1. Cell Biology
  2. Immunology and Inflammation
Download icon

Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires

  1. Yang Yang  Is a corresponding author
  2. Chunlin Wang
  3. Qunying Yang
  4. Aaron B Kantor
  5. Hiutung Chu
  6. Eliver EB Ghosn
  7. Guang Qin
  8. Sarkis K Mazmanian
  9. Jian Han  Is a corresponding author
  10. Leonore A Herzenberg  Is a corresponding author
  1. Stanford University, United States
  2. HudsonAlpha Institute for Biotechnology, United States
  3. California Institute of Technology, United States
Research Article
  • Cited 51
  • Views 3,231
  • Annotations
Cite this article as: eLife 2015;4:e09083 doi: 10.7554/eLife.09083

Abstract

Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota.

https://doi.org/10.7554/eLife.09083.001

eLife digest

Our immune system protects us by recognizing and destroying invading viruses, bacteria and other microbes. B cells are immune cells that produce protective proteins called antibodies to stop infections. These cells are activated by ‘antigens’, which are fragments of molecules from the microbes or from our own cells. When an antigen binds to a B cell, the cell matures, multiplies and produces proteins called antibodies. These antibodies can bind to the antigen, which marks the microbe for attack and removal by other cells in the immune system.

Each antibody consists of two ‘heavy chain’ and two ‘light chain’ proteins. B cells are able to produce a large variety of different antibodies due to the rearrangement of the gene segments that encode the heavy and light chains. In mice, there are two kinds of B cells – known as B-1a and B-2 cells – that play different roles in immune responses. B-1a cells have long been known to produce the ‘natural’ antibodies that are present in the blood prior to an infection. On the other hand, B-2 cells produce antibodies that are specifically stimulated by an infection and are better adapted to fighting it. Previous studies have shown that both types of antibodies are required to allow animals to successfully fight the flu virus.

Here, Yang, Wang et al. used a technique called fluorescence-activated cell sorting (or FACS) and carried out extensive genomic sequencing to study how the B-1a and B-2 populations rearrange their genes to produce heavy chains. This approach made it possible to separate the different types of B cells and then sequence the gene for the heavy chain within the individual cells. The experiments show that the “repertoire” of heavy chains in the antibodies of the B-1a cells is much less random and more repetitive than that of B-2 populations.

Furthermore, Yang, Wang et al. show that B-1a cells produce and maintain their repertoire of heavy chains in a different way to other B-2 populations. B-1a cells develop earlier and the major genetic rearrangements in the gene that encodes the heavy chain occur within the first few weeks of life. Although the gene rearrangements have mostly stopped by adulthood, the B-1a antibody repertoire continues to evolve profoundly as the B-1a cells divide over the life of the animal. On the other hand, the gene rearrangements that make the heavy chains in the B-2 cells continue throughout the life of the animal to produce the wider repertoire of antibodies found in these cells. In addition, the processes that continue to change the antibody reperotire in the B-1a cells during adulthood do not occur in the B-2 populations.

Importantly, the these reperotire-changing processes in B-1a cells also occur in mice that have been raised in germ-free conditions, which demonstrates that – unlike other B cells – the repertoire of heavy chains in B-1a cells is not influenced by antigens from microbes. Instead, it is mainly driven by antigens that are expressed by normal cells in the body. These findings open the way to future work aimed at understanding how B-1a cells help to protect us against infection, and their role in autoimmune diseases, where immune cells attack the body’s own healthy cells.

https://doi.org/10.7554/eLife.09083.002

Introduction

Follicular B (FOB), marginal zone B (MZB) and B-1a cells are the major mature B cell populations in the mouse. Although these B cell subsets all produce functionally important antibodies, they differ profoundly in function and developmental origin (Kantor and Herzenberg, 1993; Hardy and Hayakawa, 2001; Baumgarth, 2011). Previous studies have shown that B-1a cells are efficiently generated during fetal and neonatal life, and are maintained by self-replenishment in adult animals(Hayakawa et al., 1985; Montecino-Rodriguez et al., 2006; Kantor et al., 1992). In contrast, both FOB and MZB populations emerge later and are replenished throughout life by de novo development from bone marrow (BM) hematopoietic stem cells (HSC). Our recent studies show that BM HSC reconstitute FOB and MZB, but fail to reconstitute B-1a cells (Ghosn et al., 2012), which are derived from distinct progenitors at embryonic day 9 yolk sac (Yoshimoto et al., 2011).

For each B cell subset, their antibody responses are enabled by the basic processes that generate the immunoglobulin (Ig) structure. Multiple mechanisms contribute to creating the primary Ig heavy (IgH) and light chain (IgL) diversity. For IgH, these include combinatorial assortment of individual variable (V), diversity (D) and joining (J) gene segments, nucleotide(s) trimming in the D-J and V-DJ joining site, and, template-dependent (P-addition) and independent (N-addition) nucleotide(s) insertion at the joined junctions (Yancopoulos and Alt, 1986Kirkham and Schroeder, 1994). The V(D)J joining processes define the third IgH complementarity-determining region (CDR3), which often lies at the center of antigen binding site and plays a crucial role in defining antibody specificity and affinity (Xu and Davis, 2000).

After encountering antigen, “‘naïve”’ B cells are activated and can further diversify their primary antibody repertoire by activation-induced cytidine deaminase (AID)–mediated somatic hypermutation (SHM), which introduces single or multiple mutations into the IgV regions (Muramatsu et al., 2000Wagner and Neuberger, 1996). SHM commonly occurs in germinal centers (GC) (Victora and Nussenzweig, 2012), where memory B cells expressing high affinity antibodies are selected (Rajewsky, 1996Gitlin et al., 2014). Since the antigen-driven SHM-mediated secondary Ig diversification is viewed as a crucial adaptation to the environmental needs, the IgH repertoire(s) expressed by FOB, MZB and B-1a cells from non-immunized animals are thought to be free of SHM. Our studies here, however, introduce a previously unrecognized SHM mechanism that increasingly diversifies the B-1a pre-immune IgH repertoire as animals age. Importantly, the SHM operates equally in the presence or absence of microbiota influence.

The B-1a antibody repertoire is commonly thought to be ‘restricted’ with expressing germline genes, largely because the hybridomas generated from fetal and neonatal B cells, which are mainly B-1a, have few N-insertions (Carlsson and Holmberg, 1990) and preferentially express the proximal 7183, Q52 VH family genes (Perlmutter et al., 1985). The N diversity deficit is ascribed to the absence of expression of terminal deoxynucleotidyl transferase (Tdt), which adds the N nucleotides to the CDR3 junction (Gilfillan et al., 1993), during fetal life (Feeney, 1990). These early studies left the impression that the proximal VH gene usage predominates and that there is little N-addition in the B-1a IgH repertoire.

Later studies by the Rajewsky group, however, showed that although neonatal (4 day) splenic B-1a cells contain very few N-insertions, N addition is readily detected in substantial numbers of peritoneal B-1a cells from adult animals (Gu et al., 1990), indicating that B-1a cells are continuously generated after Tdt is expressed. Holmberg lab similarly found the low N-region diversity in the adult peritoneal B-1a repertoire (Tornberg and Holmberg, 1995). Our early studies confirm and extend these findings by showing that roughly two thirds of the IgH sequences from individually sorted peritoneal B-1a cells have N additions (Kantor et al. 1997). Furthermore, recent studies have shown that B-1a progenitors from both fetal liver and adult BM sources generate peritoneal B-1a cells with substantial N-addition (Holodick et al., 2014). Collectively, these findings demonstrate that the peritoneal B-1a IgH repertoire diversity is greater than previously thought.

However, these studies mainly characterized the repertories of B cells in the peritoneal cavity (PerC) and leave the questions open as to whether and how the repertoire changes throughout ontogeny in B cells at various sites of development and function. Studies here address these issues. We show that the B-1a IgH repertoire differs drastically from the repertories expressed by splenic FOB, MZB and peritoneal B-2 cells. In addition, we track the development of B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen, and elucidate the previous unrecognized somatic mechanisms that select and diversify the B-1a IgH repertoire over time. Most importantly, the potent mechanisms that uniquely act in B-1a (not in FOB and MZB cells) operate comparably in germ-free (GF) and conventional mice reared under specific pathogen free (SPF) condition, indicating that these repertoire-defining mechanisms are not driven by microbiota-derived antigens.

The dearth of these advanced understandings in the previous studies is largely due to technical difficulties that limited both their scope and depth. Studies analyzing Ig sequences from immortalized cell lines (e.g., hybridomas) or LPS-stimulated B cells had obvious sampling biases. In addition, earlier studies mainly focused on particular VH families (e.g., J558, 7183), even though the mouse IgH locus contains over 100 functional VH genes (Kirkham and Schroeder, 1994). The introduction of single cell analyses enabled higher precision and lower bias than the bulk measurements. However, they were constrained profoundly by sequencing costs and technical challenges. Indeed, our previous single cell analysis reported only 184 IgH sequences derived from 85% recovered sorted single cells representative of three types of peritoneal B subsets (Kantor et al., 1997). Thus, while the data yielded key insights, hundreds or thousands of single cells would need to be analyzed to obtain a more comprehensive view for a single B subset repertoire. Finally, difficulties in defining and cleanly sorting rare B subsets (e.g., splenic B-1a) further compromise the attempt to develop a thorough view of repertoire(s) expressed by various B cell subsets at the different anatomic location and ontogenic stage.

To overcome these obstacles, we have coupled high-dimensional (Hi-D) FACS sorting with unique IgH multiplex PCR technologies, which allow inclusive amplification of IgH transcripts for each sorted B subset and ultimate sequencing of these sequences. Using barcoded sample multiplexing, we have performed a large-scale quantitative and comparative study of the ‘pre-immune’ IgH repertoires expressed by various functionally and developmentally distinct mature B subsets (splenic FOB, MZB and B-1a; peritoneal B-2 and B-1a) from non-immune C57BL/6J mice. In addition, since microbiota are often thought to influence the Ig repertoire, we have compared the B-1a IgH repertoires in GF or conventional mice.

Results

The B-1a pre-immune IgH repertoire is far more restricted and repetitive than the repertoire expressed by FOB and MZB subsets

We sorted splenic and peritoneal B-1a (dump- CD19+ CD93-IgMhi IgDlo/- CD21-/lo CD23- CD43+ CD5+); splenic FOB and peritoneal B-2 (dump- CD19+ CD93- IgMlo IgDhi CD23+ CD43- CD5-); and splenic MZB (dump- CD19+ CD93- IgMhi IgDlo/- CD21hi CD23lo/- CD43- CD5-) from non-immune C57BL/6 mice (Figure 1). We generated and amplified IgH cDNA libraries from each subset. We then pooled the libraries, which are distinguishable by barcode, and sequenced them (Illumina MiSeq). In all, we sequenced 60 separately prepared libraries, each derived from 1-2 x104 B cells of a given subset sorted from mice at the same or different ages (from 2 days to 6 months, > 30 mice) (Table 1). Overall 18 million total clean nucleotide sequences (CNT) and about half million unique clean nucleotide sequences (CNU) were analyzed in the study (Table 1).

Figure 1 with 3 supplements see all
The B-1a IgH CDR3 sequences are much less diverse and recur more frequently than the CDR3 sequences expressed by FOB and MZB B subsets.

IgH CDR3 tree-map plots illustrating the IgH CDR3 nucleotide sequences expressed by indicated B cell subsets sorted from one 2-month old C57Bl/6 mouse. Each rectangle in a given tree-map represents a unique CDR3 nucleotide sequence and the size of each rectangle denotes the relative frequency of an individual sequence. The colors for the individual CDR3 sequences in each tree-map plot are chosen randomly thus do not match between plots. The numbers shown in the CDR3 tree-map plots highlight the highly reoccurring CDR3 sequences including PtC-binding CDR3 sequences. 1, ARFYYYGSSYAMDY, V1-55D1-1J4; 2, MRYGNYWYFDV, V11-2D2-8J1; 3, MRYSNYWYFDV, V11-2D2-6J1; 4, MRYGSSYWYFDV, V11-2D1-1J1. Lower middle panel: FACS plots showing the gating strategy used to sort the phenotypically defined each B cell subset from spleen (s) or peritoneal cavity (p). Note: peritoneal B-1a cells are well known to express CD11b, a marker expressed on many myeloid cells including macrophage and neutrophils. The level of CD11b expressed on peritoneal B-1a cells, however, is roughly 100 fold lower than the level of CD11b expressed on the myeloid cells. This drastic difference is sufficient to separate the CD11b+ B-1a cells from the myeloid cells if monoclonal anti-CD11b reagent is included in the dump channel (Figure 1—figure supplement 3).

https://doi.org/10.7554/eLife.09083.003
Table 1

Summary of the sequences for 60 separately sorted B cell populations analyzed in this study.

https://doi.org/10.7554/eLife.09083.007
SampleIdSubsetStrainAgeConditionMiceRNT*RNU*RPU*CNT*CNU*CPU*
17631FOBWT2MSPFsingle1006030151210658719034002124020470
213966FOBWT3.5MSPFsingle15081231003248011306521491114678
38706FOBWT4MSPFsingle18036553577278171597101690116568
48702FOBWT5MSPFsingle15668154195277281361011695116649
513967FOBAID KO5MSPFsingle3596714623132032772671877133
611161MZBWT1MSPFsingle3354819628127442567465846471
710658MZBWT2MSPFsingle714582697818278612581151211170
87630MZBWT2MSPFsingle1032381139832625209323532078019792
98701MZBWT4MSPFsingle21423855075264581910651546115021
108700MZBWT5MSPFsingle11886342310227941028941451714180
1113338MZBWT4MGFsingle16275439930236111416461293912605
1213343MZBWT4MGFsingle59578085497458205360721926618480
1311163pB-1aWT1MSPFpool of 3 mice458821129055964136832373007
1410660pB-1aWT2MSPFsingle22232417311863020774938913649
1513018pB-1aWT2MSPFsingle808879360311481775386847694374
167628pB-1aWT2MSPFsingle17846775945822105170623566015848
1711160pB-1aWT2WSPFpool of 8 mice653171470070255803442403704
1810655pB-1aWT3WSPFpool of 5 mice628751216266225755841803694
198705pB-1aWT4MSPFsingle310077284411188628769550634707
209870pB-1aWT4MSPFsingle229100262991046921151447454480
2111165pB-1aWT5MSPFsingle1054101952889269599444354162
228707pB-1aWT5MSPFsingle320252297861242329694647224384
239861pB-1aWT6MSPFsingle26613568332352354215211461
248704pB-1aAID KO4MSPFsingle264340337451451924594166486294
2510657pB-2WT2MSPFsingle53953230591688344986100849923
267629pB-2WT2MSPFsingle13156631234724733712382251692516065
2713969pB-2WT3.5MSPFsingle186817243041768917076890898925
289862pB-2WT4MSPFsingle225911337787371734343824357
2913973pB-2AID KO5MSPFsingle61789362319411655668261753616965
3013000sB-1aWT2dSPFpool of 8 mice29439954249252536931482758
3110651sB-1aWT5dSPFsingle123360224721083811316174535976
3210659sB-1aWT5dSPFsingle210055281401241119266273075812
339866sB-1aWT5dSPFsingle529861560068644658045953837
3410652sB-1aWT6dSPFsingle172875264371254515930476836365
359865sB-1aWT7dSPFsingle713091844687756424154824941
369868sB-1aWT7dSPFsingle201813350691447318622778476843
3710656sB-1aWT2MSPFsingle369732396031975934291494899048
3813004sB-1aWT2MSPFsingle185948279521387516852273137022
397632sB-1aWT2MSPFsingle18252181027974319017192461242811144
4011168sB-1aWT2WSPFsingle53660370201288294966711194810913
4113005sB-1aWT2WSPFsingle9801728331150018548988208207
4210654sB-1aWT3WSPFsingle14656033814196971310911199511451
4313970sB-1aWT3.5MSPFsingle17092513809928916048045134273
4413335sB-1aWT4MSPFsingle22175482234491868311311090
4513342sB-1aWT4MSPFsingle283072236681294726274453575032
468699sB-1aWT4MSPFsingle14283819151993813091543704086
479863sB-1aWT4MSPFsingle736761659987136557142334092
4811167sB-1aWT5MSPFsingle501367389121733646386375737163
498708sB-1aWT5MSPFsingle577114527232227253150891468441
509867sB-1aWT6MSPFsingle113492206121062510179145634343
5113965sB-1aAID KO4MSPFsingle177782164191228116418965396293
5213971sB-1aAID KO4MSPFsingle517141341592203148254389668395
5313968sB-1aAID KO5MSPFsingle427671308392051039697491628545
5413972sB-1aAID KO5MSPFsingle706116362172325566087492948744
5513001sB-1aWT4MGFsingle43507873448553894723182249
5613002sB-1aWT4MGFsingle47203868348204227920531965
5713003sB-1aWT4MGFsingle213347222461106819776947054449
5813017sB-1aWT4MGFsingle532250404971737550190870196398
5913337sB-1aWT4MGFsingle28559632244172404715441486
6013341sB-1aWT4MGFsingle388208289421483736072756745144
Id is a unique identifier for the sequence run
RNT*, total raw nucleotide sequences
RNU*, unique raw nucleotide sequences
RPU*, unique raw peptide sequences
CNT*, total clean nucleotide sequences
CNU*, unique clean nucleotide sequences
CPU*, unique clean peptide sequences
Sequence statisticsRNT*RNU*RPU*CNT*CNU*CPU*
Total1.9E + 072.1E + 061.1E + 061.8E + 074.9E + 054.7E + 05
Mean319865356101784829517482337762
% CV12286741256163

We also attempted to analyze the B-1a repertoire in fetal liver but found that there were too few B-1a cells to reliably sequence with our method. In essence, FACS analysis of embryonic day 19 (E19) fetal liver cells shows that IgM+ B cells represent only 0.6% of CD19+ total B cells and that only around 20% of these IgM+ B cells express the B-1a CD43+ CD5+ phenotype (Figure 1—figure supplement 1). The frequencies of IgM+ B cell in E18 fetal liver are even lower (0.2% of CD19+ B cells). These numbers are too low for us to recover enough material for sequencing from a feasible number of embryos.

The IgH CDR3 tree maps for each B cell subset show that splenic FOB and peritoneal B-2 cells express highly diversified IgH CDR3 nucleotide sequences, as do MZB cells (Figure 1). In contrast, CDR3 nucleotide sequences expressed by B-1a cells from either spleen or PerC are far less diverse and recur much more frequently (Figure 1). The recurrent CDR3 sequences include the well-studied VH11-encoded sequences specific for phosphatidylcholine (PtC) (Figure 1—figure supplement 2) and known to occur frequently in B-1a cells (Mercolino et al., 1988; Hardy et al., 1989; Seidl et al., 1997).

D50 metric analysis quantifying the IgH CDR3 nucleotide sequence diversity shows that the IgH CDR3 nucleotide sequences expressed by the FOB and MZB subsets are significantly more diverse than those expressed by splenic and peritoneal B-1a cells (p = 0.0002, Mann-Whitney-Wilcoxon Test) (Figure 2A). Consistent with this finding, IgH CDR3 peptide pairwise sharing analysis, which measures the similarity of IgH CDR3 peptide expression for each B cell subset sorted from different mice, shows that the same CDR3 peptide sequences frequently appear in both splenic and peritoneal B-1a cells from different mice whereas the common CDR3 peptides are rare in FOB and MZB subsets (Figure 2B). Taken together, these data demonstrate that the B-1a pre-immune IgH repertoire is far more restricted and repetitive than IgH repertoires expressed by FOB and MZB subsets.

The B-1a pre-immune IgH repertoire is far more restricted than the pre-immune IgH repertoires expressed by splenic FOB, MZB and peritoneal B-2 cells.

(A) D50 metric analysis quantifying the IgH CDR3 diversity for B cell subsets from mice at the indicated age. Low D50 values are associated with less diversity. Each dot represents the data for a B cell sample from an individual mouse except for the 2 day splenic B-1a data, which are derived from sorted cells pooled from 8 mice. B-1a samples are labeled with red; B-2 samples include FOB (green, n = 4), pB-2 (purple, n = 4) and MZB (yellow, n = 4). The data for germ-free (GF) animals is discussed at the end of the Result section. (B) CDR3 peptide pair-wise sharing analysis of IgH repertoire similarity among multiple samples for each B cell group (n = 5-9). Each dot represents the percentage of common CDR3 peptides in one sample that are also found in another sample within a given group. For example, to compute the similarity between sample A and B, the percentage of CDR3 peptides in sample A that are also found in sample B (pA  B ), together with the percentage of CDR3s in sample B that are also in sample A (pBA) are used as an indicator. For comparison of 6 splenic B-1a samples in 5-7 day group, there are 30 comparisons. Right upper: p values showing the statistical significance between two groups. Box plots represent the 10th, 25th, 50th, 75th and 90th percentiles here and in other figures.

https://doi.org/10.7554/eLife.09083.008

VH gene usage differs among the B-1a, FOB and MZB pre-immune IgH repertoires

We quantified the frequency of IgH sequences expressing individual VH gene for each sorted B cell sample and then compared the VH gene usage between two B cell subsets. B-1a cells are well-known to undergo self-replenishing in adult (Kantor et al., 1995). To minimize the impact of clonal expansion on the VH gene usage profile, we collected normalized data, in which we scored each distinct IgH CDR3 nucleotide sequence expressing a given VH gene as one, no matter how many times this sequence was detected.

Our approach enables detection of Ig transcripts expressing about 100 different VH genes that belong to 14 VH families (Figure 3). B-1a cells express all of these detected VH genes (Figure 3A), contrasting with earlier impressions, based largely on hybridomas sequences from fetal and neonatal mice (Malynn et al., 1990), that VH usage in the B-1a repertoire is very restricted. However, despite the broad VH usage, certain VH genes, notably V10-1 (DNA4), V6-6 (J606), V11-2 (VH11) and V2-6-8 (Q52), are expressed at a significantly higher frequency in splenic B-1a than MZB cells (p<0.05, Welch's t-test, Figure 3B).

Figure 3 with 2 supplements see all
Comparison of VH gene usage by splenic B-1a vs MZB B cells.

(A) VH gene usage profile shown as the percentage of IgH sequences expressing the listed individual VH genes for individual B cell samples. The profiles are shown for adult splenic B-1a samples (n = 9, red) and for MZB samples (n = 5, green). VH genes (from left to right) are ordered in 5’- to 3’-direction bases on chromosome location; the IMGT VH gene nomenclature is used (Lefranc, 2003). (B) VH genes showing the statistically significant differences (Welch’s t-test p<0.05) between two groups are listed and also highlighted with asterisks in the plot. To minimize the impact of the clonal expansion on the VH gene usage profile, data are presented as the normalized distribution that counts each distinct CDR3 nucleotide sequence expressing a given VH gene as one, no matter how many times the sequence was detected. Note: VH12-3 encoded IgH sequences are not detected in this study due to the technical limitations that exclude the VH12-3 primer from the set of primers designed about three years ago and used for studies presented here. We have since corrected this problem so that VH12-3 primer is now part of our new set of primers. Comparison of sequence data obtained with old vs. the new set of primers shows that, aside from now detecting VH12-3 sequences with the new set of primers, the sequences obtained with both primer sets are highly similar (Figure 3—figure supplement 2).

https://doi.org/10.7554/eLife.09083.009

Similar to MZB cells, splenic FOB and peritoneal B-2 cells show lower frequency in expressing these B-1a favored VH genes, i.e., V6-6 (J606), V11-2 (VH11) and V2-6-8 (Q52) (Figure 3—figure supplement 1B–C). Conversely, these B subsets tend to preferentially use the largest VH family, V1 (J558), located distal to DH and JH gene segments (Yancopoulos and Alt, 1986). MZB cells, in particular, have a higher tendency to express certain V1 (J558) family genes including V1-82, V1-72, V1-71, V1-42, V1-18 and V1-5 (Figure 3B).

The VH usage in the peritoneal B-1a cells is further biased toward V6-6 (J606), V9-3 (Vgam3.8), V2-9 (Q52) and V2-6-8 (Q52) genes, which are already favored in the splenic B-1a cells (Figure 3—figure supplement 1A). This finding indicates that the splenic and peritoneal B-1a populations are not in equilibrium and the latter is further enriched for cells expressing certain VH genes.

The B-1a IgH repertoire integrates rearrangements from de novo B-1a development that occur mainly during the first few weeks of life

Unlike FOB and MZB subsets, de novo B-1a development initiates prior to birth and decreases to a minimum in adult animals (Lalor et al., 1989; Barber et al., 2011). B-1a cells persist thereafter as a self-replenishing population (Kantor et al., 1995). To minimize the impact of self-replenishment on the N-addition distribution profile, and hence to weight the repertoire for de novo generated IgH sequences for B-1a cells, we collected normalized data that counts each distinct IgH sequence containing indicated N nucleotide insertions as a single sequence, regardless how many times this sequence was detected.

Consistent with Tdt expression, which is absent during the fetal life and initiates shortly after birth (Feeney, 1990; Bogue et al., 1992), N nucleotide insertion analysis of the splenic B-1a IgH repertoires demonstrate that roughly 60% of IgH sequences expressed by splenic B-1a cells from 2-–6 day mice do not contain N insertions at IgH CDR3 junction (D-J and V-DJ); about 30% contain 1–2 insertions; and, <15% contain 3–4 N-nucleotide insertions (Figure 4A,B). After 6 days, however, the frequency of sequences containing >3 N-additions progressively increases until the animals are weaned (roughly 3 weeks) (Figure 4A,B). After weaning, the N-addition pattern stabilizes, i.e., about 50% IgH sequences contain 3–7 N nucleotide insertions and about 30% have more than 8 N nucleotide insertions at IgH CDR3 junctions, and remains stable at this level for at least 5 months (Figure 4A,B).

N nucleotide insertion distribution patterns for the B-1a pre-immune IgH repertoires during ontogeny.

(A) Percentage of IgH sequences containing the indicated number of N nucleotide insertions at the IgH CDR3 junctions (V-DJ + D-J) is shown for each spleen B-1a sample from mice at indicated ages (shown at the right). To minimize the impact of self-renewal on the N-addition profile, normalized data are presented. Thus, each distinct IgH sequence containing indicated N nucleotide insertions is counted as one regardless how many times this sequence was detected. Note that the N insertion pattern changes as animals age. Colors distinguish three age-related patterns: green, D2 to D6; blue, D7 to 3W; red, 2M to 6M. (B) Percentages of IgH sequences containing the indicated N-nucleotide insertions (shown at the top) for splenic B-1a samples at the indicated ages are shown. Each dot represents data from an individual mouse, except for day 2 sample, n = 5-7.

https://doi.org/10.7554/eLife.09083.012

In essence, splenic B-1a cells from 2-6 day mice largely originate from fetal and early neonatal wave(s) of B-1a development when Tdt is poorly expressed. As newborns progress to maturity, B-1a cells, which are originated in the earlier wave(s), are ‘diluted’ by B-1a cells that emerge during later development. The high frequency of N nucleotide additions in the adult splenic B-1a IgH repertoire indicates that a higher proportion of B-1a cells are actually generated postnatally after Tdt is expressed.

Cohering with the increased N diversity in the adulthood, CDR3 peptide pairwise sharing analysis shows that the expression of common IgH CDR3 peptides is significantly more frequent in neonatal splenic B-1a cells than in adult splenic B-1a cells (p<2e-16, Mann-Whitney-Wilcoxon Test, Figure 2B). VH usage also shifts as animals mature. Splenic B-1a cells from neonatal mice (2-–7 days) preferentially express the V3 (36–60), V5 (7183) and V2 (Q52) families that are largely located proximal to D and J gene segments (Figure 3—figure supplement 1D), consistent with previous findings that hybridomas derived from fetal/neonatal B cells are bias in expressing proximal V5 (7183) and V2 (Q52) family genes (Perlmutter et al., 1985). In contrast, the splenic B-1a cells from adult animal (2–6 months) show higher frequencies in expressing distal V1 (J558) family genes including V1-75, V1-64, V1-55 and V1-53 (Figure 3—figure supplement 1D).

Collectively, we conclude that the B-1a IgH repertoire integrates rearrangements from sequential waves of de novo B-1a development that mainly occur during the first few weeks of life. The IgH repertoires defined during these waves are distinguishable both by N-additions at CDR3 junctions and by VH gene usage.

Recurring V(D)J sequences increase with age in the pre-immune B-1a IgH repertoire

Certain V(D)J nucleotide sequences become progressively more dominant with age in the B-1a repertoire. Thus, only a lower proportion of V(D)J sequences are detected at relative higher frequency in the splenic B-1a IgH repertoire before 3 weeks, after which, both the number of recurrent sequences and the frequency at which each is represented increase progressively until the animals reach 4–6 month of age (Figure 5A, Table 2). Consequently, the distribution of the splenic B-1a IgH CDR3 nucleotide sequences diversity is much less random in adults (2–6 months) than in neonates (2–7 days) (Figure 2A).

Figure 5 with 1 supplement see all
Certain V(D)J sequences increase progressively with age in the B-1a pre-immune IgH repertoire.

(A) IgH CDR3 tree map plots for splenic B-1a samples from mice at different ages are shown. Each plot represents data for an individual mouse, except for the day 2 sample. Recurrent sequences are visualized as larger contiguously-colored rectangles in each plot. (B) Relative frequencies of three PtC-binding IgH CDR3 sequences in indicated splenic B-1a sample groups (n = 5–8 for each group) are plotted with mouse age. Sequence information (peptide and V(D)J recombination) is shown at the top.

https://doi.org/10.7554/eLife.09083.013
Table 2

Top 10 highly recurring CDR3 sequences (peptide and V(D)J recombination) detected in each of the listed splenic B-1a samples.

https://doi.org/10.7554/eLife.09083.015
sB-1a samplesTop 10 IgH CDR3 sequences
IdAgePeptideV(D)J
111682 weeks1ANDYV1-53 J2
2AKHGYDAMDYV2-9 D2-9 J4
3ARRYYGSSYWYFDVV1-55 D1-1 J1
4ANWDYV1-53 D4-1 J2
5MRYSNYWYFDVV11-2 D2-6 J1
6ARDAYYWYFDVV7-1 J1
7ATDYYAMDYV1-26 J4
8ARFYYYGSSYAMDYV1-55 D1-1 J4
9AIYYLDYV1-53 D2-8 J2
10ARHYGSSYWYFDVV2-6-2 D1-1 J1
106543 weeks1ARRYYGSSYWYFDVV1-55 D1-1 J1
2ARSYSNYVMDYV1-76 D2-6 J4
3ARYYGSNYFDYV7-3 D1-1 J2
4ARGASYYSNWFAYV1-55 D2-6 J3
5ALTGTAYV1-53 D4-1 J3
6ARAGAGWYFDVV5-9 D4-1 J1
7TYSNYV6-6 D2-6 J2
8ARTGTYYFDYV1-53 D4-1 J2
9AMVDYV1-64 D2-9 J2
10ARWGTTVVGYV1-7 D1-1 J2
76322 months1MRYGNYWYFDVV11-2 D2-8 J1
2MRYSNYWYFDVV11-2 D2-6 J1
3MRYGSSYWYFDVV11-2 D1-1 J1
4ATFSYV1-55 J2
5ARFYYYGSSYAMDYV1-55 D1-1 J4
6ARIPNWVWYFDVV1-55 D4-1 J1
7ARWDTTVVAPYYFDYV1-7 D1-1 J2
8ARDYYGSSWYFDVV1-26 D1-1 J1
9TYYDYDLYAMDYV14-4 D2-4 J4
10ARFITTVVATRYWYFDVV1-9 D1-1 J1
86994 months1ARSADYGGYFDVV1-64 D2-4 J1
2ARGAYV1-80 J2
3ARSYYDYPWFAYV1-76 D2-4 J3
4ARRWLLNAMDYV1-9 D2-9 J4
5ARPYYYGSSPWFAYV1-69 D1-1 J3
6ARNDYPYWYFDVV1-4 D2-4 J1
7ARSGDYV1-64 J2
8ARVIGDYV1-53 D2-14 J4
9ARANYV1-55 J3
10AVNWDYAMDYV1-84 D4-1 J4
87085 months1ASLTYV1-55 J2
2TCNYHV14-4 D2-8 J4
3LIGRNYV1-55 D2-14 J2
4MRYSNYWYFDVV11-2 D2-6 J1
5AKQPYYGSSYWYFDVV2-3 D1-1 J1
6AGSSYAYYFDYV1-66 D1-1 J2
7ARRGIDLLWYHYYAMDYV1-26 D2-8 J4
8ARKSSGSRAMDYV7-3 D3-2 J4
9ASYAMDYV7-3 J4
10ARLYYGNSYWYFDVV1-55 D2-8 J1
98676 months1ARKYYPSWYFDVV1-55 D1-1 J1
2AREGGKFYV1-7 J2
3AKSSGYAMDYV1-55 D3-2 J4
4ARWVITTVARYFDVV1-85 D1-1 J1
5ARGFYV1-80 J2
6AKEGGYYVRAMDYV1-55 D1-2 J4
7ARSMDYV1-80 J4
8ASAMDYV1-64 J4
9TKGGYHDYDDGAWFVYV1-53 D2-4 J3
10ARKFYPSWYFDVV1-55 J3
  1. Table lists the top 10 highly recurring CDR3 sequences (peptide and V(D)J recombination) shown in the individual CDR3 tree-map plot of the splenic B-1a samples from 2 week to 6 month old mice (Figure 5A). For each splenic B-1a sample, the Id number and mouse age are shown in column 1 and column 2 respectively.

The recurrent V(D)J sequences include VH11-encoded PtC-binding V(D)J sequences, which are initially present at very low frequencies (2–6 days) but increase aggressively as animals mature to middle age (6 months) (Figure 5B). Since de novo B-1a development is minimum at adulthood, the progressive increase in the representation of the recurrent V(D)J sequences as animals reach adulthood suggests that B-1a cells are self-replenishing.

Certain V(D)J sequences are conserved by being positively selected into the shared adult B-1a pre-immune IgH repertoire

To determine to what extent the IgH CDR3 sequences (amino acid and nucleotide) expressed by each B cell subset are shared across different individuals, we carried out CDR3 sharing analysis. In the B-1a IgH repertoire, overall, we found 30 such highly shared IgH CDR3 peptides, each of which is expressed in over 80% of the splenic B-1a samples taken from more than 20 animals with nine different ages (from 2 days to 6 months) (Table 3). Each of the shared CDR3 peptides would be expected to be encoded by several convergent V(D)J recombinations, i.e., distinct V(D)J rearrangements encode the same CDR3 amino acid sequence (Venturi et al., 2008). Strikingly, we found that each of the shared CDR3 peptides is encoded by an identical V(D)J nucleotide sequence in over 70% of splenic B-1a samples from adult animals (2-6 months, 9 mice) (Table 3).

Table 3

Certain V(D)J sequences are positively selected and conserved in adult B-1a pre-immune IgH repertoires.

https://doi.org/10.7554/eLife.09083.016
CDR3 peptidePredominant V(D)JCDR3 junction diversityRepresentation in indicated repertoire
splenic B-1a
(2d-6M)
splenic B-1a (2-6M)additiondeletionPerC B-1a (2W-6M)splenic B-1a (4M germ free)FOB (2-5M)MZB (1-5M)
1TRWDY17/20V6-6 J28/9TGGJ2(8)11/115/61/80/7
2MRYSNYWYFDV17/20V11-2 D2-6 J19/90011/116/61/81/7
3MRYGNYWYFDV18/20V11-2 D2-8 J19/90011/116/61/81/7
4MRYGSSYWYFDV17/20V11-2 D1-1 J19/90011/116/61/81/7
5VRHYGSSYFDY15/20V10-1 D1-1 J25/90J2(1)11/113/60/80/7
6ARHYYGSSYYFDY19/20V5-6-1 D1-1 J29/90011/116/62/80/7
7ARLDY20/20V1-53 J27/9CTg/aJ2(8)10/114/60/81/7
8ARDYYGSSYWYFDV19/20V7-1 D1-1 J16/90V7-1(3)9/115/61/81/7
9ARDYYGSSWYFDV19/20V1-26 D1-1 J17/9GJ1(3)2/114/60/81/7
10ANWDY19/20V14-3 D4-1 J26/90V14-3(2)J2(8)5/112/60/80/7
11ATGTWFAY18/20V1-19 D4-1 J35/90V1-19(2)6/112/60/81/7
12ARYYYGSSYAMDY19/20V7-3 D1-1 J48/90V7-3(1)J4(4)10/113/63/83/7
13ARYSNYYAMDY18/20V1-39 D2-6 J46/90J4(2)8/111/60/80/7
14ARDFDY19/20V1-64 J26/9GJ2(3)1/113/61/81/7
15ARYYSNYWYFDV17/20V1-9 D2-6 J16/9004/111/60/80/7
16ARYDYDYAMDY17/20V1-39 D2-4 J46/90J4(3)7/111/60/80/7
17ARHYYGSSYWYFDV18/20V2-6-2 D1-1 J16/9006/112/61/83/7
18ARFYYYGSSYAMDY19/20V1-55 D1-1 J46/9TJ4(4)8/113/61/81/7
19ARWDFDY19/20V1-7 J26/9TGGGJ2(3)1/113/61/81/7
20ARGAY19/20V1-80 J35/9GGGJ3(8)7/116/61/81/7
21ARRFAY18/20V1-26 J37/9C/AJ3(8)9/113/61/81/7
22ARRDY18/20V1-55 J25/9AGg/aJ2(8)6/113/61/81/7
23ASYDGYYWYFDV18/20V1-55 D2-9 J18/9CTATGV1-55(1)9/115/60/80/7
24ASYAMDY16/20V7-3 J48/90V7-3(5)J4(4)9/116/60/81/7
25ARRYYFDY17/20V1-78 J27/9CGg/cT08/112/60/80/7
26ARNYYYFDY15/20V1-53 D1-2 J28/9t/a010/112/60/80/7
27ARYYGNYWYFDV15/20V3-8 D2-8 J15/9005/112/60/80/7
28ARRYYGSSYWYFDV15/20V1-55 D1-1 J17/9CGG010/115/61/81/7
29ARRLDY13/20V1-22 J27/9CGACJ2(6)8/112/60/81/7
30ARFAY18/20V1-80 J34/90J3(4)2/113/60/80/7
  1. Column 1: CDR3 peptide sequences identified to be shared in >80% of splenic B-1a samples (20 samples from mice ranging from 2 day to 6 month old); Column 2: for each shared CDR3 peptide, a single V(D)Jrearrangement sequence is selected and conserved in over 70% of adult B-1a samples (9 samples, 2-6 month old); Columns 3 and 4: nucleotides added or deleted in CDR3 junctions; Columns 5-8: the representation of each selected V(D)J sequence within the indicate repertoires (age and number of samples are shown for each group). Rows 2-4 are PtC-binding CDR3 sequences; Row 8 is CDR3 sequence for T15 Id+ anti-PC antibody. The data for germ-free animals is discussed at the end of the Result section.

These V(D)J nucleotide sequences represent the IgH structures that are positively selected into the shared adult B-1a IgH repertoire among C57BL/6 mice. Although the specificities of the majority of these selected V(D)J sequences remain to be defined, they include sequences that are specific for PtC and sequence for the T15 idiotype B-1a anti-PC antibodies (Masmoudi et al., 1990). Of note, most of these V(D)J sequences have nucleotide additions and/or deletions in the CDR3 junction (Table 3), indicating that the driving force for the selection may include, but is certainly not restricted to the germline rearrangement.

The majority of the V(D)J nucleotide sequences that are conserved in the splenic B-1a IgH repertoire are also conserved in the peritoneal B-1a IgH repertoires (2W-6M, 11 samples) (Table 3). Such V(D)J nucleotide sequences, however, are rarely detectable in FOB and MZB IgH repertoires (1-5M, 7-8 samples), either because these cells do not express these CDR3 peptides or because they use different V(D)J recombination sequences to encode them (Table 3). For example, although MZB cells express antibodies encoding the same CDR3 peptide as B-1a T15-id+, they use different V(D)J recombinations and no single V(D)J recombination dominates within the MZB IgH repertoire (Table 4). In essence, the selection of a predominant V(D)J nucleotide sequence encoding a given CDR3 peptide is unique for the B-1a IgH repertoire.

Table 4

MZB IgH repertoires use different V(D)J recombination sequences to encode the same CDR3 peptide as that of B-1a anti-PC T15Id+.

https://doi.org/10.7554/eLife.09083.017
MZB sample IdAge (Months)V(D)J recombination
76302V1-76 D1-1 J1 and V1-39 D1-1 J1
106582V1-76 D1-1 J1
87004V1-72 D1-1 J1 and V8-12 D1-1 J1
87015V1-58 D1-1 J1 and V1-61 D1-1 J1
133384V1-61 D1-1 J1 and V5-16 D1-1 J1
  1. Column 1: individual MZB samples tested; column 2: age of mouse for each MZB sample; column 3: for each MZB sample, V(D)J recombination events that encode ARDYYGSSYWYFDV, which is the CDR3 peptide associated with B-1a anti-PC T15Id+.

Multiple distinct V(D)J recombinations that encode the same CDR3 peptide in neonatal and young mice converge to a single identical V(D)J sequence in all adults

In 2–7 day animals, a few selected V(D)J nucleotide sequences, such as PtC-binding sequences, have already emerged as the predominant V(D)J recombination for their corresponding CDR3 peptide (Figure 6A, pattern II). However, most of the selected V(D)J nucleotide sequences, including T15Id+, do not initially represent the predominant recombination for their corresponding CDR3 peptide. In particular, some CDR3 peptides are each encoded by multiple different V(D)J recombinations with similar frequencies in neonate mice. However, after weaning, a particular V(D)J recombination gradually increases its representation until it dominates in the adult B-1a IgH repertoire (Figure 6A, pattern I). In essence, although multiple distinctive V(D)J recombinations encoding the same CDR3 peptide exist in the neonatal/young B-1a IgH repertoire, a single identical V(D)J recombination sequence is selected to encode the particular CDR3 peptide in adult repertoire of almost all individuals.

Figure 6 with 1 supplement see all
The level of convergent recombination in the B-1a IgH repertoire declines with age.

(A) Entropy heat map showing the diversity of V(D)J recombination events for each indicated CDR3 peptide (shown at the left) in splenic B-1a samples at different ages (shown at the bottom). The higher the entropy value, the more diverse the V(D)J recombinations for a given CDR3 peptide. CDR3 peptide sequences for T15 Id+ anti-PC (pattern I) and anti-PtC (pattern II) antibody are shown in bold. (B) The diversities of the V(D)J recombination for each CDR3 peptide for the indicated splenic B-1a samples (shown at the bottom) are quantified as entropy values (see Methods and materials), which are ranked into 4 ranges (shown at the right). For each sample, the frequencies of CDR3 peptide sequences belonging to each entropy range are shown as stacks. (C) Splenic B-1a samples are grouped based on age. For each group (n = 5–7), the frequencies of CDR3 peptide sequences belonging to each of four entropy ranges are shown. *p<0.05, Welch’s t-test.

https://doi.org/10.7554/eLife.09083.018

In accordance with this finding, quantification of the diversity of V(D)J recombination events for each CDR3 peptide reveals the profound convergent recombination in the neonatal B-1a IgH repertoire. Thus, about 30% of CDR3 peptide sequences in splenic B-1a IgH repertoire at 2–6 day are encoded by more than one V(D)J recombination (entropy >0.5, Figure 6B,C), and about 10% of CDR3 peptide sequences show the highest level of convergent recombination (entropy >1.5, Figure 6B,C, the higher the entropy value, the more diverse the V(D)J recombinations). However, the frequency of CDR3 peptides showing convergent recombinations steadily decrease until the animals reach adulthood (2 months), after which very few (<1%) CDR3 peptide sequences show the multiple V(D)J recombinations (entropy >1.5, Figure 6B,C).

The step-wise decreases in the level of convergent recombination as animals age indicate the potent selection that over-time shapes the B-1a IgH repertoire. In most cases, the related V(D)J sequences that ‘converge’ to encode the same CDR3 peptide share the same D and J segments but use distinct VH genes (Figure 6—figure supplement 1). Therefore, despite encoding the same CDR3 peptide sequence, these related V(D)J sequences differ in their upstream regions including the CDR2 (Figure 6—figure supplement 1). These upstream differences, which can contribute to ligand binding, may be central to the selection of the predominant V(D)J sequence for the corresponding CDR3 peptide.

AID-mediated SHM in pre-immune B-1a IgVH initiates after weaning and cumulatively increases the IgH repertoire diversity thereafter

Greater than 25% of splenic B-1a IgH sequences in 4–6 month old mice have at least one nucleotide change (Figure 7A). Such mutations are principally mediated by AID because they are rare (<2%) in splenic B-1a cells from age-matched AID-deficient mice (Figure 7A). The SHM even targets V(D)J sequences that are positively selected into the shared B-1a IgH repertoire in wild type mice (but not in AID-deficient mice) (Figure 7B,D). The observed mutations, most of which result in amino acid changes, are largely targeted AID hotspots, i.e., DGYW (D = A/G/T; Y = C/T; W = A/T) or WRCH (R = A/G, H = T/C/A) (Di Noia and Neuberger, 2007) (Figure 7B,C).

Figure 7 with 3 supplements see all
AID-mediated SHM accumulates on splenic B-1a IgVH with age.

(A) Percentages of sequences containing > = 1 (red) or > = 2 (green) nucleotide changes for B cell samples from mice at the indicated ages are shown (n = 3-8). Seven B cell samples from 4-5 month old AID knockout mice include sB-1a (n = 4), pB-1a (n = 1), FOB (n = 1) and pB-2 (n = 1). Sequences with the identical V(D)J recombination encoding ARGAY CDR3 peptide obtained from splenic B-1a sample from 4 month old specific pathogen free mouse, (B) germ-free mouse (C) and AID knockout mouse (D) are listed. The nucleotide substitution is analyzed at the VH region stretching from the start of CDR2 (red box) to the beginning of CDR3 (yellow box). Obtained sequence (upper line) is aligned with the reference (lower line) for V1-80 (red), J3 (blue) and constant region of IgM isotype (orange). Mutations are highlighted with triangles; asterisks indicate mutations resulting in an amino acid change; red and blue triangles denote mutations in DGYW and WRCH motifs, respectively. (E) Numbers of mutations per 104 base pairs for indicated B cell group are shown. Each dot represents data from an individual sample (n = 3–8). The data for germ-free (GF) animals is discussed at the end of the Result section. Note: The mutation profiles for the splenic B-1a IgH libraries prepared by using either old (VH12-3 deficient) or new primer set (VH12-3 included) are highly similar (Figure 7—figure supplement 3).

https://doi.org/10.7554/eLife.09083.020

In contrast, mutations are minimal in IgVH of splenic FOB, MZB and peritoneal B-2 cells from adult mice (Figure 7A). Interestingly, the frequency of mutated IgH sequences in peritoneal B-1a cells in 4-6 month old mice is substantially lower than that in age-matched splenic B-1a cells and mutations are mainly single nucleotide change (Figure 7A).

SHM in splenic B-1a IgVH initiates after weaning and the frequency of mutated IgH transcripts increases with age. Thus, mutations are minimally detectable in the IgVH of splenic B-1a cells from neonates (2–7 days) and young mice (2–3 weeks), are at lower frequencies in 2 month old mice, and are at substantially higher frequencies in 4–6 month old animals (Figure 7A). This age-dependent increase in splenic B-1a IgVH mutation argues that the detected SHM is not due to contamination with co-sorted B cells of other subsets, including GC cells, i.e., cells with the germinal center phenotype (GL7+ CD38lo CD95hi) are not detectable in the splenic B-1a population (Figure 7—figure supplement 1).

Furthermore, SHM is cumulative, becoming more pronounced with age. Thus, roughly 25% of IgH sequences from 4–6 month old splenic B-1a samples contain > = 1 nucleotide change, 19% contain > = 2 changes, and 9% contain > = 4 changes (Figure 7A and Figure 7—figure supplement 2). This translates to an average SHM rate of roughly 5 per 103 base pairs (bp) (Figure 7E), the similar range as that for SHM in GC responses, i.e., 10-3 bp per generation (Wagner and Neuberger, 1996). Both the frequency of mutated sequences and the mutation rate for splenic B-1a samples from 2 month old mice are substantially lower than those in 4–6 month old mice (Figure 7A,E), further supporting that the SHM in the splenic B-1a IgVH is an accumulative process.

Age-dependent progressive increase in the splenic B-1a IgVH mutations is accompanied by increased class-switching

Class switch recombination (CSR) is another genetic alteration process that somatically diversifies rearranged IgH genes. Both SHM and CSR are triggered by AID, which targets and introduces lesions in the IgV region for SHM and the switch regions for CSR (Muramatsu et al., 2000; Chaudhuri and Alt, 2004). Although both events require AID, SHM and CSR employ different enzymes and thus can occur independently (Li et al., 2004). Nevertheless, since they usually occur at the same differentiation stage and both are initiated by AID, the question arises as to whether the detected SHM in B-1a IgH is associated with CSR.

Our method allows detection of all different Ig isotypes. For each B cell sample, we quantified the frequency of IgH sequences expressing a given isotype and examined the relationship between the isotype profiles to the mutation status. Consistent with the close relationship between CSR and SHM, wild-type B cell samples that have minimal IgVH mutations, including the splenic FOB, MZB, peritoneal B-2, neonate splenic B-1a (2–7 days), young splenic and peritoneal B-1a (2–3 weeks), rarely express class-switched transcripts (Table 5). Similarly, for B cell populations that show lower levels of mutation, e.g., splenic B-1a from 2 month old animals and peritoneal B-1a from 2–6 month old animals, the majority of both mutated and non-mutated sequences are either IgM or IgD and thus rarely class-switched (Figure 8A, Table 5).

Progressive increase in the splenic B-1a IgVH mutation frequency with age is accompanied by increased class-switching.

(A) Left panel: The frequencies of non-mutated or mutated (> = 1 nucleotide substitution) IgH sequences obtained from indicated B cell samples are shown; Right panel: The frequencies of sequences expressing class-switched isotypes (neither IgM nor IgD) among non-mutated or mutated sequences are shown. Each dot represents data from an individual sample (n = 5–6). p values are calculated based on the Nonparametric Wilcoxon test. (B) In each plot, the IgH sequences obtained from each splenic B-1a sample from 3.5–6 month old mice are divided into five categories, based on the number of mutated nucleotides (0, 1, 2, 3, 4, > = 5) per read. In each plot, the values shown at the top are the frequencies of sequences in each category. For each category of sequences, frequencies of the distinct isotype sequences are shown as stacks. A = IgA; D = IgD; G = IgG1 + IgG3 + IgG2c + IgG2b. Each plot represents the data for a splenic B-1a sample from an individual mouse reared under either specific pathogen free (SPF) (upper plots) or germ-free (GF) (lower plots) conditions. The data for germ-free (GF) animals is discussed at the end of the Result section.

https://doi.org/10.7554/eLife.09083.024
Table 5

B cell samples that show minimal or low level mutations in IgVH rarely express class-switched transcripts.

https://doi.org/10.7554/eLife.09083.025
Sample Idsubsetagestrainnon-mutated or mutated sequences (%)IgM (%)IgD (%)IgG1(%)IgG3 (%)IgG2c (%)IgG2b (%)IgE (%)IgA (%)
13965sB-1a4MAIDKOnon-mutated98.299.50.5
mutated1.8100
13968sB-1a5MAIDKOnon-mutated99100
mutated1100
13971sB-1a4MAIDKOnon-mutated98.499.60.4
mutated1.6100
13972sB-1a4MAIDKOnon-mutated10099.90.1
8704pB-1a4MAIDKOnon-mutated98.299.90.1
mutated1.8100
13973pB-25MAIDKOnon-mutated10091.68.4
8700MZB5MWTnon-mutated99.899.90.1
8701MZB4MWTnon-mutated98.699.90.1
7630MZB2MWTnon-mutated99.599.90.1
10658MZB2MWTnon-mutated100100
8702FOB5MWTnon-mutated99.898.61.4
13966FOB3.5MWTnon-mutated99.599.70.3
7631FOB2MWTnon-mutated99.372.927.1
7629pB-24MWTnon-mutated98.488.411.5
mutated1.68911
13969pB-23.5MWTnon-mutated99.590.19.9
13974sB-1aday 2WTnon-mutated99.299.80.2
13000sB-1aday 2WTnon-mutated99.2100
10659sB-1aday 5WTnon-mutated99.2100
9866sB-1aday 5WTnon-mutated100100
10651sB-1aday 5WTnon-mutated99.7100
10652sB-1aday 6WTnon-mutated99.4100
9868sB-1aday 7WTnon-mutated99.399.90.1
9865sB-1aday 7WTnon-mutated99.599.60.4
11168sB-1a2WWTnon-mutated99.199.90.1
13005sB-1a2WWTnon-mutated99.5100
10654sB-1a3WWTnon-mutated95.8100
mutated4.2100
11160pB-1a2WWTnon-mutated99100
10655pB-1a3WWTnon-mutated99.2100
11163pB-1a1MWTnon-mutated99.199.9
7632sB-1a2MWTnon-mutated88.199.10.9
mutated11.999.90.1
10656sB-1a2MWTnon-mutated88.199.80.2
mutated11.9100
13004sB-1a2MWTnon-mutated97.799.9
mutated2.3946
13018pB-1a2MWTnon-mutated91.8100
mutated8100
13660pB-1a2MWTnon-mutated92.2100
mutated7.6100
7628pB-1a2MWTnon-mutated92.399.50.40.1
mutated7.199.60.20.2
8705pB-1a4MWTnon-mutated93.899.70.3
mutated4.499.9
9870pB-1a4MWTnon-mutated86.499.9
mutated12.699.9
11165pB-1a5MWTnon-mutated98.199.9
mutated1.5100
8707pB-1a5MWTnon-mutated91.697.20.120.50.10.1
mutated6.297.90.12
9861pB-1a6MWTnon-mutated82.499.60.4
mutated17.5100
  1. Table lists each individual B cell sample (labeled as distinct Id number) from wild-type (WT) or AID-deficient (AIDKO) mice. The mouse age and sample subset information are also shown. For each sample, the sequences are divided into non-mutated or mutated (> = 1 nucleotide change) categories, the frequencies of each category are shown. For each category, the frequencies of sequences with each isotype are also shown.

In contrast, both the mutated and non-mutated IgH sequences from splenic B-1a in 4–6 month old animals contain class-switched Ig (Figure 8). Importantly, the class-switched Ig (mainly IgG3, IgG2b, IgG2c and IgA) represents a significantly higher proportion of the mutated sequences than of the non-mutated sequences (Figure 8A, Table 6), indicating that the increased SHM with age in the splenic B-1a IgH repertoire is accompanied by increased class-switching. However, despite the increased class switching among mutated sequences, the frequency of class-switched sequences appears not to correlate with the increased number of mutations (Figure 8B). Consistent with the class-switching dependence on AID, we did not detect isotypes other than IgM and IgD in splenic B-1a cells from 4–5 month old AID-deficient mice (Table 5).

Table 6

Both the mutated and non-mutated IgH sequences obtained from splenic B-1a cells in 4-6 month old animals contain class-switched Ig.

https://doi.org/10.7554/eLife.09083.026
sample Idsubsetageconditionnon-mutated or mutated sequences (%)IgM (%)IgD (%)IgG1(%)IgG3 (%)IgG2c (%)IgG2b (%)IgE(%)IgA(%)
9867sB-1a6MSPFnon-mutated5095.82.70.60.80.1
mutated50650.0316.84.34.89.1
8699sB-1a4MSPFnon-mutated5699.50.30.10.1
mutated4489.93.421.33.4
9863sB-1a4MSPFnon-mutated74.193.90.23.611.3
mutated25.944.2419.73.71.4
13970sB-1a3.5MSPFnon-mutated74.192.70.53.71.71.3
mutated25.992.12.50.54.80.1
13342sB-1a4MSPFnon-mutated88.997.60.50.80.60.20.3
mutated11.185.20.30.27.37
13337sB-1a4MGFnon-mutated69.798.50.10.80.40.1
mutated30.37915.350.7
13003sB-1a4MGFnon-mutated74.897.20.30.20.50.21.6
mutated25.289.81.12.31.25.6
13341sB-1a4MGFnon-mutated78.2990.10.20.10.6
mutated21.872.29.65.512.60.1
13017sB-1a4MGFnon-mutated80.995.60.4211
mutated19.1790.27.93.98.90.1
13002sB-1a4MGFnon-mutated88.597.40.50.60.21.3
mutated11.563.814.88.413
  1. Table lists individual splenic B-1a cell sample sorted from 4-6 month old C57BL6/J mice reared under either specific pathogen free (SPF) or germ-free (GF) condition. For each sample, the sequences are divided into non-mutated or mutated (> = 1 nucleotide change) categories, the frequencies of each category are shown. For each category, the frequencies of sequences expressing each isotype are shown. The data for germ-free animals is discussed at the end of the result section.

The splenic B-1a cells that express class-switched Ig still express IgM on the surface, since cells were sorted as IgMhi IgDlo/- dump- CD19+ CD93- CD21-/lo CD23- CD43+ CD5+. In addition, IgM+ cells described here barely co-express other surface isotypes. Thus the class-switched transcripts are derived from IgM+ cells that apparently have already undergone class switching but have yet to turn off IgM surface protein expression. Since all of the cell preparation, staining and sorting were performed equivalently for all samples, our finding that the class-switched transcripts were selectively and predominantly detected in splenic B-1a cells from 4–6 month old mice argues that the detection of these transcripts is not due to contamination or other technical problems.

The V(D)J selection and AID-mediated diversification that uniquely act in B-1a IgH repertoire operate comparably in germ-free and conventional mice

The microbiota are often thought to participate in shaping the repertoire of ‘natural’ antibodies, which is largely produced by B-1a (Baumgarth et al., 2005). Nevertheless, we find that germ-free (GF) animals have normal numbers of B-1a cells in spleen (Figure 9—figure supplement 1). Notably, the splenic B-1a IgH repertoires in age-matched (4-5 month old) specific pathogen free (SPF) and GF mice are very similar: 1) their IgH repertoires are comparably less diversified and enriched in the recurrent V(D)J sequences (Figures 2A, 9A, Table 7); 2) their VH usage patterns show no significant differences (Figure 3—figure supplement 1E); 3) their CDR3 peptide expressions show a comparable extent of sharing between SPF and GF mice (Figure 9B); and 4) a substantial proportion of V(D)J sequences selected in the B-1a IgH repertoire in adult SPF mice are similarly selected in the B-1a IgH repertoire in GF mice (Table 3).

Figure 9 with 1 supplement see all
The B-1a IgH repertoires from mice raised in specific pathogen free condition are comparable to the B-1a IgH repertoire from age-matched germ-free mice.

(A) IgH CDR3 tree map plots for splenic B-1a cells from GF mice (upper panel), or SPF mice in Caltech animal facility (middle panel), or SPF mice in Stanford animal facility (bottom panel). Each plot represents the data for a sample from a 4 month old mouse. Recurrent CDR3 (nucleotide) sequences are visualized as larger contiguously-colored rectangles in each plot. (B) CDR3 peptide pair-wise sharing analysis of IgH repertoire similarity between multiple splenic B-1a samples from age-matched GF and SPF mice. GF mice (n = 6); SPF mice (n = 6). CDR3 peptide pair-wise analysis was conducted between GF mice (GF/GF), SPF mice (SPF/SPF) and GF vs. SPF mice (GF/SPF). Each dot represents the percentage of shared CDR3 peptide sequences between two mice. There was no statistical difference between each comparison.

https://doi.org/10.7554/eLife.09083.027
Table 7

Top 10 highly recurring CDR3 sequences (peptide and V(D)J recombination) detected in listed splenic B-1a samples from age-matched SPF and GF mice.

https://doi.org/10.7554/eLife.09083.029
sB-1a samples (4 months)Top 10 IgH CDR3 sequences
PeptideV(D)J
germ-free #11MRYGSSYWYFDVV11-2 D1-1 J1
2ARGAYV1-80 J2
3ARNPDGYYTYYYAMDYV2-2 D2-9 J4
4ARDPFYYYGSSYWYFDVV5-16 D1-1J1
5MRYSNYWYFDVV11-2 D2-6 J1
6AITRAYV1-55 J3
7ARRYYGSSYWYFDVV1-55 D1-1 J1
8ARSDYYGSSSLSYV1-26 D1-1 J2
9ASGGNYFDYV1-75 J2
10ARSLYNV1-9 J2
germ-free #21ARNYGSSYDYV1-53 D1-1 J2
2TRPSYYGSDYV14-4 D1-1 J2
3TRESYDGYYVWYAMDYV5-9-1 D2-9 J4
4ARGDYV14-3 J2
5ASNWAYV1-53 D4-1 J2
6MRYSNYWYFDVV11-2 D2-6 J1
7AKGDYYGSSYYFDYV1-9 D1-1 J2
8VRHGPRAFDYV10-1 D3-2 J2
9ARLNGDYV1-69 J2
10MRYGNYWYFDVV11-2 D2-8 J1
specific pathogen free #1 (from Caltech)1ASYSNSDVV3-6 D2-6 J1
2ARVSYSRAMDYV14-3 D2-6 J4
3ARSGNYGAMDYV1-7 D2-8 J4
4ASRLRSTFAYV2-6-8 D1-1 J3
5ARVTTVHAMDYV1-55 D1-1 J4
6ARNYGSSYWYFDVV1-53 D1-1 J1
7ARTPNWEARDYV1-55 D4-1 J4
8ARRYYGSSYWYFDVV1-55 D1-1 J1
9ARPLLYRYYFDYV1-75 D2-6 J2
10ARNYGSSYDWYFDVV1-9 D1-1 J1
specific pathogen free #2 (from Caltech)1ARGGIYYDYDEVYYYAMDYV1-55 D2-4 J4
2MRYSNYWYFDVV11-2 D2-6 J1
3ARDYYGSSWYFDVV1-26 D1-1 J1
4MRYGNYWYFDVV11-2 D2-8 J1
5MRYGSSYWYFDVV11-2 D1-1 J1
6ARYYDGYYGYYAMDYV1-26 D2-4 J4
7ALITTWYFDVV1-78 D1-2 J1
8ARHYYGSSWGYV1-53 D1-1 J2
9ARSFSPYYFDYV1-26 J2
10ARSHGYYPFDYV1-54 D2-9 J2
specific pathogen free #1 (from Stanford)1ARSADYGGYFDVV1-64 D2-4 J1
2ARGAYV1-80 J2
3ARSYYDYPWFAYV1-76 D2-4 J3
4ARRWLLNAMDYV1-9 D2-9 J4
5ARPYYYGSSPWFAYV1-69 D1-1 J3
6ARNDYPYWYFDVV1-4 D2-4 J1
7ARSGDYV1-64 J2
8ARVIGDYV1-53 D2-14 J4
9ARANYV1-55 J3
10AVNWDYAMDYV1-84 D4-1 J4
specific pathogen free #2 (from Stanford)1ARGNYV1-80 J2
2ARWVYYGSSSYWYFDVV1-54 D1-1 J1
3ARSSNYAMDYV1-78 D2-11 J4
4ARYYYGSNYAMDYV7-3 D1-1 J4
5ARGAYV1-80 J2
6ARRYYGSSYWYFDVV1-55 D1-1 J1
7ARSPYYSNYEGYFDVV1-72 D2-6 J1
8ARKNYGSSYWYFDVV1-55 D1-1 J1
9ARLEIYYGNYGRVFDVV1-80 D2-8 J2
10ARRDYYGSSYVLAYV1-9 D1-1 J3
  1. Table lists the top 10 highly recurring CDR3 sequences (peptide and V(D)J recombination) shown in each of CDR3 tree-map plot (Figure 9A).

Further, hypermutation occurs equally in the splenic B-1a IgVH in 4–6 month old SPF and GF mice, i.e., the frequency of mutated sequences and the mutation rate are comparable under two conditions (Figure 7A,E). Indeed, AID introduces mutations into the identical V(D)J sequences expressed by splenic B-1a cells from either SPF or GF mice (Figure 7B,C). Finally, similar to SPF mice, AID-mediated class-switch occurs comparably in splenic B-1a cells from GF mice (Figure 8A). Since the V(D)J selection, hypermutation and class-switching operate comparably in splenic B-1a from GF and SPF mice, we conclude that the somatic mechanisms that select and diversify B-1a IgH repertoire over time are not driven by microbiota-derived antigens.

Nevertheless, the environment has a strong impact on the isotype representation. IgA transcripts are readily detected in splenic B-1a from 4–6 month old SPF mice; however, these transcripts are minimally detected in the splenic B-1a from age-matched GF mice (Figure 8B, Table 6). This finding is consistent with the recognition that class-switching to IgA is strongly associated with the presence of gut microbiota (Kroese et al., 1989; Macpherson et al., 2000).

Discussion

Studies presented here open a new perspective on the origin and breadth of humoral immunity that protect against invading pathogens and regulate autoimmunity. Recent studies have already shown B-1a develops prior to and independent from BM HSC, which fail to generate B-1a but fully constitute FOB and MZB compartment (Ghosn et al., 2012; Yoshimoto et al., 2011). Cohering the fundamental difference in their development origin, our studies reveal two distinct IgH repertoires that develop at different times and are shaped by distinct functional mechanisms.

The first of these repertoires is expressed in B-1a cells. The de novo IgH rearrangements in this repertoire occur mainly during the first few weeks of age and largely cease thereafter. Then B-1a cells persist as a self-replenishing population. The B-1a repertoire, however, continues to evolve under stringent selection. Thus, certain V(D)J sequences increase with age, and certain V(D)J nucleotide sequences gradually emerge as the predominant recombinations encoding the specific CDR3 peptides in all adults. Furthermore, the age-dependent V(D)J selection coincides with the progressive introduction of IgVH mutation and increased class-switch. Importantly, the V(D)J selection and AID-mediated diversification occur comparably in germ-free and conventional mice, indicating that these unique repertoire-defining mechanisms are not driven by microbiota-derived antigens.

In contrast, MZB, FOB and peritoneal B-2 cells develop later, and continuously develop de novo from BM HSC throughout life and express drastically different IgH repertoire(s). Their IgH repertoires tend to preferentially utilize V1 (J558) family, are far more diverse and less repetitive and, unlike B-1a cells, show no apparent selection for particular V(D)J recombination sequences and do not show IgVH mutation and class-switch. In essence, AID introduces SHM and CSR in these B cell subsets only when they respond to their cognate antigens that are largely exogenous in nature.

These findings were enabled by employing the amplicon-rescued multiplex PCR technology, which allows the capture and amplification of Ig transcripts from a given B cell population in an inclusive and quantitative fashion. Specifically, the first RT-PCR reaction, which uses an array of gene-specific primers for almost all VH families and all constant (CH) genes, is carried out only for a few cycles. The second round of PCR is then carried out with communal primers that recognize the unique sequences tagged into each of the VH and CH primers. Since these ‘tag sequences’ were already introduced during the initial cycles, the use of the communal primers assures that all of the targets are amplified with reduced bias during the following exponential phase of amplification. Coupled with the next generation sequencing, our method is quite robust and allows detection of diverse Ig transcripts that collectively carry about 100 VH genes associated with different isotypes.

As with other bulk RNA sequence measurement, our methods cannot determine the absolute number of each Ig transcript in a given B cell population. Hence the actual number of cells expressing a certain Ig sequence is unknown. In addition, our methods do not allow determination of whether certain sequences associated with distinct isotypes belong to the same cell. Further, since the Ig transcript copy number variation among cells is unknown, the frequency of a given Ig transcript is roughly viewed as the relative index of the frequency of cells expressing this Ig transcript. This assumption is generally valid since our studies exclude plasmablast and plasma cells, which do not express surface CD5. Since B-1a cells are well-known to undergo self-replenishment in adult, the dramatic increase in certain V(D)J sequences in the B-1a IgH repertoire over time likely reflects the expansion of cells expressing this particular V(D)J sequence.

Single cell sequencing analysis has advantages in reducing technical bias and in enabling paired IgH/IgL sequencing. Nevertheless, sequencing costs are still a big hurdle to the large-scale single cell analysis, which, as our studies demonstrate, is necessary to develop a comprehensive view of the various B cell subset repertoires. Therefore, at least for the present, our approaches are more efficient and practical.

B-1a produce ‘natural’ antibodies, many of which recognize endogenous (self) antigens (Baumgarth et al., 2005) and play house-keeping roles in clearing the cellular debris or metabolic wastes (Shaw et al., 2000; Binder and Silverman, 2005). Since the natural antibodies can also react/cross-react with microorganism-derived antigens, they also participate in the first line of immune defense (Ochsenbein et al., 1999; Baumgarth et al., 2000). Germ-free mice have normal levels of circulating ‘natural’ IgM (Bos et al., 1989). Earlier immunologists have postulated that the natural antibody repertoire is selected by endogenous (self) antigens (Jerne, 1971; Coutinho et al., 1995). Our studies, which demonstrate that B-1a IgH repertoire (hence the re-activities of natural antibodies) is highly similar between individual adult C57BL/6 mice, regardless of whether the animals are reared in conventional or germ-free facilities, introduce the solid evidence supporting this argument.

Our studies also demonstrate that the B-1a IgH repertoire is selected over time. Thus, recurrent V(D)J sequences appear later, and most of the V(D)J sequences that are selected to be conserved in all individuals do not emerge until the animals reach the adulthood. As a result, the sequence composition of B-1a IgH repertoire in adult mice becomes much less random than that expressed in neonate and younger mice. Furthermore, the convergent selection of a particular V(D)J recombination sequence encoding a specific CDR3 peptide indicates that the selection is strikingly precise and occurs at both the protein and the nucleotide level.

Unexpectedly, our studies find that both SHM and CSR participate in diversifying the B-1a IgH repertoire. However, unlike GC response SHM, which occurs within a few days following antigenic stimulation, SHM in B-1a IgVH starts after weaning and is cumulative with age. The progressive increase in the SHM is also associated with increased class switching. Most importantly, SHM and CRS occur comparably in germ-free and conventional mice, indicating that SHM and CSR in the B-1a primary IgH repertoire are not driven by microbiota-derived antigen. Since B-1a cells are well-known to produce anti-self antibodies, stimulation by endogenous antigens is likely the major driving force for the AID-mediated diversification processes.

Ongoing SHM in the absence of external antigens influence have been reported in sheep B cells (Reynaud et al., 1995). The accumulation of SHM in B-1a IgVH over time likely represents a similar strategy to further diversify their restricted Ig repertoire as animal age. Such diversification may potentiate defenses against newly encountered pathogens. However, the age-dependent accumulative SHM, which is likely driven by self-antigens, may also increase the risk of autoimmune disease due to pathogenic high affinity auto-reactive antibodies. Indeed, deregulated B-1a growth have been reported in NZB/W mice, where autoantibody-associated autoimmune disease develops as animal age (Hayakawa et al., 1984).

AID-mediated mutagenesis in B-1a IgVH may occasionally introduce mutations elsewhere in the genome that facilitate dysregulated growth and neoplastic transformation, e.g., B-chronic lymphocytic leukemia (B-CLL) (Stall et al., 1988; Kipps et al., 1992; Phillips and Raveche, 1992). Although the mechanism by which the IgM+ splenic B-1a cells from older mice express class-switched Ig transcripts remains elusive, this finding suggests that certain cells are undergoing vigorous genetic alteration that may share the similar mechanisms that underlie the malignant transformation. In fact, cells with simultaneous expression IgM and class-switched Ig transcripts have been reported in B-CLL and other B cell tumors (Oppezzo et al., 2002; Kinashi et al., 1987).

The splenic and peritoneal B-1a IgH repertoires show similar characteristics. Both repertoires become more restricted with age with increased recurrent V(D)J sequences (Figure 5—figure supplement 1) and retain the positive selected V(D)J sequences in adult animals. However, our studies reveal the key repertoire differences between B-1a cells at their two native locations. Although both repertoires show extensive CDR3 sharing among individual mice, the peritoneal B-1a IgH repertoire is more similar to neonatal splenic B-1a repertoire and shows a significantly higher level of CDR3 peptide sharing among individual mice than the splenic B-1a repertoire (Figure 2B). In addition, the peritoneal B-1a IgH repertoire is more biased in using V6-6 (J606), V9-3 (Vgam3.8), V2-9 (Q52) and V2-6-8 (Q52), which are preferentially expressed in splenic B-1a from neonate and younger mice.

These findings suggest that peritoneal B-1a cells are enriched for cells that are generated during neonatal and young age of life, thus are largely consist of cells migrated from spleen into PerC when the animals were younger. This argument is further supported by the findings that the frequencies of mutated sequences in the peritoneal B-1a cells from 4-6 month old mice are substantially lower and the mutations are mainly single nucleotide changes whereas a proportion of IgH sequences with multiple mutations is detected in splenic B-1a cells from the same aged mice (Figure 7).

MZB and B-1a share many phenotypic and functional characteristics (Martin and Kearney, 2001). Our studies show that the MZB IgH repertoire differs drastically from the B-1a IgH repertoire, but is very similar to the repertoires expressed by splenic FOB and peritoneal B-2. Since MZB and FOB cells are mainly derived from BM HSC (Ghosn et al., 2012), there findings collectively support the idea that these B cells belong to the same (i.e.,B-2) developmental lineage. Nevertheless, the MZB repertoires from individual mice contain substantially higher levels of common CDR3 sequences (peptides) than the splenic FOB and peritoneal B-2 repertoires (Figure 2B).

Years ago, we postulated that B-1a and B-2 B cells belong to distinct developmental lineages that are evolved sequentially to play complementary roles in immunity (Herzenberg and Herzenberg, 1989). The sequence data presented here, which reveal the key distinctions in the repertoires as well as the repertoire-defining mechanisms between B-1a and B-2 subsets, support this argument and greatly extend our earlier version. These key distinctions provide the genetic bases for their well-known fundamental functional difference between B-1a and other B subsets. In particular, they are central to vaccine development, where the recognition that the B cells have distinct targeting antibody repertoires clearly invites attention. In addition, our findings offer insights in understanding the origins and behaviors of B cell neoplasms, particularly B-CLL, and the autoimmune diseases in which over production of autoantibodies is implicated in the pathology.

Materials and methods

Mice

C57BL/6J mice were purchased from the Jackson Laboratory. AID-deficient C57BL6/J mice were kindly provided by Dr. Michel Nussenzweig (Rockefeller University). Mice were breed and kept in the Herzenberg laboratory colony under SPF conditions at the Stanford Veterinary Service Center (VSC). Spleens from germ-free C57BL6/J mice were provided by Dr. Sarkis Mazmanian (Caltech). Germ-free mice were maintained in sterile Trexler isolators and fed autoclaved food and water. Germ-free status was assayed monthly by aerobic and anaerobic plating; and by 16s rRNA PCR. Study protocols were approved by the Stanford VSC.

Hi-dimensional FACS sorting

Request a detailed protocol

FACS staining has been previously described (Yang et al., 2012). Briefly, cell suspensions were incubated with LIVE/DEAD Aqua (Life Technologies, San Diego, CA), washed, and incubated with unconjugated anti-CD16/CD32 (FcRII/III) mAb to block Fc-receptors. Cells were then stained on ice for 20 min. with a ‘cocktail’ of fluorochrome-conjugated antibodies including: anti-CD21-FITC (Becton Dickenson, San Jose, CA), anti-CD43-PE (BD), anti-CD5-PE-Cy5 (BD), anti-CD19-PE-Cy5.5 (Life Technologies), anti-CD93 (AA41)-PE-Cy7 (eBioscience, San Diego, CA), anti-B220-APC (BD), anti-IgM-Alexa700 (Herzenberg lab), anti-IgD-APC-Cy7 (BioLegend, San Diego, CA), anti-CD23-Biotin (BD), anti-CD11b-PB (Life Technologies), anti-Gr-1-PB (Life Technologies), anti-TCRαβ-PB(Life Technologies), anti-CD11c-PB (Life Technologies), and anti-CD3?-PB (Life Technologies). After washing, cells were stained with Streptavidin-Qdot 605 (Life Technologies). Cells were sorted on FACS Aria (BD) at the Stanford Shared FACS Facility. Sorting purity was greater than 99%. Five types of B cell populations were sorted based on tissue and phenotype: splenic and peritoneal B-1a cells (dump- CD19+ CD93- IgMhi IgD-/lo CD21-/lo CD23- CD43+ CD5+); splenic FOB and peritoneal B-2 cells (dump- CD19+ CD93- IgMlo IgDhi CD23+ CD43- CD5-); splenic MZB cells (dump- CD19+ CD93- IgMhi IgD-/lo CD21hi CD23lo/- CD43- CD5-). 1-2 x 104 cells for each cell population were sorted directly into 0.5 mL Trizol LS (Life Technologies).

Amplicon rescued multiplex PCR

Request a detailed protocol

RNA was extracted according to the protocol provided by Trizol LS (Life Technologies). RT-PCR reactions were conducted using a set of sequence specific primers covering almost all of mouse VH genes (forward primers) and constant CH primers covering all of isotypes (reverse primers). Illumina paired-end sequencing communal primer B is linked to each forward VH primer. Illumina paired-end sequencing communal primer A and a barcode sequence of 6 nucleotides are linked to each reverse CH primers. In brief, cDNA was reverse transcribed from total RNA sample using mixture of forward VH and reverse CH primers and reagents from the OneStep RT-PCR kit (Qiagen, Valencia, CA). The first round of PCR was performed at: 50°C, 40 minutes; 95°C, 15 min; 94°C, 30 s, 58°C, 2 min, 72°C, 30 s, for 15 cycles; 94°C, 30 s, 72°C, 2 min, for 10 cycles; 72°C, 10 min. After the first round of PCR, primers were removed by Exonuclease I digestion at 37°C for 30 min (New England Biolabs, lpswich, MA). Then 2 μL of the first-round PCR products were used as templates for the second round of amplification using communal A and B primers and reagents from the Multiplex PCR kit (Qiagen). The second round PCR was performed as: 95°C, 15 min; 94°C, 30 s, 55°C, 30 s, 72°C, 30 s, for 40 cycles; 72°C, 5 min. About 400bp long PCR products were run on 2% agarose gels and purified using a gel extraction kit (Qiagen). The IgH libraries were pooled and sequenced with Illumina MiSeq pair-end read-length platform. The output of IgH sequence covers CDR2, CDR3 and the beginning of the constant region. The sequence information for all primers used for the library preparation can be found in US Patent Office (US9012148).

Sequence analysis

Request a detailed protocol

Sequence reads were de-multiplexed according to barcode sequences at the 5’ end of reads from the IgH constant region. Reads were then trimmed according to their base qualities with a 2-base sliding window, if either quality value in this window is lower than 20, this sequence stretches from the window to 3’ end were trimmed out from the original read. Trimmed pair-end reads were joined together through overlapping alignment with a modified Needleman-Wunsch algorithm. If paired forward and reverse reads in the overlapping region were not perfectly matched, both forward and reverse reads were thrown out without further consideration. The merged reads were mapped using a Smith-Waterman algorithm to germline V, D, J and C reference sequences downloaded from the IMGT web site (Lefranc, 2003). To define the CDR3 region, the position of CDR3 boundaries of reference sequences from the IMGT database were migrated onto reads through mapping results and the resulting CDR3 regions were extracted and translated into amino acids.

Artifacts removal

Request a detailed protocol

C57BL/6J mouse VH reference sequences were pair-wise aligned with a Smith-Waterman algorithm. Two VH reference sequences are considered related if the aligned region between them is > 200bp matched and < 6 mismatches. Two sequence reads were considered related if the best mapped VH sequences are related and the CDR3 segments have less than 1 mismatch. If two sequences are related and the frequency of the minor one is less than 5% of the dominant one, the minor one is removed from further consideration. In addition, single copy CDR3s are removed from further consideration.

To allow multiplexing of multiple samples in a single sequence run, CH primers were linked with barcodes containing 6 different nucleotides. The barcode CH primers were used in a first round RT-PCR. To compensate for potential in chemical synthetic, PCR and/or sequencing error, barcodes were designed with a Hamming distance ≥3. Given that the chemical synthetic error is roughly 5% per position, there is about a 1/8000 chance that one barcode is mistakenly synthesized as another barcode. For a CDR3 with n occurrences in one sample and the same CDR3 (nucleotide sequence) with N occurrences in another sample in the same sequencing run, we calculated the probability that such a CDR3 would occur n or more times if it were due to cross-contamination, using the following formula P = 1 - k=1n-1 e-λ · λkk! where λ is the expected number of errors given N reads and is computed by λ = N · μ and μ is the cross-contamination rate which is preset as 1/8000. CDR3s that yielded p<0.001 were considered highly unlikely to be due to cross-contamination. Sequences were obtained for 60 separately sorted cell populations (details for each population are in Table 1).

CDR3 tree map analysis

Request a detailed protocol

To draw the IgH CDR3 tree-map for each sequence sample, the entire rectangle was divided: 1st into a set of rectangles with each rectangle corresponding to a distinct VH gene segment; 2nd into a set of V-J rectangles with each rectangle corresponding to a distinct V-J; and 3rd into a set of V-J-CDR3 rectangles with each rectangle representing a distinct V-J-CDR3 combination. The rectangles are ordered based on area from largest at the bottom right to smallest at the top left. The size of an individual rectangle is proportional to the relative frequency for each V-J-CDR3 combination sequence. In order to distinguish neighboring rectangles, corners of each rectangle are rounded and each rectangles are colored randomly. Therefore, each rectangle drawn in the map represents an individual CDR3 nucleotide sequence.

CDR3 sequence diversity (D50) measurement

Request a detailed protocol

D50 is a measurement of the diversity of an immune repertoire of J individuals (total number of CDR3s) composed of S distinct CDR3s in a ranked dominance configuration, where r1 is the abundance of the most abundant CDR3, r2 is the abundance of the second most abundant CDR3, and so on. C is the minimum number of distinct CDR3s with > = 50% total sequencing reads. D50 is given by

Assume that r1r2  ri  ri+1  rss , i=1s ri=Jif i=1cri  J/2 and i=1C-1ri< J/2D50 = CS × 100

Mutation analysis

Request a detailed protocol

The forward VH primers used to amplify expressed IgH genes are located at the IgH framework region 2. To avoid primers interfering with the mutation analysis, the variable region stretching from the beginning of the CDR2 to the beginning to the CDR3 was examined for mismatches between the sequence read and the best-aligned germline reference sequence. To eliminate the impact of sequencing error on this calculation, only sequence reads with more than 4 copies were included in the mutation calculation.

Quantification of the diversities of V(D)J recombination events for a given CDR3 peptide

Request a detailed protocol

For this measurement, we introduce an entropy value as the index of diversity level. Assuming a distinct CDR3 peptide sequence X in a sample is derived from n number of distinct V(D)J recombinations (nucleotide) with each frequency as P1, P2, … Pn respectively, the entropy for X (Ex) is then calculated as: Ex = -i=1n Pi log2 Pi.

For a sample, after computing entropy values for each distinct peptide CDR3 fragments, the E values for distinct peptide CDR3 fragments are categorized into four ranges: [0, 0.5), [0.5, 1.5), [1.5, 2.5) and [2.5, + ∞). The higher the entropy value, the more diverse the V(D)J recombinations for a given CDR3 peptide.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
    Sequence homologies, n sequence insertion and JH gene utilization in VHDJH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 b cell and b-CLL progenitor generation
    1. H Gu
    2. I Förster
    3. K Rajewsky
    (1990)
    The EMBO Journal 9:2133–2140.
  17. 17
    A single VH gene is utilized predominantly in anti-BrMRBC hybridomas derived from purified Ly-1 B cells. definition of the VH11 family
    1. RR Hardy
    2. CE Carmack
    3. SA Shinton
    4. RJ Riblet
    5. K Hayakawa
    (1989)
    Journal of Immunology  142:3643–3651.
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
    An unbiased analysis of V(H)-D-J(H) sequences from B-1a, B-1b, and conventional B cells
    1. AB Kantor
    2. CE Merrill
    3. LA Herzenberg
    4. JL Hillson
    (1997)
    Journal of Immunology  158:1175–1186.
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
  50. 50
  51. 51
    B-1a, B-1b and B-2 B cells display unique VHDJH repertoires formed at different stages of ontogeny and under different selection pressures
    1. UC Tornberg
    2. D Holmberg
    (1995)
    The EMBO Journal 14:1680–1689.
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58

Decision letter

  1. Satyajit Rath
    Reviewing Editor; National Institute of Immunology, India

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires" for peer review at eLife. Your submission has been favorably evaluated by Tadatsugu Taniguchi (Senior editor) and four reviewers, one of whom is a member of our Board of Reviewing Editors. One of the reviewers, Harry Schroeder, has agreed to share his identity.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary:

The manuscript compares the IgH repertoires of mouse B-1a and conventional B cells from postnatal day 2 to adulthood using Hi-D FACS sorting, multiplex PCR, and deep sequencing. The data provide a detailed view of the global B-1a IgH repertoire diversity, and demonstrate its continuing generation and selection during the animal's lifetime. Remarkably, the data show that B-1a B cells, like conventional B cells, undergo AID-mediated somatic hypermutation (SHM) and Ig class switch recombination (CSR), yet, microbiota play no substantive role in either repertoire selection, SHM or CSR in B-1a B cells in contrast to conventional B cells. The manuscript thus represents a substantial contribution to B-1a B cell biology.

Essential revisions:

1) The manuscript treats B-1a B cells as originating in the neonatal spleen, but makes no effort to examine the IgH repertoire in immature B cells of the fetal liver (as distinct from bone marrow) vis-a-vis the later B-1a repertoire. Both positive and negative results from such a comparison would be informative.

2) The observation that the IgH repertoire of B-1a cells in adult mice contain a high frequency of TdT-dependent N nucleotide additions suggests that a large fraction of B-1a cells in adult mice are of postnatal and not fetal origin. However, this observation is consistent either with self-renewal of the cells that were generated during the first weeks after birth or with continuous output from the bone marrow, possibilities that cannot be discriminated based on the presented data. The interpretation in the manuscript suggesting that postnatal de novo B-1a B cell development only occurs during the first few weeks of life but not thereafter thus needs to be appropriately modified. In fact, the striking finding that B-1a cells in adult animals show a very high frequency of postnatal N nucleotide addition requires highlighting.

3) The manuscript argues that the germ-free mouse data indicate that there is an endogenous antigen-driven selection program at work, but does not rule out food antigens as sources for this selection. In the absence of data from antigen-free mice, this interpretation would best be appropriately modified as showing an absence of a role for microbiota rather than a definitive role for 'endogenous' antigens alone.

4) In the pairwise sharing analysis of IgH CDR3 sequences comparing various B cell subsets of individual mice (Figure 2B), splenic B-1a cells from very young mice (day 2-7) show very high CDR3 sharing. However, since 'each dot represents the data for a sample from an individual mouse except for the day 2 splenic B-1a data, which are derived from sorted cells pooled from 8 mice', it is conceivable that the very high CDR3 sequence sharing is an artifact of comparing samples of 8 mice with each other instead of individual mice like for the other developmental time points. This needs to be clarified, with exclusion of the data points of the 2-7-day-old mice if necessary. Also, the measurement of the CDR3 sequence sharing and the exact nature of the values plotted should be better explained in the text.

5) The nomenclature of VH genes used throughout the manuscript is undefined, and needs to be clarified.

6) VH11-2/Vk9-128 and VH12-3/Vk4-91 are the two most dominant PtC-binding BCRs in the IgH repertoire of B-1a cells. The authors discuss only the VH11-2 specificity, and V(D)J rearrangements involving VH12-3 segment are not even listed among conserved CDR3 sequences (Table 2). It would be useful to comment on this and clarify if this is related to technical issues and limitations.

7) A conclusion in the Results section that '...since SHM in splenic B-1a IgVH initiates later and progresses with age, these findings suggest that peritoneal B-1a in older animals are largely derived from B-1a that have migrated from the spleen when the animals were younger' while a tenable and interesting hypothesis, belongs in the Discussion, since other scenarios remain possible.

8) While germ-free mouse data are shown all along, they are only addressed at the end. This is confusing and reorganization would be useful.

9) The tree-map plots shown in Figure 1, Figure 5 and Figure 9 highlight the repertoire biases in B-1a cells in a very intuitive way. However, with randomly assigned colors, it is impossible to trace the same specificity even across panels of the same figure – yet this is crucial for following the story. It would be advisable to add numbers to the symbols of the most highly reoccurring CDR3 sequences and correlate the numbers with the actual VH gene segments at the bottom of the figure.

10) Figure 3 requires radical revisions to reduce complexity by retaining key components of FO and B-1a B cell comparisons in the main figure and moving some components to supplementary data, rectifying invisible X-axis labels and VH gene labels. Also, the fact that the data in this figure are presented 'as the normalized distribution, which counts the value of each distinct CDR3 nucleotide sequence expressing a given VH gene as one, no matter how many of this sequence are detected' needs to be included in the text as well for the sake of clarity.

11) Figure 7 needs restructuring so that its panel order follows the text. Also, it is not clear where the graphical representation of B-1a cells from AID-null mice, mentioned in the text, is shown in this figure (Figure 7E).

12) Generally, figure contents need to be comprehensively edited for readability. Similarly, the text needs proofreading as well. The Discussion could also use editing to avoid repetitions and digressions into peripheral issues such as the evolutionary origins of B-1a B cell lineage, which are not directly addressed by the present data.

https://doi.org/10.7554/eLife.09083.032

Author response

1) The manuscript treats B-1a B cells as originating in the neonatal spleen, but makes no effort to examine the IgH repertoire in immature B cells of the fetal liver (as distinct from bone marrow) vis-a-vis the later B-1a repertoire. Both positive and negative results from such a comparison would be informative.

The reviewers, we believe, are referring to our statement in the manuscript Abstract and Introduction that “we track B-1a cells from their origin in neonatal spleen...” We appreciate this comment by the reviewers since the wording we used is not what we meant. We apologize for this confusion. The correct wording is “we track B-1a cells from their early appearance in neonatal spleen”.

Actually, our laboratory is working quite intensively on the origin of B-1a cells and has shown that they arise from progenitors distinct from the progenitors that give rise to B-2 cells. We are now nearing publication of a paper tracing the B-1a progenitors in the embryo to a considerably earlier time-point than neonatal spleen.

In addition, we agree with the reviewers that it would be quite informative to compare the repertoire expressed by the immature (not fully developed) B cells in the fetal liver with the repertoire of the mature B-1a in neonates and adults. However, our methods do not give us this option. That is, we are not sequencing genomic DNA, which could be recovered equally from mature and immature B cells. Instead, we are sequencing mature IgH message, which is expected to be present, if at all, only in a very small proportion of immature B cells, whether in adult bone marrow or fetal liver.

Basically, there are too few mature IgM + B cells in fetal liver for reliable IgH sequencing with current methods. In our study, we harvested late fetal liver just prior to birth (E19) and examined the B cell development by FACS. We found that the IgM + B cells (i.e., those that have completed IgH V(D)J recombination) are detectable only at very low frequencies (0.6% of CD19 + total B cells) of which only 20% express the mature B-1a CD43 + CD5 + phenotype (see FACS plots at right). The IgM + B cell frequencies are even lower in E18 fetal liver (0.2% of CD19 + B cells; data not shown). These numbers are too low for us to obtain enough material for sequencing from a feasible number of embryos.

We will be happy to include the Figure in our paper as Figure 1—figure supplement 1 if the editors wish. It responds to a question that we feel many readers may also have.

2) The observation that the IgH repertoire of B-1a cells in adult mice contain a high frequency of TdT-dependent N nucleotide additions suggests that a large fraction of B-1a cells in adult mice are of postnatal and not fetal origin. However, this observation is consistent either with self-renewal of the cells that were generated during the first weeks after birth or with continuous output from the bone marrow, possibilities that cannot be discriminated based on the presented data. The interpretation in the manuscript suggesting that postnatal de novo B-1a B cell development only occurs during the first few weeks of life but not thereafter thus needs to be appropriately modified. In fact, the striking finding that B-1a cells in adult animals show a very high frequency of postnatal N nucleotide addition requires highlighting.

We completely agree with the reviewers that based on the information we provided, it is hard to distinguish the possibilities between the self-renewal of B-1a cells and their de novo generation from the bone marrow. This uncertainty, however, is due to our failure to clearly state in the manuscript that the N-nucleotide distribution profile that we present is based on normalized data. We use normalized data because it specifically minimizes the impact of B-1a self-replenishment on the overall N-insertion distribution profile, and hence weights the repertoire for de novo generated sequences. Thus, we score each distinct IgH sequence containing the indicated N nucleotide insertions as a single sequence, regardless how many times this sequence was detected. We have clarified this part in the text of the Result section “The B-1a IgH repertoire integrates rearrangement from de novo B-1a development that occur mainly during the first a few weeks of life” of the manuscript to make the data more understandable.

In addition, as the reviewers requested, our modified manuscript now highlights our finding that B-1a cells in adult animals show a very high frequency of N nucleotide additions, which indicates that the majority of the B-1a cells are actually generated postnatally after Tdt is expressed.

3) The manuscript argues that the germ-free mouse data indicate that there is an endogenous antigen-driven selection program at work, but does not rule out food antigens as sources for this selection. In the absence of data from antigen-free mice, this interpretation would best be appropriately modified as showing an absence of a role for microbiota rather than a definitive role for 'endogenous' antigens alone.

The reviewers are correct in pointing out that the food antigens cannot be ruled out as the sources for the selection in our studies. However, we believe that self-antigens are likely to be the major driving force, if only because B-1a cells are well-known to produce anti-self antibodies. We agree that food antigens may mimic self, or vice versa. Nevertheless, Occam’s razor suggests that the minimal hypothesis, i.e., stimulation by self-antigen, rather than microbiota, is likely the major influence on the selection of the B-1a repertoire. We have revised our manuscript to reflect this point.

4) In the pairwise sharing analysis of IgH CDR3 sequences comparing various B cell subsets of individual mice (Figure 2B), splenic B-1a cells from very young mice (day 2-7) show very high CDR3 sharing. However, since 'each dot represents the data for a sample from an individual mouse except for the day 2 splenic B-1a data, which are derived from sorted cells pooled from 8 mice', it is conceivable that the very high CDR3 sequence sharing is an artifact of comparing samples of 8 mice with each other instead of individual mice like for the other developmental time points. This needs to be clarified, with exclusion of the data points of the 2-7-day-old mice if necessary. Also, the measurement of the CDR3 sequence sharing and the exact nature of the values plotted should be better explained in the text.

In responding to the concern raised by the reviewers, we have excluded the data of the day 2 sample from the CDR3 pairwise sharing analysis. The amended Figure 2B now shows data from which the day 2 sample is excluded. In addition, as per the reviewer’s suggestion, we have now added a detailed explanation for CDR3 pair-wise sharing analysis.

5) The nomenclature of VH genes used throughout the manuscript is undefined, and needs to be clarified.

The nomenclature of IgH V, D, J genes used in this study is based on IMGT IgH gene nomenclature. We have made this statement in the paper to clarify this issue.

6) VH11-2/Vk9-128 and VH12-3/Vk4-91 are the two most dominant PtC-binding BCRs in the IgH repertoire of B-1a cells. The authors discuss only the VH11-2 specificity, and V(D)J rearrangements involving VH12-3 segment are not even listed among conserved CDR3 sequences (Table 2). It would be useful to comment on this and clarify if this is related to technical issues and limitations.

As the reviewers correctly pointed out, the sequence data we presented do not contain VH12-3 encoded sequences. The absence of these VH12-3-encoded sequences is due to a technical limitation of the methods we used. In essence, when we designed the primers for VH gene families about three years ago, we inappropriately overlooked the VH12 reference gene and only included a primer for VH12-1 gene. Since VH12-1 and VH12-3 differ in the primer-aligning region, the VH12-1 primer does not capture VH12-3-encoded sequence and hence VH12-3 encoded sequences are not included our earlier dataset, which includes the bulk of the data we report in the manuscript.

We have corrected this problem by adding VH12-3 primer in our new primer sets. While we cannot retroactively correct our initial oversight, we have now noted the problem in the text of the manuscript. We believe that this omission of VH12-3 is not fatal for the data we present. However, relevant to this issue, we have recently completed additional sequencing studies in which we compared the sequence data generated by using old version of primer set with the sequence data generated by using new version of primer set that contains the VH12-3 primer. We found that sequence results for the libraries obtained by using old and new version of primers are highly similar. In fact, the most highly represented reads from the paired data sets are identical in sequence and show similar representation order.

Specifically, we sorted two splenic B-1a populations individually from two 4 months old C57B/l6 mice. We extracted RNA from each population and divided the RNA from each into two parts. For one part, we prepared an amplified library using the old primer set and for the other, we prepared an amplified library using the new primer set. We then sequenced these amplified IgH libraries. Analysis of the resultant sequences showed that, regardless of the primer set used (old or new), the sequences obtained from the IgH library are highly similar. In essence, the top 10 highly recurring CDR3 sequences (both peptide and V(D)J recombination) are identical between each pair of libraries (shown in Figure 3-figure supplement 2). As expected, we detected VH12-3 encoded sequences from the splenic B-1a IgH libraries prepared with the new primer sets, and these VH12-3 encoded sequences included several published PtC-binding VH12-3 encode sequences, i.e., AGDYDGYWYFDV (VH12-3 D2-4 J1), AGDRDGYWYFDV (VH12-3 D3-2 J1), AGDRYGYWYFDV (VH12-3 D2-9 J1).

The mutation profiles for the libraries prepared by using either old or new primer sets are also highly similar. In two separate comparisons, we detected identical IgH sequences with identical nucleotides substitutions in each library (see Figure 7-figure supplement 3).

We hope the editor will agree that while there is a clear need to collect VH12-3 data in future studies, the absence of this sequence in our current data is not a fatal flaw. To resolve this issue for the journal readers, we would be pleased to include the comparison data presented here in the supplement data of the manuscript. Importantly, these comparisons also show the reliability of the technology we have developed for this study.

7) A conclusion in the Results section that '...since SHM in splenic B-1a IgVH initiates later and progresses with age, these findings suggest that peritoneal B-1a in older animals are largely derived from B-1a that have migrated from the spleen when the animals were younger' while a tenable and interesting hypothesis, belongs in the Discussion, since other scenarios remain possible.

We agree with reviewers and have moved this part to the Discussion section.

8) While germ-free mouse data are shown all along, they are only addressed at the end. This is confusing and reorganization would be useful.

We understand the reviewer’s concern about our decision to show the germ-free data throughout the Results section but to put off discussion of these germ-free data until the last section of the Results, in which we explicitly focus on addressing the germ-free data. We did this to efficiently utilize the space available in the manuscript. If the editor wants to relax this restriction, we will be please to discuss this material in the text when it appears. As an alternate solution, we have added the statement “The data for germ-free animals is discussed at the end of the Results section” to the current table and figure legends.

9) The tree-map plots shown in Figure 1, Figure 5 and Figure 9 highlight the repertoire biases in B-1a cells in a very intuitive way. However, with randomly assigned colors, it is impossible to trace the same specificity even across panels of the same figure – yet this is crucial for following the story. It would be advisable to add numbers to the symbols of the most highly reoccurring CDR3 sequences and correlate the numbers with the actual VH gene segments at the bottom of the figure.

Following the reviewers’ suggestion, we have revised Figure 1 by adding numbers to identify the top four highly recurring CDR3 sequences that are shared in splenic B-1a and peritoneal B-1a CDR3 tree-map plots. For Figure 5A and Figure 9A, in which there are many tree-map plots showing recurring CDR3 sequences, we added a supplementary table in which we list the top ten CDR3 sequences for the CDR3 tree-map plot that show recurring CDR3 sequences in each figure.

10) Figure 3 requires radical revisions to reduce complexity by retaining key components of FO and B-1a B cell comparisons in the main figure and moving some components to supplementary data, rectifying invisible X-axis labels and VH gene labels. Also, the fact that the data in this figure are presented 'as the normalized distribution, which counts the value of each distinct CDR3 nucleotide sequence expressing a given VH gene as one, no matter how many of this sequence are detected' needs to be included in the text as well for the sake of clarity.

We have re-done Figure 3 following the reviewers’ instruction. To better match the text, we show the comparison between MZB and splenic B-1a cells from 2-6 months old mice. In addition, as reviewers requested, we also now include the requisite statement in the text as well as in the figure legend.

11) Figure 7 needs restructuring so that its panel order follows the text. Also, it is not clear where the graphical representation of B-1a cells from AID-null mice, mentioned in the text, is shown in this figure (Figure 7E).

Following the reviewers’ advice, the panels in Figure 7 have been re-organized to correspond more closely to the text.

In Figure 7A and E, we have shown data for each B cell sample obtained from 4-5 months old AID knockout mice. As we state in the Figure 7 legend, there are a total of seven B cell samples for which data are shown. These include four splenic B-1a samples, each from an individual mouse; one peritoneal B-1a sample; one follicular B and one peritoneal B-2 samples. We have clarified the text to match.

12) Generally, figure contents need to be comprehensively edited for readability. Similarly, the text needs proofreading as well. The Discussion could also use editing to avoid repetitions and digressions into peripheral issues such as the evolutionary origins of B-1a B cell lineage, which are not directly addressed by the present data.

Following the reviewers’ suggestion, we have comprehensively edited our figure contents and text to make them clearer and more understandable. We also removed the repetitions in the Discussion section. Our sequence findings are consistent with developmental data that we and other labs have presented showing that B-1a and B-2 cells belong to evolutionarily distinct B lineages. While we agree with reviewers that our data do not directly bear on this lineage distinction, we believe that the drastic differences between the repertoire-defining mechanisms in B-1a and B-2 is consistent with, and sheds light on, the B-1a/B-2 lineage split. We agree with the reviewers that the “lineage” discussion in our manuscript should be shortened and have modified the text of the Discussion in the manuscript.

https://doi.org/10.7554/eLife.09083.033

Article and author information

Author details

  1. Yang Yang

    Genetics Department, Stanford University, Stanford, United States
    Contribution
    YY, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Chunlin Wang
    For correspondence
    yang71@stanford.edu
    Competing interests
    No competing interests declared.
  2. Chunlin Wang

    HudsonAlpha Institute for Biotechnology, Huntsville, United States
    Contribution
    CW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Yang Yang
    Competing interests
    CW and JH: Co-founder of iRepertoire.
  3. Qunying Yang

    HudsonAlpha Institute for Biotechnology, Huntsville, United States
    Contribution
    QY, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  4. Aaron B Kantor

    Genetics Department, Stanford University, Stanford, United States
    Contribution
    ABK, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  5. Hiutung Chu

    Biology and Biological Engineering Department, California Institute of Technology, Pasadena, United States
    Contribution
    HC, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Competing interests
    No competing interests declared.
  6. Eliver EB Ghosn

    Genetics Department, Stanford University, Stanford, United States
    Contribution
    EEBG, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  7. Guang Qin

    Genetics Department, Stanford University, Stanford, United States
    Contribution
    GQ, Acquisition of data, Analysis and interpretation of data
    Competing interests
    No competing interests declared.
  8. Sarkis K Mazmanian

    Biology and Biological Engineering Department, California Institute of Technology, Pasadena, United States
    Contribution
    SKM, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Competing interests
    No competing interests declared.
  9. Jian Han

    HudsonAlpha Institute for Biotechnology, Huntsville, United States
    Contribution
    JH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    jhan@irepertoire.com
    Competing interests
    CW and JH: Co-founder of iRepertoire.
  10. Leonore A Herzenberg

    Genetics Department, Stanford University, Stanford, United States
    Contribution
    LAH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    leeherz@stanford.edu
    Competing interests
    No competing interests declared.

Funding

National Institutes of Health (R01-Al1076434)

  • Leonore A Herzenberg

National Institutes of (R01-DK078938)

  • Sarkis K Mazmanian

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We acknowledge Megan Phillips, Jeffrey Waters, Jasmine Sosa, John Mantovani and the Stanford Shared FACS Facility for excellent assistance. We are grateful to Dr. Michel Nussenzweig (Rockefeller University) for generously providing Aid-/- mice. Studies are supported by the US National Institutes of Health Grants R01-AI1076434 (LAH), R01-DK078938 (SKM) and by the HudsonAlpha Institute for Biotechnology and iRepertoire (HJ).

Reviewing Editor

  1. Satyajit Rath, National Institute of Immunology, India

Publication history

  1. Received: May 30, 2015
  2. Accepted: September 30, 2015
  3. Accepted Manuscript published: September 30, 2015 (version 1)
  4. Version of Record published: November 30, 2015 (version 2)
  5. Version of Record updated: August 1, 2016 (version 3)

Copyright

© 2015, Yang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,231
    Page views
  • 909
    Downloads
  • 51
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Cell Biology
    2. Genetics and Genomics
    Maja Kneissig et al.
    Research Article Updated
    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Adriana E Golding et al.
    Research Article Updated