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Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129
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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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Extensive studies are in progress to determine the mechanisms by which
long-term acceptance of two haplotype class I mismatched renal
allografts is achieved following a 12-day course of cyclosporin A (CyA;
23 . Graft acceptance is associated with a specific deficiency of
antidonor class I T cell help during the induction and maintenance of
tolerance (23, 24, 55). Studies have also indicated that tolerance is
systemic since a second renal allograft, SLA-matched to the original
donor, is accepted without further immunosuppression if retransplanted
at the time of graftectomy (25). Recent studies have demonstrated that
tolerance involves both thymus-dependent and peripheral mechanisms
(26). The thymus is required for the induction of stable tolerance,
since animals thymectomized (thyx) before transplantation undergo acute
cellular rejection after CyA therapy is discontinued. However, in some
thymectomized animals, rejection subsides spontaneously and recipients
maintain the renal allografts long-term, indicating that peripheral
mechanisms of tolerance can also lead to graft acceptance (26).
Analysis of cytokine gene expression in renal allografts demonstrated a
correlation between graft acceptance and a differential cytokine
production consistent with a shift toward a Th2 response (27, 28).
Kidney biopsies collected from tolerant animals demonstrated high
levels of IL-10 gene expression but low levels of IFN-
gene
activation. In contrast, biopsies from rejecting animals demonstrated
marked up-regulation of the IFN- gene. These results suggested that
local production of regulatory cytokines may be involved in the
mechanism of tolerance to renal allografts.
Although peripheral T cell tolerance has been described in many systems (1, 21, 29), definitive information regarding cellular mechanisms responsible for the development of graft tolerance remains incomplete. Therefore, studies of peripheral tolerance in a preclinical large animal model may help to elucidate such mechanisms and extend these findings to clinical transplantation. The present report demonstrates that PBL from tolerant animals primed in vitro with donor Ag suppress the generation of antidonor CTL reactivity by naive recipient-matched PBL. The studies suggest that regulatory cells demonstrable in vitro may be involved in the maintenance of tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The surgical procedures, clinical course, and immunogenetic
characteristics of the miniature swine used in this study have been
described in detail elsewhere (Refs. 20, 23, and 26; Yamada et
al., manuscript in preparation). Animals received two haplotype
class I mismatched renal allografts (SLAgg (class
Ic, class IId) donor
SLAdd (class
Id, class IId) recipient) and were treated with
a 12-day course of CyA. In addition, the animals were either
nonthymectomized (nos. 11468, 12019, and 11609) or thymectomized (nos.
11446, 11384, and 11207). The swine were thymectomized on day -42
(nos. 11446 and 11384) or day 0 (no. 11207), in relation to the day of
renal transplantation. Both thymectomized and nonthymectomized swine
(hereafter referred to as thyxtolerant-to-G swine and
non-thyxtolerant-to-G swine, respectively) accepted
their SLAgg renal allografts long-term (>90 days). Two
naive SLAdd swine (nos. 10785 and 10466) were used as
control animals.
Medium
Tissue culture medium used for cell-mediated lympholysis (CML) assays consisted of RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 6% FCS (Sigma, St. Louis, MO), 100 U/ml of penicillin and 135 µg/ml of streptomycin (Life Technologies), 50 µg/ml of gentamicin (Life Technologies), 10 mM of HEPES (Fisher Scientific, Pittsburgh, PA), 2 mM of L-glutamine (Life Technologies), 1 mM of sodium pyruvate (BioWhittaker, Walkersville, MD), 0.1 mM of nonessential amino acids (BioWhittaker), and 5 x 10-5 M of 2-ME (Sigma). The effector phase of the CML assay was performed using Eagle’s basal medium (Life Technologies) supplemented with 6% controlled processed serum replacement-3 (Sigma) and 10 mM of HEPES (Fisher Scientific).
Isolation of PBL
Heparinized whole blood was diluted 
1:2 with HBSS (Life
Technologies), and the mononuclear cells were obtained by gradient
centrifugation using lymphocyte separation medium (Organon Teknika,
Durham, NC). The mononuclear cells were washed once with HBSS, and
contaminating red cells were lysed with ammonium chloride potassium
buffer (B&B Research Laboratory, Fiskeville, RI). Cells were then
washed with HBSS resuspended in tissue culture medium and kept at 4°C
until used in cellular assays or flow cytometry studies.
CML assays
Four types of CML assay systems were used in these studies.
Primary CML assays. Lymphocyte cultures containing 2 x 106 responder cells (PBL from naive SLAdd, nonthymectomized or thymectomized animals) and 4 x 106 stimulator PBL (SLAgg or third-party PBL, irradiated with 25 Gy) per well in a final volume of 2 ml of medium were incubated for 6 days at 37°C in 7.5% CO2 using 24-well flat-bottom plates (Costar, Cambridge, MA). Bulk cultures were harvested, and effectors were tested for cytotoxic activity on target lymphocytes stimulated by phytohemagglutinin (M-Form, Life Technologies) previously titrated to give optimal proliferation. Target cells were labeled with 51Cr (Amersham, Arlington Heights, IL) and incubated with the effector groups for 5.5 h using a negative control target (SLA-matched to the responders, i.e., SLAdd) and targets SLA-matched to the stimulators, which included donor-matched PBL (SLAgg: class Icc and class IIdd) and third-party stimulators (SLAhh: class Iaa and class IIdd; or SLAaa: class Iaa and class IIaa). E:T ratios of 100:1, 50:1, 25:1, and 12.5:1 were tested. Supernatants were harvested using the Skatron collection system (Skatron, Sterling, VA), and 51Cr release was determined on a gamma counter (Micromedics, Huntsville, AL). The results were expressed as percent specific lysis (PSL) and calculated as: PSL = {[experimental release (cpm) - spontaneous release (cpm)]/[maximum release (cpm) - spontaneous release (cpm)]} x 100%.
Primary CML coculture assays. Control MLC were prepared using 24-well flat-bottom plates (Costar), each well containing 2 x 106 naive SLAdd responder PBL (naive1) and 4 x 106 stimulator PBL (irradiated with 25 Gy) in a final volume of 2 ml of medium. The same source of naive SLAdd-responder PBL were used as indicator cells to detect suppression by PBL from tolerant animals in coculture assays. Similar MLC were prepared using PBL from tolerant SLAdd animals (nonthymectomized or thymectomized). Stimulator cells were donor-matched PBL (SLAgg) or third-party PBL (SLAhh or SLAaa). Cocultures contained 2 x 106 naive SLAdd-responder PBL, 2 x 106 PBL from tolerant animals, and 4 x 106 irradiated stimulator PBL per well in a final volume of 2 ml of medium. Additional cultures containing 4 x 106 responder PBL and 4 x 106 irradiated stimulator PBL were performed for naive and tolerant responder PBL to control for crowding effects due to increased numbers in the cocultures. Cultures were incubated for 6 days at 37°C in 7.5% CO2 and harvested, and effectors were tested for cytotoxic activity as described above.
Secondary coculture assays.
These assays were performed in two sequential phases (Fig. 1
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, and included: 1) PBL from nonthymectomized swine tolerant to
SLAgg renal allografts (non-thyxtolerant-to-G)
or from thymectomized swine tolerant to SLAgg
(thyxtolerant-to-G) primed with SLAgg PBL
(non-thyxtolerant-to-G-G' and
thyxtolerant-to-G-G', respectively) or with
SLAhh PBL (non-thyxtolerant-to-G-H' and
thyxtolerant-to-G-H'), respectively) (Fig. 1A,
left), and 2) PBL from a naive swine (naive2) primed with
SLAgg PBL (naive2-G') or with SLAhh PBL
(naive2-H') (Fig. 1B, left). These primed
effectors were cocultured in phase 2 as the regulator cells.
Phase 2 (coculture phase, day 7–13) was as follows. On day 7, fresh
control MLC were prepared in 24-well plates (Costar) containing 2
x 106 naive SLAdd-responder PBL (naive1 PBL)
and 4 x 106 irradiated stimulator PBL (donor-matched,
SLAgg; or third-party, SLAhh) per well in a
final volume of 2 ml of medium (Fig. 1A, right,
lines 1 and 4; Fig. 1B,
right, lines 1 and 4). The naive1 PBL
were from a different source than the naive SLAdd PBL used
in the priming phase (naive2). The naive1 PBL were used as indicator
cells for the detection of suppression by the primed cells in coculture
assays described below. The primed effectors from phase 1 were also
harvested on day 7 and washed once in complete medium before being set
up in the second phase. Control secondary MLC were performed using both
primed effector groups prepared in the first phase stimulated in a
secondary MLC with the same irradiated stimulator cells used in the
priming phase (Fig. 1A, right, lines 2
and 5; Fig. 1B, right, lines
2 and 5). Cocultures contained 2 x
106 naive1 responder SLAdd PBL, 2 x
106 primed effector cells from phase 1, and 4 x
106 irradiated stimulator PBL per well in a final volume of
2 ml of medium (Fig. 1A, right, lines
3 and 6; Fig. 1B, right,
lines 3 and 6). Control cultures for crowding
effects were set up containing 4 x 106 naive1
responder PBL and 4 x 106 irradiated stimulator PBL,
or 4 x 106 primed effectors from phase 1 and 4
x 106 irradiated stimulator PBL (not shown). Cultures in
the second phase were incubated for 6 days at 37°C in 7.5%
CO2 (day 7–13) and tested for cytotoxicity as described
above. Target cells included PBL matched to the effectors (negative
control) and PBL SLA-matched to the stimulators used in the first and
second phase of the assay.
Modifications of the secondary coculture assay. To examine the mechanism of suppression, some of the primed effector groups prepared with PBL from tolerant and naive animals at the end of phase 1 were manipulated by the following methods before setting up phase 2. 1) Preparation of nonadherent and adherent cells. The primed effector group was divided into two groups. One group of cells (unseparated) was rested overnight by the usual method in 25-cm2 culture flasks (Costar) as described above. The second group of cells was incubated overnight at 37°C in 7.5% CO2 at 4 x 106 cells/ml in 75-cm2 flasks (Costar) lying down flat to allow maximal adherence of cells to the plastic surface. On day 7, nonadherent cells were harvested and washed. Adherent cells were harvested by gentle scraping and then washed. The adherent cells were estimated to be 10% of the original primed effector group. The unseparated, nonadherent, and adherent cell populations of primed effectors from phase 1 were then set up in coculture assays (phase 2). 2) Irradiation of the primed effectors. PBL from tolerant animals primed with SLAgg PBL in phase 1 were either nonirradiated or irradiated with 25 Gy before coculturing with the naive1 PBL. 3) Transwell assays. Primed effectors from tolerant recipients and naive1 PBL were cocultured in equal numbers as described above, either in the same well or separated by a semipermeable membrane (0.4 µm) in 6-well transwells (Costar). Cell cultures included: 2 x 106 naive1 PBL, 2 x 106 PBL from tolerant swine primed with SLAgg, and 4 x 106 SLAgg stimulators per well cocultured in the same well; 2 x 106 naive1 PBL and 4 x 106 SLAgg stimulators per well cultured in the bottom transwell, and 2 x 106 PBL from tolerant swine primed with SLAgg and 4 x 106 SLAgg stimulators per well cultured in the top transwell; 2 x 106 naive1 PBL and 4 x 106 SLAgg stimulators per well in the bottom transwell and medium only in the top transwell; 2 x 106 PBL from tolerant swine primed with SLAgg and 4 x 106 SLAgg stimulators per well in the top transwell and medium only in the bottom transwell. 4) Recombinant human IL-2 (Cetus, Emeryville, CA) was added in titrated concentrations (0–50 U/ml) to the anti-SLAgg-stimulated cocultures of naive1 PBL and PBL from tolerant swine primed with SLAgg PBL. Nonspecific cytotoxicity was assessed by testing on SLAhh in addition to SLAdd targets, and background killing was subtracted to determine the specific cytotoxicity.
Three-color flow cytometry
The following directly conjugated murine anti-pig mAb were used: anti-CD25 FITC-conjugated (mAb 231-3B2, mouse IgG1; 30 , anti-CD4 biotinylated (mAb 74-12-4, mouse IgG2b; 31 , anti-CD8 conjugated to phycoerythrin (PE; mAb 76-2-11, mouse IgG2a; 31 , anti-CD2 FITC-conjugated (mAb MSA-4, mouse IgG2a; 32 , anti-CD3 FITC-conjugated (mAb 2-6-15, mouse IgG2a; Lorf et al., manuscript in preparation), anti-class I FITC-conjugated (mAb 2-27-3a, mouse IgG1; 33 , anti-class II FITC-conjugated (mAb ISCR3/anti-DR, mouse IgG2b; 34 . mAb 36-7-5 (35) FITC-conjugated (anti-murine H2 Kk, mouse IgG2a) and anti-Leu3a PE (Becton Dickinson, San Jose, CA) were used as negative control Abs. For three-color flow cytometry analysis, the staining procedure was performed as follows: 1) 106 cells per tube were incubated with purified swine IgG for 10 min to block nonspecific binding of conjugated Abs; 2) the first incubation was directly FITC-labeled Ab; 3) after a single wash, the second Ab, which was directly PE-labeled, was added; 4) after a further wash, the final biotinylated Ab was added; 5) the biotinylated Ab was developed using cy-chrome-streptavidin (PharMingen, San Diego, CA), which was incubated for 8 min with washes before and after the addition of the cy-chrome-streptavidin. mAb incubations were performed on ice for 30 min. The data acquisition was performed on a FACScan (Becton Dickinson) and analyzed using the Winlist program (Verity Software, Topsham, ME). Four PBL subpopulations were defined by staining for CD4 and CD8 using separate colors: CD4 single positive (SP), CD4/8 double positive (DP), CD4/8 double negative, and CD8 SP PBL. Three-color flow cytometric analysis was performed by simultaneous staining of CD2, CD3, class I, class II, and CD25 with a third color.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Naive animals (positive control) showed strong
anti-donor-matched (SLAgg) and anti-third-party
CTL reactivity in primary CML responses (Fig. 2A). Both nonthymectomized and
thymectomized tolerant animals demonstrated markedly lower antidonor
CTL reactivity but maintained normal third-party responses (Fig. 2A). Unprimed PBL from tolerant swine were cocultured in a
1:1 ratio with naive SLAdd PBL to determine whether the
hyporesponsive PBL from tolerant swine contained peripheral regulatory
cells that could suppress the generation of CTL among naive T cells.
Antidonor and anti-third-party CTL responses of naive animals were
not suppressed in primary coculture assays with PBL from tolerant
animals (compare Fig. 2B with the naive control responses in
Fig. 2A, left).
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We hypothesized that regulatory cells from tolerant animals may
require activation with donor Ag before any suppressive properties
could be observed. Therefore, PBL from tolerant swine, or naive swine
as controls, were prestimulated in culture for 6 days with donor Ag
(SLAgg PBL). After the priming phase, the effector groups
were then rested overnight, harvested, washed, and set up in coculture
assays as outlined in Fig. 1A (lines 1 to
3) and Fig. 1B (lines 1 to
3). Naive1 was a second naive animal that would be used in
the cocultures as indicator cells whose response might be suppressed by
putative regulatory cells from the tolerant animals. Naive1
anti-SLAgg responses (Fig. 1A, line
1 and Fig. 1B, line 1) were the positive
controls. The primed effector groups from the first phase were
stimulated by SLAgg to determine the cytotoxicity of these
effector groups alone (Fig. 1A, line 2 and Fig. 1B, line 2). Cocultures of the primed effectors
with PBL from naive1 were performed to determine whether the
anti-SLAgg responses by naive1 could be suppressed by
the primed effectors (Fig. 1A, line 3 and Fig. 1B, line 3).
The pooled results for the anti-SLAgg responses in
these assays are shown in Fig. 3. As
expected, the naive1 PBL generated strong anti-SLAgg
CTL reactivity when cultured alone (Fig. 3A). These were
similar to the cytotoxic responses of the naive PBL controls in Fig. 2.
When the primed effectors from phase 1 were stimulated a second
time in phase 2 with SLAgg PBL, the
non-thyxtolerant-to-G-G' and
thyxtolerant-to-G-G' effector groups produced minimal
antidonor CTL reactivity, while the naive2-G' effectors generated
strong secondary anti-SLAgg cytotoxic responses (Fig. 3B). Primed non-thyxtolerant-to-G-G' and
thyxtolerant-to-G-G' effectors suppressed naive1
anti-SLAgg CTL reactivity when these two groups of
cells were cocultured (Fig. 3A vs Fig. 3C). In
contrast to primed PBL from tolerant swine, naive2-G' effectors
cocultured with naive1 PBL produced increased
anti-SLAgg reactivity when compared with the
cytotoxicity of each cocultured group alone (Fig. 3C). This
result indicated that the ability to suppress CTL responses of naive
cells was specific for the tolerant animals.
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A, left and Fig. 1B, left). In the second phase, naive1
anti-third-party responses were determined as positive control
assays (Fig. 1A, line 4 and Fig. 1B,
line 4). The primed effector groups from the first phase
were stimulated by SLAhh to determine the cytotoxicity of
the primed effector groups alone (Fig. 1A, line 5
and Fig. 1B, line 5). Cocultures of the primed
effectors with naive1 were performed to determine whether the
anti-SLAhh responses by naive1 were suppressed by the
primed effectors (Fig. 1A, line 6 and Fig. 1B, line 6). The results of the
anti-SLAhh stimulated responses are shown in Fig. 4. There was no inhibition of
anti-SLAhh CTL responses when naive1 was cocultured
with non-thyxtolerant-to-G-H' or
thyxtolerant-to-G-H'or naive2-H' primed effectors (Fig. 4, A and B, vs C).
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In contrast, the suppression observed with PBL from tolerant animals
primed with donor cells was specific for tolerant animals, and also
specific for donor SLA Ag, provided the same stimulator was used in the
priming phase and coculture phase of the assay (Figs. 3 and 4).
Therefore, subsequent experiments were focused on determining the
mechanism of the regulatory response in these assays. For this purpose,
the tolerant recipient that demonstrated the greatest degree of
suppression (thymectomized animal 11446) was chosen for further study.
Suppression of naive PBL in coculture assays was cell-dose dependent
All coculture assays described above were performed with a naive
SLAdd:thyxtolerant-to-G-G' PBL ratio of 1:1. In
this analysis, the concentration of naive SLAdd PBL (2
x 106 cells/ml) was kept constant, and cocultured
with primed thyxtolerant-to-G-G' PBL added at
decremental concentrations (2 x 106 cells/ml to
0.001 x 106 cells/ml). Coculture of naive
SLAdd PBL with primed thyxtolerant-to-G-G'
effector PBL led to a titratable inhibition of the naive
anti-SLAgg cytotoxic response (Fig. 5). Significant inhibition (>50%
inhibition of the naive SLAdd antidonor cell response
alone) was still observed at a thyxtolerant-to-G-G':naive
SLAdd cell ratio of 1:4. At lower concentrations of the
regulator cells, the inhibitory effect was not detectable.
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Since previous studies suggested that miniature swine contain an
adherent cell population with suppressive properties (37, 38), naive
SLAdd PBL (2 x 106 cells/ml) were
cocultured with unseparated thyxtolerant-to-G-G' PBL
(2 x 106 cells/ml) or an adherent cell-depleted
thyxtolerant-to-G-G' effector group (2 x
106 cells/ml) (Fig. 6). The
nonadherent cell population completely inhibited the naive
anti-SLAgg response. Ten percent of the primed
thyxtolerant-to-G-G' effector group were adherent
cells, and when this number of adherent cells (0.2 x
106 cells/ml) was cocultured with naive SLAdd
responders, the naive anti-SLAgg response alone (PSL,
38.5%; E:T ratio, 100:1) was inhibited to a PSL of 25% (E:T ratio,
100:1). Therefore the suppressive effect was predominantly in the
nonadherent cell population.
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A). As controls, similar
cultures were set up with thyxtolerant-to-G-H' effectors
that were either untreated or irradiated with 25 Gy. The results showed
that the nonirradiated thyxtolerant-to-G-G', cocultured
with naive SLAdd PBL, inhibited the naive
anti-SLAgg response as expected. However, the
suppression was completely eliminated when the
thyxtolerant-to-G-G' cells were irradiated before the
coculture (Fig. 7A). In contrast, the
anti-SLAhh cytotoxic response of the naive PBL alone
and the thyxtolerant-to-G-H' primed effector group
alone were augmented when the naive PBL were cocultured with
thyxtolerant-to-G-H' (Fig. 7B). When the
primed thyxtolerant-to-G-H' effectors were irradiated
before the coculture (naive + thyxtolerant-to-G-H' 25
Gy), the cytotoxicity was similar to the cytotoxicity of the naive PBL
alone.
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Separation of the naive SLAdd PBL and the primed
thyxtolerant-to-G-G' cells by a semipermeable membrane
that allowed soluble factors, but not cells, to pass through (6-well
transwell chambers in culture plates) (Costar) was performed to
distinguish the hypothesis that
thyxtolerant-to-G-G' cells required cell-to-cell
contact with the naive PBL to inhibit the generation of CTL as opposed
to the possibility that soluble factors were involved in the mechanism
of suppression. Thymectomized PBL were prestimulated for 6 days in
phase 1 (thyxtolerant-to-G-G'), and subsequently, the
following cultures were established in phase 2, as described in
Materials and Methods: 1) naive PBL and SLAgg
stimulators in the bottom chamber of the transwell and medium only in
the top chamber, 2) thyxtolerant-to-G-G' and
SLAgg stimulators in the top chamber of the transwell and
medium only in the bottom chamber, 3) naive PBL cocultured with
thyxtolerant-to-G-G' and SLAgg stimulators
in the same well, and 4) naive PBL with SLAgg stimulators
cultured in the bottom chamber of the transwell and
thyxtolerant-to-G-G' with SLAgg stimulators
cultured in the top chamber of the transwell. These were all cultured
for 6 days, harvested, and tested for cytotoxicity. The results of one
representative experiment are presented in Fig. 8. As expected, the naive PBL alone
stimulated by SLAgg PBL generated strong
anti-SLAgg cytotoxic responses, while
thyxtolerant-to-G-G' alone stimulated by
SLAgg PBL did not generate anti-SLAgg
cytotoxic responses. Cocultures of naive PBL with
thyxtolerant-to-G-G' and SLAgg stimulators
in the same well led to complete suppression of the naive
anti-SLAgg response as described above. However,
separation of the naive and thyxtolerant-to-G-G' groups
by a transwell restored the anti-SLAgg CTL response of
the naive PBL to the level of the positive control (i.e., CTL response
of the naive PBL cultured alone). These results indicated that
cell-to-cell contact was required for the suppression of the naive PBL
and that soluble factors were not able to mediate suppression between
the transwell chambers. Control assays that examined
anti-SLAhh responses of naive PBL alone,
thyxtolerant-to-G-H' alone, and naive PBL cocultured
with thyxtolerant-to-G-H' effector groups in the same
well or across transwell membranes showed no evidence of suppression
when cocultured in the same well, or across transwells (data not
shown).
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One model for T cell-mediated suppression suggests that
Ag-specific T cells that have been rendered anergic suppress other T
cells with the same specificity by competing for IL-2 and for the
Ag-presenting cell (8). If competition or absorption of IL-2 was a
major mechanism of suppression, the 
-chain of the IL-2R might be
expected to be expressed at increased levels on the
thyxtolerant-to-G-G' effectors stimulated by
SLAgg at the end of phase 2, when compared with the control
thyxtolerant-to-G-H' effectors stimulated by
SLAhh. Flow cytometry demonstrated that
thyxtolerant-to-G-G' anti-SLAgg
effectors, which suppressed the generation of CTL by naive PBL,
expressed lower levels of IL-2R -chain (Fig. 9A) when compared with
thyxtolerant-to-G-H' anti-SLAhh
effectors, which did not demonstrate a suppressed response (Fig. 9B). The greatest difference in expression of IL-2R on the
thyxtolerant-to-G-G' anti-SLAgg
effectors and the thyxtolerant-to-G-H'
anti-SLAhh effectors was observed in the CD8 SP cell
population (Fig. 9, C vs D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Results from previous studies indicated that local regulation or suppression may play a role in the maintenance of tolerance to class I mismatched renal allografts in miniature swine (24, 40). One study demonstrated that the administration of exogenous IL-2 ("help") during the induction of tolerance on postoperative day 8, 9, and 10, led to acute rejection of allografts, suggesting that a limitation of help was an important mechanism leading to the induction of tolerance (24). In contrast, the provision of exogenous IL-2 did not abrogate tolerance in long-term acceptors. Mechanisms of tolerance that may be operating in long-term tolerant swine includes deletion, anergy, or suppression of helper cells and/or CTL. Deletion or anergy of the antidonor helper cells in the maintenance period would lead to failure to activate antidonor CTL. However, since anti-donor precursor CTL are not deleted in long-term tolerant animals (40), the provision of exogenous IL-2 during the maintenance phase would be expected to abrogate tolerance. The findings in the present study support the alternative hypothesis that regulatory cells could inhibit the generation of CTL in vivo. Further studies are required to determine whether the regulation acts at the level of the helper cell or directly on the CTL. However, the concordance of the in vivo data demonstrating that exogenous IL-2 did not abrogate tolerance in long-term acceptors (24) and the in vitro findings that IL-2 was not able to overcome the suppression in vitro in the present study, argues against a deficiency of help as the major mechanism of tolerance in the maintenance phase of tolerance.
The presence of local regulatory cells is supported by studies of cocultures of graft-infiltrating cells from miniature swine that became spontaneously tolerant to single haplotype class I mismatched renal allografts. These cells were able to suppress antidonor CTL responses of naive T cells in vitro (41, 42). However, in contrast to the regulatory cells in the present study, the graft-infiltrating cells did not require preactivation in vitro to demonstrate their inhibitory effect in CML assays, probably because these cells were purified from a site where donor Ag was at high concentration (i.e., directly from the allograft).
Studies in miniature swine have indicated that PBL from tolerant and
naive animals contain an adherent, non-T cell population that is able
to nonspecifically suppress the generation of CTL (37, 38). The data in
the present study demonstrate that the putative regulatory cells in
primed PBL of tolerant swine were distinct from the suppressive
adherent cell population described in the previous reports. Although a
minimal degree of inhibition was evident in the adherent cell
population, profound suppression of the naive antidonor response was
observed in the nonadherent cell population (Fig. 6). A further
differentiating property of the regulatory cells in the current study,
compared with the adherent suppressive cells, was the complete
inactivation of the inhibition by irradiating the
thyxtolerant-to-G-G' effector group with 25 Gy (Fig. 7). The previous adherent suppressive cells were shown to be resistant
to irradiation with 25 Gy but were sensitive to 50 Gy (37). The
suppression observed with the adherent cell population was nonspecific
and observed in naive (and tolerant) animals (43, 44), in contrast to
the suppression observed in the present study in which the inhibition
was demonstrated only in tolerant animals by priming PBL specifically
with donor Ag. Since the tolerance in this large animal model is
specific for donor Ag, it is likely that the nonadherent cells are more
important than the adherent cells in the maintenance of tolerance.
Lysis of APCs in the stimulator cell population of the secondary
cultures could induce apparent suppression by the primed tolerant-G'
effector cells in phase 2 of the CTL assays. Although this possibility
was not formally excluded, this would be an unlikely explanation since
the suppressive primed non-thyxtolerant-to-G-G' and
thyxtolerant-to-G-G' effector groups prepared in phase
1 (Fig. 1A, left) had negligible cytotoxic
reactivity against SLAgg stimulators (e.g.,
anti-SLAgg PSL of 6.0% and 0.14% for two separate
animals). In contrast, suppression of the naive PBL was not observed
when nontolerant animals that generated marked
anti-SLAgg CTL responses were the source of coculture
primed cells in the secondary cultures.
Inhibition of naive PBL responders was not observed when cells were
separated by transwells (Fig. 8). The transwell studies suggest that
either soluble factors (e.g., cytokines) are not involved in the
suppressive effect observed in vitro, or that soluble factors are
operative but only when delivered at high concentrations, requiring
cell-to-cell contact or close proximity of the regulatory cells with
the donor reactive T cell. Alternatively, soluble factors may influence
antidonor T cell responses by exerting their effects on the
Ag-presenting cell. The presence of suppression in some models may
result from differential secretion of T cell-derived cytokines by Th1
and Th2 cells (45, 46). Graft acceptance has been associated with a
preferential shift toward a Th2 response, since Th2 cytokines are able
to inhibit the Th1 program of cytokines (IL-2 and IFN-) consistently
observed in rejecting allografts (47, 48). Most attention has been
focused on CD4+ Th1/Th2 cells, however similar patterns
have been described for CD8+ cells (49). Studies from our
laboratory support the speculation that regulatory cytokines are
involved in the mechanisms leading to acceptance of class I mismatched
renal allografts in miniature swine, since tolerant animals have high
levels of IL-10 gene transcripts locally in the graft, while rejector
animals express high levels of the IFN- transcript (28). This
cytokine pattern would be consistent with the differential activation
of Th1 and Th2 cells in the tolerated allograft. Future studies will
address the cytokine production by these primed regulatory cells, which
could provide important information about soluble mediators of
suppression.
Alternative mechanisms of tolerance may require cell-to-cell contact.
Anergic T cells were shown to suppress Ag-specific T cells in vitro by
competition for locally produced IL-2 and for the Ag-presenting cell
(8). In the current study, prestimulation of PBL from tolerant animals
with donor Ag may have induced anergy of antidonor T cells, and
these anergic T cells were able to suppress the generation of
CTL in the secondary cocultures. However, the hypothesis that
inhibition could be mediated by anergic T cells expressing increased
levels of IL-2R that absorbed IL-2 (8, 50) seems an unlikely
explanation for the suppression observed in the present analysis. The
inhibitory effector group, thyxtolerant-to-G-G'
anti-SLAgg PBL, expressed relatively lower levels of
IL-2R when compared with the nonsuppressive effector groups,
thyxtolerant-to-G-H' anti-SLAhh (Fig. 9). Furthermore, anergy and suppression by anergic cells can generally
be reversed by the addition of exogenous IL-2 (8). The inhibition
by primed tolerant PBL could not be overcome by addition of
exogenous IL-2. The non-thyxtolerant-to-G-G' and
thyxtolerant-to-G-G' effectors could represent anergic
cells that suppress naive PBL by competition for the Ag-presenting cell
(8). The fact that the cell-dose dependency studies demonstrated that
the inhibition was rapidly lost after the ratio of
thyxtolerant-to-G-G':naive PBL was reduced to <1:4,
and that cell-to-cell contact was required for the inhibition, is
consistent with competition for ligand as a mechanism for the
inhibition. A similar result would be achieved if the regulatory cells
described in the current study were anti-idiotypic T cells (11), or
veto cells (17, 18) that would require cell-to-cell contact to mediate
their inhibitory effects.
Activation or maintenance of suppressor cells in mixed lymphocyte reactions has been described in rodent models (15, 51, 52, 53, 54). Regulatory cells generated from MLC were shown to prolong cardiac allograft survival when adoptively transferred in vivo using a rat model (53). Our miniature swine are inbred for the MHC but not for non-MHC loci. Inbreeding of a miniature swine line is currently underway, and adoptive transfer studies will be possible when the inbred line is established.
The data described in the present study is consistent with the hypothesis that regulatory cells are involved in the maintenance of tolerance to renal allografts in miniature swine, and concur with other studies from our laboratory suggesting that local suppressive factors are implicated in mechanism of tolerance (24, 40, 41, 42). Since the immunologic characteristics of miniature swine are similar to those of humans (20), isolation of the regulatory cells identified in the current report could provide important information for establishing donor-specific tolerance in clinical transplantation.
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Acknowledgments |
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Footnotes |
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2 F.L.I. is a recipient of a Don and Lorrain Jacquot Travelling Fellowship awarded by the Royal Australasian College of Physicians and the Australian and New Zealand Society of Nephrology.
3 Address correspondence and reprint requests to Dr. David H. Sachs, Tranplantation Biology Research Center, Massachusetts General Hospital, MGH-East, Building 149-9019, 13th Street, Boston, MA 02129.
4 Abbreviations used in this paper: SLA, swine MHC; CyA, cyclosporin A; thyx, thymectomized; CML, cell-mediated lympholysis; PSL, percent specific lysis; naive-G', PBL from naive swine stimulated with SLAgg PBL; naive-H', PBL from naive swine stimulated with SLAhh PBL; PE, phycoerythrin; SP, single positive; DP, double positive; thyxtolerant-to-G-G', PBL from thymectomized swine tolerant to a SLAgg renal allograft stimulated with SLAgg PBL; thyxtolerant-to-G-H', PBL from thymectomized swine tolerant to a SLAgg renal allograft stimulated with SLAhh PBL.
Received for publication May 8, 1998. Accepted for publication September 8, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T lymphocytes for apoptosis. Nature 353:858.[Medline]
-chain expression on CD8 peripheral blood lymphocytes of tolerant animals. Transplantation 66:454.[Medline]
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