Allogeneic Platelet MHC I Antigens Prevent CD61 Specific Cytotoxic T Cell (CTL)-Mediated Immune Thrombocytopenia (ITP)

BackgroundPrimary immune thrombocytopenia (ITP), is an autoimmune disorder characterized by isolated thrombocytopenia, either as a result of failure of self-tolerance, including increased platelet destruction mediated by autoantibodies or cytotoxic T cells, or due to impaired platelet production. With the progress in recent years, T-lymphocyte abnormalities are considered important in the pathogenesis of ITP. On the other hand, it has benn reported recently that the HLA polymorphism is responsible for ITP predisposition. Clinical evidence on platelet transfusion efficacy in ITP patients was first reported by Carr et al in 1986. Reports later on confirmed the efficacy of platelet transfusion in ITP, but the mechanism remains elusive and platelet transfusion is only recommended as an adjunctive therapy for life-threatening bleeding. Using our novel murine model which mimics both the antibody- and CTL- mediated ITP, we sought here to characterize how the different and MHC class I antigens modulate GPIIIa (CD61) specific CD8+ T cell-mediated thrombocytopenia in a murine model of immune thrombocytopenia (ITP).Objectives1. To analyze immune responsiveness against two different platelet antigens; GPIIIa (CD61) and MHC class I molecules.2. To characterize the role of CD8+ T cell in ITP using a novel murine model of perisistant active ITP.3. To characterize how the allogeneic MHC class I antigens modulate GPIIIa (CD61) specific CD8+ T cell-mediated thrombocytopenia in a murine model of immune thrombocytopenia (ITP).Meterials & Methods1. Mice Female BALB/c (H-2d), C57BL/6 (H-2b) mice, CB17(H-2d), BALB/c CD61 knockout (KO) mice, C57BL/6 CD61 KO mice and CB.17 (H-2b) severe combined immunodeficient (SCID) mice,8 to 12 weeks of age, were used as either platelet donors, platelet receipients or spleen-cell transfer recipients.2. Platelet preparation and immunization of CD61 KO mice The platelet preparation and immunization of CD61 KO mice were performed as previously described. For immunization, BALB/c CD61 KO mice were transfused with 100 uL of 108 platelets from①wildtype (WT) female BALB/c (H-2d) mice,②C57 BL/6 CD61 KO mice and③T C57BL/6 (H-2b) mice weekly for 5 weeks. Immunization was monitored by measuring IgG antiplatelet antibody production in the sera by flow cytometry. To determine the levels of platelet-specific IgG antibodies in the BALB/c CD61 KO mouse recipients were determined weekly.3. The immunized BALB/c CD61 KO mice were killed, and their spleens were removed and prepared into a splenocyte suspension. Then the indicated splenocytes (108 cells/mL cell medium) were depleted of CD 19 lymphocytes before transfer by the procedures and reagents provided by the EasySep Magnetic cell sorting kit. Depletion efficiencies were determined by flow cytometry and were greater than 96% for the specific lymphocyte subpopulations removed. Then the splenocyte were transferred into indicated BALB/c SCID mice, the phenotype of the SCID mice were observed, the peripheral platelet counting were carried out weekly and the bone marrow were harvested after 4 weeks for histology analysis.Results1. The levels of platelet-specific antibodies of the BALB/c CD61 KO recipient mice were monitered by flow cytometry. All mice responded in a similar fashion. After being transfused by indicated platelets for 5 weeks, those mice acquired high titers of IgG antibodies against①CD61 antigen (>1:12000),②MHC I antigen (1:400) and③both CD61/MHC I antigens on platelets (>1:12000).2. In anti-CD61+ group, when irradiated CD61+ SCID mice received 1.5×104 anti-CD61+ splenocytes, with or without CD19 depletion, those mice exhibited severe thromobocytopenia, which did not recover in four weeks. Histological analysis of the bone marrow showed that, the numbers of megakaryocytes were elevated and those megakaryocytes were experiencing programmed cell death (PCD).3. In contrast, in double immunity positive group (both anti-CD61+ and anti-MHC I+), after being engrafted by CD 19 depleted splenocytes with both anti-CD61 and anti-MHC I immunity, CD61+ SCID mice exhibited only transient irradiation-induced thromobocytopenia which recovered in two weeks. Moreover, the histological analysis of the bone marrow of those SCID mice showed relatively normal megakaryocytes in counting, size and morphology. The abolishment of persistent severe ITP in the double immunity positive group and the alleviation in bone marrow indicates that allogeneic platelet MHC I antigens inhibited the cytotoxic capability of CD61 specific CD8+CTL in both peripheral blood and bone marrow completely.ConclusionsIn our study, both platelet antigenic systems (MHC class I and CD61) induced anti-platelet antibodies that can mediate platelet phagocytosis. The novel mouse model could mimic the pathegenesis of both the antibodis- and cell-mediated ITP. When anti-CD61 specific CD8+ T cells interacted with allogeneic MHC antigens on platelets they were prevented from inducing CD8+ T cell-mediated thrombocytopenia. Thus, allogeneic MHC platelet transfusions may be a therapeutic option for those patients who suffer from cell-mediated ITP.