Pre-B cell loss in senescence coincides with preferential development of immature B cells characterized by partial activation and altered Vh repertoire.

Senescent mice show decline in B lymphopoiesis marked by reduced pre-B cells. Analysis of bone marrow from aged (approximately 2 years old) BALB/c mice indicates that, in senescence, an increased proportion of immature B cells exhibit a CD43/S7+ surface phenotype. This results from continued production of new CD43/S7+ B cells in aged mice from their limited pre-B cell pool while production of CD43/S7- immature B cells is highly reduced. CD43/S7 is ordinarily observed on a minor subset of immature B cells in young mice and is indicative of their partial activation. Senescent immature B cells, both ex vivo and derived in vitro, also demonstrate increased expression of VhS107 concomitant with CD43/S7. These alterations in the phenotype and Vh repertoire among senescent immature B cells likely originate prior to surface Ig expression. In aged mice with depleted pre-B and immature B cells in vivo, pre-B and immature B cells exhibited increased apoptosis in vitro. Dexamethasone-induced apoptosis among B lineage cells in young adult mice also resulted in pre-B cell loss and increased expression of CD43/S7 and VhS107 among immature B cells similar to that observed spontaneously in aged mice. These results suggest that old age, possibly due to increased apoptosis, results in loss of pre-B cells and alterations in the phenotype and Vh repertoire of newly derived B cells.

A phenotypically distinct subset of immature B cells exhibits partial activation, increased survival, and preferential expression of VhS107.

We have observed that immature B cells (IgM(low)IgD(-)) in the bone marrow of adult BALB/c mice exhibit heterogeneity, with a distinct subpopulation ( approximately 4-10%) expressing the CD43/S7 surface protein. These CD43/S7(+) immature B cells often express other surface antigens associated with B cell activation (CD5, CD11b, PD-1). Generation of optimal numbers of CD43/S7(+) immature B cells requires expression of a functional Btk protein, consistent with activation as a requisite for the CD43/S7(+) immature B cell phenotype. Like typical CD43/S7(-) immature B cells, the CD43/S7(+) immature B cells are predominantly resting cells, which are derived from cycling bone marrow B cell precursors. The CD43/S7(+) immature B cell population exhibits enhanced survival in vivo upon administration of the apoptosis-inducing corticosteroid, dexamethasone. Finally, CD43/S7(+) immature B cells show a fourfold increase in incidence of VhS107 micro heavy chain expression compared to the CD43/S7(-) immature B cells. Therefore, in adult murine bone marrow, the presence of a phenotypically distinct immature B cell population can be demonstrated which has undergone partial activation leading to increased survival and BCR-dependent Vh repertoire selection.

Aged mice exhibit distinct B cell precursor phenotypes differing in activation, proliferation and apoptosis.

Senescence in murine models is associated with a reduction, albeit heterogeneous, in bone marrow pre-B cells. We have categorized aged BALB/c mice into two phenotypes based on their patterns of pre-B/pro-B cell loss. Each phenotype is characterized by distinct responses to the growth cytokine IL-7 and capacity for survival in vitro. A 'moderate' loss of late-stage pre-B cells (25-80%) coincided with decline in proliferation to rmIL-7. This was also associated with a decrease in the frequency of pro-B cells which increased phosphotyrosine content upon IL-7 stimulation, an indicator of early activation events. A 'severe' loss of pre-B cells (>80%) resulted in a reduced pro-B cell pool which retained normal activation and proliferative responses to IL-7. B cell precursors from aged mice with severe alterations in B lymphopoiesis displayed increased susceptibility to apoptosis in comparison to both aged mice with moderate B cell precursor loss and young mice. Conceivably, during senescence, aged mice may initially accumulate B cell precursors which are poorly responsive to IL-7. Progressively, these refractory B cell precursors may be eliminated via apoptosis; however, the remaining limited pool of B cell precursors retains the capacity to respond to IL-7 stimulation.

B cell precursors in senescent mice exhibit decreased recruitment into proliferative compartments and altered expression of Bcl-2 family members.

In vitro, aged pro-B cells generally exhibit limited expansion in response to IL-7 when compared to young pro-B cells. CFSE-labeling in vitro indicated that aged mice have a lower frequency of pro-B cells which are capable of undergoing extensive proliferation upon stimulation with exogenous IL-7. Protein levels of the survival molecule Bcl-x(L) were consistently reduced in IL-7 expanded aged pro-B cells. Levels of both Bcl-2 and Baxalpha proteins were variable in aged B cell precursors. The expansion of aged pro-B cells in response to IL-7 in vitro correlated inversely with the ratio of the pro-apoptotic protein Baxalpha to the survival protein Bcl-2. Expansion of aged pro-B cells in vitro is likely dictated by both recruitment of pro-B cells into proliferative compartments and their survival; in aged B cell precursors, the latter is favored by low Baxalpha to Bcl-2 protein ratios.