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Approaches to safely induce tolerance in vascularized composite allotransplantation (VCA) with chimerism through bone marrow transplantation (BMT) are currently being pursued. However, the VCA were historically performed sequentially after donor chimerism was established. Delayed VCA is not clinically applicable due to the time constraints associated with procurement from deceased donors. A more clinically relevant approach to perform both the BMT and VCA simultaneously was evaluated.
WF (RT1Au) rats were treated with a short course of immunosuppressive therapy (anti-αβ-TCR mAb, FK-506, and anti-lymphocyte serum). One day prior to BMT, rats were treated with varying doses of total body irradiation (TBI) followed by transplantation of heterotopic osteomyocutaneous flaps from hind limbs of ACI (RT1Aabl) rats.
80% of rats conditioned with 300 cGy TBI and 40% of rats receiving 400 cGy TBI accepted the VCA. Mixed chimerism was detected in peripheral blood at one month post-VCA, but chimerism was lost in all transplant recipients by 4 months. The majority of peripheral donor cells originated from the BMT and not the VCA. Acceptors of VCA were tolerant of a donor skin graft challenge and no anti-donor antibodies were detectable, suggesting a central deletional mechanism for tolerance. Regulatory T cells (Treg) from spleens of acceptors more potently suppressed lymphocyte proliferation than Treg from rejectors in the presence of donor stimulator cells.
These studies suggest that simultaneous BMT and VCA may establish indefinite allograft survival in rats through Treg-mediated suppression and thymic deletion of alloreactive T cells.
Vascularized composite allotransplantation (VCA) refers to the transplantation of complex primarily vascularized tissues of ectodermal and mesodermal origin (1), including skin, muscle, nerve, bone, tendon, and other tissues. VCA is emerging as a potential treatment for complex tissue defects (1–3). The feasibility of the procedure has been confirmed through over 50 hand transplants, 10 facial reconstructions, and vascularized knee, esophageal, and tracheal allografts around the world to date (3,4). However, a major factor limiting VCA is the requirement for lifelong immunosuppression and the associated toxicities (5). Virtually all expected complications associated with the use of chronic immunosuppression, including renal failure, have occurred now in recipients of VCA (6). The ideal outcome to achieve permanent VCA survival would be induction of donor-specific tolerance, thereby abrogating the need for chronic immunosuppression. Experimental studies have shown that the establishment of donor-specific chimerism in organ transplant recipients can lead to donor-specific tolerance (7–10). The clinical relevance of mixed chimerism has recently been underscored by the results of a prospective clinical pilot trial in which patients became tolerant after receiving kidney and bone marrow transplantation (BMT) from the same donor (11–13).
It is hypothesized that VCA may facilitate the induction and/or maintenance of chimerism since the transplanted part includes a potential niche for bone marrow (BM) stem cells. However, conditioning of recipients to establish chimerism by BMT requires the use of myelotoxic drugs and total body irradiation (TBI) which are associated with significant toxicities. The challenge remains to develop nonmyeloablative conditioning regimens that allow maximal BM engraftment with minimal recipient toxicity. We previously reported that mixed chimerism can be established in an allogeneic rat model by nonmyeloablative conditioning with TBI as low as 300 cGy (9). This model has now been extended to assess tolerance to CTA.
Approaches to induce a tolerogenic state in transplantation often target peripheral and/or central tolerance mechanisms (14–16). Central deletional mechanisms have been demonstrated to be the major mechanism in tolerance induction and maintenance through mixed chimerism. However, tolerance has frequently been associated with suppression of alloreactive T cells by peripheral mechanisms in allograft recipients. Recent studies have shown that CD4+/CD25+/FoxP3+ regulatory T cells (Treg) play an important role in maintaining peripheral tolerance in transplant recipients by controlling alloreactive immune responses through indirect antigen recognition (18–20). Treg also have the potential to influence central tolerance through indirect presentation of donor Treg-derived alloantigens in the thymus and subsequent negative selection of alloreactive T cells, which has been shown to promote limited skin graft acceptance in mice (21). Treg frequencies are higher in BMT compared to peripheral blood (PB) stem cell transplants, which could reduce the overall alloreactivity of the graft and promote a tolerogenic state (22).
We previously used a sequential transplantation model to study VCA in nonmyeloablatively conditioned rats receiving BMT first in order to establish chimerism prior to VCA (9). Although prolongation of the MHC-disparate transplanted tissues were achieved in this study using a short course of immunosuppressive agents and systemic monoclonal antibodies (mAb), a more clinically relevant model would involve performing hematopoietic stem cell transplantation (HSCT) and VCA simultaneously. Our objectives in the present study were to establish a clinically relevant simultaneous BMT and VCA model in rats treated with a nonmyeloablative conditioning regimen, to determine whether tolerance and donor chimerism were maintained over time in graft acceptors, and to examine the role of Treg in maintenance of a tolerogenic state.
WF rats were conditioned with 100–400 cGy TBI one day prior to BMT and treated with anti-αβTCR mAb on day −3, FK-506 on days −1 to +9, and a single dose of anti-rat lymphocyte serum (ALS) on day +9 (Fig. 1). ACI flaps were transplanted into the right gluteal region of WF rats one day prior to BMT. The 5 control rats that received VCA without prior irradiation (0 cGy TBI) rejected their VCA within 25 days. Rejection occurred within 100 days in all animals receiving 100 cGy TBI, and 4 of 5 animals receiving 200 cGy TBI rejected their VCA within 125 days (Fig. 2A). Long-term VCA acceptance (>200 days post-transplant) was observed in 80% of animals receiving 300 cGy TBI and 40% of animals receiving 400 cGy TBI. The reduction in graft survival rate for recipients receiving 400 cGy TBI versus 300 cGy TBI may be the result of a small sample size rather than a direct effect. No clinical manifestations of GVHD were observed in any of the VCA recipients up to 6 months post-transplant.
None of the recipients conditioned with 0 or 100 cGy TBI engrafted with donor cells. 1 of 5 rats treated with 200 cGy TBI had > 1% donor cells in lymphocyte gate of peripheral blood (PB) and accepted their VCA. 5 out of 5 or 4 out of 5 were engrafted in 300 or 400 cGy TBI treatment groups, respectively. As no correlation between the levels of donor chimerism and the graft survival was found according to the TBI doses, recipients were divided into 2 groups as acceptors or rejectors according to the status of VCA acceptance or rejection regardless of the TBI doses received. With simultaneous BMT-VCA, there was no significant difference between the donor chimerism levels of acceptors and rejectors at one month post-transplant with 15.6 ± 10.4% and 16.3 ± 8.5%, respectively (Fig. 2B), without an obvious reason. However, there was a significant difference between donor chimerism levels at 2 months (5.6 ± 0.02% vs. 0.5 ± 0.003%; P = 0.05). Donor chimerism was lost in the PB of all chimeras that received simultaneous BMT-VCA by four months post-BMT.
In this simultaneous VCA/BMT model, the donor cells in the engrafted recipients could originate infused BMC or vascularized bones in VCA. To define the source of circulating donor cells in mixed chimeras, twelve ACI rats were treated with the same immunosuppressive therapy as described above with 300 cGy TBI. WF eGFP rats were used as the source of BM or the VCA so that donor cells could be easily detected by flow cytometry. ACI rats received that eGFP+ BMC plus eGFP− VCA showed all the donor cells circulating in PB were eGFP+ but not eGFP− cells at 1, 2, and 3 months post-transplant (Fig. 2C). Furthermore, no eGFP+ cells were detected in ACI rats received eGFP− BMC plus eGFP+ VCA and all the donor cells circulating in PB were eGFP−. The difference in donor chimerism at 1, 2, and 3 months between donor BMC from GFP+ rats and GFP− rats is most likely due to the variation between experiments. These data suggest that engrafted donor cells come from the BMT but not the VCA.
There is no method available in rats to directly detect the central deletion of alloreactive T cells, secondary antigenic challenge by skin graft was used to assess the robustness of transplantation tolerance in a chimera that accepted the first VCA graft, since the acceptance of a highly antigenic second graft strongly suggests the existence of central deletional tolerance (25,26). Second-set donor skin grafts were placed at one year post-transplant on four VCA acceptors and six rejectors. Tolerance to skin grafts was maintained in VCA acceptors throughout 120 days follow-up period (Fig. 3A), and no changes in the VCA of acceptors were observed during the secondary challenge. The rejectors rejected the skin grafts promptly within 18 days. Sera from all six VCA rejectors scored positive for anti-donor antibodies in a flow cytometric crossmatch (FCXM) assay, but no sera samples taken from VCA acceptors had circulating anti-donor antibodies (Fig. 3B).
CD4+/CD25+/FoxP3+ Treg play an important role in maintaining peripheral tolerance to transplants and may also indirectly influence central tolerance (18,19,21). To determine if the cell numbers of this important tolerogenic mediator were greater in VCA acceptors compared to rejectors, we examined tissues in 3 VCA acceptors and 3 VCA rejectors for CD4+/CD25+/FoxP3+ Treg. There was no significant difference between the percentages (Fig. 4A) or absolute numbers (Fig. 4B) of Treg measured in the hematolymphopoietic compartments of acceptors or rejectors. These data suggest that the determining factor in tolerance induction may be Treg immunomodulatory function rather than absolute cell numbers. The majority of Treg were found in the mesenteric lymph node, spleen, and thymus for both acceptors and rejectors, and a lesser percentage of Treg were found in the donor and recipient BM of VCA acceptors (Fig. 4A).
To further study the function of Treg, a MLR suppressor cell assay was used. We incubated irradiated splenic stimulator cells (either donor ACI cells or F344 third-party cells) with naïve WF responder lymph node cells and sorted CD8−/CD4+/CD25+ splenic Treg from VCA recipients. Lymphocyte proliferation was measured in samples containing a 1:1 or a 1:0.25 ratio of naïve WF responder cells to Treg (Fig. 4C and D). Splenic Treg from VCA acceptors significantly suppressed lymphocyte proliferation in the presence of donor (ACI) antigen at cell numbers that were equivalent to or even 4-fold less than the number of naïve WF responder cells (Fig. 4C, black bars; P<0.05). Treg from VCA rejectors suppressed lymphocyte proliferation in the presence of donor antigen, but this effect was lost when the concentration of Treg was reduced (Fig. 4D). These data suggest that Treg from either acceptor or rejector are functional and maintain their suppressive capabilities. When the effect of suppression reaches the maximal at a certain ratio of responder and Treg, the suppressive effect will remain the same if the number or ratio of Treg increases. That is why almost equal suppression was observed at the 1:1 ratio in both Treg from rejectors and acceptors. The Treg from acceptors still maintained the suppressive effect when fewer Treg were used, but Treg from rejectors did not, suggesting Treg from VCA acceptors have more suppressive capability to donor alloantigen compared with rejector Treg. It is also of interest to note that Treg from both acceptors and rejectors suppressed lymphocyte proliferation much less in the presence of third-party (F344) antigen compared with the donor antigen, suggesting that donor-specific Treg were present in the spleens of rejectors and acceptors.
VCA, including hand and face transplantation, significantly improves the quality of life of recipients. However, the toxicities caused by the lifelong use of immunosuppressive drugs have raised ethical concerns for these procedures. A long-term goal in VCA as well as solid organ transplantation would be to induce donor-specific transplantation tolerance to avoid the chronic use of immunosuppressive drugs. Mixed chimerism has been demonstrated to be the most clinically relevant procedure to induce donor-specific tolerance to transplanted tissues and organs (8,11). Mixed chimerism has been shown in rats to induce donor-specific tolerance to the highly antigenic composite tissue allografts (9,24,27,28). These studies demonstrated that chimerism established with a nonmyeloablative conditioning approach could promote VCA tolerance.
However, the VCA were historically performed sequentially with a more than 1 month delay after BMT to establish donor chimerism. The delayed approach for VCA is not applicable in the case of a deceased donor. The clinically relevant VCA model should be that both the BMT and VCA are performed simultaneously. Prabhune et al. reported that tolerance to hind-limb transplants can be established through simultaneous transplantation of hind-limb and BM in recipients ablatively conditioned with high levels of irradiation (27). In our current study, we have established a more clinically relevant transplantation model in rats where both BMT and VCA are performed simultaneously with reduced-intensity nonmyeloablative conditioning. The immunosuppression is halted after nine days post-transplant. Our results show that BMT with nonmyeloablative conditioning can establish long-term tolerance and indefinite survival of simultaneous VCA with measurable levels of donor chimerism at 1–3 months post-transplant. Moreover, the tolerance to grafts is maintained even after PB donor chimerism is lost.
The relationship between stem cell-driven hematolymphopoietic macrochimerism and transplantation tolerance has been well established. Recipients maintained tolerance after losing macrochimerism, which may suggest that transient engraftment exerts an immunomodulatory effect on rejection. Our data are consistent with prior observations in a sequential BMT-VCA rat model. In this study, Rahhal et al. reported that donor macrochimerism was initially achieved with nonmyeloablative conditioning, but the majority of VCA acceptors lost PB chimerism within 6 months (9). In a clinical HLA-mismatched BMT and kidney transplantation report, stable renal function was maintained for years after complete withdrawal of immunosuppressive therapy even though donor chimerism was lost within 2 weeks (11,29). Transient donor chimerism was also found to reduce the rate of acute rejection in human heart recipients (30). The mechanism responsible for this phenomenon may be due to the association between donor microchimerism and graft tolerance. Persistence of donor microchimerism was reported in organ allograft recipients and proposed to facilitate long-term graft survival (31,32).
In the report by Rahhal et al., the donor chimerism was found to be present at significantly higher levels in the transplanted bone compartment compared with other host tissues of VCA acceptors who had lost PB chimerism (9). This observation gives a good explanation to the underlying mechanisms of tolerance. The persistent high level of donor cells in donor BM establishes systemic microchimerism to facilitate tolerance maintenance. This tolerance mechanism may also apply to our current simultaneous VCA/BMT model. Moreover, central deletional tolerance is most likely present in our simultaneous model as reflected by the fact that a second set of donor skin grafts is accepted long-term and does not break tolerance to the VCA. The central tolerance that follows the induction of chimerism by BMT is associated with elimination of cells in the thymus that are reactive to donor antigen. Donor microchimerism has been reported to be sufficient to induce and maintain tolerance through central deletional mechanisms (35).
We found that the overall number of CD4+/CD25+/FoxP3+ Treg did not differ significantly between VCA acceptors and rejectors. However, our MLR assays show that VCA acceptors preferentially suppress lymphocyte proliferation in the presence of ACI antigen, and this suppressive effect was not lost when the concentration of Treg was reduced. The reduced potency of Treg in VCA rejectors points to a loss of peripheral tolerance in these animals. These data suggest that Treg in long-term VCA acceptors exhibit a stronger immunosuppressive capacity and may play an important role in maintenance of tolerance and promoting graft acceptance. The immunomodulative function of Treg seems more critical in tolerance induction and maintenance in comparison with the absolute number of Treg. Interestingly, we also reported that significant local CD4+/FoxP3+ Treg infiltration was observed in tolerant donor-allograft skin samples but not in skin samples from rejector or naïve rats (36). Taken together, these data may also suggest the importance of Treg in promoting VCA graft acceptance and maintaining tolerance in long-term VCA acceptors.
In summary, our data suggest that both Treg-mediated suppression and central tolerance mechanisms contribute to allograft acceptance in a simultaneous BMT-VCA model. Treg-mediated peripheral tolerance does not appear to be sufficient on its own to induce allograft acceptance. We observed this with our VCA rejectors: Treg from VCA rejectors were able to suppress lymphocyte proliferation in the presence donor antigen, but Treg were unable to prevent VCA or secondary skin graft rejection events. Our data support the hypothesis that maximizing the induction of central tolerance and Treg-mediated suppression, through transient but sufficient levels of donor chimerism, is necessary to achieve a tolerogenic state.
Six- to 10-week old Wistar Furth (WF; RT1Au) and August Copenhagen Irish (ACI; RT1Aabl) male rats were purchased from Harlan Sprague Dawley (Indianapolis, IN). There is fully MHC disparity between WF and ACI rats. WF enhanced green fluorescent protein (eGFP, Wistar-TgN[CAG-GFP]184Ys) male and female rats were purchased (Rat Resource and Research Center, University of Missouri, Columbia, MO) and bred (Institute for Cellular Therapeutics). Animals were housed in a specific pathogen free facility.
The T cell depleted (TCD) BMC were prepared by magnet negative selection of the TCR+ cells after incubating with mouse anti-rat αβ-TCR and γδ-TCR mAb and combining with sheep anti-mouse IgG-coated magnetic beads (Dynabeads M-450; Dynal, Lake Success, NY). The nonmyeloablative conditioning and BMT were depicted in Fig. 1.
To determine if the origin of donor cells in chimeric rats was from infused donor BMC or the bone of VCA, recipient ACI rats received grafts from WF rats with either enhanced green fluorescence protein positive (eGFP+) BMC plus eGF VCA or eGF BMC plus eGFP+ VCA.
Heterotopic osteomyocutaneous flap transplantation was performed one day prior to BMT. Donor flaps were prepared from male ACI and transplanted into the right gluteal region as previously described (9). Animals were monitored daily for two weeks following transplantation and then weekly for signs of flap rejection or graft-versus-host disease (GVHD) (9,24). Rejection was defined as loss of the skin paddle.
The percent donor cells in the periphery of transplant recipients were determined by flow cytometry at months 1–6 post-procedure. PB was stained with anti-RT1Aabl-FITC mAb (Becton Dickinson, Mountainview, CA). The number of RT1Aabl-FITC positive cells was measured in the lymphoid gate. To determine the origin of donor cells, PB was collected and analyzed by flow cytometry for the number of eGFP-positive cells in the lymphoid gate. Animals whose PB contained ≥1% donor cells were considered chimeric.
A secondary antigenic challenge was given to VCA acceptors and rejectors by grafting donor tail skin onto a bed prepared in the recipients’ mid-scapular region as previously described (9). Sera from skin graft recipients were derived from whole blood samples collected six weeks after the procedure and analyzed for anti-donor antibodies by flow cytometry.
To identify CD4+/CD25+/FoxP3+ Treg, we designed a staining panel that included anti-CD4-APC, anti-CD8-PerCP, anti-CD25-PE, anti-αβ-TCR-Pacific Orange, and anti-FoxP3-Pacific Blue (Becton Dickinson).
Briefly, stimulator cells from the spleens of WF (recipient), ACI (donor), or F344 rats (third party) were isolated and incubated overnight. The following day, stimulator cells were irradiated (2,000 cGy) and incubated with naïve WF responder lymph node cells and sorted CD8−/CD4+/CD25+ Treg from VCA recipients. Lymphocyte proliferation was measured in triplicate by (H3)-thymidine uptake.
Statistical analysis was performed using the student one-tailed t-test, and P values ≤ 0.05 are considered to be significant.
The authors thank Michael Tanner for cell sorting and technical assistance; Eiji Kobayashi for providing the eGFP rats; Carolyn DeLautre for manuscript preparation; and the staff of the University of Louisville animal facility for outstanding animal care. This research was supported in part by the following: NIH R01 DK069766 and NIH 5RO1 HL063442. This publication was also made possible by Award No.W81XWH-07-1-0185 and W81XWH-09-2-0124 from the U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD, 21702-5014 (Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the Office of Army Research); the Commonwealth of Kentucky Research Challenge Trust Fund; and The Jewish Hospital Foundation. Research was conducted in compliance with the Animal Welfare Act Regulations and other Federal statutes relating to animals and experiments involving animals and adheres to the principles set forth in the Guide for Care and Use of Laboratory Animals, National Research Council, 1996.
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1H.X. Participated in the interpretation of the data, manuscript writing, and research design; D.M.R. participated in the performance of the research, the interpretation of the data, and participated in manuscript writing; S.W. participated in the performance of the research, interpretation of the data, manuscript writing; L.D.B participated in the performance of the research, the interpretation of the data, and participated in manuscript writing; S.T.I. Participated in the interpretation of the data, manuscript writing, and research design and reviewed all data and manuscript.
S.T.I. is the founding scientist and director of Regenerex, a biotech start-up company; it has not been capitalized. L.D.B. is a research scientist and employee of Regenerex. The other authors have no conflict of interest to declare.