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The Journal of Immunology, 2008, 181, 1034 -1042
Copyright © 2008 by The American Association of Immunologists, Inc.
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Contribution of Regulatory T Cells and Effector T Cell Deletion in Tolerance Induction by Costimulation Blockadel

Bert Verbinnen*, An D. Billiau{dagger}, Jan Vermeiren*, Georgina Galicia*, Dominique M. A. Bullens*,{ddagger}, Louis Boon, Pascal Cadot*, Greet Hens*, Christiane Dewolf-Peeters§, Stefaan W. Van Gool*,{ddagger} and Jan L. Ceuppens2,*

* Division of Clinical Immunology, {dagger} Experimental Transplantation, {ddagger} Pediatrics, and § Morphology and Molecular Pathology, University Hospital, Catholic University of Leuven, Leuven, Belgium; and Bioceros, Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Blocking of costimulatory signals for T cell activation leads to tolerance in several transplantation models, but the underlying mechanisms are incompletely understood. We analyzed the involvement of regulatory T cells (Treg) and deletion of alloreactive cells in the induction and maintenance of tolerance after costimulation blockade in a mouse model of graft-vs-host reaction. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F1 offspring (C57BL/6 x C3H) induced a GVHR characterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4-Ig every 3 days during 3 wk after splenocyte injection prevented disease development and induced a long-lasting state of stable mixed chimerism (>120 days). In parallel, host-specific tolerance was achieved as demonstrated by lack of host-directed alloreactivity of donor-type T cells in vitro and in vivo. Chimerism and tolerance were also obtained after CD25+ cell-depleted splenocyte transfer, showing that CD25+ natural Treg are not essential for tolerance induction. We further show that costimulation blockade results in enhanced Treg cell activity at early time points (days 6–30) after splenocyte transfer. This was demonstrated by the presence of a high percentage of Foxp3+ cells among donor CD4+ cells in the spleen of treated animals, and our finding that isolated donor-type T cells at an early time point (day 30) after splenocyte transfer displayed suppressive capacity in vitro. At later time points (>30 days after splenocyte transfer), clonal deletion of host-reactive T cells was found to be a major mechanism responsible for tolerance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunological tolerance is a physiological mechanism intended to dampen aggressive immune responses to self-Ags. Although central tolerance is based uniquely on clonal deletion of self-reactive pre-T cells in the thymus (1), different mechanisms underlie the phenomenon of peripheral tolerance (2). These include T cell depletion through activation-induced cell death, "ignorance" of self-Ags, induction of T cell anergy, and active suppression of autoreactive T cells by regulatory T cells (Treg).3

The induction of immune tolerance can potentially be exploited to cure autoimmune or allergic diseases and to prevent detrimental responses to alloantigens after bone marrow or solid organ transplantations. One approach to induce allotolerance involves the temporary inhibition of costimulatory interactions between APC and T cells (3, 4). Costimulatory signals are required for optimal T cell activation and are assumed to regulate T cell responses as well as other aspects of the immune system (5, 6). T cell activation without proper costimulation can induce a state of Ag-specific nonresponsiveness. The most critical costimulatory signal in T cell activation results from the binding of the CD28 receptor on T cells with CD80 and CD86 ligands on APC (6). Another important costimulatory interaction results from the binding of CD154 on activated T cells with CD40, which is constitutively expressed on APC (5). Experimental blocking of the CD40-CD154 or the CD80/CD86-CD28 costimulatory interactions has been shown to prolong allograft survival in rodent models (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). However, in nonhuman primate studies, blockade of a single pathway is not enough to induce tolerance and only prevents the acute rejection of solid allografts as long as the blockade is maintained (18). Likewise, in human MLR, blockade of both costimulatory interactions is essential for the induction of nonresponsiveness (19).

Both naturally occurring (20) and adaptive Treg (21) have been demonstrated to play a role in the development of allotolerance (22, 23), including that achieved by costimulation blockade (24). Ex vivo tolerance induction via costimulation blockade is abrogated when murine natural Treg (nTreg) are depleted from the responder cell population, as measured by intact responses to alloantigen restimulation in vitro and in vivo. However, it remains unclear whether nTreg are required for tolerance induction achieved by costimulation blockade in vivo. Besides regulatory mechanisms, deletion of alloreactive T cell clones is potentially involved in the induction and maintenance of tolerance by costimulation blockade (25). In addition, complementary roles for both deletion and regulatory mechanisms, especially in the time frame before the deletion of alloreactive T cells, cannot be excluded.

In the present study, we therefore wanted to determine the relative roles of Treg and of alloreactive T cell deletion in the induction and maintenance of tolerance achieved by costimulation blockade. For this, we used a major mismatch parent-to-F1 model of allogeneic T cell activation. In this model, infusion of parental splenocytes into sublethally irradiated F1 recipients resulted in a graft-vs-host reaction (GVHR) characterized by severe pancytopenia and which caused mortality of all recipient animals. Pancytopenia is one of the consequences of GVHR and is frequently observed after the injection of lymphoid cells in sublethally irradiated F1 hybrid mice (26, 27). Moreover, it is a commonly reported manifestation of transfusion-associated GVHD (28).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Six- to 8-wk-old C57BL/6 (B6, H2Kb) female mice were used as donors and 6- to 8-wk-old (C57BL/6 x C3H)F1 (B6C, H2Kk/b) female mice as recipients. For in vitro third-party experiments, splenocytes from 6- to 8-wk-old C3H (H2Kk) or DBA/2 (H2Kd) female mice were used as stimulators. For in vivo third-party experiments, 6- to 8-wk-old (C57BL/6 x DBA/2)F1 (B6D, H2Kd/b) female mice were used. All mice were purchased from Harlan. In some experiments, thymectomies were performed on recipients 3 wk before the transfer of donor cells. These mice were obtained from Harlan and thymectomies were performed by the International Microsurgical Training Centre (Lelystad, The Netherlands). Recipient mice were housed in groups of maximum five in plastic cages, bedded with sawdust, and fitted with filter caps. The study protocol was approved by the animal ethics committee of the Katholieke Universiteit Leuven that follows international guidelines.

Reagents

The hybridoma-producing antagonistic anti-mouse CD154 mAb (MR1, hamster IgG) was obtained from the American Type Culture Collection. Murine CTLA-4Ig chimeric fusion protein (29), which blocks binding of both CD80 and CD86 to CD28, was obtained from Innogenetics. A polyclonal hamster IgG (Rockland) was used as control Ab. All treatment reagents were diluted in PBS.

Induction of GVHR, treatment, and induction of tolerance

Recipient F1 mice were conditioned on day –1 with 7 Gy of total body irradiation using a linear accelerator 18-Mev photon (General Electric) at a dose rate of 3.9 Gy/min with focus to mid-body distance of 100 cm. On the next day (day 0), recipients were given donor splenocytes administered i.v. in 250 µl of RPMI 1640. The number of total donor splenocytes necessary to induce lethality in almost all recipient animals was determined to be 10 x 106. This number of cells was used in all experiments, since lower amounts of donor splenocytes did not induce lethality in all recipients.

Changes in weight, survival, and peripheral blood cell counts of the animals were monitored.

Mice were bled 14–21 days after infusion of 10 x 106 splenocytes. RBCs were counted with a Coulter Counter DN (Analis). White blood cell (WBCs) counts and hemoglobin levels were analyzed using a Micros 60 Coulter (HoribaABX Diagnostics).

Treated animals were injected i.p. with 250 µg of MR1, 500 µg of CTLA-4Ig, or both combined. Injections started on day –1 and were repeated on days 0, 4, and 7. In some experiments, treatment was prolonged and the mice then received additional injections of half the amount of MR1 or CTLA-4Ig on days 11, 14, 17, and 21.

Treated animals were considered tolerant when they showed a gradual increase in weight, survived long term (>60 days), and developed a stable state of mixed chimerism, and when donor cells present in these treated chimeric animals were silent to recipient alloantigens upon subsequent rechallenge in vitro or in vivo.

Detection of chimerism, quantification of host-reactive T cells, and Foxp3 staining

At different intervals after cell infusion, peripheral blood lymphocytes and/or spleen cells were studied by flow cytometry using a FACSort or a FACSCanto (BD Biosciences). The cells were stained with anti-H2Kb (AF6-88.5), anti-H2Kk (36-7-5), anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD25 (PC61), anti-TCR-Vβ3 (KJ25), and anti-TCR-Vβ8.3 (1B3.3) mAb conjugated with FITC, PE or PerCP (BD Biosciences). Foxp3 staining was performed according to the protocol of the Treg staining kit from eBioscience.

Mixed lymphocyte reaction

A total of 2 x 105 responder T cells (isolated from spleen and inguinal and axillary lymph nodes) cells was plated in flat-bottom 96-well culture plates with 2 x 105 irradiated (10 Gy) spleen stimulator cells in a final volume of 200 µl. T cells were isolated by MACS following a negative selection procedure. After 96 h of incubation at 37°C and 5% CO2, cultures were pulsed with 1 µCi [3H]thymidine/well and harvested 16 h later. Triplicate cultures were set up for every condition tested. Culture medium was RPMI 1640 supplemented with 10% FCS, 2 mmol/L L-glutamine, 50 µmol/L 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Cambrex). Results are expressed as cpm.

Adoptive transfer experiments

One hundred twenty days after the transfer of donor splenocytes to B6C F1 recipients, donor-type cells were purified by H2Kk-negative selection from tolerized mice with magnetically activated cell sorting (MACS). For this, splenocytes were preincubated with anti-H2Kk-PE (BD Biosciences) and afterward anti-PE-labeled microbeads were added according to the instructions of the manufacturer (Miltenyi Biotec). For optimal depletion of recipient-type cells, a LD MACS magnetic column was used. The efficiency of the depletion using this procedure was assessed by flow cytometry. Purified donor-type cells were subsequently transferred to naive secondary B6C F1 recipients or to B6D F1 recipients.

Depletion of CD25+ cells

Depletion of CD25+ cells was achieved by MACS. Cells were preincubated with anti-CD25-PE before anti-PE-labeled microbeads were added according to the instructions of the manufacturer (Miltenyi Biotec). For optimal depletion, a LD MACS magnetic column was used. The purity of the depletion using this procedure, assessed by flow cytometry, was >98%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Transfer of parental splenocytes to sublethally irradiated F1 recipients induces a lethal GVHR that targets the hematopoietic system

A major mismatch parent-to-F1 model of GVHR was developed. Parental C57BL/6 (B6) splenocytes were injected i.v. into sublethally (7 Gy) irradiated (C57BL/6 x C3H)F1 (B6C F1) mice (Fig. 1). Transferring a high (50 x 106) or a low (10 x 106) amount of parental splenocytes induced weight loss and hunching in all recipient animals. Transferring 50 x 106 splenocytes resulted in similar clinical signs but an earlier onset of the disease. In all subsequent experiments, we used 10 x 106 splenocytes. The various control groups included F1 recipient mice that were irradiated only, nonirradiated F1 recipients injected with parental splenocytes (data not shown), and irradiated F1 recipients that received recipient-type (syngeneic) splenocytes after irradiation. All animals from control groups showed a gradual increase in weight and remained without any clinical sign of disease.


Figure 1
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  		FIGURE 1. Transfer of parental splenocytes in irradiated F1 animals causes a lethal GVHR. Recipient B6C F1 mice underwent 7 Gy of total body irradiation on day –1. On day 0, recipient animals were injected i.v. with either 10 x 106 B6 splenocytes ({blacksquare}) or 50 x 106 B6 splenocytes ({square}). Control groups consisted of recipient B6C F1 that were irradiated without splenocyte transfer ({diamondsuit}) and B6C F1 mice that were injected with 50 x 106 syngeneic splenocytes without previous irradiation ({blacktriangledown}). Mean relative weights (A) (relative to day 0) and percentage of survival (B) are shown (n = 4 in each group).

 
Diseased animals suffered from a severe blood pancytopenia. When diseased animals were sacrificed 18 days after splenocyte transfer and analyzed for peripheral blood cell counts, the animals showed a 1.3-fold reduction in WBCs and a 2.5-fold reduction in RBC counts compared with animals that were irradiated only (Table I). Serum hemoglobin levels were reduced 2.6-fold. Histological analysis revealed a marked disruption of the splenic architecture with complete depletion of lymphoid elements. In the bone marrow, phagocytosis of erythrocytes was observed. Large activated lymphoid cells were present in the blood. In most of the diseased animals, no GVHD-related lesions were found in the skin, stomach, lungs, intestines, or liver. However, animals that survived for >30 days after the transfer of donor splenocytes started to show infiltrates of lymphoid cells in their skin. Control recipient mice that were irradiated but did not receive donor splenocytes also showed a reduction in cell counts, but the degree of pancytopenia was much less pronounced (Table I), and they survived with a gradual recovery of their hematopoietic system. These results indicate that transfer of parental splenocytes to sublethally irradiated F1 recipients induces a lethal GVHR that primarily targets the hematopoietic system.


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  		Table I. Peripheral blood counts in B6C F1 mice: effect of irradiation and B6 splenocyte transfer

 
Blockade of CD40-CD154 and CD80/CD86-CD28 interactions prevents the development of GVHR

We next investigated the relative roles of the CD80/CD86-CD28 and CD40-CD154 interactions for the induction of GVHR. CTLA-4Ig fusion protein was used to block the CD80/CD86-CD28 interaction and the CD40-CD154 interaction was antagonized by a blocking mAb to CD154 (clone MR1). Treatment with CTLA-4Ig alone or with MR1 alone led to a delay in the onset of the disease, but neither of them alone was able to prevent GVHR lethality. On the other hand, we found a marked synergy between CTLA-4Ig and MR1 to inhibit GVHR induction (Fig. 2, A and B). The duration of costimulation blockade was also an important denominator in determining the outcome (Fig. 2, C and D). When treatment was stopped after 7 days, the first signs of disease were delayed, but all of the animals still developed GVHR. In contrast, animals that were treated for 21 days showed a gradual increase in body weight and did not exhibit any visible signs of GVHR. Pooled data of seven independent experiments gave a 100% survival of treated animals for at least 60 days (n = 40), in contrast to a 100% mortality in the untreated animals which died on average on day 21 (n = 40, SD = 6 days). Even on a follow up of 120 days (n = 5), no signs of GVHR developed in treated animals.


Figure 2
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  		FIGURE 2. The combination of CTLA-4Ig and MR1 prevents GVHR. Recipient B6C F1 mice (n = 5/group) underwent 7 Gy of total body irradiation on day –1. On day 0, each group of B6C F1 mice was injected i.v. with 10 x 106 B6 donor splenocytes. A and B, Mice were treated with either hamster IgG ({blacksquare}), MR1 alone ({triangledown}), CTLA-4Ig alone ({blacktriangledown}), or MR1 and CTLA-4Ig ({blacktriangleup}) for 21 days. Mean relative body weights compared with day 0 (±SD) (A) and survival rates (B) are shown. C and D, the duration of treatment with CTLA-4Ig plus MR1 was varied. Mice received hamster Ig for 21 days ({blacksquare}) or were treated with MR1 and CTLA-4Ig, either for 7 ({triangleup}) or 21 ({blacktriangleup}) days. Mean relative weights compared with day 0 (±SD) (C) and percentage of survival (D) are shown.

 
Blockade of CD40-CD154 and CD80/CD86-CD28 inhibits the proliferation of alloreactive T cells and induces mixed chimerism

To evaluate whether there was an expansion of host-reactive T cells in animals that develop GVHR, irradiated F1 mice were injected with 10 x 106 splenocytes and sacrificed 12 days later. At this time point, the percentage of TCR-Vβ3-positive CD4+ cells and of TCR-Vβ8.3-positive CD4+ cells was measured in their spleen. Recipient B6C F1 mice carry the endogenous Mtv-6 provirus, leading to thymic-negative selection of TCR-Vβ3-expressing T cells (30), a clonal deletion that does not occur in B6 donor mice. TCR-Vβ8.3-expressing T cells are present in both strains. After B6 splenocyte transfer to B6C F1 animals, a marked expansion of TCR-Vβ3-positive CD4+ cells occurred (Fig. 3A). On the other hand, the frequency of TCR-Vβ8.3-expressing CD4+ cells remained within the range of control untreated host and donor mice (Fig. 3B). Interestingly, when animals were treated with CTLA-4Ig and MR1, no expansion of host-reactive TCR-Vβ3 occurred (Fig. 3A). Thus, in diseased animals, an expansion of donor TCR-Vβ3-expressing CD4+ cells occurred, as a manifestation of allogeneic T cell activity, and this expansion could be inhibited by costimulation blockade. Moreover, CTLA-4Ig- and MR1-treated mice developed a stable state of mixed chimerism as demonstrated by the presence of H-2Kk-negative donor cells in the spleen (Fig. 3C) and in the peripheral blood (data not shown). The degree of chimerism followed up to 60 days after donor splenocyte transfer was very stable over time.


Figure 3
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  		FIGURE 3. Costimulation blockade inhibits the expansion of host-reactive T cells and induces chimerism. Recipient B6C F1 mice underwent 7 Gy of total body irradiation on day –1. On day 0, they were injected i.v. with 10 x 106 B6 splenocytes and treated with either hamster IgG (GVHR, n = 6) or with MR1 and CTLA-4Ig (treated, n = 7) for 11 days. On day 12, animals were sacrificed and splenocytes were analyzed by FACS. The percentage of donor-origin CD4+ cells expressing specific TCR-Vβ chains was determined by using PerCP-labeled CD4, PE-labeled anti-H2Kk, and FITC-labeled anti-Vβ3 TCR (A) or anti-Vβ8.3 TCR (B) mAb. As a control, the percentage of CD4+ cells expressing TCR-Vβ3 and TCR-Vβ8.3 chains was also determined in naive donor B6 mice and naive recipient B6C F1 mice (n = 4). C, The percentage of spleen chimerism in CTLA-4Ig/MR1-treated mice is shown. Recipient B6C F1 mice were treated for 21 days after splenocyte transfer. At different time intervals (range, days 28–60), mice were sacrificed and splenocytes were analyzed by FACS for the presence of H2-Kk-negative (= donor-type) B6 splenocytes. The animals (n = 14) were from three independent experiments.

 
Blockade of CD40-CD154 and CD80/CD86-CD28 interactions induces alloantigen-specific tolerance

Treatment with a combination of CTLA-4Ig and MR1 resulted in long-term survival of F1 recipients in the absence of GVHR and in the development of stable mixed chimerism. This indicates that the donor T cells were silent to the recipient alloantigens. To confirm this, spleen cells were isolated from chimeric animals (120 days after splenocyte infusion), enriched for T cells by passage over nylon wool, and subsequently restimulated in MLR with C3H (H2Kk) or third-party spleen cells (H2Kd) as stimulators (Fig. 4A). Responses to C3H cells were weak, while responses to third-party cells were strong. In addition, the adoptive transfer of purified donor-type spleen cells from these chimeric animals to third-party B6D F1 recipients resulted in an acute and lethal GVHR (Fig. 4B), while the adoptive transfer to B6C F1 recipients did not. Collectively, these data show that costimulation blockade had induced alloantigen-specific tolerance of donor T cells toward recipient alloantigens in GVHR-free long-term survivors.


Figure 4
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  		FIGURE 4. Costimulation blockade induces alloantigen-specific tolerance. Costimulation blockade was performed by CTLA-4Ig and MR1 treatment in B6C F1 recipients of B6 splenocytes. Splenocytes were collected 120 days after cell transfer. A, Anti-recipient and anti-third-party MLR were set up with T cell-enriched splenocytes from five treated chimeric animals (mice 1–5). Splenocytes were passed over nylon wool (to enrich for T cells) and were stimulated with C3H splenocytes (H2Kk) or third-party splenocytes (DBA/2, H2Kd). Thymidine incorporation was used for quantification of T cell proliferation and results are given as cpm. The percentage of cells from donor origin was 18, 67, 87, 43, and 94% for the five different chimeric animals. B, Donor-type B6 splenocytes isolated from treated chimeric animals induce GVHR upon transfer to third-party B6D F1 recipients, but not upon transfer to secondary B6C F1 recipients. Recipient B6C F1 and B6D F1 mice underwent 7 Gy of total body irradiation on day –1. On day 0, recipient B6D F1 mice were injected with 10 x 106 control B6 splenocytes ({blacktriangleup}) (n = 10) or with 10 x 106 donor-type splenocytes (H2Kb) isolated from treated chimeric animals (•) (n = 8). Recipient B6C F1 mice were injected with 10 x 106 control B6 donor-type splenocytes ({blacksquare}) (n = 10) or 10 x 106 B6 donor-type splenocytes from treated chimeric animals ({blacktriangledown}) (n = 6). Data are pooled from two independent experiments. Percentage of survival is shown.

 
CD4+CD25+ Treg are not required for the induction and maintenance of tolerance by costimulation blockade

Naturally occurring CD4+CD25+ Treg have been shown to be important for in vitro induction of tolerance against alloantigens (24). We therefore wanted to study the requirement of donor CD4+CD25+ cells in the induction and maintenance of tolerance after costimulation blockade in the current model. For this purpose, CD4+CD25+ nTreg were removed from the donor spleen population. Transfer of CD25-depleted spleen cells to irradiated B6C F1 mice resulted in a more rapid onset of GVHR as compared with total spleen cell transfer (Fig. 5). Furthermore, after transfer of 10 x 106 CD25-depleted splenocytes, GVHR could not be prevented by costimulation blockade in most of the recipient animals. However, when we reduced the number of transferred CD25-depleted splenocytes to 4 x 106 cells to have a GVHR that is comparable in onset and severity to that after transfer of 10 x 106 total spleen cells, costimulation blockade by MR1 and CTLA-4Ig efficiently prevented GVHR and induced chimerism. These results indicate that CD4+CD25+ nTreg are not essential for tolerance induction by costimulation blockade in vivo, although they dampen immune reactivity and reduce severity of GVHR.


Figure 5
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  		FIGURE 5. Tolerance induction by costimulation blockade does not depend on donor nTreg. Recipient B6C F1 mice underwent 7 Gy of total body irradiation on day –1. A and B, All mice received either 10 x 106 B6 splenocytes ({blacksquare} and {blacktriangleup}) or 10 x 106 CD25-depleted B6 splenocytes ({square} and {triangleup}) on day 0. Mice were treated with either hamster IgG ({blacksquare} and {square}) or MR1 and CTLA-4Ig ({blacktriangleup} and {triangleup}) for 21 days. Each group consisted of 10 animals. Mean relative weights compared with day 0 (±SD) (A) and percentage of survival (B) from two replicate experiments are shown. C and D, Mice received either 10 x 106 total B6 splenocytes ({blacksquare} and {blacktriangleup}) or 4 x 106 CD25-depleted B6 splenocytes ({square} and {triangleup}) on day 0. Mice were treated with either hamster IgG ({blacksquare} and {square}) or MR1 and CTLA-4Ig ({blacktriangleup} and {triangleup}) for 21 days. Each group consisted of five animals. Mean relative weights compared with day 0 (±SD) (C) and percentage of survival (D) are shown.

 
Costimulation blockade results in the expansion of Foxp3+ cells and enhanced regulatory activity

To further investigate the role of Treg in the induction of tolerance by costimulation blockade, we conducted an experiment in which at different time points after the transfer of donor splenocytes, the expression of Foxp3 was analyzed in splenocytes (Fig. 6A). At early time points after the transfer (range, days 6–30), there was a high percentage of donor-type CD4+ Foxp3-positive cells in CTLA-4Ig/MR1-treated animals. No increase in the proportion of Foxp3-positive cells was found in animals that were not treated by costimulation blockade. After day 30, the percentage of donor-type Foxp3-positive cells in the CTLA-4Ig/MR1-treated animals decreased to control levels. These results suggest that there may be an important role for regulatory cell activity early in the process of tolerance establishment induced by costimulation blockade.


Figure 6
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  		FIGURE 6. Costimulation blockade results in the expansion of Foxp3+ cells and enhanced regulatory activity. A, Recipient B6C F1 mice underwent 7 Gy of total body irradiation on day –1. On day 0, they were injected i.v. with 10 x 106 B6 splenocytes and treated with MR1 and CTLA-4Ig for 21 days. At different time intervals (days 6, 13, 19, 30, and 56), three mice were sacrificed and splenocytes were analyzed by FACS for the percentage of Foxp3+CD4+ cells from donor origin ({blacktriangleup}) and recipient origin ({blacktriangledown}). Three mice that were injected with B6 splenocytes but not treated with MR1 and CTLA-4Ig were also analyzed on days 6 and 12 for the percentage of Foxp3+CD4+ cells from donor origin ({blacksquare}). B, Recipient B6C F1 mice underwent 7 Gy of total body irradiation on day –1. On day 0, they were injected with 10 x 106 B6 splenocytes and treated with either hamster IgG (GVHR) or with MR1 and CTLA-4Ig (treated) for 11 days. Each group consisted of four animals. On day 12, the animals were sacrificed and splenocytes were analyzed by FACS. The percentage of donor-origin CD4+ cells expressing Foxp3 and TCR-Vβ chains was determined by using PerCP-labeled anti-CD4, PE-labeled anti-H2Kk, FITC-labeled anti-Vβ3 TCR, and allophycocyanin-labeled anti-Foxp3 mAb. The percentage of donor-type Foxp3-positive cells gated on CD4+ cells is shown among TCR-Vβ3-positive and TCR-Vβ3-negative cells. The percentage of CD4+ cells expressing Foxp3 was also determined in a naive donor B6 mouse and was 11.0% for TCR-Vβ3-positive cells and 10.6% for TCR-Vβ3-negative cells. C, B6C F1 recipients of B6 splenocytes were treated with CTLA-4Ig and MR1 and the splenocytes from treated mice were collected 30 or 60 days after cell transfer. Purified donor-type B6 T cells from treated mice (T1–T5; H2Kb) were added (ratio 1:1) to a MLR between responder B6 T cells (R; H2Kb) and stimulator C3H splenocytes (S; H2Kk). Thymidine incorporation was used for quantification of T cell proliferation and the results are given as cpm. The percentage of suppression for each condition is shown above each bar. Addition of T cells from a control naive B6 animal (ratio 1:1) to the MLR had no suppressive effect (data not shown).

 
Since we were able to identify a subpopulation of host-reactive T cells by TCR-Vβ3 expression, we next wondered whether the increase in Foxp3-positive cells among the donor CD4+ cells in treated animals was related to alloreactivity and not simply due to an increased ability of Foxp3-positive cells to homeostatically proliferate in lymphopenic mice. Therefore, we conducted an experiment in which at 12 days after splenocyte transfer, the Foxp3 expression on donor TCR-Vβ3-positive and TCR-Vβ3-negative cells was analyzed. Again, we found an increase in the proportion of Foxp3-positive cells in CTLA-4Ig/MR1-treated animals, but importantly, the increase was much more pronounced within the alloreactive TCR-Vβ3-positive fraction (Fig. 6B). These results illustrate that allogeneic T cell activation under costimulation-deficient conditions specifically results in an increase in the proportion of alloreactive Foxp3-positive cells. In untreated animals, no increase in the percentage of Foxp3 positivity was found, neither within the TCR-Vβ3-positive or -negative fraction.

To evaluate a functional role for these regulatory cells in tolerance induction, control B6 T cells or purified B6 donor-derived T cells from chimeric mice (isolated on day 30 or day 60) were added to a MLR between responder donor cells (H2Kb) and C3H stimulator cells (H2Kk) (Fig. 6C). Addition of day 30 donor-derived cells reduced T cell proliferation (mean suppression: 55.3 ± 12.9%), whereas addition of day 60 donor-derived cells had no influence on the proliferation. Together, these data show a transient increase in regulatory cell activity early after allogeneic cell transfer and costimulation blockade. This suggests that regulatory cell induction by costimulation-deficient APC contributes to dampening of immune alloreactivity.

Blockade of CD40-CD154 and CD80/CD86-CD28 interactions leads to the peripheral elimination of host-reactive T cells

We next wondered whether clonal deletion of host-reactive T cells might play a role in tolerance induction at later time points, when no increased percentage of Foxp3-positive cells and no suppressive activity of donor-type T cells could be found. To study this, we analyzed the expression of specific TCR-Vβ subunits on day 90 in the peripheral blood (data not shown) and in the spleen (Fig. 7) after the transfer of B6 donor splenocytes to CTLA-4Ig/MR1-treated B6C F1 recipients. At these time points, only a very low percentage of TCR-Vβ3-positive donor-type cells persisted, in contrast to a normal percentage of control TCR-Vβ8.3-positive donor-type cells. These results thus point to an elimination of host-reactive cells. However, another possibility to explain these data is that all of the injected donor CD4+ cells had died by day 60 as a result of cell aging and that the detected donor-type CD4+ cells in the chimeric animals were all newly differentiated from donor stem cells present in the splenocyte inoculum. Therefore, we repeated the same experiment in thymectomized recipients. In these animals, no new T cells can develop from donor stem cells and, as a result, all donor-type T cells detected in these animals are (or are derived from) preexisting T cells in the splenocyte injection. FACS analysis revealed that CD4+ cells from donor origin could still be found in the spleen of CTLA-4Ig/MR1-treated thymectomized animals 90 days after the splenocyte transfer. Of note, these recipients had less donor T cells in their spleen compared with euthymic recipients (6.9 ± 1.4 x 106 vs 1.4 ± 0.8 x 106). Importantly, Fig. 7 illustrates that in both euthymic and thymectomized recipients there was a comparable low percentage of TCR-Vβ3-positive host-reactive CD4+ cells and a normal percentage of TCR-Vβ8.3-positive CD4+ cells. These results suggest that donor CD4+ cells in chimeric euthymic recipients on day 90 are partly cells persisting from the donor splenocyte injection and partly are newly differentiated cells derived from donor stem cells in the splenocyte inoculum. Moreover, these data demonstrate that costimulation blockade after the transfer of splenocytes ultimately results in the peripheral elimination of host-reactive T cells that are present in the splenocyte inoculum.


Figure 7
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  		FIGURE 7. Deletion of host-reactive TCR-Vβ3+ cells in mice treated with CTLA-4Ig and MR1. Recipient euthymic (A; n = 4) and thymectomized (B; n = 6) B6C F1 mice underwent 7 Gy of total body irradiation on day –1. On day 0, they were injected i.v. with 10 x 106 B6 splenocytes and treated with MR1 and CTLA-4Ig for 21 days. On day 90, mice were sacrificed and the percentage of donor-origin splenic CD4+ T cells expressing specific TCR-Vβ chains was determined by flow cytometry using FITC-labeled anti-Vβ3-TCR or anti-Vβ8.3-TCR mAb, PerCP-labeled anti-CD4 mAb, and PE-labeled anti-H2Kk Abs. CD4+ cells expressing specific TCR-Vβ3 was 4.2% in naive B6 mice and 0.3% in naive B6C F1 mice (n = 2).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study on tolerance induction by costimulation blockade, we used a major mismatch model of GVHR induced by the transfer of B6 splenocytes to B6C F1 recipients. This model is very robust because 100% of untreated animals died within 3 wk after the transfer of donor splenocytes. In diseased animals, a dramatic decrease in WBC and RBC counts and in the serum hemoglobin content occurred, as well as in the cellularity of the bone marrow. Submyeloablative radiation of recipient animals was partly responsible for this pancytopenia. However, an additional reduction of blood cell counts resulted from the transfer of parental splenocytes (but not syngeneic splenocytes), which means that these alloreactive splenocytes target the hematopoietic system of recipient animals. Pancytopenia associated with hypoplastic bone marrow is often seen in GVHRs by the transfer of donor cells that contain few hematopoietic stem cells and in recipients that show little host-vs-graft reactivity (27, 28). This model of GVHR closely resembles that of a study by Chen et al. (26) in which parental lymph node cells were infused into sublethally irradiated F1 recipients. Affected animals also developed a fatal pancytopenia along with bone marrow destruction within 2–3 wk, without the other typical signs of GVHD. The lack of typical GVHD symptoms is probably related to the aggressiveness of the disease development with short survival in untreated animals. A high percentage of T cells in the donor inoculum, with few hematopoietic stem cells, may result in GVHR that develops very rapidly and thereby mainly targets the hematopoietic system, without allowing the time for donor cells to cause specific GVHD-related symptoms in other organs. In this respect, it is important to note that the few recipient animals that survived for longer than 30 days did indeed show lymphoid infiltrations in their skin typical of GVHD.

We here report that CTLA-4Ig and MR1 were synergistic in their activity to completely prevent the GVHR in this model. Treatment with CTLA-4Ig alone or with MR1 alone led to a delay in the onset of disease but could not prevent GVHR lethality. Our results are consistent with previous studies in GVHD models that showed that both the CD40-CD154 and the CD80/CD86-CD28 interactions have a functional role in allogeneic T cell activation. Acute GVHD could still be induced by T cells derived from CD28 knockout mice and, in these animals, blocking of the CD40-CD154 interaction prevented the CD28-independent GVHD (31). Also, the establishment of chimerism in CD154-deficient recipients was shown to require blockade of the CD80/CD86-CD28 pathway (32). Likewise, blockade of both interactions was necessary to prevent acute rejection of solid allografts (18). The rationale for combining CTLA-4Ig and MR1 has previously been reviewed (3, 4). However, the exact mechanism underlying tolerance induction is still controversial, as both Treg activity and clonal deletion have been proposed to play a role (22, 23, 24, 25).

The potential role of Treg in transplantation tolerance in general has received much attention. nTreg were demonstrated to play a role in the in vitro induction of nonresponsiveness against alloantigens based on costimulation blockade (24). We here found that the effect of costimulation blockade in preventing alloresponses in vivo was modulated by absence of nTreg activity but that tolerance still could be induced. In our model, the severity of the GVHR was increased and costimulation blockade was less effective after transfer of donor spleen cells that were depleted of nTreg. However, after reducing the amount of CD25-depleted donor cells, tolerance could be consistently induced by costimulation blockade. Thus, depletion of donor nTreg apparently lowers the threshold for GVHR induction, but when lower numbers of donor cells are infused, costimulation blockade remains efficient for tolerance induction. These findings suggest that the efficacy of prevention of allogeneic T cell activation by costimulation blockade will depend on a balance between effector and Treg.

In our model, we further found a high percentage of Foxp3-expressing donor CD4+ cells in CTLA-4Ig/MR1-treated animals during the first 4 wk after splenocyte transfer. Moreover, we demonstrated a specific increase in the percentage of TCR-Vβ3-positive cells within these Foxp3-positive donor CD4+ cells. The specific increase of Foxp3-positive T cells with host alloreactivity is of potential interest, since these cells may represent alloantigen-specific suppressor cells. In parallel with the kinetics of the Foxp3 expression, we could demonstrate suppressor activity of donor T cells isolated from chimeric animals at an early time point after splenocyte transfer, but not at a later time point. Together, these data suggest that Treg activity may transiently be involved in the establishment of tolerance by costimulation blockade. Whether the increased percentage of Foxp3-positive donor CD4+ cells is derived from preexisting nTreg or due to de novo induction of adaptive Treg is at present unknown. Several studies have already suggested that the result of costimulation blockade is at least partially based on the induction of Treg (33, 34, 35). We previously demonstrated that human T cell activation by costimulatory signal-deficient allogeneic cells induces anergic T cells with regulatory activity (36). Our present findings are therefore most compatible with Treg induction as a result of allogeneic activation by costimulation deficient APC.

Since clonal deletion has been described in some animal models as a mechanism responsible for tolerance induction by costimulation blockade (4, 25), we also looked in our model for involvement of this mechanism by analyzing the expression of certain TCR-Vβ subunits in diseased and CTLA-4Ig/MR1-treated animals. In animals developing GVHR, we found a marked expansion of host-reactive CD4+ cells as identified by TCR-Vβ3 expression at an early time point (day 12) after splenocyte transfer. This expansion was not seen in CTLA-4Ig/MR1-treated splenocyte recipient animals. Moreover, the administration of costimulatory blocking agents ultimately resulted in the elimination of these host-reactive CD4+ cells at later time points (day 90). This was also confirmed by experiments with thymectomized animals. This peripheral deletion was probably also accompanied by intrathymic deletion of newly developing CD4+ cells in recipient animals since also euthymic recipients had no TCR-Vβ3-positive donor CD4+ cells on day 90.

In conclusion, we have shown that costimulation blockade with both MR1 and CTLA-4Ig can prevent allogeneic T cell activation and induce host-specific tolerance after transfer of allogeneic donor cells to F1 recipients. Donor nTreg are not required to induce tolerance, but regulatory mechanisms as discussed above may play a role in the establishment of tolerance early after splenocyte transfer. At later time points, clonal deletion of host-reactive T cells is the predominant mechanism and the role of Treg seems to become less important. The specificity of this tolerance induction protocol makes it a promising tool in the control of GVHR or other alloresponses in transplant medicine, but also in other diseases were T cell activation plays a predominant role.


   Acknowledgments
 
We are very grateful to Lieve Coorevits and Omer Rutgeerts for excellent technical assistance and to Willy Landuyt for the irradiation of the recipient animals.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a research grant from the Research Council of the Catholic University of Leuven (OT 06-67), by research grants from the Fund for Scientific Research-Flanders (G.0509.06 and G.0255.05) and by a grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders. B.V. is a recipient of a fellowship from Institute for the Promotion of Innovation through Science and Technology in Flanders. S.W.V.G. is a senior clinical investigator of the Fund for Scientific Research-Flanders. A.D.B. and D.M.A.B. are recipients of a postdoctoral fellowship from the Fund for Scientific Research. Back

2 Address correspondence and reprint requests to Dr. Jan Ceuppens, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail address: jan.ceuppens{at}uz.kuleuven.be Back

3 Abbreviations used in this paper: Treg, regulatory T cell; nTreg, natural Treg; GVHR, graft-vs-host reaction; GVHD, graft-vs-host disease; WBC, white blood cell. Back

Received for publication February 9, 2007. Accepted for publication May 13, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hogquist, K. A., T. A. Baldwin, S. C. Jameson. 2005. Central tolerance: learning self-control in the thymus. Nat. Rev. Immunol. 5: 772-782. [Medline]
  2. Lechler, R. I., O. A. Garden, L. A. Turka. 2003. The complementary roles of deletion and regulation in transplantation tolerance. Nat. Rev. Immunol. 3: 147-158. [Medline]
  3. Van Gool, S. W., P. Vandenberghe, M. de Boer, J. L. Ceuppens. 1996. CD80, CD86 and CD40 provide accessory signals in a multiple-step T-cell activation model. Immunol. Rev. 153: 47-83. [Medline]
  4. Wekerle, T., J. Kurtz, S. Bigenzahn, Y. Takeuchi, M. Sykes. 2002. Mechanisms of transplant tolerance induction using costimulatory blockade. Curr. Opin. Immunol. 14: 592-600. [Medline]
  5. Croft, M.. 2003. Co-stimulatory members of the TNFR family: keys to effective T-cell immunity?. Nat. Rev. Immunol. 3: 609-620. [Medline]
  6. Sharpe, A. H., G. J. Freeman. 2002. The B7-CD28 superfamily. Nat. Rev. Immunol. 2: 116-126. [Medline]
  7. Blazar, B. R., P. A. Taylor, P. S. Linsley, D. A. Vallera. 1994. In vivo blockade of CD28/CTLA4: B7/BB1 interaction with CTLA4-Ig reduces lethal murine graft-versus-host disease across the major histocompatibility complex barrier in mice. Blood 83: 3815-3825. [Abstract/Free Full Text]
  8. Blazar, B. R., A. H. Sharpe, P. A. Taylor, A. Panoskaltsis-Mortari, G. S. Gray, R. Korngold, D. A. Vallera. 1996. Infusion of anti-B7.1 (CD80) and anti-B7.2 (CD86) monoclonal antibodies inhibits murine graft-versus-host disease lethality in part via direct effects on CD4+ and CD8+ T cells. J. Immunol. 157: 3250-3259. [Abstract]
  9. Blazar, B. R., P. A. Taylor, A. Panoskaltsis-Mortari, J. Buhlman, J. Xu, R. A. Flavell, R. Korngold, R. Noelle, D. A. Vallera. 1997. Blockade of CD40 ligand-CD40 interaction impairs CD4+ T cell-mediated alloreactivity by inhibiting mature donor T cell expansion and function after bone marrow transplantation. J. Immunol. 158: 29-39. [Abstract]
  10. Blazar, B. R., P. A. Taylor, R. J. Noelle, D. A. Vallera. 1998. CD4+ T cells tolerized ex vivo to host alloantigen by anti-CD40 ligand (CD40L:CD154) antibody lose their graft-versus-host disease lethality capacity but retain nominal antigen responses. J. Clin. Invest. 102: 473-482. [Medline]
  11. Foster, R. D., S. Pham, S. Li, A. Aitouche. 2003. Long-term acceptance of composite tissue allografts through mixed chimerism and CD28 blockade. Transplantation 76: 988-994. [Medline]
  12. Honey, K., S. P. Cobbold, H. Waldmann. 1999. CD40 ligand blockade induces CD4+ T cell tolerance and linked suppression. J. Immunol. 163: 4805-4810. [Abstract/Free Full Text]
  13. Larsen, C. P., D. Z. Alexander, D. Hollenbaugh, E. T. Elwood, S. C. Ritchie, A. Aruffo, R. Hendrix, T. C. Pearson. 1996. CD40-gp39 interactions play a critical role during allograft rejection: suppression of allograft rejection by blockade of the CD40-gp39 pathway. Transplantation 61: 4-9. [Medline]
  14. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, et al 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381: 434-438. [Medline]
  15. Lin, H., S. F. Bolling, P. S. Linsley, R. Q. Wei, D. Gordon, C. B. Thompson, L. A. Turka. 1993. Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion. J. Exp. Med. 178: 1801-1806. [Abstract/Free Full Text]
  16. Pan, Y., B. Luo, H. Sozen, H. Kalscheuer, B. R. Blazar, D. E. Sutherland, B. J. Hering, Z. Guo. 2003. Blockade of the CD40/CD154 pathway enhances T-cell-depleted allogeneic bone marrow engraftment under nonmyeloablative and irradiation-free conditioning therapy. Transplantation 76: 216-224. [Medline]
  17. Yin, D., L. Ma, H. Zeng, J. Shen, A. S. Chong. 2002. Allograft tolerance induced by intact active bone co-transplantation and anti-CD40L monoclonal antibody therapy. Transplantation 74: 345-354. [Medline]
  18. Haanstra, K. G., J. Ringers, E. A. Sick, S. Ramdien-Murli, E. M. Kuhn, L. Boon, M. Jonker. 2003. Prevention of kidney allograft rejection using anti-CD40 and anti-CD86 in primates. Transplantation 75: 637-643. [Medline]
  19. Van Gool, S. W., J. Vermeiren, K. Rafiq, K. Lorr, M. de Boer, J. L. Ceuppens. 1999. Blocking CD40-CD154 and CD80/CD86-CD28 interactions during primary allogeneic stimulation results in T cell anergy and high IL-10 production. Eur. J. Immunol. 29: 2367-2375. [Medline]
  20. Sakaguchi, S.. 2005. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat. Immunol. 6: 345-352. [Medline]
  21. Roncarolo, M. G., S. Gregori, M. Battaglia, R. Bacchetta, K. Fleischhauer, M. K. Levings. 2006. Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol. Rev. 212: 28-50. [Medline]
  22. Akl, A., S. Luo, K. J. Wood. 2005. Induction of transplantation tolerance-the potential of regulatory T cells. Transplant. Immunol. 14: 225-230. [Medline]
  23. Walsh, P. T., D. K. Taylor, L. A. Turka. 2004. Tregs and transplantation tolerance. J. Clin. Invest. 114: 1398-1403. [Medline]
  24. Taylor, P. A., R. J. Noelle, B. R. Blazar. 2001. CD4+CD25+ immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade. J. Exp. Med. 193: 1311-1318. [Abstract/Free Full Text]
  25. Wekerle, T., M. H. Sayegh, J. Hill, Y. Zhao, A. Chandraker, K. G. Swenson, G. Zhao, M. Sykes. 1998. Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance. J. Exp. Med. 187: 2037-2044. [Abstract/Free Full Text]
  26. Chen, J., K. Lipovsky, F. M. Ellison, R. T. Calado, N. S. Young. 2004. Bystander destruction of hematopoietic progenitor and stem cells in a mouse model of infusion-induced bone marrow failure. Blood 104: 1671-1678. [Abstract/Free Full Text]
  27. Hirabayashi, N.. 1981. Studies on graft versus host (GvH) reactions: I. Impairment of hemopoietic stroma in mice suffering from GvH disease. Exp. Hematol. 9: 101-110. [Medline]
  28. Anderson, K. C., H. J. Weinstein. 1990. Transfusion-associated graft-versus-host disease. N. Engl. J. Med. 323: 315-321. [Medline]
  29. Lane, P., W. Gerhard, S. Hubele, A. Lanzavecchia, F. McConnell. 1993. Expression and functional properties of mouse B7/BB1 using a fusion protein between mouse CTLA4 and human {gamma}1. Immunology 80: 56-61. [Medline]
  30. Tomonari, K., S. Fairchild, O. A. Rosenwasser. 1993. Influence of viral superantigens on Vβ- and V{alpha}-specific positive and negative selection. Immunol. Rev. 131: 131-168. [Medline]
  31. Saito, K., J. Sakurai, J. Ohata, T. Kohsaka, H. Hashimoto, K. Okumura, R. Abe, M. Azuma. 1998. Involvement of CD40 ligand-CD40 and CTLA4-B7 pathways in murine acute graft-versus-host disease induced by allogeneic T cells lacking CD28. J. Immunol. 160: 4225-4231. [Abstract/Free Full Text]
  32. Woodward, J. E., L. B. Zottola, A. T. Schaefer, A. J. Logar, J. K. Stazer, T. Daskivich, R. Peach, A. S. Rao. 2001. Blockade of the CD28/B7 pathway is required for the establishment of donor cell chimerism in CD40 ligand-deficient recipients. Transplant. Proc. 33: 115[Medline]
  33. Bigenzahn, S., P. Blaha, Z. Koporc, I. Pree, E. Selzer, H. Bergmeister, F. Wrba, C. Heusser, K. Wagner, F. Muehlbacher, T. Wekerle. 2005. The role of non-deletional tolerance mechanisms in a murine model of mixed chimerism with costimulation blockade. Am. J. Transplant. 5: 1237-1247. [Medline]
  34. Kurtz, J., H. Ito, T. Wekerle, J. Shaffer, M. Sykes. 2001. Mechanisms involved in the establishment of tolerance through costimulatory blockade and BMT: lack of requirement for CD40L-mediated signaling for tolerance or deletion of donor-reactive CD4+ cells. Am. J. Transplant. 1: 339-349. [Medline]
  35. Yamazaki, M., T. Pearson, M. A. Brehm, D. M. Miller, J. A. Mangada, T. G. Markees, L. D. Shultz, J. P. Mordes, A. A. Rossini, D. L. Greiner. 2007. Different mechanisms control peripheral and central tolerance in hematopoietic chimeric mice. Am. J. Transplant. 7: 1710-1721. [Medline]
  36. Vermeiren, J., J. L. Ceuppens, G. M. Van, P. Witters, D. Bullens, H. W. Mages, R. A. Kroczek, S. W. Van Gool. 2004. Human T cell activation by costimulatory signal-deficient allogeneic cells induces inducible costimulator-expressing anergic T cells with regulatory cell activity. J. Immunol. 172: 5371-5378. [Abstract/Free Full Text]




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