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with immature DCs results in differentiation to tolerance- promoting regulatory T cells (Tregs)7 or T-cell anergy.8 DC maturation is induced by pro-inflammatory stimuli (e.g., inflammatory cytokines, engagement of pattern recogni- tion receptors), and as such the “decision” regarding immunologic tolerance to FVIII may depend on whether pro-inflammatory stimuli are present during a patient’s initial exposure to FVIII.
Inhibitor risk might be reduced by avoiding pro-inflam- matory stimuli during initial exposures to FVIII.9 Patients whose first exposure is in the context of prophylactic rather than on-demand therapy may have a lower inhibitor risk.10,11 However, it is not always possible to choose the conditions of first exposure to FVIII, since bleeding that requires treatment may occur before the ini- tiation of prophylaxis. Avoiding FVIII exposure in the presence of other clinically-defined pro-inflammatory stimuli (e.g., febrile illness, vaccines, tissue injury) has been suggested to reduce inhibitor risk in an observational study,9 but these results have not been reproduced. Furthermore, this approach may be difficult to implement,11 making passive avoidance of innate immune stimulation impractical and ineffective.
Active pharmacologic suppression of inflammatory sig- nals during initial FVIII exposure would be a more control- lable strategy. However, the pro-inflammatory signals responsible for FVIII immunogenicity in HA have not been conclusively identified and therefore cannot be specifically targeted. Glucocorticoids, which affect both innate and adaptive immunity, may mediate the suppres- sion of a variety of pro-inflammatory signals and their immunological consequences.12,13 Therefore, glucocorti- coids such as dexamethasone (Dex), are attractive candi- dates for the suppression of inflammatory danger signals in the context of HA inhibitor development.
To test the ability of Dex to promote immunologic tol- erance to FVIII and investigate possible mechanisms of action, we used two murine models of HA. The first model is a severe HA mouse (knockout of exon 17 of the f8 gene) in which the murine MHCII loci were replaced with a single transgene for a chimeric human/murine MHCII allele (E17KO/hMHC). Approximately 30% of these mice develop antibodies to human FVIII after repeated exposure,14 suggesting that tolerance is possible, and perhaps inducible, in this model. The second model is a conventional severe HA mouse (knockout of exon 16 of the f8 gene) in which recombinant human FVIII exposure is immunogenic in 100% of animals (E16KO).15
We first hypothesized that E17KO/hMHC mice treated with Dex during an intense initial exposure to FVIII that did not subsequently develop anti-FVIII IgG would, on re- exposure to FVIII, be less likely to develop anti-FVIII IgG than would anti-FVIII IgG-negative mice that were initial- ly treated with FVIII alone. We then sought to determine if our treatment protocol could attenuate the anti-FVIII immune response in E16KO mice and investigate poten- tial cellular mechanisms of action.
Methods
Animals
E17KO/hMHC. HA mice with all murine MHCII alleles knocked out and expressing a single chimeric human/murine transgene of the HLADRB1*1501 allele on a mixed C57Bl6/S129
background. Male mice aged 10-14 weeks were used.14
E16KO. HA mice on a homogeneous C57Bl6 background. Mice were sex-matched across treatment groups and eight weeks of age.16 All animal procedures were in accordance with the Canadian Council on Animal Care guidelines and approved by the
Queen’s University Animal Care Committee.
Intermittent and final blood samples were obtained via retro- orbital plexus and cardiac puncture respectively, then mixed in a 1:10 ratio with 3.2% buffered citrate. Plasma was separated by centrifugation, then stored at -80°C.
Short-term treatment protocol
Initial exposure. At week zero, E17KO/hMHC or E16KO mice received FVIII and Dex (FVIII+Dex group) or FVIII alone (FVIII group) for five consecutive days (Figure 1A,B). At week five, blood samples were collected.
Re-exposure. FVIII and FVIII+Dex E17KO/hMHC mice with no evidence of anti-FVIII IgG following initial exposure received FVIII (FVIII/FVIII group and FVIII+Dex/FVIII group), or FVIII and lipopolysaccharide (LPS; FVIII/FVIII+LPS group and FVIII+Dex/FVIII+LPS group) for three consecutive days (week six, Figure 1A). At week nine, blood samples were collected.
Long-term treatment protocol
Initial exposure. E17KO/hMHC mice received FVIII and Dex (FVIII+Dex group) or FVIII alone (FVIII group) for five consecutive days (week zero, Figure 4). At week four, all mice were sampled.
Intermittent low-dose FVIII exposure and re-exposure. FVIII+Dex mice with no evidence of anti-FVIII IgG were divided into two groups. One group received FVIII for three consecutive days at week 16 (FVIII+Dex/FVIII group). The other group received inter- mittent exposures to low-dose FVIII (2IU/dose at weeks four, eight and 12) followed by FVIII (6IU/dose) for three consecutive days at week 16 (FVIII+Dex/intFVIII+FVIII group). FVIII mice with no evidence of anti-FVIII IgG received FVIII for three consecutive days at week 16 (FVIII/FVIII group). All mice were sampled before (week 14) and after (week 18) FVIII re-exposure.
Human VWF exposure. Mice received human plasma-derived VWF once weekly at weeks 18 to 21. At week 22, blood samples were collected.
Anti-FVIII IgG ELISA
Anti-FVIII IgG titers were measured via enzyme-linked immunosorbent assay (ELISA) as previously described.17 An opti- cal density (OD) cutoff of 0.3 above the OD490 of the blank sample was the criterion for positivity, and the titer was determined to be the highest dilution at which a given sample was positive. Samples with an OD490 below the cutoff at a 1:40 dilution were considered to have non-detectable anti-FVIII IgG.
Bethesda assay
FVIII inhibitory activity was measured via Bethesda assay as previously described.18 Residual FVIII activity was quantified using an automated coagulometer (STA Compact, Stago). Inhibitory activity was calculated only for samples with a residual FVIII activ-
Treatment dosing and blood sampling
Dex (Omega) (75μg/dose, ~3mg/kg) was administered intraperitoneally (IP). Recombinant human FVIII (Advate; Baxalta) (6IU/dose, ~240IU/kg unless stated otherwise), lipopolysaccharide (LPS; InvivoGen) (2μg/dose, ~8mg/kg) and ultra-pure plasma- derived human von Willebrand Factor (VWF; Biotest) (2IU/dose, ~80IU/kg) were administered intravenously (IV), via tail vein. Hank’s balanced salt solution (HBSS) was administered as vehicle control at 100μl IP and 250μl IV.
haematologica | 2018; 103(8)