An alternative conditioning regimen for induction of specific skin graft tolerance across full major histocompatibility complex barriers

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An alternative conditioning regimen for induction of specific skin graft tolerance across full major histocompatibility complex barriers
  Transphnt Immunology 1998; 6: 147 151 An aiternative conditioning regimen for induction of specific skin graft tolerance across full major histocompatibility complex barriers Anneke de Vries-van der Zwan, Marjolein A van der Pol, Leo P de Waal and Claire JP Boog Department of Transplantation mmunology Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratov for Experimental and Clinical Immunolo~ University f Amsterdam Amsterdam Received 9 February 1998; accepted as revised 2 June 1998 Ah&act: Previously, we developed a well-tolerated single-day protocol for induction of stable multilineage chimerism and permanent donor-specific tolerance across major histocompatibility complex (MHC) barri- ers, with preservation of the host’s normal immune responses. In our murine model, recipient mice were treated with a single dose of anti-CD3, anti-CD4, low dose total body irradiation ‘ISI; 3-6 Gy) and ailo- geneic bone marrow cells. An alternative cytoreductive strategy that is well-recognized in bone marrow trans- plantation, but has not been evaluated extensively in organ allograft recipients, involves the use of a combined chemotherapeutic drug treatment. The present data show that conditioning with low dose TBI, in a MHC-disparate donor-recipient combination, can be successfulIy substituted by a combined single low- dose dimethyl myleran (DMM)/cyclophosphamide (CY) therapy, resulting in both stable, mixed chlmerism and specific skin graft tolerance. Introduction linical esults after transplantation of allogeneic organs and tis- sues have improved dramatically since the 1970s. However, the success of organ transplantation has its reverse side. It means the need for chronic aspecitic immunosuppression, which is accom- panied by many complications such as opportunistic infections, malignancies, drug toxicity and chronic rejection. Therefore, induction of donor-specifk transplantation tolerance, without the need for long-term immunosuppressive therapy, is a subject of continuing interest in experimental and clinical transplantation immtmology. The capacity of bone marrow cells to induce donor- specific tolerance to tissue and organ allografts has been studied extensively in several animal models.‘” So far, the toxicity of con- Address for correspondence: CJP Boog, Department of ‘Ikansplantation Immunology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail: 0 Arnold 1998 ventional conditioning, which is currently used for bone marrow transplantation, has precluded its use in clinical organ trans- plantation. Recently, we have developed a well-tolerated non- lethal conditioning regimen for induction of permanent mixed chimerism and donor-specific tolerance for skin grafts across major histocompatiiility complex (MIX) barriers in mice. This regimen involves the use of a single dose of anti-CD3 and anti- CD4 monoclonal antibodies, low dose total body irradiation (TBI) (3-6 Gy) and allogeneic T-cell depleted bone marrow cell~.~> ‘lb avoid the potential disadvantages of TBI, and achieve more flexibility for clinical application in organ transphunation, the experiments in the present study were designed to investigate alternative ways of conditioning In humans, the most common chemotherapy conditioning prior to bone marrow transplantation includes busulphan (BU) combined with cyclophosphamide (CY). Therefore, we have investigated if low concentrations of dimethyl myleran (DMM), a dimethyl homologue of BU, in com- bination with a low dose of CY can be used for engraftment of allogeneic bone marrow cells in our tolerance-inducing protocol. 0966-3274(98)TI227OA  148 A de V&s-van der Zwan et al. The objective of the present study is to compare the efficacy of a single dose of DMM and CY with previously obtained results after low-dose TBI on induction of mixed chimerism and transplantation tolerance. Materi and methods Animals Eight to 14week-old male C57BI..00 (H-29 (BlO), BlO.D2 (H- 2d) and BlO.BR (H-23 mice were used as recipients, donors and third parties, respectively, in fully allogeneic transplantation exper- iments. H-2b/d Fl hybrids were obtained by crossing C 57/BLlO (H- 2b) females with BlO.D2, (I-&) males, and also used as donors. Animals were bred and maintained at the animal facility of the Central Laboratory of the Netherlands Red Cross Blood Bansfusion Service under specific pathogen-free conditions. Conditioning redmen Chimeras were prepared with modifications of our previously described nonlethal single day protocol BlO recipients were conditioned on day 0 with a single dose of DMM (4-U) mgkg; 2,5-hexanediol dimethanesulfonate, NSC-23890, from the Division of Cancer ‘Beatment, National Cancer Institute, NIH, Bethesda, MD, USA) and/or a single dose of CY (50-300 mg/kg; Cycloblastine, Farmitalia Carlo Erba, Brussels, Belgium) intraperitoneally (ip). Between 3 and 4 h later, conditioned mice received a single dose of 400 pg anti-CD3 mAb (145-2Cll; gen- erously provided by Dr JA Bluestone, University of Chicago, Chicago, IL, USA) ip and 15 x lo6 T cell-depleted, fully allo- geneic BlO.D2 or semi-allogeneic (BlO x BlO.D2)Fl bone mar- row cells via the tail vein. Just prior to conditioning, recipient mice were given 500 pg anti-CD4 mAb (GK1.5; American ‘Qpe Culture Collection, Rockville, MD, USA), in order to reduce the contribution of CD4+ T cells to the ‘cytokine release’ syndrome, associated with anti-CD3 treatment.4 Reparation of bone marrow cells Bone marrow cells were harvested from donor femoral and tiiial bones and resuspended in Iscove’s modified Dulbecco’s medium supplemented with 10% heat-inactivated foetal calf serum (FCS). Erythrocytes were lysed with an ammonium chloride buffer. Bone marrow cells were depleted of T cells by treatment with anti-Thyl.2 mAb (F7D5; Serotec, Wiesbaden, Germany) and complement (low toxicity M rabbit complement; Cedarlane, Homby, Ontario, Canada), and contained less than 0.2% T cells as measured by flow cytometric analysis. Recipient mice were reconstituted with 15 x 106 allogeneic bone marrow cells in 0.25 ml of saline containing 5% heat-inactivated autoiogous serum. Skin transplantation Within 1 h after completing the conditioning regimen, tail skin transplantation was performed. Each recipient mouse received a control (syngeneic), a BlO.D2 or (BlO x BlO.D2)Fl (donor) and a BlO.BR (third party) skin graft. ‘Bansplantation of sec- ond donor-type skin grafts was performed 4 months after con- ditioning. The grafts were inspected three times a week and considered to be rejected when no viable donor skin was detectable. Flow cytometric analysis The percentage of donor-type cells was assayed in peripheral blood and mesenteric lymph node cells, 2 and 4 months after Tmnsphnt Immunology 1998; 6: 147 151 conditioning, with two-colour flowcytometry on a FACScan (Becton Dickinson, Mountain View, CA, USA). All procedures were performed in phosphate-buffered saline (PBS) containing 0.2% BSA and 0.05% sodium azide. Fluorescence data were collected using logarithmic amplification on lo4 viable cells as determined by forward light scatter intensity. To quantify chimer&m, cells were stained with donor-specific (H-2D FITC) and host-specific (H-2Kb-PE) conjugated mAbs (Pharmingen, San Diego, CA, USA). For lineage analysis of chimerism, PE- conjugated mAbs against T cells (Thyl.2), B cells (B220), gran- ulocytes (GR-l), and macrophages (MAC-l) (all from Pharmingen) were used in double staining with anti-donor mAb (H-2Dd-FITC). Cell-mediated lympholysis Cell-mediated lympholysis (CML) assays were performed as described previously.4 Radioactivity was measured in a Packard gamma counter. The percentage of specitic ‘iCr release was cal- culated by the following formula: % speciftc lysis = ((cpm exper- imental - cpm background “Cr release)/(cpm 10% triton X-100 release - cpm background ‘lCr release)) x 100. All samples are performed in triplicate. Results Establishment of stable mixed chimerism in mice conditioned with DMM and/or CY We have previously demonstrated that engraftment of allogeneic bone marrow cells occurs in sublethally irradiated T cell-deplet- ed recipient mice. High levels of stable mixed and multilineage chimerism were observed in lymphoid tissues and the periph- ery. ti Present data show that, instead of low dose TBI, admin- istration of either a single dose of DMM (Figure 1A) or CY (Figure 1B) alone, can result in induction of mixed chiierism as well, High levels of donor type cells were observed after treat- ment with 20 mg/kg DMM, while 300 mg/kg CY-induced lower levels of chimerism (~14%). However, these doses of both chemotherapeutic agents, needed for chimerism induction, are still relatively high. As shown in Figure l(C), combined admin- istration of DMM and CY is also successful in achievement of a mixed chimeric state. Reduction of DMM doses (12-4 mg/kg) given in combination with a fixed amount of CY (100 mg/kg), showed a dose-response effect, such that smaller doses of DMM resulted in lower levels of donor type cells. In such a way, much lower concentrations of each agent were needed for induction of stable mixed chimerism than when they were used separate ly. In contrast, only one out of four recipients showed engraft- ment of donor-type cells (12.5% chimerism) after further reduction of CY (50 mgkg), in combination with 8 mg/kg DMM. Based on further fluorescence activated cell sorting (FACS) analysis, it appeared that the chimerism induced was multilin- eage, because donor-type cells were present in both lymphoid (T and B cells) and myeloid (macrophages and granulocytes) lin- eages (data not shown). The ratio of donor/recipient type cells is almost equal in all lineages tested. Donor-specific umespomivencss in uftro Donor-specific tolerance and immunocompetence in vitro were demonstrated using cellular cytotoxicity assays. As shown in Figure 2, splenocytes from mixed chimeras were unresponsive to either donor (BlO.D2) and host-type (BlO) target cells (mutu- al tolerance), whereas the CTL response against third party  An alternative conditioning regimen for induction of speciftc skin graft tolerance 149 C 1 8 8 4 2 2 18 DMM OwW) 1 8 8 4 2 2 CY @WW lOOA I 8 8 4 2 DMM mgkg) + 1 mgkg CY Figure 1 Percentages of donor-derived cells (H-2d) in peripheral blood of B10 (H-2b) recipients measured 4 months after injection of fully allo- geneic BlO.D2 bone marrow cells and conditioning with anti-T cell monoclonal antibodies and (Figure 1A) DMM, (Figure 1B) CY or (Figure 1C) a combination of DMM and CY. The level of H-2d cells measured in B10 controls was cl and in BlO.D2 controls ~98 . (BlO.BR) cells remained obviously present. In contrast, control untreated mice, as well as fully treated nonchimeric animals, were reactive to both donor and third party antigens. Induction of tolerance to skin allogmfte lb determine if donor-specific transplantation tolerance was induced by a combination of anti-T cell monoclonal antibodies, allogeneic bone marrow cells and sublethal doses of DMM and/or CY, conditioned mice received a host-type, donor-type and third party-type skin graft directly following conditioning. Second grafts were transplanted 4 months later. Results clearly show that the presence of stable chimerism correlated with development of spe- cific transplantation tolerance (Figure 3). All chimeric animals permanently accepted both fhst and second donor-type and host- type skin grafts, while third party grafts were uniformly rejected. In contrast, control untreated animals, as well as similarly treated recipients without detectable levels of chimerism, rapidly rejected both donor-type and third party-type skin grafts. I&ken together, these data indicate that a combined treatment with low amounts of DMM (4 mg/kg) and CY (100 mg/kg) can be used as an good alternative for TBI in our tolerance-inducing protocol. nduction of mixed chimerism in a semi-allogeneic donor*ecipient combination Because of the prospectke I-IL&matching policies widespread in kidney transplant centres, kidney transplantation across a full HLA disparity does not frequently occur. Therefore, we also tested a haplo-identical donor-recipient combination. Earlier, we reported that in this more clinically relevant situation, especially in living-related transplantation a less intensive conditioning of the recipient, 3 Gy TBI, is required for induction of stable mixed chimerism and permanent skin graft survival? Table 1 shows that similar results were obtained with DMh$/CY. In contrast to the fully allogeneic situation, we already observed stable mixed chimerism in BlO animals that received monoclonal antiies, 15 x 106 (BlO x BlO.D2)Fl (H-2Wd) T cell-depleted bone marrow cells and a single dose of as little as 100 mg/kg CY. Addition of a single low dose DMM (2-4 m&g) to the conditioning regimen resulted in higher levels of chimerism. Again all chimeras showed permanent survival of first and second donor-type and host-type skin grafts. Unrelated third party grafts were always rejected. Thus, shortly after conditioning, chimeric animals are fully reactive to third party antigens and the tolerance is donor-specific. lbble 1 Chimerism and shin graft survival. BlO hosts treated with hapto-identical bone marrow cells of (BlO x BlO.D2)Fl mice Group Skin graft survival’ (days) Host treatmenta Donor TX Third party TX DMM (mg/kg) CY (m&g) Donor-type cellsb First Second First Second 1 0 100 8.7 >125 >200 58 .4 >125 >2iHl 29 32; >125 >200 29 20 2 2 100 1:: 13:1 >125 >2OO 48 20 >125 2200 78 29 12.1 >125 >200 65 29 3 4 100 21.4 >125 >200 29 3.3 =-125 >200 EZ 25.3 >125 >200 ;; 32 Control host, untreated 0.0 23 19 32 21 Control donor 98.8 *BlO host mice were treated with anti-CD4, anti-CD3,15 x ld PI donor bone marrow cells and DMM/CY. bChimerism was determined in peripheral blood 119 days following conditioning. Donor-type cells(H-2bMd p”) + host-type cells(H-2b wdnw) was always 95-100 . ‘First transplantation (TX) of host-, donor-and third party-(BlO.BR) shin graft was performed within 1 h following conditioning, Second skin tram- plantation was performed 4 months aher the first. Syngeneic shin graft survival was 100 . Transplant mmunology 1998; 6: 147 151  15 A de Vries van der Zwan et al. anti Donor BlO.D2) anti Host BlO) Effector Target ratio anti Third Party BlO.BR) 1 5o:l 12.5:1 3.1:1 0.8:1 Effector Target ratio ‘;;_. ‘-4 - .- Control BlO.D2 5O:l 12S:l 3.1:l Effector Target ratio 0.8:1 FIgwe 2 Specitic GIL lysis of 51Cr-labelled host, donor and third party targets by splenocytes from BlO, BlO.D2, a representative chimera, pre- pared using 4 mgkg DMM, 100 mgkg CY and 15 x lo6 BlO.D2 bone marrow cells (10.0 donor-type splenocytes), and a nonchimera, conditioned with 8 mg&g DMM, 50 mgkg CY and 15 x lo6 BlO.D2 bone marrow cells. Cf’L response was examined from 10 to 12 months after conditioning. Spontaneous release was <25 . 1 irst skin graft 1 Second skin graft Days after conditioning Recipients : - ControlBlO ;n=9 -- Mixed Chimeras : n = 19 Figure3 Graft survival of donor-type (BlO.D2) shin on untreated con- trol BlO mice (n = 8) and mixed chimeras (n = 19). Fit shin trans- plantation was performed within 1 h following conditioning, and the second transplantation 4 months later. ost survival In Table 2, we demonstrate the l-year host survival following conditioning with DMM, CY or both. A 50% mortality was seen in mice conditioned with high-dose DMM (20 mg/kg) or CY (300 mgkg). In contrast, the combination of a lower dose of DMM (2-12 mg/kg) and CY (100 mgkg) was nonlethal since all animals conditioned in this way and treated with (n = 18), as well as without, bone marrow cells (n = 4) exhibited 100% Tmnsplant mmunology 1998; 6: 147 151 survival 1 year after conditioning. As expected, animals which did not receive bone marrow cells repopulated as 100% autologous. lkursfusion with allogeneic bone marrow cells resulted in mixed chimerism. Survival was excellent without evidence of graft-versus-host disease (GvHD). Discussion In humans, bone marrow transplantation is a routine treatment for a variety of haematological malignancies. The toxicity of the pretransplant preparative regimen, TBI or a combination of TBI and CY, however, still remains a major problem. Therefore, a radiation-free regimen was developed. The combination of BU and CY has been used most widely. The srcinal protocol employed a dose of 16 mg/kg BU and 200 mgkg CY, so called ‘Big BU/CY’, while many centres have preferred the ‘Small BUKY’, which employs the reduced 120 mg&g dose of CY? In several experimental animal models, both drugs has been used separately for induction of allo-tolerance.z~3 In our murine model, we show that conditioning with a combination of a single low-dose DMM (4 mg/kg) and CY (100 mgkg) together with anti-T cell mAbs and donor bone marrow cells, results in development of stable mixed multilineage chimerism, and tolerance for full MHC-disparate skin grafts, even when the skin transplantation was performed directly following conditioning. This conditioning regimen was well tolerated and alI recipient mice survived for > 1 year without clinical signs of GvHD. An important and interesting observation in the present study is the synergistic effect between treatment with DMM and CY on engraftment of fully allogeneic bone marrow cells. DMM is  An altematiue conditioning regimen for induction of specific skin graft tolerance 5 ‘lbbte 2 One-year host survival Host treatment DMM (mg/kg) CY (mgflrg) One-year survival n A. Recipients of BlO.D2 bone marrow cells 20 50% 4 16 100% 4 300 50% 4 200 100% 4 12 100 100% 4 8 100 100% 4 4 100 100% 4 4a 100 100% 3 B. Recipients of (BlO x BlO.DZ)Fl bone marrow cells 100 1OOa 2 100 4 100 ‘No bone marrow cells. 100% 5 100% 5 100% 3 100% 3 an alkylating agent that produces effective myeloablation, but little immunosuppression.s~9 It has been shown by Down et a&” that DMM is especially toxic against a more primitive stem cell subset (quiescent, high self-renewal). Depletion of this cell pop- ulation in the host seemed to be essential for achieving lasting donor marrow engraftment. CY, however, acts mainly as an immunosuppressive agent, by selectively killing proliferatin cells by the formation of cross-links between strands of DNA. lgl When mice were conditioned with a combination of DMM and CY, considerably lower doses of each drug can be used for induction of mixed chimerism than when they were treated with one of these drugs alone. Leong et a1.3 previously demonstrated that a dose of 8 mg/kg DMM was sufficient to induce chime&m and tolerance in an H-2 disparate skin transplantation model. However, their pro- tocol differs from ours in the respect that their recipients were injected three times a week, starting two days before bone mar- row transfusion with a total of seven doses of CD4 and CD8 mAbs. Skin transplantation was performed 31 days later. However, since our protocol is aimed at direct clinical applica- bility, we performed conditioning and skin transplantation on the same day, so we started immunosuppression by a single dose of CD3 and CD4 mAbs just prior to conditioning. When CY, which is mainly immunosuppressive, was added to our condi- tioning regimen, we also were able to reduce the dose of DMM (4 mg/kg). In the clinical setting, complete reconstitution with donor- type cells creates a great risk for development of GvHD. In this study, we were able to manipulate the level of chimerism by reducing the dose of DMM in the combined DMM/CY proto- col. Furthermore, by using DMMKY we were able to induce lower levels of stable donor-type chimerism than after treatment with a low dose of TBI, as previously reported.’ An explanation for this observation could be that the subset(s) of stem cells which is (are) eliminated from the host haematopoietic system plays a role in this phenomenon. We currently believe that it is not the absolute level, but more the stability of the chimerism which is important for maintaining tolerance across full MHC barriers. Depletion of primitive stem cells seemed to be essen- tial for establishing permanent engraftment of donor stem cells. Since DMM is especially toxic against long-term repopulating stem cells, treatment with this myeloablative drug may, therefore, cause lower levels of stable chimerism. We have previously demonstrated that stable mixed chimerism and donor-specific transplantation tolerance across MHC barriers can be achieved by a nonlethal conditioning approach of low dose TBI.4*5 Present data clearly show that a single treatment with low-dose DMM/CY therapy is a good alternative in replac- ing TBI, resulting in mixed chimerism and specific tolerance as well. Because this regimen does not require prolonged treat- ment of the host before transplantation, and transplantation can be performed within 1 h following conditioning, we suggest that our method can be used for induction of donor-specific toler- ance in organ transplantation. References 1 Sharabi Y, Sachs DH. Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regi- men. JExp Med 1989; 169: 494-502. 2 Mayumi H, Good Rk Long-lasting skin allograft tolerance in adult mice induced across fully agogeneic (multimajor H-2 plus multi- minor histocompatibility) antigen barriers by a tolerance-inducing method using cyclophosphamide. JExp Med 1989; 169: 213-38. 3 Leong LYW, Qin S, Cobbold SP, Waldmann H. Classical transplan- tation tolerance in the adult: the interaction between myeloablation and immunosuppression. Eur J mmunol1992; 22: 2825-30. 4 De Vries-van der Zwan A, Besseling AC, Kievits F, van Bvuyver E, de Waal LP Anti-CD3 treatment facilitates engraftment of full H-2 disparate donor bone marrow cells and subsequent skin allograft tolerance. Tmnsplantation 1994; 5S: 610-17. 5 De Vries-van der Zwan A, Besseling AC, van ‘Rvuyver E, Boog CJP, de Waal LP A substantial level of mixed chime&m is required for the induction of permanent transplantation tolerance. Tmnspl Immunol1996; 4: 232-40. 6 De Vries-van der Zwan, Besseling AC, de Waal LP, Boog CJP Specific tolerance induction and transplantation: a single day pro- tocol. Blood 1997; 89: 2596-601. 7 lbtschka PJ, Copelan EA, Klein JP. Bone marrow transplantation following a new busulfan and cyclophosphamide regimen. Blood 1987; 70: 1382-88. 8 Fkrersheim GL, Ruszkiewicz M. Bone-marrow transplantation after anti-lymphocytic serum and lethal chemotherapy. Nature 1969; 222: 854-57. 9 Lapidot T, lbrenzi A, Singer Ts, Salomon 0, Reisner Y. Enhancement by dimethyl myleran of donor type chimerism in murine recipients of bone marrow allografts. Blood 1989; 73: 2025-32. 10 Down JD, Boudewijn A, Dillingh JH, Fox BW, Ploemacher RE. Relationships between ablation of distinct haematopoietic cell sub- sets and the development of donor marrow engraftment following recipient pretreatment with different alkylating drugs. Br J Cancer 1994; 70: 611-16. 11. Mayen FH, Jawetz E, Goldfflen A. Review ofmedicaZphumuzcorogY. California: Lange Med, 1980; 83-%. Transplant Immunology 1998; 6: 147 151
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