Anti-CD3 Therapy Expands the CD4+CD25+ Compartment in Hemophilia a Mice Inducing Tregs That Potently Prevent Factor VIII Inhibitor Formation
Authors: Waters Braden , Qadura Mohammad , Burnett Erin , Chegeni Rouzbeh , Labelle Andrea , Thompson Patrick , Lillicrap David 
Published: April 2020

Abstract
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 &gt;2- fold (p&lt;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 4th 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.