The role of IL-7 in lymphoid development and T cell homeostasis has been extensively documented. However, the role of IL-7 in human B cell development remains unclear. We used a xenogeneic human cord blood stem cell/murine stromal cell culture to study the development of CD19+ B-lineage cells expressing the IL-7R. CD34+ cord blood stem cells were cultured on the MS-5 murine stromal cell line supplemented with human G-CSF and stem cell factor. Following an initial expansion of myeloid/monocytoid cells within the initial 2 wk, CD19+/pre-BCR− pro-B cells emerged, of which 25–50% expressed the IL-7R. FACS-purified CD19+/IL-7R+ cells were larger and, when replated on MS-5, underwent a dose-dependent proliferative response to exogenous human IL-7 (0.01–10.0 ng/ml). Furthermore, STAT5 phosphorylation was induced by the same concentrations of human IL-7. CD19+/IL-7R− cells were smaller and did not proliferate on MS-5 after stimulation with IL-7. In a search for cytokines that promote human B cell development in the cord blood stem cell/MS-5 culture, we made the unexpected finding that murine IL-7 plays a role. Murine IL-7 was detected in MS-5 supernatants by ELISA, recombinant murine IL-7 induced STAT5 phosphorylation in CD19+/IL-7R+ pro-B cells and human B-lineage acute lymphoblastic leukemias, and neutralizing anti-murine IL-7 inhibited development of CD19+ cells in the cord blood stem cell/MS-5 culture. Our results support a model wherein IL-7 transduces a replicative signal to normal human B-lineage cells that is complemented by additional stromal cell-derived signals essential for normal human B cell development.

We hypothesized that IFN-A would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-A induced synergistic apoptosis in melanoma and other solid tumor cell lines. Apoptosis was associated with processing of procaspase-3, procaspase-7, procaspase-8, and procaspase-9 and with cleavage of Bid and poly(ADP-ribose) polymerase. Bortezomib plus IFN-A was effective at inducing apoptosis in melanoma cells that overexpressed Bcl-2 or Mcl-1, suggesting that this treatment combination can overcome mitochondrial pathways of cell survival and resistance to apoptosis. The proapoptotic effects of this treatment combination were abrogated by a caspase-8 inhibitor, led to increased association of Fas and FADD before the onset of cell death, and were significantly reduced in cells transfected with a dominant-negative FADD construct or small interfering RNA targeting Fas. These data suggest that bortezomib and IFN-A act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-A displayed statistically significant antitumor activity compared with either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts. These data support the future clinical development of bortezomib and IFN-A for malignant melanoma. [Cancer Res 2008;68(20):8351-60]

Mammalian B-cell development can be viewed as a developmental performance with several acts. The acts are represented by checkpoints centered around commitment to the B-lineage and functional Ig gene rearrangement--culminating in expression of the pre-B-cell receptor (pre-BCR) and the BCR. Progression of cells through these checkpoints is profoundly influenced by the fetal liver and adult bone marrow (BM) stromal cell microenvironments. Our laboratory has developed a model of human B-cell development that utilizes freshly isolated/non-transformed human BM stromal cells as an in vitro microenvironment. Human CD34+ hematopoietic stem cells plated in this human BM stromal cell microenvironment commit to the B lineage and progress through the pre-BCR and BCR checkpoints. This human BM stromal cell microenvironment also provides survival signals that prevent apoptosis in human B-lineage cells. Human B-lineage cells exhibit differential expression of Notch receptors and human BM stromal cells express the Notch ligand Jagged-1. These results suggest a potential role for Notch in regulating B-lineage commitment and/or progression through the pre-BCR and BCR checkpoints.

IL-7 signaling culminates in different biological outcomes in distinct lymphoid populations, but knowledge of the biochemical signaling pathways in normal lymphoid populations is incomplete. We analyzed CD127/IL-7Rα expression and function in normal (nontransformed) human thymocytes, and human CD19+ B-lineage cells purified from xenogeneic cord blood stem cell/MS-5 murine stromal cell cultures, to further clarify the role of IL-7 in human B cell development. IL-7 stimulation of CD34+ immature thymocytes led to phosphorylation (p-) of STAT5, ERK1/2, AKT, and glycogen synthase kinase-3 β, and increased AKT enzymatic activity. In contrast, IL-7 stimulation of CD34− thymocytes (that included CD4+/CD8+ double-positive, and CD4+ and CD8+ single-positive cells) only induced p-STAT5. IL-7 stimulation of CD19+ cells led to robust induction of p-STAT5, but minimal induction of p-ERK1/2 and p-glycogen synthase kinase-3 β. However, CD19+ cells expressed endogenous p-ERK1/2, and when rested for several hours following removal from MS-5 underwent de-phosphorylation of ERK1/2. IL-7 stimulation of rested CD19+ cells resulted in robust induction of p-ERK1/2, but no induction of AKT enzymatic activity. The use of a specific JAK3 antagonist demonstrated that all IL-7 signaling pathways in CD34+ thymocytes and CD19+ B-lineage cells were JAK3-dependent. We conclude that human CD34+ thymocytes and CD19+ B-lineage cells exhibit similarities in activation of STAT5 and ERK1/2, but differences in activation of the PI3K/AKT pathway. The different induction of PI3K/AKT may at least partially explain the different requirements for IL-7 during human T and B cell development.

IL-7 is an important cytokine