- Email: [email protected]
A New Method of Bone Marrow Transplantation Leads to Extention of Skin Allograft Survival
A New Method of Bone Marrow Transplantation Leads to Extention of Skin Allograft Survival M. Siemionow, K. Ozer, D. Izycki, M. Unsal, and A. Klimczak ABSTRACT Tolerance induction through allogeneic bone marrow transplantation is an alternative method to chronic immunosuppression in maintaining long-term allograft survival. In this article, we introduce a new method of bone marrow allotransplantation, which preserves its natural microenvironment and does not require marrow processing or recipient conditioning. A total of 43 skin graft transplantations were performed in nine experimental groups between isogeneic [Lewis to Lewis (LEW, RT11)] and allogeneic [Lewis ⫻ Brown Norway (LBN ¡ F1, RT11⫹n) to Lewis] rats under 35-day protocol of ␣ T-cell receptor (TCR) monoclonal antibody (mAb) and cyclosporine (CsA) protocol. Monotherapies combined with “crude” bone marrow transplantation resulted in extended survival up to 21 days under CsA and up to 10 days under ␣-TCR mAb protocol. The use of combined protocol of ␣-TCRmAb/CsA with crude bone marrow transplantation resulted in the extension of skin allograft survival up to 65 days (P ⬍ .05). This new simple method of “crude” bone marrow allotransplantation without recipient conditioning is a promising, minimally invasive technique with a potential for direct clinical application.
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ANSPLANTATION OF COMPOSITE tissue allografts offers unique opportunities for reconstruction of upper and lower extremities and other body sites that require tissue coverage.1 In recent years, considerable interest in transplantation research focused on the development of new strategies for tolerance induction protocols without a need for chronic immunosuppression. Transplantation of hematopoietic stem cells (HSC) to establish donor-specific, hematopoietic chimerism is one of the most promising applications leading to extension of allograft survival. The concept stems from the experimental work of Billingham et al who infused replicating hematopoietic cells of the donor origin into immunologically incompetent neonates to induce tolerance.2 The establishment of stable, mixed macrochimerism provides a state of unresponsiveness to all tissue types transplanted from the same donor. Despite promising reports, the induction of hematopoietic chimerism for transplantation tolerance remained experimental rather than clinically accepted therapeutic approach. One option for tolerance induction is donor-specific bone marrow transplantation, which is a most instructive example for the curative potential of stem cell– based therapies in solid organ transplantations.3,4 Most protocols use whole bone marrow as donor-derived cells and total body irradiation, cytotoxic drugs, or both to create a “space” within the
recipient bone marrow environment to permit stem-cell engraftment. It is now widely accepted that high doses of HSC infused in the absence of recipient irradiation does not induce graft-versus-host disease (GvHD).4 The side effects of recipient conditioning, such as time needed for establishment of donor specific, mixed chimerism before organ transplantation, and the long-lasting recovery of the immune system activity, limited the clinical applicability of this method.5 As a result, investigators are still searching for clinically applicable strategies of tolerance induction without the need for recipient conditioning.6,7 In this study, we introduce an alternative method of nonvascular “crude” bone marrow transplantation directly into the recipient bone. The idea is based on avoidance of recipient conditioning and preservation of the bone marrow cell (BMC) microenvironment during transplantation. This new approach would allow for donor-derived bone marrow From The Cleveland Clinic Foundation, Department of Plastic Surgery, Cleveland, Ohio, USA. Address reprint requests to Maria Siemionow, MD, PhD, The Cleveland Clinic Foundation, Department of Plastic Surgery, 9500 Euclid Avenue A60, Cleveland, OH 44195, USA. E-mail: [email protected]
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.03.054
Transplantation Proceedings, 37, 2309 –2314 (2005)
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engraftment and establishment of donor-specific hematopoietic chimerism. This new method of “crude” bone marrow transplantation is simple and minimally invasive, does not require recipient conditioning, and can be easily implemented into the clinical practice. MATERIALS AND METHODS Animals, Experimental Groups, and Treatment
bridge. A standard compressive dressing and adhesive bandage was used for 7 days.
“Crude” Bone Marrow Transplantation Technique Harvesting of the “Crude” Bone Marrow From the Donor. The metaphyseal region of the right tibia was approached from an anterior incision. Next, on the anteromedial cortex of the tibia, a 3 mm window was created using a 1 32-inch drill (Fig 1). Following decortication, the contents of the metaphyseal parts of the bone were removed with a bone curette and were placed in container, cooled on ice. Next, the bone rongeur was used to harvest intramedullary content along the diaphysis. The weight of the harvested bone marrow was measured before transplantation, assuring that 50 mg of “crude” bone marrow would be harvested. Transplantation of the “Crude” Bone Marrow to the Recipient. The recipient’s tibia was opened in a similar fashion to the donor’s bone by exposing the anteromedial surface of the right tibia, followed by creation of the cortical window. The recipient’s cancellous bone and entire content of the bone marrow cavity were removed to create an appropriate “space” for the bone marrow engraftment. The harvested “crude” bone marrow from the donor was packed into the “space” created within the medullar cavity of the recipient’s tibia and the bone was sealed with the bone wax.
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All animals used in this study were purchased from Harlan Sprague-Dawley (Indianapolis, Ind, USA) and received humane care in compliance with the Guide for the Care and Use of Laboratory Animals. Lewis rats (LEW, RT11) served as the recipients of skin and “crude” bone marrow allografts from LewisBrown Norway donors (LBN ¡ F1, RT11⫹n). The animals were caged individually in rooms with 12-hour light/dark cycle with free access to food and water. Intraperitoneal sodium pentobarbital (50 mg/kg) was used as an analgesic during the transplantation procedure. Experimental groups, treatment protocols, and graft survival are outlined in Table 1. Three different treatment protocols were used: (1) monotherapy with cyclosporine (CsA; Bedford Laboratories, Bedford, Oh, USA) 16 mg/kg/d, subcutaneously (SC) during the first week, 8 mg/kg/d during week 2, 4 mg/kg/d at weeks 3 and 4, and 2 mg/kg/d during week 5; (2) The ␣ T-cell receptor (TCR) monoclonal antibody (mAb) monotherapy (clone: R73) (BD Pharmingen, San Diego, Calif, USA) was tapered from 250 g during the first week to 50 g within 5 weeks. (3) Combined ␣-TCR mAb and CsA (␣-TCR/CsA) protocol was applied for 5 weeks following skin and the “crude” bone allograft marrow transplantation from the same donor (LBN). All treatment protocols were administered 12 hours before transplantation and continued up to 5 weeks. Skin grafting and crude bone marrow transplantation was performed from the same donor during the same surgical procedure.
Clinical Assessment of Allograft Rejection The physical signs of skin allograft rejection, such as erythema, edema, scaling of the skin, hair loss, epidermolysis, and desquamation, were evaluated on daily basis. Rejection was defined as the destruction of over 80% of the graft.
Flow Cytometry Analysis Flow cytometry (FC) analysis was performed for evaluation of immunodepletion of T lymphocytes, phenotype of “crude” bone marrow, and presence of donor-specific chimerism for major histo compability complex (MHC) class I (RTIn) antigen.
Skin Grafting Transplantation Technique The skin grafting was performed according to the technique described by Billingham and Medawar.8 The full-thickness skin grafts, 16 mm in diameter, were taken from the donors. Graft beds were prepared by excising 18-mm circles on the lateral dorsal thoracic walls of the recipients. Care was taken to remove perniculous carnosum from the grafted skin. Both sides of the thoracic wall were used for allogeneic grafts and the midsternum was used for the syngeneic grafts. All grafts were separated by a 10-mm skin
Determination of “Crude” Bone Marrow “Crude” bone marrow taken from three LBN rats served as control for evaluation of cell content and cell viability. The “crude” bone marrow weight was measured and bone marrow was passed through a nylon filter into phosphate-buffered saline (PBS) without Mg⫹⫹ and Ca⫹⫹, centrifuged, and lysed in 0.83% NH4CI/TRIS solution for 15 minutes. Next, bone marrow cells were twice washed in PBS, resuspended, and counted.
Table 1. Experimental Groups, Treatment Protocols, and Survival Time After Application of the Novel “Crude” Bone Marrow Transplantation Technique Experimental groups
Group Group Group Group Group Group Group Group Group
1 2 3 4 5 6 7 8 9
(n (n (n (n (n (n (n (n (n
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
6) isograft 6) allograft 6) allograft 3) 3) 5) 5) 4) 5)
Transplantation procedure*
Skin Skin Skin Skin Skin Skin Skin Skin Skin
⫹ crude bone marrow
⫹ crude bone marrow ⫹ crude bone marrow ⫹ crude bone marrow
Treatment protocol
Days of survival after cessation of treatment
Survival (Mean ⫾ SD)
No No No CsA ␣-TCR mAb CsA ⫹ ␣-TCR mAb CsA ␣-TCR mAb CsA ⫹ ␣-TCR mAb
Indefinite 6, 8, 7, 6, 7, 8 7, 8, 9, 6, 7, 8 15, 16, 15 7, 9, 10 20, 21, 20, 19, 22 20, 22, 24, 21, 20 9, 12, 10, 11 67, 65, 69, 67, 71
Indefinite 7 ⫾ 0.9 7.5 ⫾ 1 15 ⫾ 0.5 8.6 ⫾ 1.5 20.4 ⫾ 1.1 21.4 ⫾ 1.7 10.5 ⫾ 1.3 68 ⫾ 4.9**
*All transplantations, except in -group 1 [isograft-control (Lew-to-Lew)], were performed between LBN and LEW rats. **P ⬍ .05.
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Fig 1. Harvesting technique of the crude bone marrow from the donor tibia for direct transplantation into the recipients’ tibia. (A) The metaphyseal region of the right tibia was opened and used as the harvesting (the donor) and transplantation (the recipient) site. (B) A hole was created using a hand-held drill on the anterior cortex of the tibia. (C) Metaphyseal region of the tibia was removed, and (D) the “crude” bone marrow was weighed before transplantation. For determination of phenotype of BMCs, two-color flow cytometry technique was used. Mouse anti-rat mAb CD90-FITC (clone OX-7, Pharmingen) in combination with CD4-PE (clone OX-35), CD8-PE (clone OX-8), CD45RA-PE (clone OX-33), and ␣-TCR-PE (clone R73) mAb (Pharmingen, San Diego, Calif, USA) were used for cells staining.
Evaluation of the Donor-Specific Chimerism For the assessment of donor-specific lymphoid chimerism in the peripheral blood of recipients, combination of mouse anti-rat CD4-PE (clone OX-35) or CD8a-PE (clone OX-8; Pharmigen) mAb with mouse anti-rat RT1n (Brown Norway MHC class I, clone MCA-156, Serotec, UK) mAb was applied. Samples were preincubated with purified anti-rat CD-32 (Fc␥II block receptor) antibody and next with 5 L of RT1n mAb for 30 minutes in 4°C. After washing samples were stained with goat anti-mouse fluorescein isothiocyanate conjugated immunoglobulin G (IgG) (rat adsorbed; Serotec) and followed was incubated with CD4-PE or CD8-PE antibodies. Negative controls included isotype-matched antibodies (IgG1/ IgG2) and PBS incubated samples. FC analyses were performed on
1 ⫻ 104 mononuclear cells by using FACS Scan (BD Pharmingen) and CellQuest software.
Statistical Analysis The survival time in treatment group was evaluated by the KaplanMeier method. The level of chimerism and efficacy of the immunosuppressive treatment was compared by independent samples t test; P ⬍ .05 was considered significant.
RESULTS The Survival of the Skin Allografts
The survival of skin allografts for each experimental group is presented in Table 1. The combined protocol of ␣-TCR/ CsA applied to the recipients of skin allografts without “crude” bone marrow significantly extended allograft survival when compared to the allograft controls without treatment (P ⬍ .05). However, following cessation of ␣TCR/CsA therapy all allografts were rejected within 20 days (P ⬍ .05). Monotherapy with CsA alone extended skin
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transplant survival over 7 days, whereas monotherapy with ␣-TCR alone resulted in rejection at the same time as rejection controls without treatment. When skin allograft transplantation was augmented by “crude” bone marrow transplantation, mean survival time of skin allograft transplants was extended over 68 days (range between 65 and 71 days; P ⬍ .05) after cessation of ␣-TCR/CsA protocol. Evaluation of “Crude” Bone Marrow
The weight of the scooped “crude” bone marrow taken from three LBN control rats was found to be in the range from 53 to 60 mg. BMCs, isolated from the “crude” bone marrow contained 28 ⫻ 106 to 33 ⫻ 106 viable cells. FC analysis showed that 75% of nucleated cells express CD90⫹ antigen specific for early progenitor cells. Double staining for CD90/CD4, CD90/CD8, and CD90/␣-TCR T lymphocytes revealed less then 1% of double-positive CD90/CD4, CD90/CD8, and CD90/␣-TCR cells. Double-positive staining for CD90/CD45RA revealed 2.9% to 4.5% of B lymphocytes to be present within whole bone marrow.
Flow Cytometry Analysis of in Vivo Depletion
FC determination of ␣-TCR expression on the lymphocytes harvested from the ␣-TCR/CsA treatment controls without skin graft and crude bone marrow transplantation and from experimental animals showed ⬎90% depletion of the ␣-TCR-positive cells in the periphery at day 7 and subsequent repopulation of the ␣-TCR⫹ cells 7 days after treatment cessation (day 42). The pretransplant level of ␣-TCR⫹ cell populations was observed 28 days after cessation of the immunosuppressive therapy (day 63; Fig 2).
SIEMIONOW, OZER, IZYCKI ET AL
Determination of the Donor-Specific Chimerism
Donor-specific chimerism was evaluated at days 0, 7, 21, 35, and 63 after transplantation in the peripheral blood of “crude” bone marrow and skin allograft recipients. Flow cytometry results at day 63 posttransplant demonstrated presence of the donor-specific chimerism ranging from 8% to 10% (RT1n-positive cells). Examination of two-color stained RT1n-FITC/CD4-PE and RT1n-FITC/CD8-PE peripheral lymphocytes revealed 3.1% and 4.6% (respectively) of double-positive T-cell subpopulations (Figs 3A, 3B). DISCUSSION
The functional results of the first series of human hand transplantations are encouraging.9 However, clinical composite tissue allotransplantation requires lifelong immunosuppression, which exposes the patients to the serious side effects. One possible approach of tolerance induction during hand transplantation would be simultaneous transplantation of the bone marrow from the hand donor. Current protocols of bone marrow transplantation require introduction of recipient conditioning regiments. The use of HSC therapy in organ transplantation is considered to promote chimerism with the aim of enhancing organ tolerance. It is now widely accepted that high doses of HSC infused in the absence of recipient irradiation do not induce GvHD.4 The first successful experiments of combined use of antilymphocyte serum (ALS) and posttransplant infusion of the donor bone marrow resulted in donor-specific unresponsiveness were performed by Wood et al.10 Studies revealing the importance of stromal cells and microenvironment in the induction of donor-specific tolerance indicated
Fig 2. FC evaluation of the peripheral blood ␣-TCR-positive cells. FC analysis at day 7 demonstrated ⬎90% depletion of the ␣-TCR⫹ cells and gradual reconstitution at day 63 in transplanted and control groups under ␣-TCR/CsA protocol. The analysis of the expression of the ␣ TCR⫹ cells in the peripheral blood of naive animals served as a control.
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Fig 3. Flow cytometric determination of the donor-originated RT1n expression on (A) CD4⫹ and (B) CD8⫹ T-cell subpopulations isolated from peripheral blood of recipients showed double-positive RT1n⫹/CD4⫹ (3.1%) and RT1n⫹/CD8⫹ (4.6%) chimeric cells subpopulation at 63 days after skin and “crude” bone marrow allograft transplantation. (C) No positive cells were found in isotype control.
that hematopoietic reconstitution following total lymphoid irradiation in rats is faster in recipients of vascularized bone marrow transplantation when compared to the recipients of cellular bone marrow transplantation.11 These findings indicate that stromal microenvironment is essential for the proliferation and differentiation of the hematopoietic progenitors. Bone marrow stromal cells play a critical role in formation of the hematopoietic microenvironment and support hematopoietic stem cell differentiation through the intercellular contact and secretion of various cytokines and growth factors.12,13 The hematopoietic microenvironment, which is preserved by transplantation of the “crude” bone marrow, allows for engraftment of hematopoietic cells at the site of transplantation. In our study, direct transplantation of the donor bone marrow in the “crude” form into the recipient’s bone marrow cavity allowed for successful bone marrow engraftment and provided natural matrix for subsequent cell repopulation and trafficking. The effectiveness of engraftment was confirmed by flow cytometry revealing 3.1% and 4.6% of the donor-specific chimerism for the RT1n-FITC/ CD4-PE and RT1n-FITC/CD8-PE, respectively, in the peripheral blood of recipients at day 63 after cessation of immunosuppression. Significantly extended skin allograft survival was achieved only in the animals receiving “crude” bone marrow in combination with short-term ␣-TCR mAb/CsA protocol. In our pilot study as well as in the current study we have found that chimerism and extended allograft survival was achieved only when the weight of transplanted crude bone marrow exceeded 50 mg. In this study we have confirmed that 50 mg of “crude” bone marrow contained 28 ⫻ 106 to 33 ⫻ 106 of the viable cells, and this correlated with higher chimerism level in the recipients of “crude” bone marrow transplant when compared to controls. These findings are
parallel to Monaco and Wood’s experiments in which they found a direct correlation between the amount of bone marrow infused and the level and duration of chimerism achieved. They concluded that 25 ⫻ 106 of the bone marrow cells were a critical to induce tolerance in ALS-treated skin-grafted mice.14 The other important component contributing to the extension of allograft survival is the specific blockage of TCR using ␣-TCR mAb. Treatment against the rat ␣TCR mAb has been shown previously to abrogate the experimentally induced adjuvant arthritis,15 prolonged cardiac allograft survival,16 and abrogated concordant cardiac xenograft rejection in a hamster-to-rat model.17 It has also been indicated that combination of ␣-TCR/CsA may exert clinically relevant and potentially important additive/synergistic in vivo effects. This is parallel to our findings in hind limb allograft model, which confirmed that a short protocol of combined ␣-TCR/CsA treatment resulted in tolerance induction (750 days).18 Timing of the initial injection of the antibody as well as bone marrow transplantation is also important for the clinical applicability of these treatment protocols. Recipient conditioning before transplantation was found to be more effective than posttransplant treatment.19,20 In this study, we have introduced ␣-TCR/CsA protocol 12 hours before transplantation of bone marrow and skin grafts to make this protocol clinically applicable. As a result significant extension of skin allograft survival was achieved without chronic immunosuppression. In conclusion, in this study we have introduced a new concept of the “crude” bone marrow transplantation directly into the recipient bone for optimal engraftment, repopulation, and chimerism induction. This minimally invasive, nonmyeloablative protocol of bone marrow transplantation may have direct clinical application in composite tissue and solid organ transplants.
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REFERENCES 1. Petit F, Minns AB, Dubernard JM, et al: Composite tissue allotransplantation and reconstructive surgery: first clinical applications. Ann Surg 237:19, 2003 2. Billingham RE, Brent L, Medawar PB: Quantitative studies on the tissue transplantation immunity. III. Actively acquired tolerance. Philos Trans R Soc Lond (Biol) 239:357, 1956 3. Ricordi C, Karatzas T, Nery J, et al: High-dose donor bone marrow infusions to enhance allograft survival: the effect of timing. Transplantation 63:7, 1997 4. Ciancio G, Miller J, Garcia–Morales RO, et al: Six-year clinical effect of donor bone marrow infusions in renal transplant patients. Transplantation 71:827, 2001 5. Sharabi Y, Sachs DH: Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen. J Exp Med 169:493, 1989 6. Sykes M, Szot GL, Swenson KA, et al: Induction of high levels of allogeneic hematopoietic reconstitution and donor specific tolerance without myelosuppressive conditioning. Nat Med 3:783, 1997 7. Wekerle T, Nikolic B, Pearson DA, et al: Minimal conditioning required in a murine model of T cell depletion, thymic irradiation and high-dose bone marrow transplantation for the induction of mixed chimerism and tolerance. Transpl Int 15:248, 2002 8. Billingham RE, Medawar PB: The technique of free skin grafting in mammals. J Exp Biol 28:385, 1951 9. Jones JW, Gruber SA, Barker JH, et al: Successful hand transplantation. One-year follow-up. Louisville Hand Transplant Team. N Engl J Med 343:468, 2000 10. Wood ML, Monaco AP, Gozzo JJ, et al: Use of homozygous allogeneic bone marrow for induction of tolerance with ALS: dose and timing. Transplant Proc 3:676, 1971
SIEMIONOW, OZER, IZYCKI ET AL 11. Janczewska S, Ziolkowska A, Durlik M, et al: Fast lymphoid reconstitution after vascularized bone marrow transplantation in lethally irradiated rats. Transplantation 68:201, 1999 12. Quesenberry PJ, Crittenden RB, Lowry P, et al: In vitro and in vivo studies of stromal niches. Blood Cells 20:97, 1994 13. Bianco P, Riminucci M, Gronthos S, et al: Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19:180, 2001 14. Monaco AP, Wood ML: Studies on heterologous antilymphocyte serum in mice. VII. Optimal cellular antigen for induction of immunologic tolerance with antilymphocyte serum. Transplant Proc 2:489, 1970 15. Yoshino S, Schlipkoter E, Kinne R, et al: Suppression and prevention of adjuvant arthritis in rats by a monoclonal antibody to the alpha/beta T cell receptor. Eur J Immunol 20:2085, 1990 16. Tsuchida M, Hirahara H, Matsumoto Y: Induction of specific unresponsiveness to cardiac allografts by short-term administration of anti-T cell receptor alpha/beta antibody. Transplantation 57:256, 1994 17. Van den Bogaerde J, White D, Roser B, et al: In vitro and in vivo effects of monoclonal antibodies against T cell subsets on allogeneic and xenogeneic responses in the rat. Transplantation 50:915, 1990 18. Siemionow M, Ortak T, Izycki D, et al: Induction of tolerance in composite-tissue allografts. Transplantation 74:1211, 2002 19. Heidecke CD, Hancock WW, Jakobs F, et al: ␣/-T cell receptor-directed therapy in rat cardiac allograft recipients. Treatment prior to alloantigen exposure prevents sensitization and abrogates accelerated rejection. Transplantation 59:78, 1995 20. Heidecke CD, Hancock WW, Westerholt S, et al: ␣/-T cell receptor-directed therapy in rat cardiac allograft recipients. Longterm survival of cardiac allografts after pretreatment with R73 mAb is associated with upregulation of Th2-type cytokines. Transplantation 61:948, 1996
T
ANSPLANTATION OF COMPOSITE tissue allografts offers unique opportunities for reconstruction of upper and lower extremities and other body sites that require tissue coverage.1 In recent years, considerable interest in transplantation research focused on the development of new strategies for tolerance induction protocols without a need for chronic immunosuppression. Transplantation of hematopoietic stem cells (HSC) to establish donor-specific, hematopoietic chimerism is one of the most promising applications leading to extension of allograft survival. The concept stems from the experimental work of Billingham et al who infused replicating hematopoietic cells of the donor origin into immunologically incompetent neonates to induce tolerance.2 The establishment of stable, mixed macrochimerism provides a state of unresponsiveness to all tissue types transplanted from the same donor. Despite promising reports, the induction of hematopoietic chimerism for transplantation tolerance remained experimental rather than clinically accepted therapeutic approach. One option for tolerance induction is donor-specific bone marrow transplantation, which is a most instructive example for the curative potential of stem cell– based therapies in solid organ transplantations.3,4 Most protocols use whole bone marrow as donor-derived cells and total body irradiation, cytotoxic drugs, or both to create a “space” within the
recipient bone marrow environment to permit stem-cell engraftment. It is now widely accepted that high doses of HSC infused in the absence of recipient irradiation does not induce graft-versus-host disease (GvHD).4 The side effects of recipient conditioning, such as time needed for establishment of donor specific, mixed chimerism before organ transplantation, and the long-lasting recovery of the immune system activity, limited the clinical applicability of this method.5 As a result, investigators are still searching for clinically applicable strategies of tolerance induction without the need for recipient conditioning.6,7 In this study, we introduce an alternative method of nonvascular “crude” bone marrow transplantation directly into the recipient bone. The idea is based on avoidance of recipient conditioning and preservation of the bone marrow cell (BMC) microenvironment during transplantation. This new approach would allow for donor-derived bone marrow From The Cleveland Clinic Foundation, Department of Plastic Surgery, Cleveland, Ohio, USA. Address reprint requests to Maria Siemionow, MD, PhD, The Cleveland Clinic Foundation, Department of Plastic Surgery, 9500 Euclid Avenue A60, Cleveland, OH 44195, USA. E-mail: [email protected]
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.03.054
Transplantation Proceedings, 37, 2309 –2314 (2005)
2309
2310
SIEMIONOW, OZER, IZYCKI ET AL
engraftment and establishment of donor-specific hematopoietic chimerism. This new method of “crude” bone marrow transplantation is simple and minimally invasive, does not require recipient conditioning, and can be easily implemented into the clinical practice. MATERIALS AND METHODS Animals, Experimental Groups, and Treatment
bridge. A standard compressive dressing and adhesive bandage was used for 7 days.
“Crude” Bone Marrow Transplantation Technique Harvesting of the “Crude” Bone Marrow From the Donor. The metaphyseal region of the right tibia was approached from an anterior incision. Next, on the anteromedial cortex of the tibia, a 3 mm window was created using a 1 32-inch drill (Fig 1). Following decortication, the contents of the metaphyseal parts of the bone were removed with a bone curette and were placed in container, cooled on ice. Next, the bone rongeur was used to harvest intramedullary content along the diaphysis. The weight of the harvested bone marrow was measured before transplantation, assuring that 50 mg of “crude” bone marrow would be harvested. Transplantation of the “Crude” Bone Marrow to the Recipient. The recipient’s tibia was opened in a similar fashion to the donor’s bone by exposing the anteromedial surface of the right tibia, followed by creation of the cortical window. The recipient’s cancellous bone and entire content of the bone marrow cavity were removed to create an appropriate “space” for the bone marrow engraftment. The harvested “crude” bone marrow from the donor was packed into the “space” created within the medullar cavity of the recipient’s tibia and the bone was sealed with the bone wax.
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All animals used in this study were purchased from Harlan Sprague-Dawley (Indianapolis, Ind, USA) and received humane care in compliance with the Guide for the Care and Use of Laboratory Animals. Lewis rats (LEW, RT11) served as the recipients of skin and “crude” bone marrow allografts from LewisBrown Norway donors (LBN ¡ F1, RT11⫹n). The animals were caged individually in rooms with 12-hour light/dark cycle with free access to food and water. Intraperitoneal sodium pentobarbital (50 mg/kg) was used as an analgesic during the transplantation procedure. Experimental groups, treatment protocols, and graft survival are outlined in Table 1. Three different treatment protocols were used: (1) monotherapy with cyclosporine (CsA; Bedford Laboratories, Bedford, Oh, USA) 16 mg/kg/d, subcutaneously (SC) during the first week, 8 mg/kg/d during week 2, 4 mg/kg/d at weeks 3 and 4, and 2 mg/kg/d during week 5; (2) The ␣ T-cell receptor (TCR) monoclonal antibody (mAb) monotherapy (clone: R73) (BD Pharmingen, San Diego, Calif, USA) was tapered from 250 g during the first week to 50 g within 5 weeks. (3) Combined ␣-TCR mAb and CsA (␣-TCR/CsA) protocol was applied for 5 weeks following skin and the “crude” bone allograft marrow transplantation from the same donor (LBN). All treatment protocols were administered 12 hours before transplantation and continued up to 5 weeks. Skin grafting and crude bone marrow transplantation was performed from the same donor during the same surgical procedure.
Clinical Assessment of Allograft Rejection The physical signs of skin allograft rejection, such as erythema, edema, scaling of the skin, hair loss, epidermolysis, and desquamation, were evaluated on daily basis. Rejection was defined as the destruction of over 80% of the graft.
Flow Cytometry Analysis Flow cytometry (FC) analysis was performed for evaluation of immunodepletion of T lymphocytes, phenotype of “crude” bone marrow, and presence of donor-specific chimerism for major histo compability complex (MHC) class I (RTIn) antigen.
Skin Grafting Transplantation Technique The skin grafting was performed according to the technique described by Billingham and Medawar.8 The full-thickness skin grafts, 16 mm in diameter, were taken from the donors. Graft beds were prepared by excising 18-mm circles on the lateral dorsal thoracic walls of the recipients. Care was taken to remove perniculous carnosum from the grafted skin. Both sides of the thoracic wall were used for allogeneic grafts and the midsternum was used for the syngeneic grafts. All grafts were separated by a 10-mm skin
Determination of “Crude” Bone Marrow “Crude” bone marrow taken from three LBN rats served as control for evaluation of cell content and cell viability. The “crude” bone marrow weight was measured and bone marrow was passed through a nylon filter into phosphate-buffered saline (PBS) without Mg⫹⫹ and Ca⫹⫹, centrifuged, and lysed in 0.83% NH4CI/TRIS solution for 15 minutes. Next, bone marrow cells were twice washed in PBS, resuspended, and counted.
Table 1. Experimental Groups, Treatment Protocols, and Survival Time After Application of the Novel “Crude” Bone Marrow Transplantation Technique Experimental groups
Group Group Group Group Group Group Group Group Group
1 2 3 4 5 6 7 8 9
(n (n (n (n (n (n (n (n (n
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
6) isograft 6) allograft 6) allograft 3) 3) 5) 5) 4) 5)
Transplantation procedure*
Skin Skin Skin Skin Skin Skin Skin Skin Skin
⫹ crude bone marrow
⫹ crude bone marrow ⫹ crude bone marrow ⫹ crude bone marrow
Treatment protocol
Days of survival after cessation of treatment
Survival (Mean ⫾ SD)
No No No CsA ␣-TCR mAb CsA ⫹ ␣-TCR mAb CsA ␣-TCR mAb CsA ⫹ ␣-TCR mAb
Indefinite 6, 8, 7, 6, 7, 8 7, 8, 9, 6, 7, 8 15, 16, 15 7, 9, 10 20, 21, 20, 19, 22 20, 22, 24, 21, 20 9, 12, 10, 11 67, 65, 69, 67, 71
Indefinite 7 ⫾ 0.9 7.5 ⫾ 1 15 ⫾ 0.5 8.6 ⫾ 1.5 20.4 ⫾ 1.1 21.4 ⫾ 1.7 10.5 ⫾ 1.3 68 ⫾ 4.9**
*All transplantations, except in -group 1 [isograft-control (Lew-to-Lew)], were performed between LBN and LEW rats. **P ⬍ .05.
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Fig 1. Harvesting technique of the crude bone marrow from the donor tibia for direct transplantation into the recipients’ tibia. (A) The metaphyseal region of the right tibia was opened and used as the harvesting (the donor) and transplantation (the recipient) site. (B) A hole was created using a hand-held drill on the anterior cortex of the tibia. (C) Metaphyseal region of the tibia was removed, and (D) the “crude” bone marrow was weighed before transplantation. For determination of phenotype of BMCs, two-color flow cytometry technique was used. Mouse anti-rat mAb CD90-FITC (clone OX-7, Pharmingen) in combination with CD4-PE (clone OX-35), CD8-PE (clone OX-8), CD45RA-PE (clone OX-33), and ␣-TCR-PE (clone R73) mAb (Pharmingen, San Diego, Calif, USA) were used for cells staining.
Evaluation of the Donor-Specific Chimerism For the assessment of donor-specific lymphoid chimerism in the peripheral blood of recipients, combination of mouse anti-rat CD4-PE (clone OX-35) or CD8a-PE (clone OX-8; Pharmigen) mAb with mouse anti-rat RT1n (Brown Norway MHC class I, clone MCA-156, Serotec, UK) mAb was applied. Samples were preincubated with purified anti-rat CD-32 (Fc␥II block receptor) antibody and next with 5 L of RT1n mAb for 30 minutes in 4°C. After washing samples were stained with goat anti-mouse fluorescein isothiocyanate conjugated immunoglobulin G (IgG) (rat adsorbed; Serotec) and followed was incubated with CD4-PE or CD8-PE antibodies. Negative controls included isotype-matched antibodies (IgG1/ IgG2) and PBS incubated samples. FC analyses were performed on
1 ⫻ 104 mononuclear cells by using FACS Scan (BD Pharmingen) and CellQuest software.
Statistical Analysis The survival time in treatment group was evaluated by the KaplanMeier method. The level of chimerism and efficacy of the immunosuppressive treatment was compared by independent samples t test; P ⬍ .05 was considered significant.
RESULTS The Survival of the Skin Allografts
The survival of skin allografts for each experimental group is presented in Table 1. The combined protocol of ␣-TCR/ CsA applied to the recipients of skin allografts without “crude” bone marrow significantly extended allograft survival when compared to the allograft controls without treatment (P ⬍ .05). However, following cessation of ␣TCR/CsA therapy all allografts were rejected within 20 days (P ⬍ .05). Monotherapy with CsA alone extended skin
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transplant survival over 7 days, whereas monotherapy with ␣-TCR alone resulted in rejection at the same time as rejection controls without treatment. When skin allograft transplantation was augmented by “crude” bone marrow transplantation, mean survival time of skin allograft transplants was extended over 68 days (range between 65 and 71 days; P ⬍ .05) after cessation of ␣-TCR/CsA protocol. Evaluation of “Crude” Bone Marrow
The weight of the scooped “crude” bone marrow taken from three LBN control rats was found to be in the range from 53 to 60 mg. BMCs, isolated from the “crude” bone marrow contained 28 ⫻ 106 to 33 ⫻ 106 viable cells. FC analysis showed that 75% of nucleated cells express CD90⫹ antigen specific for early progenitor cells. Double staining for CD90/CD4, CD90/CD8, and CD90/␣-TCR T lymphocytes revealed less then 1% of double-positive CD90/CD4, CD90/CD8, and CD90/␣-TCR cells. Double-positive staining for CD90/CD45RA revealed 2.9% to 4.5% of B lymphocytes to be present within whole bone marrow.
Flow Cytometry Analysis of in Vivo Depletion
FC determination of ␣-TCR expression on the lymphocytes harvested from the ␣-TCR/CsA treatment controls without skin graft and crude bone marrow transplantation and from experimental animals showed ⬎90% depletion of the ␣-TCR-positive cells in the periphery at day 7 and subsequent repopulation of the ␣-TCR⫹ cells 7 days after treatment cessation (day 42). The pretransplant level of ␣-TCR⫹ cell populations was observed 28 days after cessation of the immunosuppressive therapy (day 63; Fig 2).
SIEMIONOW, OZER, IZYCKI ET AL
Determination of the Donor-Specific Chimerism
Donor-specific chimerism was evaluated at days 0, 7, 21, 35, and 63 after transplantation in the peripheral blood of “crude” bone marrow and skin allograft recipients. Flow cytometry results at day 63 posttransplant demonstrated presence of the donor-specific chimerism ranging from 8% to 10% (RT1n-positive cells). Examination of two-color stained RT1n-FITC/CD4-PE and RT1n-FITC/CD8-PE peripheral lymphocytes revealed 3.1% and 4.6% (respectively) of double-positive T-cell subpopulations (Figs 3A, 3B). DISCUSSION
The functional results of the first series of human hand transplantations are encouraging.9 However, clinical composite tissue allotransplantation requires lifelong immunosuppression, which exposes the patients to the serious side effects. One possible approach of tolerance induction during hand transplantation would be simultaneous transplantation of the bone marrow from the hand donor. Current protocols of bone marrow transplantation require introduction of recipient conditioning regiments. The use of HSC therapy in organ transplantation is considered to promote chimerism with the aim of enhancing organ tolerance. It is now widely accepted that high doses of HSC infused in the absence of recipient irradiation do not induce GvHD.4 The first successful experiments of combined use of antilymphocyte serum (ALS) and posttransplant infusion of the donor bone marrow resulted in donor-specific unresponsiveness were performed by Wood et al.10 Studies revealing the importance of stromal cells and microenvironment in the induction of donor-specific tolerance indicated
Fig 2. FC evaluation of the peripheral blood ␣-TCR-positive cells. FC analysis at day 7 demonstrated ⬎90% depletion of the ␣-TCR⫹ cells and gradual reconstitution at day 63 in transplanted and control groups under ␣-TCR/CsA protocol. The analysis of the expression of the ␣ TCR⫹ cells in the peripheral blood of naive animals served as a control.
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Fig 3. Flow cytometric determination of the donor-originated RT1n expression on (A) CD4⫹ and (B) CD8⫹ T-cell subpopulations isolated from peripheral blood of recipients showed double-positive RT1n⫹/CD4⫹ (3.1%) and RT1n⫹/CD8⫹ (4.6%) chimeric cells subpopulation at 63 days after skin and “crude” bone marrow allograft transplantation. (C) No positive cells were found in isotype control.
that hematopoietic reconstitution following total lymphoid irradiation in rats is faster in recipients of vascularized bone marrow transplantation when compared to the recipients of cellular bone marrow transplantation.11 These findings indicate that stromal microenvironment is essential for the proliferation and differentiation of the hematopoietic progenitors. Bone marrow stromal cells play a critical role in formation of the hematopoietic microenvironment and support hematopoietic stem cell differentiation through the intercellular contact and secretion of various cytokines and growth factors.12,13 The hematopoietic microenvironment, which is preserved by transplantation of the “crude” bone marrow, allows for engraftment of hematopoietic cells at the site of transplantation. In our study, direct transplantation of the donor bone marrow in the “crude” form into the recipient’s bone marrow cavity allowed for successful bone marrow engraftment and provided natural matrix for subsequent cell repopulation and trafficking. The effectiveness of engraftment was confirmed by flow cytometry revealing 3.1% and 4.6% of the donor-specific chimerism for the RT1n-FITC/ CD4-PE and RT1n-FITC/CD8-PE, respectively, in the peripheral blood of recipients at day 63 after cessation of immunosuppression. Significantly extended skin allograft survival was achieved only in the animals receiving “crude” bone marrow in combination with short-term ␣-TCR mAb/CsA protocol. In our pilot study as well as in the current study we have found that chimerism and extended allograft survival was achieved only when the weight of transplanted crude bone marrow exceeded 50 mg. In this study we have confirmed that 50 mg of “crude” bone marrow contained 28 ⫻ 106 to 33 ⫻ 106 of the viable cells, and this correlated with higher chimerism level in the recipients of “crude” bone marrow transplant when compared to controls. These findings are
parallel to Monaco and Wood’s experiments in which they found a direct correlation between the amount of bone marrow infused and the level and duration of chimerism achieved. They concluded that 25 ⫻ 106 of the bone marrow cells were a critical to induce tolerance in ALS-treated skin-grafted mice.14 The other important component contributing to the extension of allograft survival is the specific blockage of TCR using ␣-TCR mAb. Treatment against the rat ␣TCR mAb has been shown previously to abrogate the experimentally induced adjuvant arthritis,15 prolonged cardiac allograft survival,16 and abrogated concordant cardiac xenograft rejection in a hamster-to-rat model.17 It has also been indicated that combination of ␣-TCR/CsA may exert clinically relevant and potentially important additive/synergistic in vivo effects. This is parallel to our findings in hind limb allograft model, which confirmed that a short protocol of combined ␣-TCR/CsA treatment resulted in tolerance induction (750 days).18 Timing of the initial injection of the antibody as well as bone marrow transplantation is also important for the clinical applicability of these treatment protocols. Recipient conditioning before transplantation was found to be more effective than posttransplant treatment.19,20 In this study, we have introduced ␣-TCR/CsA protocol 12 hours before transplantation of bone marrow and skin grafts to make this protocol clinically applicable. As a result significant extension of skin allograft survival was achieved without chronic immunosuppression. In conclusion, in this study we have introduced a new concept of the “crude” bone marrow transplantation directly into the recipient bone for optimal engraftment, repopulation, and chimerism induction. This minimally invasive, nonmyeloablative protocol of bone marrow transplantation may have direct clinical application in composite tissue and solid organ transplants.
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REFERENCES 1. Petit F, Minns AB, Dubernard JM, et al: Composite tissue allotransplantation and reconstructive surgery: first clinical applications. Ann Surg 237:19, 2003 2. Billingham RE, Brent L, Medawar PB: Quantitative studies on the tissue transplantation immunity. III. Actively acquired tolerance. Philos Trans R Soc Lond (Biol) 239:357, 1956 3. Ricordi C, Karatzas T, Nery J, et al: High-dose donor bone marrow infusions to enhance allograft survival: the effect of timing. Transplantation 63:7, 1997 4. Ciancio G, Miller J, Garcia–Morales RO, et al: Six-year clinical effect of donor bone marrow infusions in renal transplant patients. Transplantation 71:827, 2001 5. Sharabi Y, Sachs DH: Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen. J Exp Med 169:493, 1989 6. Sykes M, Szot GL, Swenson KA, et al: Induction of high levels of allogeneic hematopoietic reconstitution and donor specific tolerance without myelosuppressive conditioning. Nat Med 3:783, 1997 7. Wekerle T, Nikolic B, Pearson DA, et al: Minimal conditioning required in a murine model of T cell depletion, thymic irradiation and high-dose bone marrow transplantation for the induction of mixed chimerism and tolerance. Transpl Int 15:248, 2002 8. Billingham RE, Medawar PB: The technique of free skin grafting in mammals. J Exp Biol 28:385, 1951 9. Jones JW, Gruber SA, Barker JH, et al: Successful hand transplantation. One-year follow-up. Louisville Hand Transplant Team. N Engl J Med 343:468, 2000 10. Wood ML, Monaco AP, Gozzo JJ, et al: Use of homozygous allogeneic bone marrow for induction of tolerance with ALS: dose and timing. Transplant Proc 3:676, 1971
SIEMIONOW, OZER, IZYCKI ET AL 11. Janczewska S, Ziolkowska A, Durlik M, et al: Fast lymphoid reconstitution after vascularized bone marrow transplantation in lethally irradiated rats. Transplantation 68:201, 1999 12. Quesenberry PJ, Crittenden RB, Lowry P, et al: In vitro and in vivo studies of stromal niches. Blood Cells 20:97, 1994 13. Bianco P, Riminucci M, Gronthos S, et al: Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19:180, 2001 14. Monaco AP, Wood ML: Studies on heterologous antilymphocyte serum in mice. VII. Optimal cellular antigen for induction of immunologic tolerance with antilymphocyte serum. Transplant Proc 2:489, 1970 15. Yoshino S, Schlipkoter E, Kinne R, et al: Suppression and prevention of adjuvant arthritis in rats by a monoclonal antibody to the alpha/beta T cell receptor. Eur J Immunol 20:2085, 1990 16. Tsuchida M, Hirahara H, Matsumoto Y: Induction of specific unresponsiveness to cardiac allografts by short-term administration of anti-T cell receptor alpha/beta antibody. Transplantation 57:256, 1994 17. Van den Bogaerde J, White D, Roser B, et al: In vitro and in vivo effects of monoclonal antibodies against T cell subsets on allogeneic and xenogeneic responses in the rat. Transplantation 50:915, 1990 18. Siemionow M, Ortak T, Izycki D, et al: Induction of tolerance in composite-tissue allografts. Transplantation 74:1211, 2002 19. Heidecke CD, Hancock WW, Jakobs F, et al: ␣/-T cell receptor-directed therapy in rat cardiac allograft recipients. Treatment prior to alloantigen exposure prevents sensitization and abrogates accelerated rejection. Transplantation 59:78, 1995 20. Heidecke CD, Hancock WW, Westerholt S, et al: ␣/-T cell receptor-directed therapy in rat cardiac allograft recipients. Longterm survival of cardiac allografts after pretreatment with R73 mAb is associated with upregulation of Th2-type cytokines. Transplantation 61:948, 1996