1. Cell Biology
  2. Immunology and Inflammation

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
https://doi.org/10.7554/eLife.09083
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Cite this article
  1. Yang Yang
  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
  10. Leonore A Herzenberg
(2015)
Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires
eLife 4:e09083.
https://doi.org/10.7554/eLife.09083
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9 figures and 7 tables

Figures

Figure 1 with 3 supplements
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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
Figure 1—figure supplement 1
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FACS plots showing CD43+ CD5+ IgM+ B-1a cells in E19 fetal liver.

Live dump- (CD11b- CD11c- Gr-1- F4/80- CD3- TCRαβ-) CD45+ CD19+ cells from E19 fetal liver of C57Bl/6 mouse were gated to show IgM and IgD expression. The boundary for IgM expression was determined from fluorescence-minus-one (FMO) control in which fluorescently labeled anti-mouse IgM antibodies are omitted from the staining sets (right plot). IgM+ IgD- cells were further gated to reveal CD43+ CD5+ B-1a cells.

https://doi.org/10.7554/eLife.09083.004
Figure 1—figure supplement 2
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Recurrent VH11-encoded PtC-binding V(D)J sequences.

(A-C) lists three VH11-encoded PtC-binding V(D)J sequences. In each plot, the first line of nucleotides is the obtained sequence read while the second line refers the germline reference sequence. The underlined nucleotides are CDR2 and CDR3.

https://doi.org/10.7554/eLife.09083.005
Figure 1—figure supplement 3
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CD11b expression on peritoneal B-1a (CD5+) and B-1b (CD5-) is roughly 100-fold lower than the CD11b expression on myeloid cells.

Live cells from C57Bl/6 peritoneal cavity were gated to show CD19 and CD11b expression. The CD19 + B cells and CD11bhi myeloid cells were shown. The CD19+ B cells were gated to reveal CD5 and CD11b expression. CD11b+ B-1a and CD11b + B-1b cells were gated based on FMO control staining where anti-CD11b antibody was omitted in the staining.

https://doi.org/10.7554/eLife.09083.006
Figure 2
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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 (pB→A) 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
Figure 3 with 2 supplements
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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
Figure 3—figure supplement 1
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VH gene usage profile pair-wise comparison of B cell groups.

The colors shown at the bottom right distinguish the B cell groups (n = 4-9). VH genes showing the statistically significant differences (Welch’s t-test p<0.05) between two groups are listed on the bottom (A' to F') and also highlighted with asterisks in each plot. The data for germ-free (GF) animals is discussed at the end of the Result section.

https://doi.org/10.7554/eLife.09083.010
Figure 3—figure supplement 2
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Almost identical top 10 highly recurring CDR3 sequences are detected for splenic B-1a IgH libraries obtained either with the old or new primer set.

We sorted two splenic B-1a populations individually from two 4 month old C57BL/6J mice. We extracted RNA from each population and divided each RNA 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 the sequences obtained from the IgH libraries are highly similar, regardless of the primers used (old or new). In essence, the top 10 highly recurring CDR3 sequences (both peptide and V(D)J recombination) are almost identical and show similar representation order between each pair of libraries. As expected, we detected VH12-3 encoded sequences from the splenic B-1a IgH libraries prepared with the new primer set, and these VH12-3 encoded sequences included several published PtC-binding VH12-3 encode sequences, i.e., AGDYDGYWYFDV (VH12-3D2-4J1), AGDRDGYWYFDV (VH12-3D3-2J1), AGDRYGYWYFDV (VH12-3 D2-9 J1).

https://doi.org/10.7554/eLife.09083.011
Figure 4
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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
Figure 5 with 1 supplement
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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
Figure 5—figure supplement 1
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The peritoneal B-1a IgH repertoire is increasingly restricted during ontogeny.

IgH CDR3 tree map plots for peritoneal B-1a samples from different ontogenic stages. Each plot represents the data for a sample from an age-defined individual mouse, except for the 2 week, 3 week and 1 month samples, which are obtained from cells pooled from several mice. Recurrent sequences are visualized as larger contiguously-colored rectangles in each plot.

https://doi.org/10.7554/eLife.09083.014
Figure 6 with 1 supplement
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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
Figure 6—figure supplement 1
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Distinct V(D)J sequences encoding the same CDR3 peptide differ in VH usage.

Plots showing an example, in which four different V(D)J sequences expressed by the 5 day splenic B-1a sample all encode the same CDR3 (ANWDY). Red line denotes the V(D)J recombination. CDR2 sequences are highlighted with the blue doted lined box. The V(D)J recombination (V14-3 D4-1 J2) shown in the bottom plot is the predominant V(D)J for ANWDY identified in adult splenic B-1a IgH repertoire.

https://doi.org/10.7554/eLife.09083.019
Figure 7 with 3 supplements
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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
Figure 7—figure supplement 1
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Splenic B-1a cells do not contain cells expressing GC phenotype.

FACS analysis showing of live dump- CD19 + CD93 (AA41)- IgMHi IgD-/lo CD23- CD21-/lo B cells from spleen of 5 month old C57BL6/J mouse were gated to reveal CD43 + CD5 + B-1a cells, which were further gated to reveal GL7, CD38 and CD95 expression. GC B cells are GL7 + CD38-/lo CD95hi. The boundary for CD5 (rightmost middle plot) and GL7 (rightmost bottom plot) expression were determined from FMO controls in which fluorescently labeled anti-mouse CD5 or anti-mouse GL7 antibodies are omitted from the staining sets.

https://doi.org/10.7554/eLife.09083.021
Figure 7—figure supplement 2
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Percentage of sequences containing > = 4 nucleotides changes for each B cell group.

A, sB-1a (2-7d); B, sB-1a (2W-1M); C, sB-1a (2M); D, sB-1a (4-6M); E, sB-1a (GF, 4M); F, B cells (AIDKO, 4-5M); G, pB-1a (2-6M); H, FOB, pB-2 (2-5M); I, MZB (1-5M). Each dot represents the data for an individual B cell sample, n = 3-8.

https://doi.org/10.7554/eLife.09083.022
Figure 7—figure supplement 3
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Identical V(D)J recombination sequences containing identical mutated nucleotides are detected in sequence data sets for IgH libraries obtained by using either old or new primer set.

We sorted two splenic B-1a populations individually from two 4 month old C57BL/6J mice. We extracted RNA from each population and divided each RNA sample 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 two pair of amplified IgH libraries. In two separate comparisons, we detected identical IgH sequences containing identical nucleotides substitutions in each library. One example is shown from comparing one pair of sequence data sets. Red nucleotides are the mutated bases. Upper line of sequence is the obtained sequence reads and the lower line of sequences is the V, D and J reference sequences.

https://doi.org/10.7554/eLife.09083.023
Figure 8
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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
Figure 9 with 1 supplement
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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
Figure 9—figure supplement 1
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Normal splenic B-1a compartment in GF mice.

(A) FACS plot showing the B-1a population in spleen from SPF or GF mouse. Live dump- CD19 + CD93- IgMhi IgDlo CD23lo/- CD21- cells were gated to reveal CD5 + CD43 + B-1a cells. (B) Absolute number of splenic B-1a cells in GF and SPF mice. Each dot represents the data from an individual mouse. There is no significant difference shown between two groups.

https://doi.org/10.7554/eLife.09083.028

Tables

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
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.

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.

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+.

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.

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.

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).

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  1. Yang Yang
  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
  10. Leonore A Herzenberg
(2015)
Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires
eLife 4:e09083.
https://doi.org/10.7554/eLife.09083