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.

Severe congenital neutropenia (SCN) is a syndrome characterized by an isolated block in granulocytic differentiation and an increased risk of developing acute myeloid leukemia (AML). Recent studies have demonstrated that the majority of patients with SCN and cyclic neutropenia, a related disorder characterized by periodic oscillations in the number of circulating neutrophils, have heterozygous germline mutations in the ELA2 gene encoding neutrophil elastase (NE). To test the hypothesis that these mutations are causative for SCN, we generated transgenic mice carrying a targeted mutation of their Ela2 gene ("V72M") reproducing a mutation found in 2 unrelated patients with SCN, one of whom developed AML. Expression of mutant NE mRNA and enzymatically active protein was confirmed. Mice heterozygous and homozygous for the V72M allele have normal numbers of circulating neutrophils, and no accumulation of myeloid precursors in the bone marrow was observed. Serial blood analysis found no evidence of cycling in any of the major hematopoietic lineages. Rates of apoptosis following cytokine deprivation were similar in wild-type and mutant neutrophils, as were the frequency and cytokine responsiveness of myeloid progenitors. The stress granulopoiesis response, as measured by neutrophil recovery after cyclophosphamide-induced myelosuppression, was normal. To define the leukemogenic potential of V72M NE, a tumor watch was established.

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.

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.

The role of IL-7 in human B-cell development is still not well understood in contrast to its role in murine B-cell development. Adult mice with targeted disruptions in the IL-7 receptor (R) alpha chain or IL-7 genes completely lack B-lineage cells, while human severe combined immunodeficiency (SCID) patients with mutations in the IL-7R alpha chain have normal numbers of circulating B-lymphocytes. The goal of this study was to re-examine the functional consequences of IL-7R signaling in human B-cell precursors. This was accomplished using a xenogeneic culture system consisting of human CD34+ cord blood hematopoietic stem cells (HSC) plated on the MS-5 murine stromal cell line supplemented with G-CSF and SCF. Following the robust development of monocyte-like precursors and cells encompassing the later stages of granulocyte differentiation by 2 weeks, cells that emerged at 2.5–4 weeks were phenotypically pro-B cells (CD19+/CD10+/CD20lo/CD22+/CD24+/CD34-/pre-BCR-). The pro-B cells harbored functional heavy chain rearrangements, since re-plating them on MS-5 stromal cells supplemented with anti-CD40 and IL-4 led to the development of pre-BCR+ cells. CD19+ cells purified from HSC/MS-5 cultures underwent rapid cell death within 24 hours, and IL-7 had minimal effect on survival. Purified CD19+ cells re-plated in direct contact with MS-5 stromal cells survived for 7–10 days, but did not proliferate. However, addition of human (h) IL-7 from 0.01 to 10 ng/mL promoted dose-dependent proliferation of CD19+ cells re-plated on MS-5. Approximately one-third of the CD19+ cells that emerged at 2.5–4 weeks expressed the IL-7R. FACS-purified IL-7R+/CD19+ cells were larger and proliferated on MS-5 in response to hIL-7, while IL-7R-/CD19+ cells were smaller and did not expand on MS-5 after stimulation with hIL-7. These results suggested that the IL-7R+/CD19+ cells that emerged in the HSC/MS-5 culture might be responding to murine (m) IL-7 produced by MS-5. In order to determine if endogenous IL-7 could be contributing to B-lymphopoiesis, murine and human-specific ELISAs were used to screen HSC/MS-5 culture supernatants. Supernatants from 3 and 4 week HSC/MS-5 cultures contained 20–30 pg/mL mIL-7 and 0.1–2.0 pg/mL hIL-7. Analysis of the kinetics of mIL-7 accumulation in steady-state cultures of confluent MS-5 (no HSC present) showed an increase from 8.0 pg/mL at 2 days, to nearly 150 pg/mL after 2.5 weeks. Flow cytometric analysis of STAT5 phosphorylation in IL-7R+/CD19+ cells demonstrated that hIL-7 and mIL-7 were capable of transducing a signal through the human IL-7R resulting in STAT5 phosphorylation. Furthermore, a monoclonal antibody to the human IL-7R alpha chain blocked STAT5 phosphorylation by both hIL-7 and mIL-7. Inclusion of goat anti-mouse IL-7 neutralizing antibody in HSC/MS-5 cultures effectively reduced the concentration of bioactively available mIL-7 to less than 6 pg/mL. This resulted in a 33% and 67% reduction in the number of CD19+ cells by 3 and 4 weeks, respectively. These collective results are the first to reveal a crucial role for stromal cell-derived IL-7 in promoting the proliferative expansion of human CD19+ B-lineage cells, and support a model wherein IL-7 can transduce a proliferative/replicative signal that cooperates with a stromal cell-derived co-stimulus to regulate development of human B-lineage cells.

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The IL-7Rα chain (CD127) is expressed on some, but not all, CD19+/surface μ-B-lineage cells isolated from fetal and adult marrow, and umbilical cord blood, but the functional consequences of CD127 expression in early human B cell development during all stages of human life are unresolved. We have described a xenogeneic culture model for generating CD19+ B-lineage cells from CD34+ cord blood stem cells following a 4 wk co-culture on murine MS-5 stromal cells (Johnson SE et al., 2005 J Immunol.). The majority of these cells express a pro-B cell phenotype i.e., CD19+/CD10+/CD20lo/CD21−/CD22+/CD24+/pre-BCR-. Moreover, 1–4% express surface μ heavy chains (HC), and some express κ or λ light chains. We have also reported that 20–40% of CD19+ cells emerging in the xenogeneic culture express CD127, CD127+ cells are more blastic than CD127- cells, and development of CD19+ cells is substantially blocked in xenogeneic cultures treated with anti-murine IL-7 (Johnson SE, ibid). The goal of the present study was to further characterize the developmental potential and patterns of gene expression in CD19+/CD127+ and CD19+/CD127− cells that emerge in xenogeneic cultures. We hypothesized that CD127 expression defines a subset of CD19+ B cell precursors with heightened potential to become mature B-lymphocytes. CD19+/CD127+ and CD19+CD127− populations that emerged in 4 wk xenogeneic cultures were sorted to > 95% purity by FACS. Immunofluorescent staining showed that surface and intracellular μ HC positive cells were present in both sorted populations at frequencies of 3–4%. Using an RT-PCR-based sequencing approach, we analyzed approximately 100 IgM V region sequences from both CD19+/CD127+ and CD19+/CD127− FACS-purified populations. The results indicated that the sequences from both populations were: non-mutated and used VH4, D and J gene segments that were similar to what would be expected in a cord blood B cell repertoire, and exhibited no statistically significant difference in N segment length, CDR3 length, or CDR3 charges. These results suggest that the amplified sequences were likely derived from the 3–4% cytoplasmic μ HC+ pre-B cells present in both sorted populations. The immunofluorescent staining and VDJH rearrangement sequence results suggest that large CD19+/CD127+/cytoplasmic μ HC+ pre-B cells may differentiate into small CD19+/CD127−/cytoplasmic μ HC+ cells in the xenogeneic culture. This was further supported by showing that intra-tibial injection of FACS-purified CD19+/CD127+ and CD19+/CD127− cells into sub-lethally irradiated NOD-SCID mice led to the appearance of surface μ HC+ cells from both sorted populations. Western blotting of sorted CD19+/CD127+ and CD19+/CD127− cells showed the presence of Bcl-2, p-GSK3β (specifically p-Ser9, the residue phosphorylated by AKT) and pERK1/2 in CD19+/CD127+ cells, while these proteins/phosphoproteins were undetectable in CD19+/CD127− cells. By contrast, expression of the cell cycle inhibitor p27KIP1 was elevated in CD19+/CD127− cells compared to CD19+/CD127+ cells. Furthermore, FACS-purified CD19+/CD127+ cells survived longer than CD19+/CD127− cells when cultured in the absence of MS-5, and the survival of the former was enhanced by stimulation with IL-7. The collective results indicate that CD127 expression identifies a population of normal human CD19+ B cell precursors with a pattern of gene expression and survival/proliferation attributes consistent with a crucial role in the development of the B-lymphocyte arm of the human immune system.