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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3153-3161
IMMUNOBIOLOGY
Blockade of CD86 and CD40 induces alloantigen-specific
immunoregulatory T cells that remain anergic even after reversal of
hyporesponsiveness
Hans J. P. M. Koenen and
Irma Joosten
From the Department for Blood Transfusion and Transplantation
Immunology, University Medical Center, St Radboud, Nijmegen, The
Netherlands.
 |
Abstract |
The generation of immunoregulatory T cells that block the
B7(CD86/CD80)-CD28 and/or CD40-CD154 costimulatory pathways has great
potential for the induction of long-term transplantation tolerance. In
a human polyclonal in vitro model, combined monoclonal antibody (mAb)
blocking of the costimulatory ligands CD40 and CD86 lead to
allospecific T-cell anergy that cannot be reversed by antigenic
rechallenge in the presence of IL-2. Although antigenic restimulation
with IL-2 restored the proliferative response, subsequent antigenic
restimulation of the restored anergic cells in a tertiary mixed
lymphocyte culture still resulted in nonresponsiveness. Importantly, these anergic T cells suppress the response of naive alloreactive T cells in an antigen-specific way via linked recognition. Suppression may partially depend on local IL-10 production, while transforming growth factor- (TGF-) did not play a role.
Irrespective of the monoclonal antibody combination used, blast
formation occurred in a subset of CD4+ cells. These cells
were characterized by a sustained CD45RA expression, an increased
T-cell receptor density, and a lower level of CD4 expression. A reduced
number of CD45RO+/CD8+ T cells was observed
whenever anti-CD86 was combined with anti-CD40, which was reflected by
an even more attenuated cytotoxic T-cell function. This indicates the
importance of CD40-CD154 in the generation of cytotoxic T cells in this
transplantation model. We hypothesize that in our model, anergy is
induced in the CD4+ T-cell subset, whereby
CD8+ cytotoxic effector function is impaired by the lack
of both CD40-CD154 signaling and cytokine-mediated help. This
costimulatory ligand-directed mAb approach might well be used for the
ex vivo generation of antigen-specific immunoregulatory T cells
applicable in adoptive immunotherapy.
(Blood. 2000;95:3153-3161)
© 2000 by The American Society of Hematology.
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Introduction |
Several regulatory mechanisms are responsible for
controlling homeostasis of immunological responses and maintenance of
tolerance.1 In an attempt to prevent allograft rejection in
an antigen-specific way, many different studies deliberately evoked
these naturally occurring mechanisms to induce and maintain
allospecific T-cell tolerance. Although the mechanisms responsible for
tolerance induction in mature peripheral T cells are not completely
clear, multiple nonmutually exclusive phenomena have been indicated in
the context of transplantation.2 These include
immunological ignorance,3,4 induction of nonresponsiveness
or anergy,5 deletion,6,7 and
immunoregulation.8-13 Immunoregulatory T cells have been
proposed to act via intercellular interactions 9,13 that
are based on competition for antigen-presenting cell (APC) surface
antigens and/or locally produced cytokines.9,14 Maintenance
of the tolerant state by immunoregulatory T cells might be of clinical importance for long-term graft survival.15,16 With regard
to the mitigation of alloresponses in a polyclonal situation, an important aspect of this type of regulation is that alloreactive T-cell
clones, which are made tolerant toward a specific alloantigen, can
potentially down-regulate the response of another T cell that is
directed against a distinct second alloantigen. This is accomplished provided that the antigen is coexpressed on the same APC as the tolerance-inducing antigen. This phenomenon, called linked
suppression,17,18 might in fact be one of the first steps
in a self-sustaining form of tolerance known as infectious
tolerance.12,19
Activation of mature T lymphocytes is a multistep
phenomenon20 requiring both antigen-specific triggering of
the T-cell receptor (TcR) complex on the T cell and additional
signaling via costimulation.21 A key costimulatory signal
results from binding the CD28 receptor on T cells with CD86 (B7-2/B70)
and CD80 (B7-1/BB1) ligands on APCs.22-26 Inhibition of
this pathway in the presence of antigenic stimulation results in T-cell
anergy.20 More recently, the CD40-CD154 (CD40L) pathway was
shown to attribute to the regulation of T-cell activation, both by
independently costimulating T cells and at least in part by
up-regulating CD80/CD86 molecules on APCs.27,28
This knowledge has been successfully used in animal models to prevent
allograft rejection by blocking CD86 and/or CD80,29,30 thereby leading to long-term graft survival.25,31 Others
showed the effectiveness of blocking the CD40-CD154 (CD40L) pathway in this respect.32-36 Combined inhibition of both the B7 and
CD40 pathways showed a synergistic effect on graft survival in both rodent and primate transplant models.4,32,37,38 Human in vitro studies have shown the efficacy of blocking the costimulatory ligands in the induction of T-cell anergy in both
alloresponses24,39,40 and memory T-cell
responses.41
In the present study we elaborate on antigen specificity,
immunoregulatory features, maintenance of anergy, and the phenotype of
human alloreactive T cells, which were made anergic by monoclonal antibody (mAb) blocking of the CD86 and CD40 costimulatory ligands. The
anergic T cells were able to suppress the response of polyclonal alloreactive T cells in an antigen-specific way via linked recognition, which was mediated partially via IL-10. Importantly, the anergic state
was maintained even after restoration of the hyporesponsiveness, which
indicates a profound anergy-inducing protocol. Collectively, these data
support the therapeutic potential of anergic T cells generated by mAb
blocking of CD86 and CD40 in the polyclonal primary human mixed
lymphocyte culture (MLC).
| |
Materials and methods |
Cells
For all experiments, peripheral blood mononuclear cells (PBMCs) were
isolated by density gradient centrifugation (Lymphoprep; Nycomed
Pharma, Oslo, Norway) from buffy coats obtained from healthy blood
donors. The cells were frozen and stored in liquid nitrogen until use.
After thawing, viability of the cells was determined by trypan blue dye
exclusion. All donors were human leukocyte antigen-typed (HLA-typed),
and we developed MLCs that exploited different degrees of matching.
HLA typing
Serological HLA-A, HLA-B, HLA-DR, and HLA-DQ phenotyping (broad
specificities and splits) was performed using the standard microcytoxicity assay. Additional class I and II typing and subtyping was performed by molecular methods. Genomic DNA was prepared using the
QIA-Amp Blood Kit (Qiagen, Hilden, Germany). Low- to
intermediate-resolution typing of HLA haplotypes A, B, DR, and DQ was
performed using a polymerase chain reaction-sequence-specific primers
(PCR-SSP) technique (Pel-Freeze Clinical Systems, Deerbrook Trail, WI). HLA-DRB and HLA-DQB subtyping was performed using another PCR-SSP technique (Dynal DRB1*, B3*, B4*, B5*, and DQB subtyping kits; Dynal,
Oslo, Norway).
Mixed lymphocyte cultures
Primary one-way MLCs were performed by culturing
1 × 105 30 Gy  -irradiated stimulator PBMCs with
1 × 105 responder PBMCs in 96-well round-bottom
plates (Greiner, Frickenhausen, Germany) in 200 µL Roswell Park
Memorial Institute culture medium (RPMI-1640) and glutamax supplemented
with 0.02 mmol/L pyruvate, 100 U/mL penicillin, and 100 µg/mL
streptomycin (all from Gibco, Paisley, England) and 10%
heat-inactivated pooled human serum at 37°C, 95% humidity, and 5%
carbon dioxide (CO2). Proliferation was analyzed by
3H-thymidine incorporation at day 6 of the culture, and
0.037 MBq (1 µCi) 3H-thymidine (ICN Pharmaceuticals,
Irvine, CA; specific activity, 7.4 × 1010 Bq/mmol
[2.0 Ci/mmol]) was present during the last 18 hours.
3H-thymidine incorporation was analyzed by a gas
scintillation counter (Matrix 96 Beta counter; Canberra Packard,
Meriden, CT). The 3H incorporation is expressed as mean
counts per 5 minutes and SD of at least quadruplicate measurements.
Counts per 5 minutes by gas scintillation analysis resemble counts per
1 minute as measured by liquid scintillation analysis. For cytokine
measurements, culture supernatants were harvested on days 3 and 6.
To study the secondary response of allo-MHC-primed (allo-major
histocompatibility complex-primed) T cells, first bulk
primary MLCs were performed by culturing 1 × 106
-irradiated (30 Gy) stimulator PBMCs and 1 × 106
responder PBMCs for 7 days in 24-well culture plates (Greiner) in 2 mL
culture medium. Cells were harvested, washed, and allowed to recuperate
for 2 days. Dead cells were removed by density gradient centrifugation
(Lymphoprep, Nycomed Pharma). Subsequently, 2 × 104
recovered viable cells were restimulated with
1 × 105 -irradiated (30 Gy) stimulator PBMCs in
96-well round-bottom plates. The proliferative response of the
secondary MLCs was examined on day 3, which appeared to be the optimal
time-point.42 Antigen specificity was examined by using
completely HLA-mismatched or partially HLA-matched third-party PBMCs.
For cytokine measurements, culture supernatants were harvested after
48-72 hours.
To investigate the tertiary response, first bulk secondary MLCs were
performed in 24-well culture plates. Accordingly,
2 × 105 responder cells from a bulk primary MLC
(see above) and 1 × 106 -irradiated stimulator
PBMCs were cultured for 5 days with or without 12.5 U/mL IL-2
(Proleukine, Eurocetus, The Netherlands). Responder cells were washed
and allowed to recuperate for 2 days. Subsequently,
2 × 104 responder cells were restimulated with
1 × 105 irradiated stimulator PBMCs in 96-well
round-bottom plates. 3H incorporation was examined at day 3.
Induction of allospecific tolerance in a primary MLC
To generate allospecific anergic T cells in a primary
MLC, mAb directed against 500 ng/mL CD40 (5D12; gift from Dr M. de
Boer, Tanox Pharma, Amsterdam, The Netherlands) and 500 ng/mL CD86
(1G10; gift from Dr K. Lorré) were added, with or without 1000 ng/mL CD80 (M24; gift from Dr K. Lorré, N.V. Innogenetics, Ghent,
Belgium) at the start of the bulk primary MLC. Each of the individual
mAb dose-response titrations was performed, and the optimal inhibitory concentration was selected. In vitro tolerance was defined as hyporesponsiveness after antigen-specific restimulation and the reduced
capacity to perform a specific cytotoxic response.
Cocultures to determine the immunoregulatory potential of
anergic T cells
The regulatory capacity of anergic T cells was analyzed in an in
vitro coculture MLC, and anergic or control cells from a primary MLC
were added to a newly formed MLC. Previously we showed that both
concentration and functional state (eg, irradiated vs living) of the
added or regulatory cells are critical components in assessing the
immunoregulatory capacity.42 Cocultures were performed in
96-well round-bottom plates; 5 × 103 -irradiated (30 Gy) anergic or control cells were added to a newly formed MLC
consisting of both original responder PBMCs
(5 × 104) and -irradiated stimulator PBMCs (2.5, 5, or 10 × 104 PBMCs). All tests were performed in
quadruplicate. Antigen specificity of the regulatory phenomenon was
examined in cocultures performed with third-party stimulator PBMCs that
were either completely HLA-mismatched or partially HLA-matched (with an
isolated class I or class II mismatch) to investigate the possibility
of suppression via linked recognition. Neutralizing mAbs (5 µg/mL)
against IL-10 and TGF- (MAB217 and MAB1835, respectively; R&D
Systems, Minneapolis, MN) were added during the coculture to study the
role of these cytokines. Irrelevant isotype-matched antibodies, which
never abrogated suppression, were used to control for specificity.
To exclude that a bystander suppression occurred, we
examined the effect of anergic T cells on self-restricted recall
responses against tetanus toxoid (RIVM, Bilthoven, Lelystad, The
Netherlands) and Candida albicans extract (ARTHU Biologicals,
Lelystad, The Netherlands). PBMCs (2 × 105) were
cultured with 10 µg/mL antigen in the absence or presence of 5000 (30 Gy -irradiated) anergic or control cells, and the proliferative
response was examined on day 5.
Cytokine assays
Cytokines were measured in culture supernatants. Interferon-
(IFN-), interleukin-4 (IL-4), and IL-10 production were analyzed by
enzyme-linked immunosorbent assay (ELISA) (Pelikine-compact ELISA kit;
CLB, Amsterdam, The Netherlands), and biological active IL-2 was
determined by the IL-2 sensitive cell line (CTLL-2)
bioassay.43 The production of TGF- was measured in
culture supernatants of cells that were cultured in serum-free medium
(Stem Cell Technologies, Vancouver, British Columbia, Canada). Briefly,
soluble type II TGF- receptor (R&D Systems) was used to capture
bioactive TGF-. A standard curve of 10-2500 pg/mL TGF- (R&D
Systems) was used. Detection took place by anti-TGF-1 antibody
combined with biotinylated antichicken immunoglobulin Y (IgY) (Jackson
Immunoresearch, West Grove, PA). Color reaction was performed by a
standard high resolution horseradish peroxidase (HRPO) method
(streptavidin polyHRP mAb, CLB).
Cytotoxicity by chromium 51-release assay
The cytotoxic capacity of primed alloreactive T cells was examined
by chromium 51 (51Cr) release of labeled phytohemagglutinin
(PHA) blasts. Briefly, to generate PHA blasts, PBMCs were first
cultured with PHA-M (Boehringer Mannheim, Mannheim, Germany) and
subsequently with 50 units IL-2/mL. Target cells
(2 × 106) were labeled with 3.7 MBq (100 µ Ci)
51Cr (Amersham) and used as a target at 1000 cells per
well. Different effector/target (E/T) ratios were tested in
quadruplicate. Culture supernatants were examined for released
51Cr on a -irradiation counter (Wallac 1470 -counter;
Wallac, Turku, Finland). Cytotoxic capacity is shown as a percentage of specific lysis calculated according to the following equation, where
CPM means counts per
minute:
Flow cytometry
Cells were phenotypically analyzed by a 2-step double labeling
procedure. Briefly, cells were washed twice with phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA). The
cells were labeled first with unconjugated specific antibody, followed
by conjugate binding with goat antimouse-phycoerythrin (GAM-PE) or
GAM-fluorescein isothiocyanate (GAM-FITC) (Dako, Glostrup, Denmark).
Thereafter the cells were labeled with either CD4 or CD8 antibodies.
All incubations were for 30 minutes on ice, and thereafter the cells
were washed twice. The samples were run on a Coulter Epics XL
Flowcytometer (Beckman Coulter, Fullerton, CA), and 5000 or 10 000
events were collected based on live lymphocyte cell gating, as
indicated by 5 µg/mL propidium iodide staining. Isotype-matched
antibodies were used to define marker settings, and isotype-matched
controls were usually below background staining. Data were analyzed by
Coulter XL-2 software (Coulter Electronics, Miami, FL) and WINMDI
software (Scripps Research Institute, La Jolla, CA). CD4+
and CD8+ T cells in the live lymphocyte gate were analyzed
by the following mAbs: CD3-FITC/PE (Clone UCHT1.7), CD4-PE (MT310),
CD8-PE (DK25), CD14-FITC (TUK4), CD19-PE (HD37 [7mAb]), CD25 (ACT-1),
CD45RA (4KB5), and CD45RO (OPD41) (Dako, brand names noted
in parentheses); WT31 (anti-TcR; Dr W. Tax, Nijmegen, The Netherlands);
and L243 (anti-HLA DR ; American Type Culture Collection, Manassas, VA).
| |
Results |
Anti-CD86 mAb is a powerful inhibitor of the primary MLC, but
additional CD40 blocking attenuates the cytotoxic response
Monoclonal antibodies directed against CD40, CD80, and CD86 ligands
were tested in the polyclonal primary MLC to study their applicability
to the induction of anergy. In this study 6 distinct responder-stimulator combinations were studied; 5 combinations were
mismatched for a single HLA haplotype (ie, 1A,1B,1DR, and 1DQ
mismatch), and 1 combination was completely HLA-mismatched. Monoclonal
antibody combinations that in particular included the anti-CD86 mAb led
to a strong inhibition of proliferation (Figure 1A) and a concordant reduction in IL-2 and
IFN- production (Figure 1B).
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| Fig 1.
Blocking of CD86 inhibits the primary MLC, but additional
CD40 blocking results in a declined cytotoxic response.
Indicated mAbs were added at the start of the MLC. (A) The
proliferative response was determined by 3H incorporation
at day 6 of the cultures. (B) The presence of IL-2 (day 3) and IFN-
(day 6) was analyzed in culture supernatants. (C) Percentage-specific
lysis of allogeneic target cells is shown, in the absence of mAbs, at
different E/T ratios. Effector cells were derived from the control of
mAb-blocked primary MLCs. SD < 10%. Results are expressed as mean
and SE of quadruplicate (A, C) and duplicate (B) measurements.
Representative experiments are shown.
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Figure 1C shows the cytotoxic response of T cells primed either in the
absence (control) or presence of different mAb combinations. Clearly,
mAb blocking of CD86 in the primary MLC reduces the potential to
generate a profound antigen-specific cytotoxic effector response. Notably, although the cytotoxic potential was decreased with all mAb
combinations, the presence of anti-CD40 led to an additional reduction
of the killing capacity.
Thus, alloreactivity in heterogeneous polyclonal T-cell populations
depends merely on the interaction with the CD86 costimulatory ligand
because blocking of this ligand led to a strong reduction in
proliferation, cytokine production, and the induction of cytotoxic effector function. This was found in all HLA combinations tested, irrespective of the degree of HLA mismatch.
Alloantigen priming in the presence of mAbs against
CD86, CD40, and/or CD80 induces anergic T cells
Primary MLCs were performed in the absence (control) or presence of
mAb combinations directed against CD86+CD40, CD86+CD80, and
CD86+CD40+CD80. Viable cells were harvested and restimulated with the
original stimulator cells without mAbs (Figure
2A, B). Control T cells responded with
secondary proliferative kinetics (ie, maximum response at day 3 and
waning in time), while T cells from the mAb-blocked MLCs were
hyporesponsive (Figure 2C).The failure to proliferate was accompanied
by a seriously impaired IL-2 and IFN- production (Figure 2B), while
IL-4 and IL-10 concentrations were around detection level for both
control and hyporesponsive cells. TGF- levels were below detection
level.
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| Fig 2.
Priming in the presence of mAbs induces
hyporesponsiveness.
Primary MLCs were performed for 7 days either in the absence (control)
or presence of mAbs. Primed cells (2 × 104) were
restimulated with the original allogeneic stimulator PBMCs
(1 × 105). (A) Proliferative response at day 3 of
culture by 3H incorporation. (B) IFN- and IL-2
production were analyzed in the culture supernatants. (C) Proliferation
kinetics of secondary response of control and hyporesponsive T cells.
Results are expressed as mean and SE of quadruplicate (A, C) and
duplicate (B) measurements. Representative experiments are shown.
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Lack of priming, possibly caused by the presence of mAbs in the primary
MLC, might explain the hyporesponsiveness. To exclude this possibility,
time response kinetics of the secondary MLC were performed (Figure 2C).
If the T cells had been neglected during the primary MLC, restimulation
would have led to proliferation with primary kinetics (ie, optimal
proliferation at day 6). Restimulation of T cells from an mAb-treated
MLC showed neither a secondary nor a primary response.
Hyporesponsiveness was not due to deletion; antigenic restimulation in
the presence of exogenously added IL-2 led to a comparable response of
control and anergic T cells (Figure 3A).
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| Fig 3.
Restored hyporesponsive T cells remain anergic.
(A) Primed control or hyporesponsive T cells
(2 × 104), induced by different mAb combinations,
were restimulated with the original stimulator PBMCs
(1 × 105) in the presence of 12.5 U/mL exogenous
IL-2. The proliferative response was examined on day 3. (B) Control and
hyporesponsive T cells derived from a primary MLC were recovered with
antigen in the presence or absence of exogenously added IL-2 and
subsequently restimulated for the second time in a tertiary MLC. The
3H incorporation of this first restimulation is shown in
Table 1 (SE < 10%). Next, 2 × 104 recovered
cells were restimulated for a second time with
1 × 105 stimulator PBMCs in the absence of IL-2.
The proliferative response was examined on day 3. Results are expressed
as mean and SE of quadruplicate measurements. Representative
experiments are shown.
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Thus, combined mAb blocking of the costimulatory ligands in the primary
polyclonal MLC induces genuine T-cell hyporesponsiveness or anergy,
which is not the result of ignorance or cell death. The 3 distinct mAb
combinations led to a similar state of hyporesponsiveness.
Reversal of T-cell hyporesponsiveness by exogenous IL-2
and alloantigen does not result in anergy reversal
Various studies have described the potential of exogenously added
IL-2 in recovering the proliferative response of anergic T cells in
vitro.24,44-47 After tolerance induction we consequently analyzed the antigenic restimulation in the presence of added IL-2.
Figure 3A shows the proliferative response of anergic T cells after
antigenic restimulation either in the absence or presence of
exogenously added IL-2 and the response to IL-2 alone. The presence of
both IL-2 and alloantigen resulted in reversal of the hyporesponsive
state, while only a residual response was observed with either antigen
or IL-2 alone. This indicates that hyporesponsiveness can be restored
only if the antigen and IL-2 are present at the same time. Next, we
addressed the tertiary proliferative response (second restimulation) of
anergic T cells that were first restored with IL-2 and alloantigen in a
secondary MLC (Figure 3B). Surprisingly, these restored anergic T cells
were still nonresponsive during a subsequent encounter with alloantigen
in a tertiary MLC, indicating that anergy reversal did not occur.
Collectively, these data show that anergy is maintained even after
reversal of hyporesponsiveness by antigenic restimulation in the
presence of IL-2.
The anergic state is alloantigen-specific and not dependent on
the original APC source
Antigen specificity of the anergic T cells was studied by
restimulation experiments using third-party stimulator PBMCs that were
either fully HLA-mismatched or partially HLA-matched (match of either
HLA class I [B] or class II [DR]), with the stimulator PBMCs
originally used for priming. Primed control cells proliferated solely
when restimulated with the original PBMCs or third-party PBMCs that
shared an HLA class II (DR) antigen with the original stimulators
(Figure 4A). No antigen-specific reaction
was found against third parties that were either completely
HLA-mismatched or shared only a class I (B) locus antigen with the
original stimulator cells. As expected, the anergic T cells did not
respond irrespective of the third-party HLA type.
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| Fig 4.
Anergic T cells are alloantigen-specific.
(A) Control or hyporesponsive T cells (2 × 104)
were restimulated with 1 × 105 specific or
third-party stimulator PBMCs that were either completely mismatched or
partially matched, ie, in this case, DRB1*0401 (DR4) and B*4901 (B49).
(B) IL-2 restored the proliferative response of anergic T cells solely
when antigenic restimulation was performed with the specific stimulator
PBMCs or the third-party stimulator PBMCs with a shared DR type.
Results show the mean 3H incorporation and SE of
quadruplicate measurements on day 3 of culture. (C) The cytotoxic
response is HLA class I specific. Specific lysis (E/T = 100) is shown
against allogeneic target cells, which are either completely mismatched
(white bars) or shared MHC antigens (crossed bars) against which the
responder cells were generated (here B49 and DR4). Results are
expressed as the percentage of specific lysis. Representative
experiments are shown.
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To prove the antigen specificity of the anergic T cells, they were
antigenically restimulated in the presence of exogenous IL-2 (Figure
4B) as described previously. Hyporesponsiveness was restored solely
when antigenic restimulation, in the presence of IL-2, was performed
with either the original or third-party stimulator PBMCs with a shared
HLA class II type. Thus, anergic T cells were shown to be
alloantigen-specific by restimulation with selected third-party
stimulator PBMCs expressing the appropriate target HLA in the presence
of IL-2. The responsiveness of T cells toward third-party stimulator
cells that share antigenic determinants with the original stimulator
cell is known as linked recognition.11,12,17,18 The fact
that this phenomenon was observed only for the DR locus match and not
for the B supports the notion that proliferative and cytokine responses
in a MLC are mainly driven by class II.48
Whereas HLA class II mismatches play a major role in proliferation and
cytokine production in our experimental setup, the generation of
cytotoxic effector T cells appeared to be exclusively induced against
HLA class I mismatched antigens, and no cytotoxic response against
isolated HLA class II molecules was observed (data not shown). To
elucidate the antigen specificity of tolerized cytotoxic T cells, the T
cells were tested for their ability to kill third-party target cells
that were either completely mismatched or partially matched for HLA
class I. Target cells matched by HLA class I were lysed by the
alloprimed control T cells, while effector T cells from an mAb-blocked
MLC left them untouched. Completely mismatched third-party targets were
affected by neither control nor anergic cells (Figure 4C). Together
these cytotoxicity data indicate that priming in the presence of mAb
blocking results in disabled HLA class I specific cytotoxic T-cell function.
Immunoregulation of anergic T cells via linked recognition
The capacity of anergic T cells to affect a specific alloimmune
response was studied in an in vitro coculture MLC; anergic cells were
cultured together with a newly performed primary MLC. Previously, we
reported on the kinetics of cocultures and showed that this type of
coculture has to be performed with low numbers of irradiated anergic
cells.42 Furthermore, relative suppression was compared
with cocultures of irradiated primed control cells.
Anergic T cells were generated in the primary MLC according to the
different mAb tolerizing regimens described above. Figure 5A shows the effect of coculturing anergic
cells. The relative suppressive effect of the cocultures evoked by the
anergic cells was compared with that of cocultures with added
irradiated control cells (Table
1). Although different levels in
suppression were found, sometimes up to 60% of inhibition was found in
the following ratio: responder PBMCs:stimulator PBMCs:anergic T cells
(5:5:0.5 or 5:2.5:0.5). In general, the suppressive effect became more apparent when the number of stimulator cells was decreased, which suggests the importance of competition between anergic T cells and the
responder T cells for antigenic determinants. Antigen-specificity of this suppressive phenomenon was investigated in cocultures using
original responder cells and third-party stimulator cells that were
either completely HLA-mismatched or shared HLA antigens with the
original stimulator PBMCs (anergy-inducing antigens).
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| Fig 5.
Allospecific anergic T cells suppress naive alloreactive
T cells via linked recognition mediated partially by IL-10 and do not
confer bystander suppression.
(A) Anergic T cells suppress their specific primary MLC, as seen in 3 different representative experiments. T-cell anergy was induced by mAb
blocking in the primary MLC (see legends). Next,
5 × 103 irradiated anergic T cells were cocultured
with a newly formed MLC using 5 × 104 responder
PBMCs and 2.5-5 × 104 (experiments I and II,
respectively) or 5-10 × 104 (experiment III)
stimulator PBMCs. Control cocultures were performed with control cells
that were primed and processed in a way similar to that of the anergic
cells but in the absence of mAbs. (B) Anergic T cells mediate
suppression via linked recognition. We cocultured
5 × 103 -irradiated anergic T cells with a newly
incubated MLC consisting of 5 × 104 responder PBMCs
and 2 and 5 × 104 -irradiated stimulator PBMCs.
The stimulator PBMCs were either allospecific (left) or third-party
PBMCs being completely HLA-mismatched (middle) or partially HLA class
II matched (right) with the original stimulator cells. (C) Anergic
cells do not confer bystander suppression. Cocultures were performed by
adding 5 × 103 anergic or control cells to a
culture of 2 × 105 responder PBMCs in the presence
of 10 µg/mL tetanus toxoid or C albicans (right). As a
control, these anergic and control cells were cocultured with a newly
formed MLC consisting of 5 × 104 responder PBMCs
and 5 × 104 -irradiated stimulator PBMCs (left).
(D) Suppression partially depends on IL-10. Cocultures consisting of
5 × 103 anergic or control cells,
1 × 105 responder PBMCs, and
5 × 104 -irradiated stimulator PBMCs were
performed in the presence of 5 µg/mL TGF- and/or IL-10
neutralizing antibodies or an isotype-matched antibody. Anergic T cells
used in the experiments shown in (C, D) were generated by blocking CD40
and CD86. In all figures the proliferative response is shown as the
mean 3H incorporation and SE of quadruplicate measurements
on day 6 (except for recall responses, which were analyzed on day 5) of
the cocultures.
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Figure 5B shows that only the partially matched third-party MLCs were
suppressed to a similar level as the original specific MLC, whereas the
completely mismatched third-party MLCs were hardly affected. In the
described polyclonal system, this implies that recognition of the
specific target antigen on the surface of a third-party APC by these
anergic T cells enables them to suppress the primary reaction of
neighboring T cells. Thereby distinct alloantigens are recognized,
provided that these are present on the same APCs as the anergy-inducing
antigens. This mechanism of immunoregulation has been referred to as
linked suppression.11,12,17,18 To exclude the fact that
bystander suppression occurred, recall responses against tetanus toxoid
and C albicans were studied in the presence of either anergic
or control T cells. The proliferative response against these antigens
was left unaffected by the anergic T cells (Figure 5C). This indicates
that anergic allospecific T cells do not interfere in a nonspecific
manner in self-MHC-restricted T-cell responses.
IL-10 and TGF- have been identified important immunosuppressive
cytokines.9,10,14 To elucidate the role of
these cytokines, neutralizing antibodies against IL-10 and/or TGF-
were added to the cocultures of naive alloreactive cells, stimulator
PBMCs, and anergic or control T cells. In contrast to anti-TGF,
anti-IL-10 antibodies partially prevented suppression by the anergic T
cells (Figure 5D). The addition of anti-TGF- together with
anti-IL-10 antibodies did not affect the level of suppression caused
by IL-10 alone (Figure 5D). This indicates that IL-10, but not TGF-,
might play a role in the suppression by the anergic T cells. In some control experiments, anti-IL-10, anti-TGF-+IL-10, or
isotype-matched control antibodies themselves led to a small reduction
of the proliferative response.
Collectively, these coculture data show that anergic T cells generated
in the primary polyclonal MLCs by mAb blocking of costimulatory ligands
are able to suppress alloreactive T-cell responses directed to multiple
alloantigens, provided these antigens are coexpressed on the same APCs
as the anergy-inducing antigens. Suppression was at least partially
mediated by IL-10, and nonspecific bystander suppression was not
observed. The distinct mAb-tolerizing regimens yielded T cells with
comparable suppressive capacity, as measured in our system.
Phenotypical analysis of tolerized T cells
T cells derived from either control or mAb-blocked MLCs were
phenotypically analyzed by flow cytometry. Cells were harvested after 7 days of culture, allowed to recuperate for 2 days, and analyzed. From a
primary control MLC, 2 clear CD4+/CD3+ T-cell
populations emerged with distinct forward scatter characteristics (Figure 6A, B). A similar distribution
pattern was found after mAb blocking, and no difference was observed
between the different mAb-blocking combinations. However, the
blast-like large-sized CD4+ T-cell population had markedly
reduced cell numbers (Figure 6A). Notably, the large-sized T cells from
the mAb-blocked MLC showed an increase in TcR expression and a decrease
in CD4 expression (Figure 6B) as compared with large-sized T cells from
a control MLC. Irrespective of the presence or absence of mAbs in the
primary MLC, the majority of the blast-like CD4+ T-cell
population was CD45RO+, but in the case of the mAb-blocked
MLC, a clear relative increase in CD45RA+ large-sized
CD4+ cells was observed (Figure 6B). Large-sized
CD4+ T cells from both the control and mAb-blocked MLCs
were HLA class II positive, and they generally expressed CD25 to
similar levels (not shown).
View larger version (3742K):
[in this window]
[in a new window]
| Fig 6.
Phenotypical analysis of tolerized T-cell populations.
After a 7-day primary MLC in the absence or presence of mAbs, the
responder lymphocytes were harvested, allowed to rest, and analyzed by
flow cytometry. (A) Contour plots showing forward scatter (linear
scale) and either CD4 (upper 2 panels) or CD8 (lower 2 panels)
expression (fluorescence log scale) in the live lymphocyte gate. The
left panel shows T cells from a control MLC, and the right panel shows
T cells from an mAb-blocked MLC. (There were no observed differences
between mAb combinations). The percentages in the upper 2 panels
indicate the relative number of CD4+ T cells with a large
blast-like appearance, while in the lower 2 panels, the total
percentage of CD8+ T cells is indicated. (B) The upper
panel shows the size difference in a forward scatter histogram. The 3 lower panels show the expression of CD4, TcR (WT31), and CD45RA on the
large-sized backgated CD4+ T-cell population, which was
derived from either a control (shaded histogram) or an mAb-blocked MLC
(open histogram). The histograms show the number of events on the
vertical axis (lin scale) and fluorescence intensity on the horizontal
axis (log scale). (C) CD8+ cells from an MLC with
simultaneous blocking of CD86 and CD40 showed a decrease in the number
of CD45RO expressing cells and CD45RO intensity. Additional CD80
blocking had no effect. Cells were derived from either a control MLC or
an mAb-blocked MLC, where the indicated mAbs were present. The contour
plots show the fluorescence intensity of CD45RO on CD8-expressing T
cells. Note that the Y axis shows a log scale from 102 to
104, which indicates that all cells shown are
CD8+.
|
|
The CD8+ population from both the control and mAb-blocked
MLCs comprised 1 uniformly sized population, with similar
CD8+ cell numbers and CD8 expression levels (Figure 6A).
CD8+ cells derived from an MLC blocked with anti-CD86+CD40
or anti-CD86+CD40+CD80 showed a reduced number (and expression) of
CD45RO+/CD8+ T cells, which was not observed in the CD8+ T
cells from the CD80+CD86 blocked MLC (Figure 1C). This indicates the
importance of the CD154-CD40 interaction in the activation of
CD8+ T cells and might explain the relatively higher
cytotoxic response of T cells derived from a CD80+CD86 MLC as compared
with T cells from an mAb-blocked MLC where CD40 was also blocked
(Figure 1C). These data support an important role for the CD154-CD40
pathway in the generation of cytotoxic effector function in the human polyclonal MLC, as was recently demonstrated in murine
models.49-51
In addition, phenotypical analysis excluded the presence of putatively
tolerizing APCs (ie, the presence of B cells or monocytes with
cell-surface bound anti-CD80, anti-CD86, and/or anti-CD40 mAbs) that might interfere during restimulation and would inadvertently lead to the observed hyporesponsiveness (data not shown).
| |
Discussion |
Alloantigen-specific immunosuppression is one of the main goals in
preventing graft rejection. Here we demonstrate the ex vivo generation
of anergic allospecific T cells from a primary polyclonal MLC by mAb
blocking of CD86, CD40, and/or CD80. These anergic T cells have an
antigen-specific immunoregulatory function because they are able to
suppress the response of naive alloreactive T cells via linked
recognition. Importantly, although hyporesponsiveness of these T cells
was recovered by antigenic restimulation with exogenous IL-2, this did
not extend to anergy reversal; nonresponsiveness was still observed in
a tertiary MLC, implying that anergy was maintained. Anergic
immunoregulatory T cells generated by this anti-CD86+CD40-based
tolerizing protocol might be a putative tool for antigen-specific
adoptive immunotherapy in transplant medicine.15
During the anergy induction phase, there was a strong inhibition of the
primary MLC, especially by anti-CD86. This dominant effect, sorted on
proliferation and cytokine production, might be explained by the
constitutive expression of CD86 on the majority of APCs. Activation
through costimulatory ligands also appeared to be essential for the
induction of allospecific cytotoxic effector T-cell function because
combined mAb blocking of both CD86 and CD40 in the primary MLC resulted
in a strongly affected cytotoxic response. This inability was not the
result of differences in CD8+ T-cell numbers, but rather it
reflects an intrinsic defect caused either directly or indirectly by
the lack of help. Moreover, although minimal cytolytic activity was
found after anti-CD86 blocking of the primary MLC, the cytotoxic
response was even more attenuated after additional blocking with
anti-CD40 mAb. This fits the notion that the CD40-CD154 pathway
actively contributes to the induction of cytotoxic effector
function.49-51 Of interest here is the change observed in
CD45RO expression in the CD8+ population; CD8+
cells from a CD40+CD86-blocked MLC clearly showed a decreased number
of CD45RO+ cells, which suggests that CD40-CD154 ligation
delivers an important signal for differentiation into cytotoxic
effector cells.
The large-sized, blast-like CD4+ T cells derived from the
mAb-blocked MLCs all expressed the CD45RO marker, indicating that activation occurred. In contrast to the CD4+ blastoid cells
from the control MLC, blast-like CD4+ T cells from the
tolerized MLCs had higher CD45RA numbers, which might be characteristic
of anergized cells in our model. These large-sized tolerized
CD4+ T cells are indulged to spot antigen, as judged by
their increased TcR expression. At the same time, however, the decrease
in CD4 coreceptor density might result in the loss of proliferation. This is reminiscent of the data described by Madrenas et
al52 showing that partial activation is the result of
inefficient CD4 recruitment to the TcR.
Recovery of the proliferative response of anergic T cells by
exogenously added IL-2 was previously demonstrated in distinct experimental settings, albeit using 2 distinct approaches. Either anergic T cells were restimulated in the presence of
IL-2,24,44 as in our experiments, or alternatively, anergic
T cells were first left in culture medium with exogenously added IL-2
only and subsequently restimulated.45,46 We show that
hyporesponsive T cells were recovered by antigenic restimulation in the
presence of IL-2. This, however, did not lead to reversal of the
anergic state as such because a subsequent antigenic restimulation of these recovered cells in a tertiary MLC still left these cells nonresponsive, and an anergy persisted. This particular characteristic was also found in a T cell to T cell (T-T) presentation
model,53 revealing the importance of the absence of
costimulation in this type of anergy.
The implications of this recovery-sensitive persistent anergy for the
in vivo situation are as yet speculative, but it might serve the
purpose of specific tolerance induction after transplantation. After
the initial period of trauma, the transplanted organ itself could serve
as a source to maintain tolerance, and there would be little risk of
reversing the anergic state of circulating anergic allospecific T cells
upon encounter with IL-2 far from the site of transplantation (in the
absence of antigen). The proliferative capacity of the anergic cells
could be reestablished only if local inflammation occurs and IL-2 is
produced at the transplant site. However, because these cells remain
dependent on IL-2, suppression of this particular cytokine would
restore the tolerant state.
It is difficult to demonstrate antigen specificity of anergic T cells
in a polyclonal MLC with stimulator cells carrying many potential
target antigens. To circumvent this problem we used the characteristics
of anergic cells to recover their response upon antigenic restimulation
in the presence of exogenously added IL-2. We reasoned that if the
anergy is antigen-specific, this state can be recovered only by
third-party stimulator PBMCs that share a specific HLA with the
stimulator cells which were used for anergy induction. In our
experimental system it appeared that the proliferative response was
directed against class II and not class I antigens, and consequently
that anergy was induced against the former. This is not entirely
surprising because we and others48 have obtained evidence
that proliferation and cytokine production in a primary MLC are mainly
driven by the class II antigens. An isolated class I mismatch appears
to be an insufficient trigger for a substantial response to take place
during a standard 7-day MLC.
Recently, it was shown that in vivo anergized T cells displayed a
phenotype of regulatory cells which were not able to proliferate. Nevertheless, the cells produced high levels of IL-10 after in vitro
stimulation.54 Others also showed that IL-10 and TGF- were generated in anergic or regulatory T-cell
subsets.10,14,39,55 In our model we did not observe
elevated IL-10 or TGF- production after restimulation of the anergic
T cells. This is probably the consequence of the limited number of
anergic cells per well due to the heterogeneous cellular composition in
our polyclonal system. In fact only a small percentage of the T cells
present will have specificity for the stimulator cells used, and
therefore the bulk will be left untouched. However, IL-10 did play a
role in suppression; neutralizing IL-10 antibody counteracted the
suppression caused by the anergic T cells. Apparently very small
amounts of IL-10, which confer a suppressive function in the
microenvironment comprising anergic T cells, naive alloreactive T
cells, and stimulator PBMCs, are produced.
Nonspecific suppression in a system like this can occur in several
ways. Previously we have reported on the kinetics of this in vitro
coculture model. It appeared that the addition of more than
1 × 104 control cells (live or irradiated)
prohibited the response of the responding cells.42
Irradiation proved necessary because primed T cells respond with
secondary kinetics upon subsequent encounter with the antigen and thus
prohibit a newly cultured primary MLC simply by consuming culture
nutrients and overcrowding. Consequently, to detect an
immunoregulatory phenomenon in a primary MLC, small numbers of
irradiated anergic or control cells were cocultured with freshly
isolated responder and irradiated stimulator cells.
Others40 demonstrated suppression of primary MLCs with large numbers of nonirradiated anergic T cells, and their anergic T
cells:responder PBMCs:stimulator PBMCs ratio had a setting of 3:1:1 × 105 compared with our setting of
5 × 103:5 × 104:2.5-5 × 104.
This raises questions about the mechanism and specificity of the
suppression described by this group.40 In addition, even when using small numbers, we and others56 observed that
control T cells added to a culture can affect the response of naive T cells in a nonspecific manner, therefore the antigen-specific immunoregulatory effect can only be deduced after comparison with appropriate control cultures. Consequently, we compared cocultures of
anergic cells with antigen-primed control T cells and showed that
HLA-specific suppression occurs. The anergic T cells suppressed naive
responder T cells directed toward third-party HLAs only when
third-party stimulators shared an HLA with the stimulator PBMCs used
for anergy induction. This antigen-specific manner of suppression has
previously been shown to occur locally via linked
recognition,11,12,17,18 and it implies that suppression will occur when direct contact is obtained between T cells of different
specificities.9,13 Lombardi et al9 showed that local competition for the antigen binding site might be one of the
suppressive mechanisms; indeed reduction of stimulator APC numbers in
our study led to an increase in suppression in most cocultures.
Furthermore, specificity of the response in this kind of system is not
always easy to confirm because linked recognition might also lead to
third-party tolerance via minor antigens.17
Finally, from our model we hypothesize that blocking the CD86-CD28
pathway induces anergy in the CD4+ T-cell subset, which as
a consequence provides insufficient cytokine mediated help for complete
activation of CD8+ cytotoxic effector T cells. The
CD40-CD154 interaction predominantly controls the activation of
CD8+ cytotoxic T cells in a direct way because the
cytolytic response was attenuated by mAb blocking of CD40.
| |
Acknowledgment |
We are indebted to Alwin Scharstuhl for analyzing TGF-.
| |
Footnotes |
Submitted August 23, 1999; accepted January 9, 1999.
Reprints: Irma Joosten, Department for Blood
Transfusion and Transplantation Immunology/OV603, University Medical
Center, St Radboud, 6500 HB Nijmegen, The Netherlands; e-mail:
i.joosten{at}utdts.azn.nl.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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J. K. Davies, J. G. Gribben, L. L. Brennan, D. Yuk, L. M. Nadler, and E. C. Guinan
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Top
Abstract
Introduction