Page 172 - Haematologica August 2018

M.T. Georgescu et al.
Administration of Dex during initial FVIII exposure induces tolerance to FVIII in E17KO/hMHC mice, even when co-administered with LPS
Next, we wanted to determine whether E17KO/hMHC mice that did not develop anti-FVIII IgG after initial expo- sure would be immunologically tolerant upon re-exposure to FVIII. Anti-FVIII IgG negative mice from both the FVIII and FVIII+Dex groups were re-exposed to FVIII alone or in combination with LPS (week 6, Figure 1A).
At week nine, 7% of FVIII+Dex/FVIII mice compared to 52% of FVIII/FVIII mice had evidence of plasma anti-FVIII IgG (P=0.0050, Figure 3A). The single FVIII+Dex/FVIII mouse with evidence of anti-FVIII IgG had a lower titer than most of the FVIII/FVIII mice (Figure 3B). Furthermore, 0% of FVIII+Dex/FVIII mice compared to 35% of FVIII/FVIII mice showed evidence of FVIII inhibitors by Bethesda assay (P=0.0132, Figure 3C,D).
At week nine, 100% of FVIII/FVIII+LPS mice compared to 30% of FVIII+Dex/FVIII+LPS mice had evidence of anti-FVIII IgG (P=0.0699, Figure 3E). FVIII+Dex/FVIII+LPS mice also had a trend towards lower anti-FVIII IgG titers than FVIII/FVIII+LPS mice (P=0.1536, Figure 3F). Furthermore, 20% of FVIII+Dex/FVIII+LPS compared to 100% of FVIII/FVIII+LPS mice (P=0.0150, Figure 3G) had evidence of FVIII inhibitors and FVIII+Dex/FVIII+LPS mice had lower FVIII inhibitor levels than FVIII/FVIII+LPS (Figure 3H). These data suggest that Dex can promote per- sistent tolerance to FVIII in the E17KO/hMHC murine
model of HA, and that this tolerance is robust enough to withstand re-exposure to FVIII when co-delivered with LPS, a potent adjuvant.
Administration of Dex during initial FVIII exposure induces durable, antigen-specific tolerance to FVIII in E17KO/hMHC mice
We next sought to investigate the durability of Dex- induced tolerance to FVIII, using the long-term treatment protocol described (Figure 4). At week four, 0% of FVIII+Dex E17KO/hMHC mice compared to 64% of FVIII E17KO/hMHC mice had evidence of anti-FVIII IgG (P=0.0001, Figure 5A,B). Mice with evidence of anti-FVIII IgG at week four were excluded from the remainder of the experiment. At week 14, all remaining mice had main- tained their anti-FVIII IgG negative status. At week 18, two weeks after re-exposure to FVIII, 7% of FVIII+Dex/FVIII mice and 27% of FVIII+Dex/intFVIII+FVIII mice had evidence of anti-FVIII IgG compared to 50% of FVIII/FVIII mice (P=0.0970 FVIII/FVIII vs. FVIII+Dex/FVIII; P=0.5573 FVIII/FVIII vs. FVIII+Dex/intFVIII+FVIII; P=0.3295 FVIII+Dex/FVIII vs. FVIII+Dex/intFVIII+FVIII, Figure 5C). No apparent differ- ences were seen between the titers of the few mice from each group positive for anti-FVIII IgG (Figure 5D). These data indicate that administration of Dex during initial FVIII exposure confers tolerance that persists for at least 18 weeks, and that ongoing intermittent FVIII exposure is
Figure 4. Long-term treatment protocol. E17KO/hMHC mice received FVIII (6IU IV) alone or in combination with Dex (75μg IP) for five consecutive days. At week four, blood was collected and plasma anti-FVIII IgG titers were measured. Mice with evidence of anti-FVIII IgG were excluded from the remainder of the study. FVIII+Dex mice with no evidence of anti-FVIII IgG received FVIII (6IU IV) for three consecutive days at week 16, or single intermittent exposures to low-dose FVIII (2IU IV) at weeks four, eight and 12, followed by FVIII (6IU IV) for three consecutive days at week 16. FVIII treated mice with no evidence of anti-FVIII IgG at week four received FVIII for three consecutive days at week 16. All mice were sampled two weeks before and two weeks after FVIII re-exposure. Plasma anti-FVIII IgG titers were determined at these time points. At week 18 mice started receiving weekly human VWF injections for four consecutive weeks. At week 22, blood was collected and plasma FVIII inhibitory activity and the presence of plasma anti-human VWF IgG were determined. VWF: von Willebrand Factor; FVIII: factor VIII; Dex: dexametha- sone; Wk: week. : anti-FVIII IgG negative mice; : anti-FVIII IgG positive mice; : injection; : blood collection.
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