Nisha Shah

Murine and Human IL-7 Activate STAT5 and Induce Proliferation of Normal Human Pro-B Cells

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.

Microenvironmental influences on human B-cell development

Summary: 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.

IFN-alpha and bortezomib overcome Bcl-2 and Mcl-1 overexpression in melanoma cells by stimulating the extrinsic pathway of apoptosis.

We hypothesized that IFN-alpha would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-alpha 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-alpha 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-alpha act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-alpha 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-alpha for malignant melanoma.

IL-7 Activates the Phosphatidylinositol 3-Kinase/AKT Pathway in Normal Human Thymocytes but Not Normal Human B Cell Precursors

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-7R expression and IL-7 signaling confer a distinct phenotype on developing human B-lineage cells

IL-7 is an important cytokine for lymphocyte differentiation. Similar to what occurs in vivo, human CD19⁺ cells developing in human/murine xenogeneic cultures show differential expression of the IL-7 receptor α (IL-7Rα) chain (CD127). We now describe the relationship between CD127 expression/signaling and Ig gene rearrangement. In the present study, < 10% of CD19⁺CD127⁺ and CD19⁺CD127⁻ populations had complete VDJ(H) rearrangements. IGH locus conformation measurements by 3D FISH revealed that CD127⁺ and CD127⁻ cells were less contracted than pediatric BM pro-B cells that actively rearrange the IGH locus. Complete IGH rearrangements in CD127⁺ and CD127⁻ cells had smaller CDR3 lengths and fewer N-nucleotide insertions than pediatric BM B-lineage cells. Despite the paucity of VDJ(H) rearrangements, microarray analysis indicated that CD127⁺ cells resembled large pre-B cells, which is consistent with their low level of Ig light-chain rearrangements. Unexpectedly, CD127⁻ cells showed extensive Ig light-chain rearrangements in the absence of IGH rearrangements and resembled small pre-B cells. Neutralization of IL-7 in xenogeneic cultures led to an increase in Ig light-chain rearrangements in CD127⁺ cells, but no change in complete IGH rearrangements. We conclude that IL-7-mediated suppression of premature Ig light-chain rearrangement is the most definitive function yet described for IL-7 in human B-cell development.

B-cell development in the presence of the MLL/AF4 oncoprotein proceeds in the absence of HOX A7 and HOX A9 expression

Infant acute lymphoblastic leukemia (ALL) is frequently characterized by the t(4;11)(q21;q23) cytogenetic abnormality encoding the MLL/AF4 oncogene, increased HOX gene expression and a pro-B/monocytoid phenotype. We have previously established a novel MLL/AF4-positive cell line, B-lineage 3 (BLIN-3), which retains several features of normal B-lineage development (functional Ig gene rearrangement and apoptotic sensitivity to stromal cell withdrawal) not generally observed in infant ALL. We now use microarray analysis to identify patterns of gene expression in BLIN-3 that may modulate MLL/AF4 oncogenesis and contribute to the retention of normal B-lineage developmental characteristics. Comparison of 6815 expressed genes in BLIN-3 with published microarray data on leukemic blasts from t(4;11) patients indicated that BLIN-3 was unique in lacking the expression of certain HOX-A cluster genes. These results were validated by RT-PCR showing no expression of HOX A7 or HOX A9 in BLIN-3. A HOX C8 promoter reporter was active in BLIN-3, indicating that lack of HOX gene expression in BLIN-3 was not due to a nonfunctional MLL/AF4. Our results suggest that B-lineage development can proceed in t(4;11) leukemic blasts in the absence of HOX-A gene expression.