin grafts were
rejected (data not shown). Cotransfer of 5 x
105
CD25-CD4+ T cells from
tolerant mice did not prevent rejection of B10 skin grafts (Fig. 3B). Taken together, these findings indicate that
CD45RBlow and CD25 can be used to enrich a
subpopulation of CD4+ T cells in tolerant mice
that are capable of transferring specific unresponsiveness.
Immunoregulation by CD45RBlow CD4+ T cells from tolerant mice can be demonstrated in vitro and is mediated via the indirect pathway of allorecognition
To evaluate the regulatory activity of
CD45RBlow CD4+ cells in
vitro, 2 x 105
CD45RBhigh CD4+ cells from
naive mice were cultured with T cell-depleted APC from the spleen of
either (CBA x B10)F1 (direct and indirect
pathway) or B10 (direct pathway) mice in the presence or absence of
1 x 105 CD45RBlow
CD4+ cells (2:1
CD45RBhigh:CD45RBlow is the
physiological ratio). Data from a representative experiment are shown
in Fig. 4.
CD45RBlow CD4+ T cells from
tolerant mice inhibited the proliferation of naive
CD45RBhigh CD4+ cells
significantly in response to stimulation by (CBA x
B10)F1 APCs (Fig. 4A). Moreover, IL-2
and IFN- production were also significantly reduced (11.3% and
44.0%, respectively) (Fig. 4, B and C).
Increasing the number of CD45RBlow
CD4+ cells from tolerant mice such that the ratio
of CD45RBlow CD4+ cells
from tolerant mice to naive CD45RBhigh
CD4+ cells was 1:1 resulted in more marked
suppression of proliferation as well as IL-2 and IFN- production
(12%, 0.1%, 4%, respectively; data not shown). In contrast, when B10
APCs were used as stimulators, CD45RBlow
CD4+ cells from tolerant mice were unable to
inhibit proliferation or cytokine production by naive
CD45RBhigh CD4+ T cells.
These data suggest that indirect presentation of peptides processed
from H-2b molecules and B10 minor Ags and
presented by H-2k class II molecules expressed by
(CBA x B10)F1 APC is important for
immunoregulation by CD45RBlow
CD4+ T cells from the tolerant mice.
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production by naive CD45RBhigh
CD4+ T cells when stimulated by either (CBA
x B10)F1 or B10 APCs (Fig. 4, A and
C; open bars). Indeed, proliferation was enhanced in some
experiments (Fig. 4A), but this was not a consistent
finding. In contrast, IL-2 production was inhibited when
CD45RBlow CD4+ cells from
either naive or tolerant mice were added to the cultures (Fig. 4B; closed bars), indicating that inhibition of IL-2
production or consumption of IL-2 by CD45RBlow
CD4+ T cells was not dependent on previous
exposure to alloantigen. Immune suppression transferred by tolerant CD45RBlow CD4+ T cells is dependent on IL-10, but independent of IL-4 in vivo
Strategies that induce tolerance to alloantigens have in
some circumstances been shown to be dependent on IL-4 and to involve
induction of Th2 cells. To analyze the role of both IL-4 and IL-10 in
the mechanism of immune suppression transferred by tolerant
CD45RBlow CD4+ T cells,
these cells were transferred to MR mice and the recipients were treated
weekly with either isotype control mAb or a combination of
anti-IL-4 and anti-IL-10 mAbs. Neutralization of IL-4 and IL-10
completely abrogated the transfer of tolerance toward B10 alloantigens;
B10 skin grafts were rejected by six of seven mice in this group
(p < 0.05; vs GL113), with a tempo similar to
MR mice. In contrast, grafts remained healthy for up to 100 days in the
majority of MR mice restored with tolerant
CD45RBlow CD4+ T cells and
treated with isotype control mAb (Fig. 5A).
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B). In this latter case, B10 skin grafts were
rejected by five of six mice (p < 0.01; vs
GL113-treated group). This result is not attributable to the inability
of the mAb to effectively neutralize IL-4, as this same batch of Ab was
shown to impair the induction of tolerance (38) and this
mAb has been used to inhibit Th2 responses in vivo (41).
These results indicate that IL-10, but not IL-4, is required for the
function of CD4+ T cells capable of transferring
tolerance to alloantigens. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In contrast, cells contained within the
CD45RBlow population failed to mount a
proliferative response or secrete cytokines in response to alloantigen
in vitro and failed to induce allograft rejection in vivo (Fig. 1).
However, this population was far from inactive, as addition of these
cells to cultures of naive CD45RBhigh
CD4+ T cells and allogeneic APCs led to
inhibition of the mixed leukocyte reaction (Fig. 4). Significantly,
cotransfer of CD45RBlow
CD4+ T cells from tolerant mice to T
cell-deficient mice prevented skin graft rejection initiated by naive
CD45RBhigh CD4+ T cells
(Fig. 3A). Further fractionation of
CD4+ T cells revealed that the regulatory cells
were contained within the CD25+ subset (Fig. 3B). The regulatory mechanism involved IL-10, but not IL-4,
as administration of anti-IL-10 but not anti-IL-4 mAbs
abrogated immune suppression transferred by alloreactive
CD45RBlow CD4+ T cells in
vivo (Fig. 5). These data demonstrate that in mice rendered long-term
tolerant to alloantigens, regulatory T cell populations capable of
inhibiting graft rejection in vivo are contained within the
CD25+ and CD45RBlow
CD4+ subset. It seems likely that this T
cell-dependent mechanism of active suppression, as opposed to clonal
deletion, plays a dominant role in the maintenance of tolerance to
alloantigen in this model.
In vitro analysis showed that alloreactive T cells, capable of
responding to donor alloantigens presented via the direct or indirect
pathways, are present at similar levels within the
CD45RBhigh CD4+ population
in naive and tolerant mice with long-term surviving B10 cardiac
allografts (Fig. 1). Interestingly, despite the continuous presence of
donor alloantigens, i.e., for 100 days since transplantation, these
cells were contained within the CD45RBhigh
CD4+ T cell subset. This is in contrast to
findings in the rat in which after blood transfusion alone the
CD45RC+ population was depleted of cells reactive
with blood donor alloantigens (42). The prolonged
presence, 100 vs 14 days, as well as the source of donor alloantigen, a
cardiac allograft vs blood transfusion, may account for this
difference, but it may also reflect functional differences between T
cell subsets identified by expression of different isoforms of
CD45.
Despite clear evidence that immune suppressive
CD4+ T cells play a role in tolerance to
alloantigens (6, 8, 13, 43), little is known about their
mechanism of action and allorecognition properties. The findings
presented in this work identify IL-10 as an essential component of the
mechanism of immune suppression in mice with long-term surviving
allografts. The alloreactive regulatory CD4+ T
cells identified in this study resemble regulatory T cells present in
normal mice that control inflammatory responses to intestinal Ags.
These latter cells are also contained within the
CD25+ CD45RBlow
CD4+ T cell population and are dependent on IL-10
(29) as well as TGF- for their function
(30). Interestingly, neutralization of TGF- has
recently been shown to abrogate tolerance to alloantigens in the rat
(44) and in the mouse (45).
Alloantigen-specific regulatory T cells dependent on IL-10 have also
been found in patients (16). Similarly, regulatory T cells
that can control pathogenic responses toward self Ags have been
identified in the T cell repertoire of normal individuals and are
thought to be responsible for preventing autoimmune disease (32, 33, 46, 47). These populations may be related to regulatory T
cell clones that have been generated by chronic activation of both
mouse and human CD4+ T cells in the presence of
IL-10 in vitro (48). The fact that regulatory T cells with
similar properties control pathologic responses against Ags as diverse
as alloantigens, autoantigens, and intestinal Ags highlights the
importance of this mechanism in the regulation of the immune
response.
The finding that IL-10 is required for the function of this population of regulatory T cells that inhibit graft rejection in vivo is in accord with the known anti-inflammatory properties of this cytokine. IL-10 has been shown to inhibit Ag-induced proliferation and cytokine synthesis by T cells most probably through its effects on APCs, particularly down-regulation of molecules involved in T cell costimulation (49). However, IL-10 is pleiotropic, and contradictory results concerning its role in the regulation of immune responses to transplantation Ags have been reported. In vitro, IL-10 was shown to inhibit alloantigen-induced proliferation in a dose-dependent manner (50). Somewhat surprisingly, systemic injection or local high concentrations of IL-10 was found to accelerate graft rejection (51). In contrast, expression of viral IL-10 in nonvascularized cardiac allografts by retroviral transduction of the graft before transplantation resulted in graft prolongation (52), and neutralization of IL-10 by anti-IL-10 therapy abrogated skin graft prolongation induced by portal venous injection of allogeneic splenocytes (53). Moreover, high levels of IL-10 have been described as associated with tolerance to HLA-mismatched bone marrow stem cells (54), and in renal transplant patients with EBV-induced posttransplant lymphoproliferative disorder increased levels of IL-10 correlated with operational tolerance to the graft in aciclovir-treated patients (55). These conflicting data may be explained by the observation that there are dose-dependent differences in the effect of IL-10 in vivo (56).
Neutralizing IL-4 failed to affect the transfer of tolerance to alloantigens by CD45RBlow CD4+ T cells in vivo in this study. Consistent with this, CD45RBlow CD4+ T cells from tolerant mice failed to produce detectable levels of IL-4 in response to stimulation with the tolerogen in vitro. There are conflicting reports on the role of Th2 cells and IL-4 in tolerance to alloantigens. Induction of tolerance in a number of model systems has been shown to correlate with increases in Th2 cytokines (12, 57), and IL-4 has been shown to be absolutely required for neonatal tolerance induction (58, 59). However, in adult animals there is no direct evidence that Th2 cells are involved in the induction or maintenance of tolerance (14). Indeed, Th2 cells have been shown to trigger, not prevent graft rejection (60). Although Th2 cells per se may not be responsible for tolerance induction, the cytokines they produce or stimulate may be critical. Thus, although the majority of experiments using IL-4 knockout mice have suggested that IL-4 does not play a role in long-term graft acceptance (61), other studies reveal that IL-4 does play a role when conditions are limiting (11, 38). Taken together, these data suggest that IL-4 may be involved in the induction, but not the maintenance of tolerance, playing a role in the growth or expansion of regulatory T cells, but not their effector function (38). The precise role that particular immune suppressive cytokines play in the mechanism of action of regulatory T cells responsive to either self or alloantigens remains controversial and may in part be dependent on the phase of the tolerance process (induction vs maintenance) examined.
CD45RBlow CD4+ T cells from long-term tolerant mice were able to regulate responses to alloantigen in vitro only when donor alloantigens were presented via the indirect pathway of allorecognition by the APCs present in the culture. This suggests that tolerant CD45RBlow CD4+ T cells function predominantly by recognizing alloantigen via the indirect pathway of allorecognition. Teleologically, this makes a great deal of sense; in tolerant recipients with long-term surviving allografts, the main route of alloantigen presentation under normal circumstances will be via the indirect pathway. After transplantation, passenger leukocytes migrate out of the graft into the draining lymphoid tissue and initiate the rejection response (62). Administration of immunosuppressive agents or the induction of tolerance to donor alloantigens before transplantation will inhibit rejection, but in most cases does not inhibit leukocyte migration. Thus, long-term surviving grafts have few passenger leukocytes, and the transplanted tissue itself may not possess the costimulatory capacity to stimulate naive T cells to respond (63). In contrast, T cells capable of responding to alloantigens via the indirect pathway will be stimulated continuously after transplantation (64). In support of this conclusion, it has been shown that regulatory T cells require constant stimulation by donor alloantigens to maintain their function both following infusion of alloantigen (65, 66) and after transplantation (67). Thus, presentation of donor-derived allopeptides by recipient APC is sufficient to maintain the functional activity of CD45RBlow CD4+ T cells in tolerant recipients and is required for the detection of the functional activity of these cells in vitro.
In summary, we have demonstrated that tolerance to alloantigens in vivo is maintained by a subpopulation of CD4+ T cells that is characterized by expression of CD45RBlow or CD25+, requires IL-10 to function in vivo, and responds to donor alloantigen via the indirect pathway of allorecognition. The characterization of both the phenotype and function of the regulatory T cells responsible for both the induction and maintenance of tolerance to alloantigens is key for the development and implementation of new strategies for inducing tolerance in transplantation and autoimmune disease as well as for identifying markers of tolerance that can be used to assess transplant patients with long-term surviving grafts. In many experimental and clinical situations in which operational tolerance to donor alloantigens can be demonstrated, in vivo antidonor reactivity remains detectable in vitro. It is possible that the presence of regulatory cells in these situations may be masked by naive, CD45RBhigh cells responding to donor Ag. These observations may therefore allow transplant patients with long-term surviving to be assessed more effectively for the level of tolerance to donor Ags they exhibit.
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2 Address correspondence and reprint requests to Prof. Kathryn Wood, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, U.K.
3 Abbreviation used in this paper: MR, minimally reconstituted; MST, median survival time.
Received for publication October 5, 2000. Accepted for publication January 3, 2001.
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but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J. Exp. Med. 183:2669.
chains (CD25): breakdown of a single mechanism of self tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
in donor transfusion induced allograft tolerance. J. Clin. Invest. 102:1920.[Medline]
and interleukin-10 subvert delayed type hypersensitivity in cardiac allograft acceptor mice. Transplantation 69:1517.[Medline]
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T.-C. Chen, S. P. Cobbold, P. J. Fairchild, and H. Waldmann Generation of Anergic and Regulatory T Cells following Prolonged Exposure to a Harmless Antigen J. Immunol., May 15, 2004; 172(10): 5900 - 5907. [Abstract] [Full Text] [PDF]
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P. L. Vieira, J. R. Christensen, S. Minaee, E. J. O'Neill, F. J. Barrat, A. Boonstra, T. Barthlott, B. Stockinger, D. C. Wraith, and A. O'Garra IL-10-Secreting Regulatory T Cells Do Not Express Foxp3 but Have Comparable Regulatory Function to Naturally Occurring CD4+CD25+ Regulatory T Cells J. Immunol., May 15, 2004; 172(10): 5986 - 5993. [Abstract] [Full Text] [PDF]
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L. Aurelian Herpes Simplex Virus Type 2 Vaccines: New Ground for Optimism? Clin. Vaccine Immunol., May 1, 2004; 11(3): 437 - 445. [Abstract] [Full Text] [PDF]
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B. K. Choi, J. S. Bae, E. M. Choi, W. J. Kang, S. Sakaguchi, D. S. Vinay, and B. S. Kwon 4-1BB-dependent inhibition of immunosuppression by activated CD4+CD25+ T cells J. Leukoc. Biol., May 1, 2004; 75(5): 785 - 791. [Abstract] [Full Text] [PDF]
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J. R. Torrealba, M. Katayama, J. H. Fechner Jr., E. Jankowska-Gan, S. Kusaka, Q. Xu, J. M. Schultz, T. D. Oberley, H. Hu, M. M. Hamawy, et al. Metastable Tolerance to Rhesus Monkey Renal Transplants Is Correlated with Allograft TGF-{beta}1+CD4+ T Regulatory Cell Infiltrates J. Immunol., May 1, 2004; 172(9): 5753 - 5764. [Abstract] [Full Text] [PDF]
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E. S. Morris, K. P. A. MacDonald, V. Rowe, D. H. Johnson, T. Banovic, A. D. Clouston, and G. R. Hill Donor treatment with pegylated G-CSF augments the generation of IL-10-producing regulatory T cells and promotes transplantation tolerance Blood, May 1, 2004; 103(9): 3573 - 3581. [Abstract] [Full Text] [PDF]
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J. Cai, J. Lee, E. Jankowska-Gan, R. Derks, J. Pool, T. Mutis, E. Goulmy, and W. J. Burlingham Minor H Antigen HA-1-specific Regulator and Effector CD8+ T Cells, and HA-1 Microchimerism, in Allograft Tolerance J. Exp. Med., April 5, 2004; 199(7): 1017 - 1023. [Abstract] [Full Text] [PDF]
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B. Martin, A. Banz, B. Bienvenu, C. Cordier, N. Dautigny, C. Becourt, and B. Lucas Suppression of CD4+ T Lymphocyte Effector Functions by CD4+CD25+ Cells In Vivo J. Immunol., March 15, 2004; 172(6): 3391 - 3398. [Abstract] [Full Text] [PDF]
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 E. M. Aandahl, J. Michaelsson, W. J. Moretto, F. M. Hecht, and D. F. Nixon Human CD4+ CD25+ Regulatory T Cells Control T-Cell Responses to Human Immunodeficiency Virus and Cytomegalovirus Antigens J. Virol., March 1, 2004; 78(5): 2454 - 2459. [Abstract] [Full Text] [PDF]
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X. Zhang, D. N. Koldzic, L. Izikson, J. Reddy, R. F. Nazareno, S. Sakaguchi, V. K. Kuchroo, and H. L. Weiner IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells Int. Immunol., February 1, 2004; 16(2): 249 - 256. [Abstract] [Full Text] [PDF]
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L. A. Stephens, A. N. Barclay, and D. Mason Phenotypic characterization of regulatory CD4+CD25+ T cells in rats Int. Immunol., February 1, 2004; 16(2): 365 - 375. [Abstract] [Full Text] [PDF]
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M. Karim, C. I. Kingsley, A. R. Bushell, B. S. Sawitzki, and K. J. Wood Alloantigen-Induced CD25+CD4+ Regulatory T Cells Can Develop In Vivo from CD25-CD4+ Precursors in a Thymus-Independent Process J. Immunol., January 15, 2004; 172(2): 923 - 928. [Abstract] [Full Text] [PDF]
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L. E. Marra, Z. X. Zhang, B. Joe, J. Campbell, G. A. Levy, J. Penninger, and L. Zhang IL-10 Induces Regulatory T Cell Apoptosis by Up-Regulation of the Membrane Form of TNF-{alpha} J. Immunol., January 15, 2004; 172(2): 1028 - 1035. [Abstract] [Full Text] [PDF]
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J. Andrassy, S. Kusaka, E. Jankowska-Gan, J. R. Torrealba, L. D. Haynes, B. R. Marthaler, R. C. Tam, B. M.-W. Illigens, N. Anosova, G. Benichou, et al. Tolerance to Noninherited Maternal MHC Antigens in Mice J. Immunol., November 15, 2003; 171(10): 5554 - 5561. [Abstract] [Full Text] [PDF]
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S. Bozza, K. Perruccio, C. Montagnoli, R. Gaziano, S. Bellocchio, E. Burchielli, G. Nkwanyuo, L. Pitzurra, A. Velardi, and L. Romani A dendritic cell vaccine against invasive aspergillosis in allogeneic hematopoietic transplantation Blood, November 15, 2003; 102(10): 3807 - 3814. [Abstract] [Full Text] [PDF]
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S. Gregori, N. Giarratana, S. Smiroldo, and L. Adorini Dynamics of Pathogenic and Suppressor T Cells in Autoimmune Diabetes Development J. Immunol., October 15, 2003; 171(8): 4040 - 4047. [Abstract] [Full Text] [PDF]
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H. Nishikawa, T. Kato, K. Tanida, A. Hiasa, I. Tawara, H. Ikeda, Y. Ikarashi, H. Wakasugi, M. Kronenberg, T. Nakayama, et al. CD4+ CD25+ T cells responding to serologically defined autoantigens suppress antitumor immune responses PNAS, September 16, 2003; 100(19): 10902 - 10906. [Abstract] [Full Text] [PDF]
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S. Jiang, N. Camara, G. Lombardi, and R. I. Lechler Induction of allopeptide-specific human CD4+CD25+ regulatory T cells ex vivo Blood, September 15, 2003; 102(6): 2180 - 2186. [Abstract] [Full Text] [PDF]
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S. Fisson, G. Darrasse-Jeze, E. Litvinova, F. Septier, D. Klatzmann, R. Liblau, and B. L. Salomon Continuous Activation of Autoreactive CD4+ CD25+ Regulatory T Cells in the Steady State J. Exp. Med., September 2, 2003; 198(5): 737 - 746. [Abstract] [Full Text] [PDF]
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C. Asseman, S. Read, and F. Powrie Colitogenic Th1 Cells Are Present in the Antigen-Experienced T Cell Pool in Normal Mice: Control by CD4+ Regulatory T Cells and IL-10 J. Immunol., July 15, 2003; 171(2): 971 - 978. [Abstract] [Full Text] [PDF]
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T. Pearson, T. G. Markees, D. V. Serreze, M. A. Pierce, M. P. Marron, L. S. Wicker, L. B. Peterson, L. D. Shultz, J. P. Mordes, A. A. Rossini, et al. Genetic Disassociation of Autoimmunity and Resistance to Costimulation Blockade-Induced Transplantation Tolerance in Nonobese Diabetic Mice J. Immunol., July 1, 2003; 171(1): 185 - 195. [Abstract] [Full Text] [PDF]
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M. Kataoka, J. A. Margenthaler, G. Ku, and M. W. Flye Development of Infectious Tolerance After Donor-Specific Transfusion and Rat Heart Transplantation J. Immunol., July 1, 2003; 171(1): 204 - 211. [Abstract] [Full Text] [PDF]
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C. Seifarth, S. Pop, B. Liu, C. P. Wong, and R. Tisch More Stringent Conditions of Plasmid DNA Vaccination Are Required to Protect Grafted Versus Endogenous Islets in Nonobese Diabetic Mice J. Immunol., July 1, 2003; 171(1): 469 - 476. [Abstract] [Full Text] [PDF]
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 T. D. Zorina, V. M. Subbotin, S. Bertera, A. M. Alexander, C. Haluszczak, B. Gambrell, R. Bottino, A. J. Styche, and M. Trucco Recovery of the Endogenous {beta} Cell Function in the NOD Model of Autoimmune Diabetes Stem Cells, July 1, 2003; 21(4): 377 - 388. [Abstract] [Full Text] [PDF]
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 K. Asadullah, W. Sterry, and H. D. Volk Interleukin-10 Therapy--Review of a New Approach Pharmacol. Rev., June 1, 2003; 55(2): 241 - 269. [Abstract] [Full Text] [PDF]
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A. D. Salama, N. Najafian, M. R. Clarkson, W. E. Harmon, and M. H. Sayegh Regulatory CD25+ T Cells in Human Kidney Transplant Recipients J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1643 - 1651. [Abstract] [Full Text] [PDF]
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Y. Zhai and J. W. Kupiec-Weglinski Regulatory T Cells in Kidney Transplant Recipients: Active Players but to What Extent? J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1706 - 1708. [Full Text] [PDF]
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C. Guillot, S. Menoret, C. Guillonneau, C. Braudeau, M. G. Castro, P. Lowenstein, and I. Anegon Active suppression of allogeneic proliferative responses by dendritic cells after induction of long-term allograft survival by CTLA4Ig Blood, April 15, 2003; 101(8): 3325 - 3333. [Abstract] [Full Text] [PDF]
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N. E. Phillips, T. G. Markees, J. P. Mordes, D. L. Greiner, and A. A. Rossini Blockade of CD40-Mediated Signaling Is Sufficient for Inducing Islet But Not Skin Transplantation Tolerance J. Immunol., March 15, 2003; 170(6): 3015 - 3023. [Abstract] [Full Text] [PDF]
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A. Sundstedt, E. J. O'Neill, K. S. Nicolson, and D. C. Wraith Role for IL-10 in Suppression Mediated by Peptide-Induced Regulatory T Cells In Vivo J. Immunol., February 1, 2003; 170(3): 1240 - 1248. [Abstract] [Full Text] [PDF]
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W.-P. Min, D. Zhou, T. E. Ichim, G. H. Strejan, X. Xia, J. Yang, X. Huang, B. Garcia, D. White, P. Dutartre, et al. Inhibitory Feedback Loop Between Tolerogenic Dendritic Cells and Regulatory T Cells in Transplant Tolerance J. Immunol., February 1, 2003; 170(3): 1304 - 1312. [Abstract] [Full Text] [PDF]
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D. Yin, N. Dujovny, L. Ma, A. Varghese, J. Shen, D. K. Bishop, and A. S. Chong IFN-{gamma} Production Is Specifically Regulated by IL-10 in Mice Made Tolerant with Anti-CD40 Ligand Antibody and Intact Active Bone J. Immunol., January 15, 2003; 170(2): 853 - 860. [Abstract] [Full Text] [PDF]
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M. Sho, A. Yamada, N. Najafian, A. D. Salama, H. Harada, S. E. Sandner, A. Sanchez-Fueyo, X. X. Zheng, T. B. Strom, and M. H. Sayegh Physiological Mechanisms of Regulating Alloimmunity: Cytokines, CTLA-4, CD25+ Cells, and the Alloreactive T Cell Clone Size J. Immunol., October 1, 2002; 169(7): 3744 - 3751. [Abstract] [Full Text] [PDF]
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J. L. Cohen, A. Trenado, D. Vasey, D. Klatzmann, and B. L. Salomon CD4+CD25+ Immunoregulatory T Cells: New Therapeutics for Graft-Versus-Host Disease J. Exp. Med., August 5, 2002; 196(3): 401 - 406. [Abstract] [Full Text] [PDF]
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A. Banz, C. Pontoux, and M. Papiernik Modulation of Fas-Dependent Apoptosis: A Dynamic Process Controlling Both the Persistence and Death of CD4 Regulatory T Cells and Effector T Cells J. Immunol., July 15, 2002; 169(2): 750 - 757. [Abstract] [Full Text] [PDF]
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 P. M. Cobelens, A. Kavelaars, R. van der Zee, W. van Eden, and C. J. Heijnen Dynamics of mycobacterial HSP65-induced T-cell cytokine expression during oral tolerance induction in adjuvant arthritis Rheumatology, July 1, 2002; 41(7): 775 - 779. [Abstract] [Full Text] [PDF]
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S. Hori, M. Haury, A. Coutinho, and J. Demengeot Specificity requirements for selection and effector functions of CD25+4+ regulatory T cells in anti-myelin basic protein T cell receptor transgenic mice PNAS, June 11, 2002; 99(12): 8213 - 8218. [Abstract] [Full Text] [PDF]
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L. Graca, S. Thompson, C.-Y. Lin, E. Adams, S. P. Cobbold, and H. Waldmann Both CD4+CD25+ and CD4+CD25- Regulatory Cells Mediate Dominant Transplantation Tolerance J. Immunol., June 1, 2002; 168(11): 5558 - 5565. [Abstract] [Full Text] [PDF]
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P. A. Taylor, T. M. Friedman, R. Korngold, R. J. Noelle, and B. R. Blazar Tolerance induction of alloreactive T cells via ex vivo blockade of the CD40:CD40L costimulatory pathway results in the generation of a potent immune regulatory cell Blood, May 29, 2002; 99(12): 4601 - 4609. [Abstract] [Full Text] [PDF]
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), (CBA x B10)F1
(
), or (CBA x BALB/c)F1 (
). C
(naive) and D (B10 tolerant), Cytokine production in
response to (CBA x B10)F1 stimulators was assessed in
the supernatant after 72 h (IL-2, 
) or 96 h (IFN-
)
of culture. IL-4 and IL-10 levels were below the limit of sensitivity
of the assay. Data are expressed as mean ± SEM of triplicate
wells. Two additional experiments gave similar results.
; n = 7; median survival
time (MST) 19 days). Cotransfer of 5 x 105
CD45RBlow CD4+ T cells purified from CBA mice
with long-term surviving B10 cardiac allografts prevented rejection of
B10 (
;
n = 7; MST 19 days) did not prevent rejection.
Repeat experiments gave similar results. B,
CD25+ CD4+ T cells from tolerant mice prevent
skin graft rejection by naive CD45RBhigh CD4+ T
cells. All T cell-depleted CBA mice were reconstituted with 1 x
105 naive CD45RBhigh CD4+ T cells.
B10 skin grafts transplanted onto T cell-depleted CBA mice
reconstituted with 1 x 105 naive
CD45RBhigh CD4+ T cells alone were rejected
(
