Age-associated B cells (ABC) inhibit B lymphopoiesis and alter antibody repertoires in old age.

With old age (∼2y old), mice show substantial differences in B cell composition within the lymphoid tissues. In particular, a novel subset of IgM+ CD21/35lo/- CD23- mature B cells, the age-associated B cells or ABC, increases numerically and proportionately. This occurs at the expense of other B cell subsets, including B2 follicular B cells in spleen and recirculating primary B cells in bone marrow. Our studies suggest that ABC have a distinctive antibody repertoire, as evidenced by relatively high reactivity to the self-antigens phosphorylcholine (PC) and malondialdehyde (MDA). While PC and MDA are found on apoptotic cells and oxidized lipoproteins, antibodies to these antigens are also cross-reactive with epitopes on bacterial species. In old mice, ABC express TNFα and are pro-inflammatory. ABC can inhibit growth and/or survival in pro-B cells as well as common lymphoid progenitors (CLP). In particular, ABC cause apoptosis in pro-B cells with relatively high levels of the surrogate light chain (SLC) and, consequently, promote an "SLC low" pathway of B cell differentiation in old mice. SLC together with µ heavy chain comprises the pre-B cell receptor (preBCR) critical for pre-B cell expansion and selection of the µ heavy chain Vh repertoire. The low level of SLC likely impairs normal preBCR driven proliferation and alters µ heavy chain Vh selection thereby affecting the antibody specificities of new B cells. In this manner, ABC may contribute to both qualitative and quantitative disruptions of normal B lymphopoiesis in old age.

In old BALB/c mice, bone marrow pre-B cell and surrogate light chain reduction is associated with increased B cell reactivity to phosphorylcholine, but reduced T15 idiotype dominance.

In young adult BALB/c mice, antibodies to phosphorylcholine (PC) bearing the T15 (TEPC 15) idiotype confer protection against pneumococcal infections. In old age, even though PC reactive B cells are often increased, the proportion of T15+ antibodies declines. We hypothesize that limited surrogate light chain (SLC) and compromise of the pre-B cell receptor checkpoint in old mice contribute to both reduced new B cell generation and changes in the anti-PC antibodies seen in old age. In old mice: 1) early pre-B cell loss is most pronounced at the preBCR checkpoint; however, the reduced pool of early pre-B cells continues to proliferate consistent with preBCR signaling; 2) increased PC reactivity is seen in bone marrow immature B cells; 3) deficient SLC promotes increased B cell PC reactivity and diminished T15 idiotype expression; and 4) as pre-B cell losses and reduced SLC become progressively more severe, increased T15 negative PC reactive B cells occur. These results associate a reduction in pre-B cells, imposed at the preBCR checkpoint, with increased reactivity to PC, but more limited expression of the protective T15 idiotype among PC reactive antibodies in old age.

Impaired B lymphopoiesis in old age: a role for inflammatory B cells?

Continued generation of new B cells within the bone marrow is required throughout life. However, in old age, B lymphopoiesis is inhibited at multiple developmental stages from hematopoietic stem cells through the late stages of new B cell generation. While changes in B cell precursor subsets, as well as alterations in the supporting bone marrow microenvironment, in old age have been known for the last 20 years, only more recently have insights into the cellular and molecular mechanisms responsible become clarified. Our recent discovery that B cells in aged mice are pro-inflammatory and can diminish B cell generation within the bone marrow suggests a potential mechanism of inappropriate "B cell feedback" which contributes to a bone marrow microenvironment unfavorable to B lymphopoiesis. We hypothesize that the consequences of a pro-inflammatory microenvironment in old age are (1) reduced B cell generation and (2) alteration in the "read-out" of the antibody repertoire. Both of these likely ensue from reduced expression of the surrogate light chain (λ5 + VpreB) and consequently reduced expression of the pre-B cell receptor (preBCR), critical to pre-B cell expansion and Vh selection. In old age, B cell development may progressively be diverted into a preBCR-compromised pathway. These abnormalities in B lymphopoiesis likely contribute to the poor humoral immunity seen in old age.

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.

A molecular mechanism for TNF-α-mediated downregulation of B cell responses.

B cell function with age is decreased in class switch recombination (CSR), activation-induced cytidine deaminase (AID), and stability of E47 mRNA. The latter is regulated, at least in part, by tristetraprolin (TTP), which is increased in aged B cells and also negatively regulates TNF-α. In this study, we investigated whether B cells produce TNF-α, whether this changes with age, and how this affects their function upon stimulation. Our hypothesis is that in aging there is a feedback mechanism of autocrine inflammatory cytokines (TNF-α) that lowers the expression of AID and CSR. Our results showed that unstimulated B cells from old BALB/c mice make significantly more TNF-α mRNA and protein than do B cells from young mice, but after stimulation the old make less than the young; thus, they are refractory to stimulation. The increase in TNF-α made by old B cells is primarily due to follicular, but not minor, subsets of B cells. Incubation of B cells with TNF-α before LPS stimulation decreased both young and old B cell responses. Importantly, B cell function was restored by adding anti-TNF-α Ab to cultured B cells. To address a molecular mechanism, we found that incubation of B cells with TNF-α before LPS stimulation induced TTP, a physiological regulator of mRNA stability of the transcription factor E47, which is crucial for CSR. Finally, anti-TNF-α given in vivo increased B cell function in old, but not in young, follicular B cells. These results suggest new molecular mechanisms that contribute to reduced Ab responses in aging.

E47 retroviral rescue of intrinsic B-cell defects in senescent mice.

In aging, immune responses are dramatically impaired, specifically the ability to produce protective antibodies. We previously showed that with age there is a B-cell intrinsic decrease in class switch recombination (CSR) because of a decrease in activation-induced cytidine deaminase (AID). One mechanism we have demonstrated for decreased AID includes increased mRNA degradation of the transcription factor E47, critical for AID transcription. Here, we show by means of a retroviral construct containing the DsRED reporter and the 3'UTR of E47 that the 3'UTR lowers mRNA expression, and particularly in B cells from old mice. This is the first demonstration that the E47 3'UTR directly regulates its degradation. The AID mRNA was not differentially regulated by degradation in aging. Therefore, we have here further established critical components for decreased AID with age. The major aim of this study was to establish conditions for the rescue of the intrinsic defect of aged B cells with retroviral addition of the coding region of E47 in splenic B cells to restore their ability to produce optimal AID and class switch to IgG. In this study, we show that young and old primary B cells overexpressing a stable E47 mRNA up-regulate E47, AID, and CSR and improve B-cell immune responses in senescent murine B cells. Our results provide a proof of principle for the rescue of intrinsic B-cell defects and the humoral immune response in senescence.

B cells and aging: molecules and mechanisms.

Recent advances allow aging-associated changes in B-cell function to be approached at a mechanistic level. Reduced expression of genes crucial to lineage commitment and differentiation yield diminished B-cell production. Moreover, intrinsic differences in the repertoire generated by B-cell precursors in aged individuals, coupled with falling B-cell generation rates and life-long homeostatic competition, result in narrowed clonotypic diversity. Similarly, reductions in gene products crucial for immunoglobulin class switch recombination and somatic hypermutation impact the efficacy of humoral immune responses. Together, these findings set the stage for integrated analyses of how age-related changes at the molecular, cellular and population levels interact to yield the overall aging phenotype.

NK cells in the CD19- B220+ bone marrow fraction are increased in senescence and reduce E2A and surrogate light chain proteins in B cell precursors.

E2A encoded proteins, key transcriptional regulators in B lineage specification and commitment, have been shown to decrease in B cell precursors in old age. E2A regulates genes encoding the surrogate light chain proteins lambda5 and VpreB. In old age, B cell precursors express less surrogate light chain and this results in compromised pre-B cell receptor function and diminished expansion of new pre-B cells in senescence. Herein, we show that aged bone marrow has increased Hardy Fraction A (CD19(-) B220(+)) cells, including NK cells, that can inhibit both E47 (E2A) protein and surrogate light chain protein expression in B cell precursors. In vitro, NK-associated inhibition of E47 protein is contact-independent and partially reversed by neutralization of TNFalpha. In vivo, depletion of NK cells in aged mice by treatment with anti-asialo GM1 antibody led to restoration of surrogate light chain protein levels to that typical of young B cell precursors. These studies suggest that NK cells, within the CD19(-) B220(+) bone marrow cell fraction, may contribute to a bone marrow microenvironment that has the potential to negatively regulate E47 (E2A) as well as surrogate light chain levels in B cell precursors in old age.

Old mice retain bone marrow B1 progenitors, but lose B2 precursors, and exhibit altered immature B cell phenotype and light chain usage.

The bone marrow of old adult mice ( approximately 2 years old) has reduced B lymphopoiesis; however, whether the B1 pathway in adult bone marrow is also compromised in senescence is not known. Herein, we show that phenotypic (IgM(-)Lin(-)CD93(+)[AA4.1(+)] CD19(+)B220(low/-)) B1 progenitors are retained in old bone marrow even as B2 B cell precursors are reduced. Moreover, B1 progenitors from both young adult and old mice generated new B cells in vitro enriched for CD43 expression, likely due to their activation, and exhibited increased lambda light chain usage and diminished levels of kappa light chain expression. B1 progenitors were shown to have lower surrogate light chain (lambda5) protein levels than did B2 pro-B cells in young mice and these levels decreased in both B1 and B2 precursor pools in old age. These results indicate that the B1 B cell pathway persists during old age in contrast to the B2 pathway. Moreover, B1 B cell progenitors generated new B cells in the adult bone marrow that have distinct surface phenotype and light chain usage. This is associated with decreased surrogate light chain expression, a characteristic held in common by B1 progenitors as well as B2 precursors in old mice.

Deviation of the B cell pathway in senescent mice is associated with reduced surrogate light chain expression and altered immature B cell generation, phenotype, and light chain expression.

B lymphopoiesis in aged mice is characterized by reduced B cell precursors and an altered Ab repertoire. This likely results, in part, from reduced surrogate L chains in senescent B cell precursors and compromised pre-BCR checkpoints. Herein, we show that aged mice maintain an ordinarily minor pool of early c-kit(+) pre-B cells, indicative of poor pre-BCR expression, even as pre-BCR competent early pre-B cells are significantly reduced. Therefore, in aged mice, B2 B lymphopoiesis shifts from dependency on pre-BCR expansion and selection to more pre-BCR-deficient pathways. B2 c-kit(+) B cell precursors, from either young or aged mice, generate new B cells in vitro that are biased to larger size, higher levels of CD43, and decreased kappa L chain expression. Notably, immature B cells in aged bone marrow exhibit a similar phenotype in vivo. We hypothesize that reduced surrogate L chain expression contributes to decreased pre-B cells in aged mice. The B2 pathway is partially blocked with limited B cell development and reduced pre-BCR expression and signaling. In old age, B2 pathways have limited surrogate L chain and increasingly generate new B cells with altered phenotype and L chain expression.

Mechanisms for decreased function of B cells in aged mice and humans.

The immune system has been known for some time to be compromised in aged individuals, e.g., both mice and humans, and in both humoral and cellular responses. Our studies have begun to elucidate intrinsic B lymphocyte defects in Ig class switch recombination, activation-induced cytidine deaminase, and E47 transcription factor expression. These defects occur in both mice and humans. Our studies have also shown that tristetraprolin is one of the key players in regulating the decreased E47 mRNA stability in aged B lymphocytes. These and current studies should lead to improvements in B lymphocyte function in aged populations.

Accelerated Notch-dependent degradation of E47 proteins in aged B cell precursors is associated with increased ERK MAPK activation.

The transcriptional regulator E47, encoded by the E2A gene, is crucial to B lymphopoiesis. In BALB/c senescent mice (approximately 2 years old), the incidence of E47-expressing pro-B cells in vivo and E47 protein steady state levels in B cell precursors in vitro were reduced. Poor expression of E47 protein was a consequence of accelerated proteasome-mediated turnover and was associated with heightened ubiquitin modification of E2A-encoded proteins in aged B cell precursors. Both MAPK and Notch activity have been previously associated with E2A-encoded protein stability in lymphocytes. Aged B cell precursors exhibited heightened levels of MAPK activity reflected in increased levels of phospho-ERK proteins. Phosphorylation of E2A-encoded proteins was also increased in aged B cell precursors and pharmacologic inhibition of MEK-1 resulted in a partial restoration of their E47 protein. Both Notch proteins and their Delta-like ligands were detected comparably in young and aged B cell precursors. Either inhibition of Notch activation via gamma-secretase or Ab blockade of Notch-Delta-like ligand interactions partially restored E47 expression in aged B cell precursors. We hypothesize that increased MAPK activity promotes phosphorylation of E2A-encoded protein in aged B cell precursors. Subsequently, E2A-encoded proteins undergo ubiquitination and accelerated degradation in a Notch-dependent process. The dysregulation of E2A-encoded protein expression may contribute to the reductions seen in early B lymphopoiesis during murine senescence.

Aging murine B cells have decreased class switch induced by anti-CD40 or BAFF.

We previously demonstrated that in vitro stimulated splenic B cells from senescent mice are deficient in production of multiple class switch isotypes, class switch recombination (CSR), induction of the E2A-encoded transcription factor E47, and activation-induced cytidine deaminase (AID) which is necessary for CSR and somatic hypermutation. Both anti-CD40 as well as BAFF have been shown to be able to induce CSR. We have investigated the ability of BAFF/IL-4, as compared to anti-CD40/IL-4, to induce CSR to gamma(1) in splenic B cells from young and old mice. We found that anti-CD40/IL-4 is a better CSR stimulus than BAFF/IL-4 in young B cells, as measured by RT-PCR of post-switch transcripts and flow cytometry. CSR is reduced in old B cells and this is independent of the stimulus. AID and gamma(1)PSTs are significantly reduced in old B cells stimulated with anti-CD40/IL-4, but only slightly reduced with BAFF/IL-4. BAFF receptor mRNA expression (BAFF-R, TACI, and BCMA) is not affected by aging. The age-related decrease in CSR induced by anti-CD40/IL-4 is primarily associated with a decrease in E47, whereas the less affected response to BAFF/IL-4 is associated with decreases in both E47 and NF-kappaB. Therefore, NF-kappaB is not involved in the decreased response of old B cells to anti-CD40/IL-4. These differences in B cell responses to CD40/IL-4 and BAFF/IL-4 may help to explain the maintenance of TI vs TD responses in senescent mice.

Humoral immune response and B-cell functions including immunoglobulin class switch are downregulated in aged mice and humans.

Vaccinations are powerful tools for combating infections. Because of the age-related impairment in immune functions, the currently available vaccines are protecting only a small proportion of the elderly population. We, here, provide an overview of age-related changes in innate and adaptive immunity with particular emphasis to changes in antibody production with aging. We also summarize our results showing that the E2A-encoded transcription factor E47, which regulates many B cell functions including class switch recombination (CSR) and somatic hypermutation (SHM), is downregulated in splenic B cells from old mice. This leads to a reduction in the activation-induced cytidine deaminase (AID), which directly induces CSR and SHM, and, in turn, to reduced amounts of switched antibodies produced by splenic activated B cells. Our preliminary results in humans indicate similar reductions: we show herein that the expression of E2A and AID progressively decline with age. Our results provide a possible molecular basis for a decrease in the humoral immune response in aging mice and humans.

Deficient B lymphopoiesis in murine senescence: potential roles for dysregulation of E2A, Pax-5, and STAT5.

B lymphopoiesis in senescent mice is typically diminished and characterized by low pre-B cell numbers. The transcription factors E2A, Pax-5, and STAT5 have been implicated in the differentiation, proliferation, and survival of B cell precursors. In this review, we discuss the impairment of B lymphopoiesis during old age in the context of mechanisms at the molecular level responsible for the handling and turnover of these key transcriptional proteins. Alterations in the expression of E2A, Pax-5, and STAT5 may affect multiple stages of B cell development, contribute to reduced B lymphopoiesis, and preface changes in the "read-out" of the BCR repertoire during murine senescence.

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.

Decreased E47 in senescent B cell precursors is stage specific and regulated posttranslationally by protein turnover.

The E2A-encoded transcription factor E47 is crucial to B lymphopoiesis. Senescent BALB/c mice ( approximately 2 years old) had reduced pre-B cells ex vivo. Pro-B/early pre-B cells from these aged mice, both ex vivo and in vitro, were deficient in E47 protein. In vitro, IL-7 expanded pro-B/early pre-B cells from young BALB/c mice expressed E47 protein that was relatively stable over a 5-h period. Cultured senescent pro-B/early pre-B cells exhibited reduced E47 protein stability with approximately 50-90% loss of E47 over the same time period. Degradation of E47 was effectively blocked by the proteasome inhibitor lactacystin as well as calpain I and II inhibitors; E2A proteins were also shown to undergo ubiquitination. Although senescent B cell precursors expressed less E47 protein, E47 mRNA levels and turnover were normal. Therefore, E47 protein levels are reduced relatively early in B lineage differentiation in senescence and the decline in E47 protein occurs via increased protein degradation by proteasome and, possibly, calpain pathways. In contrast, normal E47 protein levels were observed within the highly reduced pre-B cell pool in aged mice. This suggests that pre-B cells in senescence undergo selection based on E47 expression. Increased degradation rates and lower steady-state levels were also observed for the transcription factors Pax-5/BSAP, Bob-1, and Ikaros, but this was not a general property of all proteins in aged B cell precursors. Therefore, altered turnover of multiple, select proteins crucial to B cell development may contribute to diminished B lymphopoiesis in old age.

Age-related differences in the E2A-encoded transcription factor E47 in bone marrow-derived B cell precursors and in splenic B cells.

We have investigated the effects of aging on the E2A-encoded transcription factor E47, a key regulator of B cell functions, in B cell precursors and in splenic B cells. Here, we show that old mice can be classified as severely depleted, moderately depleted or not depleted mice, according to the percentage of pre-B cells in their bone marrow. IL-7-expanded populations of pro-B/early pre-B cells from bone marrow of both severely depleted and moderately depleted old mice exhibit a reduced E47 DNA-binding and expression compared to young mice, and this defect in severely depleted old mice is more dramatic than that in moderately depleted old mice. However, mRNA levels were comparable, suggesting that E47 in the bone marrow is not transcriptionally regulated. In the spleen, activated B cells from both severely depleted and moderately depleted old mice show a lower E47 DNA-binding and expression than young mice. However, in contrast to precursor B cells, E47 DNA-binding and expression are similarly and only moderately reduced in both severely depleted and in moderately depleted mice. The mRNA levels were found to be decreased in stimulated splenic B cells from old as compared to young mice, suggesting that E47 mRNA in the spleen may be both transcriptionally and/or post-transcriptionally regulated.

Reduced Ig class switch in aged mice correlates with decreased E47 and activation-induced cytidine deaminase.

The capacity to class switch the IgH chain is critical to the effectiveness of humoral immune responses. We show that in vitro-stimulated splenic B cells from senescent mice are deficient in production of multiple class switch isotypes (IgG1, G2a, G3, and E), class switch recombination (CSR), and induction of the E2A-encoded transcription factor E47. E47 has previously been shown to be required for CSR, at least in part via expression of the activation-induced cytidine deaminase. Our studies show that impaired induction of E47, and subsequently activation-induced cytidine deaminase, contribute to poor CSR and production of secondary isotypes in senescence.

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.