Transplant tolerance is the “Grail” of all transplantologists, since the pioneering work of Medawar and coworkers (1). In humans, however, only two circumstances have been able to produce full tolerance: (a) when the recipient of the transplant is not immunologically competent or mature and (b) when the transplant involves total replacement of host lymphocytes with donor lymphocytes.
Organ transplantation often involves immunologically mature patients in whom one may legitimately be reluctant to induce total lymphocyte destruction and immunological reconstitution with donor lymphocytes. To avoid the risks associated with such therapies, attempts have been made to produce split lymphocyte chimerism but have been hampered by a high risk of graft-versus-host reaction. To this end, Ildstad et al. (2) and Kawai et al. (3) recently reported the successful induction of tolerance through the promotion of mixed chimerism in patients who had received a human leukocyte antigen-incompatible bone marrow transplant together with a nonmyeloablative preparative regimen before organ transplantation. These patients became tolerant to a kidney transplant from the same donor, to the extent that they did not need any immunosuppressive therapy, while their renal function remained stable for 2.0 to 5.3 years after transplantation. Other clinical trials also successfully achieved tolerance through mixed chimerism (4, 5). Thus, allogeneic hematopoietic stem cells (HSCs) are liable to facilitate and improve allogeneic transplantation through the induction of tolerance to transplanted cells, tissues, or organs (6).
We have analyzed experimentally the capacity of interleukin (IL)-10-transduced HSCs to survive rejection reactions by the host and to induce transplant tolerance. Indeed, IL-10, which is produced by macrophages and Th2 cells, is a cytokine that inhibits the production of Th1 cytokines, in particular γ interferon (6, 7), and down-regulates the expression of class II major histocompatibility complex (MHC) determinants in antigen-presenting cells (8). Several studies have demonstrated that local injection of an exogenous viral IL-10 (vIL-10) gene into the transplanted organ improved graft survival through impaired antigen presentation, reduction of immunogenicity, inhibition of leukocyte infiltration, and prevention of rejection (9–13). Moreover, Qin et al. (11) and Zhao et al. (14) have shown that retroviral vectors that encode vIL-10 prolonged graft survival for a longer period than adenoviral vectors (39.4±2.5 vs. 18.4±1.2 days, respectively). However, the effects of vIL-10 in these studies were limited in time due to the induction of a recipient antiviral immune response.
Taking the promising results of these studies into account, we previously proposed that transduction of donor HSC with exogenous human IL-10 (hIL-10) would induce mixed chimerism together with propagation of the exogenous IL-10 gene on the basis that IL-10-secreting HSCs would migrate to the lymphoid organs that are involved in the induction of tolerance. Stem cells from fetal liver have been used because their proliferative capacity is twice as high as that of bone marrow HSCs, thus facilitating the integration of genetic material (15). In a previous study (16), we showed that total fetal liver stem cells (TFLs) and their progenitors have a greater ability to proliferate in vitro than HSCs from adult bone marrow. Similarly, TFLs have shown graft superiority over adult bone marrow cells in vivo (17). We previously demonstrated the feasibility of maintaining donor lymphocytes in recipient mice using IL-10-transduced TFLs (IL-10-TFLs) from the same donor. Moreover, we have also shown that continued production of IL-10 in vivo in the vicinity of the transplanted cells seemed to protect them from rejection by host T lymphocytes. This transient chimerism was associated with some degree of transplant tolerance: skin allografts from the same donor strain were specifically accepted and were only rejected when donor cells were no longer present (16). In this study, we show the high efficiency of repeated injections of hIL-10-TFLs in the induction of long-term survival of heart allografts.
Mixed Leukocyte Reaction
Our aim was to evaluate the effect of TFLs transduced with hIL-10 (hIL-10-TFLs) on the survival of organs harvested from C57Bl/6 mice and grafted into Balb/c mice. Therefore, we first tested the effect of hIL-10 on the suppression of the allogeneic response mediated between these two mouse strains in mixed leukocyte reaction (MLR) experiments. As shown in Figure 1, in vitro proliferation of T cells from Balb/c mice was induced by allostimulation with irradiated C57Bl/6 mononuclear cells (one-way MLR). Higher rates of proliferation were observed when the stimulating cells were not irradiated (two-way MLR), demonstrating a strong MHC class II incompatibility between these two strains. Under each condition, addition of 1000 U/mL hIL-10 strongly inhibited the alloresponse. The MLR response decreased to levels as low as those of the negative control, which was a coculture of irradiated peripheral blood mononuclear cells from the two strains.
These results demonstrated that hIL-10 is a potent inhibitor of the allogeneic response induced between Balb/c and C57Bl/6 strains of mice and can therefore be considered as a good candidate to prevent the rejection of C57Bl/6 cells or organs by Balb/c mice in vivo.
Survival of Skin Allografts
To investigate whether injection of hIL-10-TFLs could mitigate donor-specific rejection, we prepared TFLs from C57Bl/6 mice and grafted Balb/c mice with the skin of C57Bl/6 or CBA (a third-party strain) mice. The hIL-10-TFLs were injected 2 days before transplantation. The results of the experiment demonstrated that, in the absence of hIL-10-TFLs, irradiated Balb/c mice rejected C57Bl/6 or CBA skin grafts within 9.0±0.4 days or 9.3±0.4 days, respectively. When these mice received 107 hIL-10-TFLs before transplantation, C57Bl/6 skin grafts survived for up to 17.8±0.6 days, whereas CBA skin grafts were rejected within 9.5±0.3 days (P<0.001; Fig. 2,I)
Thus, the skin graft results demonstrated that a single injection of hIL-10-TFLs is sufficient to delay graft rejection significantly in this model. Moreover, they showed that prolongation of skin graft survival after injection of hIL-10-TFLis donor specific.
Survival of Heart Allografts
We then investigated whether hIL-10-TFLs could also delay the rejection of allogeneic hearts transplanted into Balb/c mice and whether one or several injections would be required. The hearts from C57Bl/6 mice were grafted into Balb/c mice under two different conditions. In the first series of experiments, 107 TFLs were prepared from C57Bl/6 mice, were transduced with hIL-10 or were untreated, and were injected at day 0 (the day of transplantation); in the second series, several such injections were given every 20 days, starting from day 0, and their effects were compared with those of a single injection. As a control, Balb/c mice were transplanted with isogenic hearts (positive control) or with hearts from C57Bl/6, without being irradiated before transplantation and with no preventive cell therapy (negative control).
In the first series, the results in the control groups showed that allogeneic heart transplants were rejected by irradiated MHC-mismatched recipients within a mean of 13.5±1.0 days, whereas the control isogenic hearts were still beating more than 100 days after transplantation. When recipient mice were irradiated at 5 Gy before transplantation and injected with 107 untransduced TFLs from donor mice, allografted heart survival was prolonged for up to 28.5±1.8 days. When 107 hIL-10-TFLs were injected, the rejection was delayed for up to 52.1±7.2 days; this difference was statistically significant (P<0.01; Fig. 2,IIA).
In the second series of mice that were irradiated at 5 Gy before transplantation and injected every 20 days with hIL-10-TFLs, allogeneic heart transplants were rejected within a mean of 86.25±13.8 days, whereas they were rejected after 46.3±4.6 days after a single injection of hIL-10-TFLs (Fig. 2,IIC). It is noteworthy that the two mice with the longest survival (83 and 140 days) had no apparent rejection of the heart transplant but died as the result of the injections; indeed, their transplanted hearts were still beating normally before the last injection. As a control, transplanted hearts in mice that received a single injection of untransduced TFLs survived 28.1±1.5 days and those of mice that received injections of untransduced TFLs every 20 days survived 38.8±2.1days.
These results showed that even a single injection of hIL-10-TFLs can induce a longer delay in graft rejection than several injections of untransduced TFLs (46.3 vs. 38.8 days). This single injection of hIL-10 TFLs gave results that were statistically superior to those of a single injection of untransduced TFLs (Fig. 2,IIB). Moreover, several injections, when compared with a single injection, of hIL-10-TFLs, resulted in a greater increase in graft survival, which was statistically significant (P<0.05; Fig. 2,IID).
To perform histological analyses of the transplanted hearts, the hearts were removed on day 14 after transplantation or when the mice died. The organs were fixed, cut, and stained with hematoxylin-eosin and fluorescent anti-CD8 monoclonal antibodies (mAb). As shown in Figure 3A, no mononuclear cell infiltration was observed when isogenic hearts were transplanted (control group). In contrast, many inflammatory foci and necrotic cells were observed when allografts were transplanted into mice that did not receive any preventive cell therapy (Fig. 3B). However, when hIL-10-TFLs were injected on the day of the allogeneic heart transplantation, only a few inflammatory foci were present at day 14, with no sign of necrosis (Fig. 3C). Moreover, when several injections of hIL-10-TFLs were given, histological analysis of the allogeneic heart of the mouse that survived up to 140 days showed that myocardial cells were still present, although some fibrosis was observed, indicating the absence of complete rejection (Fig. 3D).
Furthermore, when heart tissues were stained with anti-CD8 mAb, we observed that CD8+ cells were expressed at much higher levels in the allogeneic heart of untreated mice compared with those of mice treated with hIL-10-TFLs (Figs. 3E and F, respectively). Therefore, these histological results supported our clinical data and confirmed the attenuation of allograft rejection in mice that received heart allografts together with a single or several injections of hIL-10-TFLs.
Evaluation of the Microchimerism
Because donor cells express the H-2b gene, the presence of this gene was examined in recipient mice. DNA was extracted from the thymus, spleen, liver, lung, and heterotopic heart of recipient mice, and H-2b gene expression was measured by polymerase chain reaction (PCR). The results for three different mice are reported in this study. Mouse A had been allografted with no preventive cell therapy and rejected its graft at day 14 (Fig. 4A); mice B and C had received a single or repeated injections of hIL-10-TFLs, respectively, and were analyzed at day 14 and day 140, respectively (Fig. 4B). Three other mice from the same three groups have been analyzed, and they provided results comparable to those of mice A, B, and C.
As shown in Figure 4A, in the mouse that had not received hIL-10-TFLs, expression of the H-2b gene was found in the allogeneic heart but not in the spleen, liver, thymus, or lung. As a positive control, DNA from donor cells showed expression of the H-2b gene (Fig. 4A, lane 6). In contrast, when mice were injected with hIL-10-TFLs, the H-2b gene was detected in the allogeneic heart, thymus, liver, and lung of the two other mice but not in their spleen, thus demonstrating the presence of a microchimerism due to the persistence of donor cells in many organs of the recipient mice for a long period of time after transplantation (Fig. 4B).
Production of hIL-10 Protein and Expression of Its Gene in Allotransplanted Mice That Received hIL-10-TFLs
We then investigated whether the organs of the two mice that showed expression of H-2b also expressed hIL-10. Expression of the hIL-10 gene was measured by PCR. In accordance with H-2b gene expression, no hIL-10 expression was found in the spleen of either mouse, whereas the hIL-10 gene was found in the liver, thymus, lung, and heart of both mice (Fig. 4C). Moreover, when production of hIL-10 was measured by an enzyme-linked immunosorbent assay (ELISA) in the sera of mice that received hIL-10-TFLs, the protein was found at days 7 and 14 after transplantation at levels of 290 and 150 pg/mL, respectively (data not shown) but was no longer detected at day 21.
These results clearly demonstrated that injection with hIL-10-TFLs resulted in the production of hIL-10 protein in the blood soon after transplantation and in the expression of this gene in organs such as the thymus and liver, which probably play a role in the education of T cells toward tolerance. Interestingly, the hIL-10 gene was also expressed in the transplanted organ as late as 140 days after transplantation.
FoxP3+ Cells Are Present at High Levels Inside the Allotransplanted Heart of Mice That Received Repeated Injections of hIL-10-TFLs
Because we found FoxP3+ T cells inside a long-term human skin allograft several years after transplantation (18), we investigated whether these cells could play a role in the long-term persistence of the allografted heart in mice injected with hIL-10-TFLs. The allogeneic hearts of mice that had or had not received hIL-10-TFLs were extracted, fixed, cut, and stained with anti-FoxP3 mAb. As shown in Figures 5, IA and IB, the presence of FoxP3+ cells was observed around the vessels in the allografted hearts at day 14 after transplantation, whether the recipients had been injected with TFLs. In contrast, in the mouse that survived up to 140 days, FoxP3+ cells not only were found around the vessels but also in the interstitium of the transplanted heart at rather high levels (Fig. 5,IC), suggesting that FoxP3+ cells might have infiltrated the allogeneic heart, and then possibly proliferated inside the organ. Simultaneous CD4 labeling was weaker than FoxP3 labeling under the technical conditions of the study, but it definitely included all FoxP3+ cells and some FoxP3− cells (data not shown). To ensure that FoxP3+CD4+ cells were implicated in the delay of organ rejection, we then analyzed the kinetics of their migration in a skin transplant model. We first measured the expression of CD4+FoxP3+ cells by cytofluorometry in Balb/c mice that had received a single injection of TFLs or hIL-10-TFLs (or no TFLs as a control) before a skin transplant from C57Bl/6 mice. Mice were killed at day 5, and organs were harvested. CD4+FoxP3+ cells were found in the thymus and predominantly in the spleen in each group. It must be pointed out, however, that the percentage of spleen cells that expressed FoxP3 increased after injection of TFLs and to a greater extent after injection of IL10-TFLs. We then measured the expression of FoxP3 in mice repeatedly injected with hIL-10-TFLs at days 5, 35, and 45 and observed that the percentage of CD4+FoxP3+ cells decreased in the spleen and increased in the thymus over time (Fig. 5,II). These results may suggest that CD4+FoxP3+ cells migrated from the spleen toward the thymus after organ transplantation and repeated injections with hIL-10-TFLs.
We previously reported that transduction of the hIL-10 gene into mouse hematopoietic fetal liver cells contributes to the prolongation of both skin and heart allograft survival in a sublethally irradiated host (16, 19). The fact that such transplants are ultimately rejected suggests a lack of permanent tolerance due to elimination of the donor stem cells. Indeed, days or weeks before transplant rejection, H-2b hematopoietic cells were no longer detectable (16). It was assumed that the stem-cell graft, although facilitated by local IL-10 production, was only transient and was followed by rejection of all donor cells. Interestingly, the level of hIL-10 in mouse serum also decreased over time.
We demonstrated in this study that repeated injections of hIL-10-TFLs resulted in considerably longer survival of donor stem cells, prolonged hIL-10 production, and, therefore, a “prope” tolerance to heart allografts. After each administration of hIL-10-TFLs, IL-10 protein was detectable for 2 weeks. After repeated injections of hIL-10-TFLs, IL-10 production was maintained; it was detectable at day 140 in the organs of a recipient mouse with an unrejected heart allograft. Donor H-2b cells were also present in the same organs at this time. Moreover, we showed that this “prope” tolerance was specific for the histocompatibility antigens expressed by the donor fetal liver cells, as indicated by the absence of allograft prolongation in mice that received an allogeneic skin graft from a third-party strain.
The potential benefit of IL-10 in the prevention of rejection of allogeneic HSCs was suggested by previous investigations in which we showed that tolerance to MHC-mismatched HSCs was associated with the production of IL-10 in vivo in children treated with an HSC transplant for severe combined immunodeficiency disease (20).
Because the experimental data on the capacity of IL-10 treatment or local IL-10 gene therapy to induce genuine and long-lasting transplantation tolerance were not convincing, we took another approach. In our study, IL-10 gene therapy was only used to ensure survival of donor-type stem cells, because tolerance to the organ transplant was thought to be a result of the maintenance of donor TFLs in the recipient. Sustained chimerism has long been known to induce tolerance in the specific donor (6, 21).
In the case of partial chimerism or microchimerism only, it is less clear what is the cause and what is the consequence of the survival of donor TFLs and lymphoid cells and that of donor organs (22, 23). The tolerance in the latter condition can be considered as a “prope” tolerance, which is less complete and stable than “full” tolerance.
In our experiments with repeated administration of donor-type hIL-10TFLs to recipient mice, we observed prolonged tolerance in some animals and delayed rejection only in others. Whether this difference is associated with various degrees of chimerism (microchimerism or partial chimerism) is still not clear. The hIL-10 gene was found to be present throughout the experiment, but the production of detectable levels of hIL-10 protein in the blood lasted less than 3 weeks after administration of hIL-10-TFLs. Similarly, the presence of a few H-2b donor cells in various organs was shown by PCR throughout the observation period, but circulating donor cells (identified by immunofluorescence) were scarce after 8 days. It is, therefore, possible that, because of a selective partiality to host cells over donor cells in hematopoietic niches, only microchimerism was achieved 8 days after each injection of hIL-10-TFLs. This level of microchimerism may have been sufficient in some (but not all) animals to provide a definitive and full tolerance.
Various forms of transplant tolerance can be induced in experimental animals in the central or peripheral immune system (2, 24, 25). The most complete forms of tolerance involve replacement of the host immune system with that of donor after transplantation of donor-type TFLs into a recipient that is fully devoid of lymphoid cells (e.g., by lethal irradiation then reconstitution with donor stem cells) (26). A less complete but more operational form is the tolerance induced by manipulation of the host immune system by various means (27). One of the mechanisms of such an “operational tolerance” is related to the presence of sufficient numbers of regulatory T cells (Tregs) in the transplant, the lymphoid organs, and the circulatory system (28, 29).
Tregs belong to several cell subsets that are generated by the induction of FoxP3 expression in CD4+ CD25− FoxP3− or CD4+ CD25+ FoxP3− T cells (24, 30, 31). Inhibitory cytokines, and especially IL-10, tumor growth factor-β, and IL-35, seem to play an important role (32, 33). Tregs can suppress many steps of the immune response: inhibition of CD4+ and CD8+ T-cell activation (by strongly reducing the induction of IL-2 mRNA and by consuming IL-2) (34) and modulation of the function of antigen-presenting cells, natural killer cells, natural killer T cells, and mast cells (35).
Tregs may have contributed to the operational tolerance to heart allografts observed in our mice. Indeed, as reported by others (36), we also found Tregs in the transplants, even after rejection. Interestingly, Tregs with the FoxP3+ phenotype were also detected within the interstitium of the allogeneic hearts that survived for prolonged periods after repeated injections of hIL-10-TFLs. It may be postulated that there are two successive stages of FoxP3+ cell infiltration, that is, migration from the blood and then diffusion or proliferation inside the allogeneic tissue. The second stage only might account for the regulatory action of FoxP3+ T cells, in agreement with reports that have shown that FoxP3 levels are related with successful reversal of acute rejection in kidney allografts (37). FoxP3+ T cells have also been observed inside well-tolerated composite tissue allografts (18).
Interestingly, in the skin transplant model, we also observed two stages in FoxP3+ cell infiltration. In the first, CD4+FoxP3+ cells were present in the spleen of mice at day 5 after skin transplantation, irrespective of the injection of hIL-10TFLs (Fig. 5,II). In the second, FoxP3+ Treg cells were presumed to migrate from the spleen to the thymus, where they possibly contributed to the induction of prope tolerance.
In conclusion, operational tolerance can be induced, probably due to both partial chimerism and the development of Tregs, in adult animals that receive allogeneic stem cells transduced with the hIL-10 gene. Such a procedure is less dangerous for the recipient than induction of complete chimerism but may require repeated administration of the specific donor stem cells to maintain the state of tolerance definitively.
MATERIALS AND METHODS
See SDC 1 (http://links.lww.com/TP/A638).
The authors are grateful to Marcelo Lopez Lastra (Ecole Normale Supérieure, Lyon) who produced the GPE86 hIL-10 cell line, and to Maria-Grazia Roncarolo and Kathryn Wood for help and advice. The authors wish to thank Véronique Barbalat (Institut BioMérieux, Lyon) and Kamel Wahbi (Laboratoire Pathologique, Centre Hospitalier Lyon-Sud) for valuable assistance in PCR analyses. They also thank Marie-Angélique Cazalis (Institut BioMérieux, Lyon) for aid in statistics, and Jane Mitchell for help in editing the manuscript.
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