C1c2c3c4

FIGURE 4 (See color insert) Primary and secondary structure of PF4 in relation to HIT neoepitopes. (Top) 3D representation of the PF4 tetramer, indicating two neoepitope sites (per monomer). The "ring of positive charge" is formed by lysine residues in the C-terminus (light blue) and other lysine and arginine residues (dark blue). (Bottom) The linear sequence of the 70-amino acid polypeptide of a single PF4 molecule is shown. Abbreviation: PF4, platelet factor 4. Source: From Li et al., 2002.

conditions such as systemic lupus erythematosus, systemic sclerosis, and insulin autoimmune syndrome (Ito et al., 1993; Crow et al., 1994; Kuwana et al., 1995a). In both lupus and scleroderma, T-helper cells mediate antigen-specific autoanti-body production by B cells (Adams et al., 1991; Mohan et al., 1993; Kuwana et al., 1995b).

We hypothesized that the PF4-heparin complex not only is the target for antibody but also is the stimulus for T-cell activation, and have used T-cell receptor (TCR) spectratyping (Maslanka et al., 1995), also called immunoscope (Cochet et al., 1992; Pannetier et al., 1993), and clonotyping (Maslanka et al., 1996) to characterize the T-cell response to PF4-heparin complexes in HIT.

Culture of peripheral blood mononuclear cells (PBMC) from patients experiencing HIT incubated with PF4-heparin complexes, but not PF4 or heparin alone, leads to selective expansion of T-cell subsets (Liu et al., 2000; Bacsi et al., 2001). On in vitro culturing of PBMC from two HIT patients, the PF4-heparin complexes preferentially stimulated CD4 T cells expressing TCR with b-chains of the V 5.1 family, with a shared core CDR3 region amino acid motif (PGTG) (Bacsi et al., 1999). In a study of a third HIT patient, we found PF4-heparin-specific expansion of several bV 17 TCR clonotypes with yet another shared core CDR3 region amino acid motif (TSG) (Bacsi et al., 2001). However, T-cell lines derived from this third patient and maintained in culture in the presence of PF4-heparin demonstrated selective expansion of the b 6.1 and 17 families sharing the conserved core GTG motif previously identified in the bV 5.1 family of the first two HIT patients (Liu et al., 2001).

These findings provide evidence for the existence of T-cell subpopulations specific to PF4-heparin complexes in the peripheral blood of patients experiencing HIT and suggest that a common CDR3 TCR motif may be important for recognition of a peptide derived from PF4 processed by antigen-presenting cells (APCs) in the presence of heparin.

Despite the intriguing implications of these findings, however, T-cell lines and clones derived from these cultures and from those derived from two more HIT patients proliferated very poorly or not at all in the presence of PF4-heparin. Several reports have identified PF4 as an inhibitor of EC proliferation and angiogenesis (Broxmeyer et al., 1993; Strieter et al., 1995). Furthermore, PF4 recently has been shown to inhibit strongly T-cell proliferation, interferon-y (IFN-g), and IL-2 release of isolated T cells (Fleischer et al., 2002). We wondered if similar inhibitory effects of PF4 could be observed on specific T-cell subsets, e.g., T regulatory (Tr) cells.

It has been recently reported in a murine model of HIT that the induction of anti-PF4-heparin antibodies requires involvement of T cells that have been educated in the thymus (Suvarna et al., 2005). On the contrary, a reported clinical evidence of lack of immunologic memory (Warkentin and Kelton, 2001) together with the antigenicity of high molecular weight PF4-heparin complexes (Visentin et al., 2001; Rauova et al., 2005, 2006) is consistent with the possibility that HIT can be induced independently of T-cell help.

Autoreactive T and B cells can be detected in healthy individuals but are normally kept in check by regulatory mechanisms. Among those is active suppression of naïve T cells by endogenous Tr cells. Several types of Tr cells exist, including CD4+ T cells that express the IL-2 receptor a chain (CD25) constitu-tively, do not secrete IL-10, and suppress immune responses via direct cell-to-cell interactions. CD4+CD25+ Tr cells represent 5-10% of the endogenous CD4+ T-cell subset and are able to suppress CD4+ and CD8+ T-cell responses in vitro and in vivo upon TCR engagement. The recent finding that human PF4 inhibits the proliferative response of human CD4+CD25~ T cells, while inducing expansion of CD4+CD25+ Tr cells, and that PF4-induced CD4+CD25+ Tr cells lose their potent suppressor function in vitro (Fig. 5), suggests a previously unrecognized role of PF4 in the regulation of immune responsiveness (Liu et al., 2005).

It is presently unclear why patients with HIT mount a brisk humoral immune response to an autologous protein (PF4). PF4 is undoubtedly processed, under normal circumstances, by antigen-APCs without triggering immunity.

It appears that multiple factors influence the formation of antibodies specific to PF4-heparin complexes in patients receiving heparin. Currently, there is no evidence to support genetic predisposition as a basis for antibody formation in patients receiving heparin. Unlike the situation in alloimmune thrombocytopenia (de Waal et al., 1986; Mueller-Eckhardt et al., 1989), no connection between HIT and human leukocyte antigens (HLA) has been found (Greinacher and Mueller-Eckhardt, 1993). IgM antibodies specific to PF4-heparin complexes are a common finding in HIT (Visentin et al., 1994), indicating a primary immune response, and it could be speculated that patients who received UFH previously may be at

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