The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia major.

Beta-thalassemia major is characterized by ineffective erythropoiesis leading to severe anemia and extensive erythroid expansion. The ineffective erythropoiesis is in part due to accelerated apoptosis of the thalassemic erythroid precursors; however, the extent of apoptosis is surprisingly variable. To understand this variability as well as the fact that some patients undergoing allogeneic marrow transplantation are resistant to the myeloablative program, we attempted more quantitative analyses. Two groups of patients totaling 44 were studied, along with 25 healthy controls, and 7 patients with hemolysis and/or ineffective erythropoeisis. By 2 flow cytometric methods, thalassemic erythroid precursors underwent apoptosis at a rate that was 3 to 4 times normal. Because thalassemic marrow has between 5- to 6-fold more erythroid precursors than healthy marrow, this translated into an absolute increase in erythroid precursor apoptosis of about 15-fold above our healthy controls. In searching for the causes of the variability in thalassemic erythroid precursor apoptosis, we discovered tight direct correlations between the relative and absolute extent of apoptosis and the extent of erythroid expansion as measured either by the absolute number of marrow erythroid precursors or by serum soluble transferrin receptor levels. These results could mean that the most extreme rates of erythroid proliferation lend themselves to cellular errors that turn on apoptotic programs. Alternatively, extreme rates of erythroid hyperplasia and apoptosis might be characteristic of more severely affected patients. Lastly, extreme erythroid hyperplasia could generate such numbers of apoptotic erythroid precursors that marrow macrophages are overwhelmed, leaving more apoptotic cells in the sample.

Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation.

Twenty-six transplanted thalassemic patients out of 295 analyzed, showed the presence of persistent mixed chimerism, over a period of time varying between 2 and 11 years after BMT. Despite the presence of large numbers of residual host cells, these transplanted thalassemic patients no longer require red blood cell transfusions and have a functional graft, producing sufficient levels of hemoglobin A ranging from 8.3-14.7 g/dl. These ex-thalassemic patients with persistent mixed chimerism, although they did not achieve complete donor engraftment are no longer exposed to the risk of graft rejection. The mechanisms underlying this apparent state of tolerance or education in these patients are at the present time unknown. However, these observations may be useful for physicians involved in defining optimal strategies for clinical gene therapy, in utero hematopoietic stem cell transplantation and adoption of less toxic conditioning regimens in mini-transplantation.

Coexistence of two functioning T-cell repertoires in healthy ex-thalassemics bearing a persistent mixed chimerism years after bone marrow transplantation.

Bone marrow transplantation (BMT) from an HLA-identical donor is an established therapy to cure homozygous beta-thalassemia. Approximately 10% of thalassemic patients developed a persistent mixed chimerism (PMC) after BMT characterized by stable coexistence of host and donor cells in all hematopoietic compartments. Interestingly, in the erythrocytic lineage, close to normal levels of hemoglobin can be observed in the absence of complete donor engraftment. In the lymphocytic lineage, the striking feature is the coexistence of immune cells. This implies a state of tolerance or anergy, raising the issue of immunocompetence of the host. To understand the state of the T cells in PMC, repertoire analysis and functional studies were performed on cells from 3 ex-thalassemics. Repertoire analysis showed a profound skewing. This was due to an expansion of some T cells and not to a collapse of the repertoire, because phytohemagglutinin stimulation showed the presence of a complex repertoire. The immunocompetence of the chimeric immune systems was further established by showing responses to alloantigens and recall antigens in vitro. Both host and donor lymphocytes were observed in the cultures. These data suggest that the expanded T cells play a role in specific tolerance while allowing a normal immune status in these patients.

Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia.

Molecular genetic techniques permit sensitive assessment of host hematopoiesis after marrow transplantation for thalassemia. Information on this persistence and the cell lines in which it occurs may permit therapeutic intervention in patients at high risk for rejection and/or relapse. The objective of this study, therefore, was to determine the evolution and cell line distribution of persistent mixed chimerism detected in 55 patients treated for beta thalassemia. Our findings indicated that rejection occurred in 20 patients, the host component disappeared in 20, and mixed chimerism without transfusion need persisted for 1 to 7 years in 15. In three patients with stable mixed chimerism for 4, 5, and 7 years, host hematopoiesis fluctuated between 25% and 75%. Despite this, donor pattern beta-globin chain synthesis maintained hemoglobin levels between 10 and 13.5 g/dL without transfusion. In these three patients, the polymerase chain reaction of the VNTR and the fluorescent in situ hybridization analysis revealed the coexistence of donor and host cells in the different peripheral blood cell subpopulations and precursors studied (CD2+, CD4+, CD8+, and CD19+ granulocytes; glycophorin-A+, erythroid burst-forming units, CD33+, granulocyte-macrophage colony-forming units). We found that rejection and disease recurrence occur in approximately one third of patients with early mixed chimerism. High levels of host type hematopoiesis can be present in patients not requiring transfusion.

Soluble transferrin receptor following bone marrow transplantation from donors heterozygous for beta thalassemia.

This study analyzes the serum transferrin receptor (sTfR) levels in a series of 184 ex-thalassemic patients with a follow-up of 1 to 9 years after bone marrow transplantation (BMT) for homozygous beta thalassemia. A significant inverse correlation between sTfR and Hb levels was observed (r = -0.36, p < 0.001). Patients who received the marrow from an HLA-identical sibling donor heterozygous for beta thalassemia displayed significantly higher levels of sTfR than patients transplanted from a normal sibling donor (p < 0.001). A cut-off value of 2600 ng/mL of sTfR was established. Only 3 out of 56 (5%) patients who received the marrow from a normal sibling, reached a sTfR value above the cut-off level, while 64 out of 128 (50%) patients transplanted from a heterozygous sibling donor showed sTfR values > 2600 ng/mL (p < 0.001). These results suggest that the level of sTfR helps to identify ex-thalassemic patients with enhanced or normal erythropoietic activity among those transplanted from HLA-identical sibling donors heterozygous for beta thalassemia. The physiologic and clinical significance of different patterns of sTfR levels in ex-thalassemic patients with beta thalassemia trait deserves to be investigated.