Role Of B Cell Tolerance In PF4/Heparin Antibody Production

Abstract

Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of heparin, platelet factor 4 (PF4) and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, heparin, a glycosoaminoglycan, and PF4 are normal body constituents and it is as yet unclear what triggers the initial induction of pathogenic antibodies. Here we described detection of B cells among peripheral blood mononuclear cells (PBMCs) from each of 9 healthy adults that produced PF4/heparin-specific IgM antibodies following in vitro stimulation with ubiquitous pro-inflammatory molecules containing unmethylated CpG dinucleotides derived from bacterial and viral DNA. PF4/heparin-specific IgM-generating B cells were present at a frequency of at least 0.03 to 1 per thousand B cells present in the PBMC population. Similarly, splenic B cells isolated from unmanipulated wild-type mice consistently produced PF4/heparin-reactive antibodies following in vitro stimulation with CpG. In addition, wild-type mice produced PF4/heparin-reactive antibodies upon in vivo challenge with CpG whereas unchallenged wild-type mice did not. These findings demonstrate that both humans and mice possess pre-existing, inactive and tolerant PF4/heparin-specific B cells. We suggest that tolerance can be broken by a strong inflammatory stimulus, leading to activation of these B cells and production of antibodies that recognize PF4/heparin in vitro and in vivo. Consistent with this concept, mice lacking protein kinase Cd (PKCd), a signaling molecule of the B-cell survival factor BAFF (B-cell activation factor), that are known to have breakdown of B-cell tolerance to self-antigens, spontaneously produced anti-PF4/heparin antibodies in the absence of an inflammatory stimulus. Taken together, these findings demonstrate that breakdown of tolerance can lead to PF4/heparin-specific antibody production and that B-cell tolerance plays an important role in HIT pathogenesis. Disclosures: White II: Bayer: Membership on an entity’s Board of Directors or advisory committees; CSL-Behring: Membership on an entity’s Board of Directors or advisory committees; NIH: Membership on an entity’s Board of Directors or advisory committees; Asklepios: Membership on an entity’s Board of Directors or advisory committees; Wyeth: Membership on an entity’s Board of Directors or advisory committees; Entegrion: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees.