Assessment of T regulatory cells in patients with moderate and severe hemophilia type A and its relation with development of Factor VIII inhibitors

Abstract

SUMMARY H emophilia is an X-linked bleeding disorder caused by mutations in coagulation factor VIII (hemophilia A) or IX (hemophilia B). Severe disease is defined by protein levels below 1% of normal, and typically results in frequent spontaneous joints and soft tissue bleeding. Bleeding into critical closed spaces (e.g. intracranial) can be fatal. The genes encoding FVIII and FIX are on the long arm of the X chromosome. Hemophilia A and B are the only hereditary clotting diseases inherited in a sex-linked recessive pattern. All female children of a father with hemophilia will be carriers, whereas none of his sons will be affected. Further, the sons of a carrier mother have a 50% chance of disease, whereas the daughters will have a 50% chance of being a carrier. Hemophiliacs have the heterogeneous phenotypic presentation depending upon its severity described in the Table 1. Severe hemophilia usually present in neonatal period and early infancy, while moderate hemophilia in toddlers and mild hemophilia in late childhood or adolescent and adult often incidental or following major trauma . Bleeding is the hallmark of hemophilia, sites and pattern of bleeding varies over life time. The optimal treatment is recombinant factor replacement to prevent bleeding; however, this treatment has many barriers. The most serious complication of treatment is the development inhibitors to factor products. The development of neutralizing antibodies remains a frequent and serious complication of hemophilia replacement therapy. Most patients with hemophilia do not mount a clinically measurable immune response toward FVIII. In ~30% of patients, however, such FVIII antibodies develop, rendering FVIII treatment ineffective and impairing the functional status of patients. Recently, James et al. characterized T cell responses of a patient with mild hemophilia A (missense genotype A2201P) for one year following his initial inhibitor response, with the goals to define primary epitopes and their MHC Class II restriction. This patient developed a high-titer inhibitor (250 BU/ml) that declined over time to 8 BU/ml. His clotting activity was initially impaired (3%), but returned to baseline (8-10%) within 4 weeks. Interestingly, the responsiveness of CD25-depleted CD4+ cells to an A2201 peptide was enhanced 11 and 19 weeks following inhibitor detection, suggesting involvement of Treg in modulating the immune response. Treg have also been shown to suppress FVIII-induced proliferation in healthy individuals. Therefore, depletion of Treg may enhance FVIII-specific T-cell responses in non-hemophilic individuals. The aim of this study was to determine the frequency of regulatory T cells (Tregs) in children and adolescents with Hemophilia A and to assess their relation to the clinical characteristics of patients as well as Factor VIII inhibitors. We conducted a case-control study included 30 patients with Hemophilia A recruited from the regular attendants of the Pediatric Hematology Clinic, Pediatric Hospital, Ain Shams University from 2013 to November 2014. Another group of 30 age- and sex-matched healthy subjects were enrolled as a control group. An informed consent was obtained from each patient or control or their legal guardians before enrollment in the study. This study was approved from the local ethical committee of Ain Shams University. We found that the mean age of patients was 7.6 ± 4.1 years (range, 3-17 years) while that of controls was 6.9 ± 3.7 years (range, 3-16 years). The median disease duration was 5 years (IQR, 3-9 years). Fifteen patients (50%) received cryotherapy and 6.7% received FVIII alone while 13 patients (43.3%) were on combined cryo and FVII therapy. According to residual FVIII activity levels, 20 patients (66.7%) had mild and moderate hemophilia A while 10 (33.3%) patients had severe hemophilia In this study, the frequency and MFI of Tregs was significantly decreased in all the studied hemophilia A patients whether with or without FVIII inhibitors compared with healthy controls and the lowest levels were found among FVIII inhibitors-positive group. This could be explained by the fact that Tregs are a critical component of self-tolerance and many forms of induced tolerance to exogenous antigens, including tolerance to coagulation factors in gene- or protein-based therapies. Moreover, Tregs suppress B and T cell responses to coagulation factors and cells expressing these therapeutic proteins. Tregs have also been shown to suppress FVIII-induced proliferation in healthy individuals and their depletion may enhance FVIII-specific T-cell responses in non-hemophilic individuals.