Expression of CD28 by bone marrow stromal cells and its involvement in B lymphopoiesis

Young mice lacking CD28 have normal numbers of peripheral B cells; however, abnormalities exist in the humoral immune response that may result from an intrinsic defect in the B cells. The goal of this study was to assess whether CD28 could be involved in the development of B cells. CD28 mRNA was detected preferentially in the fraction of bone marrow enriched for stromal cells. Flow cytometry and RT-PCR analysis demonstrated that CD28 was also expressed by primary-cultured stromal cells that supported B lymphopoiesis. Confocal microscopy revealed that in the presence of B-lineage cells, CD28 was localized at the contact interface between B cell precursors and stromal cells. In addition, CD80 was detected on 2-6% of freshly isolated pro- and pre-B cells, and IL-7 stimulation led to induction of CD86 on 15-20% of pro- and pre-B cells. We also observed that stromal cell-dependent production of B-lineage cells in vitro was greater on stromal cells that lacked CD28. Finally, the frequencies of B-lineage precursors in the marrow from young (4- to 8-wk-old) CD28(-/-) mice were similar to those in wild-type mice; however, older CD28(-/-) mice (15-19 mo old) exhibited a 30% decrease in pro-B cells and a 50% decrease in pre-B cells vs age-matched controls. Our results suggest that CD28 on bone marrow stromal cells participates in stromal-dependent regulation of B-lineage cells in the bone marrow. The localization of CD28 at the stromal cell:B cell precursor interface suggests that molecules important for T cell:B cell interactions in the periphery may also participate in stromal cell:B cell precursor interactions in the bone marrow.     

B lymphopoiesis is upregulated after orchiectomy and is correlated with estradiol but not testosterone serum levels in aged male rats.

Previous studies have shown that B lymphopoiesis is augmented after androgen withdrawal in male mice. As an analogy to the skeletal system, some effects of androgens on the proliferation of B cells may be mediated via aromatization into estrogens in vivo. The aim of the present study was to assess the effects of androgen withdrawal on B lymphopoiesis in bone marrow of aged male rats sequentially over a period of 9 months, and to correlate the flow-cytometric findings with changes in systemic levels of sex steroids. We first showed that androgen withdrawal is associated with enhanced B lymphopoiesis in bone marrow of 4-month-old male orchiectomized (ORX) rats, and that the changes in the bone marrow B cell compartment in ORX animals can be reversed by testosterone supplementation. In a subsequent, sequential experiment, we found that orchiectomy induced a sustained rise in Thy 1.1+/CD45R+ bone marrow cells committed for the B cell lineage that lasted for several months in 13-month-old aged rats. In a stepwise model of multiple regression analysis using estradiol, free and total testosterone as independent variables, estradiol was the strongest predictor of the percentage of B precursor cells in bone marrow in aged SHAM and ORX rats. Free and total testosterone did not correlate with B lymphopoiesis in aged SHAM rats. The current experiment has clearly shown that androgen withdrawal upregulates the number of B lineage cells over several months in rat bone marrow. Furthermore, our results provide evidence that estradiol may play an important role as a physiological suppressor of B lymphopoiesis in aged male rats.     

Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment

Cell-intrinsic checkpoints limit the proliferative capacity of primary cells in response to telomere dysfunction. It is not known, however, whether telomere dysfunction contributes to cell-extrinsic alterations that impair stem cell function and organ homeostasis. Here we show that telomere dysfunction provokes defects of the hematopoietic environment that impair B lymphopoiesis but increase myeloid proliferation in aging telomerase knockout (Terc(-/-)) mice. Moreover, the dysfunctional environment limited the engraftment of transplanted wild-type hematopoietic stem cells (HSCs). Dysfunction of the hematopoietic environment was age dependent and correlated with progressive telomere shortening in bone marrow stromal cells. Telomere dysfunction impaired mesenchymal progenitor cell function, reduced the capacity of bone marrow stromal cells to maintain functional HSCs, and increased the expression of various cytokines, including granulocyte colony-stimulating factor (G-CSF), in the plasma of aging mice. Administration of G-CSF to wild-type mice mimicked some of the defects seen in aging Terc(-/-) mice, including impairment of B lymphopoiesis and HSC engraftment. Conversely, inhibition of G-CSF improved HSC engraftment in aged Terc(-/-) mice. Taken together, these results show that telomere dysfunction induces alterations of the environment that can have implications for organismal aging and cell transplantation therapies.     

Somatically diversified and proliferating transitional B cells: implications for peripheral B cell homeostasis

The peripheral B cell compartment in mice and humans is maintained by continuous production of transitional B cells in the bone marrow. In other species, however, including rabbits, B lymphopoiesis in the bone marrow abates early in life, and it is unclear how the peripheral B cell compartment is maintained. We identified transitional B cells in rabbits and classified them into T1 (CD24(high)CD21(low)) and T2 (CD24(high)CD21(+)) B cell subsets. By neutralizing B cell-activating factor in vivo, we found an arrest in peripheral B cell development at the T1 B cell stage. Surprisingly, T1 B cells were present in GALT, blood, and spleen of adult rabbits, long after B lymphopoiesis was arrested. T1 B cells were distinct from their counterparts in other species because they are proliferating and the Ig genes are somatically diversified. We designate these newly described cells as T1d B cells and propose a model in which they develop in GALT, self renew, continuously differentiate into mature B cells, and thereby maintain peripheral B cell homeostasis in adults in the absence of B lymphopoiesis.     

The role of endogenous glucocorticoids in lymphocyte development in melanocortin receptor 2-deficient mice

Glucocorticoids are extensively used in anti-inflammatory therapy and are thought to contribute to the steady-state regulation of hematopoiesis and lymphopoiesis. We have previously established MC2R(-/-) mice, a model of familial glucocorticoid deficiency, that show several similarities to patients with this disease, including undetectable levels of corticosterone, despite high levels of ACTH and unresponsiveness to ACTH. In this study, we analyzed the possible roles of endogenous glucocorticoids in hematopoiesis and lymphopoiesis in MC2R(-/-) and CRH(-/-) mice as models of chronic adrenal insufficiency. Our analysis of total peripheral blood cell counts revealed that the number of lymphocytes was increased and the number of erythrocytes was slightly, but significantly, decreased in MC2R(-/-) mice. Numbers of immature double negative (CD4(-) CD8(-)) thymocytes, transitional type 1 B cells in the spleen, and pre-B cells in the bone marrow, were significantly increased in MC2R(-/-) mice, suggesting that endogenous glucocorticoids contribute to steady-state regulation of lymphopoiesis. Oral glucocorticoid supplementation reversed peripheral blood cell counts and reduced numbers of T and B cells in the thymus and the spleen. T cells in the thymus and B cells in the spleen were also increased in CRH(-/-) mice, another animal model of chronic adrenal insufficiency. MC2R(-/-) mice were sensitive to age-related thymic involution, but they were resistant to fasting-associated thymic involution. Our data support the idea that endogenous glucocorticoids contribute to stress-induced as well as steady-state regulation of hematopoiesis and lymphopoiesis.     

B cell deficiency progresses with lineage maturation in nude.X-linked immunodeficient mice B cell deficiency progresses with lineage maturation.

Previously we showed that unlike normal, nude, or X-linked immune deficient (xid) mice, nude.xid mice are deficient in bone marrow pre-B cell targets for Abelson murine leukemia virus transformation. We show that nude.xid bone marrow is deficient in both CD45(B220)+ and CD45(B220)- surface (s)IgM- progenitors that give rise to B cell colonies in Whitlock-Witte cultures. CD45(B220)+ precursors had normal differentiation potential in vitro. CD45(B220)- precursors differentiated into CD45(B220)+ cells at the same rate as normal controls, but acquired sIgM at a much slower rate. These results correlated with the observation that in nude.xid mice the severity of B lineage defects correlates with maturity: a profound (ninefold) deficit of sIgM+, CD45(B220)+ mature B cells, a fivefold deficit in the sIgM-, CD45(B220)+ precursors of short term B cell colonies (colonies forming within 4-5 days in Whitlock-Witte cultures), and a moderate (twofold) decrease in the frequency of sIgM-, CD45(B220)- (less mature) precursors of long term B cell colonies (colonies forming after 14 days of Whitlock-Witte culture. Thus the combination of the nude and xid mutations produces a deficiency in early B cell progenitors and the deficiency becomes more profound with further maturation. Therefore the lack of mature B cells is the result of a cascade effect. Inasmuch as bone marrow progenitors are affected, and these are the source of the vast majority of B cells, most B cells are affected by the xid mutation and the xid defect cannot be attributed to a loss of a fetal lineage of B cells. These results suggest that xid affected cells lack the capacity to progress efficiently through differentiation in the absence of an exogenous factor(s) that is dependent on the product of a normal allele at the nude locus. This product might be supplied in vivo by a T cell or T cell-dependent source and/or epithelial elements such as bone marrow stromal cells all of which are known to be affected by the nude mutation.     

Abnormalities of B lineage cells are demonstrable in long term lymphoid bone marrow cultures of New Zealand black mice

The formation of B lymphocytes in young New Zealand Black (NZB) mice proceeds at an accelerated rate resulting in a deficiency of B lineage precursors in adult (greater than 15 wk old) animals. To study the characteristics of B lineage cells in young (4 wk) and old (6 mo) NZB mice, bone marrow from these animals was used to initiate long term lymphoid bone marrow cultures (LBMC) that permit the long term maintenance of B cells and their precursors. Age-matched cultures from BALB/c mice and NZB.xid marrow were established in parallel. Primary LBMC were readily established from these strains and showed similar patterns of growth for the 3-mo observation period. No significant differences in numbers of 14.8 positive cells were observed. However, NZB mice at both ages had a higher percentage of membrane IgM (mIgM)-expressing cells. Significant levels of supernatant IgM were found only in cultures of 6-mo NZB and BALB/c mice; levels were highest in NZB culture supernatants and were often more than 500 ng/ml; significant, although much lower, levels of IgG were likewise detected. Lymphoid cells from NZB.xid mice were unable to generate significant levels of IgM in supernatant fluids indicating the effects of the xid gene were displayed in vitro. Autoantibodies were not detected in any of the culture supernatants. Additional evidence for NZB hyperactivity in primary B lymphopoiesis was observed upon initiation of primary myeloid bone marrow cultures (MBMC) from these strains of mice and subsequently transferring them to LBMC conditions. This results in the cessation of myelopoiesis at the initiation of B lymphopoiesis. At the time of converting MBMC to LBMC, cultures of NZB and BALB/c mice morphologically resembled myeloid cultures and had neither B cell colony-forming units nor cells that expressed 14.8 or mIgM. However, following the switch, NZB mice had a 5-fold higher number of B cell colony-forming units. Further, MBMC established from NZB bone marrow cells had a reduced capacity to form colonies in the granulocyte-macrophage colony-forming unit assay. These studies indicate that defects of NZB hemopoietic cells are manifest in vitro and suggest the use of in vitro long term cultures as a valuable technique to further dissect the hematopoietic abnormalities of NZB mice and possible underlying microenvironmental defects.     

CD80 expression on B cells regulates murine T follicular helper development, germinal center B cell survival, and plasma cell generation

Germinal center (GC) B cells and T follicular helper (T(FH)) cells interact in the production of high-affinity long-lived plasma cells (PCs) and memory B cells, although the mechanisms regulating the formation of these long-lived populations remain unclear. Because CD80 is one of the few markers shared by human and murine memory B cells, we investigated its role in the development of GCs, memory cells, and PCs. In CD80-deficient mice, fewer long-lived PCs were generated upon immunization compared with that in B6 controls. In concert, the absence of CD80 resulted in an increase in apoptotic GC B cells during the contraction phase of the GC. CD80(-/-) mice had fewer T(FH) cells compared with that of B6, and residual T(FH) cells failed to mature, with decreased ICOS and PD-1 expression and decreased synthesis of IL-21 mRNA. Mixed bone marrow chimeras demonstrated a B cell-intrinsic requirement for CD80 expression for normal T(FH) cell and PC development. Therefore, B cell expression of CD80 plays a critical role in regulating B-T interactions in both early and late GC responses. This, in turn, results in impaired ability to produce long-lived PCs. These data provide new insights into the development of GCs and Ab-forming cells and the functions of CD80 in humoral immunity.     

Maintenance of long-lived plasma cells and serological memory despite mature and memory B cell depletion during CD20 immunotherapy in mice

CD20 mAb-mediated B cell depletion is an effective treatment for B cell malignancies and some autoimmune diseases. However, the full effects of B cell depletion on natural, primary, and secondary Ab responses and the maintenance of Ag-specific serum Ig levels are largely unknown. The relationship between memory B cells, long-lived plasma cells, and long-lived humoral immunity also remains controversial. To address the roles of B cell subsets in the longevity of humoral responses, mature B cells were depleted in mice using CD20 mAb. Peritoneal B cell depletion reduced natural and Ag-induced IgM responses. Otherwise, CD20+ B cell depletion prevented humoral immune responses and class switching and depleted existing and adoptively transferred B cell memory. Nonetheless, B cell depletion did not affect serum Ig levels, Ag-specific Ab titers, or bone marrow Ab-secreting plasma cell numbers. Coblockade of LFA-1 and VLA-4 adhesion molecules temporarily depleted long-lived plasma cells from the bone marrow. CD20+ B cell depletion plus LFA-1/VLA-4 mAb treatment significantly prolonged Ag-specific plasma cell depletion from the bone marrow, with a significant decrease in Ag-specific serum IgG. Collectively, these results support previous claims that bone marrow plasma cells are intrinsically long-lived. Furthermore, these studies now demonstrate that mature and memory B cells are not required for maintaining bone marrow plasma cell numbers, but are required for repopulation of plasma cell-deficient bone marrow. Thereby, depleting mature and memory B cells does not have a dramatic negative effect on preexisting Ab levels.     

Relatively normal human lymphopoiesis but rapid turnover of newly formed B cells in transplanted nonobese diabetic/SCID mice.

Human B lineage lymphocyte precursors in chimeric nonobese diabetic/SCID mice transplanted with umbilical cord blood cells were directly compared with those present in normal bone marrow. All precursor subsets were represented and in nearly normal proportions. Cell cycle activity and population dynamics were investigated by staining for the Ki-67 nuclear Ag as well as by incorporation experiments using 5-bromo-2'-deoxyuridine. Again, this revealed that human B lymphopoiesis in chimeras parallels that in normal marrow with respect to replication and progression through the lineage. Moreover, sequencing of Ig gene rearrangement products showed that a diverse repertoire of V(H) genes was utilized by the newly formed lymphocytes but there was no evidence for somatic hypermutation. The newly formed B cells frequently acquired the CD5 Ag and had a short life span in the periphery. Thus, all molecular requirements for normal B lymphocyte formation are present in nonobese diabetic/SCID mice, but additional factors are needed for recruitment of B cells into a fully mature, long-lived pool. The model can now be exploited to learn about species restricted and conserved environmental cues for human B lymphocyte production.     

The age-dependent loss of bone marrow B cell precursors in autoimmune NZ mice results from decreased mitotic activity, but not from inherent stromal cell defects

The formation of B lymphocytes is abnormal in autoimmune NZB and (NZB x NZW)F1 mice. With age, the proportion of sIg- Ly-5(220)+ pre-B cells and less mature B cell progenitors in the bone marrow progressively declines, reaching only approximately one-third of normal levels in 20-wk-old NZ mice. To determine the mechanisms responsible for the deficiency of NZ B lineage precursors, the mitotic activity of sIg- Ly-5(220)+ bone marrow cells in vivo was determined in NZ and conventional inbred mice as a function of age. The proportion of sIg- Ly-5(220)+ B cell precursors in (S + G2/M) stages of the cell cycle steadily decreased with age in NZ autoimmune mice. Furthermore, upon metaphase arrest, the rate of entry of sIg- Ly-5(220)+ bone marrow cells into G2/M also decreased with age in NZ mice. Therefore, the mitotic activity of sIg- Ly-5(220)+ B cell precursors is substantially decreased in NZ mice greater than or equal to 20 wk of age. The capacity of the bone marrow stromal microenvironment of NZ mice to support B lineage precursor growth was tested in two ways: 1) the capacity of preformed NZ bone marrow stroma to support B lineage cell growth in long term bone marrow cell culture under lymphopoietic conditions was assessed and 2) the capacity of NZ bone marrow B lineage precursors to expand in vivo after sublethal (200 rad) whole body irradiation was determined. Stroma derived from adult NZ mice supported the growth and development of B lineage lymphocytes in long term bone marrow cell culture to a greater extent than did age-matched conventional murine stroma. Furthermore, sublethal irradiation of older adult NZ mice resulted in some expansion of bone marrow sIg- Ly-5(220)+ B cell precursors in vivo. Therefore, the deficiency of B cell progenitors in the bone marrow of older NZ autoimmune mice is associated with diminished mitotic activity. However, this does not result from defects in the capacity of NZ bone marrow stroma to permit B lineage cell expansion as determined by both in vitro and in vivo experiments. In the absence of a detectable stromal cell defect, it is possible that an active inhibitory process within the bone marrow influences the mitotic activity of B cell precursors in NZ mice.     

Cutting edge: B cell linker protein is dispensable for the allelic exclusion of immunoglobulin heavy chain locus but required for the persistence of CD5+ B cells

The pre-B cell receptor (pre-BCR) and the BCR are required for B lymphopoiesis and for the allelic exclusion of Ig genes. Mice lacking B cell linker (BLNK) protein that is a component of the BCR signaling pathway have impaired B cell development. In this report, we show that allelic exclusion is intact in BLNK(-/-) mice harboring a V(H)12 transgene. This differs from mice lacking the tyrosine kinase Syk that is upstream of BLNK in BCR signaling and contrasts with mice lacking SLP-76 that is the equivalent adaptor molecule in TCR-signal transduction. We also show that, whereas most wild-type V(H)12-expressing B cells are CD5(+), the majority of the splenic V(H)12-expressing BLNK(-/-) B cells are CD5(-). A small population of V(H)12-expressing, BLNK(-/-) CD5(+) B cells is detectable in the peritoneal cavity of younger but not older mice. This suggests that BLNK deficiency affects not only the generation but also the persistence of B-1 cells.     

Aging-dependent exclusion of antigen-inexperienced cells from the peripheral B cell repertoire

Aging is accompanied by greatly reduced B cell production in the bone marrow, yet peripheral B cell numbers do not decline. We hypothesize that this may reflect filling of the peripheral pool with B cells that are long-lived as a consequence of specificity for, and chronic stimulation by, environmental Ags. To begin to explore this possibility, we analyzed the effects of aging on B cell population dynamics in the anti-H2(k/b) 3-83 mu-delta Ig-transgenic mouse. We predicted that, because they presumably do not bind environmental Ags, B cells bearing the transgenic receptor may be lost in aged animals. As seen in nontransgenic animals, total splenic B cell numbers remained constant with age in the Ig-transgenic animals despite reduced B cell production. Importantly, although the few newly produced B cells in the bone marrow of aged mice are 3-83 positive, the peripheral compartment of these mice is dominated by B cells that express endogenous Ig genes rather than the transgenes. This population includes large numbers of marginal zone-like and CD21(low/-)CD23(low/-)IgM(low) B cells, as well as elevated numbers of CD5+ B cells. Many of these cells express only non-B220 CD45 isoforms, suggesting that they may be memory cells. A significant proportion of aged transgenic animals produce autoantibodies that are reactive with ssDNA, dsDNA, or histones. Results support the hypothesis that, in the face of severely reduced production with age, B cells are selected based on reactivity to environmental Ags, accumulate, and display activated phenotypes. Cells bearing 3-83-transgenic receptors are excluded from this population due to their specificity. Beyond their importance in aging, these findings define a novel form of receptor revision in which B cells are selected rather than deleted based on Ag reactivity.     

B lymphocyte development in rabbit: progenitor B cells and waning of B lymphopoiesis

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-mu and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2'-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one lambda5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and lambda5 coimmunoprecipitated with the mu-chain in lysates of 293T cells transfected with VpreB, lambda5, and mu, indicating that VpreB, lambda5, and mu-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.     

Maintenance of long term gamma-herpesvirus B cell latency is dependent on CD40-mediated development of memory B cells

It has been proposed that the gamma-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40(+) and CD40(-) B cells from CD40(+)CD40(-) mixed bone marrow chimera mice after infection with a murine gamma-herpesvirus, MHV-68. CD40(+) B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40(-) B cells and preferentially maintained in the long-lived, isotype-switched CD40(+) B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency.     

Differential role for cyclic AMP response element binding protein-1 in multiple stages of B cell development, differentiation, and survival

CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser(119-ala) phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed approximately 70% increase in pre-B1 (CD43(+)B220(+)CD24(+(int))) and approximately 60% decreased pre-BII (CD43(+)B220(+)CD24(++(high))) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed approximately 4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into RAG-2(-/-) mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43(+)B220(+)CD24(high) population that is normally absent in RAG-2(-/-) mice. In the periphery, the Tg mice exhibited decreased CD21(dim)CD23(high)IgM(+) follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the first evidence for a differential role for CRE-binding proteins in multiple stages of B cell development, functional maturation, and B1 and B2 B cells.     

Age-dependent changes in B lymphocyte lineage cell populations of autoimmune-prone BXSB mice

BXSB mice, a recently developed autoimmune strain, develop a human lupus-like disease with B cell hyperplasia in peripheral lymphoid organs. Unlike other experimental models of autoimmunity and human lupus, BXSB male mice manifest accelerated autoimmune phenomena through the influence of a Y chromosome-linked enhancing factor. The present studies were performed to investigate the features of B lymphopoiesis in BXSB mice and to determine whether differences exist between BXSB males and females in this respect. B lineage cell populations in the marrow of BXSB mice were identified phenotypically by studying the cytoplasmic mu-heavy chains of IgM (c mu), and functionally by their ability to acquire clonability and sIg in short-term liquid cultures. Male BXSB mice became deficient in both the precursors of functional B cells and c mu + pre-B cells by the age of 8 to 12 wk. This followed a transient increase in this population, which peaked when the mice were 2 to 4 wk old. In females, substantial numbers of functional B cell precursors and c mu + cells were maintained until more than 4 mo of age. Cells lacking Ig but bearing a B lineage cell antigen (14.8) were elevated in numbers in both BXSB males and females until 16 wk of age when compared to normal strains of mice. At the time pre-B cells and functional B precursors were elevated in numbers, some sIg- cells were shown to form colonies in mitogen-stimulated semisolid agar cultures without a period of preculture. Most of these sIg- cells seemed to bear the B lineage cell antigen (14.8). They were independent of both G-10 adherent regulatory cells and Thy-1+ cells for their colony formation. These results indicate that B lymphocyte formation may be maintained in a hyperactive state in BXSB females, whereas males become deficient in B cell precursors very early in life. This early decline might be related to the accelerated development of autoimmune disease in BXSB mice. Bone marrow transplantation studies showed that these unusual characteristics of B lymphopoiesis were reciprocally transferable with unseparated bone marrow cells between BXSB males and females. This finding indicates that sex hormones are not a critical variable in abnormal B lymphocyte formation in this strain, and that the premature deficiency of immediate B precursors in males may be regulated by a genetic factor(s) located on the Y chromosome.     

In senescence,age‐associated B cells secrete TNFα and inhibit survival of B‐cell precursors*

Aged mice exhibit ~ 5–10‐fold increases in an ordinarily minor CD21/35^? CD23^? mature B‐cell subset termed age‐associated B cells (ABCs). ABCs from old, but not young, mice induce apoptosis in pro‐B cells directly through secretion of TNFα. In addition, aged ABCs, via TNFα, stimulate bone marrow cells to suppress pro‐B‐cell growth. ABC effects can be prevented by the anti‐inflammatory cytokine IL‐10. Notably, CD21/35⁺ CD23⁺ follicular (FO) splenic and FO‐like recirculating bone marrow B cells in both young and aged mice contain a subpopulation that produces IL‐10. Unlike young adult FO B cells, old FO B cells also produce TNFα; however, secretion of IL‐10 within this B‐cell population ameliorates the TNFα‐mediated effects on B‐cell precursors. Loss of B‐cell precursors in the bone marrow of old mice in vivo was significantly associated with increased ABC relative to recirculating FO‐like B cells. Adoptive transfer of aged ABC into RAG‐2 KO recipients resulted in significant losses of pro‐B cells within the bone marrow. These results suggest that alterations in B‐cell composition during old age, in particular, the increase in ABC within the B‐cell compartments, contribute to a pro‐inflammatory environment within the bone marrow. This provides a mechanism of inappropriate B‐cell ‘feedback’ that promotes down‐regulation of B lymphopoiesis in old age.