Anti-CD3 Therapy Expands the CD4⁺CD25⁺ Compartment in Hemophilia a Mice Inducing Tregs That Potently Prevent Factor VIII Inhibitor Formation

Non-Fc-Receptor binding anti-CD3 Ab therapy, in the setting of several different autoimmune disorders, can induce antigen-specific and long-lasting immunological tolerance. As FVIII inhibitor formation is the most serious treatmentrelated 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. Mice were treated intravenously with 50 μg anti-CD3 daily for 5 consecutive days. One, 8 and 15 days after the final anti-CD3 treatment spleens were harvested (n=5/time point) and total splenocytes were analyzed by flow cytometry. One day after anti-CD3 treatment the effector CD4⁺ and CD8⁺ populations decreased by >2- fold (p<0.00009), while CD4⁺CD25⁺ Treg cells increased 2.2-fold (p=0.0002) compared to untreated control mice. This was maintained at 8 days post anti-CD3 treatment, and by day 15, all T cell populations returned to pre-treatment levels. Interestingly, we observed that smaller doses of anti-CD3 were the most effective at preventing FVIII inhibitor formation. In four separate experiments this optimized dose of anti-CD3 (10 μg/d for 5 d), was administered to Balb/c hemophilia A mice, and 3 days later these mice were immunized with 0.2 μg FVIII (50 U/kg) weekly for 4 weeks. By 1 week after the final FVIII immunization, 98% of the HBSS-treated control (n = 30) and 22% of anti-CD3-treated mice (n = 33) were inhibitor positive. Twenty-seven of 33 anti-CD3-treated mice were inhibitor negative (p = 0.000065). Importantly, the mean inhibitor titer in the anti-CD3-treated mice was 1.6 ± 1.1 BU/mL compared to the HBSS-treated mice at 50.1 ± 14.9 BU/mL (p = 0.0014). We depleted CD4⁺CD25⁺ T cells in vivo to test their importance in the FVIII tolerogenic process. Hemophilia A Balb/c mice were injected intraperitoneally with anti-CD25 (clone PC61) or isotype control (Rat IgG1) on days 0 and 5. Next, mice received 10 μg/d anti- CD3 on days 3–7 and on day 10, the first of 4 weekly FVIII immunizations was begun. One week after the 4^th FVIII immunization, plasma was assessed for inhibitor formation by the Bethesda assay. Tolerance to FVIII was completely ablated in the anti-CD25/anti-CD3- treated mice (mean titer 110 ± 36 BU/mL), while the isotype control/anti-CD3-treated mice continued to show markedly reduced inhibitor levels (1.2 ± 1.9 BU/mL, p = 0.00003). To directly test the tolerogenic capacity of CD4⁺CD25⁺ population from tolerant animals, this population was cocultured, in the presence of FVIII, with splenocytes from FVIII immunized mice that were not treated with anti-CD3. Cells were either in direct contact or were physically separated by a transwell membrane. We observed a contact-dependent 30% and 20% reduction of IFN-γ and IL-10, respectively, in the coculture studies when compared to cytokine secretion by splenocytes cultured alone. The suppressive function was localized to the CD4⁺CD25⁺ compartment because coculture of CD4⁺CD25⁻ cells from tolerant animals with splenocytes caused a 6.5- and 1.9-fold increase in IFN-γ and IL-10, respectively, compared to splenocytes alone. Phenotypic characterization of regulatory cells in tolerant mice showed a consistently higher number of CD4⁺GITR⁺ and CD4⁺FoxP3⁺ cells in both strains of mouse compared to HBSS-treated control mice. Additionally, in tolerant C57Bl/6 mice we observed an increase in CD4⁺CD25⁺CTLA-4⁺ and CD4⁺CD25⁺mTGF-β1⁺ cells. 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 occurs through the generation of several distinct regulatory T cell lineages and by influencing cytokine production and profile. In light of these results, a short course of low-dose anti-CD3 treatment in patients at high risk of inhibitor development, with concomitant FVIII exposure administered prophylactically, in the absence of potentially “inflammatory” bleeding, may reduce the likelihood of inhibitor development.