Our investigations show that Dlk1−/−
mice display several abnormalities in B cell development and function. The most dramatic observations were changes in splenic B cell subsets of Dlk1−/−
mice, including the increased numbers of Tr and MZ B cell populations. An especially intriguing observation was that the numbers of Tr2 B cells was the same in Dlk1−/−
and control mice. This population also contains most of the recently identified MZ precursors (sIgMhi
) as well as Tr3 cells (sIgMint
]. If either Tr3 or MZ precursor populations had expanded to the same degree as the MZ B cell population (four-fold or more), we most likely would have detected this in our analysis of the Tr2 population. Because the Tr2 populations were the same in knockout and control mice, the expansion of the MZ B cell population in Dlk1−/−
mice is not matched by a similar expansion of the MZ precursor population. The expansion of MZ B cells could be due to increased longevity or proliferation of MZ B cells. Alternatively, Dlk1−/−
MZ precursors may be subject to more rapid kinetics of formation and differentiation than in wild-type mice. Accumulation of transitional B cells in the spleen of Dlk1−/−
mice could be due to decreased negative selection, because this is the population of cells targeted for negative selection in spleen [37
]. Future experiments will better characterize the Tr/MZ precursor populations to understand more fully the importance of dlk in splenic B cell development. Also, Dlk1−/−
mice had a consistent > four-fold increase in a small B220+
population of cells that needs further analysis because it has not been previously characterized and appears to be expanded due to lack of dlk.
dlk may influence the cell fate choice for MZ B cell development. The Notch2 signaling pathway regulates development of marginal zone B cells [11
]. dlk is expressed on B cells in the spleen () and has been shown to act as a negative regulator of Notch signaling [40
]. If dlk inhibits Notch2, lack of dlk could explain the expansion of MZ B cells in Dlk1−/−
mice. Further work is in progress to determine whether dlk influences Notch2 signaling in B cells.
We also observed that young Dlk1−/−
mice have increased numbers of Fr A cells (). Our observation that Dlk1
is expressed in both B lineage cells and stromal cells in the bone marrow suggests that dlk may play an important role in the cell–cell interactions that are crucial for early B cell development in bone marrow [30
Several possibilities could explain the observation that lack of dlk results in increased numbers of Fr A cells in bone marrow. One possibility is increased proliferation of Fr A cells. Our observations () and prior evidence indicate that Fr A cells are not highly proliferative [4
]. Our results show that Fr A proliferates minimally in the presence of IL-7 and certainly not better when dlk is lacking, so our experiment does not support the possibility that Fr A cells are more proliferative in Dlk1−/−
mice. However, our observation that normal Dlk1+/+
pro-B cells can proliferate slowly without IL-7 on stroma lacking dlk () agrees with our previous findings [24
A second possibility is that the transition from Fr A to Fr B is suppressed in the absence of dlk. Indeed, we observed that the absence of dlk on both stroma and B lineage cells leads to poorer differentiation from Fr A in vitro both in the presence and absence of IL-7. It is possible that this effect allows for the accumulation of Fr A observed in vivo.
A third possibility is that lack of dlk results in more frequent production of common lymphoid progenitors or more frequent commitment to formation of Fr A cells. Our experiment did not directly address these scenarios, but we did not observe increases in T cell numbers in Dlk1−/−
mice that might be expected due to more progenitors, or decreases if the existing progenitors more frequently are directed to the B cell lineage. It remains possible that dlk inhibits differentiation of hematopoietic stem cells (HSCs) and that its absence resulted in increased differentiation of HSCs into Fr A pro-B cells. dlk may play a similar role in human hematopoiesis because it has been established that dlk is expressed in certain fetal stromal cell lines and is necessary for proliferation of undifferentiated hematopoietic stem cells (HSCs) [20
hematopoietic stem cells from low-risk myelodysplastic syndrome (MDS) patients have increased expression of dlk in bone marrow [27
], and the primary MDS condition results in defective differentiation and ineffective hematopoiesis [27
The increased number of Fr A cells in 4-week-old Dlk1−/− mice decreased to normal levels by 6 weeks of age. Dlk1 RNA was found to be expressed in neonatal liver () and bone marrow stroma and B cells (). These observations suggest that the influence of dlk on differentiation of stem cells into early B lineage progenitors is more important for embryonic and neonatal B cell differentiation.
Our preliminary in vitro differentiation studies suggested that the generation of Fr C cells is at least equivalent, if not better, in bone marrow cultures from 4-week-old Dlk1−/− mice compared to controls. When we used a less heterogeneous culture system, we observed that Fr B cells are quite proliferative and capable of generating Fr C cells in the presence of IL-7. In the absence of IL-7, Fr B cells did not proliferate well and differentiation to Fr C was inhibited. This might explain the trend toward less Fr C cells as the Dlk1−/− mice aged. It is possible that the young Dlk1−/− bone marrow microenvironment may not produce sufficient IL-7 to support differentiation of Fr C efficiently. The dramatic response of Dlk1−/− bone marrow cells to IL-7 stimulation in vitro shows that there is no B cell-intrinsic defect in IL-7 response in Dlk1−/− mice, supporting the idea that changes occur in the bone marrow microenvironment.
Interestingly, there was a decrease of the Fr F mature recirculating B cell population in bone marrow of 8-week-old Dlk1−/−
mice, at an age where increases in Tr and MZ B-cells were observed. The reduced number of FO B cells and reduced memory response suggest the possibility that Fr F production is inhibited in Dlk1−/−
mice. Another possibility is that homing or stromal support are decreased when dlk is lacking. It has been shown that survival of mature B/plasma cells in the bone marrow is dependent on stromal cells [44
] and homing of plasma cells to the bone marrow is dependent upon cell–cell interactions between plasma cells and stromal cells [45
]. Further studies are necessary to distinguish among these possibilities.
Our results show that lack of dlk not only affects bone marrow B cell differentiation but also perturbs peripheral B cell compartments, resulting in increased numbers of Tr and MZ B cells and decreases in FO B cell numbers. Studies in IL-7, E2A, and λ5
knockout mice have also shown that defective early B cell development often results in an expanded MZ as a compensatory mechanism to attain a complete natural antibody repertoire [46
]. An interesting question is whether the peripheral B cell changes in Dlk1−/−
mice are due to the alterations in early B cell development or peripheral influences on B cell development.
Changes in B cell development are reflected in the altered immunoglobulin levels in preimmune and immunized animals. Whereas FO B cells are important for the germinal center reaction and formation of long-term memory cells [47
], MZ B cells act as a first line of defense [48
] and mediate T-independent immune responses [49
]. In preimmune animals at 8 weeks of age, there were elevated levels of IgG1
, isotypes associated with MZ B cells (). At 8 weeks of age, MZ B cell numbers are increased compared to those of the wild-type control mice () and could have led to the observed increases in these Ig isotypes in Dlk1−/−
Our observation that the primary T-dependent antibody response () is increased in Dlk1−/−
mice may indicate that both FO and MZ B cells could have contributed to it. It has been recently shown that MZ B cells are capable of mounting a T-dependent response [50
]. The decrease in IgG1
secondary responses are explained by several observations including the decreased number of FO B cells, the primary source of memory B cells. Also, the MZ B cells, most likely the source of the increased primary antibody, do not generally form memory cells [33
] and might not participate in the secondary response and could favor the generation of antibody-forming cells (AFCs) over memory cells.
Recently, published observations show that dlk plays a role in B cell development in vivo. Sakajiri et al. [27
] reported impaired in vitro pre-B cell growth as evidenced by a pre-B colony assay. In contrast, our observations suggest there are no defects in pre-B cell formation (Fr C and Fr D) and no defect in the in vitro response to IL-7 stimulation. However, the differences in those results could be due to differences in the targeting construct and technical details of assays. Our study used more molecular markers to characterize in vitro and in vivo B cell populations in Dlk1−/−
Different stages of B cell development require cell–cell interactions for growth and differentiation. We have shown that mice lacking dlk have altered patterns in stromal-dependent stages of bone marrow B cell development. We also saw that splenic B cell populations are altered in Dlk1−/− mice and that the humoral immune response in dlk-deficient mice is altered. Altogether, these results show that dlk is an important molecule that regulates B cell development.