T Lymphocyte Proliferative Responses Induced by Recombinant Factor VIII in Hemophilia A Patients with Inhibitors

Abstract

SummaryIn this study we sought to determine whether factor VUI-reactive T lymphocytes were present in hemophilia A patients with inhibitor antibodies. Peripheral blood mononuclear cells (MNC) were obtained from 12 severe hemophilia A patients having high titer inhibitors, 4 severe hemophilia A patients without inhibitors and 5 normal male subjects. B cell-depleted MNC were cultured in serum-free medium in the absence or presence of 2 µg of recombinant human factor VIII (rFVIII) per ml, and cellular proliferation was assessed after 5 days of culture by measuring 3H-thymidine incorporation. rFVIII induced marked cellular proliferation in cultures of 4 of 12 inhibitor-positive hemophilia patients: fold increase over background (stimulation index, SI) of 7.8 to 23.3. The remaining 8 inhibitor-positive patients, the 4 hemophilia patients without inhibitors and the 5 normal subjects, all had lower proliferative responses to rFVIII, SI range = 1.6 to 6.0. As a group, the inhibitor-positive subjects had significantly higher proliferative responses to rFVIII than did the inhibitor-negative and normal subjects (p < 0.05 by t-test). Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation. Thus, rFVIII-reactive T lymphocytes are present in the peripheral circulation of some inhibitor-positive hemophilia A patients. These T cells may recognize FVIII in an antigen-specific manner and play a central role in the regulation of inhibitor antibody production

CD4+ T-cell clones specific for wild-type factor VIII: a molecular mechanism responsible for a higher incidence of inhibitor formation in mild/moderate hemophilia A

“…13,14 The epitopes recognized by such T cells were located by incubating T cells with peptides overlapping the whole FVIII molecule. 14 In severe hemophilia A patients, FVIII-specific T cells recognizing a large array of peptides covering the entire FVIII molecule were detected.…”

“…A complementary approach to generate FVIII-specific T-cell clones would therefore be to use proliferation assays as a screening method. 13,14 The FVIII amounts administered for substitution therapy in hemophilia A patients are relatively low, with FVIII concentrations peaking between 0.1 to 0.2 g/mL plasma. This raises the question of how the immune system efficiently presents such small amounts of antigen to T lymphocytes.…”

“…12 FVIII-specific T cells have been identified in the peripheral blood of hemophilia A patients with inhibitor using T-cell proliferation assays with native FVIII. 13 The epitopes recognized by such T cells were studied using synthetic peptides covering the entire FVIII molecule. 14 Epitopes were mapped in all domains of the FVIII molecule.…”

A Cellular Viewpoint of Anti-FVIII Immune Response in Hemophilia A

“…Tolerance to FVIII is no exception to this general rule. Indeed, the proliferation of circulating CD4 + T cells from healthy individuals upon exposure to FVIII has been described more than 10 years ago [76]. It has since been confirmed using, as antigens, both intact FVIII and pools of synthetic peptides that span different domains of the FVIII molecule.…”

Dynamics of factor VIII interactions determine its immunologic fate in hemophilia A

“…The proliferation of circulating CD4 ϩ T cells from healthy individuals upon exposure to FVIII was described more than 10 years ago. 44 It has since been confirmed using, as antigens, both intact FVIII and pools of synthetic peptides that span different domains of the FVIII molecule. Depending on the reports, circulating CD4 ϩ T cells from 52% to 78% of the donors proliferated when incubated with FVIII in vitro.…”