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.
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.
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