R EED O. D INGMAN S OCIETY - Department of Surgery - University ...

Options for reconstruction of peripheral nerve
gaps are currently limited. In clinical practice,
long nerve gaps following injury or tumor
resection that cannot be repaired primarily are
repaired with autologous nerve grafts. This
procedure requires sacrifice of healthy nerves
with permanent functional impairments. In
some clinical situations, there may not be
sufficient autologous nerve available for
reconstruction of larger, more complex nerve
defects. As a result, some investigators and
clinicians have turned to peripheral nerve
allografting as an alternative (MacKinnon
1992). The main obstacle to widespread clinical
use of peripheral nerve allografts however has
been T cell mediated immune rejection
(Ansselin 1990). Although potent
immunosuppressive drugs are currently
available to prevent graft rejection, they are
associated with many toxicities. In addition,
chronic global suppression of the immune
system with these medications may result in
opportunistic infections or secondary
malignancies and their routine use in nerve
allografting is not justified (Elster 2000).
Therefore more selective immunomodulatory
strategies to prevent graft rejection of peripheral
nerve allografts such as induction of specific
immune tolerance will need to be developed
before this technique can be routinely used
clinically.
The CD40-CD40L co-stimulatory
pathway has been shown to play a crucial role in
allograft rejection. CD40 is a 50-kDa
membrane glycoprotein found on a variety of
antigen-presenting cells (APCs) of grafted
tissue. The ligand for this membrane receptor
is CD40L, a 39-kDa glycoprotein that is
preferentially expressed on activated CD4+ T
helper cells of the recipient. The CD40-
CD40L interaction mediates T cell immune
responses by enhancing the co-stimulatory
pathway which leads to rejection (Steurer
2001). Manipulation of the CD40-CD40L
co-stimulatory pathway may be beneficial in
preventing allograft rejection. Indeed,
T HE CD40-CD40L CO - STIMULATORY P ATHWAY
IN P ERIPHERAL N ERVE A LLOGRAFT R EJECTION
-Paul S Cederna, M.D., Anil K Mungara, M.D., Sherri Y Wood, Keith D Bishop, PhD.
consistent with its central role in cell mediated
immunity, blockade of CD40-CD40L by anti-
CD40L monoclonal antibody (mAb) has been
shown to prevent rejection of solid organ
transplants such as cardiac and renal allografts
(Kirk 1997, 1999,Larsen 1996,Kenyon 1999,
Pierson 1999).
Current thinking about the mechanisms
leading to tissue rejection following
transplantation revolve around T cell receptor
(TCR) binding to genetically disparate major
histocompatibility class II antigens (MHC II Ag)
on APCs. The binding of the TCR to the
MHC II antigen leads to release of cytokines
from the T cell that lead to rejection of the
grafted tissue. The most important cytokines
released in this process are interferon gamma
(INF- g) and interleukins (IL) 2, 4, and 5.
INF- g and IL-2 are designated as TH1
responses and mediate cellular rejection by
activating macrophages as well as helper T and
cytotoxic T cells. IL-4 and IL-5 are designated
as TH2 responses and mediate humoral
rejection by transforming B lymphocytes into
plasma cells with subsequent antibody
production.
In addition to the MHC II Ag, APCs of
the transplanted tissue also express a molecule
referred to as CD40 on their cell surface.
When T cells encounter the foreign MHC II Ag
of grafted tissue they become activated and
produce the ligand for the CD40 molecule,
referred to as CD40L on their cell surface.
Interaction of CD40 with CD40L is essential
for the TCR binding to the transplanted MHC
II antigen. Without this CD40-CD40L costimulatory
pathway, the T cell receptor cannot
bind to the MHC II antigen of the grafted
tissue, and cytokine production is inhibited,
averting the cellular and humoral response
cascades. This data has led many investigators to
block this pathway using a monoclonal antibody
directed against the CD40L molecule of
activated T cells in an attempt to induce
tolerance to transplanted tissue. This approach
has been shown to be highly effective in
12 R E E D O . D I N G M A N S O C I E T Y
preventing rejection in cardiac and renal
transplantation models (Kirk 1997, Pierson
1999). However, induction of tolerance to
nerve allografts using this method has yet to be
determined.
In previous work from our laboratory, we
have used sciatic nerve grafting between
genetically disparate strains of mice such as
BALB/c and C57BL/6 as a model of peripheral
nerve allografting. Sciatic nerves from BALB/c
donor mice are placed into a subcutaneous
pocket in the back of recipient C57BL/6 mice.
Following nerve grafting and post-op recovery,
we harvest splenocytes and brachial lymph node
cells and expose these cells to the alloantigens
from the peripheral nerve graft donor.
Cytokine production (INF- g, ILs-2, 4, and5)
by these cells is then measured by the ELISPOT
technique as a measure of cellular rejection.
Humoral rejection is evaluated by measuring
serum IgM and IgG levels by mean channel
fluorescence. In this model, we have found that
treatment of mice with a 3 day course of anti-
CD40L mAb at the time of nerve allografting
significantly reduces both T cell mediated TH1
and TH2 responses as well as alloantibody
production. The reduction in cytokine
production is demonstrated in T cells isolated
from the spleen as well as the draining lymph
node basin. In addition, this hyporesponsiveness
may be tissue specific as
rechallenge with nerve allograft maintains an
attenuated response while rechallenge with
cardiac allografts results in a more dramatic
response.
In cardiac and renal allograft models, the
reported timing and duration of the anti-
CD40L mAb is quite variable. When
treatment is delayed until 5 days after surgery
in the mouse model of cardiac
transplantation, no prolongation of graft
survival has been observed (Larsen 1996).
Because sensitization to nerve allografts occurs
within the first several weeks following
transplantation, coverage of the host with
anti-CD40L mAb at least during this period