New insights in heparin-induced thrombocytopenia by the use of fluid-phase assays to detect specifically platelet factor 4/heparin complex antibodies and antibody-secreting cells.

INTRODUCTION: The key feature of heparin-induced thrombocytopenia (HIT) is the production of antibodies (Ab) against the platelet factor 4 (PF4)/heparin complex. These Ab are directed against neoepitopes of the PF4 tetramer, which are induced by the complex formation with heparin. To study this humoral immune response in greater detail, either in a murine immunization model or in human blood samples, reliable and specific immune assays to detect specifically Ab against the PF4/heparin complexes, but not PF4 alone are required. MATERIALS AND METHODS: We established fluid-phase enzyme-immunoassays in which the soluble biotinylated antigen, PF4/heparin, is firstly captured by specific Ab, and secondly directly detected with enzyme-conjugated streptavidin. RESULTS: The use of this fluid-phase principle allowed a higher specificity than the traditional solid-phase enzyme-immunoassays in terms of Ab binding to murine PF4/heparin compared to murine PF4 alone. This fluid-phase approach applied to the detection of specific murine PF4/heparin Ab-secreting cells (ASC) identified the spleen as the main lymphatic organ that contributes to the PF4/heparin Ab response in mice. IgG ASC specific for PF4/heparin are very transiently detectable in mice, which might explain why anti-PF4/heparin IgG Ab typically disappear within 100 days in humans. Furthermore, this fluid-phase approach was successfully transferred to detect human PF4/heparin-specific Ab. CONCLUSION: The fluid-phase principle for the specific detection of anti-PF4/heparin IgG and IgM Ab enables new and improved assays for HIT research in men and mice. At least in mice PF4/heparin antibodies are produced by transient B cells.

Desmoplakin and KIF20B as target antigens in patients with paroxysmal nocturnal haemoglobinuria.

At least two hypotheses have been proposed to explain the mechanism of clonal expansion of mutant cells in paroxysmal nocturnal haemoglobinuria (PNH). One hypothesis assumes an immune escape mechanism and another proposes an intrinsic second mutational event within clonal cells. We hypothesised that autoantibodies detected in PNH patients could identify antigens that might play a role in the pathophysiology of this disease and screened a human fetal liver cDNA library for serological reactivity against haematopoietic stem/progenitor cells antigens using the SEREX approach. Two antigens were identified that are constitutively expressed in CD34(+) cells. Three and four of 10 PNH patients showed antibody responses against kinesin family member 20B (KIF20B) (previously termed M-phase phosphoprotein 1, MPP1) and desmoplakin (DSP) respectively. We also found an antibody response in one of 20 healthy volunteers against desmoplakin, yet at a much lower titre than in PNH patients. No response to KIF20B or desmoplakin was detected in five patients with aplastic anaemia without a glycosyl phosphatidyl inositol -deficient clone. We conclude that KIF20B and desmoplakin have been shown to be the first known auto-antigens to be recognised by the immune system of patients with PNH. The analysis of the mechanisms underlying the autoimmune response might contribute to our understanding of the clonal expansion in PNH.

Spontaneous high-titered IgG antibody responses against BCL-2 in patients with aggressive lymphomas.

PURPOSE: Molecules which are exclusively or preferentially expressed by malignant cells are potential targets for immunotherapeutic approaches to the treatment of cancer. METHODS: We therefore tested serum samples of 95 patients with aggressive B-cell lymphomas for antibodies against lymphoma-associated molecules using SEREX for antibodies against BCL-2, BCL-XL, MCL-1, survivin, BCL-6, CD20, LM02, PDE4B, cyclin-D2, actinin-alpha1, SCYA3, PKCbeta2, E2IG3, and HGAL. RESULTS: One patient had low-titered (1:100) antibodies against PKCbeta2. Antibodies against BCL-2 were detected in the sera of five patients (5.3%), with responses found more frequently in follicular lymphoma grade 3 (3/26 or 11.5%) than in diffuse large B-cell lymphomas (2/67 or 3%). Anti-BCL-2 antibodies were of the IgG class and titers ranged from 1:1,000 to 1:100,000 with highest titers before treatment and decreasing slowly during remission. CONCLUSION: The spontaneous immunogenicity of BCL-2 makes this molecule a prime candidate for vaccine approaches in BCL-2 positive B-cell lymphomas.