AAHS ASPN ASRM - 2013 Annual Meeting - American Association ...

Perfusing with Anti-alpha-beta-T Cell Receptor Monoclonal Antibody in Composite Osteomyocutaneous Tissue
Allotransplantation Avoids Graft-versus-Host Disease in the Lethally Irradiated Recipient Rats
Institution where the work was prepared: Chung Rong Ho, Tao-yuan, Taiwan
Chung-Rong Ho, MD; Wei-Chao Huang, MD; Jeng-Yee Lin; Nai-Jen Chang; Yu-Hsuan Hsieh; Fu-Chan Wei; Chang Gung Memorial Hospital
Introduction:
Graft-versus-host disease (GvHD) can be a major hazard to the recipient in composite tissue allotransplantation (CTA). Lymphadenectomy and irradiation of
the allotransplants have been used to reduce the severity of GvHD. This study is to develop an alternative method to prevent the occurrence of GvHD by utilizing
graft perfusion with anti-alpha-beta-T cell receptor monoclonal antibody (anti-alpha-beta-TCR mab) and manipulating the bone graft of CTA in the
lethally irradiated recipient rats.
Materials and Methods:
Male (6- to 8-week old) donor Brown Norway (BN, RT1Ac) and (10- to 12-week old) recipient Lewis (RT1Al) rats weighing between 220 and 300g were used.
Lewis rats were preconditioned with 950 cGy total body irradiation (TBI) on Day-1. Osteomyocutaneous (OMC) flap weighted around 10±2.4g from the
hindlimbs were transplanted on Day0. No immunosuppressant was used. The experiment was grouped as shown in table1. In the study group (group4), the
medullary cavity of the bone was flushed with 10cc heparinized normal saline and curettaged with 22 gauge needle to remove bone marrow cells and then
perfused with pure 1mg anti-alpha-beta-TCR mab and 9cc normal saline via artery of the pedicle after flap harvest and before transplantation. Body weight
was checked weekly. Chimerism level was assessed by flow cytometry on 15, 30, 60, 90, 120, and 150 days after flap transplantation. Sections of the auricle
and the graft skin were taken for pathology on Day18. In vitro T-cell responses were evaluated by mixed lymphocyte reactivity assays.
Results:
All of the rats in group 1 died within 12 days. Syngeneic and allogeneic hindlimb OMC flap could prolong the life of the recipient rats after lethal irradiation.
The allogeneic hindlimb OMC flap transplantion group (group3) developed GvHD, and animal died around 3 weeks. The average of chimerism level in group
3 and group 4 were 98.85% and 99.77%. In contrast to group 3, the group 4 had 83% long term survival of the allotransplants and the animals didn't lose
body weight (Figure1). The flow cytometry showed engraftment of the donor hematopoietic cell with reconstitution of multilineaged hematolymphoid cells
(Figure2). In vitro mixed lymphocyte reaction assay in group 4 showed hypo-responsiveness to donor antigen, but hyper-responsiveness to third-party antigen.
Conclusion:
Allogeneic OMC flap transplantation could induce GvHD in the recipients with full myeloablative conditioning. GvHD can be prevented by the flap with antialpha-beta-TCR
mab perfusion and manipulation the bone graft. This immunomodulation can successfully induce donor-specific tolerance to CTA.
Fludarabine Facilitates the Nonmyeloablative Strategy and Creation of Mixed Chimerism to Induce Immune
Tolerance to Composite Tissue Allograft
Institution where the work was prepared: Chang Gung Memorial Hospital, Tayoyuan, Taiwan
Jeng-Yee Lin, MD; Wei-Chao Huang; Chung-Rong Ho; Fu-Chan Wei; David CC Chuang; Ming-Huei Cheng; Chang Gung Memorial Hospital
Background:
It has been proved that tolerance induction to composite tissue allotransplantation (CTA) through mixed allogeneic chimerism (MAC) is feasible in rats. Bone
marrow transplantation (BMT) to create MAC inevitably involves total body irradiation (TBI) for successful engraftment. However, TBI-based strategy for CTA
can not be justified clinically. Fludarabine can suppress lymphocyte proliferation and has been reported to reduce TBI doseage necessary for BMT engraftment.
The purpose of this study is to investigate if fludarabine can facilitate MAC to induce immune tolerance in rat CTA model with low-dose TBI. Materials and
Methods:
Thirty male (8-to 10-week old) Lewis rats (RTA1l) were equally categorized into 6 groups as recipients. Male Brown Norway (BN) rats (8-to-10 week old RTA1c)
were the donors. Recipients were irradiated with different dosages of TBI one day before BMT. Group I: 950 cGy; Group II: 600 cGy; Group III: 400 cGy; Group
IV: 200cGy; Group V: 400 cGy plus fludarabine (50mg/kg), intraperitoneally (IP); Group VI: 200 cGy plus fludarabine, IP. The recipients from group II to VI
were treated with one dose of 5 mg antilymphocyte serum (ALS), one day before BMT, cyclosporine 16 mg/kg/d from days 0 to 10 and one dose of 5 mg ALS,
10 days after BMT. Recipients were transplanted with 100 x 10^6 bone marrow (BM) cells with alpha beta-TCR+ and gamma delta-TCR+ T-cell depletion (TCD).
A hindlimb osteomyocutaneous flap allotransplantation (BN to MAC rat) were performed 28 days after BMT. The level of chimerism and multilineage were
assessed by flowcytometry 28 days after BMT and 15, 30, 60, 120, and 150 days following CTA. Rejection and graft versus host disease (GVHD) were examined
clinically and pathologically. In vitro T proliferation was assessed by mixed lymphocyte reaction assay 150 days after CTA.
Results:
Recipients in all groups were 100 % engrafted with donor BM. The level of chimerism in each group 28 days after BMT were: Group I: 98%; Group II:81 %;
Group III: 49.7 %; Group IV: 27%; Group V: 51.2%; Group VI: 41%. GVHD didn't occur after BMT with TCD, but its incidence after CTA increased with the
increasing TBI doseage . Graft acceptance rate (without GVHD) were: Group II:100%; Group III: 80%; Group IV: 20%; Group V:80%; Group VI.:60%.
Conclusion:
Partial conditioning and BMT with TCD can successfully create MAC to achieve immune tolerance in rats. Addition of fludarabine in the immunosuppression
regimen significantly increases MAC and graft tolerance rate.
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