Μοριακή ανάλυση των λεμφοκυττάρων και των προγονικών αιμοποιητικών κυττάρων σε ασθενείς με απλαστική αναιμία πριν και μετά τη θεραπεία

Abstract

Immune dysfunction, which leads to suppression of haemopoiesis through cytokines secreted by activated T lymphocytes, is considered to play a key role in the pathogenesis of acquired aplastic anaemia (AAA). The peripheral blood lymphocyte subpopulations were calculated by flow cytometry. Lymphopenia, mainly of CD4+ subset, was observed in both patients with active disease and in patients in remission. There was also a significant increase in activated CD4+ and CD8+ T cells in active disease but also in remission. We also studied the intracytoplasmic cytokines of type-1 and type-2 immune responses in CD4+ and CD8+ cells before and after in vitro activation in AAA patients and controls. Untreated or refractory patients had a significantly higher proportion of unstimulated CD4+ and CD8+ cells that produced IFNg and IL-2, while the production of IL-4 and IL-10 did not differ from that of controls. In patients in remission, the proportion of IFN-γ-producing unstimulated CD4+ and CD8+ cells was also increased with a parallel increased production of IL-4 and IL-10. In newly diagnosed or refractory patients, the balance of type-1 /type-2 cytokines, as it has been shown by the ratio of IFNγ/IL-4, shows a polarization towards type-1 response. In patients in remission, despite the increased INF-γ production by unstimulated T cells, the balance returns to that of controls. Polarization of CD4+ cells towards a type-1 response may be essential for the pathogenesis of AAA, leading to activation of cytotoxic CD8+ cells and stem cell destruction. A small proportion of patients with AAA in hematologic remission after immunosuppressive therapy develop a secondary clonal abnormality (e.g. paroxysmal nocturnal hemoglobinuria, karyotypic abnormality). A significantly higher proportion of unstimulated CD4+ cells produced IL-2 and IFNγ in patients with a normal karyotype. The balance between IFNγ/IL-4 is normalized in unstimulated CD4+ cells of AA patients with an abnormal clone, whereas in patients with normal karyotype this ratio remains significantly higher compared to the group with clonal abnormality. According to the above data, the possibility exists that an ongoing type-1 reaction, especially in CD4+ cells in a cohort of AA patients after successful immunotherapy “protects” these patients from the emergence of an abnormal clone. Increased apoptosis is a possible pathogenetic mechanism for the damage of hematopoietic stem cells in aplastic anemia (AA), but the regulation of apoptotic machinery remains unclear. We examined the presence of apoptosis in the marrow cells with flow cytometry (FC) analysis. Bone marrow was obtained from aplastic anemia patients and healthy volunteers of similar age. The AA patients had active disease (untreated or refractory) or complete or partial hematological remission long after immunosuppressive therapy was discontinuated. Two-or three-color FC analysis was used for quantitative measurement of cell surface expression of Apo-1/Fas receptor in CD34+ cells and Annexin+/PI- total BMMNC and CD34+ cells. Two-color FC analysis revealed a significant increase in Fas expression CD34+ in active disease patients as well as in patients in remission compared to controls. CD34+/Annexin+/PI- cells are significantly increased in both active disease and in remission patients compared to controls. In conclusion, a type-1 immune response dominates in aplastic anemia patients, especially in patients with active disease. This polarization of lymphocytes correlates with increased apoptosis in CD34+ bone marrow cells.