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*For correspondence:

jhan@

irepertoire.com (JH); leeherz@

stanford.edu (LAH)

†

These authors contributed

equally to this work

Competing interest:

See

page 29

Funding:

See page 29

Received:

30 June 2015

Accepted:

30 September 2015

Published:

30 September 2015

Reviewing editor:

Satyajit Rath,

National Institute of

Immunology, India

article is distributed under the

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Creative Commons

Attribution License,

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permits unrestricted use and

redistribution provided that the

original author and source are

credited.

Distinct mechanisms define murine B cell

lineage immunoglobulin heavy chain (IgH)

repertoires

Yang Yang

1†

, Chunlin Wang

2†

, Qunying Yang

, Aaron B Kantor

, Hiutung Chu

Eliver EB Ghosn

, Guang Qin

, Sarkis K Mazmanian

, Jian Han

Leonore A Herzenberg

Genetics Department, Stanford University, Stanford, United States;

HudsonAlpha

Institute for Biotechnology, Huntsville, United States;

Biology and Biological

Engineering Department, California Institute of Technology, Pasadena, United

States

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.

DOI: 10.7554/eLife.09083.001

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 Haya-

kawa, 2001

;

Baumgarth, 2011

). Previous studies have shown that B-1a cells are efficiently gener-

ated 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

develop-

ment 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 var-

iable (V), diversity (D) and joining (J) gene segments, nucleotide(s) trimming in the D-J and V-DJ join-

ing site, and, template-dependent (P-addition) and independent (N-addition) nucleotide(s) insertion

at the joined junctions (

Yancopoulos and Alt, 1986

;

Kirkham and Schroeder, 1994

). The V(D)J join-

ing processes define the third IgH complementarity-determining region (CDR3), which often lies at

Yang

etal

. eLife 2015;4:e09083.

DOI: 10.7554/eLife.09083

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RESEARCH ARTICLE

the center of antigen binding site and plays a crucial role in defining antibody specificity and affinity

(

Xu and Davis, 2000

After encountering antigen, “‘naı

ve”’ B cells are activated and can further diversify their primary

antibody repertoire by activation-induced cytidine deaminase (AID)–mediated somatic hypermuta-

tion (SHM), which introduces single or multiple mutations into the IgV regions (

Muramatsu et al.,

2000

;

Wagner 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, 1996

;

Gitlin et al., 2014

). Since the antigen-driven SHM-mediated second-

ary 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 increas-

ingly 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

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.

DOI: 10.7554/eLife.09083.002

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Cell biology

Immunology

7183, Q52 V

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 V

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 peri-

toneal B-1a cells from adult animals (

Gu et al., 1990

), indicating that B-1a cells are continuously gen-

erated after

Tdt

is expressed. Holmberg lab similarly found the low N-region diversity in the adult

Figure 1.

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

DOI: 10.7554/eLife.09083.003

The following figure supplements are available for figure 1:

Figure supplement 1.

FACS plots showing CD43

CD5

IgM

B-1a cells in E19 fetal liver.

DOI: 10.7554/eLife.09083.004

Figure supplement 2.

Recurrent V

11-encoded PtC-binding V(D)J sequences.

DOI: 10.7554/eLife.09083.005

Figure supplement 3.

CD11b expression on peritoneal B-1a (CD5

) and B-1b (CD5

) is roughly 100-fold lower

than the CD11b expression on myeloid cells.

DOI: 10.7554/eLife.09083.006

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Table 1.

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

Sample

Subset

Strain

Age

Condition

Mice

RNT*

RNU*

RPU*

CNT*

CNU*

CPU*

7631

FOB

SPF

single

1006030

151210

65871

903400

21240

20470

13966

FOB

3.5M

SPF

single

150812

31003

24801

130652

14911

14678

8706

FOB

SPF

single

180365

53577

27817

159710

16901

16568

8702

FOB

SPF

single

156681

54195

27728

136101

16951

16649

13967

FOB

AID KO

SPF

single

35967

14623

13203

27726

7187

7133

11161

MZB

SPF

single

33548

19628

12744

25674

6584

6471

10658

MZB

SPF

single

71458

26978

18278

61258

11512

11170

7630

MZB

SPF

single

1032381

139832

62520

932353

20780

19792

8701

MZB

SPF

single

214238

55075

26458

191065

15461

15021

8700

MZB

SPF

single

118863

42310

22794

102894

14517

14180

13338

MZB

single

162754

39930

23611

141646

12939

12605

13343

MZB

single

595780

85497

45820

536072

19266

18480

11163

pB-1a

SPF

pool of 3 mice

45882

11290

5596

41368

3237

3007

10660

pB-1a

SPF

single

222324

17311

8630

207749

3891

3649

13018

pB-1a

SPF

single

808879

36031

14817

753868

4769

4374

7628

pB-1a

SPF

single

1784677

59458

22105

1706235

6601

5848

11160

pB-1a

SPF

pool of 8 mice

65317

14700

7025

58034

4240

3704

10655

pB-1a

SPF

pool of 5 mice

62875

12162

6622

57558

4180

3694

8705

pB-1a

SPF

single

310077

28441

11886

287695

5063

4707

9870

pB-1a

SPF

single

229100

26299

10469

211514

4745

4480

11165

pB-1a

SPF

single

105410

19528

8926

95994

4435

4162

8707

pB-1a

SPF

single

320252

29786

12423

296946

4722

4384

9861

pB-1a

SPF

single

26613

5683

3235

23542

1521

1461

8704

pB-1a

AID KO

SPF

single

264340

33745

14519

245941

6648

6294

10657

pB-2

SPF

single

53953

23059

16883

44986

10084

9923

7629

pB-2

SPF

single

1315663

123472

47337

1238225

16925

16065

13969

pB-2

3.5M

SPF

single

186817

24304

17689

170768

9089

8925

9862

pB-2

SPF

single

22591

13377

8737

17343

4382

4357

13973

pB-2

AID KO

SPF

single

617893

62319

41165

566826

17536

16965

13000

sB-1a

SPF

pool of 8 mice

29439

9542

4925

25369

3148

2758

10651

sB-1a

SPF

single

123360

22472

10838

113161

7453

5976

10659

sB-1a

SPF

single

210055

28140

12411

192662

7307

5812

9866

sB-1a

SPF

single

52986

15600

6864

46580

4595

3837

10652

sB-1a

SPF

single

172875

26437

12545

159304

7683

6365

9865

sB-1a

SPF

single

71309

18446

8775

64241

5482

4941

9868

sB-1a

SPF

single

201813

35069

14473

186227

7847

6843

Table 1 continued on next page

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Table 1 continued

Sample

Subset

Strain

Age

Condition

Mice

RNT*

RNU*

RPU*

CNT*

CNU*

CPU*

10656

sB-1a

SPF

single

369732

39603

19759

342914

9489

9048

13004

sB-1a

SPF

single

185948

27952

13875

168522

7313

7022

7632

sB-1a

SPF

single

1825218

102797

43190

1719246

12428

11144

11168

sB-1a

SPF

single

536603

70201

28829

496671

11948

10913

13005

sB-1a

SPF

single

98017

28331

15001

85489

8820

8207

10654

sB-1a

SPF

single

146560

33814

19697

131091

11995

11451

13970

sB-1a

3.5M

SPF

single

170925

13809

9289

160480

4513

4273

13335

sB-1a

SPF

single

22175

4822

3449

18683

1131

1090

13342

sB-1a

SPF

single

283072

23668

12947

262744

5357

5032

8699

sB-1a

SPF

single

142838

19151

9938

130915

4370

4086

9863

sB-1a

SPF

single

73676

16599

8713

65571

4233

4092

11167

sB-1a

SPF

single

501367

38912

17336

463863

7573

7163

8708

sB-1a

SPF

single

577114

52723

22272

531508

9146

8441

9867

sB-1a

SPF

single

113492

20612

10625

101791

4563

4343

13965

sB-1a

AID KO

SPF

single

177782

16419

12281

164189

6539

6293

13971

sB-1a

AID KO

SPF

single

517141

34159

22031

482543

8966

8395

13968

sB-1a

AID KO

SPF

single

427671

30839

20510

396974

9162

8545

13972

sB-1a

AID KO

SPF

single

706116

36217

23255

660874

9294

8744

13001

sB-1a

single

43507

8734

4855

38947

2318

2249

13002

sB-1a

single

47203

8683

4820

42279

2053

1965

13003

sB-1a

single

213347

22246

11068

197769

4705

4449

13017

sB-1a

single

532250

40497

17375

501908

7019

6398

13337

sB-1a

single

28559

6322

4417

24047

1544

1486

13341

sB-1a

single

388208

28942

14837

360727

5674

5144

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 statistics

RNT*

RNU*

RPU*

CNT*

CNU*

CPU*

Total

1.9E + 07

2.1E + 06

1.1E + 06

1.8E + 07

4.9E + 05

4.7E + 05

Mean

319865

35610

17848

295174

8233

7762

% CV

122

125

DOI: 10.7554/eLife.09083.007

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Immunology

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 peri-

toneal 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 peri-

toneal 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 immortal-

ized cell lines (e.g., hybridomas) or LPS-stimulated B cells had obvious sampling biases. In addition,

earlier studies mainly focused on particular V

families (e.g., J558, 7183), even though the mouse

IgH locus contains over 100 functional V

genes (

Kirkham and Schroeder, 1994

). The introduction

of single cell analyses enabled higher precision and lower bias than the bulk measurements. How-

ever, 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 reper-

toires 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 micro-

biota 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

IgM

IgD

lo/-

CD21

-/lo

CD23

CD43

CD5

); splenic FOB and peritoneal B-2 (dump

CD19

CD93

IgM

IgD

CD23

CD43

CD5

); and

splenic MZB (dump

CD19

CD93

IgM

IgD

lo/-

CD21

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 x10

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

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

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(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

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

11-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 perito-

neal 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 reper-

toire is far more restricted and repetitive than IgH repertoires expressed by FOB and MZB subsets.

Figure 2.

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. (

) 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. (

) 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 (

together with the percentage of CDR3s in sample B that are also in sample 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 10

, 25

, 50

, 75

and 90

percentiles

here and in other figures.

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Table 2.

Top 10 highly recurring CDR3 sequences (peptide and V(D)J recombination) detected in

each of the listed splenic B-1a samples.

sB-1a samples

Top 10 IgH CDR3 sequences

Age

Peptide

V(D)J

11168

2 weeks

ANDY

V1-53 J2

AKHGYDAMDY

V2-9 D2-9 J4

ARRYYGSSYWYFDV

V1-55 D1-1 J1

ANWDY

V1-53 D4-1 J2

MRYSNYWYFDV

V11-2 D2-6 J1

ARDAYYWYFDV

V7-1 J1

ATDYYAMDY

V1-26 J4

ARFYYYGSSYAMDY

V1-55 D1-1 J4

AIYYLDY

V1-53 D2-8 J2

ARHYGSSYWYFDV

V2-6-2 D1-1 J1

10654

3 weeks

ARRYYGSSYWYFDV

V1-55 D1-1 J1

ARSYSNYVMDY

V1-76 D2-6 J4

ARYYGSNYFDY

V7-3 D1-1 J2

ARGASYYSNWFAY

V1-55 D2-6 J3

ALTGTAY

V1-53 D4-1 J3

ARAGAGWYFDV

V5-9 D4-1 J1

TYSNY

V6-6 D2-6 J2

ARTGTYYFDY

V1-53 D4-1 J2

AMVDY

V1-64 D2-9 J2

ARWGTTVVGY

V1-7 D1-1 J2

7632

2 months

MRYGNYWYFDV

V11-2 D2-8 J1

MRYSNYWYFDV

V11-2 D2-6 J1

MRYGSSYWYFDV

V11-2 D1-1 J1

ATFSY

V1-55 J2

ARFYYYGSSYAMDY

V1-55 D1-1 J4

ARIPNWVWYFDV

V1-55 D4-1 J1

ARWDTTVVAPYYFDY

V1-7 D1-1 J2

ARDYYGSSWYFDV

V1-26 D1-1 J1

TYYDYDLYAMDY

V14-4 D2-4 J4

ARFITTVVATRYWYFDV

V1-9 D1-1 J1

8699

4 months

ARSADYGGYFDV

V1-64 D2-4 J1

ARGAY

V1-80 J2

ARSYYDYPWFAY

V1-76 D2-4 J3

ARRWLLNAMDY

V1-9 D2-9 J4

ARPYYYGSSPWFAY

V1-69 D1-1 J3

ARNDYPYWYFDV

V1-4 D2-4 J1

ARSGDY

V1-64 J2

ARVIGDY

V1-53 D2-14 J4

ARANY

V1-55 J3

AVNWDYAMDY

V1-84 D4-1 J4

Table 2 continued on next page

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

repertoires

We quantified the frequency of IgH sequences expressing individual V

gene for each sorted B cell

sample and then compared the V

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 expan-

sion on the V

gene usage profile, we collected normalized data, in which we scored each distinct

IgH CDR3 nucleotide sequence expressing a given V

gene as one, no matter how many times this

sequence was detected.

Our approach enables detection of Ig transcripts expressing about 100 different V

genes that

belong to 14 V

families (

Figure 3

). B-1a cells express all of these detected V

genes (

Figure 3A

contrasting with earlier impressions, based largely on hybridomas sequences from fetal and neonatal

mice (

Malynn et al., 1990

), that V

usage in the B-1a repertoire is very restricted. However, despite

the broad V

usage, certain V

genes, notably V10-1 (DNA4), V6-6 (J606), V11-2 (V

11) 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

Similar to MZB cells, splenic FOB and peritoneal B-2 cells show lower frequency in expressing

these B-1a favored V

genes, i.e., V6-6 (J606), V11-2 (V

11) and V2-6-8 (Q52) (

Figure 3—figure sup-

plement 1B–C

). Conversely, these B subsets tend to preferentially use the largest V

family, V1

(J558), located distal to D

and J

gene segments (

Yancopoulos and Alt, 1986

). MZB cells, in par-

ticular, 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

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—

Table 2 continued

sB-1a samples

Top 10 IgH CDR3 sequences

Age

Peptide

V(D)J

8708

5 months

ASLTY

V1-55 J2

TCNYH

V14-4 D2-8 J4

LIGRNY

V1-55 D2-14 J2

MRYSNYWYFDV

V11-2 D2-6 J1

AKQPYYGSSYWYFDV

V2-3 D1-1 J1

AGSSYAYYFDY

V1-66 D1-1 J2

ARRGIDLLWYHYYAMDY

V1-26 D2-8 J4

ARKSSGSRAMDY

V7-3 D3-2 J4

ASYAMDY

V7-3 J4

ARLYYGNSYWYFDV

V1-55 D2-8 J1

9867

6 months

ARKYYPSWYFDV

V1-55 D1-1 J1

AREGGKFY

V1-7 J2

AKSSGYAMDY

V1-55 D3-2 J4

ARWVITTVARYFDV

V1-85 D1-1 J1

ARGFY

V1-80 J2

AKEGGYYVRAMDY

V1-55 D1-2 J4

ARSMDY

V1-80 J4

ASAMDY

V1-64 J4

TKGGYHDYDDGAWFVY

V1-53 D2-4 J3

ARKFYPSWYFDV

V1-55 J3

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.

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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

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 reper-

toires 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 nucleo-

tide insertions at IgH CDR3 junctions, and remains stable at this level for at least 5 months

(

Figure 4A,B

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

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

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 proxi-

mal 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 rep-

ertoires defined during these waves are distinguishable both by N-additions at CDR3 junctions and

by V

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 rep-

ertoire. 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 sequen-

ces 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

The recurrent V(D)J sequences include V

11-encoded PtC-binding V(D)J sequences, which are ini-

tially present at very low frequencies (2–6 days) but increase aggressively as animals mature to mid-

dle age (6 months) (

Figure 5B

). Since de novo B-1a development is minimum at adulthood, the

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progressive increase in the representation of the recurrent V(D)J sequences as animals reach adult-

hood 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 dif-

ferent 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 rearrange-

ments 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.

Figure 3.

Comparison of V

gene usage by splenic B-1a vs MZB B cells. (

) V

gene usage profile shown as the percentage of IgH sequences

expressing the listed individual V

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). V

genes (from left to right) are ordered in 5’- to 3’-direction bases on chromosome location; the IMGT V

gene nomenclature

is used (

Lefranc, 2003

). (

) V

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 V

gene usage profile, data are presented as the

normalized distribution that counts each distinct CDR3 nucleotide sequence expressing a given V

gene as one, no matter how many times the

sequence was detected. Note: V

12-3 encoded IgH sequences are not detected in this study due to the technical limitations that exclude the V

12-3

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

12-3 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 V

12-3 sequences with the new set of primers, the sequences obtained with both primer sets are highly similar (

Figure 3—figure

supplement 2

DOI: 10.7554/eLife.09083.009

The following figure supplements are available for figure 3:

Figure supplement 1.

gene usage profile pair-wise comparison of B cell groups.

DOI: 10.7554/eLife.09083.010

Figure supplement 2.

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.

DOI: 10.7554/eLife.09083.011

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Table 3.

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

CDR3 peptide

Predominant V(D)

CDR3 junction

diversity

Representation in indicated repertoire

splenic B-1a

(2d-6M)

splenic B-1a (2-6M) addition deletion

PerC B-1a (2W-

6M)

splenic

B-1a (4M

germ free)

FOB (2-

5M)

MZB (1-

5M)

1 TRWDY

17/

V6-6 J2

TGG

J2(8)

11/11

5/6

1/8

0/7

2 MRYSNYWYFDV

17/

V11-2 D2-6 J1 9/

11/11

6/6

1/8

1/7

3 MRYGNYWYFDV

18/

V11-2 D2-8 J1 9/

11/11

6/6

1/8

1/7

4 MRYGSSYWYFDV

17/

V11-2 D1-1 J1 9/

11/11

6/6

1/8

1/7

5 VRHYGSSYFDY

15/

V10-1 D1-1 J2 5/

J2(1)

11/11

3/6

0/8

0/7

6 ARHYYGSSYYFDY

19/

V5-6-1 D1-1

11/11

6/6

2/8

0/7

7 ARLDY

20/

V1-53 J2

CTg/a

J2(8)

10/11

4/6

0/8

1/7

8 ARDYYGSSYWYFDV 19/

V7-1 D1-1 J1 6/

V7-1(3)

9/11

5/6

1/8

1/7

9 ARDYYGSSWYFDV 19/

V1-26 D1-1 J1 7/

J1(3)

2/11

4/6

0/8

1/7

10 ANWDY

19/

V14-3 D4-1 J2 6/

V14-3(2)J2

(8)

5/11

2/6

0/8

0/7

11 ATGTWFAY

18/

V1-19 D4-1 J3 5/

V1-19(2)

6/11

2/6

0/8

1/7

12 ARYYYGSSYAMDY 19/

V7-3 D1-1 J4 8/

V7-3(1)J4(4) 10/11

3/6

3/8

3/7

13 ARYSNYYAMDY

18/

V1-39 D2-6 J4 6/

J4(2)

8/11

1/6

0/8

0/7

14 ARDFDY

19/

V1-64 J2

J2(3)

1/11

3/6

1/8

1/7

15 ARYYSNYWYFDV

17/

V1-9 D2-6 J1 6/

4/11

1/6

0/8

0/7

16 ARYDYDYAMDY

17/

V1-39 D2-4 J4 6/

J4(3)

7/11

1/6

0/8

0/7

17 ARHYYGSSYWYFDV 18/

V2-6-2 D1-1

6/11

2/6

1/8

3/7

18 ARFYYYGSSYAMDY 19/

V1-55 D1-1 J4 6/

J4(4)

8/11

3/6

1/8

1/7

19 ARWDFDY

19/

V1-7 J2

TGGG J2(3)

1/11

3/6

1/8

1/7

20 ARGAY

19/

V1-80 J3

GGG

J3(8)

7/11

6/6

1/8

1/7

21 ARRFAY

18/

V1-26 J3

C/A

J3(8)

9/11

3/6

1/8

1/7

22 ARRDY

18/

V1-55 J2

AGg/a J2(8)

6/11

3/6

1/8

1/7

ASYDGYYWYFDV

18/

V1-55 D2-9 J1 8/

CTATG V1-55(1)

9/11

5/6

0/8

0/7

24 ASYAMDY

16/

V7-3 J4

V7-3(5)J4(4) 9/11

6/6

0/8

1/7

25 ARRYYFDY

17/

V1-78 J2

CGg/cT 0

8/11

2/6

0/8

0/7

Table 3 continued on next page

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The majority of the V(D)J nucleotide sequences that are conserved in the splenic B-1a IgH reper-

toire 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.

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 grad-

ually increases its representation until it dominates in the adult B-1a IgH repertoire (

Figure 6A

, pat-

tern 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.

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 reper-

toire. 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 ani-

mals 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

Table 3 continued

CDR3 peptide

Predominant V(D)

CDR3 junction

diversity

Representation in indicated repertoire

26 ARNYYYFDY

15/

V1-53 D1-2 J2 8/

t/a

10/11

2/6

0/8

0/7

27 ARYYGNYWYFDV

15/

V3-8 D2-8 J1 5/

5/11

2/6

0/8

0/7

ARRYYGSSYWYFDV

15/

V1-55 D1-1 J1 7/

CGG

10/11

5/6

1/8

1/7

29 ARRLDY

13/

V1-22 J2

CGAC J2(6)

8/11

2/6

0/8

1/7

ARFAY

18/

V1-80 J3

J3(4)

2/11

3/6

0/8

0/7

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.

DOI: 10.7554/eLife.09083.016

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sequences that ‘converge’ to encode the same CDR3 peptide share the same D and J segments but

use distinct V

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 corre-

sponding CDR3 peptide.

AID-mediated SHM in pre-immune B-1a IgV

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

In contrast, mutations are minimal in IgV

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

Figure 4.

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

(

) 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. (

) 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.

DOI: 10.7554/eLife.09083.012

Yang

etal

. eLife 2015;4:e09083.

DOI: 10.7554/eLife.09083

14 of 32

Research article

Cell biology

Immunology

SHM in splenic B-1a IgV

initiates after weaning and the frequency of mutated IgH transcripts

increases with age. Thus, mutations are minimally detectable in the IgV

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

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 pheno-

type (GL7

CD38

CD95

) are not detectable in the splenic B-1a population (

Figure 7—figure sup-

plement 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 10

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

is an accumulative process.

Age-dependent progressive increase in the splenic B-1a IgV

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

Figure 5.

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

) 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. (

) 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.

DOI: 10.7554/eLife.09083.013

The following figure supplement is available for figure 5:

Figure supplement 1.

The peritoneal B-1a IgH repertoire is increasingly restricted during ontogeny.

DOI: 10.7554/eLife.09083.014

Yang

etal

. eLife 2015;4:e09083.

DOI: 10.7554/eLife.09083

15 of 32

Research article

Cell biology

Immunology