Anti-CD3 prevents factor VIII inhibitor development in hemophilia A mice by a regulatory CD4+CD25+-dependent mechanism and by shifting cytokine production to favor a Th1 response

Non-Fc-receptor binding anti-CD3 Ab therapy, in the setting of several different autoimmune disorders, can induce antigen-specific and long-lasting immunologic tolerance. Because factor VIII (FVIII) inhibitor formation is the most serious treatment-related complication for hemophilia A patients, we tested the efficacy of anti-CD3 to prevent FVIII inhibitor formation in hemophilia A BALB/c and C57BL/6 mice. A short course of low-dose anti-CD3 significantly increased expression of CD25 and the proportion of CD4(+)CD25(+) regulatory T cells in the spleen and potently prevented the production of inhibitory and non-neutralizing anti-FVIII antibodies in both strains of mouse. Depleting the CD4(+)CD25(+) cells during anti-CD3 therapy completely ablated tolerance to FVIII. Further phenotypic characterization of regulatory cells in tolerant mice showed a consistently higher number of CD4(+)GITR(+) and CD4(+)FoxP3(+) cells in both strains of mice. In addition, in tolerant C57BL/6 mice we observed an increase in CD4(+)CD25(+)CTLA-4(+) and CD4(+)CD25(+)mTGF-beta 1(+) cells. Finally, in vitro cytokine profiling demonstrated that splenocytes from tolerant BALB/c and C57BL/6 were polarized toward a Th1-immune response. Taken together, these findings indicate that anti-CD3 induces tolerance to FVIII and that the mechanism(s) regulating this response almost certainly occurs through the generation of several distinct regulatory T-cell lineages and by influencing cytokine production and profile. (Blood. 2009; 113: 193-203)