B cell development is a highly ordered process that requires cells to progress through multiple checkpoints during maturation. B cells originate from the hematopoietic stem cell (HSC) pool and pass through multiple stages of development before becoming committed to the B lineage (1
). Prior to B lineage commitment, potential B cells exist as common lymphoid progenitor cells (CLPs). Although CLPs maintain the capacity to differentiate into other non-B lineages, they are considered to be B lineage specified and are actively undergoing DH
IgHC gene rearrangement. In addition, CLPs express the IL-7 receptor, which is required for progression along the B lineage. CLPs progress to the pre-pro-B cell stage where expression of B220 can first be detected. At this stage DH
IgHC gene rearrangement is nearly complete, and these cells are considered B lineage committed although lineage plasticity remains (4
Transition from the pre-pro-B to pro-B stage is accompanied by surface expression of CD19. The IgHC locus then begins to undergo VH
). If an in-frame coding region is produced from these recombination events, the IgHC protein is expressed on the cell surface along with components of the surrogate light chain (SLC), including the λ5 and VpreB proteins and the Igα/Igβ heterodimer. This forms the pre-B cell receptor (pre-BCR). Proper expression and intracellular signal transmission of the pre-BCR is crucial for progression from the pro-B to pre-B stage, and only a third of pro-B cells successfully complete this process (5
). Disruption of components of the pre-BCR (such as in Rag1
-/- mice) can lead to a complete block at the pre-BCR checkpoint. (6
). A functional antigen receptor is also required for mature B cell maintenance in the periphery, since deletion of the B cell receptor in mature splenic B cells results in rapid cell death (9
). Successful signaling through the pre-BCR leads to survival, proliferation, and developmental progression. Once cells advance to the late pre-B stage, the recombination machinery initiates recombination of the light chain locus (10
). The light chain pairs with IgHC and the Igα/Igβ heterodimer is expressed on the BCR in immature B cells.
Functionally immature B cells leave the bone marrow via the bloodstream and travel to the periphery where they undergo further maturation (11
). Immature B cells in the spleen are denoted transitional B cells and show a high rate of turnover in vivo
with rapid cell loss (12
). A significant amount of negative selection occurs at the T1-T2 transition when immature B cells from the bone marrow encounter new antigens (13
). Once cells reach the T2 stage, they progress to the mature B pool after positive selection. Three distinct types of mature B cells exist: follicular (FO), marginal zone (MZ), and B-1 cells, and there are different requirements for the development of each cell type. One current hypothesis for the formation of different mature B cell populations is the signal strength hypothesis. This hypothesis suggests that strong signaling from the BCR skews development to the FO B developmental pathway whereas weaker signaling through the BCR results in MZ B cell formation (14
Ras signaling to Raf is required for B development as well as signaling through the BCR. Overexpression of a dominant negative Ras (H-ras
N17) in early B cells blocked B cell development at the pre-pro-B to early pro-B transition (15
). This block was partially rescued by expression of Raf-CAAX, which mimics Ras-induced activation of Raf. Transgenic mice that express dominant inhibitory Ras (Asn-17 HA-Ras) in the late stages of pro-B development showed decreased survival of B lymphocytes although proliferation was not impaired (as assessed by BrdU incorporation in the bone marrow) (16
). Interestingly, expression of an activated Ras (c-HA-rasV12) on a Rag deficient background generated B lineage cells in peripheral tissues, suggesting that activated Ras was able to induce pro-B cells to differentiate beyond the pre-BCR checkpoint in the absence of the μ heavy chain (17
). Thus, Ras plays important roles in B cell development both prior to and during the pre-BCR checkpoint.
Cytokines and their cognate receptors have been shown to play critical roles in B cell development prior to the pre-B cell receptor checkpoint (18
). Deletion of cytokine receptors can inhibit B cell development at the earliest stages by blocking proliferation, differentiation, and/or survival of developing B lymphocytes. IL-7 and its cognate receptor are particularly important at the pre-pro-B to pro-B transition. Inhibiting the IL-7 receptor by injection of blocking antibodies (21
) or genetic knockout (22
) severely disrupts B cell development at the pre-pro-B to pro-B stage. This block has been linked to loss of commitment to the B lineage through decreased expression of the transcription factor EBF (early B factor) (23
). In addition to B lineage commitment, IL-7 also functions to promote survival (24
) and proliferation (25
) of early B subsets.
Proper intracellular transmission of receptor signals through appropriate cytoplasmic signaling intermediates is also required for successful B lymphopoiesis. The adaptor protein Shc signals downstream of multiple receptors, including antigen and cytokine receptors (27
). During receptor signal transduction the p52 isoform of ShcA (hereinafter Shc) is phosphorylated on three critical tyrosine residues (Y239, Y240, and Y317), allowing the binding/recruitment of Grb2/Sos proteins leading to the activation of Ras, and in turn the MAPK cascade (28
). Shc protein expression and Shc tyrosine phosphorylation have been shown to be critical for thymocyte development at the β selection checkpoint. Impaired Shc-mediated signaling blocks T cell development by inhibiting the proliferative burst that accompanies successful pre-TCR signal transduction (30
). However the role of Shc in B cell development remains unknown.
In this report, attempt to address the role of Shc during B cell development by conditionally expressing a dominant negative Shc protein with mutation of its three critical tyrosine residues (hereinafter ShcFFF), or conditional loss Shc protein expression, in early B lymphocytes. Mice conditionally expressing ShcFFF had severely reduced pre-B cell numbers. However, we also noticed a surprising defect in cellularity of the pro-B population. Upon further analysis, we identified Shc as an important player in signaling downstream of the IL-7 receptor and in providing survival signals to pro-B cells. In vivo and ex vivo studies indicate a critical role for Shc during early B cell development that is distinct from its role during T cell development.