The Comparison of the Cytobiological Role of Bone Marrow CD4⁺ T Cells in Aplastic Anemia Patients and Healthy Subjects.

Recent studies indicate that immune-associated aplastic anemia (AA) resembles such autoimmune diseases as insulin-dependent diabetes and chronic autoimmune thyroiditis that belong to organ-specific autoimmune diseases. Many independent investigation groups have successfully isolated the pathopoiesis-associated T cell clone causing hematopoiesis failure with a CD4 phenotype from AA patients peripheral blood and bone marrow (BM). In the current study, BM CD4⁺T cells were isolated from both AA patients and healthy subjects with immunomagnetic beads sorting and were compared with regard to proliferation capability, apoptosis features and the impacts of their secreted cytokines on hematopoiesis stem/progenitor cells. With the improved MTT method, BM CD4⁺T cells of AA group presented 1.6 times more enhanced reproductive activity than those of normal group. Induced by high concentrational CD3 monoclonal antibody for 18h, evident apoptosis cells could be seen under the electron microscope in both normal group and AA group. Quantified by flow cytometry using Annexin-V FITC/PI dual staining method, apoptosis rates in the early and advanced stages of AA group were more than those of normal group (P<0.01). Apoptosis rate in early stage: normal group(7.03±0.86)%, AA group(16.11±1.37)%; apoptosis rate in advanced stage: normal group(2.07±0.42)%, AA group(8.05±0.36)%. The influence of BM CD4⁺T cell culture supernatant on CFU-GM formation of cord blood CD34⁺ hematopoiesis stem/progenitor cells: the CFU-GM count in AA group was: (74.50±9.50)/10⁴ cells; the CFU-GM count in normal group was: (124.25±19.80)/10⁴ cells, observing under an inverted microscope. A significant difference was present in the comparison between the two groups (P<0.01). CyclinD3 mRNA and protein expression levels of cord blood CD34⁺ cells were both down-regulated induced by BM CD4⁺ T cell culture supernatant of AA patients. These results indicate a strong likelihood of an abnormally proliferative and activated state of BM CD4⁺T cells among AA patients and its intimate correlation with AA hematopoiesis failure by secreting some soluble humoral agents that are able to inhibit CyclinD3 and consequently suppress hematopoietic stem cell in proliferation.