Summary
The analysis of anti-factor VIII (FVIII) antibody-secreting cells (ASC) at different
anatomic sites provides valuable information about the nature of the anti-FVIII immune
response in hemophilic mice after treatment with human FVIII. An Elispot system is
described that is suitable for analyzing frequencies and IgG subclasses of anti-FVIII
ASC at the single-cell level. Hemophilic mice were treated with four doses of FVIII.
Anti-FVIII antibodies in blood as well as anti-FVIII ASC in spleen and bone marrow
were analyzed after each dose of FVIII and subsequently up to 22 weeks after termination
of the FVIII treatment. Anti-FVIII ASC first appeared in the spleen where they were
detectable after two intravenous doses of FVIII. Their appearance correlated with
that of anti-FVIII antibodies in blood plasma. Anti- FVIII ASC in bone marrow were
detectable after three doses of FVIII and were probably cells that initially formed
in the spleen and subsequently migrated to the bone marrow. Whereas the frequency
of anti- FVIII ASC in the spleen increased up to the fourth dose of FVIII and declined
thereafter, in the bone marrow it remained constant for up to at least 22 weeks after
the termination of the FVIII treatment. Titers of anti-FVIII antibodies in blood plasma
increased up to the fourth dose of FVIII, then remained high constantly for 14 weeks
and decreased but the antibodies were still detectable for up to at least 22 weeks
after the fourth dose of FVIII. The IgG-subclass distribution of anti-FVIII ASC was
similar in spleen and bone marrow and matched the subclasses of anti-FVIII antibodies
in blood plasma indicating that both organs contribute to circulating antibodies in
the blood.
Keywords
Hemophilia A - factor VIII - antibody-secreting cells - Elispot - animal model
