Critical role of CD4 T cells in PF4/heparin antibody production in mice

Abstract

Antibodies specific for platelet factor 4 (PF4)/heparin complexes are central to the pathogenesis of heparin-induced thrombocytopenia. Marginal zone B cells appear to be the source of such antibodies, but whether T-cell help is required is unclear. Here, we showed that induction of PF4/heparin-specific antibodies by PF4/heparin complexes was markedly impaired in mice depleted of CD4 T cells by anti-CD4 antibodies. Furthermore, Rag1-deficient recipient mice produced PF4/heparin-specific antibodies upon PF4/heparin challenge when reconstituted with a mixture of wild-type splenic B cells and splenocytes from B-cell-deficient (mMT) mice but not splenocytes from T-and B-cell-deficient (Rag1 knockout) mice. Lastly, mice with B cells lacking CD40, a B-cell costimulatory molecule that helps T-cell-dependent B-cell responses, displayed a marked reduction of PF4/ heparin-specific antibody production following PF4/heparin challenge. Together, these findings show that helper T cells play a critical role in production of PF4/heparin-specific antibodies. (Blood. 2015;125(11):1826-1829 Introduction Heparin-induced thrombocytopenia (HIT) is the most common druginduced, antibody-mediated thrombocytopenia and occurs 3 to 6 days following heparin treatment.1,2 HIT patients develop antibodies quickly, however, which are typically undetectable in a few months. Platelet factor 4 (PF4)/heparin-specific antibodies, central to the pathogenesis of HIT, are predominantly of the immunoglobulin G1 (IgG1) isotype with some IgG2 in humans.2-4 IgG/PF4/heparin immune complexes bind FcgRIIA on the platelet surface and induce platelet activation, leading to thrombocytopenia and a high risk of arterial and/or venous thrombosis/thromboembolism. 5,6 Long-lived mature B cells comprise 3 subsets: marginal zone (MZ), B1, and follicular B cells. 7,8 The MZ subset has been shown to be critical for production of PF4/heparin-specific antibodies.9 Typically, MZ B cells produce IgM or IgG antibodies independent of T-cell help. [10][11][12] Indeed, HIT patients have features of a T-cell-independent humoral immune response, characterized by rapid onset and decline of antibodies and apparent absence of immunologic memory.1 However, patients with severe HIT possess T cells that have a T-cell receptor with highly restricted complementarity determining region 3 regions and are responsive to PF4/heparin, suggesting a role of T cells in HIT pathogenesis. 13,14 Nonetheless, direct evidence for a role of T cells in HIT pathogenesis has not been reported. Here, we describe studies to define the role of T-cell help in regulating production of PF4/heparin-specific antibodies. Study design MiceEight-to 10-week-old Rag1-deficient, CD40-deficient, mMT, and wild-type C57BL/6 mice from The Jackson Laboratory were maintained in the Biological Resource Center at the Medical College of Wisconsin (MCW). Animal protocols were approved by the MCW Institutional Animal Care and Use Committee. In vivo depletion of CD4 T cellsWild-type C57BL/6 mice were injected intraperitoneally with ...

Use of a whole‐cell ELISA to detect additional antibodies in setting of suspected heparin‐induced thrombocytopenia

“…42 At the immune cell level, CD4 + T-helper cells play a critical role in the production of HIT antibodies while marginal zone B cells produce the relevant antibodies. 43 However, heparin exposure is not always necessary for some anti-PF4 antibody formation. 32 These results suggest that antibodies generated in the presence of heparin may be distinct from those generated in its absence, leading to the possibility that distinct or different immunologic determinants on platelets may lead to a HIT-like clinical presentation.…”

Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

“…Thus, all B-and T-cells were originated from the Drd3 -/-(or Drd3 +/+ in the control group) BM. On the other hand, the 4:1 ratio (Rag1 -/--to-Drd3 -/or Rag1 -/--to-Drd3 +/+ ) in the donor BM [48] ensure that all the other hematopoietic lineages different from lymphocytes were predominantly originated from the Rag1 -/-BM, and therefore were Drd3 sufficient. To restrict genetic Drd3deficiency only to B-cells, irradiated µMT mice received a BM mix composed of 80% from µMT mice and 20% from Drd3 -/mice.…”

“…To address the role of DRD3 in the adaptive immune system in the development of CNS autoimmunity we generated chimeric mice bearing Drd3-deficient adaptive immune system and Drd3-sufficient innate immune system. For this purpose, recombination-activating gene 1 knockout (Rag1 -/-) mice, which are devoid of T and B lymphocytes, were g-irradiated with a myeloablative dose and then received the transfer of donor bone marrow (BM) obtained from Rag1 -/mice and Drd3 -/mice mixed in a 4:1 ratio Rag1 -/--to-Drd3 -/as described before [48]. Thus, after BM reconstitution, Drd3-deficiency was confined to T and B lymphocytes, bearing Drd3-deficient adaptive immune system ( Fig.…”

The unique immunological features of heparin‐induced thrombocytopenia

“…Pregnancy, which leads to a suppression of T cell function (Doria et al , ), is also associated with a low incidence of HIT (Fausett et al , ). Murine models have shown that T cells expressing both CD4 and CD40 ligand are required to generate anti‐PF4/heparin IgG antibodies (Zheng et al , ); however, these antibodies were not investigated for their potential to induce Fc‐receptor‐specific platelet activation and cause HIT. In typical antibody responses, follicular T H cells express CD40 ligand, which engages CD40 on cognate B cells to induce class switch recombination, germinal centre formation, and memory cell generation (Elgueta et al , ).…”