In adults, B cells are generated continuously from bone marrow (BM) hematopoietic stem cells (HSCs) (Box 1
). Descriptive studies have revealed substantial changes in the functional potential and sizes of developing B-cell subsets with age. For example, the frequency of precursors capable of generating B cells in vitro
is reduced [13
], and the pre-B and immature (IMM) BM pools are smaller [16
]. These findings prompted the question of whether such changes reflect upstream shifts in B-lineage commitment; cell-intrinsic changes in mediators of key differentiation steps or deterioration of microenvironmental cues required for successful differentiation. Further, they suggested that B-cell production might wane with age, resulting in diminished BM output and altered turnover properties in mature B-cell subsets. Advances in the resolution of early B-lineage progenitors, insights into the genetic events required for B-lineage specification and the advent of tools to assess the dynamics of developing populations have allowed interrogation of these possibilities.
Box 1. Bone marrow B-cell development
Specification and commitment to the B-cell lineage involves key transcription factor networks [62
], which in concert yield early B-cell progenitors. Lineage commitment is followed by recombination activating gene (RAG)-mediated IgH (heavy chain) gene rearrangement in the pro-B-cell stage. On successful IgH rearrangement, the Ig heavy chain is expressed on the cell surface associated with surrogate light chain (lambda-5/Vpre-B) and the Ig-α and Ig-β signaling complex. This initiates the pre-B-cell stage where, after brief proliferation, successful light chain rearrangement allows surface expression of a complete B-cell receptor, marking entrance to the immature marrow B-cell stage. At each stage, marrow stromal elements and products, such as interleukin 7, play key roles in sustaining differentiation.
There is increasing evidence that the differentiative potential of HSCs changes with age [17
]. HSCs from aged mice show numerous changes in gene expression, resulting from an apparent breakdown of epigenetic regulation [21
]. Other cell-intrinsic changes include increased HSC self-renewal and diminished lymphoid potential [18
]. This is accompanied by downregulation of genes that mediate lymphoid specification and function – and enhanced expression of genes specifying myeloid development [18
]. Together, these findings suggest epigenetic changes in HSCs that occur in aged individuals might impact all subsequent downstream subsets and differentiative events. Consistent with this, recent studies show that early B-cell progenitors (EBPs) are reduced with age [22
]. Also in accord with this idea, the expression of transcriptional regulators essential to generating pro-B cells, including E2A gene products such as E47, are reduced [23
]. Similarly, the expression of genes crucial to passage through the pro- and pre-B cell stages, including RAG (recombination activating gene) enzymes and lambda-5, is diminished in developing B cells from aged individuals [26
]. Recent studies using a RAG reporter system coupled with flow cytometry demonstrated such reductions at the single cell level [29
], strengthening the notion that intrinsic epigenetic changes in HSCs and developing B-cell subsets play a role in shifting the dynamics and quality of BM B-cell output. All of these findings suggest that both B-lineage commitment and transit through early developmental stages are compromised with advancing age, implying that BM B-cell output should fall. Indeed, in vivo
labeling has confirmed that production rates in the pro-, pre- and immature BM B-cell pools are diminished with age [29
], reflecting decreased ability to successfully complete each differentiation stage and transit to the next.
Determining the relative contributions of cell intrinsic versus microenvironmental changes has proven complex, because both mechanisms are involved. For example, BM stromal cells from aged mice provide less interleukin 7 (IL-7) in vitro
, but B-cell progenitors from aged mice also respond less efficiently to IL-7 [32
]. Similarly, reciprocal BM chimeras indicate that aged BM HSCs recapitulate young adult B-cell production kinetics when transferred in large numbers to young adults [29
]; however, limiting the numbers of purified HSCs reveals cell-intrinsic reductions in B lineage–specified progenitors under the same conditions [22
Together, these findings suggest interplay between intrinsic and microenvironmental factors, whereby the levels of gene products vital to lineage commitment and subsequent differentiation are altered. It is tempting to speculate that the overall B-cell aging phenotype derives from a ‘snowball’ effect, whereby failures in crucial upstream events – beginning with epigenetic changes in HSCs – yield consecutive, escalating downstream consequences. Accordingly, determining how levels of key gene products are controlled and how decreased BM output impacts the selection and homeostasis of pre-immune B-cell pools is a key next step.
An exciting possibility is that post-transcriptional mechanisms involved in mRNA and protein turnover regulate molecules involved in B-lineage development and function. In vitro
studies indicate that the reduced E2A protein levels seen in B-cell precursors from aged hosts reflect increased protein turnover [33
]. Under normal conditions, the turnover of E2A proteins is regulated by mitogen activated protein kinase (MAPK) activity and requires Notch [35
]. B-cell precursors in aged hosts show increased extracellular signal-regulated kinase (ERK) MAPK activity, coinciding with heightened phosphorylation, ubiquitination and accelerated turnover of E2A (E47) proteins [34
]. In contrast to E2A dysregulation in B-cell precursors from aged hosts, mature splenic B cells from aged mice and humans display reduced E2A due to increased mRNA degradation [9
]. Therefore, expression of E2A, and possibly other key regulatory molecules, is compromised in aged B-lineage cells, but the mechanisms probably differ depending on their developmental or activation stage. Although decreased E2A levels will directly compromise lineage specification and developmental progression, the diminished expression of E2A regulated genes (e.g. RAGs or surrogate light chain) will further impair or alter B-cell repertoire establishment and selection, particularly at the pre-B-cell stage [37
It is important to note that multiple pathways exist for B-cell generation in the adult BM, including the predominant B2 cell pathway, as well as a second pathway devoted to the B1 cell lineage [38
]. B1 cells predominate in peritoneal and pleural cavities and represent a self-renewing pool that is established early in life. In addition, B1 cells express a specificity repertoire different from B2s that is generally characterized by polyspecificity and low-affinity self-reactivity [39
]. B1 B cells are largely responsible for so-called natural antibodies and are skewed toward responses to parasite and bacterial antigens. The relative contributions of these two developmental pathways vary with age: in prenatal and neonatal life, the B1 pathway predominates, whereas in young adult life, the B2 pathway is dominant. However, as B2 production wanes with age, the proportional contribution of the B1 pathway again increases [40
]. The exact consequences of this reversion in the proportional representation of B1 versus B2 output remain unclear but might contribute to some repertoire differences observed with advancing age.