CD40 Costimulation Blockade in Limb and Nerve Transplantation
Thomas H. Tung, MD, Susan E. Mackinnon, MD.
Introduction: Hand and other nonvital transplantation procedures are controversial because of the
risks associated with long-term immunosuppression and the likelihood of chronic allograft
rejection. Recent investigation has focused on antibody-based therapies such as CD40
costimulation blockade that can reliably induce tolerance in experimental models of organ
transplantation. This strategy has not been previously applied to composite tissue transplantation
and may facilitate the universal acceptance of nonvital transplantation. This essay summarizes
the current understanding of costimulation theory and our study of the efficacy of CD40 pathway
blockade in composite tissue transplantation.
Methods: Whole limb transplants were performed in genetically disparate mice. Balb/c mice (H2Kd) (Jackson Laboratory, Bar Harbour, ME) were recipients of limb allografts from C57Bl/6
mice (H-2Kb)inset in a heterotopic subcutaneous position in the groin. Recipients received no
treatment or 500 μg MR1 (hamster anti-mouse anti-CD40L mAb; Bio Express, Inc., West
Lebanon, NH) administered by intra-peritoneal (IP) injection on days 0, 2, 4, 6, 14, 28 and 60.
Another group received vascularized myocutaneous allografts consisting of gracilis muscle and
groin skin, and received the same MR1 therapy. The purpose of this group was to study the role
of the bone marrow component of the limb allograft.
The efficacy of anti-CD40L mAb was also studied in the murine nerve allograft model.
C3H and C57BL/6 mice were chosen for full MHC-disparity. The study groups were composed
of C3H recipients of nerve allografts from C57BL donors. The animals received either no
treatment or anti-CD40L antibody (MR1, TSD Bioservices, Newark, DE) both topically to the
graft and by IP injection on days 1, 3, 10, 18, and 28. Half of the animals in each group were
randomly selected for sacrifice at three weeks post-engraftment, and the remaining animals were
sacrificed at six weeks post-engraftment.
Four genetically mismatched pairs of monkeys (Macaca fascicularis) received fivecentimeter ulnar nerve allografts. Treated animals received 10mg/kg of anti-CD40L mAb [IDEC
Pharmaceuticals Corp., San Diego, CA] directly applied to the graft, and subsequent intravenous
injections of 20mg/kg on post-op days 0, 1, 3, 10, 18, and 28, and monthly thereafter for a total
of 6 months. Untreated allografts served as negative controls and autografts served as positive
controls. Two of the treated and both untreated animals received 1cm2 skin auto-and allografts
(SG) to allow monitoring of the rejection response. Electrophysiologic testing of the nerve grafts
was performed at 6 months postoperatively, and the specimens were then harvested for
histomorphometric analysis.
Results: The transplantation of whole limb and nerve allografts was performed in experimental
models across a major histocompatibity barrier. In a newly developed model of heterotopic limb
transplantation in the mouse, therapy with anti-CD40 ligand (L) antibody produced long-term
survival (>300 days) of whole limb allografts with differential rejection of the skin component.
The bone marrow component appeared to play a significant role in prolonging allograft survival..
In the murine and non-human primate models of nerve allotransplantation, anti-CD40L antibody
allowed axonal regeneration through nerve allografts across a long gap comparable to that seen
in iso- and autografts.
Conclusions: These findings suggest that CD40 blockade may significantly reduce the morbidity
associated with immune modulation for successful composite tissue allotransplantation. This
strategy has significant potential as the basis of combined regimens for the induction of donorspecific tolerance and could eliminate the possibility of chronic rejection in the long term. As
single-agent therapy, CD40 blockade produces immune unresponsiveness that is well suited to
the temporary immunosuppression required for nerve allograft transplantation.
Table 1. Murine Limb Allograft Study
Group
1. Allogeneic Limb
Transplant
Treatment
Survival
None
8, 9, 9, 10, 11 days
2. Allogeneic Limb
Transplant
MR1 500 g IP on
days 0,2,4,6,14,28,60
3. Allogeneic Skin/Muscle
Transplant
MR1 500 g IP on
days 0,2,4,6,14,28,60
Skin component:
90, 102, 82, 59, 40 days
Musculoskeletal component:
>300, >300, 240, 150, 120 days
15, 18, 19 days
Fiber Count
Fiber Width (m.)
2000
1500
1000
500
0
4
3
2
1
0
21 days
42 days
21 days
2
Neural Density (fibers/mm )
Percent Nerve
20
30000
15
20000
10
10000
5
0
42 days
0
21 days
Isograft
42 days
Anti-CD40L-treated Allograft
21 days
42 days
Untreated Allograft
Figure 1. Murine Nerve Allograft Study: Histomorphometric data including nerve fiber count, fiber
width, neural density and percent nerve, demonstrate axonal regeneration in anti-CD40L mAb-treated
allografts comparable to isografts. No regeneration seen in untreated allografts at 21-day timepoint.
Primate Nerve Allograft Study: Nerve Graft Fiber Count
#
mAb
SG
Prox. auto
Mid auto
Prox. allo
Mid allo
1
(+)
(-)
13722
22762
2642
362
2
(+)
(-)
13324
15665
9920
1051
3
(+)
(-)
7200
6300
7526
9417
4
(+)
(-)
9105
9584
8034
6212
5
(+)
(+)
6328
4783
0
0
6
(-)
(+)
1429
322
0
0
7
(-)
(-)
4553
2217
n/a
n/a
8
(-)
(-)
7402
7231
n/a
n/a
9
(+)
(+)
n/a
n/a
0
0
10
(-)
(+)
n/a
n/a
0
0
Table 2. Primate Nerve Allograft Study: Summary of histomorphometric data (fiber count)
demonstrating good axonal regeneration in anti-CD40L mAb-treated allografts (without concomitant skin
allograft) versus no regeneration in allograft recipients that were either untreated or treated but received a
skin allograft.