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,25-Dihydroxyvitamin D3 and Mycophenolate Mofetil Treatment Mediate Transplantation Tolerance1
*
Roche Milano Ricerche, and
Department of Medicine, University Vita-Salute, Milan, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-Dihydroxyvitamin D3, the active form of vitamin
D3, and mycophenolate mofetil, a selective inhibitor of T
and B cell proliferation, modulate APC function and induce dendritic
cells (DCs) with a tolerogenic phenotype. Here we show that a short
treatment with these agents induces tolerance to fully mismatched mouse
islet allografts that is stable to challenge with donor-type spleen
cells and allows acceptance of donor-type vascularized heart grafts.
Peritransplant macrophages and DCs from tolerant mice express
down-regulated CD40, CD80, and CD86 costimulatory molecules. In
addition, DCs from the graft area of tolerant mice secrete, upon
stimulation with CD4+ cells, 10-fold lower levels of IL-12
compared with DCs from acutely rejecting mice, and induce a
CD4+ T cell response characterized by selective abrogation
of IFN-
production. CD4+ but not CD8+ or
class II+ cells from tolerant mice, transferred into naive
syngeneic recipients, prevent rejection of donor-type islet grafts.
Graft acceptance is associated with impaired development of
IFN--producing type 1 CD4+ and CD8+ cells
and an increased percentage of CD4+CD25+
regulatory cells expressing CD152 in the spleen and in the
transplant-draining lymph node. Transfer of
CD4+CD25+ cells from tolerant but not naive
mice protects 100% of the syngeneic recipients from islet allograft
rejection. These results demonstrate that a short treatment with
immunosuppressive agents, such as 1,25-dihydroxyvitamin
D3/mycophenolate mofetil, induces tolerance to islet
allografts associated with an increased frequency of
CD4+CD25+ regulatory cells that can adoptively
transfer transplantation tolerance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interference with the CD80/CD86-CD28 costimulatory pathway by administration of the fusion protein CD152-Ig induces long-term allograft survival in rodents (7), although it is less effective in prolonging islet allograft survival in nonhuman primates (8). Blocking the CD40/CD154 pathway with anti-CD154 mAb has also been demonstrated to prevent rejection of heart, skin (9), and islet (10) allografts in rodents. Moreover, administration of anti-CD154 prevents acute renal allograft rejection (11) and induces long-term survival of islet allografts (12, 13) in nonhuman primates. Simultaneous blockade of the CD28 and CD40 pathways most effectively promotes long-term graft survival and inhibits the development of chronic rejection (9). Activation-induced cell death is an important mechanism of the prolonged allograft survival induced by anti-CD154 mAb (14, 15), but in addition to deletion of alloreactive T cells this treatment also induces infectious transplantation tolerance (16).
1,25-Dihydroxyvitamin D3
(1,25(OH)2D3),3
the activated form of vitamin D3, not only has a
vital role in bone and calcium metabolism, but also modulates the
immune response via specific receptors expressed in APCs and activated
T cells (17).
1,25(OH)2D3 and its analogs
have been shown to inhibit autoimmune diseases and graft rejection in
several experimental models (18). Intriguingly, renal
graft loss has been found decelerated in patients treated with
1,25(OH)2D3
(19).
1,25(OH)2D3 inhibits
Ag-induced T cell proliferation (20) and cytokine
production (21), and prevents Th1 cell development
(22, 23). APCs and, in particular, dendritic cells (DCs),
are primary targets for the immunosuppressive activity of
1,25(OH)2D3.
1,25(OH)2D3 inhibits the
differentiation and maturation of human DCs, leading to down-regulated
expression of CD40, CD80, and CD86 costimulatory molecules and to
inhibition of alloreactive T cell activation (24, 25, 26, 27). In
addition, 1,25(OH)2D3
inhibits IL-12 and enhances IL-10 production by DCs, leading to
induction of CD4+ cells characterized by
up-regulated CD152 expression and hyporesponsiveness to alloantigens
(24). Based on these results, we have analyzed the ability
of 1,25(OH)2D3,
administered alone or in combination with mycophenolate mofetil (MMF),
to induce transplantation tolerance. MMF, an immunosuppressive agent
clinically used to inhibit allograft rejection (28, 29),
inhibits both T and B cell proliferation to mitogenic and allogenic
stimulation (30), and also directly affects DCs by
inhibiting costimulatory molecule expression and IL-12 production
(31).
Results in this paper demonstrate that 1,25(OH)2D3/MMF treatment induces donor-specific transplantation tolerance to islet allografts. CD4+ cells from tolerant mice transferred into diabetic syngeneic recipients prevent donor-type islet allograft rejection, and tolerant mice display an increased percentage of CD4+CD25+ regulatory T cells, suggesting an active mechanism of suppression. Active suppression of islet allograft rejection is shown by the capacity of CD4+CD25+ cells to transfer transplantation tolerance. These results demonstrate that low m.w. immunosuppressive agents, like a combination of 1,25(OH)2D3 and MMF, can induce in vivo DCs with a tolerogenic phenotype, enhance the frequency of CD4+CD25+ regulatory cells, and promote peripheral tolerance to allografts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c, C57BL/6 (B6), and C3H female mice were purchased from Charles River Laboratories (Calco, Italy), and kept under specific pathogen-free conditions.
Transplantation
Hand-picked B6 islets were transplanted (350/mouse), after
overnight culture at 37°C, under the kidney capsule of BALB/c
recipient mice rendered diabetic by a single i.v. injection of
streptozotocin (250 mg/kg; Sigma, St. Louis, MO), as described
(32). Two consecutive nonfasting blood glucose levels of
200 mg/dl defined islet graft rejection. Fully vascularized hearts
were grafted into the abdomen of recipient mice as described
(33). Graft survival was monitored by palpation, and
rejection was confirmed by direct graft inspection.
Immunosuppressive treatments
MMF, a prodrug of mycophenolic acid, was dissolved in a vehicle consisting of sodium chloride (0.9%), sodium carboxymethylcellulose (0.5%), polysorbate 80 (0.4%), benzyl alcohol (0.9%) (all obtained from Sigma), and distilled water (97.3%). 1,25(OH)2D3 was dissolved in ethanol (1 mg/ml) and then diluted in miglyol. Mice were dosed orally with MMF (100 mg/kg/day) and/or 1,25(OH)2D3 (5 µg/kg p.o. three times per week), from day –1 to day 30 relative to the time of grafting. Control mice were dosed with vehicles only. Purified anti-CD4 GK1.5 mAb (American Type Culture Collection (ATCC), Manassas, VA) was injected i.p. (10 mg/kg) at days –1, 0, 1, and 2 relative to islet transplantation.
Histology
Kidney poles containing islet grafts were snap-frozen in Tissue Tek (Miles Laboratories, Elkhart, IN) and stored at -70°C. Frozen sections, 5 µm thick, were stained with biotinylated mAb against CD4, CD8, B220, CD11b (all purchased from PharMingen, San Diego, CA), or CD11c (N418; ATCC), followed by streptavidin-peroxidase conjugate. 3-amino-9-ethylcarbazole (DAKO, Carpenteria, CA) was used as chromogen, and hematoxylin as a counterstain. Cryostat sections were also stained with anti-porcine insulin (Sigma) followed by peroxidase-anti-peroxidase (Sigma).
Intracellular staining for cytokine production
Cells were stained for IFN-, IL-4, IL-10, and IL-2 as
previously described (34). Reagents for intracytoplasmic
staining contained 1% FCS, 0.5% saponin (Sigma), and 0.1% sodium
azide. All incubations were performed at room temperature. Cells were
washed, preincubated for 10 min with PBS/FCS/saponin, and then
incubated with FITC-labeled rat anti-mouse IFN- (XMG1.2) and
PE-labeled rat anti-mouse IL-4 (11B11), PE-labeled rat
anti-mouse IL-10 (JES5-16E3), or PE-labeled rat anti-IL-2
(JE56-5H4), all obtained from PharMingen. Isotype controls were FITC-
and PE-labeled rat IgG1 (R3-34). After 30 min, cells were washed twice
with PBS/FCS/saponin and then with PBS containing 5% FCS without
saponin to allow membrane closure. The cell surface was then stained
with CyChrome-labeled anti-CD4 (L3T4) for 15 min at room
temperature. Analysis was performed with a FACScan flow cytometer
equipped with CellQuest software (BD Biosciences, Mountain View,
CA).
Flow cytometric analysis
Flow cytometric analysis was performed on single-cell suspensions of graft-associated cells, isolated by mechanical disruption. Stainings were performed in the presence of 100 µg/ml mouse IgG using the following mAbs, all obtained from PharMingen: CyChrome-labeled anti-CD45 (30-F11), FITC-labeled anti-CD11b (M1/70), FITC-labeled anti-CD40 (HM40-3), FITC-labeled anti-CD80 (16-10A1), PE-labeled anti-CD11c (HL-3), FITC-labeled anti-CD25 (7D4), and FITC-labeled anti-CD38 (H1.2F3). Cells were analyzed with a FACScan flow cytometer equipped with CellQuest software (BD Biosciences).
Cell cultures
To obtain graft-associated CD11c+ cells,
kidney poles containing the islet grafts were injected with 1 mg/ml
collagenase D (Boehringer Mannheim, Mannheim, Germany) and 0.02 mg/ml
DNase I (Sigma), and incubated at 37°C for 15 min. Single cell
suspensions were collected after diluting the enzyme with ice-cold HBSS
containing 2% FCS, 2.5 mM HEPES, and 5 mM EDTA and removing the
aggregates by settling for 2 min on ice. Aggregates were further
digested with collagenase D and DNase I for 5 min. Single cell
suspensions were washed three times, and CD11c+
cells were purified by positive selection on MiniMACS (Miltenyi Biotec,
Auburn, CA). CD11c+ cells (4 x
103 cells/well) from tolerant or acutely
rejecting BALB/c mice were cultured in 96-well plates with splenic
CD4+ cells (2 x 105
cells/well) from donor-type B6 mice, positively selected by MiniMACS as
described (35), in synthetic HL-1 medium (Ventrex
Laboratories, Portland, ME) supplemented with 2 mM
L-glutamine and 50 µg/ml gentamicin (Sigma). After 2
days, IL-12p75, IL-12p40, IFN-, IL-2, and IL-4 were quantified in
culture supernatants.
Quantification of secreted cytokines
Secreted IL-12p75, IL-12p40, IFN-, IL-4, and IL-2 were
quantified by two-site sandwich ELISA, as described
(35, 36, 37). For IL-12p40 and IFN-, polyvinyl microtiter
plates (Falcon 3012) were coated with 100 µl of 10F6 anti-mouse
IL-12p40 (IL-12p40) and AN-18.17.24 (IFN-) in carbonate buffer.
Samples were titrated in test solution (PBS containing 5% FCS and 1
g/L phenol) and incubated overnight at 4°C. To detect bound
cytokines, plates were then incubated with biotinylated goat
anti-mouse IL-12 (IL-12p40) or XMG1.2 mAb (IFN-) in test
solution. After washing, the bound biotinylated Abs were revealed by an
additional 30-min incubation with alkaline phosphatase-conjugated
streptavidin (Jackson ImmunoResearch Laboratories, Avondale, PA)
diluted 1/5000. The plates were washed again and incubated with the
developing substrate p-nitrophenylphosphate disodium (Sigma)
in diethanolamine buffer (pH 9.6; 100 µl/well). For IL-12p75
determination, plates were coated with 100 µl of the rat
anti-mouse IL-12 heterodimer 9A5 mAb in carbonate buffer. After
blocking, samples were incubated with 50 ml peroxidase-conjugated 5C3
mAb (rat anti-mouse IL-12p40 subunit). Anti-IL-12 Abs were provided
by Dr. M. K. Gately (Hoffmann-LaRoche, Nutley, NJ). After
overnight incubation at room temperature, bound peroxidase was detected
by 3,3',5,5'-tetramethylbenzidine (Fluka Chemicals, Ronkonkoma, NY),
and absorbance was read at 450 nm with an automated microplate ELISA
reader (MR5000; Dynatech Laboratories, Chantilly, VA). For IL-2 and
IL-4 determination, two-site ELISAs were performed with paired mAbs
obtained from PharMingen. For capture, JES6-1A12 (anti-IL-2) and
BVD4-1D11 or 11B11 (anti-IL-4) mAbs were used. Samples were
titrated in test solution and incubated overnight at 4°C. To detect
bound cytokines, plates were then incubated with the biotinylated mAb
JES6-5H4 (anti-IL-2) or BVD6-24G2 (anti-IL-4) in test solution.
After washing, the bound biotinylated mAbs were revealed by an
additional 30-min incubation with alkaline phosphatase-conjugated
streptavidin (Jackson ImmunoResearch Laboratories) diluted 1/5000. The
plates were washed again and incubated with the developing substrate
p-nitrophenylphosphate disodium (Sigma) in diethanolamine
buffer (pH 9.6, 100 µl/well). The reaction was stopped by adding 50
µl/well NaOH 3N, and absorbance was read at 405 nm. Cytokines were
quantified from two to three titration points using standard curves
generated by purified recombinant mouse cytokines, and results were
expressed as cytokine concentration in picograms per milliliter.
Detection limits were 15 pg/ml for IFN-, IL-2, and IL-4, and 5 pg/ml
for IL-12p40 and IL-12p75.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first analyzed the ability of
1,25(OH)2D3 and MMF,
administered alone or in combination, to inhibit islet allograft
rejection. Pancreatic islets isolated from B6 mice were transplanted
under the kidney capsule of BALB/c mice rendered diabetic by a single
injection of streptozotocin. Recipient mice were treated from day –1
to day 30 with a nonhypercalcemic dose of
1,25(OH)2D3 (5 µg/kg p.o.
three times per week) and/or MMF (100 mg/kg p.o. daily). The mean
rejection time in vehicle-treated recipients was 23 ± 3 days.
1,25(OH)2D3 and MMF
administered alone prolonged islet graft survival, but only in 
50%
of the recipients. Conversely, over 80% of the mice treated with both
drugs showed long-term (>70 days) islet graft acceptance (Fig. 1
A). Next, we challenged
BALB/c recipients showing long-term (>70 days) allograft acceptance
with i.p. injection of 106 donor-type B6 spleen
cells (Fig. 1B). Recipient mice treated with peritransplant
administration of anti-CD4 mAb accommodated the islet graft but
were not tolerant, because all mice rejected the graft after challenge
with a mean survival time of 14 ± 2.4 days. Indeed, induction of
transplantation tolerance in fully mismatched combinations by
anti-CD4 mAb has been found to require additional treatments, such
as CD152-Ig (38) or donor-specific transfusion
(39).
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B). Mice that continued to show islet
graft function for 4 wk after challenge were transplanted, 100 days
after the initial islet graft, with a vascularized heart from B6
(donor-type) or C3H (third party) mice. Results
in Fig. 1C show that naive BALB/c mice rejected B6 heart
grafts in 10 days, whereas only one tolerant mouse of five rejected the
heart graft 25 days after transplant. In contrast, tolerant BALB/c mice
rejected a third-party heart in 10–12 days. These results demonstrate
that a short-term treatment with
1,25(OH)2D3 and MMF can
induce donor-specific tolerance to allografts. Tolerant mice display a massive peritransplant infiltrate
Islet grafts were analyzed, 140 days after the initial transplant,
in BALB/c mice accepting B6 heart grafts. Immunohistochemical analysis
in mice rendered tolerant by MMF and
1,25(OH)2D3 treatment (Fig. 2A) showed a massive
infiltrate around the islet grafts comprising B cells (b),
CD4+ (c) and
CD8+ (d) T cells, macrophages
(e), and DCs (f). Only a few scattered
cells were observed within the islet grafts. Thus, graft function, as
demonstrated by the positive staining for insulin (a),
persisted despite substantial peritransplant infiltration. Few
I-Ab positive cells were found only within the B6
islets (g), whereas graft-surrounding
lymphomononuclear cells expressed the recipient
I-Ad molecule (h), indicating the
recruitment of recipient cells. This implies that recipient-derived
APCs could interact with recipient T cells via the indirect pathway of
Ag presentation. The cells surrounding the tolerated grafts were
analyzed by flow cytometry in mice rendered tolerant with MMF alone or
1,25(OH)2D3/MMF treatment
and compared with acutely rejected islet grafts 30 days after
transplantation (Fig. 2B). CD45+ cells
recovered from the graft area of BALB/c mice rendered tolerant with
1,25(OH)2D3/MMF treatment
(3 x 106/mouse) were 10-fold higher
compared with mice acutely rejecting B6 islet grafts (0.4 x
106/mouse). In contrast,
106 CD45+ cells/mouse were
recovered from the graft area of mice rendered tolerant with MMF alone.
This increase was mostly due to perigraft accumulation of
CD4+ cells and, in part, of B and
CD8+ cells. Conversely, the total numbers of
macrophages and DCs were not increased (Fig. 2B, left
panel). No difference in the percentage of
CD4+, CD8+, and B cells was
found between the two groups (Fig. 2B, right panel).
Strikingly, the percentages of CD11b+ macrophages
and CD11c+ DCs were reduced 5-fold in mice
rendered tolerant under the cover of MMF and
1,25(OH)2D3 compared with
infiltrates in acutely rejecting mice or in mice rendered tolerant
under the cover of MMF alone (Fig. 2B, right panel).
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Costimulatory molecule expression on macrophages and DCs was
further analyzed in tolerant and acutely rejecting mice. Down-regulated
CD40, CD80, and CD86 costimulatory molecules were observed on perigraft
CD11c+ DCs from mice rendered tolerant under the
cover of MMF and
1,25(OH)2D3 treatment, as
demonstrated by the representative experiment shown in Fig. 3, A, left panel, and
B. The percentage of both macrophages and DCs was reduced in
grafts from tolerant
(CD11b+/CD11c- 5%,
CD11c+ 2%) compared with acutely rejecting
(CD11b+/CD11c- 14%,
CD11c+ 8%) mice (Fig. 3B). CD40,
CD80, and CD86 expression was 3- to 7-fold reduced on DCs and
macrophages surrounding tolerated islet grafts compared with acutely
rejecting ones. Results (means ± SE) from four experiments showed
a significant reduction from 4.4 ± 0.7 to 0.7 ± 0.3 in the
percentage of CD11c+ DCs expressing CD40
(p < 0.05 by Mann-Whitney U test).
Similarly, DCs expressing CD80 were reduced from 9.2 ± 1.5 to
0.9 ± 0.4 (p < 0.05), and DCs expressing
CD86 molecules from 4.1 ± 0.5 to 0.8 ± 0.4
(p < 0.05). The percentage of graft-associated
macrophages and DCs, as well as their expression of costimulatory
molecules, in rejected grafts from untreated mice or from the 27% of
1,25(OH)2D3/MMF-treated
mice that failed to become tolerant, was indistinguishable (data not
shown). Intriguingly, the expression of CD40 on B cells was not
significantly down-regulated (Fig. 3B). Results (means
± SE) from four experiments showed a reduction from 25.0 ± 4.7
to 19.5 ± 1.7 (p = 0.7) in the percentage
of CD40+ B cells surrounding acutely rejected vs
tolerated islet grafts.
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A). Thus,
1,25(OH)2D3 treatment is
required to induce long-lasting down-regulation of costimulatory
molecules. Interestingly, tolerant mice displayed an increased
percentage of CD11c+ DCs (3.9 vs 0.6% in acutely
rejecting mice) and CD11b+ macrophages (10 vs
2.7%) in the kidney lymph nodes draining the islet graft (Fig. 3C). CD40, CD80, and CD86 costimulatory molecules were
up-regulated in the kidney lymph node cells (Fig. 3, A, right
panel, and C) and in the spleen or mesenteric lymph
node cells of tolerant compared with acutely rejecting mice (data not
shown). This indicates a selective effect of
1,25(OH)2D3 treatment on
APCs recruited to the graft area. The tolerogenic phenotype, as defined
by down-regulated costimulatory molecules, expressed by perigraft DCs
from tolerant mice, prompted us to test their function in terms of
IL-12 production and capacity to activate donor-type
CD4+ cells. CD11c+ cells
surrounding the islet grafts of tolerant and acutely rejecting BALB/c
mice were purified and cultured with CD4+ spleen
cells from donor-type B6 mice. Results in Fig. 4 demonstrate that DCs from the graft
area of tolerant mice secrete, upon stimulation with
CD4+ cells, 10-fold lower levels of IL-12p75 and
IL-12p40 compared with DCs from acutely rejecting mice. In the same
culture supernatants, the T cell-derived cytokines IFN-, IL-2, and
IL-4 were also measured. Consistent with the profound decrease of IL-12
secretion by DCs, IFN- production by CD4+
cells was selectively abrogated. Thus, perigraft DCs from tolerant
mice, as predicted by their reduced expression of CD40 molecules, are
less sensitive to IL-12-inducing signals from
CD4+ cells. In turn, they induce only partial
activation of CD4+ cells, characterized by lack
of IFN- production.
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The tolerant state of recipients with long-term accepted grafts
following 1,25(OH)2D3/MMF
treatment was characterized by a reduced percentage of
IFN--producing CD4+ and
CD8+ cells in the kidney lymph nodes draining the
islet allografts. In contrast, IL-2-producing cells were reduced only
among CD8+ cells. IL-4- and IL-10-producing cells
were comparably low in acutely rejecting and tolerant mice (Fig. 5A). To assess whether
transplantation tolerance could be transferred to naive syngeneic
recipients, total spleen cells from tolerant BALB/c mice were injected
into diabetic BALB/c mice (20 x
106/recipient) 2 days before transplantation of
either B6 (donor-type) or C3H (third party)
islets. Transfer of unfractionated spleen cells from tolerant BALB/c
mice inhibited donor-type (B6) allograft rejection and induced in 50%
of the recipients long-term (>70 days) graft survival. In contrast,
third party (C3H) islets were rejected with a
mean graft survival time of 16 ± 2 days, similar to that of B6
islets transplanted in naive diabetic BALB/c mice (Fig. 5B).
Following transfer of spleen cells from tolerant mice, the percentage
of IFN--producing CD4+ and
CD8+ cells in the kidney lymph nodes draining the
islet graft of recipients with long-term accepted allograft was 3- to
6-fold reduced compared with recipients that rejected the islet graft
(Fig. 5C). Conversely, the percentage of IL-4- or
IL-10-producing cells was very low in both groups. Similar results were
obtained with spleen cells (data not shown). Thus, the patterns of
cytokine production by CD4+ and
CD8+ cells from primarily tolerant mice and
recipients rendered tolerant by adoptive transfer of spleen cells were
essentially superimposable.
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demonstrate that CD4+ spleen cells from tolerant
mice, equivalent in number to those contained in 20 x
106 spleen cells, prevented donor-type islet
allograft rejection in all recipients, indicating an active and
effective tolerogenic mechanism. Interestingly, mice transferred with
CD8+ spleen cells isolated from tolerant mice
displayed a primary nonfunction of the islet grafts and never returned
normoglycemic. CD4+CD25+ cells from tolerant mice transfer transplantation tolerance
Tolerant mice displayed an increased percentage of
CD4+CD25+ regulatory cells
both in draining lymph nodes (13.3 vs 6.7%) and in the spleen (32.8 vs
16.4%) compared with acutely rejecting mice, without a corresponding
increase of CD4+CD69+ cells.
CD4+CD38+ cells were also higher in tolerant
mice, in particular in the spleen. Moreover, tolerant mice presented a
lower percentage of CD45RBhigh cells and an
increased percentage of CD45RBlow cells compared with
acutely rejecting mice (Table II). An
increased expression of CD152 was found in
CD4+CD25+ T cells isolated
from tolerant mice compared with acutely rejecting mice (Fig. 6), both in draining lymph nodes (37 vs
17%) and spleens (35.5 vs 20%). To evaluate the ability of
CD4+CD25+ T cells to
prevent allograft rejection,
CD4+CD25- (4 x
106/mouse) or CD4+CD25+ (5 x
105/mouse) T cells, equivalent in number to those contained
in 20 x 106 spleen cells isolated from naive or
tolerant mice, were transferred into naive diabetic BALB/c mice. Two
days later, these recipients were transplanted with donor-type (B6)
islets. Results in Fig. 7A
show that neither C4+CD25- nor
CD4+CD25+ cells from naive BALB/c mice could
protect, at least at the dose transferred, from islet allograft
rejection. Conversely, results in Fig. 7B show that
CD4+CD25+ cells prevented islet allograft
rejection, whereas CD4+CD25- cells did not,
indicating an active mechanism of tolerance induction mediated by
CD4+CD25+ regulatory T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing
type 1 CD4+ and CD8+ cells.
Recipients transferred with CD4+ cells from mice
rendered tolerant with
1,25(OH)2D3/MMF treatment
show an increased percentage of
CD4+CD25+ regulatory T
cells expressing CD152, and these cells can transfer tolerance to islet
allografts. The rationale of this study was to induce tolerance to allografts by a combined short-term targeting of APCs and T cells. 1,25(OH)2D3 is an immunomodulator inhibiting, in different models, autoimmune diseases and allograft rejection (18). 1,25(OH)2D3 inhibits alloreactive T cell activation by targeting APCs, and in particular DCs (24, 25, 26, 27). Notably, 1,25(OH)2D3 inhibits the differentiation, maturation, and activation of DCs in vitro while promoting their apoptosis (24). DCs matured in the presence of 1,25(OH)2D3 display reduced levels of MHC class II and CD40, CD80, and CD86 costimulatory molecules, and induce hyporesponsiveness in alloreactive T cells (24). DC modulation by 1,25(OH)2D3 has recently been shown to require a vitamin D receptor-dependent pathway that could promote a persistent inhibition of DC maturation in vitro and in vivo (41). MMF has also been shown to affect DCs, reducing the expression of costimulatory molecules and inhibiting the production of IL-12 (31). In addition, MMF inhibits the proliferation of activated T and B cells. Orally administered MMF is hydrolyzed by esterases in the intestine and blood to release mycophenolic acid, a potent, selective, noncompetitive inhibitor of the type 2 isoform of inosine monophosphate dehydrogenase, and therefore inhibits the de novo pathway of guanosine nucleotide synthesis (30). Because T and B lymphocytes are critically dependent for their proliferation on de novo purine synthesis, whereas other cell types can use salvage pathways, MMF is a selective lymphocyte inhibitor.
As shown here,
1,25(OH)2D3/MMF treatment
induces similar effects in vivo, leading to a reduction in the
percentage of macrophages and DCs surrounding the tolerated graft, both
showing down-regulated expression of CD40, CD80, and CD86 costimulatory
molecules. 1,25(OH)2D3 has
also been shown to inhibit IL-12 production in vitro (42),
via inhibition of NF-
B (43), and in vivo
(23). The long-lasting down-modulation of costimulatory
molecules and the profoundly reduced IL-12 production by DCs recruited
to the graft area of tolerant mice, clearly seen over 100 days after
treatment withdrawal, cannot be the direct effect of
1,25(OH)2D3/MMF treatment.
Rather, this reflects the early disruption of APC-T cell interactions
with long-lasting effects, consistent with the observation that both T
cell and DC persistence in vivo is dependent on CD40-CD154 interactions
(44). It is likely that the inhibition of alloreactive T
cell activation fails, in turn, to provide APC-activating signals
(45), thus explaining the persistent tolerogenic phenotype
of macrophages and DCs recruited to the graft area. Perigraft DCs from
tolerant mice induce only partial activation of alloreactive
CD4+ cells, characterized by the absence of
IFN- but conserved IL-2 and IL-4 secretion. The partial activation
of CD4+ cells lacking IFN- production can be
easily explained by the low levels of IL-12 produced by DCs, and
provides an example of disruption of APC-activating signals induced by
1,25(OH)2D3/MMF
treatment.
Our results highlight the important role of APCs with tolerogenic properties in transplantation tolerance (46, 47, 48). Notably, they indicate that a tolerogenic phenotype, characterized by down-regulated costimulatory molecules and profoundly reduced IL-12 production, can be induced in graft-associated macrophages and DCs by a short course of treatment with low m.w. immunomodulatory agents. Importantly, the immature-like tolerogenic phenotype of DCs is maintained for over 100 days after drug withdrawal. A long-term reduction in mature DCs, associated with transplantation tolerance, could also be induced by peritransplant administration of other immunosuppressive agents, like deoxyspergualin combined with anti-CD3 immunotoxin (49).
Mechanistically, immature DCs could favor peripheral tolerance not only by decreasing the activation of effector T cells but also by inducing the differentiation of T regulatory cells (50, 51). Different types of T regulatory cells have been described, all with the ability to regulate both in vitro and in vivo T cell activation (1, 52). CD4+ cells defined by constitutive expression of CD25 (53, 54) represent the most characterized subset of regulatory T cells. CD4+CD25+ peripheral regulatory T cells derive from thymic precursors (55), and their functional development involves an extrathymic phase dependent on the presence of relevant Ags (56, 57). Although the Ag specificity of CD4+CD25+ regulatory T cells is still unclear, our results indicate that priming by alloantigen is required for their induction, because cells from tolerant but not from naive mice could transfer protection from allograft rejection. CD4+CD25+ cells suppress immune responses via non-Ag specific mechanisms that appear to be independent from the production of immunoregulatory cytokines (4, 58), although the alloantigen-specific regulatory T cells contained within the CD45RBlow population have been found to require IL-10 for functional activity (6). Rather, the suppressive function of CD4+CD25+ regulatory T cells has been associated with up-regulated expression of CD152 (5, 59). Signaling via CD152 is required for immune suppression in vitro (59), and blockade of CD152 signaling increases T cell-mediated responses in several models (60). In our study, the percentage of CD4+CD25+ regulatory T cells expressing CD152 was increased in tolerant mice and they could transfer very effectively transplantation tolerance, suggesting their relevance in tolerance to allografts. Indeed, regulatory T cells have been documented in patients with long-term surviving allografts (61, 62), and isolated examples of operational tolerance have been noted in graft recipients treated with immunosuppressive drugs (63).
In conclusion, our results demonstrate that a short treatment with 1,25(OH)2D3/MMF can induce transferable tolerance to islet grafts. Tolerance is associated with the induction of macrophages and DCs with a tolerogenic phenotype and with an increase of CD4+CD25+ regulatory T cells. The enhancement of CD4+CD25+ regulatory T cells by low m.w. immunosuppressive agents, such as a combination of 1,25(OH)2D3 and MMF, may provide a new paradigm, applicable to the prevention of allograft rejection in humans and to the treatment of autoimmune diseases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Luciano Adorini, Roche Milano Ricerche, Via Olgettina 58, I-20132 Milan, Italy. E-mail address: luciano.adorini{at}roche.com
3 Abbreviations used in this paper: 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; MMF, mycophenolate mofetil; DC, dendritic cell.
Received for publication April 10, 2001. Accepted for publication June 13, 2001.
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