The Role of Interleukin-7 in the Regulation of Normal Human B Lymphopoiesis
The role of IL-7 in murine lymphoid development has been extensively documented, but the role in human B cell development remains unclear. Studies of human blood cell development have been facilitated by the use of in vitro stromal cell co-cultures. One murine stromal cell line, MS-5, was previously used to support human lymphohematopoiesis from cord blood CD34+ hematopoietic progenitors. The initial goal of this thesis was to identify the cytokines synthesized by MS-5 that support human B cell development. When cord blood CD34+ hematopoietic progenitors were plated on MS-5, a population of CD19+ pro-B cells developed of which ∼30% expressed the IL-7R (CD127). Murine IL-7 was detected in MS-5 supernatants and demonstrated to be biologically active for human CD19+ pro-B cells. Limiting the biological activity of mIL-7 inhibited the development of CD19+ cells in this model. However, IL-7 by itself could not promote proliferation. Although the complete identity of the MS-5 gene products that cooperate with IL-7 was not resolved, insulin-like growth factor 1 may play at least a partial role. The functional consequence of CD127 expression on human B-lineage cells was examined by two approaches. The first compared IL-7 signaling in human CD34+ thymocytes and CD19+ B-lineage cells. These two populations exhibited similarities in activation of STAT5 and ERK1/2, but differences in activation of the PI3K/AKT pathway. The second compared the survival, Ig gene rearrangement and protein expression characteristics of FACS-purified CD19+/CD127+ and CD19+/CD127- cells. CD19+/CD127+ cells from CD34+ cell/MS-5 cultures had enhanced survival capacity, DJH but few VDJH rearrangements, and essentially no IgL rearrangements. In contrast, CD19+/CD127- cells exhibited poor survival characteristics, similar IgH rearrangements, but extensive rearrangement at the IgL loci. These collective results indicate that IL-7 transduces a replicative signal to human B-lineage cells that is complemented by additional stromal cell-derived signals, and that signaling through CD127 is incompatible with the onset of IgL rearrangement. The xenogeneic model can now be used as a biological platform upon which future studies of the role of IL-7 and other cytokines in normal and abnormal (i.e., immune deficiencies and leukemia) human B cell development can be based.
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Characterization of the CD34+ cellMS5 xenogeneic
Murine and human IL7 activate STAT5 and induce
Stromal cell derived interleukin7 transduces a signal
alleles assay B-cell BD Biosciences bone marrow CD127 expression CD19+ B-lineage cells CD19+ cells CD34-enriched cells CD34+ cell/MS-5 culture CD34+ HSC CD34+ thymocytes cell development cells expressed cells Fig cells that emerged chemokine clone cord blood cord blood CD34+ CXCR4 cytokine data not shown detected differentiation fetal flow cytometry FLT3 gene rearrangement hematopoietic stem cells HSC/MS-5 cultures human B cell human B lymphopoiesis human B-lineage cells human CD19+ human IL-7 IgL rearrangements IL-7 induced IL-7 signaling IL-7 stimulation immunoglobulin Immunol incubated interleukin-7 Invitrogen Jak3 LeBien lineage lymphocytes lymphohematopoietic cells lymphoid Materials and Methods mFLT3L mice MS-5 stromal cells murine murine B lymphopoiesis murine IL-7 ng/ml number of CD19+ p-AKT phosphorylation PI3K plated pre-B pro-B cells proliferation protein receptor role of IL-7 siRNA staining STAT5 stromal cell stromal cell line supernatants transcription factor VDJH rearrangements vitro Western blotted wk CD34+ cell/MS-5 xenogeneic cultures µg/mL µHC