Human-only co-cultures support in vitro human B lymphopoiesis
We developed a novel, human-only in vitro model of B cell development based on co-culturing nonfetal human hematopoietic progenitors with primary human stroma from adult BM (). Initial experiments used CB as a source of HSCs. While CB contains fetally-generated B cells, the stem cells present in CB are of interest because they give rise to B cells in the neonatal period and are increasingly used clinically as a source of HSCs for transplant.
B cell production in human-only cultures was compared to that in an established hybrid co-culture model (45
) consisting of human HSCs and murine S17 stroma. (). The emergence of human B lineage cells in human-only cultures paralleled that observed in hybrid cultures with murine S17 stroma. CD19+ B lineage cells were undetectable at one week and rare at two weeks (data not shown). At three weeks, CD19+ B lineage cells were detectable in both human-only and hybrid S17 cultures under control conditions. However the frequency of B lineage cells was low in human-only cultures (, middle panel). With the addition of human IL-7, human-only cultures produced a substantial population of CD19+ cells (, middle panel). At 3 weeks, the CD19+ B lineage cells present in human-only cultures, like those in hybrid cultures, were B cell precursors that did not express the cytoplasmic mu (cμ) Ig heavy chain (, middle and left panels) or surface immunoglobulin (Ig) as evidenced by the absence of kappa and lambda Ig light chains (data not shown). At 5 weeks, surface IgM+ cells were observed in both human-only (, right panels) and hybrid co-cultures (data not shown), and while the production of cμ+ and IgM+ B cells in human-only cultures was inefficient (~11 to 16% of CD19+ cells), it was consistently higher than that observed in hybrid co-cultures (~3% of CD19+ cells). These data provide evidence that co-cultures with primary human BM stromal cells support the production of human B cells and establish human-only cultures as an in vitro
model for studying early stages of human B lymphopoiesis.
Exogenous IL-7 increases B cell production in human-only, but not hybrid co-cultures with murine stromal cell lines
Using human-only and hybrid S17 cultures we characterized the effect of IL-7 on the production of human B cell precursors from CB HSCs. The addition of IL-7 to human-only, but not hybrid S17 cultures, significantly increased the production of CD19+ B lineage cells as compared to cultures without IL-7 (). Stem cell factor (SCF) had no significant effect on human B cell production, either alone or in combination with IL-7 in human-only or hybrid S17 cultures (). Thus, human-only cultures revealed specific, IL-7 effects on human B cell production.
A titration of IL-7 into human-only cultures showed that IL-7-induced increases in the production of B lineage cells were dose-dependent and specific to CD19+ cells, achieving maximal effects (43-fold) at 10 ng/ml of IL-7 (). In contrast, IL-7-induced increases observed in hybrid S17 cultures at 3 weeks never averaged more than 3-fold at any concentration of IL-7 (). These data provide evidence that IL-7 induces a lineage-specific increase in the in vitro production of human B cell precursors that is not detectable in hybrid cultures with the murine S17 stromal cell line.
To determine if the inability to support IL-7-induced increases in human B cell production is specific to murine S17 stroma, we examined IL-7 effects in hybrid cultures with the MS-5 murine stromal cell line, another established model (42
) for in vitro
human B lymphopoiesis. As with S17 stroma, the addition of IL-7 did not significantly increase the production of human B cell precursors in hybrid MS-5 cultures (data not shown). However, substantial numbers of human B lineage cells were produced in the absence of exogenous IL-7 in hybrid cultures with murine S17 and MS-5 stroma ( and data not shown) suggesting that another factor replaces human IL-7 in hybrid human-mouse co-cultures.
IL-7 activity is critical for B cell production in hybrid S17 and MS-5 cultures
While early studies suggested that mouse IL-7 does not act on human cells (52
), more recent reports indicate that this is not the case (22
). A titration of anti-mouse IL-7 neutralizing antibodies, but not control antibodies, into hybrid cultures with murine stroma gave a dose-dependent reduction in human B cell production. At the highest concentrations, anti-mouse IL-7 neutralizing antibodies decreased B cell production from CB HSCs in hybrid S17 and MS-5 cultures by 10 and 17 fold, respectively, as compared to co-cultures with no neutralizing antibody (). The production of non-B lineage (CD19−) cells was not affected by anti-mouse IL-7 neutralizing antibodies (). These data provide evidence that endogenously produced murine IL-7 is critical for the production of human B cell precursors in hybrid co-cultures with murine S17 and MS-5 stroma.
Production of IL-7 by primary human and murine BM stroma
To determine if stromal cells can serve as a source of IL-7 for in vivo human B cell development we examined the ability of primary human BM stroma to produce IL-7. IL-7 mRNA was detected in primary adult human BM stroma as well as primary murine BM stroma and murine stromal cell lines (). Next we compared the levels of IL-7 protein produced by murine and human stroma grown as in co-cultures. Weekly assessment of IL-7 by ELISA showed that primary adult human BM stroma produce low levels of IL-7 in vitro (ranging from .15 to 1.02 pg/ml during a five week culture period) and that IL-7 production is not affected by sub-lethal irradiation prior to plating for co-culture. In contrast, the murine S17 and MS-5 stromal cell lines produced levels of IL-7 that were at least 40 fold higher than that of primary human BM stroma (). However, in vitro IL-7 production by primary adult murine BM stroma, either with or without sub-lethal irradiation, was below the threshold of detection by the ELISA for mouse IL-7. Thus, the high level of in vitro IL-7 production by murine S17 and MS-5 murine stromal cell lines does not reflect that of primary murine BM stroma that support in vivo IL-7-dependent B cell development in the mouse.
While the production of IL-7 from primary adult human BM stroma was detectable with the much more sensitive ELISA assay for human IL-7, those levels would have been below the sensitivity of the mouse IL-7 ELISA assay. These data provide evidence that primary human adult BM stroma produce IL-7 and that the level of in vitro
IL-7 production by these cells is consistent with that observed for mouse, an animal model where the physiological relevance of IL-7 in B cell development has been demonstrated in vivo
IL-7 is essential for the production of B lineage cells from HSCs in adult BM, but not from HSCs in CB
To determine the extent to which B cell production is dependent on IL-7 and how this might change during ontogeny, we compared the production of B cell precursors from CB and adult BM HSCs in human-only cultures supplemented with anti-human IL-7 neutralizing antibodies or with IL-7 (). A small population of CD19+ B lineage cells were generated from CB HSCs in cultures where IL-7 activity was neutralized. In contrast, B lineage cells derived from adult human BM were almost totally absent from cultures with anti-IL-7 antibodies, (). These data suggest that HSCs in CB but not adult BM have the potential for IL-7-independent human B lymphopoiesis.
A comparison of human-only cultures initiated with CB and adult BM HSCs showed that IL-7-induced increases in both cases were similar in magnitude and specific to CD19+ cells (). However, the fraction of culture progeny comprised of B lineage cells was significantly less in cultures with HSCs from adult BM (). Therefore, we compared the generative capacities of CB and BM HSCs with respect to B lymphoid (CD19+) and non-B lymphoid (CD19−) progeny and the effects of IL-7 on these capacities (). For each Lin−CD34+ CB cell plated at culture initiation, an average of 5.7 CD19+ cells were generated at 3 weeks in human-only cultures with IL-7. Surprisingly, the average B lymphoid generative capacity of adult BM HSCs in identical conditions was only .11 CD19+ cells/HSC plated. The capacities of CB and adult BM HSCs to generate CD19− progeny were not significantly different and were unaffected by IL-7. Thus, the in vitro B lymphoid generative capacity of adult BM HSCs is more than 50 fold less than that of CB HSCs and this decreased generative capacity is specific to CD19+ cells. These data suggest that IL-7 is more critical in maintaining human B cell production during adult life than during the neonatal period and that this may be due in part to a selective decrease in the B lymphoid generative capacity of HSCs in adult BM as compared to CB.
Flt3 ligand, but not TSLP is required for the IL-7-independent production of B lineage cells from CB HSCs
Studies of mouse B cell development suggest that TSLP, a cytokine that signals through the IL-7Rα and/or Flt3 ligand support the production of fetal/neonatal B cells in the absence of IL-7 (4
). Therefore we examined the extent to which the IL-7-independent production of B lineage cells from CB HSCs in our human-only cultures ( left panel) is dependent on Flt3 ligand and TSLP. The production of CD19+ cells from CB HSCs in human-only cultures supplemented with Flt-ligand and anti-IL-7 was compared to that observed with neutralizing anti-Flt-ligand, neutralizing anti-TSLP, or both. As shown in , the addition of anti-TSLP antibodies had no effect on IL-7 independent production of B lineage cells, while CD19+ B lineage cells were essentially absent in cultures that contained anti-Flt3 ligand. These data show that Flt3 ligand, but not TSLP, is required for the IL-7-independent production of B lineage cells from CB HSCs that we observed in human-only cultures.
B lineage cells produced in the absence of IL-7 are capable of subsequent IL-7 responses
IL-7 has been reported to increase the expression of CD19 on fetally-derived human B cell precursors (55
) and in the presence of IL-7, IL-7Rα+ B cell precursors are larger in size than B cell precursors that lack the IL-7Rα (22
). Consistent with this report, flow cytometry analysis showed that the B lineage cells generated from CB and BM HSCs in human-only cultures expressed high levels of CD19 in the presence of IL-7, while cultures supplemented with anti-IL-7 neutralizing antibodies expressed much lower levels of CD19 (). Human-only cultures supplemented with IL-7 also showed higher frequencies of B cells that were larger (as indicated by increased forward scatter (FSC) in flow cytometry analysis) as compared to those produced in cultures with anti-IL-7 neutralizing anti-bodies (see below).
To determine if the B lineage cells generated in the absence of IL-7 were capable of responding to IL-7 by upregulating CD19 and exhibiting increased cells size characteristic of cycling cells, we switched the culture progeny produced from CB HSCs in human-only cultures supplemented with anti-IL-7 neutralizing antibodies to human stromal cell cultures with IL-7 (anti-IL-7 to IL-7 switch cultures). After 3 days, anti-IL-7 to IL-7 switch cultures were compared to cultures that had been switched to human stroma and anti-IL-7 (anti-IL-7 to anti-IL-7 switch cultures) or to human-only cultures that had been both initiated with and switched to IL-7 supplementation (IL-7 to IL-7 switch cultures). The percentage of cells expressing high levels of CD19 (CD19HI) was increased in anti-IL-7 to IL-7 switch cultures ( middle panel), as compared to anti-IL-7 to anti-IL-7 switch cultures ( top panel), however, the percentages were not as high as those observed among B lineage cells that were produced under continuous exposure to IL-7 (, bottom panel, and ). Cultures with B cell precursors that were never exposed to IL-7 (anti-IL-7 to anti-IL-7 switch cultures) showed a low frequency of cells with the high levels of FSC characteristic of cycling cells. The switch from anti-IL-7 to IL-7 resulted in a significant increase in the frequency of cells that were FSCHI and an FSC histogram profile that was similar to that seen for IL-7 to IL-7 switch cultures.
IL-7R-signaling, partially via
upregulation of EBF, has been reported to be a regulator of the transcription factor network that controls B cell development in the mouse (2
). Expression of EBF in the absence of IL-7 in fetal and neonatal mice allows IL-7-indepdent B cell differentiation at early points in ontogeny (9
). Therefore we were interested in examining expression of EBF and its downstream target PAX-5 (56
) in the early B lineage cells generated in our human-only cultures. CD19+ cells generated in switch cultures were FACS-sorted for RT-PCR. Real time RT-PCR showed that B lineage cells generated in all three switch culture conditions expressed mRNA for CD19, PAX-5, and EBF, (Supplemental Data
) however the small number of cells generated in cultures initiated with anti-IL-7 precluded a quantitative comparison of mRNA levels. Taken together these data provide evidence that the CD19LO
Cells generated from CB HSCs in the absence of IL-7 are B lineage cells and have the capacity to respond to subsequent IL-7 stimulation.
IL-7 directly targets B cell precursors in human-only cultures
To identify the cellular targets of IL-7 we used flow cytometry to examine IL-7Rα expression among cells present during in vitro B lymphopoiesis (). IL-7Rα was not detectable on primary human BM stromal cells used in co-cultures (). Approximately 35% of CD19+ B cell precursors produced in co-culture expressed the IL-7Rα, regardless of whether they arose from HSCs in CB or adult BM or whether IL-7 was present ( right panel). We gated CD19− cells into a CD34− subset that included more mature non B-lineage cells and into a CD34+ subset that included HSCs and progenitors present in the 3-week human-only cultures. Of the CD19−CD34− non B lineage cells generated from CB and BM HSCs in human-only cultures, 3-12 % expressed the IL-7Rα ( left panel) while 4-14% of the of the CD19−CD34+ HSC and progenitor subset was IL-7Rα+ ( middle panel). The percentage of cells in each subset that expressed IL-7Rα was not significantly different in cultures initiated with CB and BM HSCs, and the presence or absence of IL-7 activity had no significant impact on the percentage of cells expressing the IL-7Rα in any of the subsets (). These data show that CD19+ B cell precursors represent the major IL-7Rα+ population in human-only cultures and are therefore a potential IL-7 target during in vitro B cell development.
FIGURE 5 IL-7 directly targets B lineage cells. (A) Human-only cultures were initiated with FACS-sorted Lin−CD34+ HSCs from CB or BM under control conditions (3, F) and supplemented with anti-IL-7 neutralizing antibodies or exogenous human IL-7. At 3 weeks (more ...)
To determine if IL-7 acts directly on B cell precursors we used flow cytometry to assess phosphorylation of STAT5, an event downstream of IL-7R signaling (35
). CD19+ B cell precursors were isolated from human-only cultures and assessed by flow cytometry for purity (based on CD19 expression) and for IL-7Rα expression (, left panel) and then for STAT5 phosphorylation following incubation with or without IL-7 (, right panel). Phosphorylated STAT5 was detected in B cell precursors incubated with IL-7, but not in B cell precursors cultured without IL-7 ( right panel). The percentage of B cell precursors positive for phosphorylated STAT5 following incubation with IL-7 was virtually identical to the percentage of cells expressing IL-7Rα ( compare left and right panels). These data provide evidence that B cell precursors are directly targeted by IL-7 in human-only cultures.
IL-7-induced increases in B cell production are mediated through increased proliferation of B cell precursors
To determine if increased cell survival is involved in the IL-7-induced increases observed in our co-cultures, we examined the effect of IL-7 on apoptosis among culture-generated human B cell precursors. Human-only cultures initiated with CB HSCs were harvested at 3 weeks and CD19+ cells (either enriched or unseparated from CD19− cells) were placed in co-culture with fresh human stroma and supplemented with anti-IL-7 neutralizing antibodies, control antibodies, or IL-7 Cells were harvested at daily time points and flow cytometric analysis of staining with the Annexin V apoptosis indicator was used to determine the percentages of CD19+ B cell precursors undergoing apoptosis. IL-7 had no effect on the percentages of CD19+ that were Annexin V+, whether they were in co-culture alone or together with CD19− cells (data not shown). Similarly IL-7 had no effect on apoptosis among in vivo-generated B cell precursors (CD19+K/L−) that were FACS sorted from adult BM and assessed in identical assays (data not shown). These results suggest that IL-7 does not mediate protection from apoptosis and that increased survival of CD19+ B cell precursors is unlikely to be a major mechanism by which IL-7 increases human B cell production.
To determine if increased proliferation is a mechanism for IL-7-induced increases in human B cell production, we used flow cytometry to examine expression of the Ki-67 proliferation antigen in cells harvested from human-only cultures supplemented with IL-7 or with anti-IL-7 neutralizing antibodies (). IL-7 did not change the percentage of CD19− cells that were Ki-67+ in cultures of CB or BM HSCs (). In contrast, in CB cultures the percentage of CD19+ cells expressing the Ki-67 was significantly higher with IL-7 supplementation (). Cultures of BM HSCs generated too few B cell precursors to be assessed for Ki-67 when anti-IL-7 antibodies were added. However, the percentage of B cell precursors expressing Ki-67 was the same in CB and BM cultures with IL-7 ().
FIGURE 6 IL-7-induced increases in the production of B lineage cells are mediated through increased proliferation. Human-only cultures were initiated with FACS-sorted Lin−CD34+ HSCs from CB or BM under control conditions (3, F) and supplemented with anti-human (more ...)
To verify that the increases in proliferation observed in cultures supplemented with IL-7 were due to the direct action of IL-7 on B cell precursors, we compared Ki-67 expression () and size (indicated by FSC, ) in IL-7Rα+ and IL-7Rα− B cell precursors generated in cultures with IL-7. For both CB and BM cultures, average cell size and the percentages of cells expressing Ki-67 was significantly higher for IL-7Rα+ than for L-7Rα− B cell precursors. These data provide further evidence that IL-7 increases the production of human B lineage cells through increased proliferation due to direct targeting of B cell precursors by IL-7.
Expression of the IL-7Rα in context of human B cell development in vivo
To determine whether the IL-7 targets that we identified in our human-only cultures are reflective of human B cell development in vivo, we examined the expression of IL-7Rα in early B lineage cells that were generated in vivo in adult human bone marrow. The average frequency of IL-7Rα+ cells among in vivo-generated B cell precursors (CD19+K/L−) in adult human BM (, Table 1) was similar (32%) to that observed for B cell precursors generated in vitro (35%) from HSCs in CB and adult BM in human-only cultures (). The percentages of in vivo-generated CD19+K/L− B cell precursors that we assessed in human subjects (Table 1) exhibited a broad range in the percentage of cells that were IL-7Rα+ (17%-56%) and showed no obvious correlation with age, gender, or race, suggesting that there is substantial natural variation among adult individuals. Thus, the IL-7Rα+ population of B cell precursors observed in human-only cultures is reflective of in vivo B cell development in adult human BM and suggests a physiological role for IL-7 in human B cell development.
FIGURE 7 The distribution of IL-7Rα+ cells in adult human B cell precursor populations produced in vivo. (A) Primary adult human BM mononuclear cells, either fresh or frozen and then thawed, were stained for flow cytometry to assess co-expression of CD19, (more ...)
A substantial portion of the B cell precursors generated in human-only cultures initiated with CB and BM HSCs were IL-7Rα−. This was also the case for CD19+ K/L−cells in adult BM. To determine if the expression of IL-7Rα correlated with the expression of developmental markers during in vivo B cell differentiation we examined IL-7Rα expression among developmental subsets of CD19+ K/L− B cell precursors identified based on expression of CD34+ or cμ (). The percentages of IL-7Rα+ cells were similar for all developmental subsets (cμ+, cμ−, CD34−, and CD34+) of CD19+K/L− cells. Thus, the expression of IL-7Rα among B cell precursors produced in vivo does not appear to correlate with particular stages of development that can be identified based on expression of CD34 or with successful rearrangement of the Ig heavy chain as indicated by the expression of cμ. ().
Precursor populations targeted by IL-7-induced expansion in human-only cultures
In the mouse, IL-7R signaling has been associated with distinct differentiation and proliferation signals. Thus, while the expression of IL-7Rα may not correlate with markers of development in CD19+ B cell precursors, it is possible that IL-7 may directly target distinct developmental populations of IL-7Rα+ B lineage precursors for proliferation or for differentiation. To identify the developmental targets of IL-7-induced proliferation we examined IL-7-induced increases and expression of the Ki-67 proliferation antigen in four subsets that include developmentally sequential populations of B cell precursors. FACS analysis of the cells present at 3 weeks in human-only cultures was used to identify four populations (): 1) The most primitive IL-7R−HSCs and progenitors (HSC) through which IL-7 effects might be indirectly mediated, 2) IL-7Rα+ progenitors (IL-7R+ P) that would include the earliest CD19− lymphoid-committed progenitors that express the IL-7Rα (57
) ; 3) CD34+ pro-B cells (CD34+ B) and 4) CD34− pro-B cells (CD34− B).
Human-only cultures initiated with Lin−CD34+ cells from CB and BM and supplemented with IL-7 showed significantly increased numbers of cells in the CD34+ B and CD34− B subsets at three weeks when compared to cultures with anti-IL-7. The relative numbers of CD34+ B cells increased by only 5 to 14-fold (CB and BM cultures respectively) when IL-7 was present while CD34− B cells went up by 194 to 90-fold (CB and BM cultures respectively). In contrast, the relative numbers of HSCs, and IL-7R+ P were not significantly increased in cultures with IL-7 (). Consistent with the IL-7 effects that we observed on cell numbers, CB cultures supplemented with IL-7 showed significantly higher percentages of Ki-67+ cells among the CD34+ B and CD34− B, but not among the HSC or IL-7R+ P subsets (). The percentage of Ki-67+ B cells among CD34− B cells in BM cultures was similar to that observed in CB cultures, however there were too few CD19+ cells to assess in Ki-67 expression in any BM cultures with anti-IL-7 or in the CD34+ B cells in BM cultures where IL-7 was present ( right panel). Taken together these data provide evidence that IL-7 targets both CD34+ and CD34− pro-B cells for increased proliferation. In contrast, in our culture model we were unable to detect any significant changes in proliferation due to IL-7 effects among developmental subsets that would include CD19− B cell precursors.
In we showed that IL-7 is particularly critical for the production of B lineage cells from adult BM because the capacity of adult BM to generate B lineage cells is greatly reduced as compared to CB. To get a better understanding of the dynamics and potential mechanisms that might be responsible for the changes in B cell development that occur during ontogeny, we compared the ability of CB and adult BM to generate the developmental subsets observed in vitro in human-only cultures. In cultures with adult BM the HSC generative capacity was about half, and the IL-7R+ P generative capacity about 10 fold less than that observed for CB cultures (, Note the 10X scale reduction in the right panel). In cultures with IL-7, the adult BM generative capacities for cells in the CD34+ B and CD34− B subsets were ~40-fold and ~50-fold less, respectively than those observed for CB. While BM cultures generated detectable CD19−subsets, and the capacity to generate these cells was unaffected by IL-7, B lineage precursors that reached the CD19+ stage of development were essentially absent in cultures with anti-IL-7. These data suggest that there is a progressive loss of generative capacity during the course of B lineage differentiation in adult BM, as compared to CB, and that as a consequence of this reduced B lineage generative capacity, IL-7 is more critical for B cell development from HSCs in adult BM.