Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 Mar 1.
Published in final edited form as: J Immunol. 2017 Jan 16;198(5):1921–1927. doi: 10.4049/jimmunol.1601106

Age-associated B cells express a diverse repertoire of VH and Vκ genes with somatic hypermutation

Lisa M Russell Knode *, Martin S Naradikian †, Arpita Myles †, Jean L Scholz †, Yi Hao †,1, Danya Liu ‡, Mandy L Ford ‡, John W Tobias §, Michael P Cancro †,2, Patricia J Gearhart *,2
PMCID: PMC5322232  NIHMSID: NIHMS839153  PMID: 28093524

Abstract

The origin and nature of age-associated B cells (ABCs) in mice is poorly understood. Here we show that their emergence required MHC class II and CD40/CD40L interactions. Young donor B cells were adoptively transferred into congenic recipients and allowed to remain for one month in the absence of external antigen. B cells expressing the T-bet transcription factor, a marker for ABCs, were generated after multiple cell divisions from C57BL/6 donors, but not from MHC class II- or CD40-deficient donors. Furthermore, old CD154 (CD40L)-deficient mice did not accrue ABCs, confirming that they arise primarily through T-dependent interactions. To determine what immunoglobulins ABCs express, we sequenced VH and Vκ rearranged genes from unimmunized 22-month old C57BL/6 mice, and showed that they had a heterogeneous repertoire, which was comparable to that seen in old follicular and marginal zone B cell subsets. However, in contrast to the follicular and marginal zone cells, ABCs displayed significant somatic hypermutation. The mutation frequency was lower than found in germinal center cells after deliberate immunization, suggesting ABCs have undergone mild stimulation from endogenous antigens over time. These observations show that quiescent ABCs are antigen-experienced cells that accumulate during T-cell dependent responses to diverse antigens during the life of an individual.

Introduction

Profound changes in the composition and dynamics of lymphoid populations occur with age, likely contributing to the decline in immune status, collectively termed immune senescence. For example, B cell production from bone marrow steadily decreases with age, yet the numbers of peripheral B cells remain relatively constant, due to slowed turnover and altered representation of naive and antigen-experienced B cell subsets (1-8). A novel B cell subset that accumulates with age, termed age-associated B cells (ABCs), was recently identified (9-12). These cells have unique features, that include preferential responsiveness to TLR7 and TLR9 ligands, surface markers consistent with prior antigen activation, and expression of the T-box transcription factor, Tbx21 (T-bet), which is required for their accumulation (13). Some ABCs also express Itgax (CD11c), an integrin that potentiates their ability to present antigen to T cells (14). ABCs are associated with the onset and severity of humoral autoimmunity in both animal models and humans (10, 15, 16). Further, these cells play roles in age-associated immune dysfunctions, including elevated inflammatory cytokine levels and reduced B cell generation rates (11). Finally, a growing literature suggests that B cells with similar characteristics arise during some viral, bacterial, and parasitic infections (13, 17-21), implying a role for ABCs in normal immune function.

Despite these observations, the origin and nature of ABCs remain poorly understood. Here we investigate their formation, immunoglobulin repertoire, and level of somatic hypermutation. The results indicate a polyclonal, antigen-experienced B cell population that arises primarily through T-dependent immune responses to diverse endogenous antigens.

Materials and Methods

Mice

All mice used for experiments were females on a C57BL/6 background. Old mice were obtained from the Charles River aged mouse colony at 18 months of age and used at 22 months. Cd154-/- (B6.129S2-Cd40lgtm1Imx/J) mice were purchased from the Jackson Laboratory and kept until 22 months of age. Young (2-4 month) CD45.1 and CD45.2 mice were obtained from the Jackson Laboratory. Young I-Ab-/- mice were from Terri Laufer (University of Pennsylvania), and Cd40-/- spleens from young mice were sent from M. Ford's colony. Aid -/- mice were bred in the NIA colony. Animal protocols were reviewed and approved by the Animal Care and Use Committees at the National Institute on Aging and the University of Pennsylvania.

Adoptive transfers

CD23+ splenic B cells from 2 month-old CD45.2 mice were enriched by positive selection using the MACS bead system (Miltenyi Biotec). Cells were then labeled with CFSE (eBioscience) according to the manufacturer's instructions, and 8 million cells were transferred into each CD45.1 congenic host by retro-orbital injection.

Flow cytometry and FACS sorting

Single cell suspensions were prepared from spleens and stained with fluorochrome-conjugated antibodies. For flow cytometry of the adoptive transfer and influenza experiments, Live/Dead Zombie Aqua, anti-CD45.1-AF700 (A20), anti-CD45.2-BV421 (104), anti-CD19-BV785 (6D5), and anti-CD23 biotin (B3B4), and anti-CD11c (N418) were from Biolegend. Anti-CD43-PE (S7) was from BD Biosciences. Cells were analyzed on an LSRII, and data analyzed using FlowJo software (Tree Star). Intracellular stains for T-bet were performed with anti-T-bet-APC (4B10) from Biolegend and the Foxp3 transcription factor kit (eBioscience) according to manufacturer's instructions. For FACS sorting to isolate subsets, anti-CD43-APC (S7) was from BD Biosciences. Anti-CD23-PE Cy7 (B3B4), anti-CD21/CD35-eFluor 450 (4E3), anti-CD45R-FITC (B220, RA3-6B2), and anti-CD93 (AA4.1)-APC were from eBioscience. Stained splenocytes were analyzed with a BD FACSCanto II, or sorted using a BD FACSAria III, BD FACSAria Fusion, iCyt Reflection (Sony Biotechnology), or Beckman Coulter MoFlo. Follicular (FO) B cells were isolated as CD93 (AA4.1)- CD43- B220+ CD21/35+ CD23+. Marginal zone (MZ) B cells were isolated as CD93 (AA4.1)- CD43- B220+ CD21/35+ CD23Lo. ABCs were isolated as CD93 (AA4.1)- CD43- B220+ CD21/35- CD23-. Analyses were done using FlowJo software.

V gene identification and mutation analyses

Sorted cells were lysed in Trizol and RNA was prepared. cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). Immunoglobulin heavy (IgH) chain variable, diverse, and joining (VDJ) genes, and kappa light (Igκ) chain VJ genes were amplified using Taq polymerase (TaKaRa, Clontech) with 5′ degenerate primers specific to framework 1 of V genes and 3′ primers located in IgM or Igκ constant regions as previously described (22). PCR products were then cloned into Strataclone TA cloning vector (Agilent Technologies) and sequenced. Only sequences with unique VDJ or VJ joins were counted. The sequences were blasted against the mouse Ig loci using IgBLAST from NCBI to identify V, D, and J gene segment usage and mutations. For mutational analysis of the JH4 intron, DNA was prepared, and a 492-bp intronic region downstream of JH4 from rearranged VHJ558 genes was amplified using nested PCR. The first round used forward primer J558 5′-AGCCTGACATCTGAGGAC-3′ and reverse primer V1.8NR4R 5′-TCCATACACATACTTCTGTGTTCCT-3′, and the second round used the same J558 forward primer listed above and reverse primer JH2827Bam 5′-CGCGGATCCGATGCCTTTCTCCCTTGACTC-3′. DNA was amplified using Herculase II Fusion DNA polymerase (Agilent Technologies). The amplified PCR products were then cloned into StrataClone Blunt PCR Cloning vector (Agilent Technologies), and sequenced.

Influenza virus infection and analysis

4-month old C57BL/6 mice were either uninfected or infected intranasally with 30 tissue culture infectious dose50 of influenza strain A/Puerto Rico/8/1934 (PR8), which was provided by Dr. Scott Hensley (University of Pennsylvania). Both uninfected and infected mice were sacrificed 100 days later. To detect hemagglutinin reactive B cells, we used a phycoerythrin (PE)-labelled probe that recognizes H1 hemagglutinin of PR8 (HA-PE) (23). The probe was used at a concentration of 1:500, and data acquisition and analysis were performed as described (23).

Results

ABC generation requires B cell expression of MHC class II and CD40

We showed previously that ABCs could arise from FO B cells after in vivo expansion in adoptive hosts (9). This extensive division may reflect homeostatic expansion, or could instead implicate antigen-driven activation involving T cell help and co-stimulation. To distinguish these possibilities, we modified our adoptive transfer model with CFSE-labeled donor B cells to use MHC class II- or CD40-deficient donor B cells. The rationale was that homeostatic expansion should be independent of both antigen presentation and co-stimulation, whereas antigen-driven events should not. As shown in Fig. 1A, after one month in the absence of immunization, a small proportion of C57BL/6 donor B cells, ∼0.2%, underwent 5-8 rounds of division, likely in response to stimulation by endogenous antigens. These extensively-divided CFSElo cells were CD23-negative and T-bet-positive, which are markers for ABCs. Although the events occurred in only a month, they represent a snapshot of the slow accumulation of ABCs with time. In contrast, B cells from MHC class II-deficient (I-Ab-/-) and CD40-deficient (Cd40-/-) mice underwent fewer divisions with far less T-bet expression than C57BL/6 cells of the same division cohort. Analyses of multiple mice in Fig. 1B confirmed a significant increase in T-bet mean fluorescence intensity in CFSElo cells compared to CFSEhi cells from C57BL/6 donors, whereas cells from I-Ab-/- and Cd40-/- donors had no increase. The data suggest that ABCs arise from B cells involved in immune responses to T-dependent antigens, because both cognate antigen-presenting capacity and competence to receive CD40 co-stimulation are required. This interpretation further predicts that CD154-deficient mice, which lack the CD40 ligand, should have reduced ABC accumulation. Consistent with this expectation, analysis of splenic B cells from 22-month old CD154-deficient mice revealed a paucity of ABCs (Fig. 1C), despite no change in FO and MZ compartments compared to controls (Fig. 1D). Collectively, these results show that ABCs are generated slowly with time after endogenous antigen presentation via MHC class II, and co-stimulation via the CD40 receptor with the CD40 ligand on T cells. The notion that ABCs are derived from T-dependent immune responses raises questions about the breadth and nature of potential antigens involved in their generation, and whether they bear hallmarks of germinal center participation. Accordingly, we interrogated both immunoglobulin variable (V) gene usage and levels of somatic hypermutation among quiescent, naturally occurring ABCs from old mice.

Figure 1.

Figure 1

Open in a new tab

Interactions with MHC class II and CD40 drive the accumulation of ABCs. (A) CD23+ FO B cells from 2-month old donor mice (CD45.2) were labeled with CFSE and adoptively transferred into young congenic CD45.1 hosts. Recipient mice were analyzed one month later. Shown are a representative plot of the gating strategy, and representative dot plots of CFSE dilution in C57BL/6, I-Ab-/-, and Cd40-/- cells. Numbers depict the percentage of cells in each box. Cells with multiple rounds of proliferation (CFSElo) are boxed in red. Histograms show intracellular staining for T-bet in CFSElo cells (red). (B) Analyses of T-bet change in mean fluorescence intensity (ΔMFI) are summarized in 3 independent experiments for a total of 12 mice for C57BL/6, 6 mice for I-Ab-/-, and 12 mice for Cd40-/-. P value was calculated by an unpaired, equal variance Student t test. (C) Spleen cells from 22-month old Cd154-/- mice were gated on live B220+ cells. A representative dot plot shows the absence of ABCs (CD23-CD21-). Numbers represent the percentage of B cells in each population. (D) Absolute B cell numbers of the indicated cell subset from old C57BL/6 and Cd154-/- mice are shown. Error bars signify the standard deviation of values from 31 C57BL/6 mice, and 5 Cd154-/- mice. P value was calculated by an unpaired, equal variance Student t test.

ABCs exhibit a diverse V gene segment repertoire

ABC accumulation may reflect the aggregate of immune responses to a large and diverse class of endogenous antigens, and thus involve a broad array of clonotypic specificities. Alternatively, accumulation could instead be mediated by common exposure to a limited array of self or environmental ligands that generate oligoclonal expansions with limited repertoire diversity. To differentiate these possibilities, we sorted FO, MZ, and ABC B cell subsets from 22-month old mice and compared VH and Vκ gene segment usage. Since the majority of ABCs express IgM (9), sequencing analyses for heavy chain genes were done on cDNA amplified with a Cμ 3′ primer and degenerate VH 5′ primers. Likewise, kappa light chain genes were identified by amplifying cDNA with a Cκ 3′ primer and degenerate Vκ 5′ primers. Some 2400 unique sequences for both loci were collected and analyzed. Overall, the usage of VH and Vκ gene segments was similar between all three subsets. For VH genes, 85 genes from 12 families were identified, and their frequencies were measured within the subsets. ABCs were separately compared to FO (Fig. 2A) and MZ (Fig. 2B) cells, and significant differences in over- or under-utilization were seen in only 2-4 individual genes. For Vκ genes, 69 genes from 15 families were found, and when ABCs were compared to FO (Fig. 3A) or MZ (Fig. 3B) cells, only 3-5 genes were significantly over- or under-utilized. Thus, there was no evidence for strong repertoire skewing, arguing against a restricted antigen-driven response. We also did not observe significant selection for amino acid replacement changes in complementarity determining regions for IgH and Igκ chains from the ABC population (data not shown). These results suggest that ABCs develop in response to a broad range of antigens.

Figure 2.

Figure 2

Open in a new tab

Diverse VH gene segment usage in ABCs. The frequency of gene expression within the indicated B cell population in old mice (n = 18-24 mice for FO, MZ, and ABC subsets) was determined using RT-PCR. For each subset, about 400 VH sequences were analyzed. V genes were grouped by family, which is indicated numerically below the graph. Significant differences in V gene usage between either ABC and FO (A) or ABC and MZ (B) subsets were calculated using the Fisher exact test, and are shown below the gene name in the yellow bar. The P value heat map scale is shown.

Figure 3.

Figure 3

Open in a new tab

Diverse Vκ gene segment usage in ABCs. Details are similar to Fig. 2 legend.

ABC V genes have undergone somatic hypermutation

The requirement for CD40-CD154 interactions in ABC accumulation suggests that most ABCs are products of activation involving cognate T cell help. If so, the V genes of ABCs should contain increased frequencies of mutations compared to other subsets. To address this, we counted the number of mutations in VDJ heavy and VJ kappa light exons amplified from FO, MZ, and ABC B cell subsets from 22-month old mice used for the repertoire analysis. Sequences of V, D, and J gene segments were compared to their germline counterparts to identify mutations. VDJ and VJ genes from ABCs had a significant four-fold increase in mutations compared to FO cells and a significant two-fold increase compared to MZ cells (Fig. 4A and B). As a control, V exons were sequenced from FO and MZ cells from young Aid -/- mice, which cannot undergo hypermutation because the activation-induced deaminase (AID) protein is absent. The mutation frequency was approximately 2 × 10-3 mutations per bp for AID-deficient cells, which represents the background frequency of errors produced during cDNA synthesis and PCR amplification. The distribution of mutations per sequence is shown in Fig. 4C, which shows that two-thirds of sequences from ABCs had mutations, indicating that most of these B cells have encountered some type of antigen during their existence. An examination of the types of nucleotide substitutions in the cadre of over 2,700 mutations from VDJ and VJ genes from the ABC sequences showed no difference compared to FO and MZ substitutions (data not shown). Because the error rate for sequencing cDNA clones from RNA is elevated due to errors from the low-fidelity reverse transcriptase used to make cDNA, we also analyzed mutations in the 492-bp JH4 intronic region directly from DNA, using a high-fidelity polymerase. FO, MZ, and ABC B cell subsets were sorted as described above, and the JH4 region was amplified from genomic DNA. As shown in Fig. 4D-F, there was a significant increase in mutation frequency from ABCs compared to those from FO and MZ cells, confirming that ABCs have undergone somatic hypermutation. As a control, introns were sequenced from germinal center B cells of young mice taken 4 weeks after immunization with (4-hydroxy-3-nitrophenyl)-acetyl (NP)-chicken gamma globulin (24), and the frequency was five-fold higher than in ABCs. This comparison places ABCs in the middle range between naïve and germinal center cells, suggesting they undergo mild chronic stimulation by endogenous antigens vs. acute stimulation by immunization.

Figure 4.

Figure 4

Open in a new tab

ABCs have increased somatic hypermutation. (A-C) Exon sequences from Figs. 2 and 3 were analyzed for mutations in rearranged VDJ genes for the heavy chain, and VJ genes for the κ light chain. (A) The numbers of unique sequences, nucleotides, mutations, and frequencies are shown. (B) Mean mutation frequencies (mutations/nucleotides) in the exons of each B cell subset were calculated. The dotted line indicates the mutation frequency in Aid -/- FO and MZ B cells from young mice. *, P < 0.0001, Chi-squared test. (C) Distribution of mutations per sequence. The number of sequences is shown in the center of each circle. Segments represent the proportion of sequences that contain the indicated number of mutations. (D-F) JH4 intron sequences were analyzed from genomic DNA of 11-13 mice for each subset. (D). Numbers of sequences, nucleotides, mutations and frequencies. (E) Mean mutation frequencies; the dotted line represents the frequency in germinal centers from young immunized mice. *, P< 0.0001, Chi-squared test. (F) Distribution of mutations per sequence.

Some antigen-specific B cells express T-bet and CD11c

Our results suggest an antigen-driven origin for ABCs that, coupled with their continuous accumulation, BLyS independence, and resting state (9, 25), lends credence to the idea that ABCs are an unusual subset of B cells. To further interrogate the provenance of ABCs, we compared their gene expression profiles to those from FO B cells sorted from old and young mice. ABC uniqueness is shown by a subset of 70 genes with at least five-fold higher expression in old ABCs compared to old and young FO B cells (Fig. S1A and Table S1). Both T-bet and CD11c were overexpressed in ABCs, confirming previous reports (13, 14). Principal component analysis was then used to visualize inter-sample variation among all the genes from sorted subsets, and illustrated that old ABCs have distinct gene expression profiles compared to FO B cells from old and young mice (Fig. S1B).

Based on their accumulation of somatic hypermutation, we hypothesized that ABCs represent a subset of antigen-experienced B cells, whose accretion reflects the cumulative aggregate of challenges that drive their formation. To demonstrate that another subset of antigen-experienced B cells arising from deliberate infection also express T-bet and CD11c, we infected young mice with influenza. HA-specific B cells were tracked by binding to fluorescent labeled HA-PE. Prior to infection, the frequency of HA-reactive B cells was low (Fig. 5A), consistent with prior estimates of ∼1 per 50,000 splenic B cells (26). Following infection, mice displayed the expected weight loss, and fully recovered 30 days later (Fig. 5B), indicating that the virus was cleared. At day 100 post infection, HA-reactive B cells increased, and about 25% of these were T-bet+CD11c+ (Fig. 5C and D). Collectively, these observations on influenza-infected mice support the analogy that some long-lived, antigen-experienced cells express the phenotype associated with ABCs.

Figure 5.

Figure 5

Open in a new tab

B cells infected with influenza HA express T-bet and CD11c. Mice were infected intranasally with PR8, and spleens were harvested 100 days post infection. (A) Representative profile of HA-PE binding B cells (CD19+) in an uninfected spleen. Number shows the percent cells in highlighted box. (B) Weight loss and recovery after infection. Error bars signify standard deviation from 8 mice. (C) Upper panel: Gating profile for HA-binding B cells in spleens at day 100. Number in box reflects the percent cells. Lower panel: A portion of HA-binding B cells express T-bet and CD11c. (D) Number of B cells that bind HA. Total (T-bet-CD11c- and T-bet+CD11c+ cells) are compared to only T-bet+CD11c+ cells. Data is from 7 uninfected and 8 day 100 infected mice collected in three independent experiments. Significance between uninfected and infected groups was calculated by an unpaired, equal variance Student t test; *, P<0.01.

Discussion

These studies probe the origin and nature of ABCs, a B cell subset that steadily accumulates with age, and whose surface phenotype and transcriptional signature has been associated with both humoral autoimmunity and anti-pathogen immune responses. We provide three lines of evidence that indicate ABCs are a unique B cell subset. First, adoptive transfer studies using MHC class II and CD40-deficient cells confirm our prior report that young FO B cells can give rise to ABCs after extensive division (9), and extend this observation in several ways. Notably, they demonstrate that ABCs can be generated with cognate T cell help, which is substantiated by the lack of ABCs in old CD154-/- mice. These findings also significantly connect T-bet expression with these extensively divided cells, a feature that is well established in ABC genesis (12, 13). In addition, our results indicate that homeostatic expansion is unlikely to be the major source of ABCs. The transfer experiments involved replete hosts, with minimal space for donor cells to fill by division, and the relatively few transferred cells from MHC class II- and CD40-deficient donors that divided did not express T-bet. Overall, our data support the hypothesis that most ABCs are the cumulative result of enduring environmental antigen stimulation through T-dependent mechanisms. However, the results do not exclude a Toll-like receptor-mediated origin for some ABCs that may respond to viral or autoimmune stimuli (27).

Second, analyses of V gene segment use and somatic hypermutation indicate encounters with multiple antigens. The breadth of V-gene usage speaks against a monolithic origin in terms of antigen or epitope recognition, and demonstrates that ABCs represent a cross section of responses to a broad array of antigens. Numerous early reports suggested that the total B cell repertoire was restricted in old mice (5, 28-31). However, our extensive analysis of 85 VH gene segments and 69 Vκ gene segments revealed a rich spectrum of V-gene usage by FO, MZ, and ABCs from old mice, indicating that global sequencing generates a more comprehensive view of the repertoire than limited studies of antigen-specific cells. Only a handful of genes were over- or under-utilized by ABCs, but overall, there was no significant difference when ABCs were compared to either FO or MZ repertoires. Such results would be expected if multiple, heterogeneous antigens generated the diverse repertoire. Possible candidates are self-antigens or antigens of the microbiota environment. Concerning self-antigens, a previous report found that mice stimulated chronically with TLR7 agonists developed ABCs expressing high titers of anti-Smith autoimmune antibodies (10). However, the few genes that were over-utilized in ABCs in this study did not possess positively-charged CDR3 regions in their rearranged sequences, which are common in self-reactive antibodies (32). It appears that healthy old mice without deliberate immunization develop a heterogeneous repertoire, without propensity for autoimmune antibodies. Although the repertoires of ABCs, FO cells, and MZ cells were similar, the results of the mutation analyses were strikingly different. ABCs showed clear evidence of mutation in both the VH and Vκ exons compared to FO and MZ B cells. While FO B cells had the lowest mutation frequency, consistent with their pre-immune status, the frequency was two-fold higher in MZ cells, which have likely encountered microbial antigens during circulation, and four-fold higher in ABCs. In ABCs, mutations did not accumulate in complementarity-determining regions, consistent with the lack of selection of certain V genes in the repertoire analysis. Furthermore, there was a significant increase in mutation frequency in ABCs in the non-coding JH4 intron, which is a broad substrate for hypermutation in the absence of selection (33). However, it remains uncertain if all ABCs are the products of germinal center reactions. For example, most ABCs have IgM receptors and continue to express the surface receptor, TACI (9), both of which are inconsistent with the germinal center B cell phenotypes (34). Moreover, the mutation frequency in ABCs was lower compared to germinal center B cells from immunized mice (24). There is precedent for mutated IgM-bearing cells occurring in the absence of germinal centers (35, 36). Alternatively, ABCs may represent early germinal center emigrants, which exit before concerted selection (37, 38).

Third, microarray analyses of gene expression show that ABCs from old mice are unique in relation to FO cells from young and old mice. Transcription analysis was also performed by Rubtsov et al. (10) to compare old ABCs to old FO, old B1, and young B1 cells. However, the two analyses profile different sets of genes because the cells were isolated under different conditions. The ABCs from Rubtsov et al. (10) were sorted for CD11b+ expression, and the ABCs analyzed here were sorted as CD21- CD23-. Nonetheless, both analyses show that T-bet and CD11c are greatly increased in old ABC relative to old FO. We found this signature was also present in some long-lived B cells following influenza infection 100 days later, confirming our previous report (39). By analogy, ABCs are antigen-experienced, because they have increased somatic hypermutation, and they require T cell interactions for their generation. The cells presumably arise from chronic stimulation by endogenous antigens, but it is important to note that ABCs are resting cells that persist over time. Whether they can undergo recall responses when they encounter cognate antigens remains to be determined.

Supplementary Material

1

Acknowledgments

We thank Robert Maul, Ranjan Sen, and Diana Castiblanco for stimulating conversations and advice; William Yang for technical assistance; and Kate Turlington, Haleigh Larson, and Arielle Kilner for experiments. Robert Wersto, Jade Scheers, Tonya Wallace, and Cuong Nguyen at the NIA flow cytometry core facility assisted in cell sorting. Michael Paley and E. John Wherry at the University of Pennsylvania assisted with the ABC/FO microarray and initial analyses.

This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging (L.M.R.K., P.J.G.); and by grants PR130769 (Dept. of the Army) and RO1 AG030227 (NIA) to M.P.C. M.S.N. was supported in part by T32 AI055428 (NIAID).

Abbreviations used in this article

ABC

age-associated B cell

AID

activation-induced deaminase

FO

follicular

HA

hemagglutinin

MZ

marginal zone

Footnotes

L.M.R.K., M.S.N., M.P.C., and P.J.G. designed experiments; L.M.R.K., M.S.N., A.M., Y.H., J.L.S., and J.W.T. performed research and analyzed data; D.L. and M.L.F. contributed reagents; L.M.R.K., M.S.N., J.L.S, J.W.T., M.P.C., and P.J.G. wrote the paper.

References

  • 1.Miller JP, Allman D. The decline in B lymphopoiesis in aged mice reflects loss of very early B-lineage precursors. J Immunol. 2003;171:2326–2330. doi: 10.4049/jimmunol.171.5.2326. [DOI] [PubMed] [Google Scholar]
  • 2.Cancro MP. B cells and aging: gauging the interplay of generative, selective, and homeostatic events. Immunol Rev. 2005;205:48–59. doi: 10.1111/j.0105-2896.2005.00272.x. [DOI] [PubMed] [Google Scholar]
  • 3.Miller JP, Cancro MP. B cells and aging: balancing the homeostatic equation. Experimental gerontology. 2007;42:396–399. doi: 10.1016/j.exger.2007.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kline GH, Hayden TA, Klinman NR. B cell maintenance in aged mice reflects both increased B cell longevity and decreased B cell generation. J Immunol. 1999;162:3342–3349. [PubMed] [Google Scholar]
  • 5.Johnson SA, Rozzo SJ, Cambier JC. Aging-dependent exclusion of antigen-inexperienced cells from the peripheral B cell repertoire. J Immunol. 2002;168:5014–5023. doi: 10.4049/jimmunol.168.10.5014. [DOI] [PubMed] [Google Scholar]
  • 6.Zheng B, Han S, Takahashi Y, Kelsoe G. Immunosenescence and germinal center reaction. Immunol Rev. 1997;160:63–77. doi: 10.1111/j.1600-065x.1997.tb01028.x. [DOI] [PubMed] [Google Scholar]
  • 7.Frasca D, Blomberg BB. Aging impairs murine B cell differentiation and function in primary and secondary lymphoid tissues. Aging and disease. 2011;2:361–373. [PMC free article] [PubMed] [Google Scholar]
  • 8.Goenka R, Scholz JL, Naradikian MS, Cancro MP. Memory B cells form in aged mice despite impaired affinity maturation and germinal center kinetics. Experimental gerontology. 2014;54:109–115. doi: 10.1016/j.exger.2013.12.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Hao Y, O'Neill P, Naradikian MS, Scholz JL, Cancro MP. A B-cell subset uniquely responsive to innate stimuli accumulates in aged mice. Blood. 2011;118:1294–1304. doi: 10.1182/blood-2011-01-330530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Rubtsov AV, Rubtsova K, Fischer A, Meehan RT, Gillis JZ, Kappler JW, Marrack P. Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c(+) B-cell population is important for the development of autoimmunity. Blood. 2011;118:1305–1315. doi: 10.1182/blood-2011-01-331462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ratliff M, Alter S, Frasca D, Blomberg BB, Riley RL. In senescence, age-associated B cells secrete TNFalpha and inhibit survival of B-cell precursors. Aging Cell. 2013;12:303–311. doi: 10.1111/acel.12055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Rubtsova K, Rubtsov AV, Cancro MP, Marrack P. Age-Associated B Cells: A T-bet-Dependent Effector with Roles in Protective and Pathogenic Immunity. J Immunol. 2015;195:1933–1937. doi: 10.4049/jimmunol.1501209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Rubtsova K, Rubtsov AV, van Dyk LF, Kappler JW, Marrack P. T-box transcription factor T-bet, a key player in a unique type of B-cell activation essential for effective viral clearance. Proc Natl Acad Sci U S A. 2013;110:E3216–3224. doi: 10.1073/pnas.1312348110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Rubtsov AV, Rubtsova K, Kappler JW, Jacobelli J, Friedman RS, Marrack P. CD11c-Expressing B Cells Are Located at the T Cell/B Cell Border in Spleen and Are Potent APCs. J Immunol. 2015;195:71–79. doi: 10.4049/jimmunol.1500055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ratliff M, Alter S, McAvoy K, Frasca D, Wright JA, Zinkel SS, Khan WN, Blomberg BB, Riley RL. In aged mice, low surrogate light chain promotes pro-B-cell apoptotic resistance, compromises the PreBCR checkpoint, and favors generation of autoreactive, phosphorylcholine-specific B cells. Aging cell. 2015;14:382–390. doi: 10.1111/acel.12302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Claes N, Fraussen J, Vanheusden M, Hellings N, Stinissen P, Van Wijmeersch B, Hupperts R, Somers V. Age-Associated B Cells with Proinflammatory Characteristics Are Expanded in a Proportion of Multiple Sclerosis Patients. J Immunol. 2016;197:4576–4583. doi: 10.4049/jimmunol.1502448. [DOI] [PubMed] [Google Scholar]
  • 17.Moir S, Ho J, Malaspina A, Wang W, DiPoto AC, O'Shea MA, Roby G, Kottilil S, Arthos J, Proschan MA, Chun TW, Fauci AS. Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals. J Exp Med. 2008;205:1797–1805. doi: 10.1084/jem.20072683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Weiss GE, Crompton PD, Li S, Walsh LA, Moir S, Traore B, Kayentao K, Ongoiba A, Doumbo OK, Pierce SK. Atypical memory B cells are greatly expanded in individuals living in a malaria-endemic area. J Immunol. 2009;183:2176–2182. doi: 10.4049/jimmunol.0901297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Zinocker S, Schindler CE, Skinner J, Rogosch T, Waisberg M, Schickel JN, Meffre E, Kayentao K, Ongoiba A, Traore B, Pierce SK. The V gene repertoires of classical and atypical memory B cells in malaria-susceptible West African children. J Immunol. 2015;194:929–939. doi: 10.4049/jimmunol.1402168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Portugal S, Tipton CM, Sohn H, Kone Y, Wang J, Li S, Skinner J, Virtaneva K, Sturdevant DE, Porcella SF, Doumbo OK, Doumbo S, Kayentao K, Ongoiba A, Traore B, Sanz I, Pierce SK, Crompton PD. Malaria-associated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function. eLife. 2015;4 doi: 10.7554/eLife.07218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Yates JL, Racine R, McBride KM, Winslow GM. T cell-dependent IgM memory B cells generated during bacterial infection are required for IgG responses to antigen challenge. J Immunol. 2013;191:1240–1249. doi: 10.4049/jimmunol.1300062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Wang Z, Raifu M, Howard M, Smith L, Hansen D, Goldsby R, Ratner D. Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3′ to 5′ exonuclease activity. J Immunol Methods. 2000;233:167–177. doi: 10.1016/s0022-1759(99)00184-2. [DOI] [PubMed] [Google Scholar]
  • 23.Whittle JR, Wheatley AK, Wu L, Lingwood D, Kanekiyo M, Ma SS, Narpala SR, Yassine HM, Frank GM, Yewdell JW, Ledgerwood JE, Wei CJ, McDermott AB, Graham BS, Koup RA, Nabel GJ. Flow cytometry reveals that H5N1 vaccination elicits cross-reactive stem-directed antibodies from multiple Ig heavy-chain lineages. J Virol. 2014;88:4047–4057. doi: 10.1128/JVI.03422-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Zanotti KJ, Maul RW, Castiblanco DP, Yang W, Choi YJ, Fox JT, Myung K, Saribasak H, Gearhart PJ. ATAD5 deficiency decreases B cell division and Igh recombination. J Immunol. 2015;194:35–42. doi: 10.4049/jimmunol.1401158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Scholz JL, Crowley JE, Tomayko MM, Steinel N, O'Neill PJ, Quinn WJ, 3rd, Goenka R, Miller JP, Cho YH, Long V, Ward C, Migone TS, Shlomchik MJ, Cancro MP. BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact. Proc Natl Acad Sci U S A. 2008;105:15517–15522. doi: 10.1073/pnas.0807841105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Cancro MP, Gerhard W, Klinman NR. The diversity of the influenza-specific primary B-cell repertoire in BALB/c mice. J Exp Med. 1978;147:776–787. doi: 10.1084/jem.147.3.776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Naradikian MS, Hao Y, Cancro MP. Age-associated B cells: key mediators of both protective and autoreactive humoral responses. Immunol Rev. 2016;269:118–129. doi: 10.1111/imr.12380. [DOI] [PubMed] [Google Scholar]
  • 28.Viale AC, Chies JA, Huetz F, Malenchere E, Weksler M, Freitas AA, Coutinho A. VH-gene family dominance in ageing mice. Scand J Immunol. 1994;39:184–188. doi: 10.1111/j.1365-3083.1994.tb03358.x. [DOI] [PubMed] [Google Scholar]
  • 29.Ben-Yehuda A, Szabo P, LeMaoult J, Manavalan JS, Weksler ME. Increased VH 11 and VH Q52 gene use by splenic B cells in old mice associated with oligoclonal expansions of CD5 + B cells. Mech Ageing Dev. 1998;103:111–121. doi: 10.1016/s0047-6374(98)00004-9. [DOI] [PubMed] [Google Scholar]
  • 30.Riley SC, Froscher BG, Linton PJ, Zharhary D, Marcu K, Klinman NR. Altered VH gene segment utilization in the response to phosphorylcholine by aged mice. J Immunol. 1989;143:3798–3805. [PubMed] [Google Scholar]
  • 31.LeMaoult J, Delassus S, Dyall R, Nikolic-Zugic J, Kourilsky P, Weksler ME. Clonal expansions of B lymphocytes in old mice. J Immunol. 1997;159:3866–3874. [PubMed] [Google Scholar]
  • 32.Wardemann H, Yurasov S, Schaefer A, Young JW, Meffre E, Nussenzweig MC. Predominant autoantibody production by early human B cell precursors. Science. 2003;301:1374–1377. doi: 10.1126/science.1086907. [DOI] [PubMed] [Google Scholar]
  • 33.Lebecque SG, Gearhart PJ. Boundaries of somatic mutation in rearranged immunoglobulin genes: 5′ boundary is near the promoter, and 3′ boundary is approximately 1 kb from V(D)J gene. J Exp Med. 1990;172:1717–1727. doi: 10.1084/jem.172.6.1717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Goenka R, Matthews AH, Zhang B, O'Neill PJ, Scholz JL, Migone TS, Leonard WJ, Stohl W, Hershberg U, Cancro MP. Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation. J Exp Med. 2014;211:45–56. doi: 10.1084/jem.20130505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Voigt I, Camacho SA, de Boer BA, Lipp M, Forster R, Berek C. CXCR5-deficient mice develop functional germinal centers in the splenic T cell zone. Eur J Immunol. 2000;30:560–567. doi: 10.1002/1521-4141(200002)30:2<560::AID-IMMU560>3.0.CO;2-T. [DOI] [PubMed] [Google Scholar]
  • 36.Di Niro R, Lee SJ, Vander Heiden JA, Elsner RA, Trivedi N, Bannock JM, Gupta NT, Kleinstein SH, Vigneault F, Gilbert TJ, Meffre E, McSorley SJ, Shlomchik MJ. Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation. Immunity. 2015;43:120–131. doi: 10.1016/j.immuni.2015.06.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Pape KA, Taylor JJ, Maul RW, Gearhart PJ, Jenkins MK. Different B cell populations mediate early and late memory during an endogenous immune response. Science. 2011;331:1203–1207. doi: 10.1126/science.1201730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Weisel FJ, Zuccarino-Catania GV, Chikina M, Shlomchik MJ. A Temporal Switch in the Germinal Center Determines Differential Output of Memory B and Plasma Cells. Immunity. 2016;44:116–130. doi: 10.1016/j.immuni.2015.12.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Naradikian MS, Myles A, Beiting DP, Roberts KJ, Dawson L, Herati RS, Bengsch B, Linderman SL, Stelekati E, Spolski R, Wherry EJ, Hunter C, Hensley SE, Leonard WJ, Cancro MP. Cutting Edge: IL-4, IL-21, and IFN-gamma Interact To Govern T-bet and CD11c Expression in TLR-Activated B Cells. J Immunol. 2016;197:1023–1028. doi: 10.4049/jimmunol.1600522. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS839153-supplement-1.pdf (299.2KB, pdf)

RESOURCES