Abstract
ITP is an autoimmune disease mediated by autoantibodies to platelet membrane glycoproteins, such as GPIIb-IIIa. We recently identified CD4+ T cells reactive with GPIIb-IIIa in patients with chronic ITP, and these T cells are considered pathogenic because they help B cells produce antibodies that bind normal platelet surfaces in vitro. GPIIb-IIIa-reactive T cells respond to tryptic peptides of GPIIb-IIIa or recombinant GPIIb-IIIa fragments produced in bacteria, but not to native GPIIb-IIIa, indicating that the epitopes they recognized are ’cryptic’ determinants, generated at a subthreshold level by the processing of native GPIIb-IIIa under normal circumstances. We have found that activation of T cells and subsequent anti-platelet antibody production occur primarily in the spleen of ITP patients, but mechanisms that induce the processing and presentation of cryptic peptides of GPIIb-IIIa remains unknown. In this study, antigen-presenting cells (APCs) presenting the GPIIb-IIIa cryptic peptides was evaluated by their ability to induce a specific response of GPIIb-IIIa-reactive CD4+ T-cell lines generated from ITP patients undergoing splenectomy. All 6 T-cell lines used were HLA-DR-restricted Th0 cells with various antigenic specificity and 5 of them had helper activity. To identify splenic APCs responsible for presentation of the GPIIb-IIIa cryptic peptides, GPIIb-IIIa-reactive T-cell lines were cultured with freshly isolated autologous splenic APCs, including CD14+ macrophages, CD19+ B cells, or Lin−CD11c+ dendritic cells, in the presence or absence of GPIIb-IIIa tryptic peptides. All APCs induced a T-cell proliferation in response to the antigen. Interestingly, macrophages stimulated GPIIb-IIIa-reactive T-cell lines without any exogenous antigen, but B cells or dendritic cells did not. This response was blocked by anti-HLA-DR monoclonal antibody, indicating presentation of GPIIb-IIIa cryptic peptides by splenic macrophages in vivo in ITP patients. To further examine whether uptake of opsonized platelets by macrophages results in presentation of the GPIIb-IIIa cryptic peptides, GPIIb-IIIa-reactive T-cell lines were cultured with autologous macrophages, which were prepared by culturing peripheral blood monocytes with M-CSF, in the presence of platelets derived from ITP patients and healthy individuals. Cultured macrophages required preincubation of GPIIb-IIIa tryptic peptides to stimulate GPIIb-IIIa-reactive T-cell lines. As expected, cultured macrophages preincubated with autologous or allogeneic ITP platelets, but not with healthy platelets, were able to stimulate GPIIb-IIIa-reactive T-cell lines. A response of the T-cell line was also induced by macrophages carrying healthy platelets pre-treated with ITP platelet eluates. This response induced by macrophages carrying ITP platelets was completely inhibited by anti-FcγRI, but not by anti-FcγRII monoclonal antibody. Finally, GPIIb-IIIa-reactive T-cell lines failed to induce anti-GPIIb-IIIa antibody production in culture with autologous B cells and platelets, but anti-GPIIb-IIIa antibody production was observed when this culture was carried out on autologous macrophages. In summary, splenic macrophages are a source of the GPIIb-IIIa cryrptic peptides in ITP patients. It is likely that splenic macrophages that phagocytose opsonized platelets via FcγRI have the ability to efficiently concentrate small quantities of platelet antigens that were previously cryptic, and to maintain continuous production of pathogenic anti-platelet antibody.
T and B cells that recognize the same antigen do not transcribe similar heavy chain variable region gene segments.
