Abstract
In contrast to normal hematopoiesis and acute leukemias, research in CLL still is hampered by the lack of a reliable in vivo model for primary B-CLL. We here report highly reproducible engraftment of B-CLL cells, when 1x10^8 MNC derived from the peripheral blood of CLL patients were transplanted i.v and i.p. into NOD/SCID mice. So far, 14 different CLL samples were investigated in 41 mice. At weeks 4, 8 or 12 mice were sacrificed and bone marrow (BM), spleen, and peritoneal fluid (PF) were analyzed by FACS for human CD19/CD5/CD23/CD45 (B-CLL) cells and CD45/CD3/CD5 (T) cells. Additionally, HE- and immunostaining was performed on spleen sections.
Analysis at week 4 revealed engraftment in NOD/SCID mice for 13/14 samples (spleen: 13/14, BM: 4/14, PF: 12/14). B-CLL cells were observed predominantly in the spleen (8.9±2.4% or 9.1±4.4x10^5 cells) and PF (19.0±4.4% or 3.4±1.8x10^5 cells) with much lower engraftment in BM (0.6±0.3% or 0.1±0.1x10^5 cells). Detection of B-CLL cells in peripheral blood could be obtained in 3/14 experiments. Also substantial engraftment of human T-cells was observed in 13/14 experiments (spleen: 13/14, BM: 8/14, PF: 11/14). T-cells engraftment was highest in the spleen (23.8±9.8% or 28.7±13.1x10^5 cells) and somewhat lower in PF (16.4±8.2% or 3.0±1.6x10^5 cells) and BM (7.3±3.8% or 2.9±1.1x10^5 cells). Subpopulation analysis revealed a CD4+ phenotype in 65, 59 and 72 % of T-cells within spleen, PF and BM, respectively. Noteworthy, immunohistological analysis of HE stained spleen sections of engrafted animals revealed a pseudofollicular infiltration with human CD45LCA+ cells along splenic arterioles. Within these pseudofollicles human B-CLL but also CD3+ T-cells were detected. Contribution of B-CLL and T-cells to individual follicles was highly variable ranging from 5–95% for both cell types. When engraftment was analysed separately for the i.p and the i.v. route, engraftment of transplanted cells in PF seemed to be depended on the i.p. route whereas splenic engraftment was obtained following i.v. as well as i.p. injection. Sustained B-CLL engraftment was seen after 8 weeks (spleen: 3.1±1.4% or 7.3±3.1x10^5 total cells; PF: 57.6±23.3% or 1.0±0.5x10^5 cells; n=3 mice) and 12 weeks (spleen: 1.4±1.3% or 0.3±0.3x10^5 cells; PF: 10.2±7.3% or 0.5±0.5x10^5 cells; n=2 mice).
Thus, we have shown efficient engraftment of human B-CLL cells in the spleen and PF of NOD/SCID mice. This in vivo model should significantly help to understand B-CLL biology and to test novel therapeutic approaches. The observed pseudofolicular pattern of splenic infiltration supports the theory of T-cells creating a “microenvironment” sustaining the growth of the leukemic B cell clone.
In vitro preacdvated human T cells engraft in SCID mice and migrate to murine lymphoid tissues
