Abstract
B-cell type chronic lymphocytic leukemia (B-CLL), an incurable disease of unknown etiology, results from the clonal expansion of a CD5+CD19+ B lymphocyte. Progress into defining the cell of origin of the disease and identifying a stem cell reservoir has been impeded because of the lack of reproducible models for growing B-CLL cells in vitro and in vivo. To date, attempts to adoptively transfer B-CLL cells into immune deficient mice and achieve engraftment and growth are sub-optimal. At least one possible cause for this is the murine microenvironment’s inability to support B-CLL survival and proliferation. We have attempted to overcome this barrier by creating a human hematopoietic microenvironment by reconstituting the tibiae of NOD/SCID/γcnull mice by intrabone (ib) injection of 1–3 × 105 CD34+ cord blood cells along with ~106 bone marrow-derived human mesenchymal stem cells (hMSCs). When human engraftment of 1–10% CD45+ cells was documented in the blood by immunofluorescence using flow cytometry, a total of 108 PBMCs from individual B-CLL patients were injected into the same bones. Before injection, B-CLL PBMCs were labeled with CFSE to permit distinction of leukemic B cells from normal B cells that might arise from the injected CD34+ cells. CFSE labeling also permitted tracking initial rounds of cell division in vivo. Every two weeks after B-CLL injection, peripheral blood from the mice was examined for the presence of cells bearing human CD45, CFSE, and various human lineage markers by flow cytometry. In the presence of a human hematopoietic microenvironment, CD5+CD19+ leukemic cells underwent at least 6 cell doublings, after which CFSE fluorescence was no longer detectable. Timing of B-CLL cell division varied among patients, occurring between 2 to 6 weeks after the injection of PBMC. In contrast, leukemic cells injected into mice that were not reconstituted by ib injection with hCD34+ cells and hMSCs or were reconstituted with only hMSCs failed to proliferate. Moreover the number of CFSE+CD5+CD19+ cells detected in the blood of mice with a human hematopoietic microenvironment far exceeded that in mice receiving only hMSC. Robust T-cell expansion occurred in several mice receiving CD34+ cells; in some instances T-cell growth was also found without hCD34+ cell injection, although in these cases it was usually less extensive. Based on genome-wide SNP analyses, the T cells were of B-CLL patient origin and not from hCD34+ cells. Furthermore, most of the mice with significant T-cell overexpansion died within 6 weeks of B-CLL cell injection from apparent graft vs. host disease. Therefore in subsequent experiments, we eliminated T cells by injecting an anti-CD3 antibody (OKT3); this treatment led to an inhibition of B-CLL cell proliferation. Moreover the percentage of CD38+ cells in the CFSE+CD5+CD19+ cell fraction was similar to that in the donor patient inoculum only in the mice in which T-cell-mediated B-CLL cell proliferation occurred. The percentage and intensity of CD38− expressing B-CLL cells was higher in the spleen and bone marrow (BM) of mice not treated with OKT3 antibody. Finally, the percentage of CFSE+CD5+CD19+ cells in the spleen far exceeded that in the blood, BM, liver and peritoneum, even when leukemic cells were no longer present in the blood and other organs; these findings suggest that the spleen is better at supporting B-CLL cell viability and proliferation than the other anatomic sites. These studies demonstrate conditions making adoptive xenogeneic transfer and clonal expansion of B-CLL cells into a mouse model possible. Factors conferring an advantage in this model include both a human hematopoietic environment and autologous T cell growth. Increased numbers of CD38+ B-CLL cells, similar to those in the patient, were only found when leukemic B cell division occurred. The optimal site for B-CLL cell growth was murine spleen. Since non-genetic factors promoting B-CLL expansion are not completely known, this model will be useful in discovering these as well as for studying the basic biology of this disease, such as if leukemic stem cells exist and also to conduct preclinical tests on possible therapeutics.
In Vivo Analysis of Cell Division, Cell Growth, and Differentiation at the Shoot Apical Meristem in Arabidopsis
