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.
The role of IL-7 and its receptor subunits, the IL-7Rα chain and the common γ (γc)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-7Rα chain (6) genes led to profound disruption of B cell development in adult mice. Targeted disruption of the genes encoding the γc 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-7Rα 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 γc 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-7Rα chain revealed the presence of normal/elevated numbers of peripheral blood B cells (17). Two recent reviews have reported that mutations in the IL-7Rα 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
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 × 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 (1× 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 × 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 × 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 × 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 × 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-7Rα 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 × 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 × 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.
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 × 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.
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.
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. 1 A portray the emergence of CD19+ cells over a 1-mo period. Following an input of 200 CD34+ HSC, the cultures reached ∼3 × 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).
Fig. 1,B 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 β1 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. 1,B). 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. 1 B) 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. 1,B). 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. 2,A). 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. 2 A). 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.
Because the IL-7R was expressed on a subpopulation of CD19+ cells (Fig. 1,B), 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. 2,B 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. 2 A). 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.
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. 4,A 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. 4 B 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.
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.
|.||Week 1 .||Week 2 .||Week 3 .||Week 4 .|
|.||Murine/Humana .||Murine/Human .||Murine/Human .||Murine/Human .|
|MS-5 (G + S)||NS/NS||14/NS||32/NS||29/NS|
|CD34+/MS-5 (G + S)||NS/NS||NS/NS||20/0.1||19/0.2|
|CD34+ (G + S)||NS/NS||NS/NS||NS/NS||NS/NS|
|.||Week 1 .||Week 2 .||Week 3 .||Week 4 .|
|.||Murine/Humana .||Murine/Human .||Murine/Human .||Murine/Human .|
|MS-5 (G + S)||NS/NS||14/NS||32/NS||29/NS|
|CD34+/MS-5 (G + S)||NS/NS||NS/NS||20/0.1||19/0.2|
|CD34+ (G + S)||NS/NS||NS/NS||NS/NS||NS/NS|
All values are in picograms per milliliter.
NS, Not significantly above sensitivity threshold (0.1 pg/ml for hIL-7, 10.0 pg/ml for mIL-7).
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. 5,A). 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. 5 B 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.
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.
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. 1,B, 3, and 4 A). 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. 2,B), 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 β-chain (45). We do not know whether hepatocyte growth factor β-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), γc (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 γc 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).
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.
The authors have no financial conflict of interest.
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.
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.
Abbreviations used in this paper: γc, common γ; FLT, fms-like tyrosine kinase; XSCID, X-linked SCID; BM, bone marrow; HSC, hemopoietic stem cell; SCF, stem cell factor; ALL, acute lymphoblastic leukemia.