We sorted splenic and peritoneal B-1a (dump^- CD19⁺ CD93^-IgM^hi IgD^lo/- CD21^-/lo CD23^- CD43⁺ CD5⁺); splenic FOB and peritoneal B-2 (dump^- CD19⁺ CD93^- IgM^lo IgD^hi CD23⁺ CD43^- CD5^-); and splenic MZB (dump^- CD19⁺ CD93^- IgM^hi IgD^lo/- CD21^hi CD23^lo/- 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).

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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 V_H11-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.

Figure 2

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We quantified the frequency of IgH sequences expressing individual V_H gene for each sorted B cell sample and then compared the V_H 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 V_H gene usage profile, we collected normalized data, in which we scored each distinct IgH CDR3 nucleotide sequence expressing a given V_H gene as one, no matter how many times this sequence was detected.

Our approach enables detection of Ig transcripts expressing about 100 different V_H genes that belong to 14 V_H families (Figure 3). B-1a cells express all of these detected V_H genes (Figure 3A), contrasting with earlier impressions, based largely on hybridomas sequences from fetal and neonatal mice (Malynn et al., 1990), that V_H usage in the B-1a repertoire is very restricted. However, despite the broad V_H usage, certain V_H genes, notably V10-1 (DNA4), V6-6 (J606), V11-2 (V_H11) 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).

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Similar to MZB cells, splenic FOB and peritoneal B-2 cells show lower frequency in expressing these B-1a favored V_H genes, i.e., V6-6 (J606), V11-2 (V_H11) and V2-6-8 (Q52) (Figure 3—figure supplement 1B–C). Conversely, these B subsets tend to preferentially use the largest V_H family, V1 (J558), located distal to D_H and J_H 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 V_H 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 V_H genes.

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).

Figure 4

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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). V_H 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 V_H gene usage.

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).

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The recurrent V(D)J sequences include V_H11-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.

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).

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.

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.

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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 V_H 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.

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).

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In contrast, mutations are minimal in IgV_H 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 IgV_H initiates after weaning and the frequency of mutated IgH transcripts increases with age. Thus, mutations are minimally detectable in the IgV_H 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 IgV_H 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⁺ CD38^lo CD95^hi) 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 IgV_H is an accumulative process.

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 IgV_H 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).

Figure 8

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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).

The splenic B-1a cells that express class-switched Ig still express IgM on the surface, since cells were sorted as IgM^hi IgD^lo/- 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 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 V_H 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).

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Further, hypermutation occurs equally in the splenic B-1a IgV_H 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).

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 IgV_H 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 IgV_H 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 V_H families and all constant (C_H) 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 V_H and C_H 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 V_H 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 IgV_H 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 IgV_H 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 IgV_H 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.

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.

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^- IgM^hi IgD^-/lo CD21^-/lo CD23^- CD43⁺ CD5⁺); splenic FOB and peritoneal B-2 cells (dump^- CD19⁺ CD93^- IgM^lo IgD^hi CD23⁺ CD43^- CD5^-); splenic MZB cells (dump^- CD19⁺ CD93^- IgM^hi IgD^-/lo CD21^hi CD23^lo/- CD43^- CD5^-). 1-2 x 10⁴ cells for each cell population were sorted directly into 0.5 mL Trizol LS (Life Technologies).

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 V_H genes (forward primers) and constant C_H primers covering all of isotypes (reverse primers). Illumina paired-end sequencing communal primer B is linked to each forward V_H primer. Illumina paired-end sequencing communal primer A and a barcode sequence of 6 nucleotides are linked to each reverse C_H primers. In brief, cDNA was reverse transcribed from total RNA sample using mixture of forward V_H and reverse C_H 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 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.

C57BL/6J mouse V_H reference sequences were pair-wise aligned with a Smith-Waterman algorithm. Two V_H 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 V_H 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, C_H primers were linked with barcodes containing 6 different nucleotides. The barcode C_H 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 $P = 1 - \sum _{k=1}^{n-1} \frac{e^{-\lambda } · {\lambda }^k}{k!}$ where λ is the expected number of errors given N reads and is computed by $\lambda = N · \mu$ 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).

To draw the IgH CDR3 tree-map for each sequence sample, the entire rectangle was divided: 1^st into a set of rectangles with each rectangle corresponding to a distinct V_H gene segment; 2^nd into a set of V-J rectangles with each rectangle corresponding to a distinct V-J; and 3^rd 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.

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

The forward V_H 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.

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, … P_n respectively, the entropy for X (E_x) is then calculated as: $E_x = -\sum _{i=1}^n P_i {\log }_2 P_i$.

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.

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

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).

© 2015, Yang et al.

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