Abstract
Despite important advances in the refinement of therapy for acute lymphoblastic leukaemia (ALL), a significant proportion, ~30%, relapse due to failure to eradicate the disease. ALL is thought to be maintained by a subpopulation of cells with extensive proliferative capacity, unlike the majority of blasts, which have limited proliferative ability. This sub-population may be resistant to drug regimens designed to kill the bulk ALL population and subsequent relapses may arise from these cells. Hence, identification and characterisation of these putative ALL stem cells is essential for the development of more effective therapeutic strategies. We have demonstrated that ALL cells capable of long-term proliferation in vitro and in vivo are CD34+/CD10−/CD19−, suggesting that cells with an immature phenotype, rather than committed B-lymphoid cells, are the targets for transformation in B cell leukaemias. Here we have attempted to further define these ALL progenitor cells by investigating the expression of CD133, the stem cell marker, on cells with long-term proliferative ability in vitro and in vivo. ALL cells from 6 pts at diagnosis (4 c-ALL, 2 pre B) and 3 c-ALL in relapse were sorted for expression of CD133 and CD19 and evaluated in a suspension culture (SC) assay. The majority of cells at sorting were CD133−/CD19+ (59±7%) and the CD133+/CD19+ and CD133+/CD19− subfractions represented only (9±6%) and (0.8±0.3%) respectively. However, after 3 weeks in SC, the majority of cells were derived from the CD133+/CD19− subfraction (62±8%). This trend continued with 77±7% of proliferating cells derived from the CD133+/CD19− subfraction by week 6. In the cultures of CD133+/CD19− cells there was a 4 - > 4 log fold expansion in cell numbers, starting from an average 9.3x104 cells at initiation to an average 1.8x106 cells at week 6. Unsorted cells and cells sorted for expression of CD133 and CD19 from these 9 pts were evaluated for their ability to repopulate sublethally irradiated NOD/SCID mice. Engraftment was achieved in each case using unsorted cells (0.7–38% CD45+, with 2x106–107 cells). The only sorted subfraction that engrafted were CD133+/CD19− cells (range 0.8–70% CD45+, using 103–5x104 cells). There was no engraftment with the other subfractions despite injecting significantly higher cell numbers. Secondary transplantation experiments to evaluate the self-renewal potential of the CD133+/CD19− cells are ongoing. Cytogenetic analyses of CD133+/CD19− cells have revealed that they contain translocations such as TEL-AML1 suggesting the translocations occurred as early leukaemogenic events rather than as the blast cells differentiate. IgH rearrangements present in the bulk ALL population at diagnosis were also detected in the CD133+/CD19− cells by PCR analyses. These data suggest that ALL cells with long-term proliferative ability and NOD/SCID repopulating ability express CD133 and lack expression of CD19 providing further evidence for the existence of a hierarchy of progenitor cells in ALL. More precise definition of these ALL progenitor cells should improve MRD immunosurveillance techniques, that are based on the phenotype of the total cell population and may not detect the leukaemic progenitor cells, and permit investigation of the efficacy of therapeutic agents on the cells that may be responsible for disease relapse.
CELL-MEDIATED CYTOTOXICITY DURING REJECTION AND ENHANCEMENT OF ALLOGENEIC SKIN GRAFTS IN RATS
