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. 2011 Aug;9(8):1524-33.
doi: 10.1111/j.1538-7836.2011.04351.x.

Induction of tolerance to factor VIII by transient co-administration with rapamycin

B Moghimi  1 , B K Sack, S Nayak, D M Markusic, C S Mah, R W Herzog
Affiliations

Induction of tolerance to factor VIII by transient co-administration with rapamycin

B Moghimi et al. J Thromb Haemost. 2011 Aug.

Abstract

Background: Formation of inhibitory antibodies is a frequent and serious complication of factor (F) VIII replacement therapy for the X-linked bleeding disorder hemophilia A. Similarly, hemophilia A mice develop high-titer inhibitors to recombinant human FVIII after a few intravenous injections.

Objective: Using the murine model, the study sought to develop a short regimen capable of inducing tolerance to FVIII.

Methods: A 1-month immunomodulatory protocol, consisting of FVIII administration combined with oral delivery of rapamycin, was developed.

Results: The protocol effectively prevented formation of inhibitors to FVIII upon subsequent intravenous treatment (weekly for 3.5 months). Control mice formed high-titer inhibitors and had CD4(+) T effector cell responses characterized by expression of IL-2, IL-4 and IL-6. Tolerized mice instead had a CD4(+)CD25(+)FoxP3(+) T cell response to FVIII that suppressed antibody formation upon adoptive transfer, indicating a shift from Th2 to Treg if FVIII antigen was introduced to T cells during inhibition with rapamycin. CD4(+) T cells from tolerized mice also expressed TGF-β1 and CTLA4, but not IL-10. The presence of FVIII antigen during the time of rapamycin administration was required for specific tolerance induction.

Conclusions: The study shows that a prophylactic immune tolerance protocol for FVIII can be developed using rapamycin, a drug that is already widely in clinical application. Immune suppression with rapamycin was mild and highly transient, as the mice regained immune competence within a few weeks.

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Figures

Fig. 1
Fig. 1
Prophylaxis against inhibitor formation against BDD-F.VIII in hemophilia A mice (C57BL/6/129). A. Tolerance induction and treatment schedule. B. Inhibitor titers as a function of time in mice that received rapamycin (rapa) plus BDD-F.VIII (0.3 IU/mouse/dose) followed by treatment with BDD-F.VIII (circles, n=5). Open triangle: mice that received rapamycin without F.VIII followed by treatment with BDD-F.VIII (n=4). Solid triangle: mice that received no rapamycin followed by treatment with BDD-F.VIII (n=4). C. BDD-F.VIII-specific IgG1 titers in the same animals. Data are average±SD. Statistically significant differences for 5-week time point are indicated as described in Methods. Fold-differences between control groups and tolerized mice are also indicated. D. Schedule for long-term assessment of tolerance to BDD-F.VIII. E. Inhibitor titers and F. IgG1 titers after long-term treatment (n=8). Non-tolerized control mice formed high-titer antibodies (n=4). G. Correction of aPTT 10 min after IV administration of 2 IU BDD-F.VIII in i) naïve mice, ii) mice that had been tolerized and received long-term treatment, and iii) control mice with inhibitors (n=5 per group).
Fig. 2
Fig. 2
Prophylaxis against inhibitor formation against FL-F.VIII in hemophilia A mice (C57BL/6/129). The protocol was identical as in Fig. 1A, except that FL-F.VIII was used instead of BDD-F.VIII. A. Inhibitor titers in mice that received rapamycin plus FL-F.VIII followed by treatment with FL-F.VIII (0.3 IU/mouse/dose; circles, n=5). Solid triangle: mice that received no rapamycin followed by treatment with full-length F.VIII (n=9). B. FL-F.VIII-specific IgG1 titers in the same animals. Data are average ±SD.
Fig. 3
Fig. 3
Formation of NAB to adenovirus in sera of control mice or of mice that had been treated with rapamycin. Virus was administered 5 weeks after rapamycin had been discontinued, and NAB titers measured 3 weeks later. Shown are the ranges and means of NAB titers in a box-and-whisker plot (n=4 per group).
Fig. 4
Fig. 4
T cell responses to BDD-F.VIII. Relative levels of transcripts for cytokine and T cell marker genes in F.VIII- vs. mock-stimulated A. total splenocytes, B. splenic CD3+CD4+ T cells (purified from stimulated splenocyte cultures by flow sorting), and C. non-T cells (i.e. remaining cells after purification of T cells by sorting). Each data point is average for n=4 mice per group ±SD. Hemophilia A mice (C57BL/6/129) had been assayed individually, and all transcript levels were determined by quantitative RT-PCR and normalized for GAPDH expression. Open bars are mice tolerized to BDD-F.VIII followed by treatment with BDD-F.VIII. Black bars are for control mice treated with BDD-F.VIII. D. Purity of sorted CD3+CD4+ T cells and CD3− cells.
Fig. 5
Fig. 5
Frequencies of CD4+CD25+FoxP3+ T cells following in vitro stimulation of splenocytes with hF.VIII. A. Examples of results from tolerized and control hemophilia A mice stimulated with hF.VIII (first two panel; third panel: mock stimulated splenocytes from tolerized mouse). Control mice had been challenged with F.VIII without prior tolerance induction. Upper panels: CD25 and FoxP3 stains gated on CD4+ cells. Lower panels: corresponding histograms for CD4+ staining. B. Summary of results for n=4 mice per group. Data are average ±SD. **: statistically significant difference with P<0.01.
Fig. 6
Fig. 6
Enhanced phosphorylation of Stat3 (A) and Stat5 (B) one week after last F.VIII/rapamycin treatment of hemophilia A C57BL/6/129 mice compared to control mice (treated with identical dose of F.VIII without rapamycin). Following staining with antibodies specific for the phosphorylated forms, results are reported as mean geometric MFI (mean fluorescence intensity) ±SD (n=4 per group).
Fig. 7
Fig. 7
Suppression of anti-BDD-F.VIII formation upon adoptive transfer of Treg from tolerized mice. A. Tolerance induction and treatment protocol for hemophilia A BALB/c mice (n=7) followed by adoptive transfer of Treg. B. Anti-F.VIII titers and C. inhibitor titers in hemophilia A BALB/c mice that were immunized with BDD-deleted F.VIII/CFA 24 hrs after receiving no cells or 1×106 control CD4+CD25+ Treg (from naïve donors), or CD4+CD25+ or CD4+CD25− splenocytes from tolerized mice (n=5/group). C. Magnetically isolated CD4+ splenocytes were >98% pure after negative selection, and subsequent positive selection for CD4+CD25+ cells resulted in >85% CD4+CD25+ cells (85-90% of which were also FoxP3+).
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