The development of platelet factor 4 (PF4)/heparin (H) antibodies initiates the morbidity of heparin-induced thrombocytopenia (HIT). This application seeks to elucidate the cellular basis of the PF4/H immune resposne using an optimized murine immunization model developed in our laboratory. Studies with this model have shown that PF4/H ultra large complexes (ULCs) potently induce PF4/H antibody (Ab) formation, directly activate DCs and induce proliferation of antigen-naive T-cells. Based on these observations, we hypothesize that PF4/H ULCs are not processed and presented as conventional antigens, but activate the immune system as SAGS. To test this hypothesis, we propose the following specific aims:
Specific Aim 1. Mechanisms of APC activation by PF4/H ULCs. Our studies show Ab responses to mPF4/H depend on the structural features of the antigen, that PF4/H complexes interact with variety of cell-surfaces through charge-dependent interactions, and that DCs are directly activated by PF4/H ULCs. Based on these observations, we hypothesize that biophysical attributes of PF4/H ULCs (charge and size) are critical for its SAG-like properties, enabling intact antigen to directly activate: DCs and T-cells without need for MHC-restricted antigen processing and presentation. To test this hypothesis, we will examine: effects of antigen composition (size and charge) on DC activation and DC binding, requirements for cell-surface MHC class II molecules on DCs and role of ULCs on promoting heterocellular interactions.
Specific Aim 2. Effects of PF4/H ULCs on T-cell activation, T/B cell interactions and T-cell memory. Our preliminary data show that PF4/H ULCs, like SAGs, elicit APC-dependent T-cell activation and proliferation, and that proliferation is CD28 dependent. In this aim, we hypothesize that PF4/H ULCs, as SAGs, elicit polyclonal activation of Vp restricted T-cell subsets and potentiate T-cell helper activity, but fail to produce memory T-cells. To test this hypothesis, we will examine proliferation of V(3-restricted T-cell subsets, T-helper activity leading to in vivo cytokine production, and T-cell memory.
Specific Aim 3. Germinal center formation and B- and T-cell fate in HIT. In this aim, we show that despite robust GC formation and production of isotype-switched Abs, immune recall is impaired in animals receiving mPF4/H ULCs. Based on these findings, we hypothesize that impaired immune recall in our murine model and human HIT is consistent with SAG induced T-cell anergy and failure to form memory B-cells. To test this hypothesis, we will investigate mechanisms of T-cell anergy and examine the fate of GC activated B-cells in the HIT immune response. With the availability of an optimized murine model, we are poised to address fundamental questions regarding the HIT immune response, specifically, how PF4, a self-antigen, becomes a potent immunogen in the presence of heparin, why antibody responses occur commonly in certain settings and why the immune response appears to be self-limited. We hope that insights from these studies will lead to novel interventions that will maintain anticoagulation but mitigate PF4/H Ab formation.

Public Health Relevance

These proposed studies address why this allergic response occurs in response to a commonly used blood-thinner (heparin). In the context of this Program Project, these studies may lead to new preventative or diagnostic test and better therapies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL110860-03
Application #
8695463
Study Section
Heart, Lung, and Blood Program Project Review Committee (HLBP)
Project Start
Project End
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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