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*
Transplantation Biology Research Center and
Division of Cardiac Surgery, Department of Surgery, Massachusetts General Hospital, Boston, MA 02114;
Repligen Corporation, Needham, MA 02494; and
Laboratory of Immunogenetics and Transplantation, Renal Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
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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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T cell anergy has traditionally been defined as the absence of a T cell response, when the TCR is engaged with an MHC receptor but a second costimulatory signal is not delivered (12). However, the conventional understanding of T cell anergy has been challenged by the recent observations by Lechler and others that anergic T cells can suppress the responses of other T cells (13, 14, 15, 16). Thus, instead of simply not responding, certain anergic T cells can actively inhibit the alloresponses of other naive T cells. For instance, T cell clones rendered anergic either by soluble peptide cultures or immobilized anti-CD3 mAb have been shown to actively suppress the response of other T cell clones to Ags in an MHC-linked fashion (13, 14, 15). The ability of some anergic T cells to actively inhibit the response of other naive T cells represents a novel form of immune regulation and represents a potentially important strategy for achieving immune tolerance. However, the precise nature of this immune regulation is unknown.
The effect of CTLA4Ig in large animals is not well studied (2). We have recently investigated the in vitro effects of CTLA4Ig in functional porcine T cell assays and demonstrated that CTLA4Ig was able to induce Ag-specific T cell hyporesponsiveness in secondary allogeneic pig mixed lymphocyte cultures (3). We refer to these cells as hyporesponsive T cells as opposed to anergic T cells, as there is controversy concerning whether anergy requires signaling through CTLA4 (17). In this report, we extend previous findings by showing that Ag-specific hyporesponsive T (ASHT)34 (3) cells induced by CTLA4Ig in mixed lymphocyte cultures were able to actively inhibit the allogeneic responses of naive, but not primed, porcine T cells. This inhibition occurred in an MHC-linked manner, required cell-cell contact, and appeared to be IL-10 dependent. These results suggest that the profound immunosuppressive effects of CTLA4Ig on T cell alloresponses is mediated in part through the generation of regulatory T cells using IL-10-dependent mechanisms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CTLA4Ig used in these studies was human CTLA4IgG4 purified from plasmid-transformed NSO cells. CTLA4IgG4 is a fusion protein that combines the human T cell surface receptor CTLA4 with the constant region of human (h) IgG4. The extracellular domain of hCTLA4 was cloned as a fusion protein to hinge, CH2, and CH3 domains of a mutant hIgG4 deficient in Fc receptor binding (data not shown). This hIgG4 sequence contains L235G and G237A missense mutations. CTLA4IgG4 cross-reacts with the porcine B7 molecule as previously described (3). Isotype control Abs included h60.1, a humanized anti-CD11b IgG4 that binds human, but not porcine, CD11b.
Animals
The inbred miniature swine used in this study have been described in detail previously (18, 19). Presently, inbred swine of three homozygous MHC (swine leukocyte Ag (SLA) in swine) haplotypes are maintained: SLAa (IaIIa), SLAc (IcIIc), and SLAd (IdIId). In addition, four intra-MHC recombinants of the SLAg (IcIId), SLAh (IaIId), SLAj (IaIIc), and SLAk (IdIIc) haplotypes have been derived by spontaneous recombination events during the breeding of heterozygotes as part of the breeding program. All animal care and procedures were in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animals Resources and published by the National Institutes of Health.
Preparation of PBMC
For preparation of pig PBMC from freshly collected, heparinized whole blood, the blood was diluted 2/3 with HBSS (Life Technologies, Grand Island, NY), and 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 RBC were lysed with ACK lysing buffer (BioWhittaker, Walkersville, MD). Cells were washed again with HBSS and resuspended in complete tissue culture medium.
Mixed leukocyte reaction
Responder PBMC (2–4 x 105) and irradiated (2500 cGy) PBMC (2–4 x 105) were added to 200 µl of complete tissue culture medium in U-bottom wells in triplicate. Complete tissue culture medium for MLR assays consisted of RPMI 1640 (Life Technologies) supplemented with 6% fetal porcine sera (Sigma, St. Louis, MO), 100 U/ml penicillin, 135 µg/ml streptomycin (Life Technologies), 50 µg/ml gentamicin (Life Technologies), 10 mM HEPES (Cellgro, Agawam, MA), 2 mM L-glutamine (Life Technologies), 1 mM sodium pyruvate (BioWhittaker), nonessential amino acids (BioWhittaker), and 5 x 10-5 M 2-ME (Sigma). The cultures were incubated at 37°C in humidified air containing 7% CO2 for 1–5 days. [3H]Thymidine (1 µCi/well; New England Nuclear, Boston, MA) was added for a 5- to 6-h period at the end of the culture on appropriate days. The samples were harvested onto glass-fiber filter mats, and [3H]thymidine incorporation was measured by beta scintillation counting on a liquid scintillation counter. Results are expressed as the mean counts per minute with SE bars.
Secondary MLR and coculture MLR
Secondary MLRs were performed in two culture phases, termed
primary and secondary MLR cultures. In the primary MLR culture (priming
phase, days 0–7), 4 x 106 responder PBMC
and 4 x 106 irradiated (2500 cGy)
stimulator PBMC were preincubated in 2 ml of MLR medium in 24-well
plates (Costar, Cambridge, MA) with either CTLA4IgG4 (50 µg/ml) or
control IgG4 (h60.1; 50 µg/ml) at 4°C for 30 min. Plates were then
incubated for 7 days at 37°C in 7% CO2. On day
7, cells were harvested, washed, and reconstituted in fresh MLR medium.
Cells were then rested for 3 days at 4 x
106 cells/ml in 25-cm2
flasks at 37°C in 7% CO2. The secondary MLR
culture (days 10–14) was initiated on day 10 when the responder cells
were collected, washed, and reconstituted in fresh MLR medium at
appropriate concentrations. Responder cells (2–4 x
105) were replated in 96-well U-bottom plates
with the appropriate irradiated (2500 cGy) stimulators (2–4 x
106) in the absence of Ab unless indicated in the
figure. The secondary MLR cultures were incubated at 37°C in
humidified air containing 7% CO2 for 5 days,
then assayed for [3H]thymidine incorporation as
indicated above. In some assays mouse anti-swine IL-10 mAb
(IgG1
; 5–20 µg/ml; 945A 4C4 37B1, BioSource International,
Camarillo, CA), rabbit polyclonal anti-swine IL-4 mAb (10 µg/ml;
BioSource), swine IFN-
(1000 U/ml; BioSource International), control
mouse IgG1 (MOPC-1; Sigma), or recombinant human IL-2 (20 IU/ml; Cetus,
Emeryville, CA) were added to secondary MLR cultures.
For coculture assays, unirradiated or irradiated Ag-specific hyporesponsive T cells induced by CTLA4Ig (2 x 105) were added to naive or primed responder T cells (2 x 105) in the same well with stimulator cells (2–4 x 105) in 96-well U-bottom plates. For Transwell assays, naive or primed responder T cells (2 x 105) and the appropriate stimulator (2 x 105) were cocultured with ASHT cells (2 x 105) and the appropriate stimulator (2 x 105) was separated by a semipermeable membrane using Anopore eight-well Transwell inserts (0.2 µm; Nalge Nunc, Rochester, NY). Recombinant human IL-2 (100 IU/ml) alone was added to the top well for a positive control, while medium alone was used as a negative control. The cultures were incubated at 37°C in humidified air containing 7% CO2 for 5 days. [3H]Thymidine incorporation was measured as described above. Results are expressed as the mean counts per minute with SE bars.
ELISAs
ELISA kits specific for swine IFN- and IL-10 were purchased
from BioSource International. Supernatants harvested on day 4 of
incubation were tested for IFN- and IL-10 according to the
manufacturer’s instructions. Appropriate standard controls were
tested, and linear regression analysis was performed. Supernatants and
standards were tested in duplicate.
SLA class Ic peptides
One peptide, 25 aa in length, spanning the polymorphic region of
the 
1 domain (aa 3–27) of the pig class I
gene PC1 was purchased from Quality Controlled Biochemicals
(Hopkinton, MA). Peptides were synthesized based on previously
published swine class I sequences (20). The amino acid
sequence of the PC1 peptide used in these experiments (aa 3–27) was
HSLRYFDTAVSRPDRRKPRFISVGY. Peptide purity was >90% as verified by
HPLC and mass spectrometry.
Immunization of pigs with SLA class Ic peptides
Five hundred micrograms of the PC1 peptide (aa 3–27) in 0.25 ml of PBS was injected s.c. in the neck of an anesthetized SLAdd pig in CFA (Sigma; 1/1, v/v).
In vitro peptide proliferation assay
Approximately 2 wk after immunization with the PC1 allogeneic peptide, PBMCs from the peptide-immunized pig were tested against the same PC1 allogeneic peptide (aa 3–27) in 96-well U-bottom plates at 37°C in 7% CO2. Naive syngeneic SLAdd (IdIId) or (SLAcd)F1 (IcIIc x IdIId) nylon wool-adherent PBMCs, used as APCs (2–4 x 105), were preincubated with 50 µg of the class I allopeptide for 2 h at 37°C in 7% CO2. After incubation, peptide-loaded APCs were washed in fresh medium and added to the appropriate wells (2–4 x 105). After 5 days of incubation, [3H]thymidine incorporation was measured as the mean counts per minute as described above.
Flow cytometry
Flow cytometry was analyzed using a Becton Dickinson FACScan
microfluorometer (San Jose, CA). Swine IgG was used to block FcR
binding for porcine cells. Apoptotic cells were detected using TdT- and
FITC-labeled nucleotides (ApopTag; Intergen, Purchase, NY) according to
the manufacturer’s instructions. Biotinylated mouse anti-class
Id (2.12.3) (21) was used to stain
for SLA class Id expression. PE-avidin (Becton
Dickinson) was used as the secondary staining reagent. 74-12-4-PE or
CyChrome Ab was used to stain swine CD4 cells, and 76-2-11-PE or FITC
Ab was used to stain swine CD8 cells (22). Naive
SLAdd PBMCs, irradiated (600 cGy)
SLAdd PBMCs (6 h after irradiation), MLCs
containing naive SLAdd PBMC (4 x
106) and irradiated (2500 cGy) stimulator
SLAcc PBMCs (4 x
106), and cocultures containing CTLA4Ig-induced
hyporesponsive SLAdd PBMCs (4 x
106) and irradiated (2500 cGy) stimulator
SLAcc PBMCs (4 x 106)
were analyzed for apoptosis. Cells taken from culture were analyzed 60
and 80 h after incubation at 37°C in 7%
CO2. For staining, 5–10 x
105 cells/tube of porcine or human cells were
resuspended in 100 µl of HBSS (Life Technologies) containing 0.1%
BSA and 0.1% NaN3 (FACS medium). Ten microliters
of primary or isotype control Ab at 
1 µg/1 x
106 cells was added to the appropriate tubes for
30 min at 4°C. After two washes, a saturating concentration of
secondary Ab was added and incubated for 30 min at 4°C. Cells were
washed with FACS medium twice and then were analyzed by double-color
flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that miniature swine T cells cultured with complete SLA-mismatched stimulators in the presence of CTLA4Ig were hyporesponsive to specific stimulators upon restimulation in the absence of CTLA4Ig, but maintained primary responses to third-party allogeneic stimulators (3). For clarity, we have labeled these CTLA4Ig-modulated T cells ASHT cells (3).
To further characterize ASHT cells that were generated after exposure
to CTLA4Ig, secondary MLRs were performed (Fig. 1
A).
SLAdd
(IdIId) T cells were
incubated with fully allogeneic SLAcc
(IcIIc) stimulators in the
presence of CTLA4Ig or isotype control IgG4 during the primary MLR
culture. After 7 days of incubation, responder cells were collected,
washed, and rested in fresh medium. Subsequently, the responders were
replated in secondary MLR cultures in the absence of any Ab.
SLAdd T cells incubated with allogeneic
SLAcc stimulators in the presence of CTLA4Ig were
hyporesponsive to specific SLAcc stimulators upon
restimulation in the absence of CTLA4Ig compared with responders
incubated originally with control IgG4 (Fig. 1B). This
inhibition was significant compared with the control IgG4 group;
however, a residual proliferative response could be detected on days 2
and 3 of the secondary MLR. This residual response could not be
eliminated in multiple assays, even with higher titrations of CTLA4Ig,
suggesting that either CD28-negative populations of T cells were
responding or alternative pathways of costimulation were being
used.
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A). In fact, the kinetics and magnitude of
proliferation to (SLAac)F1
stimulators mirrored that observed against SLAcc
stimulators, suggesting that the presence of the
SLAc Ag on the APC prevented a primary response
from T cells that would otherwise react to the
SLAa Ag (linked Ag). This inhibition was not
observed when a 1/1 mixture of SLAaa and
SLAcc stimulators (unlinked Ag) was used (Fig. 1A). The proliferative curve against the 1/1 mixture
occurred earlier than that observed with SLAaa
stimulators. Although the reason for this change in kinetics was
unclear, the result was observed consistently in four independent
assays. Perhaps, the residual proliferative response of the ASHT cells
to SLAcc stimulators generated early production
of IL-2 and facilitated an accelerated response of naive cells to
SLAaa stimulators through the action of secreted
cytokines.
In contrast, SLAdd T cells incubated with
specific SLAcc stimulators in the presence of
control IgG4 during the primary MLC showed brisk and vigorous secondary
responses on days 2 and 3 to SLAcc,
(SLAac)F1, and a 1/1
mixture of SLAaa and SLAcc
stimulators (Fig. 1B). The primary response of the control
group to third-party SLAaa stimulators peaked on
day 4 (Fig. 1B). Of note, the lack of a primary response to
SLAac cells was not due to the immunodominance of
SLAc Ags on the
(SLAac)F1 cells, as
SLAdd responders primed with
(SLAac)F1 cells in vitro
responded to both SLAaa cells and
SLAcc cells upon restimulation (data not
shown).
ASHT cells inhibited allogeneic proliferative responses of cocultured naive T cells to specific and linked Ags
One trivial interpretation of these data is that the response of
the bulk culture to third-party SLAaa stimulators
was not due to the proliferation of naive
anti-SLAaa T cell clones present in the bulk
culture, but, rather, was due to cross-reactive responses of the ASHT
cells to SLAaa stimulators. This interpretation
could explain why the response to SLAac resembled
that to SLAcc. To address this concern, we
cocultured SLAdd ASHT cells with naive
SLAdd T cells and tested the allogeneic responses
in a primary MLR. Fig. 2A
shows that the addition of SLAdd ASHT cells
(A-DD, Fig. 2A) in a 1/1 mixture with naive
SLAdd T cells led to inhibition of the
anti-SLAcc response of naive T cells, as no
proliferation was observed on days 4 and 5, whereas a primary response
was observed with naive T cells cultured without ASHT cells.
SLAdd ASHT cells also inhibited naive
SLAdd T cell responses to
(SLAac)F1 stimulators,
which link the specific SLAc Ags to third-party
SLAa Ags on the same cell (Fig. 2B).
In contrast, naive responses to a 1/1 mixture of
SLAaa and SLAcc stimulators
were not inhibited in coculture assays with SLAdd
ASHT cells (Fig. 2B), although the presence of ASHT cells
did accelerate the kinetics of response to the 1/1 mixture of
SLAaa and SLAcc stimulators
as shown in Fig. 1A. The precise reason for this
acceleration was not apparent; perhaps, Ag-experienced cells from the
original culture secreted low levels of IL-2 early in the culture
period. Allogeneic inhibition was observed even when the number of
specific stimulators was doubled during the coculture assays (CC-4,
Fig. 2A), arguing against Ag competition as a mechanism of
inhibition. Naive T cells cultured without ASHT cells responded
normally to (SLAac)F1,
stimulators with peak proliferation on day 5 (Fig. 2B).
Naive T cells cultured with or without ASHT cells did not respond to
third-party SLAaa stimulators alone (Fig. 2B). The early proliferation in response to
SLAcc stimulators in cocultures of ASHT cells
with naive T cells (Fig. 2A) observed on days 2 and 3 was
probably due to the residual response of the ASHT cells, consistent
with the data in Fig. 1A. Thus, ASHT cells were able to
regulate the response of naive T cells to both specific and third-party
MHC Ags provided that the third-party Ags were expressed on the same
cell as the original Ag, i.e., linked regulation.
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We analyzed the phenotype of the ASHT cells after 7 days of
incubation with fully allogeneic stimulators and CTLA4Ig. Double
staining for CD4 and CD8 (22) revealed that 44.1% of the
population were CD8+ T cells, 3.3% were
CD4+ CD8low T cells, and
49.7% were double-positive T cells (Fig. 3A). The simultaneous
expression of CD4 and CD8 Ags by a large population of resting
peripheral T lymphocytes has been well documented in swine (6–60% in
swine vs 0.5–8% in humans) (23, 24). The porcine
CD8+CD4+ T cell subset
contains both mature resting T cells (25) and Ag-activated
memory Th cells (26). Furthermore, after Ag exposure,
single-positive CD4+ T cells in swine may acquire
a double-positive CD4+CD8+
phenotype (27). Consistent with the literature, we found a
6-fold increase (6.8–38.4%) in the double-positive population after
Ag exposure (Fig. 3, C and B). The presence of a
significant population of
CD8+CD4+ T cells in swine
and the dynamic nature of the expression of CD4 and CD8 after Ag
exposure made definitive T cell subset phenotyping of ASHT cells
difficult to interpret.
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To define the specificity of ASHT cells,
SLAdd responders were cultured with either class
I-disparate SLAgg
(IcIId) or class
II-disparate SLAkk
(IdIIc) stimulators in the
presence of CTLA4Ig during the primary MLR culture. After 3 days of
rest, the responder cells were cocultured with naive
SLAdd cells and restimulated in a secondary MLR
culture without Ab using allogeneic stimulators that were linked with
the original Ag or a third-party Ag. Fig. 4A demonstrates that ASHT
cells were generated after incubation of SLAdd
cells with class I-disparate SLAgg stimulators in
the presence of CTLA4Ig during the primary culture. The proliferative
response of cocultured naive SLAdd cells against
(SLAag)F1,
(IaIIa) x
(IcIId), stimulators, which
linked the original class Ic Ag to third-party
SLAaa Ags, was markedly inhibited. However, the
response to (SLAak)F1,
(IaIIa) x
(IdIIc) stimulators, which
presented a new class IIc Ag, was comparable to
that observed with third-party SLAaa and
SLAkk cells.
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B). The primary response of cocultured naive
SLAdd cells against
(SLAak)F1 and
SLAkk stimulators, but not
(SLAag)F1 stimulators, was
inhibited by the SLAdd ASHT cells. The primary
response to third-party SLAaa cells remained
intact. As the specificity of CD8+ and
CD4+ cells is primarily restricted to class I and
class II Ags, respectively, these data suggest that CTLA4Ig can
generate CD8+ and CD4+ (or
CD4+CD8+) ASHT cells that
mediate the linked regulation of naive T cell alloresponses.
Furthermore, the finding that the regulation was MHC class I or class
II specific (a specific characteristic of T cells) supports our
hypothesis that T cells were mediating suppression. Flow cytometric
analysis revealed that the majority of ASHT cells were CD25 negative
(>90%) and CD154 negative (>99%; data not shown), suggesting that
CTLA4Ig did prevent Ag activation. Inhibition mediated by ASHT cells was radiosensitive
We next cocultured naive T cells with irradiated (250 cGy)
SLAdd ASHT cells and tested them against specific
SLAcc stimulators. Fig. 2A
demonstrates that irradiation of the SLAdd ASHT
cells before coculture with naive SLAdd T cells
completely eliminated the inhibition of naive T cell responses to
specific stimulators. Of note, irradiation of the ASHT cells also
eliminated the early proliferation on days 2 and 3, confirming the idea
that the residual response of the ASHT cells to
SLAcc was responsible for the early proliferation
in the cocultures. The inhibition of naive allogeneic responses was
probably not due to overcrowding, as the cell counts of naive cells
cocultured with ASHT and stimulator cells on day 4 were comparable with
those of naive cells cultured with stimulators alone (data not
shown).
Inhibition mediated by ASHT cells was dependent on cell-cell contact
To determine whether this regulatory phenomenon was contingent on
cell-cell contact, coculture assays with Transwells were performed.
SLAdd ASHT cells and SLAcc
stimulators were cultured in the upper wells, while naive
SLAdd T cells were cultured with
SLAcc stimulators in the lower wells. Fig. 5 demonstrates that naive
SLAdd T cells were able to respond to
SLAcc stimulators when the
SLAdd ASHT cells were separated from responders
by a semipermeable membrane. In contrast, when the ASHT cells were
cocultured with naive SLAdd T cells in the
absence of a membrane, full inhibition of the primary allogeneic MLR
was observed (Fig. 5). In addition, no inhibition was observed when
naive SLAdd T cells were tested against
third-party SLAaa stimulators in the lower wells.
For a positive control, exogenous IL-2 (100 IU/ml) was added to the
upper well. This led to maximal proliferation to
SLAcc stimulators on day 5 (Fig. 5). Thus, the
regulation of naive T cells by ASHT cells required cell-cell
contact.
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Next, we investigated whether T cells rendered hyporesponsive by
CTLA4Ig could inhibit the response of in vitro and in vivo primed T
cells. When SLAdd T cells that were primed in
vitro against SLAcc stimulators were cocultured
with ASHT cells in a secondary MLR, the response against specific
SLAcc stimulators at the same or double the
responder concentration (CC-2 or CC-4) was not inhibited (Fig. 6A). Instead, the Ag-activated
T cells displayed primed MLR responses against specific allogeneic
stimulators, with brisk and robust responses on days 2 and 3 after
restimulation (Fig. 6A). These results suggested that,
unlike naive T cells, directly primed T cells were resistant to the
regulation mediated by CTLA4Ig-induced ASHT cells.
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1 domain. These in vivo peptide-immunized
SLAdd T cells were cocultured in a peptide
proliferation assay with SLAdd ASHT cells
(SLAdd hyporesponsive to
SLAcc) and
(SLAcd)F1 stimulators
loaded with PC1 allospecific peptide. Peptide-loaded
(SLAcd)F1 stimulators
presented the specific class Ic Ag linked with
the allogeneic peptide presented in the context of syngeneic class
IId molecules. Fig. 6B shows that
peptide-immunized SLAdd responders demonstrated
vigorous proliferative responses to the specific allopeptide presented
by syngeneic SLAdd cells, confirming
sensitization to the allogeneic peptide (Fig. 6B, left
panel). When, SLAdd ASHT cells were added to
the coculture, the primary response to SLAcc
stimulators by the peptide-immunized SLAdd
responders was inhibited (Fig. 6B, center panel).
However, the response to
(SLAcd)F1 stimulators
loaded with specific class I peptide remained intact (Fig. 6B, center panel). Unimmunized, naive
SLAdd responders failed to respond to
peptide-loaded syngeneic APCs, as expected (Fig. 6B,
right panel). Interestingly, the response of the
peptide-immunized SLAdd cells to
(SLAcd)F1 stimulators
without loaded peptide was higher than the response directed against
SLAcc stimulators, but less than the response
directed against peptide-loaded
(SLAcd)F1 cells. This
suggests that (SLAcd)F1
cells naturally present class Ic peptides in
association with class IId molecules, which is
consistent with previous studies (28). Thus, ASHT cells
were not able to inhibit the allogeneic responses of T cells primed in
vivo through the indirect pathway of Ag presentation, although it is
not known whether this failure of inhibition was due to the fact that
the response was a primed one or an indirect one. Effects of IL-2 on ASHT cells
The absence of a secondary proliferative response to
SLAcc or SLAac stimulators
by SLAdd ASHT cells could be due to deletion of
responding cells, which would not be expected to be sensitive to
exogenous IL-2. To test this hypothesis, IL-2 was added to appropriate
wells during the secondary MLR. The addition of exogenous IL-2 restored
proliferative responses of SLAdd ASHT cells to
specific SLAcc stimulators, with peak
proliferation 4 days postrestimulation (Fig. 7). Exogenous IL-2 also restored the
primary response of the bulk culture to
(SLAac)F1 stimulators,
which presented a linked Ag. In the absence of IL-2, specific
hyporesponsiveness to SLAcc stimulators was
observed (Fig. 7). The kinetics of the proliferative response appeared
to be accelerated by 1 day compared with the normal naive response (cf
Figs. 1A and 7). The reason for this acceleration was not
apparent. Possibly, the ASHT cells were more responsive to exogenous
IL-2 because of previous exposure to allogeneic stimulators. In any
case, the ability of IL-2 to restore the proliferative response to
specific and linked stimulator argues against a deletional mechanism of
T cell regulation. Furthermore, regulation mediated by the ASHT cells
was not simply due to consumption of nutrients and IL-2 within the
medium during the early response of the ASHT cells (Fig. 1A), as supernatants from MLCs were harvested on day 4 and
used to replate primary allogeneic MLRs. Vigorous primary allogeneic
proliferative responses were observed after plating MLRs with the
culture supernatants (data not shown).
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The in vivo administration of CTLA4Ig has been shown to suppress
Th1 responses but spare Th2
responses (7). To ascertain the phenotype of
CTLA4Ig-induced, ASHT cells, we examined the cytokine profile of the
ASHT T cells in thepresence of specific SLAcc
stimulators. Fig. 8A
demonstrates that ASHT T cells secreted sustained low levels of IL-10
(15–25 pg/ml) throughout the culture period, with peak production 2
days after restimulation. The level of IL-10 production was
approximately one-tenth of the amount secreted by primed T cells in
response to specific SLAcc stimulator (Fig. 8B). In contrast, ASHT cells produced only minimal amounts
of IFN- throughout the culture period (Fig. 8C), while
control primed responders generated high levels of IFN- for the
duration of the culture period (Fig. 8D). This cytokine
profile was consistent with a Th2 or possibly a Th3 phenotype
(29). Further analysis of the cytokine profile of the ASHT
cells was precluded by the lack of available swine-specific
reagents.
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Since ASHT cells secreted IL-10, but not IFN-, neutralization
and supplementation MLRs were performed to further characterize the
nature of the T cell regulation mediated by these cells. Fig. 9A demonstrates that the
addition of anti-swine IL-10 mAb (5 µg/ml) to cocultures
containing ASHT cells and naive SLAdd T cells
restored the primary allogeneic response to SLAac
stimulators. The primary response to third-party
SLAaa stimulators was also maintained. In
contrast, anti-swine IL-4 Ab (10 µg/ml), exogenous swine IFN-
(1000 U/ml), and control IgG had no effect on reversing the inhibition
(Fig. 9, B–D). Thus, the immunoregulatory effects mediated
by the ASHT cells appeared to be IL-10 dependent.
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The effects of anti-IL-10 mAb might be contributing to the direct
reversal of hyporesponsiveness, which would, in effect, lead to the
reversal of suppression indirectly. To distinguish whether
anti-IL-10 mAb was directly reversing the suppression mediated by
the regulatory cells or actually reversing the hyporesponsiveness of
the ASHT cells, neutralization MLRs were performed using ASHT cells for
responders and specific SLAcc or linked
SLAac stimulators. If anti-IL-10 mAb were
reversing hyporesponsiveness directly, then a primary MLR should be
detected against both specific and linked stimulators. If, however,
anti-IL-10 mAb were reversing the suppression, then a primary MLR
should be observed for only the linked stimulator. Fig. 10 demonstrates the addition of
anti-IL-10 mAb restored the proliferative response to linked
SLAac stimulators, but not to specific
SLAcc stimulators, while the response to
third-party SLAaa stimulators remained intact.
Thus, anti-IL-10 was effecting the reversal of suppression and not
hyporesponsiveness.
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Another possible hypothesis to explain the mechanism of immune
regulation mediated by ASHT cells is the deletion of naive cells by
either the APC or the regulatory T cells themselves. This would be
similar to a veto-like mechanism found in mice (31, 32).
To address this hypothesis, we examined whether the level of apoptosis
in responder class Id+ cells after coculture with
ASHT cells was increased relative to that in cultures with naive
responders and stimulator cells alone. Two-color flow cytometric
analysis was performed using a mouse mAb specific for class
Id (responder haplotype; 2.12.3)
(21) and TdT- and FITC-labeled nucleotides, which indicate
DNA fragmentation, the hallmark of apoptosis. Fig. 11 demonstrates that the percentage of
responder class Id+ cells that stained brightly
for dNTP-FITC after coculture of naive SLAdd
cells with SLAdd ASHT cells and irradiated
SLAcc stimulators was 3% at 60 h of
culture (Fig. 10D) and 10% at 84 h of culture (Fig. 11F). These levels were actually lower than those observed
after culturing naive SLAdd cells with irradiated
SLAcc stimulators alone (8.5% at 60 h (Fig. 11C) and 30.6% at 84 h (Fig. 11E)).
Irradiated (600 cGy) SLAdd cells were analyzed
after 6 h of incubation for a positive apoptotic control (Fig. 11B), while naive SLAdd cells served
as a negative control (Fig. 11A). The failure to observe a
significant population of apoptotic class
Id-negative cells (i.e.,
SLAcc irradiated stimulators) in the cultures was
probably due to complete cell death and fragmentation by 60 and 84
h of incubation. Flow cytometric analysis revealed very few class
Ic+ cells (<7%) remaining as early as 12 h
postculture using irradiated (2500 cGy) SLAcc
cells (data not shown). Finally, SLAdd ASHT cells
did not lyse specific SLAcc or syngeneic
SLAdd targets in chromium release assays (data
not shown), arguing against a direct cytotoxic mechanism of
regulation.
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The immunoregulatory effects of IL-10 are well documented (30). IL-10 directly inhibits T cell proliferation and cytokine production in response to Ag (34) and indirectly inhibits T cell function through its effects on the APC (30). Regulatory CD4+ T cells cells are characterized by the production of high levels of IL-10, but no IL-4 (29), and IL-10 not only leads to the generation of regulatory T cells, but also serves as a mechanism through which regulatory T cells exert their effect (29). Recently, a surface-bound form of IL-10 was described on human PBMCs (35). The existence of a surface form of IL-10 might explain our seemingly paradoxical finding that anti-IL-10 mAb could reverse the inhibition mediated by CTLA4Ig-induced regulatory cells, yet cell-cell contact was required for the regulatory cells to effect inhibition. Perhaps, the regulatory T cells interacted with APCs and delivered a negative signal through surface-bound IL-10, which led to inactivation of the APC and prevention of a primary response by a naive T cell interacting with that APC. One would expect the effects of this regulation to be very local, since the surface-bound IL-10 would be required to interact with a surface receptor. An alternative interpretation is that the regulatory T cells inactivated naive T cells after direct cell-cell contact with the naive T cell through a veto-like mechanism. While this is formally possible, we think that this is unlikely, as a 1/1 mixture of SLAaa and SLAcc stimulators did not lead to suppression. In bulk culture it seems statistically unlikely that a T cell interacting with SLAaa APC would not also be in contact with a T cell that is recognizing SLAcc APC. Furthermore, no evidence for a deletional or cytotoxic mechanism was found.
To our knowledge, this represents the first report describing the ability of blocking of the B7-CD28 pathway alone with CTLA4Ig to mediate linked regulation of allogeneic T cell responses. Early models of anergic T cells with regulatory properties were based on the use of immobilized anti-CD3 Ab, soluble peptides, or T cell presentation of Ag and were not dependent upon inhibitory cytokines, such as IL-10 (13, 14, 15, 16). Although there are some similarities between these models of immune regulation and our own, our model appears to be distinct because 1) primed cells were resistant to regulation, 2) bulk populations of cells were used to generate regulatory cells and not Ag-specific T cell clones, and 3) linked regulation was dependent upon the actions of IL-10 (13, 14, 15, 16). Blockade of the CD40-CD154 pathway alone has been shown to induce linked suppression to MHC-matched, minor Ag-disparate skin grafts in mice, but anti-CD8 mAb was also required for graft prolongation (36). Very recently, blockade of both CD40 and CD86 pathways was shown to induce regulatory human T cells; however, the degree of inhibition was only partial (up to 60% maximal) compared with the level of inhibition in our studies (37). In the human study regulation required the use of both anti-CD40 and anti-CD86 mAbs (CTLA4Ig was not addressed), while our study showed complete suppression with the use of CTLA4Ig alone. IL-10 also appeared to play a role in the mechanism of regulation in the human study; however, neutralization of IL-10 only partially restored the allogeneic response in that study. These differences suggest that the characteristics of CTLA4Ig-induced regulatory cells and the nature of their regulation are probably distinct from those exhibited by the anergic regulatory T cells previously described.
Evidence for the in vivo generation of a regulatory T cells by CTLA4Ig has been suggested in several rodent models of allotransplantation. The adoptive transfer of CTLA4Ig-treated CD4+ cells along with naive T cells led to donor-specific tolerance of mouse allogeneic islets (38). Rat cardiac allograft recipients treated with CTLA4Ig and donor-specific transfusion led to indefinite graft survival in 50% of recipients (39). Furthermore, the transfer of cells from the CTLA4Ig-treated rats led to infectious tolerance in naive hosts (39). The ability of costimulatory blockade with CTLA4Ig to mediate immune regulation in a linked fashion is reminiscent of previous reports demonstrating linked suppression through the use of nondepleting CD4 Abs in vivo (40, 41). Indeed, linked suppression mediated by regulatory T cells may be a general mechanism of immune regulation that contributes to various models of peripheral tolerance (42).
Our model could also explain the observation that immune modulation by CTLA4Ig often does not require continued administration or persistent circulating Ab, since Ag-specific hyporesponsive T cells induced by CTLA4Ig in our system were able to regulate the responses of naive T cells even after cessation of therapy. In light of the recent phase I clinical trial of CTLA4Ig in psoriasis vulgaris patients (11) and the use of CTLA4Ig to prevent graft-vs-host disease in bone marrow transplant recipients (43), the ability of CTLA4Ig-induced regulatory cells to suppress the naive T cell response could have significant implications for clinical transplantation and therapies to treat autoimmunity (43).
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Acknowledgments |
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2 Address correspondence and reprint requests to Dr. Joren C. Madsen, EDR 105, Department of Surgery, Massachusetts General Hospital, Boston, MA 02114.
3 Abbreviations used in this paper: ASHT cell, Ag-specific hyporesponsive T cell; h, human; SLA, swine leukocyte Ag.
4 We are using the nomenclature of ASHT only to simplify the description of these cells in this paper and are not suggesting that the term necessarily be adopted for use beyond this purpose.
Received for publication June 12, 2000. Accepted for publication November 2, 2000.
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