The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Johnson, S. E.
Right arrow Articles by LeBien, T. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Johnson, S. E.
Right arrow Articles by LeBien, T. W.
The Journal of Immunology, 2005, 175: 7325-7331.
Copyright © 2005 by The American Association of Immunologists

Murine and Human IL-7 Activate STAT5 and Induce Proliferation of Normal Human Pro-B Cells1

Sonja E. Johnson*, Nisha Shah*, Angela Panoskaltsis-Mortari*,{dagger} and Tucker W. LeBien2,*,{ddagger}

* The Cancer Center, {dagger} Department of Pediatrics, and {ddagger} Department of Laboratory Medicine/Pathology, University of Minnesota Medical School, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of IL-7 and its receptor subunits, the IL-7R{alpha} chain and the common {gamma} ({gamma}c)3 chain, have been studied in murine and human lymphopoiesis for a number of years (1, 2). We have previously discussed the similarities and differences in how IL-7 regulates murine and human B cell development in studies published through 2000 (3). Following the original description as a murine B-lineage stimulatory molecule in 1988 (4), the role of IL-7 was clarified by the demonstration that targeted disruption of the IL-7 (5) or IL-7R{alpha} chain (6) genes led to profound disruption of B cell development in adult mice. Targeted disruption of the genes encoding the {gamma}c subunit of the receptors for IL-2, 4, 7, 9, 15, and 21 (7, 8), and the Jak3 tyrosine kinase (9, 10) also led to severe disruptions in B cell development, consistent with the known structure of the IL-7 receptor and the activation of Jak3 following IL-7 stimulation. However, more recent studies have led to some modification of the earlier conclusions. Carvalho et al. (11) showed that IL-7-deficient mice undergo normal fetal and perinatal B lymphopoiesis, as reflected by normal B1 cell development and a normal-sized marginal zone B cell compartment. Thus, B cell development in fetal and neonatal mice is largely IL-7 independent. A difference in cytokine dependency in fetal vs adult murine B lymphopoiesis was revealed by the observation that thymic stromal lymphopoietin can induce proliferation in fetal pro-B cells (12). Furthermore, mice deficient for fms-like tyrosine kinase (FLT) 3 and the IL-7R{alpha} chain fail to develop fetal and neonatal B-lineage cells (13). Thus, IL-7, FLT3 ligand and thymic stromal lymphopoietin are the three cytokines that orchestrate fetal and adult murine B cell development.

Insight into the role of IL-7 in human B cell development has been aided by analysis of congenital immunodeficiency patients. Patients with X-linked SCID (XSCID) have mutations in the {gamma}c chain and have severe defects in T and NK cell development. However, they have normal or even elevated numbers of peripheral blood B cells (14). Furthermore, patients with autosomal recessive mutations in the Jak3 tyrosine kinase exhibit a developmental phenotype indistinguishable from XSCID, including normal numbers of peripheral blood B cells (15, 16). An initial report on two patients with mutations in the IL-7R{alpha} chain revealed the presence of normal/elevated numbers of peripheral blood B cells (17). Two recent reviews have reported that mutations in the IL-7R{alpha} chain are the third most common cause of SCID (18, 19), with the total number of such patients now close to 40. As with the initial report (17), an increase in the percentage or absolute number of peripheral blood B cells was observed in essentially all these patients (18, 19). In an interesting potential physiologic contrast, a subgroup of common variable immunodeficiency patients with elevated plasma levels of IL-7 (mechanism unknown) did not exhibit an increase in peripheral blood B cells (20). Using an in vitro human bone marrow (BM) (3) stromal cell culture, we demonstrated that human CD34+ hemopoietic stem cells (HSC) developed into B-lineage cells independent of IL-7 stimulation (21). These collective results indicated that IL-7 signaling was not essential for development of B-lineage cells in selected SCID patients and an in vitro model, but did not clarify the role of IL-7 in steady-state human B lymphopoiesis in vivo.

Human B cell development has also been studied using several murine stromal cell lines as an in vitro microenvironment, including: S17 (22, 23, 24), MS-5 (25, 26, 27, 28), and AFT-024 (29, 30). Although the origin of the murine stromal cells, the population of initiating human HSC and the culture conditions varied among these reports, several general conclusions could be drawn. First, plating cord blood CD34+ cells or sorted CD34+/CD38–/LIN– HSC onto the murine stromal cell lines S17 or MS-5 led to the appearance of CD19+ B-lineage cells that generally peaked at 4–6 wk (22, 26, 27, 28). Second, several different cytokine combinations were crucial for enhancing the appearance of CD19+ B-lineage cells, with the combination of G-CSF and stem cell factor (SCF) proving highly effective using the MS-5 stromal cell line (26, 27). Third, although the degree of B cell maturation was somewhat variable, in general, CD19+ B-lineage cells expressing the cytoplasmic µ H chain and/or the cell surface pre-BCR were rare. When additional stimuli such as IL-4 and CD40L were included, B cell differentiation to the immature/activated B cell stage occurred (23, 30). Notably, in none of these studies was the identity of the murine stromal cell-derived molecule (or molecules) essential for promoting development and/or proliferation of human CD19+ B-lineage cells characterized. In the present study, we report a crucial role for stromal cell-derived IL-7 in promoting the proliferative expansion of human CD19+ B-lineage cells, and propose a model wherein IL-7 cooperates with a costimulus to regulate development of human B-lineage cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Origin and isolation of cells

Human umbilical cord blood was obtained from the laboratory of Dr. J. Miller (Department of Medicine, University of Minnesota, Minneapolis, MN), following approval by the Institutional Review Board at the University of Minnesota. Cord blood was diluted in PBS, overlaid on Histopaque 1077 (Sigma-Aldrich) and centrifuged at 400 x g for 30 min. The upper plasma fraction was removed and the interface cells were collected, pooled, and washed twice with PBS. Cells were subsequently washed once in MACS buffer (1x PBS, 0.3% (w/v) BSA) and incubated for 30 min on ice with 0.5 µl of FcR block and 0.5 µl of anti-CD34 microbeads (Direct CD34 Progenitor Cell Isolation kit; Miltenyi Biotec) per 1 x 106 cells. After incubation, cells were washed twice, resuspended in MACS buffer, and passed through a MACS LS separation column (Miltenyi Biotec). CD34+ HSC were collected in MEM/10% FBS (Invitrogen Life Technologies).

CD19+ B-lineage cells were isolated from HSC/MS-5 cultures (see below) by initially harvesting culture supernatants containing loosely adherent cells, and then disrupting the entire content of the flasks using 0.25% trypsin (Invitrogen Life Technologies). This step was essential because examination of the cultures by inverted light microscopy revealed that many of the lymphohemopoietic cells had transmigrated underneath MS-5. After one MACS buffer wash, cells were incubated on ice for 30 min with 0.5 µl of anti-human CD19 microbeads (Miltenyi Biotec) per 1 x 106 cells. Cells were washed twice, resuspended in MACS buffer, and passed through a MACS LS separation column. CD19+ cells were collected in MEM/10% FBS. Alternatively, CD19+ B-lineage cells were isolated by negative selection. HSC/MS-5 cultures were harvested as described above, washed once with MACS buffer, resuspended at 1 x 107 cells/ml in MACS buffer, and incubated with biotinylated anti-CD11b (clone OKM-1) Ab at 12 µg/ml for 20 min at 4°C on a rotating mixer. Cells were washed once and incubated with 2 µl of mouse anti-biotin beads (Miltenyi Biotec) per 1 x 106 cells for 15 min on ice. Cells were washed twice, resuspended in MACS buffer, and passed over a MACS LS separation column. The flow-through fraction contained CD11b– cells that were very similar to CD19+ cells with respect to phenotype and light scatter (see below).

The murine stromal cell line MS-5 (31) was provided by Dr. P. Kincade, Oklahoma Medical Research Foundation (Oklahoma City, OK). MS-5 stromal cells were maintained in DMEM supplemented with 10% FBS. The BLIN-1 pre-B acute lymphoblastic leukemia (ALL) cell line was maintained in RPMI 1640/10% FBS (32).

Flow cytometry/cell sorting

All flow cytometry was performed on a FACSCalibur (BD Biosciences Immunocytometry Systems) using standard single or multicolor immunofluorescent staining protocols (33). Mouse mAb against the following human cell surface molecules and their fluorescent labels were purchased from BD Biosciences/BD Pharmingen: CD5-FITC (BD 347303), CD10-biotin (custom made), CD14-FITC (BD 555397), CD15-FITC (BD 347423), CD20-PE (BD 34767), CD21-PE (BD 555422), CD33-PE (BD 347785), CD34-FITC (BD 348053), CD45RA-biotin (custom made), CD135-PE (BD 558996), CD184-PE (BD 555974). Anti-CD23-FITC (product no. 9580-02) was obtained from Southern Biotechnology Associates. A PE-conjugated mAb to the IL-7R{alpha} chain (CD127) was obtained from Beckman Coulter. Mouse mAb against the following cell surface molecules were purified and conjugated in our laboratory: CD11b-biotin (clone OKM-1), CD19-allophycocyanin (clone 25C1), CD24 (clone BA-1), CD40 (clone EA-5), CD49d-biotin (clone P4C2), and human µ H chain-biotin (clone HB57). The human VpreB-specific mAb VpreB8 (conjugated to biotin in our laboratory) was a gift from M. Cooper, University of Alabama at Birmingham (Birmingham, AL) (34). FITC goat anti-mouse Ig (catalog no. 1031-02; Southern Biotechnology Associates) and streptavidin-PE (Molecular Probes) were used to detect unconjugated and biotinylated mAb, respectively.

Phosphoprotein flow cytometry was used to detect phosphorylation of STAT5 using the protocols published by Nolan and colleagues (35, 36). The mAb used to detect phosphorylated STAT5 was anti-phospho-STAT5 (Y694) conjugated to Alexa 488 (catalog no. 612598; BD Biosciences). Staining was conducted according to the manufacturer’s protocol.

CD19+ cells isolated by positive selection were adjusted to 1 x 107 cells/ml and stained with PE-anti-human IL-7R/CD127 for 20 min at 4°C. IL-7R+ and IL-7R– cells were then sorted on a triple laser FACSAria (BD Immunocytometry Systems).

HSC/MS-5 culture system

Freshly isolated CD34+ HSC were plated on confluent monolayers of MS-5 stromal cells in T25 filter-top tissue culture flasks (BD Biosciences) at 5 x 104 cells/flask in 5 ml of MEM/10% FBS. Cultures were supplemented with 10 ng/ml G-CSF and 10 ng/ml SCF (PeproTech), as previously described by others (26, 27). After 7 days, 4 ml of fresh MEM/10% FBS+ cytokines was added, and cultures were subsequently fed twice weekly.

Proliferation assay

CD19+ B-lineage cells were isolated by positive or negative selection, and plated on confluent monolayers of MS-5 stromal cells in 96-well flat-bottom plates (BD Biosciences) in 0.2 ml of MEM/10% FBS. Human and murine IL-7 (both from PeproTech) were added at the concentrations described in individual experiments. At harvest times, supernatants containing loosely adherent cells were pooled with trypsinized contents from each well, washed once in FACS buffer (PBS containing 2.5% FBS), and stained with allophycocyanin-conjugated anti-CD19. Following staining on ice for 15 min, a known quantity of Polybead 6-µm polystyrene Microspheres (Polysciences) was added to each tube. Cells were analyzed by flow cytometry and the total number of CD19+ cells/well was calculated as previously described (21).

Neutralizing Ab cultures

Human cord blood CD34+ HSC were isolated as described above and plated at ~1 x 103 cells/well in a 96-well flat-bottom plate containing confluent monolayers of MS-5. Alternatively, magnetic bead purified CD34+ HSC were held in Iscove’s medium at 4°C overnight, stained with anti-CD34-allophycocyanin Ab (BD Pharmingen), and sorted with a FACSVantage (BD Immunocytometry Systems) into individual wells (500 cells/well) of a 96-well flat-bottom plate containing confluent monolayers of MS-5. Feedings consisted of half-medium changes twice weekly, followed by addition of either 10 µg/ml goat anti-human IL-7 or 10 µg/ml goat anti-mouse IL-7 (both Abs from R&D Systems), commencing at day 7. The 50% neutralization dose for these two reagents was essentially identical: 1–2 µg/ml for goat anti-human IL-7 and 0.5–1.5 µg/ml for anti-mouse IL-7. Six wells per condition were harvested at 3 and 4 wk, and quantitation of CD19+ cells was conducted as described above.

ELISA

Human IL-7 was detected using a high sensitivity human-specific IL-7 ELISA kit (R&D Systems; assay sensitivity 0.1 pg/ml). Murine IL-7 was detected using a sandwich ELISA (developed by the Cytokine Reference Laboratory, University of Minnesota), using commercially available murine-specific reagents. Briefly, 96-well plates were coated with polyclonal goat anti-mouse IL-7 capture Ab (no. AF407; R&D Systems) at 5 µg/ml in PBS overnight. Wells were then blocked for 1 h in PBS containing 1% BSA and 5% sucrose, and samples (neat or diluted culture supernatants) were incubated for 2 h. Wells were then incubated with biotinylated polyclonal goat anti-mouse IL-7 sandwich Ab (no. BAF407; R&D Systems) at 300 ng/ml in TBS-Tween 20, 0.1% BSA for 2 h, followed by HRP-labeled streptavidin for 20 min. Color was developed using trimethylbenzidine substrate and the reaction was terminated with H2SO4. OD measurements were read at 450 nm with a correction wavelength of 540 nm on a Bio-Rad 550 plate reader. IL-7 values were interpolated from a standard curve using recombinant murine IL-7 (R&D Systems). All incubations were conducted at room temperature with washes between steps using PBS/0.05% Tween 20. The sensitivity of the murine IL-7 ELISA was 10 pg/ml.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Development of CD19+ cells in the HSC/MS-5 culture system

Several laboratories have used the murine MS-5 stromal cell line to support development of CD19+ B-lineage cells from various populations of cord blood CD34+ HSC (25, 26, 27, 28), but none of these reports described the expression and function of the IL-7R. The results in Fig. 1A portray the emergence of CD19+ cells over a 1-mo period. Following an input of 200 CD34+ HSC, the cultures reached ~3 x 105 total hemopoietic cells/ml by 2 wk, and remained at that density through 4 wk. The initial burst of hemopoiesis resulted in the appearance of monocyte-like precursors and cells in the later stages of granulocyte differentiation. CD19+ B-lineage cells were detected 2 wk following culture initiation and underwent a >2-log increase between 2 and 4 wk. After 3–4 wk, CD19+ cells represented 21 ± 11% (range = 5–42%, n = 17 donors) of total hemopoietic cells. Limiting dilution analysis revealed that myeloid and/or B-lineage lymphoid expansion occurred following plating of CD34+ HSC at 10 or 1 cell/well, with cloning efficiencies of ~25% (data not shown).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 1. Development and phenotype of CD19+ B-lineage cells from cord blood CD34+ HSC cultured on the murine stromal cell line MS-5. A, Cord blood CD34+ HSC were isolated with anti-CD34 magnetic beads, and plated into 96-well flat-bottom plates containing MS-5 monolayers in MEM/10% FBS supplemented with 10 ng/ml each of G-CSF and SCF. Total lymphohemopoietic cells (total cells) and CD19+ cells were quantified on a weekly basis by evaluating the entire contents of each well. Each value represents the mean ± SD of triplicate values. The arrow on the y-axis shows the input number of cells (200 cells/well). Note the y-axis is shown on a log scale. B, Phenotype of CD19+ B-lineage cells isolated from 3 to 4 wk cord blood CD34+ HSC/MS-5 stromal cell cultures. Filled histograms represent the isotype-staining controls and thin lines represent specific Ab staining. The lower right corner shows the light scatter profile of CD19+ cells isolated with anti-CD19 magnetic beads. All fluorescent stainings were analyzed on the gated cells with lymphoid light scatter characteristics shown in the lower right corner dot plot.

 
Fig. 1B shows a representative phenotype of CD19+ cells purified from 3 to 4 wk HSC/MS-5 cultures. Essentially all cells expressed CD10, CD19, CD22, CD24, and the {beta}1 integrin VLA-4/CD49d. CD19+ cells that emerged in cultures initiated with CD34+ HSC from multiple cord blood donors heterogeneously expressed other cell surface molecules including CD20, CD33, CD40, and CD45RA; however, the staining pattern was invariant from donor to donor. CD19+ cells did not express CD5, CD11b, CD34, or the µ H chain and VpreB subunits of the pre-BCR. However, when µ H chain and VpreB expression were examined by dot plot, a small number (< 5%) of pre-BCR+ cells were occasionally present. Analysis of the IL-7R and CXCR4 indicated variable expression on CD19+ cells (Fig. 1B). The aggregate data indicated that 32 ± 15% (range = 5–66%, n = 30 donors) of CD19+ cells expressed the IL-7R, and 39 ± 26% (range = 11–91%, n = 9 donors) of CD19+ cells expressed CXCR4. FLT3 was very weakly expressed (Fig. 1B) and in some donors was not detectable.

Influence of IL-7 on CD19+ cell survival and proliferation

We next evaluated whether the expression of IL-7R had functional consequences for survival/proliferation of CD19+ B-lineage cells. CD19+ cells were enriched to 90–95% purity following removal from HSC/MS-5 cultures (Fig. 1B). When CD19+ cells were cultured overnight they underwent apoptosis and were >75% dead after 24 h; in the absence or presence of IL-7 (data not shown). When CD19+ cells were replated on fresh MS-5 without cytokine supplementation they survived for 7 days, but did not proliferate (Fig. 2A). However, survival on MS-5 was somewhat variable. In other experiments, the CD19+ cell number gradually decreased over 7 days to 20–30% of input. CD19+ cells replated on fresh MS-5 stromal cells supplemented with increasing concentrations of human IL-7 underwent a dose-dependent proliferative expansion (Fig. 2A). This result was observed in six similar experiments. CD19+ cells consistently responded to 100 pg/ml human IL-7 and in some experiments responded to 10 pg/ml. Therefore, although IL-7 had no effect on the survival of CD19+ cells in the absence of MS-5, CD19+ cells were very sensitive to IL-7 signaling in the presence of MS-5.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 2. CD19+ cells proliferate in response to IL-7 in contact with MS-5 stromal cells. A, CD19+ cells were purified from a 4-wk HSC/MS-5 culture with anti-CD19 magnetic beads and plated on MS-5 in MEM/10% FBS, without or with the indicated concentrations of human IL-7. CD19+ cells were quantified at the time points shown by harvesting the entire contents of each well. Each bar represents the mean ± SD of triplicate values. B, Survival and proliferation of CD19+/IL-7R+ cells. The FACS-purified cells from Fig. 3 were plated on fresh MS-5 stromal cells in MEM/10% FBS. IL-7R+ and IL-7R– cells were then incubated in medium alone, or supplemented with the indicated concentrations of human IL-7. CD19+ cell numbers were quantified at day 7 by harvesting the entire contents of each well. Each bar represents the mean ± SD of triplicate values. The arrow on the y-axis shows the input number of cells (1.8 x 104 cells/well). * and * indicate statistically significant (p < 0.01) differences compared with IL-7R+ and IL-7R– cells, respectively, incubated in medium alone for 7 days.

 
Because the IL-7R was expressed on a subpopulation of CD19+ cells (Fig. 1B), we determined whether IL-7R+ and IL-7R– cells had different biologic characteristics. CD19+ cells were stained with anti-IL-7R and sorted into CD19+/IL-7R+ and CD19+/IL-7R– populations. As shown in Fig. 3, the two populations exhibited distinct light scatter profiles. CD19+/IL-7R– cells were predominantly small cells with low forward and side scatter characteristics, whereas CD19+/IL-7R+ cells were distinctively larger with greater heterogeneity in forward scatter. Fig. 3 also shows that >95% purity was achieved by sorting CD19+ cells into IL-7R+ and IL-7R– populations. The sorted populations were then plated on fresh MS-5 monolayers and stimulated with IL-7 concentrations ranging from 0.01–100 ng/ml. The results in Fig. 2B demonstrate that IL-7R+ cells showed only a small (~30%) decrease in survival after 7 days on MS-5, and responded to IL-7 similarly to total CD19+ cells (Fig. 2A). In contrast, CD19+/IL-7R– cells did not proliferate in response to IL-7 and were markedly impaired in their capacity to survive on MS-5 alone.



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 3. FACS purification of CD19+/IL-7R+ cells. CD19+ cells were purified with anti-CD19 magnetic beads, stained with PE-conjugated anti-human IL-7R/CD127 and sorted on a BD FACSAria. The IL-7R staining of the presorted CD19+ cells is shown in the upper left dot plot (inset shows control staining), and the light scatter characteristics of the IL-7R+ cells (black dots) and IL-7R– cells (gray dots) are shown in the upper right dot plot. The postsort purities of the IL-7R– and IL-7R+ populations are shown as contour plots (generated by reanalysis on the FACSAria).

 
IL-7 signaling

Previous studies have shown that IL-7 binding to the IL-7R induces phosphorylation (PO4) of STAT5 in B-lineage cells (37, 38, 39). In preliminary experiments, we confirmed the capacity of IL-7 to induce concentration and time-dependent STAT5-PO4 in the IL-7R+ BLIN-1 pre-B ALL cell line, with maximum STAT5-PO4 occurring at 50 ng/ml for 15 min (data not shown).

We next determined whether CD19+/IL-7R+ cells that emerged in HSC/MS-5 cultures underwent STAT5-PO4 following IL-7 signaling. Fig. 4A shows that CD19+ cells exhibited a dose-dependent STAT5-PO4 response to IL-7. Subtle, but detectable, STAT5-PO4 occurred at 0.1 ng/ml human IL-7. Results in Fig. 4B indicated that STAT5-PO4 was detected in IL-7-stimulated CD19+ cells within 5 min, peaked at 30–60 min, and remained constant at 120 min.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 4. IL-7 stimulation of STAT5-PO4 in normal CD19+ B-lineage cells isolated from HSC/MS-5 cultures. CD19+ cells were isolated from 4 wk HSC/MS-5 cultures using anti-CD19 magnetic beads and resuspended in MEM/10% FBS. A, CD19+ cells were stimulated with the indicated concentrations of IL-7 (ng/ml) for 30 min and assayed for STAT5-PO4 by phosphoprotein flow cytometry. The shaded histograms and thin lines represent unstimulated and IL-7-stimulated CD19+ cells, respectively. The values in each histogram correspond to the percentage of total CD19+ cells that underwent STAT5-PO4. The histogram to the left shows the expression of the IL-7R (thin line) on CD19+ cells used in the STAT5-PO4 analysis. B, CD19+ cells were stimulated with 50 ng/ml IL-7 for the indicated times and assayed for STAT5-PO4 by phosphoprotein flow cytometry. The shaded histograms and thin lines represent unstimulated and IL-7-stimulated CD19+ cells, respectively. The values in each histogram correspond to the percentage of total CD19+ cells that underwent STAT5-PO4.

 
Role of murine IL-7 signaling

Given the results in Figs. 2 and 3 showing the differences between CD19+/IL-7R+ and CD19+/IL-7R– B-lineage cells in light scatter profile and IL-7 responsiveness, we considered the possibility that endogenous IL-7 was being produced in the HSC/MS-5 culture. As shown in Table I, murine IL-7 was detected in concentrations ranging from 14 to 38 pg/ml in MS-5 supernatants or supernatants from MS-5 stromal cells incubated with G-CSF and SCF. Murine IL-7 was also detected in 3 and 4 wk HSC/MS-5 cultures, in the absence or presence of G-CSF and SCF. Very small quantities of human IL-7 were detected at 3 and 4 wk in HSC/MS-5 culture supernatants (Table I), and in other experiments 2 pg/ml was detected. As controls for the species specificity of the IL-7 ELISA, human IL-7 was not detected in MS-5 stromal cell supernatants and murine IL-7 was not detected in CD34+ HSC incubated with G-CSF and SCF.


View this table:
[in this window]
[in a new window]
  		Table I. Human and murine IL-7 are present in HSC/MS-5 cultures

 
We then determined whether murine IL-7 would induce STAT5-PO4 in human CD19+/IL-7R+ cells that emerged in HSC/MS-5 cultures. A side-by-side comparison of murine and human IL-7 showed that murine IL-7 induced STAT5-PO4, although on a concentration basis it was slightly less effective than human IL-7 (Fig. 5A). Similar results were obtained using the BLIN-1 pre-B ALL cell line (data not shown). As an additional approach, we tested whether a mouse anti-human IL-7R Ab would inhibit STAT5-PO4. The results in Fig. 5B indicated that preincubation of BLIN-1 cells with anti-IL-7R inhibited STAT5-PO4 by murine and human IL-7. These collective results indicate that murine IL-7 can bind to the human IL-7R and initiate signaling.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 5. Normal CD19+ B-lineage cells undergo STAT5-PO4 in response to murine and human IL-7. A, CD19+ cells were isolated from a 4 wk HSC/MS-5 culture using anti-CD19 magnetic beads and resuspended in MEM/10% FBS. The cells were then stimulated with the indicated concentrations of murine or human IL-7 (ng/ml) for 30 min and assayed for STAT5-PO4 by phosphoprotein flow cytometry. The shaded histograms and thin lines represent unstimulated and IL-7-stimulated CD19+ cells, respectively. The values in each histogram correspond to the percentage of total CD19+ cells that underwent STAT5-PO4. B, The BLIN-1 pre-B ALL cell line was preincubated with IgG1 control (G1) or mouse anti-human IL-7R ({alpha}7R) for 20 min at 37°C. Cells were then stimulated with 50 ng/ml murine or human IL-7 or medium alone (no IL-7) for 60 min. STAT5-PO4 was then assayed by phosphoprotein flow cytometry.

 
To assess whether murine IL-7 produced in the HSC/MS-5 coculture had a functional role in the development of CD19+ B-lineage cells from CD34+ cord blood HSC, we asked whether a goat anti-murine IL-7 Ab would block human B cell development in this model. As shown in Fig. 6, treatment of HSC/MS-5 cultures with the goat anti-murine IL-7 neutralizing Ab dramatically decreased the number of CD19+ cells emerging after 4 wk. In three independent experiments, goat anti-mouse IL-7 inhibited the number of CD19+ B-lineage cells by 33 ± 7% (range 26–39%) at 3 wk and 67 ± 15% (range 55–84%) at 4 wk. In contrast, goat anti-human IL-7 at the identical concentration had no effect.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 6. Development of CD19+ B-lineage cells in HSC/MS-5 cultures is inhibited by goat anti-murine IL-7. Cord blood CD34+ HSC/MS-5 cultures were established as described in Fig. 1A. Beginning at 1 wk, and then twice per week thereafter, goat anti-mouse IL-7 or goat anti-human IL-7 were added at 10 µg/ml. CD19+ cells were quantified at 4 wk as described in Materials and Methods. Each bar represents the mean ± SD of six replicate wells. The input number of CD34+ HSC at culture initiation was 1 x 103 cells/well. *, p < 0.005 compared with medium alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Development of mammalian B-lineage cells in primary lymphoid tissue requires the functional expression of genes that regulate H chain and L chain gene rearrangement, and functional rearrangements that lead to sequential expression of the pre-BCR and BCR. In concert with these gene rearrangement requirements is the essential role of cytokines in regulating survival, proliferation, and differentiation of B-lineage cells. Murine (4) and human (40) IL-7 were discovered over 15 years ago. Yet, our understanding of their role in B and T cell development remains incomplete. The objective of this study was to re-examine the consequences of IL-7 signaling during human B cell development by focusing on the functional significance of IL-7R expression. We chose the previously described cord blood CD34+ HSC/MS-5 xenogeneic culture system (25, 26, 27, 28) because it supports robust myelopoiesis and lymphopoiesis, thereby providing an excellent model of hemopoiesis.

The results of our study demonstrate that the developmental kinetics and phenotype of CD19+ cells are similar to published studies using the murine MS-5 stromal cell line (25, 26, 27, 28), although we did not detect the degree of human B cell differentiation (i.e., development of BCR+ cells) described by others (27). A consistent outcome of cord blood CD34+ HSC differentiation into CD19+ B-lineage cells was the expression of the IL-7R on 25–50% of CD19+ cells (Figs. 1B, 3, and 4A). We obtained similar results analyzing IL-7R expression on freshly isolated fetal BM B-lineage cells with (41). Because cord blood CD34+ HSC are of fetal origin (indeed, possibly derived from fetal marrow), the expression of IL-7R on CD19+ B-lineage cells that develop in our cord blood HSC/MS-5 model may reflect what occurs during fetal B cell development in vivo. In contrast, a study of adult BM indicated that the IL-7R was virtually undetectable on CD19+ pro-B cells, but showed greater expression on CD34+/CD19– lymphoid progenitors (42). The collective results suggest that IL-7R expression/function may vary between fetal and adult human B cell development. Future studies comparing IL-7R signaling in the development of CD19+ B-lineage cells from cord blood and adult BM CD34+ HSC will provide further clarification.

An important observation in the current study was the signaling sensitivity of CD19+/IL-7R+ cells that developed in the CD34+ HSC/MS-5 xenogeneic culture. CD19+/IL-7R+ cells responded to picogram concentrations of IL-7 when replated on MS-5 stromal cells (Fig. 2B), and underwent STAT5-PO4 in an IL-7 dose-dependent manner (Fig. 5). The pronounced difference in light scatter characteristics between CD19+/IL-7R+ and CD19+/IL-7R– cells (Fig. 3) could be explained by endogenous stimulation of the CD19+/IL-7R+ cells by murine IL-7. Data in Figs. 5 and 6 provide strong evidence that murine IL-7 can transduce signals through the IL-7R on human B-lineage cells. Reduction of CD19+ cell numbers by goat anti-mouse IL-7 (Fig. 6) may reflect the neutralization of all available murine IL-7, and the residual CD19+ cells might then respond to some other cytokine. Alternatively, the neutralizing Ab may be only partially effective and/or some IL-7 may be inaccessible to the neutralizing Ab due to sequestration in the HSC/MS-5 culture. Optimal IL-7 signaling has been reported to depend upon IL-7 binding to heparan sulfate proteoglycans (43), and MS-5 stromal cells synthesize a complex array of cell matrix components (44). It is therefore possible that murine IL-7 synthesized by MS-5 is presented to human CD19+ B-lineage cells by heparan sulfate proteoglycans. Another study reported the proliferation of murine pro-B cells in response to a hybrid cytokine consisting of IL-7 and the hepatocyte growth factor {beta}-chain (45). We do not know whether hepatocyte growth factor {beta}-chain is present in HSC/MS-5 cultures and, if so, whether it binds to IL-7. Finally, some human stromal cells express the IL-7R (46). Thus, although unlikely, we cannot rule out the possibility that murine IL-7 binds to IL-7-binding proteins (IL-7R?) on MS-5. This interaction would trigger a feedback loop culminating in production of MS-5-derived cytokines that stimulate human B-lineage cells.

In one of the initial papers in the field, murine IL-7 was reported to be inactive against human cells (40). This conclusion was reinforced by enthalpy calculations derived from molecular modeling, arguing that murine IL-7 binds weakly to the human IL-7R (47). We were therefore surprised to discover that murine IL-7 can transduce signals through the IL-7R on human B-lineage cells. A previously published study offers a possible explanation (48). A virtual construction of a three-dimensional model of IL-7 structure was created, and a sequence bounded by Cys131 and Cys143 containing a potential hydrophobic surface was hypothesized to be important for protein interactions. Comparison of this region indicates that murine and human IL-7 share 12 of the 13 aa residues, with the only difference being an arginine in position 138 in humans and a glutamine in comparable position in the mouse (40). Thus, it is conceivable that this sequence (designated helix D) confers upon murine IL-7 the capacity to bind to the human IL-7R.

In conclusion, we have identified functional consequences of IL-7 signaling in human CD19+/IL-7R+ B-lineage cells. This is the first report to show that a stromal cell synthesizing endogenous IL-7 can regulate the development of normal human B-lineage cells from HSC. How do we reconcile results in the current study with the presence of circulating B cells in SCID patients with mutations in the IL-7R (17, 18, 19), {gamma}c (14), and Jak3 (15, 16)? We favor a model wherein IL-7 does play an important role in human B cell development–exerting a replicative stimulus upon pro-B cells that, however, requires an additional cytokine provided by stromal cells in the BM microenvironment. This unknown cytokine acts independent of the {gamma}c subunit family of IL receptors, and may compensate for the absence of signaling through the IL-7R in SCID patients. We emphasize that only limited information is available on the status of B cell development in SCID patients with IL-7R mutations, largely restricted to quantitation of circulating B cells (18, 19). Thus, although their circulating B cell numbers are normal, other IL-7-dependent events may be absent or reduced, but go undetected. Finally, because we have shown that murine IL-7 stimulates human B-lineage cells, our results may provide at least a partial explanation for why human CD34+ HSC transplanted into NOD-SCID mice can develop into human CD19+ B-lineage cells (49, 50, 51, 52).


   Acknowledgments
 
We gratefully acknowledge the technical assistance of Rebecca Asch and Nicole Owen. We thank Melinda Koch for assistance with the ELISA, and Joel Sederstrom and Greg Veltri (Flow Cytometry Core, University of Minnesota Cancer Center) for cell sorting.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant RO1 CA31685 and the Leukemia Research Fund. S.E.J. was supported by Immunology Training Grant T32 AI07313. T.W.L. is the Apogee Enterprises Chair in Cancer Research. Back

2 Address correspondence and reprint requests to Dr. Tucker W. LeBien, Department of Laboratory Medicine/Pathology and The Cancer Center, University of Minnesota, MMC806, 420 Delaware Street SE, Minneapolis, MN 55455. E-mail address: lebie001{at}umn.edu Back

3 Abbreviations used in this paper: {gamma}c, common {gamma}; FLT, fms-like tyrosine kinase; XSCID, X-linked SCID; BM, bone marrow; HSC, hemopoietic stem cell; SCF, stem cell factor; ALL, acute lymphoblastic leukemia. Back

Received for publication June 17, 2005. Accepted for publication September 19, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kang, J., S. D. Der. 2004. Cytokine functions in the formative stages of a lymphocyte’s life. Curr. Opin. Immunol. 16: 180-190. [Medline]
  2. Fry, T. J., C. L. Mackall. 2002. Interleukin-7: from bench to clinic. Blood 99: 3892-3904. [Free Full Text]
  3. LeBien, T. W.. 2000. Fates of human B-cell precursors. Blood 96: 9-23. [Abstract/Free Full Text]
  4. Namen, A. E., S. Lupton, K. Hjerrild, J. Wignall, D. Y. Mochizuki, A. Schmierer, B. Mosley, C. J. March, D. Urdal, S. Gillis, et al 1988. Stimulation of B-cell progenitors by cloned murine interleukin-7. Nature 333: 571-573. [Medline]
  5. von Freeden-Jeffrey, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181: 1519-1526. [Abstract/Free Full Text]
  6. Peschon, J. J., P. J. Morrissey, K. H. Grabstein, F. J. Ramsdell, E. Maraskovsky, B. C. Gliniak, L. S. Park, S. F. Ziegler, D. E. Williams, C. B. Ware, et al 1994. Early lymphocyte expansion is severely impaired in interleukin-7 receptor-deficient mice. J. Exp. Med. 180: 1955-1960. [Abstract/Free Full Text]
  7. Cao, X., E. W. Shores, J. Hu-Li, M. R. Anver, B. L. Kelsall, S. M. Russell, J. Drago, M. Noguchi, A. Grinberg, E. T. Bloom, et al 1995. Defective lymphoid development in mice lacking expression of the common cytokine receptor {gamma} chain. Immunity 2: 223-238. [Medline]
  8. DiSanto, J. P., W. Muller, D. Guy-Grand, A. Fischer, K. Rajewsky. 1995. Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor {gamma} chain. Proc. Natl. Acad. Sci. USA 92: 377-381. [Abstract/Free Full Text]
  9. Nosaka, T., J. M. van Deursen, R. A. Tripp, W. E. Thierfelder, B. A. Witthuhn, A. P. McMickle, P. C. Doherty, G. C. Grosveld, J. N. Ihle. 1995. Defective lymphoid development in mice lacking Jak3. [Published erratum appears in 1996 Science 271:17]. Science 270: 800-802. [Abstract/Free Full Text]
  10. Thomis, D. C., C. B. Gurniak, E. Tivol, A. H. Sharpe, L. J. Berg. 1995. Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3. Science 270: 794-797. [Abstract/Free Full Text]
  11. Carvalho, T. L., T. Mota-Santos, A. Cumano, J. Demengeot, P. Vieira. 2001. Arrested B lymphopoiesis and persistence of activated B cells in adult interleukin 7(–/–) mice. J. Exp. Med. 194: 1141-1150. [Abstract/Free Full Text]
  12. Vosshenrich, C. A., A. Cumano, W. Muller, J. P. Di Santo, P. Vieira. 2003. Thymic stromal-derived lymphopoietin distinguishes fetal from adult B cell development. Nat. Immunol. 4: 773-779. [Medline]
  13. Sitnicka, E., C. Brakebusch, I. L. Martensson, M. Svensson, W. W. Agace, M. Sigvardsson, N. Buza-Vidas, D. Bryder, C. M. Cilio, H. Ahlenius, et al 2003. Complementary signaling through flt3 and interleukin-7 receptor {alpha} is indispensable for fetal and adult B cell genesis. J. Exp. Med. 198: 1495-1506. [Abstract/Free Full Text]
  14. Noguchi, M., H. Yi, H. M. Rosenblatt, A. H. Filipovich, S. Adelstein, W.S. Modi, O. W. McBride, W. J. Leonard. 1993. Interleukin-2 receptor {gamma} chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 73: 147-157. [Medline]
  15. Macchi, P., A. Villa, S. Giliani, M. G. Sacco, A. Frattini, F. Porta, A. G. Ugazio, J. A. Johnston, F. Candotti, J. J. O’Shea. 1995. Mutations of Jak3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature 377: 65-68. [Medline]
  16. Russell, S. M., N. Tayebi, H. Nakajima, M. C. Riedy, J. L. Roberts, M. J. Aman, T. S. Migone, M. Noguchi, M. L. Markert, R. H. Buckley, et al 1995. Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development. Science 270: 797-800. [Abstract/Free Full Text]
  17. Puel, A., S. F. Ziegler, R. H. Buckley, W. J. Leonard. 1998. Defective IL7R expression in T–B+NK+ severe combined immunodeficiency. Nat. Genet. 20: 394-397. [Medline]
  18. Buckley, R. H.. 2004. Molecular defects in human severe combined immunodeficiency and approaches to immune reconstitution. Annu. Rev. Immunol. 22: 625-655. [Medline]
  19. Gillianim, S., L. Mori, G. de Saint Basile, F. Le Diest, C. Rodriguez-Perez, C. Forino, E. Mazzolari, S. Dupuis, R. Elhasid, A. Kessel, et al 2005. Interleukin-7 receptor a (IL-7Ra) deficiency; cellular and molecular bases: analysis of clinical, immunological, and molecular features in 16 novel patients. Immunol. Rev. 203: 110-126. [Medline]
  20. Holm, A. M., P. Aukrust, J. K. Damas, F. Muller, B. Halvorson, S. S. Froland. 2005. Abnormal interleukin-7 function in common variable immunodeficiency. Blood 105: 2887-2890. [Abstract/Free Full Text]
  21. Pribyl, J. A., T. W. LeBien. 1996. IL-7 independent development of human B cells. Proc. Natl. Acad. Sci. USA 93: 10348-10353. [Abstract/Free Full Text]
  22. Rawlings, D. J., S. G. Quan, R. M. Kato, O. N. Witte. 1995. Long-term culture system for selective growth of human B-cell progenitors. Proc. Natl. Acad. Sci. USA 92: 1570-1574. [Abstract/Free Full Text]
  23. Fluckiger, A. C., E. Sanz, M. Garcia-Lloret, T. Su, Q. L. Hao, S. Quan, A. de la Hera, G. M. Crooks, O. N. Witte, D. A. Rawlings. 1998. In vitro reconstitution of human B-cell ontogeny: from CD34+ multipotent progenitors to Ig-secreting cells. Blood 92: 4509-4520. [Abstract/Free Full Text]
  24. Hao, Q. L., J. Zhu, M. A. Price, K. J. Payne, L. W. Barsky, G. M. Crooks. 2001. Identification of a novel, human multilymphoid progenitor in cord blood. Blood 97: 3683-3690. [Abstract/Free Full Text]
  25. Berardi, A. C., E. Meffre, F. Pflumio, A. Katz, W. Vainchenker, C. Schiff, L. Coulombel. 1997. Individual CD34+CD38lowCD19–CD10– progenitor cells from human cord blood generate B lymphocytes and granulocytes. Blood 89: 3554-3564. [Abstract/Free Full Text]
  26. Nishihara, M., Y. Wada, K. Ogami, Y. Ebihara, T. Ishii, K. Tsuji, H. Ueno, S. Asano, T. Nakahata, T. Maekawa. 1998. A combination of stem cell factor and granulocyte colony-stimulating factor enhances the growth of human progenitor B cells supported by murine stromal cell line MS-5. Eur. J. Immunol. 28: 855-864. [Medline]
  27. Ohkawara, J. I., K. Ikebuchi, M. Fujihara, N. Sato, F. Hirayama, M. Yamaguchi, K. J. Mori, S. Sekiguchi. 1998. Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors. Leukemia 12: 764-771. [Medline]
  28. Rossi, M. I., T. Yokota, K. L. Medina, K. P. Garrett, P. C. Comp, A. H. Schipul, P. W. Kincade. 2003. B lymphopoiesis is active throughout human life, but there are developmental age-related changes. Blood 101: 576-584. [Abstract/Free Full Text]
  29. Miller, J. S., V. McCullar, M. Punzel, I. R. Lemischka, K. A. Moore. 1999. Single adult human CD34+/Lin–/CD38– progenitors give rise to natural killer cells, B-lineage cells, dendritic cells, and myeloid cells. Blood 93: 96-106. [Abstract/Free Full Text]
  30. Barker, J., C. M. Verfaillie. 2000. A novel in vitro model of early human adult B lymphopoiesis that allows proliferation of pro-B cells and differentiation to mature B lymphocytes. Leukemia 14: 1614-1620. [Medline]
  31. Suzuki, J., J. Fujita, S. Taniguchi, K. Sugimoto, K. J. Mori. 1992. Characterization of murine hemopoietic-supportive (MS-1 and MS-5) and non-supportive (MS-K) cell lines. Leukemia 6: 452-458. [Medline]
  32. Wormann, B., J. M. Anderson, J. A. Liberty, K. Gajl-Peczalska, R. D. Brunning, T. L. Silberman, D. C. Arthur, T. W. LeBien. 1989. Establishment of a leukemic cell model for studying pre-B to B cell differentiation. J. Immunol. 142: 110-117. [Abstract]
  33. Bertrand, F. E., C. Vogtenhuber, N. Shah, T. W. LeBien. 2001. Pro-B cell to pre-B-cell development in B-lineage acute lymphoblastic leukemia expressing the MLL/AF4 fusion protein. Blood 98: 3398-3405. [Abstract/Free Full Text]
  34. Wang, Y. H., J. Nomura, O. M. Faye-Peterson, M. D. Cooper. 1998. Surrogate light chain production during B cell differentiation: differential intracellular versus cell surface expression. J. Immunol. 161: 1132-1139. [Abstract/Free Full Text]
  35. Krutzik, P. O., G. P. Nolan. 2003. Intracellular phosphor-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry 55A: 61-70. [Medline]
  36. Irish, J. M., R. Hovland, P. O. Krutzik, O. D. Perez, O. Bruserud, B. T. Gjertsen, G. P. Nolan. 2004. Single cell profiling of potentiated phosphor-protein networks in cancer cells. Cell 118: 217-228. [Medline]
  37. van der Plas, D. C., F. Smiers, K. Pouwels, L. H. Hoefsloot, B. Lowenberg, I. P. Touw. 1996. Interleukin-7 signaling in human B cell precursor acute lymphoblastic leukemia cells and murine BAF3 cells involves activation of STAT1 and STAT5 mediated by the interleukin-7 receptor {alpha} chain. Leukemia 10: 1317-1325. [Medline]
  38. Jiang, Q., W. Q. Li, R. R. Hofmeister, H. A. Young, D. R. Hodge, J. R. Keller, A. R. Khaled, S. K. Durum. 2004. Distinct regions of the interleukin-7 receptor regulate different Bcl2 family members. Mol. Cell Biol. 24: 6501-6513. [Abstract/Free Full Text]
  39. Stanton, M. L., P. H. Brodeur. 2005. Stat5 mediates the IL-7-induced accessibility of a representative D-distal VH gene. J. Immunol. 174: 3164-3168. [Abstract/Free Full Text]
  40. Goodwin, R. G., S. Lupton, A. Schmierer, K. J. Hjerrild, R. Jerzy, W. Clevenger, S. Gillis, D. Cosman, A. E. Namen. 1989. Human interleukin 7: molecular cloning and growth factor activity on human and murine B-lineage cells. Proc. Natl. Acad. Sci. USA 86: 302-306. [Abstract/Free Full Text]
  41. Dittel, B. N., T. W. LeBien. 1995. The growth response to IL-7 during normal human B cell ontogeny is restricted to B-lineage cells expressing CD34. J. Immunol. 154: 58-67. [Abstract]
  42. Ryan, D. H., B. L. Nuccie, I. Ritterman, J. L. Liesveld, C. N. Abboud, R. A. Insel. 1997. Expression of interleukin-7 receptor by lineage-negative human bone marrow progenitors with enhanced lymphoid proliferative potential and B-lineage differentiation capacity. Blood 89: 929-940. [Abstract/Free Full Text]
  43. Borghesi, L. A., Y. Yamashita, P. W. Kincade. 1999. Heparan sulfate proteoglycans mediate interleukin-7-dependent B lymphopoiesis. Blood 93: 140-148. [Abstract/Free Full Text]
  44. Drzeniek, Z., B. Siebertz, G. Stocker, U. Just, W. Ostertag, H. Greiling, H.-D. Haubeck. 1997. Proteoglycan synthesis in hematopoietic cells: isolation and characterization of heparan sulphate proteoglycans expressed by the bone marrow stromal cell line MS-5. Biochem. J. 327: 473-480.
  45. Lai, L., I. Goldschneider. 2001. Cutting edge: identification of a hybrid cytokine consisting of IL-7 and the {beta}-chain of the hepatocyte growth factor/scatter factor. J. Immunol. 167: 3550-3554. [Abstract/Free Full Text]
  46. Pilllai, M., B. Torok-Storb, M. Iwata. Expression and function of IL-7 receptors in marrow stromall cells. Leuk. Lymphoma. 45: 2403-2408.
  47. Kroemer, R. T., R. Kroncke, J. Gerdes, W. G. Richards. 1998. Comparison of the 3D models of four different human IL-7 isoforms with human and murine IL-7. Protein Engin. 11: 31-40. [Abstract/Free Full Text]
  48. Cosenza, L., A. Rosenbach, J. V. White, J. R. Murphy, T. S. Smith. 2000. Comparative model building of interleukin-7 using interleukin-4 as a template: a structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation. Protein Science 9: 916-926. [Abstract]
  49. Pflumio, F., B. Izac, A. Katz, L. D. Schultz, W. Vainchenker, L. Coulombel. 1996. Phenotype and function of human hematopoietic cells engrafting immune-deficient CB17-severe combined immunodeficiency mice and nonobese diabetic severe combined immunodeficiency mice after transplantation of human cord blood mononuclear cells. Blood 88: 3731-3740. [Abstract/Free Full Text]
  50. Bhatia, M., J. C. Y. Wang, U. Kapp, D. Bonnet, J. E. Dick. 1997. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc. Natl. Acad. Sci. USA 94: 5320-5325. [Abstract/Free Full Text]
  51. Hogan, C. J., E. J. Shpall, O. McNulty, I. McNiece, J. E. Dick, L. D. Schulz, G. Keller. 1997. Engraftment and development of human CD34+-enriched cells from umbilical cord blood in NOD/LtSz-scid/scid mice. Blood 90: 85-96. [Abstract/Free Full Text]
  52. Rossi, M. I., K. L. Medina, K. Garrett, G. Kolar, P. C. Comp, L. D. Schultz, J. D. Capra, P. Wilson, A. Schipul, P. W. Kincade. 2001. Relatively normal human hematopoiesis but rapid turnover of newly formed B cells in transplanted nonobese diabetic/SCID mice. J. Immunol. 167: 3033-3042. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. E. Johnson, N. Shah, A. A. Bajer, and T. W. LeBien
IL-7 Activates the Phosphatidylinositol 3-Kinase/AKT Pathway in Normal Human Thymocytes but Not Normal Human B Cell Precursors
J. Immunol., June 15, 2008; 180(12): 8109 - 8117.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. R. Simpson-Abelson, G. F. Sonnenberg, H. Takita, S. J. Yokota, T. F. Conway Jr., R. J. Kelleher Jr., L. D. Shultz, M. Barcos, and R. B. Bankert
Long-Term Engraftment and Expansion of Tumor-Derived Memory T Cells Following the Implantation of Non-Disrupted Pieces of Human Lung Tumor into NOD-scid IL2R{gamma}null Mice
J. Immunol., May 15, 2008; 180(10): 7009 - 7018.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
D. de Totero, R. Meazza, M. Capaia, M. Fabbi, B. Azzarone, E. Balleari, M. Gobbi, G. Cutrona, M. Ferrarini, and S. Ferrini
The opposite effects of IL-15 and IL-21 on CLL B cells correlate with differential activation of the JAK/STAT and ERK1/2 pathways
Blood, January 15, 2008; 111(2): 517 - 524.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. W. Lim and C. H. Kim
Loss of IL-7 Receptor {alpha} on CD4+ T Cells Defines Terminally Differentiated B Cell-Helping Effector T Cells in a B Cell-Rich Lymphoid Tissue
J. Immunol., December 1, 2007; 179(11): 7448 - 7456.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
V. I. Brown, J. Hulitt, J. Fish, C. Sheen, M. Bruno, Q. Xu, M. Carroll, J. Fang, D. Teachey, and S. A. Grupp
Thymic Stromal-Derived Lymphopoietin Induces Proliferation of Pre-B Leukemia and Antagonizes mTOR Inhibitors, Suggesting a Role for Interleukin-7R{alpha} Signaling
Cancer Res., October 15, 2007; 67(20): 9963 - 9970.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
S. L. Kalis, S.-K. Zhai, P.-C. Yam, P. L. Witte, and K. L. Knight
Suppression of B lymphopoiesis at a lymphoid progenitor stage in adult rabbits
Int. Immunol., June 1, 2007; 19(6): 801 - 811.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
T. W. LeBien
Battling bone marrow B-lineage cells
Blood, August 15, 2006; 108(4): 1120 - 1120.
[Full Text] [PDF]

Home page
 BloodHome page
A. Liu, C. A. J. Vosshenrich, C. Lagresle-Peyrou, M. Malassis-Seris, C. Hue, A. Fischer, J. P. Di Santo, and M. Cavazzana-Calvo
Competition within the early B-cell compartment conditions B-cell reconstitution after hematopoietic stem cell transplantation in nonirradiated recipients
Blood, August 15, 2006; 108(4): 1123 - 1128.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me