Haematologica 2000;85:supplement to no. 10 - Supplements ...

Immune Tolerance and the Treatment of Hemophilacs with an Inhibitor 105
Figure 1. The anti-FVIII activity, evaluated in a chromogenic
assay, of purified anti-FVIII Abs from SCID
mice reconstituted with cells from healthy donors. Similar
results were obtained with the anti-FVIII Ab fractions
from the source donors. 12
SCID mice and anti-CD40 ligand
Anti-FVIII antibodies can only be produced
when efficient help is provided by specific T-cells.
This help includes 2 signals besides the production
of cytokines. The first signal is the presentation
of a FVIII peptide to the TCR in the frame
of MHC class II molecules. In the absence of a
second signal, the B-T cell interaction results in
anergy rather than activation. The second signal
is constituted by interaction of antigen-nonspecific,
accessory molecules, complementary
structures on the membrane surface. One
important accessory molecule interaction is the
binding of CD40L on T-cells to CD40. CD40L is
transiently expressed (2 to 24 hours) on helper
(CD4 + ) T-lymphocytes following early activation
(usually mediated through the TCR). 14-16 The
molecule plays an early and important role in
the interaction of these activated T-lymphocytes
with cells expressing CD40. CD40-bearing cells
include B-ymphocytes, vascular endothelial cells,
macrophages, dendritic cells, various epithelial
cell types, and fibroblasts, inter alia.
Administration of an antibody blocking the
CD40L-CD40 interaction during immunization
with protein antigens can specifically block the
antibody response to that antigen in a mouse
model. 17 Total serum Ig and antibody responses
to proteins administered outside of the window
of CD40L blockade are not affected. Preclinical
studies in a variety of autoimmune disease
models have shown that blockade of this
pathway with antibodies to CD40L can substantially
reduce symptoms of disease and, in
some cases, reduce mortality. 18-21 The antibodies
have also been used to induce long-term graft
acceptance in allogeneically mismatched transplant
settings. 22-24 The impressive activity of anti-
CD40L antibody therapy in a variety of both
antibody-and cell-mediated autoimmune diseases,
and in organ transplantation, suggests
that blockade of the CD40L-CD40 interaction
will be therapeutically useful in human disease.
This is why we have evaluated this CD40L-
CD40 interaction to block the anti-FVIII
humoral response in SCID mice reconstituted
with cells of a hemophilia A patient with
inhibitors. Preliminary experiments showed an
important decrease in the anti-FVIII antibody
production in reconstituted mice injected with
FVIII and anti-CD40L antibody when compared
to control group.
SCID-FVIII-knock-out mice
Direct detection of natural specific antibodies
in mouse serum is not possible, whereas antibodies
made by mice reconstituted with cells of
hemophilia patient with a high inhibitor titer can
be detected. There are several possible reasons
for this phenomenon. Anti-FVIII antibodies
could be neutralized by anti-idiotypic antibodies
produced in reconstituted mice or the specific
anti-FVIII antibody concentration could be too
low to be detectable in plasma. Attempts to
detect anti-idiotypic activity in mice serum were
unsuccessful, using a method described previously
for the characterization of anti-idiotypic
antibodies specific for anti-FVIII antibodies
made by healthy donors. This suggest that the
time period during which mice were followed
was too short. The fact that specific antibodies
are undetectable in mouse plasma may be related
to the close homology between murine and
human FVIII, 25 suggesting that anti-FVIII antibodies
produced in SCID mice could bind to
FVIII and thereby render the anti-FVIII antibodies
undetectable in the plasma. To avoid such a
problem, we have created SCID-FVIII-knock-out
mice by cross-breeding the two species, creating
a model in which we will be able to explore
the different conditions under which anti-FVIII
antibodies are produced.
Data obtained with SCID mice and preliminary
data generated by the SCID-FVIII-knockout
mice indicate that such models are appropriate
to study the regulation of tolerance
against self antigens in normal subjects and
could be extended to diseases such as acquired
hemophilia in which unresponsiveness to self
has been lost. These models are also suitable for
the pre-clinical evaluation of immune therapies
such as immunomodulation (e.g. by administration
of anti-CD40L antibodies) or immune
regulation, which includes the evaluation of idio-
Haematologica vol. 85(supplement to n. 10):October 2000