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

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J.G. Gilles et al.
Table 1. The capacity to inhibit 50% recombinant or plasma-derived
FVIII function by the serum of mice reconstituted
with PBL of hemophilia A patients with
inhibitors is similar to the capacity of the purified anti-
FVIII Ab fraction from the corresponding hemophiliacs.
Table 2. The isotypic distributions of total IgG and anti-
FVIII Ab produced in SCID mice are identical to total IgG
and anti-FVIII Ab produced by healthy source donors,
except for the percentage of the IgG4 isotype which is
significantly increased.
rec FVIII
IgG (µg/mL)
purified FVIII
IgG (µg/mL)
IgG subclasses (%)
IgG1 IgG2 IgG3 IgG4
Patients’ total IgG 100 100
Patients’ specific IgG (immunopurified) 0.5 0.5
Mice’s serum IgG (8) 0.16-2 0.12-1.5
results show that the SCID mice can be used to
study a human anti-FVIII secondary immune
response in vivo. As several mice can be reconstituted
with cells from a single patient, this model
should allow for direct comparative studies of the
immunogenicity of different FVIII preparations.
SCID mice and healthy donors
The presence of antibodies directed towards
self-antigens in the serum of healthy individuals
has already been demonstrated in a number of
situations. 11 One of the most representative
examples of this is the presence of anti-factor
VIII antibodies in the serum of healthy donors. 12
Several studies have demonstrated the presence
of such antibodies, which include not only
non-functional anti-FVIII antibodies but also
high-affinity antibodies able to inhibit FVIII function
with the same efficiency as antibodies produced
by hemophilia A patients under FVIII infusion
therapy. Natural auto-antibodies (NAA) are
only detectable after passing a total IgG fraction
through a specific FVIII immunosorbent.
The specificity of the purified antibodies was
confirmed by their capacity to recognize soluble
and insolubilized FVIII. Epitope mapping using
monoclonal mouse anti-FVIII antibodies
demonstrated that in most cases the A3 and the
C2 domains of the FVIII light chain were recognized,
while the reactivity towards heavy chain
epitopes differed from one patient to another.
The reason why FVIII cofactor activity is not
inhibited is still conjectural, but could be in part
due to the presence of anti-idiotypic antibodies.
The latter have indeed been detected in healthy
donor's plasma and in a larger amount in the
plasma of hemophilia A patients who had developed
inhibitors and were successfully desensitized
by administration of high doses of FVIII. 13
In an attempt to further characterize NAA to
FVIII and to study the conditions under which
auto-immunity to FVIII can develop, SCID mice
were reconstituted with immunocompetent cells
Total IgG
Healthy donors 59.4±4.2 30.0±4.3 5.1±1.6 5.2±1.7
SCID mice 63.4±7.1 28.7+6.6 4.0±1.2 3.9±2.4
Affinity-purified antibodies
Healthy donors 52.9±3.3 39.9±3.8 3.0±0.7 4.4±1.1
SCID mice 62.0±6.6 28.2±6.4 4.0±1.0 5.7±2.7
Normal distribution 70 20 8 2
of human origin, namely peripheral blood
mononuclear cells of unrelated healthy blood
donors. The production of human anti-FVIII
antibodies was detected within a month following
reconstitution. Specific and functional antibodies
were undetectable in mouse plasma, as
was the case in healthy donors, 12 and therefore
affinity purification on a FVIII immunosorbent
had to be carried out. Importantly, administration
of human FVIII did not increase the production
of such NAA in comparison with salinetreated
mice. This spontaneous production of
anti-FVIII antibodies in mice indicates that tolerance
towards FVIII had been transferred with
cell reconstitution. A thorough analysis showed
that the isotypic distribution (Table 2) and the
capacity to inhibit FVIII of antibodies produced
in SCID mice was identical to that of antibodies
produced by healthy source donors (Figure 1).
Moreover, immunoprecipitation experiments
with recombinant FVIII fragments showed that
the specificity of the anti-FVIII antibodies was
restricted to the light chain and more specifically
to the C1 and C2 domains. Binding of antibodies
to the heavy chain could however not be
totally excluded but major epitopes were located
in the light chain. A prominent mechanism of
FVIII inhibition by these antibodies is identical to
that observed with inhibitors produced by hemophilia
patients, namely prevention of FVIII binding
to phospholipids. Since antibodies of interest
can be found in the SCID mouse model and,
moreover, since they are qualitatively comparable
to the source donor's antibodies, the model
provides appropriate experimental conditions to
study the regulation of tolerance against FVIII in
normal subjects.
Haematologica vol. 85(supplement to n. 10):October 2000