Heparin-Induced Thrombocytopenia Is a Thymus (T cell) Dependent Disorder.

Heparin-Induced Thrombocytopenia (HIT) is a drug-dependent immune disorder caused by autoantibodies to Platelet Factor 4 (PF4) and heparin. The immune basis of HIT is poorly understood. Recent studies describing transient antibody responses and absence of immunologic memory in HIT suggest that PF4/heparin autoantibodies may develop independently of T cell help. To investigate the cellular basis of the HIT immune response, we have developed a murine autoimmune model in which anti-murine PF4/heparin (anti-mP+H) arise de novo. Cohorts of BALB/c mice were immunized daily either intravenously (IV, n=10) or intraperitoneally (IP, n=10) for five days with complexes of murine (m) PF4/heparin (IV, n=5 or IP, n=5), heparin alone (IV, n= 2) or buffer (IV, n=3 or IP, n=5). Mice were screened for anti-mP+H for four weeks after immunization using a murine PF4/heparin ELISA. Peak antibody responses to antigen were seen at 11–15 days in 2/5 mice injected with IV mP+H (Day 11, mouse IV P+H #0 peak A_450nm =0.34±0.01; Day15 mouse IV P+H #2 peak A_450nm =0.69±0.01), and at days 22–25 in 2/5 mice injected by IP route (Day 22, mouse IP P+H #0 peak A_450nm =0.37±0.01; Day 25 mouse IP P+H #2 peak A_450nm =0.78±0.02). Anti-mP+H were not detected in mice injected with heparin alone or buffer alone at any time point (peak maximum IV A_450nm= 0.1±0.001, Control #2; mouse IP P+H Control #2 A_450nm =0.04±0.002). Serologically, murine autoantibodies were similar to anti-human (h) PF4/heparin. Anti-mP+H reactivity was specific for murine antigen (mouse IV P+H #2 A_450nm=0.65±0.06), and was reduced with antigen in the presence of excess heparin (A_450nm=0.38±0.01). Minimal reactivity was seen with wells coated with hP+H (A_450nm=0.09±0.005), albumin (mouse IV P+H #2 A_450nm=0.15±0.03), or PBS alone (mouse IV P+H #2 A_450nm =0.16±0.01). Similar to human HIT antibodies, anti-mP+H were of IgG₁ subclass. To determine if T cells are required for development of anti-mP+H, mice lacking T cell function (BIG:BALB/c-Nu, n=10) were injected IV with mP+H daily for five days. Unlike euthymic mice, nude mice did not manifest any antibody responses to IV injections of mP+H. In summary, we have developed a novel murine autoimmune model of anti-PF4/heparin that recapitulates many salient features of the human immune syndrome. Using this murine model, we demonstrate that T cells are essential for development PF4/heparin autoantibodies. Studies are currently underway to delineate mechanisms of T cell regulation and peripheral tolerance in HIT.