Title: Induction Of Tolerance To Composite Tissue Allografts In The
Rat With Short-Term Administration Of Deoxyspergualin And
Anti-T Lymphocyte Monoclonal Antibody
Authors:
Fabio Quatra, MD, Oreste M. Romeo, MD, Dave Lowenberg, MD,
Darrell Brooks, MD, Lee Ann Baxter-Lowe, PhD, Michele R.
Colonna, MD, Francesco Stagno d'Alcontres, MD, and Harry J.
Buncke, MD
Short-term combination therapy with an anti-TcR alpha/beta monoclonal
antibody (clone R73) and 15-deoxyspergualine (DOS) was tested in
inducing long-term survival of composite tissue allografts.
Materials and Methods: Hindlimbs were transplanted across a fully
mismatched MHC barrier (RT1-1 / RT1-n) from Brown Norway to Lewis
rats. Anaesthesia was obtained with Sodium Pentobarbital 30-40 mg/Kg
i.p., perioperative analgesia with Butorphanol 1 mg/Kg i.p q6hrs.
Six groups were entered: Group A was a no-treatment control group.
Group B received R73 at 500 mcg/Kg i.p on the day of surgery. Group C
received R73 (250 mcg/Kg i.p.) on the day of surgery and DOS (2.0
mg/Kg i.p.) for 15 days starting on the day of surgery. Group D
received R73 (500 mcg, day of surgery) and DOS (4.0 mg/Kg i.p) for 20
days. Group E and F received a sham operation (limb amputation and
replant) respectively with or without a 15-day treatment with DOS, to
record weight loss, drug-related toxicity and nerve regeneration.
Onset of rejection was assessed clinically. Long-term survivors
received a skin graft from a new Brown-Norway donor and from a thirdparty, unrelated strain (Dark Agouti) on postop day 60, to assess
donor-specific tolerance. Tissues were harvested for histology and
blood drawn for assessment of chimerism with flow cytometry. Red
blood cells were removed by a gradient of sedimentation (Lympholyte®,
Cederlane Lab, Ontario). After centrifugation for 20 min at 1500 rpm,
the pellet was resuspended, washed, filtered and cells counted with a
fluorescent microscope after staining with acridin orange. About
500,000 cells were washed in Phosphate Buffered Solution and double
labeled with an anti CD34 monoclonal antibody (FITC conjugated) as a
marker for stem cells and an anti T-cell receptor antibody (PE
conjugated) as a marker for mature T-lymphocytes (both antibodies
from Cederlane Lab., Ontario) and counted on a Beckton-Dickinson
FACSort flow cytometer.
Results: Rejection became evident for groups A-C as follows: Group
A=6-8 days, Group B=12-13 days, Group C=22-25 days, while limbs in
Group D survived over 100 days (Fig.1). Weigh loss during the
treatment period peeked to 20% of preoperative weight, without
histological signs of Graft versus Host Disease. Skin grafts from the
same donor strain remained viable while grafts from the third party
were rejected within 10 days (Fig.1). Flow citometry analysis
suggested a minimal number of T-cells in the peripheral blood, less
than 1%
Conclusions: Combination therapy with R73/DOG showed to reliably
prevent rejection in all the animals during the treatment period
although a significant weight loss was observed. Treatment extended
to 20 days was able to prolong survival well beyond DOS withdrawal,
up to the 100-day scheduled endpoint, with persistent donor-specific
tolerance.
Fig.1: A limb without clinical signs of rejection at Post-Operative
day 72.
Fig. 2: Flow cytometry analysis of peripheral blood from rat #27
showing minimal amount of donor-derived cells (upper and lower right
quadrants).
References
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