Myeloma clonotypic B cells are hampered in their ability to undergo B-cell differentiation in vitro

In the peripheral blood (PB) of multiple myeloma (MM) patients, clonotypic B cells are present that express the identical V( D) J rearrangements as the malignant plasma cells in the bone marrow. In the present study, the proliferative capacity of clonotypic B cells from MM patients (n = 10) and the ability to differentiate in vitro was determined using the CD40-culturing system. For six patients, the presence of clonotypic B cells expressing variant immunoglobulin (Ig) isotypes was assessed by Ig isotype-specific allele-specific oligonucleotide reverse transcription polymerase chain reaction (ASO-RT-PCR) after culturing with CD40L and interleukin 4 (IL-4). In three out of six patients, clonotypic B cells expressing variant isotypes were detected both before and after culturing. The ability of clonotypic B cells to undergo B-cell differentiation was studied by abrogating CD40 signalling accompanied by IL-10 and IL-2 stimulation, enhancing differentiation towards Ig-secreting cells. The numbers of clonotypic B cells were determined by quantitative ASO-PCR. An increase in cell number was observed upon CD40L and IL-4 stimulation, whereas the relative number of clonotypic B cells was unaltered. In contrast, upon B-cell differentiation the relative number of clonotypic B cells decreased. In conclusion, clonotypic B cells can be cultured and isolated in vitro using the CD40 system. Clonotypic B cells responded to CD40 triggering in a similar fashion as to non-clonotypic normal B cells. However, the ability of clonotypic B cells to undergo in vitro activation and differentiation into Ig-secreting cells is hampered

Mobilized human CD34+hematopoietic stem cells enhance tumor growth in a nonobese diabetic/severe combined immunodeficient mouse model of human non-Hodgkin's lymphoma

Autologous peripheral blood stem cell mobilization is increasingly applied in the treatment of hematological malignancies. Despite the frequent clinical use in a setting of residual disease, it is not known whether mobilization of hematopoietic stem cells might facilitate tumor outgrowth in vivo. In the bone marrow, a bipotential precursor for hematopoietic and endothelial cells called hemangioblast exists. This hemangioblast, characterized by the expression of CD34 and vascular endothelial growth factor receptor (VEGFR)-2, is released from the bone marrow by mobilization and might be able to result in not only the generation of peripheral blood cells but vasculogenesis due to differentiation of the hemangioblast along the endothelial lineage [in addition to VEGFR-2 expression, angiopoietin-2 (ANG-2) expression can also be found in this stage]. New vessel formation in the tumor is critical for tumor growth. A xenotransplant model was established with 10 X 10(6) Daudi cells (non-Hodgkin's lymphoma) s.c. injected in the neck region of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, who were sublethally irradiated with 2 Gy. At day 10 after tumor inoculation, half of the mice were given 0.5 X 10(6) human CD34+ cells i.v., whereas the other half were given PBS i.v. The human CD34+ cells were obtained from leukapheresis samples of myeloma patients undergoing autologous peripheral blood stem cell mobilization. We compared tumor growth and human-specific VEGFR-2 and ANG-2 expression in the two groups. Tumor growth is enhanced 2-fold when mobilized hematopoietic human CD34+ cells are given compared with PBS controls (P = 0.004). In addition, the human-specific VEGFR-2 and ANG-2 reverse transcription-PCR was only positive in the tumors of mice i.v. injected with human CD34+ cells. This indicates that the injected human CD34+ cells home to the tumors and differentiate along the endothelial lineage. In the present study, we demonstrate that mobilized human CD34+ hematopoietic cells injected i.v. might facilitate the outgrowth of tumors in the setting of minimal residual disease. Malignant tumors are capable of incorporating human CD34+ hematopoietic cells. This study questions the safety of leukapheresis in patients with (residual) tumor and has important implications for further development of intensive chemotherapy protocols with autologous stem cell rescue