Page 173 - Haematologica August 2018

Dexamethasone promotes tolerance to factor VIII
not required to maintain this tolerance.
To determine whether the observed effect is antigen
specific, we injected all mice with a structurally unrelated antigen (human VWF) (week 18-21). At week 22, of the anti-FVIII IgG negative mice, 100% of FVIII+Dex/FVIII, 89% of FVIII+Dex/intFVIII+FVIII and 100% of FVIII/FVIII mice had evidence of anti-human VWF IgG (Online Supplementary Figure S1). We conclude that Dex treatment during initial FVIII exposure does not result in general immunosuppression but rather promotes antigen-specific tolerance to FVIII, and does not impair immune responses to other antigens.
At week 22, we also measured FVIII inhibitory activity. 8% of FVIII+Dex/FVIII, 8% of FVIII+Dex/intFVIII+FVIII and 50% of FVIII/FVIII mice had evidence of FVIII inhibitors (P=0.1206 FVIII/FVIII vs. FVIII+Dex/FVIII; P=0.1357 FVIII/FVIII vs. FVIII+Dex/intFVIII+FVIII; P=1 FVIII+Dex/FVIII vs. FVIII+Dex/intFVIII+FVIII, Figure 5E). No apparent differences were seen between the titers of the few mice from each group positive for inhibitors (Figure 5F).
Administration of Dex during initial FVIII exposure causes early changes in lymphocyte populations of E16KO mice
To elucidate possible cellular mechanisms of our treat- ment protocol, we determined the percentage of key lym- phocyte populations in the thymus, spleen and blood via flow cytometry. Three days after treatment, FVIII+Dex mice had a decreased percentage of both splenic (47.22% vs. 53.62%, P=0.0395) and blood (18.27% vs. 29.11%, P=0.0050) B cells (CD19+ lymphocytes) compared to FVIII
mice (Figure 6A). At this time point, no significant changes in the percentages of T cells (CD4+CD8– lymphocytes) were observed across the three tissues (Figure 6B). However, FVIII+Dex mice showed a significant increase in the percentage of thymic Tregs (CD25 and FoxP3 expressing CD4+CD8– lymphocytes, 12.06% vs. 4.73%, P<0.0010, Figure 6C). Similar trends were observed when comparing Dex and HBSS mice (Figure 6A-C). This sug- gests that Dex promotes tolerance to FVIII partly by decreasing the percentage of splenic and circulating B cells as well as skewing the distribution of lymphocytes in the thymus towards a regulatory phenotype early after treat- ment.
Three weeks after treatment, FVIII+Dex mice had no significant differences in thymic and splenic lymphocyte populations when compared to FVIII mice (Figure 6D-F). We did however observe an increase in the percentage of blood B cells (36.33% vs. 21.33%, P=0.028) in FVIII+Dex mice compared to FVIII mice (Figure 6D). No significant differences in thymic, splenic or blood lymphocyte popu- lations were detected when comparing Dex and HBSS mice (Figure 6D-F). This suggests that the effects of Dex likely occur early on and have no major lasting impact on lymphocyte populations despite the long-term FVIII toler- ance.
Administration of Dex during initial FVIII exposures alters the thymic but not splenic transcript profile of E16KO mice
We also assessed the effects of our treatment protocol on thymic and splenic mRNA transcription profiles of E16KO mice. In the thymus, a total of 54 genes had
ACE
P=0.0001
P=0.5573
P=0.3295
P=0.1357 P=0.1206
P=0.0970
BDF
P=1
Figure 5. Administration of Dex during initial FVIII exposure induces durable tolerance to FVIII in E17KO/hMHC mice. A. Anti-FVIII IgG incidence and B. anti-FVIII IgG titers following initial exposure with FVIII or FVIII+Dex. C. Anti-FVIII IgG incidence, D. anti-FVIII IgG titers, E. FVIII inhibitor incidence and F. FVIII inhibitory activity fol- lowing FVIII re-exposure in E17KO/hMHC mice initially treated with FVIII or FVIII+Dex and with no evidence of anti-FVIII IgG at week four. Some statistical comparisons could not be carried out because fewer than three mice had evidence of antibodies and/or inhibitors. ND: not detectable; FVIII: factor VIII; Dex: dexamethasone; IgG: immunoglobulin G.
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