Original Basic ScienceçGeneral

Methyl-Guanine-Methyl-Transferase Transgenic Bone Marrow Transplantation Allows N,N-bis (2-chloroethyl)-Nitrosourea Driven Donor Mixed-Chimerism Without Graft-Versus-Host Disease, and With Donor-Specific Allograft Tolerance Min Hu, PhD,1,2 Belinda Kramer, PhD,3 Geoff Y. Zhang, MMed,1 Yuan Min Wang, PhD,1 Debbie Watson, PhD,1,4 Brian Howden, PhD,5 Geoff McCowage, FRACP,3 Ian E. Alexander, PhD, FRACP, FAHMS,6 Peter Gunning, PhD,7 and Stephen I. Alexander, FRACP1 Background. Transplant tolerance has been achieved by mixed chimerism in animal models and in a limited number of kidney transplant patients. However, these mixed-chimerism strategies were limited either by loss of long-term mixed chimerism or risk of graft-versus-host disease (GVHD). Selective bone marrow (BM) engraftment using marrow protective strategies are currently reaching clinical use. In this study, we tested the utility of methyl-guanine-methyl-transferase (MGMT)-transgenic-C57BL/6 BM into a major histocompatibility complex mismatched-BALB/c model followed by N,N-bis(2-chloroethyl)-nitrosourea (BCNU) treatment to enhance donor-cell engraftment and then evaluated transplant tolerance induction. Methods. A single-dose of anti-CD8 antibody and busulfan was administered into BALB/c-host-mice at day 1. The BALB/c-mice also received costimulatory blockade through multiple-doses of anti-CD40L antibody. 10  106 BM-cells from MGMT-transgenic-mice were transplanted into host BALB/c mice at day 0. The BCNU was administered at 4 time points after BM transplantation (BMT). Heterotopic donor C57BL/6 cardiac allografts were performed at day 243 after BMT. Skin transplantation with third-party CBA, host BALB/c and donor C57BL/6 grafts was performed at day 358 after BMT. Results. The BALB/c-mice showed long-term stable and high-level donor-cell engraftment with MGMT transgenic C57BL/6 BMT after BCNU treatment, demonstrating full reconstitution and donor cardiac-allograft tolerance and no GVHD with expanded donor and host Foxp3+ T regulatory cells. Further, skin grafts from donor, host, and third party showed good immune function with rejection of third-party grafts from all mice and benefit from enhanced chimerism after BCNU with less cell infiltrate and no chronic rejection in the donor skin grafts of BCNU treated mice compared no BCNU treated mice. Conclusions. High-level mixed chimerism without GVHD can be achieved using MGMT transgenic BM in a mixed-chimerism model receiving BCNU across a major histocompatibility complex mismatch. Enhanced mixed chimerism leads to long-term donor-specific allograft tolerance.

(Transplantation 2015;99: 2476–2484)

T

olerance strategies have been investigated for over 15 years in animal models, and more recently in human studies.1 A major reason has been to reduce the need for Received 1 January 2015. Revision received 14 April 2015. Accepted 22 April 2015. 1 Centre for Kidney Research, The Children's Hospital at Westmead, University of Sydney, NSW, Australia. 2

Centre for Transplant and Renal Research, Westmead Millennium Institute for Medical Research, University of Sydney, NSW, Australia. 3

Children's Cancer Research Unit, The Children's Hospital at Westmead, University of Sydney, NSW, Australia.

4

Centre for Medical and Molecular Bioscience, University of Wollongong, NSW, Australia.

5

Australia Microsurgical Consultants, Melbourne, VIC, Australia.

6 Gene Therapy Research Unit, The Children’s Hospital at Westmead and Children's Medical Research Institute, University of Sydney, NSW, Australia. 7 Oncology Research Unit, School of Medical Science, University of New South Wales, NSW Australia.

This work was supported by NHMRC grants 512246 and1029601 and NHMRC Training Fellowship APP1013185 (to M.H.).

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immunosuppression in solid organ transplantation, while inducing donor-specific tolerance, which has been most successfully achieved using mixed-chimerism approaches.1-4 Long-term tolerance has been achieved by strategies aimed at developing full chimerism or mixed chimerism in animal models5,6 and HLA-identical and HLA single-haplotype The authors declare no conflicts of interest. M.H. designed project and performed experiments, analyzed research data and wrote the article. B.K., G.Y.Z., Y.M.W., D.W. contributed to design of experiments and discussion of the research data. B.H. performed heterotopic heart transplant. G.M, I.A., P.G. contributed to discussion and edited the article. S.I.A. designed the project, edited and reviewed the manuscript and approval final article for publication. Correspondence: Stephen I. Alexander, MBBS, MPH, FRACP, Centre for Kidney Research, The Children's Hospital at Westmead, University of Sydney, Westmead, NSW 2145, Australia; and Min Hu, MD, PhD, Centre for Transplant and Renal Research, Westmead Millennium Institute, University of Sydney, Westmead, NSW 2145, Australia. ([email protected] and [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0041-1337/15/9912-2476 DOI: 10.1097/TP.0000000000000825

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mismatched patients.2,7,8 However, the most longstanding mixed-chimerism strategies, while inducing tolerance, do not maintain long-term mixed chimerism.9-11 In clinical transplantation, renal allograft tolerance can be achieved in HLA-identical and HLA-mismatched mixed chimerism transplant patients who were transplanted with combined kidney and nonmyeloablative bone marrow (BM) transplantation (BMT), though with loss of chimerism in the HLA mismatched patients.4 Other strategies using facilitator cells and more potent induction have achieved full chimerism in HLAmismatched transplantation12 but with limited follow-up and concerns regarding the risk of graft-versus-host disease (GVHD) and infection.4 However, post-BMT cyclophosphamide has been successful in haplomismatched transplants in achieving chimerism though still with some signs of GVHD, and this approach is entering solid organ clinical trials.13,14 Improving engrafting donor-cell survival has been used to enhance chimerism. Methyl-guanine-methyl-transferase (MGMT) is a DNA repair protein that removes N,N-bis(2-chloroethyl)nitrosourea (BCNU) from guanine and limits BCNU toxicity, enhancing engraftment of hematopoietic cells that express it, as shown in a minor major histocompatibility complex (MHC)-mismatch murine model.15 The critical challenge for the induction of transplant tolerance combining mixed chimerism clinically is to control GVHD while crossing HLA barriers. In the current study, we tested whether (1) MGMT transgenic C57BL/6 BM-cells into BALB/c mice, with subsequent BCNU treatment enhanced chimerism without GVHD in a fully MHCmismatched, mixed-chimerism model; and whether 2) allograft tolerance could be achieved. Clinical tolerance was assessed using vascularized C57BL/6 cardiac allografts and skin grafts.

MATERIALS AND METHODS Animals

C57BL/6 (H-2b), BALB/c (H-2d) and CBA (H-2k) mice (8–10 weeks) were obtained from the Animal Resource Centre (Perth, WA) and MGMT-transgenic-C57BL/6 mice as established.15 Experiments were approved by the Animal Ethics Committee of Children's Hospital at Westmead. Mixed Chimerism and Mouse Heart and Skin Transplants

A dose (300 μg/mouse) of depleting CD8 antibody (Clone-2.43; Bioexpress, Kaysville, UT) and dose (15 mg/kg) of busulfan (Orphan Medical, Minnetonka, MN) were administered intraperitoneally into host BALB/c at day −1 BMT. The mice received anti-CD40L antibody (MR.1) (Bioexpress) at day −1 (500 μg/mouse), days 0 to 5, 8, 12, and 15 (200 μg/mouse). Bone marrow was from MGMT transgenic C57BL/6 mice (10–14 weeks),15 and 10  106 BM cells were transplanted into BALB/c mice at day 0. BCNU (Bristol-Myer Squibb, Mt Waverley, Vic) was given at day 48 (5 mg/kg), at days 88, 140, and 189 (2.5 mg/kg) after BMT. Heterotopic donor-C57BL/6 cardiac-allografts were performed at 243 days after BMT as previously described.16 Graft function was assessed by daily palpation, with rejection defined as loss of palpable beating.16 Skin transplantation with third-party CBA, host-BALB/c and donor-C57BL/6 grafts was performed at day 358 post-BMT

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(over 100 days after heart transplantation), with rejection assessed as previously described.17 Flow Cytometric Analysis

Flow cytometric analysis was performed by FACScan (BD Biosciences, San Jose, CA) for engraftment and immune profile.16 Peripheral blood (PB) was collected before BMT and days 9, 10, 16, 27, 34, 40, 42, 68, 133, 166, 211, 224, 308, 458 after BMT. Splenocytes were harvested and Foxp3 + CD4+ T regulatory (Treg) cells were measured at days 460 to 487 after BMT. Antibodies comprised of PE-H2kd, FITCH2kb, Percp/PE-CD3, Percp-CD4, FITC-CD8, FITC-CD19, FITC-CD25, Percp-CD45, PE-NK1.1, (BD Biosciences) and PE-Foxp3, (eBioscience, San Diego, CA). Histological Examination

Skin, small intestine, and liver of host-BALB/c, skin grafts and cardiac allografts were fixed in 10% neutral-buffered formalin and paraffin embedded before staining with hematoxylin-eosin.16,17 Enzyme-Linked Immunosorbent Spot Assay for IFN-g Production

The IFN-γ production by the host splenocytes from day 487 after BMT was determined by enzyme-linked immunosorbent spot (ELISPOT) (Mabtech, AB, NackeStrand, Sweden) according to the manufacturer's instructions.18 Plates were counted using the computer program AID Elispot6.0iSpot ELISPOT reader (AIDGmbH, Strassberg, Germany). Statistical Analysis

Data are presented as mean ± SD. PRISM 4.0 software (GraphPad, San Diego, CA) was used for statistical analysis for comparisons of matched groups, P values less than 0.05 were considered significant. The log-rank test compared survival data between groups. Statistical differences between the 2 groups were analyzed by unpaired 2-tailed t tests and multiple groups by the Kruskal-Wallis 1-way analysis of variance test. The ELISPOT data used 2-tailed Student t test with a Bonferroni correction. RESULTS Long-Term Stable and High Level Mixed Chimerism With MGMT BMT and Treatment with BCNU

We assessed chimerism of donor-C57BL/6 cells and hostBALB/c cells using H-2Kb and H-2Kd staining, respectively, by flow cytometry, after gating on CD45+ cells in PB of the mixed-chimeric mice. In this study, use of BCNU led to enhanced, long-term stable mixed chimerism in the fully MHC-mismatched MGMT transgenic C57BL/6 BMT into host-BALB/c mice. Host CD8+ T cells, although initially depleted, reconstituted rapidly after BMT (Figure 1A). Longterm stable allogeneic MGMT donor cells engraftment was achieved. After administration of low doses of BCNU at 4 time points, at day 211 after BMT, the proportion of engrafted donor H2Kb cells was significantly higher in PB of host mice (52.9% ± 22.2, n = 8) compared to the nonBCNU group (22.8% ± 17.1, n = 7, **P < 0.01)(Figure 1B). Absence of GVHD in the Presence of Mixed Chimerism

Graft-versus-host disease is a major limitation in BMT.9,19 To determine whether mixed-chimeric mice developed GVHD,

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wild-type C57BL/6 mice was achieved, with multilineage reconstitution of B, T, and NK cells found in PB of mixedchimeric mice (Figure 2C) and in the engrafting donor C57BL/6 cells (Figure 2D). Long-Term Stable Mixed-Chimerism Resulted in Donor-Specific Tolerance to Cardiac Allografts in both Groups but Absence of Chronic Damage in Donor Skin Grafts in the BCNU Group

FIGURE 1. Long-term stable and high level mixed-chimerism was achieved in a fully MHC-mismatched MGMT transgenic C57BL/6 BMT to BALB/c murine model with BCNU treatment. A, A representative flow cytometry dot plot showing that the percentage of CD8+ T cells was 34.5% before using the regimen to 0% at days 9 to 16, and then returned to 11.5% at day 34, and 13.2% at day 42 after BMT in PB of the mice. B, Engraftment of H2Kb donor-cell in BALB/C host. After gating on CD45+ cells, the percentage of donor H2Kb cells was 52.9% ± 22.2 (n = 8) in PB of host mice after 4 doses of BCNU compared to the non-BCNU group (22.8% ± 17.1)(**P < 0.01) (n = 7) at 211 days after BMT.

body weight was monitored weekly, and mice were evaluated histologically. The mixed-chimeric mice showed an increase in body weight in both BCNU and non-BCNU groups (Figure 2A) and with no loss of fur or skin inflammation in the mice (data not shown). Further histology of skin, small intestine, and liver showed no evidence of GVHD at day 487 after BMT in both groups compared to naive BALB/c mice (Figure 2B). Full Reconstitution of PB Components Including B, T, and Natural Killer Cells From Host and Donor-Cells

To assess multilineage reconstitution of lymphocytes, T, B, and natural killer (NK) cells were investigated by flow cytometric analysis. Equivalent reconstitution of T, B, and NK cells was found in both groups of mixed-chimeric mice. By day 458 after BMT lymphocyte reconstitution equivalent to

To assess donor-specific tolerance in the mixed-chimerism model, transplantation of heterotopic donor C57BL/6 cardiac allografts was performed at day 243 after BMT, then skin transplantation at day 358 after BMT (over 100 days after heart transplantation) with third-party CBA, host BALB/c, and donor C57BL/6 skin grafts. Donor C57BL/6 cardiac allografts had prolonged survival with median survival time (MST) >200 days in both groups (Figure 3A). Further, donor C57BL/6 skin allografts had significant and specific prolonged survival with MST greater than 100 days similar to host BALB/c control grafts compared to third-party CBA allografts with MST of 12 days (**P < 0.01) in the BCNU group and in the non-BCNU group with MST of 11 days (*P < 0.05) (Figure 3B). Histological examination showed donor C57BL/6 cardiac allografts had less cellular infiltration in the BCNU group, compared to the non-BCNU group (Figure 3C). This indication of persistent alloreactivity in the mice with less engraftment was reflected in the donor C57BL/6 skin allografts from the non-BCNU group that showed chronic rejection with cellular infiltration as compared to donor C57BL/6 skin allografts from the BCNU group that had no infiltrate (Figure 3D). In both groups, the third-party CBA allografts showed acute rejection (Figure 3B and D). This suggests that in the mixed-chimerism BCNU-MGMT model, with greater levels of engraftment, there was complete donorspecific tolerance and maintenance of immune function as demonstrated by third-party allograft rejection. High Level of Mixed-Chimerism Maintained in the BCNU Group

Long-term stable mixed chimerism was confirmed after heart transplantation and after skin transplantation. This high level of donor-cell engraftment was maintained after heart transplantation and after skin transplantation in the BCNU group but not in the non-BCNU group (Figure 4A). The proportion of donor H-2Kb cells was significantly higher in the BCNU group at day 65 after heart transplantation (day 308 after BMT) (P < 0.05) and at day 95 after skin transplantation (day 456 post BMT) (P < 0.05) compared to the non-BCNU group (Figure 4A). Role of Treg Cells and Potential CD25− Effector Subset of Treg Cells in the BCNU Group

The Treg cells play an important role in mixed chimerism, both in limiting GVHD and in maintaining chimerism with host Treg cells involved in preventing chronic allograft rejection6,20,21 and donor Treg cells controlling GVHD.20 We investigated the number, proportion, and phenotype of Foxp3+ Treg cells in this model. Foxp3 Treg cells were assessed at days 460 to 487 after BMT. The proportion of CD4+Foxp3 + Treg cells in the CD4+ T cell of spleens from the mixedchimerism mice was significantly higher in the non-BCNU group (P < 0.001) and the BCNU group (P < 0.05) compared

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FIGURE 2. Absence of GVHD and B, T, and NK cells components in the fully MHC mismatched mixed chimerism model. A, Increased body weight for host mice in both the BCNU and non-BCNU groups. After 4 doses of BCNU, body weight was 17.5 g ± 1.0 g at day −1 and 22.8 ± 1.3 g at day 240 after BMT (n = 8), whereas non-BCNU group showed a gain in body weight from 17.4 ± 0.6 at day −1 to 23.8 ± 1.6 (n = 7). B, Histology of skin, small intestine and liver (H&E) for naive BALB/c mice, representative of a non-BCNU and BCNU host-BALB/c mice. There were no GVHD features found in either group at day 487 after BMT compared to naive BALB/c mice. C, The proportion of CD3+ T, CD19+ B and NK1.1+ NK cells in PB of mixed-chimerism mice and (D) engrafted donor-cells at day >308 post BMT examined by flow cytometry. CD3+, CD19+ and NK1.1+ cells examined in mixed-chimerism mice and engrafted H2Kb donor-cells of both non-BCNU (n = 3) and BCNU (n = 5) showed no significant differences compared to naive C57BL/6 mice. H&E indicates hematoxylin-eosin.

to naive BALB/c (Figure 4B). The proportion of CD4 + Foxp3+ Treg cells in donor H-2Kb cells was also significantly higher in both the BCNU (P < 0.05) and nonBCNU groups (P < 0.05) compared to naive C57BL/6 mice (Figure 4B). Both donor and host Foxp3+ Treg cells are increased as a proportion of total CD4+ T cells, suggesting that both donor and host Treg cells are playing a role in maintaining chimerism and limiting GVHD. CD25−Foxp3+ Treg cells as a proportion of CD4+ T cells were more in the BCNU group than the non-BCNU group (P < 0.05) or naive BALB/c mice (P < 0.05) (Figure 4C). CD25 is downregulated

in Foxp3+ Treg cells in both mixed chimerism groups but this is greater in the BCNU group (Figure 4C), suggesting CD25 − Foxp3+ Treg cells may be a functionally active or allospecific Treg cell subset.22 Assessment of Donor, Recipient, and Third-Party Alloreactivity by IFN-g ELISPOT Assay Showed Maintenance of Third Party Responses With Limited Donor and Host Responses in the BCNU Group

We tested donor and third-party alloreactivity using the IFN-γ ELISPOT assay. The IFN-γ is important in acute T

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FIGURE 3. BCNU treatment enhanced donor-cell engraftment and resulted in donor-specific allograft tolerance in this model. A, Mixed chimerism was associated with long-term survival of donor-strain cardiac allografts in both the non-BCNU and BCNU groups. C57BL/6 cardiac allografts had allograft survival of >200 days (MST) in both the non-BCNU group (n = 3)(white triangle) and the BCNU group(n = 5)(black squares) groups. B, Skin allograft survival curve for both non-BCNU and BCNU groups. Non-BCNU group showed third-party CBA allografts (n = 3) (white circle) were rejected with MST of 11 days, whereas donor-C57Bl/6 allografts (n = 3) (white diamond) (*P < 0.05) had prolonged survival with MST greater than 100 days as well as host BALB/c (n = 3) (white triangle) (*P < 0.05). In the BCNU group C57BL/6 skin allografts (n = 5) (black squares) (**P < 0.01) had significantly and specifically prolonged survival with MST greater than 100 days as did host BALB/c control allografts (n = 5) (black diamond) (**P < 0.01) compared to third-party allografts CBA (n = 5) (black circle) which were rejected with MST = 12 days. C, Histology of naive BALB/c heart, host hearts, and C57BL/6 cardiac-allografts and (D) naive BALB/c and C57BL/6 skin, host skin, and C57BL/6 skin allografts in both groups and third-party CBA skin allograft from the non-BCNU group (H&E staining).

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FIGURE 4. Foxp3 Treg cells played a role in the mixed-chimerism model. A, The high-level of donor H2Kb cells was maintained in the BCNU group (70.6% ± 23.1) (P < 0.05) at day 65 after heart transplantation (PHTx) (day 308 after BMT) compared to the non-BCNU group (15.9% ± 7.5); and the BCNU group (85.1% ± 8.4) (P < 0.05) at day 98 after skin transplantation (PSTx) (day 456 after BMT), compared to non-BCNU group (14.7% ± 4.7). B, The proportion of CD4+Foxp3+ Treg cells in CD4+ Tcells of mixed-chimerism mouse spleens were significantly higher in the non-BCNU (22.3% ± 1.1) (P < 0.001)(n = 3) and BCNU group (23.7% ± 4.9) (P < 0.05) (n = 3) at later than day 460 after BMT compared to naive BALB/c group (14.7% ± 1) (n = 5). There were also a significantly higher CD4+Foxp3+ Treg cells H2Kb-donor-cell proportion of CD4+ T cells in the non-BCNU (42.0% ± 8.6) (P < 0.05) and BCNU (P < 0.05) groups compared to naive C57BL/6 mice (27.3% ± 3.9) (n = 5). C, Among these CD4+ T cells CD25+Foxp3+ Treg cells were less in the BCNU group (2.8% ± 1.4) (P < 0.05) compared to non-BCNU group(8.3% ± 1.2) and naïve BALB/c mice (8.2% ± 0.7).

cell–mediated allograft rejection,23 and also required for tolerance.24 The IFN-γ is also a major contributor to GVHD.25 The BCNU group, showed limited responses to both host and donor stimulation while maintaining strong third-party responses to CBA stimulators (Figure 5 A and C). However, the non-BCNU group had significantly stronger responses to donor and host stimulation with equivalent responses to third-party stimulation (Figure 5 A and B). These data were consistent with the histological findings of chronic rejection in the donor C57BL/6 skin allografts on non-BCNU mice compared to the intact tolerant skin allografts on BCNUtreated mice.23 DISCUSSION Donor-specific transplant tolerance in organ transplantation has been achieved using mixed-chimerism approaches but with either loss of donor cell chimerism or risks of GVHD. In the present study, we demonstrated the utility of the MGMT transgenic C57BL/6 mice BM given to fully MHC mismatched host BALB/c mice and subsequent BCNU treatment without irradiation to achieve high-level and stable mixed-chimerism without GVHD. These mice, after BCNU, demonstrate donor-specific allograft tolerance for both skin

and cardiac allografts while retaining the capacity to reject third-party grafts. Induction of mixed-chimerism allows for donor-specific transplantation tolerance in rodents,5,6 large animals, and recently in a clinical trials of renal transplant patients.4 However, many of the protocols to establish mixed chimerism use a myelosuppressive and/or nonmyeloablative total body irradiation. Although T-cell depletion of donor BM reduces GVHD, it limits MHC-mismatched donor cell engraftment and requires increased host conditioning.26,27 Clinical organ transplantation is routinely performed across extensive HLA barriers. Therefore, limiting GVHD, when crossing HLA barriers, is the major challenge for the induction of mixed chimerism and transplant tolerance. T cell–depleted BMT combined with costimulatory blockade and busulfan have previously been shown, in mice, to allow mixed chimerism and skin allograft tolerance.28 However, our protocol achieved mixed chimerism with only CD8 T-cell depletion and without the development of GVHD, suggesting a potential role for Treg cells.4 In solid organ transplants, Treg cells have been shown to be important including liver transplantation and chronic allograft rejection in an animal model.29 In human studies using mixed chimerism after nonmyeloablative BMT, the development of host

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FIGURE 5. IFN-γ production was maintained in response to third-party stimulators and a limited response to donor and host stimulators observed in the BCNU group. A, IFN-γ ELISPOT production of representative wells of IFN-γ ELISPOTspots from the host splenocytes of the nonBCNU and BCNU groups at day 487 after BMT. Responder cells from the non-BCNU group showed increased IFN-γ production in response to BALB/c, C57BL/6, host splenocytes (mixed) and third-party CBA compared to responder cells only (medium). The BCNU group had reduced IFN-γ production when directly stimulated with donor-C57BL/6 as well as BALB/c or host splenocytes compared to third-party. B, The splenocytes of the non-BCNU group had increased IFN-γ production to donor C57BL/6 (B6) (217.3 ± 56.6 spots)(P < 0.05*), thirdparty CBA (3rd) (258.7 ± 42.7 spots) (P < 0.01**), host splenocytes (mix) (388.3 ± 110 spots)(P < 0.05*) and BALB/c (B/c) (152.7 ± 45.6 spots) compared to responder cells only (M) (48.7 ± 8.4). C, The splenocytes from the BCNU group had significantly reduced IFN-γ production when directly stimulated with donor-C57BL/6 (B6) (101.3 ± 11 spots) compared with third-party CBA (3rd) stimulators (304 ± 3.5 spots) (P < 0.001***). Reduced IFN-γ production was also observed when stimulated with host splenocytes (mix) (136.3 ± 25.1 spots) (P < 0.01**), or BALB/c (B/c) (62.7 ± 12.5 spots) (P < 0.001***) compared to the third-party CBA stimulators.

Treg cells may play a role in maintaining tolerance in kidney transplant patients even after loss of donor chimerism.2,21 Although 4 of 5 patients with combined BM and kidney transplants from HLA single-haplotype mismatched living related donor at Massachusetts General Hospital achieved kidney transplant tolerance even after loss of donor chimerism, high level of Foxp3 Treg cells in kidney transplant tolerance patients suggested that Treg cells induced by the initial mixed chimerism and potentially deletion of alloreactive T cells played a role in maintaining tolerance in these kidney transplant patients. Animal work has shown an important role for host Treg cells in the mouse model of nonmyeloablative BMT and costimulatory blockade and total body irradiation in tolerance induction in a mixed chimerism model.6 In other animal models, findings suggest that newly generated host Treg cells can prevent chronic allograft rejection in a mixed chimerism combined with skin transplant murine model.30 Here, depletion of host Treg cells did not affect mixed chimerism but led to rapid loss of skin allografts.30 Moreover, donors Foxp3+ Treg cells limit GVHD in mixed-chimerism murine models.20 Further, the studies by Pilat et al31,32 demonstrated Treg cell therapy–enhanced donor cell engraftment and allowed transplant tolerance. In our study, only host CD8 T cells were temporarily depleted in addition to busulfan, compared to other regimes which use donor T cell depletion to limit GVHD. In the setting of mixed chimerism we also found a high proportion of

donor and host Treg cells in both the BCNU and non-BCNU groups which suggest that the limitation of GVHD and the limitation of rejection of the donor graft might be occurring simultaneously through active suppression by Treg cells. Skin allograft tolerance in the BCNU group and chronic skin allograft rejection in the non-BCNU group were observed in our model along with rejection of third party grafts by both groups. The presence of CD25−Foxp3+ Treg cells in the BCNU group may represent antigen-specific or more potent Treg cells, explaining the observations of more robust tolerance in this group. Functional assays using IFN-γ ELISPOTs to assess function in the chimeric mice demonstrated both specificity of reduction of IFN-γ while maintaining strong third-party responses, potentially involving Treg cells suppressing the type 1T helper effector pathway. Thus, although IFN- γ is critical in acute T cell–mediated allograft rejection, tolerance, and GVHD, in our model, IFN- γ is suppressed, and this effect is strongest in the BCNU-treated group.23 The possibility that the reduction in IFN-γ has limited Treg cells is not supported by the data showing an increase in Treg cells in both mixedchimerism groups. However, this leaves open the question of whether IFN-γ might alter chimerism or limit tolerance in models of mixed chimerism. In the clinical setting, crossing HLA barriers is still a major challenge for the induction of mixed chimerism and transplant tolerance. The Stanford group has shown that renal

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allograft tolerance can be achieved in HLA-identical patients receiving combined kidney and BMT who were treated with total lymphoid irradiation-based conditioning regimen and developed mixed chimerism.8,33 However, a similar regimen has been less successful in achieving tolerance in HLAmismatched recipients.34 Clinical trials at Massachusetts General Hospital in patients with renal failure secondary to refractory multiple myeloma who received HLA-identical combined kidney and BMT using cyclophosphamide, T cell depletion plus thymic irradiation regimen achieved kidney transplant tolerance in 5 of 7 patients.7,35,36 However, a similar regimen in HLA-mismatched renal transplant patients, although successful in achieving long-term tolerance in many patients, had complications of disease recurrence and rejection as well as loss of chimerism.2,4 These results point to the difference in chimerism between mouse models and primate/human studies and the potential difficulties in translating our model into primate and human studies. A third clinical approach has recently been reported to achieve mixed or full donor chimerism without GVHD in 8 HLA-mismatched kidney transplant recipients with a regimen consisting of total body irradiation (200 Gy), fludarabine, and high-dose cyclophosphamide, both before and after BMT, together with administration of donor hematopoietic stem cells and concurrent treatment with “facilitator” cells.37 Our study is in keeping with these clinical studies, using less intensive induction but still inducing mixed chimerism and then further enhancing mixed chimerism with BCNU, allowing organ transplant tolerance across a major MHC barrier that subsequent skin transplants demonstrate is specific. In conclusion, high-level and stable mixed chimerism without GVHD can be achieved using donor marrow containing a protective MGMT transgene in a mixed-chimerism model receiving BCNU. Enhanced chimerism demonstrated enhanced long-term donor-specific allograft tolerance in the skin grafts. The expansion of Treg cells in the mixed chimeras suggests these may play an important part in maintaining chimerism and protecting against GVHD. ACKNOWLEDGMENTS The authors thank the Children's Medical Research Institute Animal House for animal care. REFERENCES 1. Lechler RI, Sykes M, Thomson AW, et al. Organ transplantation—how much of the promise has been realized? Nat Med. 2005;11:605–613. 2. Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med. 2008;358:353–361. 3. Pilat N, Wekerle T. Transplantation tolerance through mixed chimerism. Nat Rev Nephrol. 2010;6:594–605. 4. Sachs DH, Kawai T, Sykes M. Induction of tolerance through mixed chimerism. Cold Spring Harb Perspect Med. 2014;4:a015529. 5. Wekerle T, Kurtz J, Ito H, et al. Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat Med. 2000;6:464–469. 6. Bigenzahn S, Blaha P, Koporc Z, et al. The role of non-deletional tolerance mechanisms in a murine model of mixed chimerism with costimulation blockade. Am J Transplant. 2005;5:1237–1247. 7. Spitzer TR, Sykes M, Tolkoff-Rubin N, et al. Long-term follow-up of recipients of combined human leukocyte antigen-matched bone marrow and kidney transplantation for multiple myeloma with end-stage renal disease. Transplantation. 2011;91:672–676.

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8. Scandling JD, Busque S, Dejbakhsh-Jones S, et al. Tolerance and chimerism after renal and hematopoietic-cell transplantation. N Engl J Med. 2008;358:362–368. 9. Starzl TE. Chimerism and tolerance in transplantation. Proc Natl Acad Sci U S A. 2004;101(Suppl 2):14607–14614. 10. Fehr T, Sykes M. Clinical experience with mixed chimerism to induce transplantation tolerance. Transpl Int. 2008;21:1118–1135. 11. Kawai T, Sachs DH. Tolerance induction: hematopoietic chimerism. Curr Opin Organ Transplant. 2013;18:402–407. 12. Leventhal J, Abecassis M, Miller J, et al. Tolerance induction in HLA disparate living donor kidney transplantation by donor stem cell infusion: durable chimerism predicts outcome. Transplantation. 2013;95:169–176. 13. Luznik L, O'Donnell PV, Symons HJ, et al. HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide. Biol Blood Marrow Transplant. 2008;14:641–650. 14. Kasamon YL, Luznik L, Leffell MS, et al. Nonmyeloablative HLAhaploidentical bone marrow transplantation with high-dose posttransplantation cyclophosphamide: effect of HLA disparity on outcome. Biol Blood Marrow Transplant. 2010;16:482–489. 15. Kramer BA, Lemckert FA, Alexander IE, et al. Characterisation of a P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT)-expressing transgenic mouse line with drug-selectable bone marrow. J Gene Med. 2006;8:1071–1085. 16. Hu M, Watson D, Zhang GY, et al. Long-term cardiac allograft survival across an MHC mismatch after “pruning” of alloreactive CD4 T cells. J Immunol. 2008;180:6593–6603. 17. Watson D, Zhang GY, Sartor M, et al. “Pruning” of alloreactive CD4+ Tcells using 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester prolongs skin allograft survival. J Immunol. 2004;173:6574–6582. 18. Hu M, Wu J, Zhang GY, et al. Selective depletion of alloreactive T cells leads to long-term islet allograft survival across a major histocompatibility complex mismatch in diabetic mice. Cell Transplant. 2013;22:1929–1941. 19. Mapara MY. The quest for the optimal conditioning regimen: some answers, more questions. Biol Blood Marrow Transplant. 2013;19:1275–1276. 20. Koyama M, Kuns RD, Olver SD, et al. Promoting regulation via the inhibition of DNAM-1 after transplantation. Blood. 2013;121:3511–3520. 21. Pilat N, Wekerle T. Mechanistic and therapeutic role of regulatory T cells in tolerance through mixed chimerism. Curr Opin Organ Transplant. 2010;15:725–730. 22. Graca L, Thompson S, Lin CY, et al. Both CD4(+)CD25(+) and CD4(+) CD25(−) regulatory cells mediate dominant transplantation tolerance. J Immunol. 2002;168:5558–5565. 23. Wiseman AC, Pietra BA, Kelly BP, et al. Donor IFN-gamma receptors are critical for acute CD4(+) T cell-mediated cardiac allograft rejection. J Immunol. 2001;167:5457–5463. 24. Sawitzki B, Kingsley CI, Oliveira V, et al. IFN-gamma production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. J Exp Med. 2005;201:1925–1935. 25. Robb RJ, Hill GR. The interferon-dependent orchestration of innate and adaptive immunity after transplantation. Blood. 2012;119:5351–5358. 26. Fleischhauer K, Kernan NA, O'Reilly RJ, et al. Bone marrow-allograft rejection by T lymphocytes recognizing a single amino acid difference in HLA-B44. N Engl J Med. 1990;323:1818–1822. 27. Martin PJ, Hansen JA, Buckner CD, et al. Effects of in vitro depletion of T cells in HLA-identical allogeneic marrow grafts. Blood. 1985;66:664–672. 28. Adams AB, Durham MM, Kean L, et al. Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression. J Immunol. 2001;167:1103–1111. 29. Demirkiran A, Bosma BM, Kok A, et al. Allosuppressive donor CD4 + CD25+ regulatory T cells detach from the graft and circulate in recipients after liver transplantation. J Immunol. 2007;178:6066–6072. 30. Pasquet L, Douet JY, Sparwasser T, et al. Long-term prevention of chronic allograft rejection by regulatory T-cell immunotherapy involves host Foxp3expressing T cells. Blood. 2013;121:4303–4310. 31. Pilat N, Baranyi U, Klaus C, et al. Treg-therapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning. Am J Transplant. 2010;10:751–762. 32. Pilat N, Klaus C, Gattringer M, et al. Therapeutic efficacy of polyclonal Tregs does not require rapamycin in a low-dose irradiation bone marrow transplantation model. Transplantation. 2011;92:280–288. 33. Scandling JD, Busque S, Dejbakhsh-Jones S, et al. Tolerance and withdrawal of immunosuppressive drugs in patients given kidney

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and hematopoietic cell transplants. Am J Transplant. 2012; 12: 1133–1145. 34. Millan MT, Shizuru JA, Hoffmann P, et al. Mixed chimerism and immunosuppressive drug withdrawal after HLA-mismatched kidney and hematopoietic progenitor transplantation. Transplantation. 2002; 73: 1386–1391. 35. Spitzer TR, Delmonico F, Tolkoff-Rubin N, et al. Combined histocompatibility leukocyte antigen-matched donor bone marrow and renal transplantation for multiple myeloma with end stage renal disease: the

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induction of allograft tolerance through mixed lymphohematopoietic chimerism. Transplantation. 1999; 68: 480–484. 36. Buhler LH, Spitzer TR, Sykes M, et al. Induction of kidney allograft tolerance after transient lymphohematopoietic chimerism in patients with multiple myeloma and end-stage renal disease. Transplantation. 2002; 74:1405–1409. 37. Leventhal J, Abecassis M, Miller J, et al. Chimerism and tolerance without GVHD or engraftment syndrome in HLA-mismatched combined kidney and hematopoietic stem cell transplantation. Sci Transl Med. 2012; 4: 124ra28.

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Methyl-Guanine-Methyl-Transferase Transgenic Bone Marrow Transplantation Allows N,N-bis(2-chloroethyl)-Nitrosourea Driven Donor Mixed-Chimerism Without Graft-Versus-Host Disease, and With Donor-Specific Allograft Tolerance.

Transplant tolerance has been achieved by mixed chimerism in animal models and in a limited number of kidney transplant patients. However, these mixed...
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