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We have shown previously that donor-derived splenocytes can replace recipients' bone marrow and induce donor-specific tolerance (DST). We have also shown the usefulness of the chimeric state for the induction of DST. Further analysis of mixed splenocytes chimera, especially the role of each T cells in mixed splenocytes chimera, is indispensable issue for its clinical use. A chimeric state has been shown to achieve long-term survival in major histocompatibility complex (MHC)-mismatched grafts. The donor-derived splenocytes can replace recipients' bone marrow and induce DST. The long-term survival of allogeneic skin grafts was achieved without immunosuppressants. In this study we show the role of each T cell type in a splenocyte mixed chimera. This review provides a short summary of our original work, adding some supplemental interpretations. Mixed chimerism is thus considered an attractive approach for the induction of DST without the use of immunosuppressants. In this paper, we summarize some of the findings on mixed splenocyte chimeras and review mixed chimerism in recent organ transplantation.
Mixed chimerism has been considered an attractive approach for the induction of donor-specific tolerance (DST) [1-5]. By inducing DST the host's immune system is rendered unresponsive to specific donor-transplantation antigens, while the overall immune function of the individual is maintained, thereby avoiding immunosuppressant-related complications [6-9].
A recent report proposed a new therapeutic approach for organ transplantation with the use of mixed chimerism . Five patients with end-stage renal disease received simultaneous bone marrow and kidney transplants from single-haplotype mismatched human leucocyte antigen (HLA) donors. Only one patient had irreversible rejection. Moreover, patients without rejection could discontinue immunosuppressant therapy. This approach has been extended successfully from non-humans to humans . Learning from this phenomenon, we prepared a splenocyte mixed chimera model to investigate the roles of each type of T cells in mixed chimerism.
We have shown previously that splenocytes, a rich source of lymphocytes and haematopoietic cells, can replace bone marrow (BM) chimeric cells and maintain allogeneic skin graft tolerance without the use of immunosuppressants [4, 12-14]. This model was suitable for analysing (i) the ability of splenocytes to replace the recipient haematopoietic system with donor-derived non-bone marrow cells, (ii) the safety of partially major histocompatibility complex (MHC)-matched organs in mixed splenocyte chimera and (iii) the role of T cells in mixed splenocyte chimera. Splenocyte chimeras are thus an attractive model for analysing the mechanism of the development of mixed chimerism.
Usefulness of mixed chimerism in solid organ transplantation
Recently, epoch-making studies of transplantation were reported by Kawai et al. in Massachusetts General Hospital (MGH) [15, 16]. They induced renal allograft tolerance successfully using bone marrow mixed chimerism methods. Initially, this strategy required treatment 6 days before solid organ transplantation. Thereafter, they developed another method to induce delayed tolerance. This method used mixed chimerism to induce allograft tolerance in post-transplantation recipients . They added anti-CD8 monoclonal antibody to the conditioning regimen and found that inactivation of CD8 facilitates the development of mixed chimerism.
CD4+CD25+ regulatory T cells (Treg) play essential roles in the induction and maintenance of self-tolerance. Recently, the extra-depletion or activation of CD4+CD25+ Treg was shown to accelerate or prevent graft-versus-host-disease (GVHD) . These results suggested that CD4+CD25+ Treg also has an important role in the generation of mixed chimerism. Most previous work has focused on the roles of CD4+CD25+ Treg in recipients. One very small study assessed donor-derived CD4+CD25+ Treg in a mixed chimera model [3-5]. Our splenocyte chimera model is unique, because it can be used to assess the induction phase of mixed chimera by non-bone marrow cells. In addition, our model can assess the contribution of the mixed chimeric state in the maintenance phase.
Animal model of splenocyte chimera
First, we made a model of mixed splenocyte chimera and investigated whether splenocyte mixed chimera can be used to maintain chimerism and to prolong partially MHC-type matched skin graft survival . We used C57BL/6J (MHC class I: H-2Kb), C3H/He (H-2Kk), first-generation offspring (F1) from crosses of B6 and DBA/2 mice: B6D2F1 (H-2Kb/d) and F1 from crosses of B6 and C3H strains: B6C3F1 (H-2Kb/k).
Mixed BM was established in this chimeric model by irradiating recipient C3H/He (H-2Kk) mice with a dose of 1000 rad (Fig. 1a). To make a mixed BM chimera, a mixture of BM from C3H/He and B6D2F1 was injected into the recipient mice 30 days before skin grafting. Skin grafts were obtained from C57BL/6 mice. The splenocytes from C3H/He (syngeneic to the recipients), B6D2F1 (syngeneic to the BM donors) and B6C3F1 (with both recipient and skin donor MHC types) mice were administered to the chimeric mice 30 days after skin grafting.
BM-derived cells were replaced by each injected type of splenocyte. Skin graft rejection depends upon MHC class I splenocytes. The B6D2F1- and B6C3F1-derived splenocytes can replace peripheral blood cells and maintain the C57BL/6 skin graft at least 200 days after splenocyte injection, thereby maintaining the new chimerism (Fig. 1b). The B6 skin graft was rejected 30 days after C3H/He splenocyte infusion (group A). The B6 skin grafts survived after partially HLA-matched splenocyte infusion (group B: B6D2F1, group C: B6C3F1) (Fig. 1c).
We found that when the recipient peripheral blood and skin graft MHC were partially matched, specific tolerance to the skin graft was induced and graft survival could be maintained by a mixed chimeric state. The newly constructed splenocyte chimeras can maintain allogeneic grafts without immunosuppressants for a long time. The model is suitable for analysing the mechanisms involved in switching a recipient immune system to another source of haematopoietic cells.
Role of each type of T cell in splenocyte chimera
Splenocytes contain a rich source of lymphocytes and haematopoietic cells. T cells, in particular, have been shown to play a key role in the replacement of the chimeric state [18-20]. Some previous work has revealed that chimerism is destroyed by host-derived T cells for chimeric cell depletion, and donor-derived T cells are essential to prevent the rejection of donor-derived haematopoietic cells. However, it remains unclear which populations of splenocyte T cells control the replacement and maintenance of the chimeric state in our model. We analysed this issue with the use of a splenocyte mixed chimera model with subtraction of each T cell fraction.
Roles of CD8+ and CD4+ T cells in splenocyte chimera
According to our previous protocol, a C3H/B6D2F1 mixed BM chimera with skin grafting from C57/BL6 was established (Fig. 2a) [12, 13]. Splenocytes from B6C3F1 mice were transplanted to establish splenocytic BM chimeras using total splenocytes, CD90+-depleted (total T cell) splenocytes, CD4+-depleted splenocytes or CD8+-depleted splenocytes 30 days after skin grafting.
We identified three different patterns in the development of splenocyte-derived BM chimeras (Fig. 2b). The first was seen using total splenocytes (control): the proportion of H-2Kb+H-2Kk− cells decreased over the first 2 weeks, reaching a minimum on day 14 after the administration of splenocytes, while the number of H-2Kb+H-2Kk+ double-positive cells increased simultaneously.
The second pattern was seen with either CD90+ or CD8+ T cell-depleted splenocytes. The proportions of H-2Kb+H-2Kk− and H-2Kb+H-2Kk+ double-positive cells remained unchanged. The replacement of BM chimeric cells by splenocytes and the establishment of splenocyte chimeras did not occur in the absence of CD8+ T cells.
The third pattern was seen using CD4+ T cell-depleted splenocytes. The proportion of H-2Kb+H-2Kk− cells decreased gradually, while the increase in H-2Kb+H-2Kk+double-positive cells was only partial and peaked at an intermediate level between the other two patterns. The formation of a splenocyte chimera was incomplete in the absence of CD4+ T cells.
These results reflect an indispensable role of CD8+ T cells in splenocytes (Fig. 3a). T helper type 1 (Th1) cells can activate naive CD8+ T cells to become cytotoxic T lymphocytes (CTLs) and can maintain and expand the memory CD8+ T cell population during resensitization [21, 22]. Naive CD8+ T cells differentiate into CTLs on exposure to antigens. In the absence of CD8+ T cells in splenocytes, no activation of cytotoxic activity occurred. The incomplete cytotoxic activity of CD8+ T cells might have been caused by the lack of cell-to-cell interactions with CD4+ T cells in splenocytes (Fig. 3b). The remaining naive CD8+ T cells that survive the exclusion of CTLs could potentially mediate an incomplete replacement of BM.
The role of CD4+CD25+ Treg in splenocyte chimera
CD4+CD25+ Treg are known to suppress or accelerate various immune responses and maintain normal immune function [23, 24]. Thus, proliferation and functional enhancement of CD4+CD25+ Treg may enable the control of graft rejection. We investigated whether donor-derived CD4+CD25+ Treg cells regulate the speed of reconstitution in the recipient immune system.
The same splenocyte chimera model described above was used to investigate the role of CD4+CD25+ Treg in splenocytes [12, 13]. Donor-derived CD4+CD25+ Treg was depleted from splenocytes. The development pattern of splenocyte-derived BM chimeras was assessed .
The initial increase in the proportion of H-2Kb+H-2Kk+ cells in the CD4+CD25+ Treg-depleted group was faster than that in the total splenocyte injection group (control) (Fig. 4a). The proportion of H-2Kb+H-2Kk+ cells in the control group caught up by day 21. Our results showed that the depletion of CD4+CD25+ Treg in donor cells can accelerate splenocytic chimera. This phenomenon suggests that the enhanced cytotoxic activity of CD8+ T cells was led by the lack of cell-to-cell interactions with CD4+CD25+ Treg in splenocytes (Fig. 4b). Enhancement of the pathway from CD8+ T cells to CTLs resulted in accelerated replacement by donor-derived splenocytes.
Chimerism has been considered an attractive approach to induce DST without immunosuppressants, as described first by Medawar et al.  This stable immune condition was confirmed in human orthotopic hepatic allografts with long-term survival by Starzl et al. [2, 3] Moreover, Monaco et al. demonstrated that chimerism plays an active role in DST induced by BM transplantation [4, 5]. A stable chimeric state has enabled long-term survival of MHC-mismatched grafts in some studies [11, 25-29].
In our mixed chimera model, the transfer of splenocytes from allogeneic mice to recipient mice resulted in the substitution of chimeric cells in the peripheral blood. Thereafter, the chimerism was maintained over a prolonged period by a mixed chimeric state in which the recipient and graft MHC are partially matched. Kanamoto and Maki reported that a small number of injected splenocytes, which shared MHC with skin grafts, persisted in mice in which a mixed chimeric state had been established  and seemed to protect grafted skin against rejection. They advocated that the existence of a small number of partially MHC-matched cells, i.e. ‘micro-mixed chimerism’, was sufficient to maintain allograft tolerance.
T cells, in particular, have been shown to play a key role in the replacement of the chimeric state . However, it remains unclear which populations of splenocyte T cells control the replacement and maintenance of the chimeric state. We examined splenocyte populations to determine which cells play key roles in the replacement and maintenance of splenocyte chimeras. First, we showed that partially MHC-matched CD8+ donor T cell splenocytes were essential for the formation of splenocyte chimeras. Without this population, the mixed chimeric state could not be established. Secondly, we investigated whether the speed of construction of the chimeric state could be controlled.
CD4+CD25+ Treg is known to suppress or accelerate various immune responses and maintain normal immune function . The depletion of CD4+CD25+ Treg from recipients has been shown to decrease the graft survival rate , and increased CD4+CD25+ Treg has been demonstrated in recipients with established immune tolerance . Bigenzahn et al. reported that the depletion of CD25+ T cells at the time of BM transplantation with anti-CD154 and CTLA4Ig prevents the development of tolerance . In contrast, delayed depletion of CD25+ T cells failed to prevent the development of tolerance. These findings suggest that Treg is indispensable to generation of a chimeric state, especially in the induction phase. Our protocol assessed various donor-derived fractions of T cells, including CD4+CD25+ Treg. In particular, the activities of donor-derived CD4+CD25+ Treg cells were analysed by depleting total splenocytes from this population. Our results showed that reconstitution of the recipient immune system from BM cells to splenocytes was achieved more promptly in the induction phase.
Recently, Hu et al. have shown that donor-derived BM transfusion produces mixed chimerism in a murine model . They assessed the immune reaction of donor-derived BM cells in simultaneous small-bowel transplantation. The donor-derived cell rate in peripheral blood and spleen was significantly higher, and small-bowel survival was significantly better in the donor-derived BM transplantation group than in the FK506 and control groups. These results also suggested that donor-derived cells may be one option for inducing graft tolerance via mixed chimerism.
In contrast, Taylor showed that depletion of donor CD4+CD25+ Treg from BM accelerates GVHD in MHC-mismatched recipients . They confirmed ex vivo that expanded and activated CD4+CD25+ Treg in BM inhibits GVHD significantly. Moreover, they showed that the depletion of CD25+ cells from donor splenocytes resulted in an acceleration of GVHD, whereas splenocytes expanded insufficiently ex vivo, even in the presence of high-dose interleukin (IL)-2. These results suggested that the depletion of donor CD4+CD25+ Treg affects the generation of a mixed chimera negatively. In contrast to their model, however, MHC class I is matched partially in our splenocyte mixed chimera model, and the proportions of donor and recipient cells are well balanced. Because of these differences, our results do not agree with the findings of Taylor, who concluded that the depletion of donor CD4+CD25+ Treg affects the generation of a mixed chimera negatively. We believe that the rapid and stable reconstitution of recipient haematopoiesis, including the immune system, due to a splenocytic chimera may contribute to the prevention of skin graft rejection.
Rapid and stable reconstitution of the recipient immune system in association with chimerism may contribute to the prevention of skin-graft rejection. We believe that this novel strategy can contribute to the prevention of graft rejection by controlling immune reactions in recipients. Clinical application of the phenomena described here represents an important step in inducing tolerance.
Transplantation protocols involving mixed chimerism are more common in blood transplantation and leukaemia therapy. In solid organ transplantation, a mixed chimera will hopefully facilitate the development of tolerance protocols to overcome the usage of immunosuppressants. Further investigations are needed to clarify which mechanisms activate the generation and maintenance of mixed chimerism.
We have shown a unique experimental model for analysing mixed splenocyte chimera. The model is suitable for analysing the mechanisms involved in switching a recipient immune system to another source of haematopoietic cells. Induction of tolerance in skin graft transplantation has been achieved via mixed splenocyte chimerism approaches. Improvements in the consistency of tolerance induction by the present protocol will lead to the next steps, permitting a wider range of clinical applications.
This study was supported by a grant from the 2013 Hagiwara Fund, Nihon University School of Medicine. The authors thank Mrs Naoko Kutsuna for her technical advice in drafting this paper.
The authors have no conflicts of interest to declare.