|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on December 19, 2002; DOI 10.1182/blood-2002-07-2076.
TRANSPLANTATION
From the Institut national de la santé et de la
recherche médicale (INSERM) U 437, Nantes,
France; the Institut de Transplantation et Recherche en
Transplantation (ITERT), Nantes, France; the Centre
Hospitalier Universitaire de Nantes, Nantes, France; and
the Gene Therapeutics Research Institute Cedars-Sinai Medical Center,
Los Angeles, CA.
Costimulatory blockade using cytotoxic T lymphocyte-associated
antigen 4 immunoglobulin (CTLA4Ig) efficiently down-regulates immune responses in animal models and is currently used in autoimmune and transplantation clinical trials, but the precise cellular and
molecular mechanisms involved remain unclear. Rats that received allogeneic heart transplants and were treated with adenoviruses coding
for CTLA4Ig show long-term allograft survival. The immune mechanisms
regulating induction of long-term allograft acceptance were analyzed in
splenocytes using mixed leukocyte reactions (MLRs). MLRs of splenocytes
but not purified T cells from CTLA4Ig-treated rats showed higher
than 75% inhibition compared with controls. Splenocytes from
CTLA4Ig-treated rats inhibited proliferation of naive and
allogeneically primed splenocytes or T cells. MLR suppression was
dependent on soluble secreted product(s). Production of soluble
inhibitory product(s) was triggered by a donor antigen-specific stimulation and inhibited proliferation in an antigen-nonspecific manner. CTLA4Ig levels in the culture supernatant were undetectable and
neither interleukin-10 (IL-10), transforming growth factor Therapies targeted to inhibit activation of
recipient T cells can induce specific tolerance to donor allografts in
rodent models.1 Depending on the model used, different
mechanisms such as deletion, anergy, and suppression have been proposed
to account for the induced tolerance,1 but the precise
cellular and molecular pathways involved are not completely understood.
Activation of T cells specific for donor alloantigens is central to the
development of allograft rejection. T cells become activated by a first
set of T-cell receptor (TCR)-mediated stimulatory signals following
recognition of peptides derived from donor alloantigens and by a second
set of costimulatory signals.1 Both types of signals are
originated from interactions of T cells with antigen-presenting cells
(APCs). In transplantation, donor antigens are presented both by APCs
from donor and recipient origin (by direct and indirect antigen
presentation, respectively).2
Immature or mature dendritic cells (DCs) can act as initiators both of
immune responses to foreign antigens and of tolerance to
self-antigens.3 In allotransplantation, administration of immature DCs or CD8 Regulatory CD4+ T cells with immunosuppressive function
constitute a heterogeneous group of cells for which the relationship to
each other and their underlying molecular mechanisms of action still
need to be clarified.16-24 Regulatory/suppressor
CD8+ T cells have also been
described.12,25
Suppressor CD8+ T cells12 or anergic
CD4+ T cells19,26 can inhibit allogeneic
immune responses through modulation of DC function indicating a mutual
regulation between regulatory T cells and
DCs.3,16,27,28
CD80/CD86 interaction with CD28 plays a central role in T-cell
costimulation and development of immune responses.29
Blocking this pathway has allowed dramatic improvement in
transplantation and autoimmune experimental models and in clinical
studies.29,30 The use of cytotoxic T
lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) or
anti-CD80 or CD86 monoclonal antibody (MAb) as sole treatment
has resulted in long-term allograft survival in many animal models, but
evidence of immune tolerance has been described in only some of these
models.31,32 Little is known about the mechanisms
underlying immune tolerance induced by CTLA4Ig blockade of
CD80/CD86-CD28 costimulation in vivo. Blockade of CD80/CD86-CD28 or
CD40-CD40L interactions in vitro induces regulatory T
cells15,21,22 and suppressive macrophages20
against alloantigens.
We have previously reported that prolonged exposure to CTLA4Ig via gene
transfer allowed long-term cardiac allograft survival in
rats32 and that this survival was associated with
acceptance of second cardiac grafts from first- but not third-party
donors. The present study aimed to analyze in vitro the cellular and
molecular mechanisms underlying the inductive phase of this in
vivo tolerance.
Animals, heart transplantation, and gene transfer with an
adenovirus encoding the extracellular domain of mouse CTLA4 fused to
the Fc portion of human IgG1 (AdCTLA4Ig)
AdCTLA4Ig and the noncoding adenoviral vector Addl324 were
purified and injected into the heart as previously
described.32,33 Rats treated with AdCTLA4Ig and Addl324
are defined as the CTLA4Ig-treated and control groups, respectively.
Cell purification
APCs were enriched from spleen fragments digested with collagenase D as
described.32,34 DCs were further purified (> 90% as
assessed using MAbs specific for major histocompatibility complex [MHC] class II antigens, T cells, B cells, and macrophages)
by positive selection using the OX62 MAb ( MLR cultures LEW.1A responder cells from naive (did not receive transplant), CTLA4Ig-treated, or Addl324-treated (control) rats that underwent transplantation were seeded (105 cells/well) onto round-bottomed 96-well plates (Nunc, Roskilde, Denmark) in triplicate and evaluated for their proliferative response against -irradiated
(35 Gy) APCs (5 × 104 cells/well) from LEW.1W, Brown
Norway (BN, haplotype RT1n), or F1 hybrids
(LEW.1W × LEW.1A).
For coculture MLRs, splenocytes, T cells, or DCs from CTLA4Ig-treated or control rats (5 × 104 cells) were added to splenocytes or T cells from naive LEW.1A rats (5 × 104 cells) and irradiated LEW.1W or BN APCs (5 × 104 cells) in round-bottomed 96-well plates. For transwell MLRs, the lower and upper compartments of 24-well plates were separated by a 0.45-µm pore size membrane (Becton Dickinson, Le Pont de Claix, France). The lower compartment contained splenocytes from CTLA4Ig-treated or control rats (6 × 105 cells) and irradiated APCs (3 × 105 cells). The upper compartment contained naive or primed splenocytes from untreated LEW.1A rats (6 × 105 cells) and irradiated APCs (3 × 105 cells). Cells were cultured at 37°C in humidified air containing 5% CO2 for 3, 4, and 5 days, and, for the final 8 hours of culture, 1 µCi (0.037 MBq) and 5 µCi (0.185 MBq) [3H] thymidine deoxyribose ([3H]Tdr) were added to each 96- and 24-well plate, respectively. For experiments performed in 96-well plates, [3H]Tdr was directly assessed in the 96-well plates and results were expressed as the delta mean cpm ± SD after subtraction of proliferation in the presence of culture medium. For transwell cultures, [3H]Tdr was assessed after transferring cells into 96-well plates and results were expressed as counts per minute. Modulation of MLRs by bioreagents MLRs were cultured in the presence of the following mouse MAbs at 25 µg/mL: anti-rat interleukin (IL)-4 (OX81; ECACC, Wiltshire, United Kingdom), anti-transforming growth factor 1 (TGF1; clone 2G7),38 agonistic anti-CD28
(JJ31939; T. Hüning, University of Würzburg,
Germany), anti-human IL-13 (Diaclone, Besançon, France), and an
irrelevant control (3G8, anti-human CD16; American Type Culture
Collection, Bethesda, MD). Rabbit neutralizing anti-rat IL-10
and rabbit IgG (both kindly provided by J. Khalife, Institut Pasteur,
Lille, France) were used at 10 µg/mL. The extracellular domain of
tumor necrosis factor (TNF)-related activation-induced cytokine
(TRANCE) fused to the extracellular domain of human CD8 (TRANCE-CD8; kindly provided by Y. Choi, University of Pennsylvania, Philadelphia, PA) was used at 10 µg/mL. The supernatant of COS cells
transfected with a coding plasmid (provided by Y. Choi)40 was used as a source of the fusion molecule CD40L-CD8.
NG-monomethyl-L-arginine (L-NMMA) and its
inactive analog, NG-monomethyl-D-arginine
(D-NMMA; Sigma) were diluted (5 mM) in culture medium. Human
recombinant IL-2 was used as indicated in most experiments at 100 U/mL,
or 500 U/mL in others.
Enzyme-linked immunosorbent assays (ELISAs) CTLA4Ig was detected in culture supernatant using a sandwich ELISA (sensitivity, 1 ng/mL) as described.32 Cytokines were analyzed in duplicate serially diluted supernatants harvested on day 3 MLRs. IL-10 (PharMingen, Franklin Lakes, NJ), IL-13, IL-2, interferon (IFN) (Biosource, Camarillo, CA), and
TGF1 (Promega, Madison, WI) were analyzed using ELISA kits according to manufacturers' instructions. The ELISA for IL-4 was performed using
the OX81 and the biotinylated B11-3 (PharMingen) anti-IL-4 MAbs, and
recombinant rat IL-4 (PharMingen) was used as a standard. The ELISA
sensitivity was 0.015 ng/mL.
Flow cytometry Cells were stained with the following biotin or fluorescein isothiocyanate (FITC)-conjugated MAbs: anti-CD3 (G4.18; PharMingen), anti-CD11b/c (OX42),41 anti-CD4 (W3/25), anti-CD8 chain (OX8), antimonomorphic class II MHC antigens
(OX6), anti-CD25 (OX39), anti-CD8 chain (3.4.1), anti-CD161, and
anti-CD161 (3.2.3) (ECACC); anti-CD4036 (PharMingen); a
mixture of anti-CD80 (3H5) and anti-CD86 (24F) (PharMingen); anti-CD28
(JJ319); anti-CD40L (AH.F5; kindly provided by C. Benjamin, Biogen,
Boston, MA); and an irrelevant mouse MAb (3G8, antihuman CD16). Binding
of CTLA4Ig was revealed with a biotin-conjugated rat IgG-absorbed
F(ab)'2 donkey anti-human IgG Fc antibody (Jackson Laboratories, West
Grove, PA). Biotin or FITC-labeled mouse IgG1 was used as negative
control (Immunotech, Marseille, France). Binding of biotin-labeled
antibodies was detected by incubation with phycoerythrin
(PE)-conjugated streptavidin (Immunotech). A FACScalibur
cytofluorimeter was used and data were analyzed using CellQuest
software (Becton Dickinson, Mountain View, CA).
Statistical analysis Student t test was used for group comparisons; P values less than .05 were considered significant.
Lack of allogeneic proliferation of splenocytes, but not T cells, from animals treated with AdCTLA4Ig We have previously shown that treatment of LEW.1A rats with CTLA4Ig through gene transfer using recombinant adenoviruses results in long-term (> 150 days) survival of LEW.1W heart allografts, and that this was associated with a lack of MLR proliferation at day 3 of culture.32 The results in Figure 1 confirm these previous results and show that MLRs using splenocytes of CTLA4Ig-treated rats were still profoundly decreased on days 4 and 5 of culture compared with that of splenocytes from control rats treated with a noncoding adenovirus. Lack of allogeneic proliferation was observed against first- and third-party alloantigens (Figure 1A,C).
Unlike total splenocytes, purified splenic T cells from the same CTLA4Ig-treated rats had similar proliferation against first- and third-party alloantigens compared with that of control animals (Figure 1B,D). Allogeneic proliferative responses of splenocytes are triggered by alloantigens presented by both donor and recipient APCs (direct and indirect antigen presentation, respectively), whereas proliferation of purified splenic T cells is triggered by only donor APCs. Therefore, these results suggest that recipient APCs inhibited T-cell proliferation against alloantigens. Lack of allogeneic proliferation of splenocytes from animals treated with AdCTLA4Ig is not due to indirect antigen presentation The inhibition of MLRs using splenocytes from CTLA4Ig-treated rats may be due to either an inhibitory activity of recipient APCs or to the development of regulatory T cells by indirect alloantigen presentation by recipient APCs, as previously described.42To evaluate this possibility, we performed MLRs using as stimulators APCs from F1 animals derived from crossing LEW.1W and LEW.1A rats, and T cells and splenocytes from LEW.1A CTLA4Ig-treated rats as responders. APCs from F1 rats express LEW.1W MHC molecules (direct alloantigen presentation) and display LEW.1W allopeptides in the context of LEW.1A MHC molecules (indirect alloantigen presentation). If indirect alloantigen presentation was responsible for activation of the recipient regulatory T cells, proliferation of T cells against F1 APCs should be inhibited. As expected, splenocytes from CTLA4Ig-treated rats did not proliferate in response to F1 APCs (Figure 1E). Importantly, T-cell proliferation against F1 APCs was not inhibited and was comparable with that of T cells from control rats (Figure 1F), thus ruling out the possibility that indirect alloantigen presentation was responsible for inhibition of proliferation. We conclude that in vivo costimulatory blockade through treatment with CTLA4Ig resulted in a modification of APC-T-cell interactions and the suppression of T-cell proliferation by recipient APCs. Inhibition of MLRs by soluble inhibitory products Transwell culture experiments were performed to determine whether the lack of proliferation in response to alloantigens depends on production of soluble products or cell contact. Proliferation of naive MLR was assessed in the upper compartments of transwell plates in the presence, in the lower compartment, of splenocytes from control or CTLA4Ig-treated animals that underwent transplantation, cultured either with medium (nonstimulated) or irradiated donor or third-party APCs. These experiments showed that proliferation of naive MLRs was similar in the presence of unstimulated splenocytes from control or CTLA4Ig-treated animals (Figure 2A). In contrast, proliferation was inhibited in the presence of splenocytes from CTLA4Ig-treated animals stimulated with APCs from donor (Figure 2B) but not from third-party origin (Figure 2C). The same transwell MLR experiments using naive purified T cells also showed decreased proliferation in the presence of MLRs from splenocytes of CTLA4Ig-treated rats (data not shown). Similarly, MLRs using naive splenocytes were inhibited by more than 90% upon addition of 50% cell-free culture supernatant of MLRs from CTLA4Ig-treated rats, in which CTLA4Ig was undetectable (data not shown). This suggests the presence of soluble inhibitory product(s) other than CTLA4Ig. Proliferation against third-party APCs in the upper compartment was inhibited in the presence of MLRs using splenocytes from CTLA4Ig-treated rats and donor APCs (Figure 2D).
Altogether these results show that stimulated splenocytes from CTLA4Ig-treated rats are able to inhibit the allogeneic proliferation of naive splenocytes and that this is due to the presence of inhibitory soluble products. The production of inhibitory products is induced by donor-specific alloantigens and has an antigen nonspecific bystander inhibitory activity. Inhibition of naive MLRs is abrogated by splenocyte irradiation and is exerted on allogeneically primed splenocytes and T cells To evaluate the effect of irradiation on the inhibitory function of splenocytes from CTLA4Ig-treated animals, MLRs using naive splenocytes were cocultured with irradiated (35 Gy) splenocytes from CTLA4Ig-treated or control rats. The inhibition of proliferation of naive MLRs by coculture with splenocytes from CTLA4Ig-treated animals (Figure 3A) was reverted by prior irradiation (Figure 3B).
We then asked whether inhibition of MLRs would also be exerted on in vivo allogeneically primed cells. Proliferation of splenocytes (Figure 3C) and T cells (Figure 3D) against APCs from donor origin was inhibited when cocultured with splenocytes from CTLA4Ig-treated animals. Similarly, proliferation of allogeneically primed T cells against donor APCs was inhibited in transwell experiments (data not shown). Thus, splenocytes from CTLA4Ig-treated animals are capable of inhibiting not only naive but also allogeneically primed T cells in coculture and transwell culture systems. Inhibition of MLRs of splenocytes from CTLA4Ig-treated rats is dependent on NO production and reduced IL-2 synthesis T-cell proliferative responses can be inhibited by NO.43 Addition of L-NMMA to MLRs against donor antigens performed in 96-well plates resulted in a small increase in proliferation of splenocytes from CTLA4Ig-treated rats on days 3 and 4 of culture. However, their responses were still lower than those of splenocytes from control rats treated with L-NMMA, by 75% and 95% on days 4 and 5 of culture, respectively (Figure 4A).
The lack of proliferation of splenocytes from CTLA4Ig-treated animals against third-party APCs in the presence of D-NMMA was completely reverted in the presence of L-NMMA (Figure 4B). Similar to the results obtained in 96-well plates, experiments in transwell cultures showed that the inhibition of naive MLRs by splenocytes from CTLA4Ig-treated rats was not reverted by addition of L-NMMA (Figure 4C). These results indicate that in the absence of NO, inhibition of proliferation of splenocytes from CTLA4Ig-treated rats was LEW.1W antigen-specific and dependent on other soluble inhibitory products. Although NO played a role in the inhibition of MLRs when splenocytes from CTLA4Ig-treated rats and naive splenocytes were in close proximity (coculture), this was less prominent in transwell cultures (probably due to the short half-life and range of action of NO44), indicating that additional inhibitory soluble molecules were produced upon donor antigen-specific stimulation. The simultaneous production of NO and additional inhibitory soluble molecule(s) may also account for the apparent paradox whereby donor antigen-specific inhibition of MLRs was observed in transwell MLRs, but not in 96-well MLR experiments, in which both donor and third-party responses were inhibited. We then asked whether concomitant production of NO and limited IL-2 production could account for lack of proliferation observed for splenocytes from CTLA4Ig-treated recipients. Simultaneous addition of IL-2 and L-NMMA to MLRs performed in transwell plates increased proliferation of both CTLA4Ig-treated and control splenocytes that showed comparable proliferation on days 3 and 5 of culture (Figure 4D). Compared with MLRs from control splenocytes (n = 7), supernatants
harvested at day 3 of culture from MLRs performed with splenocytes from
CTLA4Ig-treated rats (n = 7) showed significantly decreased levels of
IL-2 (535 ± 62 pg/mL vs 287 ± 71, P = .022) and
IFN Thus, production of NO and inhibition of IL-2 production by soluble inhibitory product(s), are responsible for the reduced donor-specific T-cell proliferative responses of CTLA4Ig-treated animals. Supernatants harvested at day 3 of culture from MLRs performed with
splenocytes from CTLA4Ig-treated rats (n = 4) or controls (n = 4)
contained comparable levels of IL-13 (7-20 pg/mL), IL-10 (50-125 pg/mL), and TGF Phenotype of splenocytes from CTLA4Ig-treated rats The percentages of CD3+, CD4+, CD8+, B, and CD161+ natural killer
(NK) cells among splenocytes from CTLA4Ig-treated rats were similar to those observed in control rats (data not shown). The phenotype of CD3+ T cells and CD11b/c+ cells
(macrophages and DCs)41 was analyzed in 2-color
immunofluorescence. Splenocytes from CTLA4Ig-treated rats contained
fewer CD3+CD25+ cells compared with control
splenocytes (Figure 5) and
CD4+CD25+ cell population was similarly reduced
(data not shown). CD3+CD40L+ and
CD3+MHC-II+ cell populations were also reduced
in splenocytes from CTLA4Ig-treated rats compared with control
splenocytes (Figure 5). Expression of CD28 (Figure 5) or of CD45RC
(data not shown) was identical among CD3+ cells from
CTLA4Ig-treated rats and controls.
The percentages of CD11b/c+ cells were comparable between splenocytes from CTLA4Ig-treated rats and control animals. CD11b/c+ cells expressed lower levels of CD40, MHC class II, and B7s molecules (Figure 5). Analysis of CTLA4Ig binding to different splenocyte populations showed that CD11b/c+ (Figure 5) but not CD3+ (data not shown) cells displayed CTLA4Ig on their surface. Thus, both T cells and CD11 b/c+ cells from rats that underwent transplantation and were treated with CTLA4Ig expressed levels of activation molecules lower than those on the corresponding cells from control animals. Activation of T cells and/or DCs restores allogeneic proliferation of splenocytes T cells and APCs from CTLA4Ig-treated rats showed a phenotype of resting cells, and we reasoned that inhibition of allogeneic proliferation could be reverted by T and/or APC activation. Therefore, we evaluated MLRs in the presence of L-NMMA to block NO production with or without added IL-2 and anti-CD28 agonistic antibodies to effectively stimulate T cells and/or TRANCE-CD8 or CD40L-CD8 to stimulate APCs.Stimulation of T cells through CD28 enhanced proliferation of
splenocytes from CTLA4Ig-treated animals at days 3 and 5 of culture,
but this was 25% to 50% lower than that of control cells similarly
treated. In the presence of anti-CD28 and IL-2 the proliferation of
splenocytes from CTLA4Ig-treated animals was comparable with those of
control cells in the same conditions (Figure
6).
Stimulation of APCs using TRANCE-CD8 or CD40L-CD8 enhanced proliferation of splenocytes from CTLA4Ig-treated animals to levels comparable with those of control cells, but with increased proliferation at day 5 for CTLA4Ig versus day 3 for control cells. This increase of proliferation was enhanced in the presence of IL-2 and followed the same kinetics as observed in its absence (Figure 6). Simultaneous stimulation of T cells and APCs using anti-CD28 and TRANCE-CD8 or CD40L-CD8 resulted in proliferation of CTLA4Ig-treated cells comparable and higher at days 3 and 5 with that of control cells. Addition of IL-2 further enhanced this phenomenon (Figure 6). These results indicate that inhibition of allogeneic proliferative responses against donor APCs by splenocytes from CTLA4Ig-treated rats is reverted by T-cell and/or APC costimulation. DCs from CTLA4Ig-treated rats inhibit MLRs To characterize the type of cells responsible for inhibition of MLRs of splenocytes from CTLA4Ig-treated rats we performed coculture experiments in which naive MLRs were evaluated in the presence of T cells or DCs purified from splenocytes of CTLA4Ig-treated rats. Proliferation of naive cells on day 5 of culture in the presence of L-NMMA was inhibited by more than 50% by coculture with total splenocytes from CTLA4Ig-treated animals, but not with cells from control animals that underwent transplantation (Figure 7A). Importantly, coculture with DCs from CTLA4Ig-treated rats but not from control rats in the presence of L-NMMA resulted on day 5 in more than 70% inhibition of MLRs (Figure 7B). DCs also inhibited proliferation of naive T cells from 2 of 3 animals (data not shown). T cells purified from splenocytes of CTLA4Ig-treated rats did not inhibit MLRs (Figure 7C).
Thus, DCs from CTLA4Ig-treated rats are capable of inhibiting naive T-cell proliferation in response to alloantigens.
Our results indicate that in vivo treatment with CTLA4Ig in a transplantation model promotes development of donor-specific tolerogenic mechanisms. Immune tolerance is critical for the prevention of autoimmunity and maintenance of immune homeostasis. The mechanisms involved in immune tolerance could be exploited to induce tolerance in pathologic conditions such as graft rejection and autoimmune diseases.2,3 The in vitro activation of T cells in the absence of costimulation
results in T-cell anergy,45 the development of regulatory T cells,20-22 and suppressive
macrophages,15 but the tolerogenic effects of
costimulatory blockade in vivo are still ill-defined. One potential
mechanism is the development of cells capable of inhibiting immune
responses. These cells can be T cells or APCs, as both cell types are
capable of reciprocal regulation resulting in
immunosuppression.2,10,16 To our knowledge, the results reported in this work represent the first detailed analysis of tolerogenic cells induced by CTLA4Ig treatment in vivo. Our
observations suggest that in vivo blockade of the B7/CD28 pathway with
CTLA4Ig can result in tolerance to alloantigens by at least 2 mutually nonexclusive mechanisms. First, binding of CTLA4Ig to DCs results in
the generation of tolerogenic DCs. Second, CTLA4Ig disrupting normal
APC-T cell interactions results in the development of regulatory T
cells, which induce tolerogenic DCs. It has already been shown that in
vitro blockade of B7/CD28 costimulation induces generation of
alternatively activated macrophages through IL-10 production by T
cells.15 Similarly, T cells can regulate DC function;
anergic T cells19,26 and T
CD8+CD28 The generation of regulatory T cells or APCs inducing tolerance and not
pathogenic immune responses depends on factors influencing APCs-T cells
interactions, such as the type and level of costimulatory signals or
cytokines expressed by each cell type and presented antigenic
determinants.3,9-11,26 The development of tolerogenic DCs
has been described after in vitro treatment with IL-10, corticoids, vitamin D3, low doses of granulocyte-macrophage
colony-stimulating factor, TNF Central thymic tolerance is mediated by DCs, and it has been suggested
that certain forms of peripheral tolerance, either by deletion, anergy,
or suppression, are mediated by them.3,13,53 The
prevailing view is that steady-state or immature DCs expressing lower
amounts of surface MHC and costimulatory molecules tolerize T cells,
whereas activated or mature DCs, expressing higher levels of surface
MHC and costimulatory molecules induce immune
response.2-6,8 However, recent evidence shows that mature
DCs can also generate tolerance and that the overall response
(tolerance vs immune response) depends on the balance between yet
unidentified tolerogenic signals and CD40-CD40L
signaling.5,13,14 Tolerogenic DCs with NK characteristics
generated in vivo after blockade of CD40-CD40L signals have been
described only very recently in a diabetes autoimmune model.54 Tolerogenic macrophages or DCs are functionally
characterized by increased expression of scavenger receptors,
anti-inflammatory mediators (PGE2 and IL-10),3,9,10,48,55
immunoglobulin-like transcript 3 and 4 (ILT3 and ILT4)12
and IDO,56 reduced expression of IL-1 Regulatory T cells have been isolated in vivo from naturally occurring CD4+CD25+ cells16 or after treatment with oral tolerance protocols (Th3),24 anti-CD4,1,16,58 or donor-specific blood transfusion.25 In vitro development of regulatory T cells has been obtained using costimulation blockers20-22; soluble antigen or immobilized antibody to CD317-19; allogeneic immature DCs6; IL-10-treated DCs46; or IL-10-treated T cells (Tr1).23 The fact that inhibition of proliferation by cells from CTLA4Ig-treated rats was triggered in a donor alloantigen-dependent manner argues either for regulatory T cells being implicated or for the generation of tolerogenic DCs of donor origin, but further work is needed to explore these possibilities. IL-10-producing T cells are implicated in the generation of suppressive macrophages after in vitro blockade of MLRs by CTLA4Ig.15 Despite the fact that addition of exogenous IL-2 was needed to abrogate inhibition of splenocyte proliferation, suggesting the induction of anergy, T cells from CTLA4Ig-treated rats proliferated in response to alloantigen rechallenge when isolated. The existence of other tolerogenic mechanisms is possible. Proliferation may proceed normally through the first rounds of cell division and effector mechanisms may be gradually reduced by an increase in cell apoptosis, as recently demonstrated in other in vitro13 and in vivo53 tolerance models induced by DCs. Alternatively, other CD4+ or CD8+ T cell functions, such as cytokine production or cytotoxic activity, may be down-regulated in CTLA4Ig-treated rats. Previous publications have shown that the activity of regulatory T cells is thwarted by cell irradiation in certain21 but not all19,20,26 models. Irradiation (35 Gy) abrogated the inhibitory activity of splenocytes from CTLA4Ig-treated rats. This irradiation dose can result in not only blockade of proliferation but also in cell death.59 Therefore, inhibition of MLRs by splenocytes from CTLA4Ig-treated rats is dependent on at least cell metabolic activity and/or proliferation but not on passive release of preformed molecules. These results raise the question of whether the inhibitory activity on naive MLRs could be due to culture overcrowding by splenocytes primed in vivo by donor antigens. The fact that splenocytes from control animals that underwent transplantation did not show this effect, that inhibition was cell-contact independent, and that the suppressive effect of splenocytes from CTLA4Ig-treated rat versus responding naive splenocytes was observed with ratios as low as 1:5 (data not shown) argue against this hypothesis. Bystander inhibition of immune responses was observed as splenocytes
stimulated with donor cells inhibited proliferative responses against
third-party antigens. This phenomenon is in keeping with the
observation that inhibition depended on the secretion of soluble products. Inhibition of immune responses by secreted products has been
described in vitro for Tr1 cells and Th3 cells (producers of IL-10 and
TGF Inhibition of NO enabled partial and complete restoration of
proliferation against donor and third-party alloantigens, respectively. NO is produced by macrophages and DCs,44,55 and its
production is decreased in alternatively activated macrophages
generated by B7/CD28 blockade.15 NO inhibits T-cell
proliferation by interfering with the IL-2R intracellular signaling
pathway without affecting expression levels of IL-2 or
IL-2R.43,44 Our results show that nearly complete
restoration of proliferation was obtained upon addition of NO
inhibitors and IL-2, thus suggesting that IL-2 production was inhibited
by secreted product(s). This inhibition of IL-2 production was not
mediated by known cytokines with immunosuppressive activities, such as
IL-4, IL-10, TGF Therefore, CTLA4Ig in vivo treatment promotes T-cell unresponsiveness not only by blocking T-cell activation through CD28 but also by generating tolerogenic DCs.
We are grateful to Claire Usal, Helga Smit, and Emmanuel Merieau for heart transplantation; to all researchers who contributed reagents; and to Bice Perussia and Robert Lechler for critically reading the manuscript. We thank the Vector Core of the University Hospital of Nantes, supported by the Association Française contre les Myopathies (AFM), for producing the adenoviral vectors used in this study.
Submitted July 15, 2002; accepted December 10, 2002.
Prepublished online as Blood First Edition Paper, December 19, 2002; DOI 10.1182/blood-2002-07-2076 .
Supported in part by Fondation Transvie and EEC grant Biomed2 BMM-CT98-3277.
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.
Reprints: Ignacio Anegon, INSERM U437, 30 boulevard Jean Monnet, 44093 Nantes, France; e-mail: ianegon{at}nantes.inserm.fr.
1.
Waldmann H.
Transplantation tolerance 2. Lechler R, Ng W, Steinman R. Dendritic cells in transplantation: friend or foe. Immunity. 2001;14:357-368. 3. Steinman R, Nussenzweig M. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc Natl Acad Sci U S A. 2002;99:351-358. 4. Lutz M, Suri R, Niimi M, et al. Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo. Eur J Immunol. 2000;30:1813-1822. 5. O'Connell P, Li W, Wang Z, et al. Immature and mature CD8 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
chain) MAb reactive cells
using magnetic beads (Dynal, Compiègne, France). Culture medium
was RPMI 1640 supplemented with 10% heat-inactivated fetal calf
serum, 2 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 1 mM sodium pyruvate, 1% nonessential amino acids,
1% HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), and 5 × 10
5 M 2-
-integrin expressed by 80% of splenic DCs)
) or control (
) LEW.1A rats that received LEW.1W
heart transplants. Total splenocytes or T cells isolated from
splenocytes of the same animal were assayed against donor (LEW.1W),
third-party (BN), or F1 LEW.1W × LEW.1A APCs at the indicated time
points. (A) Splenocyte proliferation against LEW.1W cells. (B) T-cell
proliferation against BN cells. (C) Splenocyte proliferation against
LEW.1W cells. (D) T-cell proliferation against BN cells. (E)
Splenocyte proliferation against LEW.1W × LEW.1A cells. (F) T-cell
proliferation against LEW.1W × LEW.1A cells. Results are
expressed as delta (allogeneic 
spontaneous) cpm ± SD.
Results are representative of 3 experiments with identical
results.
where do we stand?
Nat Med.
1999;5:1245-1248.