Publication Date:
2012-11-16
Description:
Abstract 13 Hemophilia A and B result from deficiency in clotting factor VIII (FVIII) or IX (FIX), respectively. In a subset of patients, treatment by factor replacement therapy is limited by formation of inhibitory antibodies to the clotting factors, representing a serious complication that increases risks of morbidity and mortality. Immune responses to the therapeutic coagulation factors are also a concern in newly emerging gene therapies. Regulatory T cells (Treg) offer to be a novel alternative pathway toward immune tolerance. Treg has been identified as a major component of immune tolerance to coagulation factors in pre-clinical studies. Therefore, we hypothesize that ex vivo expanded autologous Treg can suppress inhibitor formation. Our study seeks to test this approach in hemophilic mice. Initially, we optimized in vitro expansion of murine BALB/c-derived Treg. Using flow sorting, GFP+ cells were purified (〉98% purity) from spleens of BALB/c knock-in mice containing a GFP reporter linked to FoxP3 expression with an IRES sequence. Sorted cells were stimulated in culture using anti-CD28/anti-CD3 beads in the presence of high-level IL-2 (1000 U/ml). IL-2 was replenished every second day in culture. After ∼1 week, cells were freshly stimulated. At the end of 2 weeks, viability, purity, and FoxP3/GFP expression was confirmed. Greater than 30-fold expansion was repeatedly accomplished. Assumming a dose of 1×106 Treg/mouse, expansion is sufficiently robust to treat 〉30 mice starting with Treg from 2–3 donor mice. Ex vivo expanded Tregs were adoptively transferred to male hemophilia A mice (BALB/c F8e16 −/−), which were then treated with F.VIII (1 IU human B domain-deleted F.VIII, IV, once per week) for two months. Bethesda assays demonstrated that Treg transplant had effectively suppressed inhibitor formation. Inhibitor titers in control mice were 15–20 BU at 1 month and 30–40 BU at two months. In contrast, Treg treated mice (n=5 per group) formed at most low-titer inhibitors (2–3 BU for both time points). By 2 months, peripheral Treg frequencies had returned to near baseline. To further demonstrate presence of a Treg population capable of suppressing antibody formation against F.VIII, a secondary transfer of sorted CD4+CD25+ splenocytes was performed. Recipient hemophilia A mice were immunized against F.VIII in adjuvant. Compared to mice receiving control Treg, there was significant (P
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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