Figures and data in Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires

Version of Record: August 1, 2016
Version of Record: November 30, 2015
Accepted Manuscript: September 30, 2015

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Yang Yang

Chunlin Wang

Qunying Yang

Aaron B Kantor

Hiutung Chu

Eliver EB Ghosn

Guang Qin

Sarkis K Mazmanian

Jian Han

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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Figures
Tables

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 …
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 …
https://doi.org/10.7554/eLife.09083.004

Figure 1—figure supplement 2

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Recurrent V_H11-encoded PtC-binding V(D)J sequences.

(**A-C**) lists three V_H11-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. …
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 CD11b^hi myeloid cells were shown. The CD19⁺ B cells were gated to reveal CD5 and CD11b …
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 …
https://doi.org/10.7554/eLife.09083.008

Figure 3 with 2 supplements

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Comparison of V_H gene usage by splenic B-1a vs MZB B cells.

(A) V_H gene usage profile shown as the percentage of IgH sequences expressing the listed individual V_H genes for individual B cell samples. The profiles are shown for adult splenic B-1a samples (n = …
https://doi.org/10.7554/eLife.09083.009

Figure 3—figure supplement 1

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V_H 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). V_H genes showing the statistically significant differences (Welch’s t-test p<0.05) between two groups are listed on the …
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 …
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 …
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 …
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 …
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 …
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 V_H 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 …
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 IgV_H 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 …
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)^- IgM^Hi 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 …
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 …
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 …
https://doi.org/10.7554/eLife.09083.023

Figure 8

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Progressive increase in the splenic B-1a IgV_H 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 …
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 pane*l). Each …
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^- IgM^hi IgD^lo CD23^lo/- CD21^- cells were gated to reveal CD5 ⁺ CD43 ⁺ B-1a cells. (B) Absolute number …
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

Sample	Id	Subset	Strain	Age	Condition	Mice	RNT*	RNU*	RPU*	CNT*	CNU*	CPU*
1	7631	FOB	WT	2M	SPF	single	1006030	151210	65871	903400	21240	20470
2	13966	FOB	WT	3.5M	SPF	single	150812	31003	24801	130652	14911	14678
3	8706	FOB	WT	4M	SPF	single	180365	53577	27817	159710	16901	16568
4	8702	FOB	WT	5M	SPF	single	156681	54195	27728	136101	16951	16649
5	13967	FOB	AID KO	5M	SPF	single	35967	14623	13203	27726	7187	7133
6	11161	MZB	WT	1M	SPF	single	33548	19628	12744	25674	6584	6471
7	10658	MZB	WT	2M	SPF	single	71458	26978	18278	61258	11512	11170
8	7630	MZB	WT	2M	SPF	single	1032381	139832	62520	932353	20780	19792
9	8701	MZB	WT	4M	SPF	single	214238	55075	26458	191065	15461	15021
10	8700	MZB	WT	5M	SPF	single	118863	42310	22794	102894	14517	14180
11	13338	MZB	WT	4M	GF	single	162754	39930	23611	141646	12939	12605
12	13343	MZB	WT	4M	GF	single	595780	85497	45820	536072	19266	18480
13	11163	pB-1a	WT	1M	SPF	pool of 3 mice	45882	11290	5596	41368	3237	3007
14	10660	pB-1a	WT	2M	SPF	single	222324	17311	8630	207749	3891	3649
15	13018	pB-1a	WT	2M	SPF	single	808879	36031	14817	753868	4769	4374
16	7628	pB-1a	WT	2M	SPF	single	1784677	59458	22105	1706235	6601	5848
17	11160	pB-1a	WT	2W	SPF	pool of 8 mice	65317	14700	7025	58034	4240	3704
18	10655	pB-1a	WT	3W	SPF	pool of 5 mice	62875	12162	6622	57558	4180	3694
19	8705	pB-1a	WT	4M	SPF	single	310077	28441	11886	287695	5063	4707
20	9870	pB-1a	WT	4M	SPF	single	229100	26299	10469	211514	4745	4480
21	11165	pB-1a	WT	5M	SPF	single	105410	19528	8926	95994	4435	4162
22	8707	pB-1a	WT	5M	SPF	single	320252	29786	12423	296946	4722	4384
23	9861	pB-1a	WT	6M	SPF	single	26613	5683	3235	23542	1521	1461
24	8704	pB-1a	AID KO	4M	SPF	single	264340	33745	14519	245941	6648	6294
25	10657	pB-2	WT	2M	SPF	single	53953	23059	16883	44986	10084	9923
26	7629	pB-2	WT	2M	SPF	single	1315663	123472	47337	1238225	16925	16065
27	13969	pB-2	WT	3.5M	SPF	single	186817	24304	17689	170768	9089	8925
28	9862	pB-2	WT	4M	SPF	single	22591	13377	8737	17343	4382	4357
29	13973	pB-2	AID KO	5M	SPF	single	617893	62319	41165	566826	17536	16965
30	13000	sB-1a	WT	2d	SPF	pool of 8 mice	29439	9542	4925	25369	3148	2758
31	10651	sB-1a	WT	5d	SPF	single	123360	22472	10838	113161	7453	5976
32	10659	sB-1a	WT	5d	SPF	single	210055	28140	12411	192662	7307	5812
33	9866	sB-1a	WT	5d	SPF	single	52986	15600	6864	46580	4595	3837
34	10652	sB-1a	WT	6d	SPF	single	172875	26437	12545	159304	7683	6365
35	9865	sB-1a	WT	7d	SPF	single	71309	18446	8775	64241	5482	4941
36	9868	sB-1a	WT	7d	SPF	single	201813	35069	14473	186227	7847	6843
37	10656	sB-1a	WT	2M	SPF	single	369732	39603	19759	342914	9489	9048
38	13004	sB-1a	WT	2M	SPF	single	185948	27952	13875	168522	7313	7022
39	7632	sB-1a	WT	2M	SPF	single	1825218	102797	43190	1719246	12428	11144
40	11168	sB-1a	WT	2W	SPF	single	536603	70201	28829	496671	11948	10913
41	13005	sB-1a	WT	2W	SPF	single	98017	28331	15001	85489	8820	8207
42	10654	sB-1a	WT	3W	SPF	single	146560	33814	19697	131091	11995	11451
43	13970	sB-1a	WT	3.5M	SPF	single	170925	13809	9289	160480	4513	4273
44	13335	sB-1a	WT	4M	SPF	single	22175	4822	3449	18683	1131	1090
45	13342	sB-1a	WT	4M	SPF	single	283072	23668	12947	262744	5357	5032
46	8699	sB-1a	WT	4M	SPF	single	142838	19151	9938	130915	4370	4086
47	9863	sB-1a	WT	4M	SPF	single	73676	16599	8713	65571	4233	4092
48	11167	sB-1a	WT	5M	SPF	single	501367	38912	17336	463863	7573	7163
49	8708	sB-1a	WT	5M	SPF	single	577114	52723	22272	531508	9146	8441
50	9867	sB-1a	WT	6M	SPF	single	113492	20612	10625	101791	4563	4343
51	13965	sB-1a	AID KO	4M	SPF	single	177782	16419	12281	164189	6539	6293
52	13971	sB-1a	AID KO	4M	SPF	single	517141	34159	22031	482543	8966	8395
53	13968	sB-1a	AID KO	5M	SPF	single	427671	30839	20510	396974	9162	8545
54	13972	sB-1a	AID KO	5M	SPF	single	706116	36217	23255	660874	9294	8744
55	13001	sB-1a	WT	4M	GF	single	43507	8734	4855	38947	2318	2249
56	13002	sB-1a	WT	4M	GF	single	47203	8683	4820	42279	2053	1965
57	13003	sB-1a	WT	4M	GF	single	213347	22246	11068	197769	4705	4449
58	13017	sB-1a	WT	4M	GF	single	532250	40497	17375	501908	7019	6398
59	13337	sB-1a	WT	4M	GF	single	28559	6322	4417	24047	1544	1486
60	13341	sB-1a	WT	4M	GF	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	86	74	125	61	63

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 samples		Top 10 IgH CDR3 sequences
Id	Age		Peptide	V(D)J
11168	2 weeks	1	ANDY	V1-53 J2
		2	AKHGYDAMDY	V2-9 D2-9 J4
		3	ARRYYGSSYWYFDV	V1-55 D1-1 J1
		4	ANWDY	V1-53 D4-1 J2
		5	MRYSNYWYFDV	V11-2 D2-6 J1
		6	ARDAYYWYFDV	V7-1 J1
		7	ATDYYAMDY	V1-26 J4
		8	ARFYYYGSSYAMDY	V1-55 D1-1 J4
		9	AIYYLDY	V1-53 D2-8 J2
		10	ARHYGSSYWYFDV	V2-6-2 D1-1 J1
10654	3 weeks	1	ARRYYGSSYWYFDV	V1-55 D1-1 J1
		2	ARSYSNYVMDY	V1-76 D2-6 J4
		3	ARYYGSNYFDY	V7-3 D1-1 J2
		4	ARGASYYSNWFAY	V1-55 D2-6 J3
		5	ALTGTAY	V1-53 D4-1 J3
		6	ARAGAGWYFDV	V5-9 D4-1 J1
		7	TYSNY	V6-6 D2-6 J2
		8	ARTGTYYFDY	V1-53 D4-1 J2
		9	AMVDY	V1-64 D2-9 J2
		10	ARWGTTVVGY	V1-7 D1-1 J2
7632	2 months	1	MRYGNYWYFDV	V11-2 D2-8 J1
		2	MRYSNYWYFDV	V11-2 D2-6 J1
		3	MRYGSSYWYFDV	V11-2 D1-1 J1
		4	ATFSY	V1-55 J2
		5	ARFYYYGSSYAMDY	V1-55 D1-1 J4
		6	ARIPNWVWYFDV	V1-55 D4-1 J1
		7	ARWDTTVVAPYYFDY	V1-7 D1-1 J2
		8	ARDYYGSSWYFDV	V1-26 D1-1 J1
		9	TYYDYDLYAMDY	V14-4 D2-4 J4
		10	ARFITTVVATRYWYFDV	V1-9 D1-1 J1
8699	4 months	1	ARSADYGGYFDV	V1-64 D2-4 J1
		2	ARGAY	V1-80 J2
		3	ARSYYDYPWFAY	V1-76 D2-4 J3
		4	ARRWLLNAMDY	V1-9 D2-9 J4
		5	ARPYYYGSSPWFAY	V1-69 D1-1 J3
		6	ARNDYPYWYFDV	V1-4 D2-4 J1
		7	ARSGDY	V1-64 J2
		8	ARVIGDY	V1-53 D2-14 J4
		9	ARANY	V1-55 J3
		10	AVNWDYAMDY	V1-84 D4-1 J4
8708	5 months	1	ASLTY	V1-55 J2
		2	TCNYH	V14-4 D2-8 J4
		3	LIGRNY	V1-55 D2-14 J2
		4	MRYSNYWYFDV	V11-2 D2-6 J1
		5	AKQPYYGSSYWYFDV	V2-3 D1-1 J1
		6	AGSSYAYYFDY	V1-66 D1-1 J2
		7	ARRGIDLLWYHYYAMDY	V1-26 D2-8 J4
		8	ARKSSGSRAMDY	V7-3 D3-2 J4
		9	ASYAMDY	V7-3 J4
		10	ARLYYGNSYWYFDV	V1-55 D2-8 J1
9867	6 months	1	ARKYYPSWYFDV	V1-55 D1-1 J1
		2	AREGGKFY	V1-7 J2
		3	AKSSGYAMDY	V1-55 D3-2 J4
		4	ARWVITTVARYFDV	V1-85 D1-1 J1
		5	ARGFY	V1-80 J2
		6	AKEGGYYVRAMDY	V1-55 D1-2 J4
		7	ARSMDY	V1-80 J4
		8	ASAMDY	V1-64 J4
		9	TKGGYHDYDDGAWFVY	V1-53 D2-4 J3
		10	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.

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 peptide		Predominant V(D)J		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	17/20	V6-6 J2	8/9	TGG	J2(8)	11/11	5/6	1/8	0/7
2	MRYSNYWYFDV	17/20	V11-2 D2-6 J1	9/9	0	0	11/11	6/6	1/8	1/7
3	MRYGNYWYFDV	18/20	V11-2 D2-8 J1	9/9	0	0	11/11	6/6	1/8	1/7
4	MRYGSSYWYFDV	17/20	V11-2 D1-1 J1	9/9	0	0	11/11	6/6	1/8	1/7
5	VRHYGSSYFDY	15/20	V10-1 D1-1 J2	5/9	0	J2(1)	11/11	3/6	0/8	0/7
6	ARHYYGSSYYFDY	19/20	V5-6-1 D1-1 J2	9/9	0	0	11/11	6/6	2/8	0/7
7	ARLDY	20/20	V1-53 J2	7/9	CTg/a	J2(8)	10/11	4/6	0/8	1/7
8	ARDYYGSSYWYFDV	19/20	V7-1 D1-1 J1	6/9	0	V7-1(3)	9/11	5/6	1/8	1/7
9	ARDYYGSSWYFDV	19/20	V1-26 D1-1 J1	7/9	G	J1(3)	2/11	4/6	0/8	1/7
10	ANWDY	19/20	V14-3 D4-1 J2	6/9	0	V14-3(2)J2(8)	5/11	2/6	0/8	0/7
11	ATGTWFAY	18/20	V1-19 D4-1 J3	5/9	0	V1-19(2)	6/11	2/6	0/8	1/7
12	ARYYYGSSYAMDY	19/20	V7-3 D1-1 J4	8/9	0	V7-3(1)J4(4)	10/11	3/6	3/8	3/7
13	ARYSNYYAMDY	18/20	V1-39 D2-6 J4	6/9	0	J4(2)	8/11	1/6	0/8	0/7
14	ARDFDY	19/20	V1-64 J2	6/9	G	J2(3)	1/11	3/6	1/8	1/7
15	ARYYSNYWYFDV	17/20	V1-9 D2-6 J1	6/9	0	0	4/11	1/6	0/8	0/7
16	ARYDYDYAMDY	17/20	V1-39 D2-4 J4	6/9	0	J4(3)	7/11	1/6	0/8	0/7
17	ARHYYGSSYWYFDV	18/20	V2-6-2 D1-1 J1	6/9	0	0	6/11	2/6	1/8	3/7
18	ARFYYYGSSYAMDY	19/20	V1-55 D1-1 J4	6/9	T	J4(4)	8/11	3/6	1/8	1/7
19	ARWDFDY	19/20	V1-7 J2	6/9	TGGG	J2(3)	1/11	3/6	1/8	1/7
20	ARGAY	19/20	V1-80 J3	5/9	GGG	J3(8)	7/11	6/6	1/8	1/7
21	ARRFAY	18/20	V1-26 J3	7/9	C/A	J3(8)	9/11	3/6	1/8	1/7
22	ARRDY	18/20	V1-55 J2	5/9	AGg/a	J2(8)	6/11	3/6	1/8	1/7
23	ASYDGYYWYFDV	18/20	V1-55 D2-9 J1	8/9	CTATG	V1-55(1)	9/11	5/6	0/8	0/7
24	ASYAMDY	16/20	V7-3 J4	8/9	0	V7-3(5)J4(4)	9/11	6/6	0/8	1/7
25	ARRYYFDY	17/20	V1-78 J2	7/9	CGg/cT	0	8/11	2/6	0/8	0/7
26	ARNYYYFDY	15/20	V1-53 D1-2 J2	8/9	t/a	0	10/11	2/6	0/8	0/7
27	ARYYGNYWYFDV	15/20	V3-8 D2-8 J1	5/9	0	0	5/11	2/6	0/8	0/7
28	ARRYYGSSYWYFDV	15/20	V1-55 D1-1 J1	7/9	CGG	0	10/11	5/6	1/8	1/7
29	ARRLDY	13/20	V1-22 J2	7/9	CGAC	J2(6)	8/11	2/6	0/8	1/7
30	ARFAY	18/20	V1-80 J3	4/9	0	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.

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 Id	Age (Months)	V(D)J recombination
7630	2	V1-76 D1-1 J1 and V1-39 D1-1 J1
10658	2	V1-76 D1-1 J1
8700	4	V1-72 D1-1 J1 and V8-12 D1-1 J1
8701	5	V1-58 D1-1 J1 and V1-61 D1-1 J1
13338	4	V1-61 D1-1 J1 and V5-16 D1-1 J1

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 IgV_H rarely express class-switched transcripts.

https://doi.org/10.7554/eLife.09083.025

Sample Id	subset	age	strain	non-mutated or mutated sequences (%)		IgM (%)	IgD (%)	IgG3 (%)	IgG2c (%)	IgG2b (%)	IgA (%)
13965	sB-1a	4M	AIDKO	non-mutated	98.2	99.5	0.5
13965	sB-1a	4M	AIDKO	mutated	1.8	100
13968	sB-1a	5M	AIDKO	non-mutated	99	100
13968	sB-1a	5M	AIDKO	mutated	1	100
13971	sB-1a	4M	AIDKO	non-mutated	98.4	99.6	0.4
13971	sB-1a	4M	AIDKO	mutated	1.6	100
13972	sB-1a	4M	AIDKO	non-mutated	100	99.9	0.1
8704	pB-1a	4M	AIDKO	non-mutated	98.2	99.9	0.1
8704	pB-1a	4M	AIDKO	mutated	1.8	100
13973	pB-2	5M	AIDKO	non-mutated	100	91.6	8.4
8700	MZB	5M	WT	non-mutated	99.8	99.9	0.1
8701	MZB	4M	WT	non-mutated	98.6	99.9	0.1
7630	MZB	2M	WT	non-mutated	99.5	99.9	0.1
10658	MZB	2M	WT	non-mutated	100	100
8702	FOB	5M	WT	non-mutated	99.8	98.6	1.4
13966	FOB	3.5M	WT	non-mutated	99.5	99.7	0.3
7631	FOB	2M	WT	non-mutated	99.3	72.9	27.1
7629	pB-2	4M	WT	non-mutated	98.4	88.4	11.5
7629	pB-2	4M	WT	mutated	1.6	89	11
13969	pB-2	3.5M	WT	non-mutated	99.5	90.1	9.9
13974	sB-1a	day 2	WT	non-mutated	99.2	99.8	0.2
13000	sB-1a	day 2	WT	non-mutated	99.2	100
10659	sB-1a	day 5	WT	non-mutated	99.2	100
9866	sB-1a	day 5	WT	non-mutated	100	100
10651	sB-1a	day 5	WT	non-mutated	99.7	100
10652	sB-1a	day 6	WT	non-mutated	99.4	100
9868	sB-1a	day 7	WT	non-mutated	99.3	99.9	0.1
9865	sB-1a	day 7	WT	non-mutated	99.5	99.6	0.4
11168	sB-1a	2W	WT	non-mutated	99.1	99.9	0.1
13005	sB-1a	2W	WT	non-mutated	99.5	100
10654	sB-1a	3W	WT	non-mutated	95.8	100
10654	sB-1a	3W	WT	mutated	4.2	100
11160	pB-1a	2W	WT	non-mutated	99	100
10655	pB-1a	3W	WT	non-mutated	99.2	100
11163	pB-1a	1M	WT	non-mutated	99.1	99.9
7632	sB-1a	2M	WT	non-mutated	88.1	99.1	0.9
7632	sB-1a	2M	WT	mutated	11.9	99.9		0.1
10656	sB-1a	2M	WT	non-mutated	88.1	99.8	0.2
10656	sB-1a	2M	WT	mutated	11.9	100
13004	sB-1a	2M	WT	non-mutated	97.7	99.9
13004	sB-1a	2M	WT	mutated	2.3	94					6
13018	pB-1a	2M	WT	non-mutated	91.8	100
13018	pB-1a	2M	WT	mutated	8	100
13660	pB-1a	2M	WT	non-mutated	92.2	100
13660	pB-1a	2M	WT	mutated	7.6	100
7628	pB-1a	2M	WT	non-mutated	92.3	99.5	0.4	0.1
7628	pB-1a	2M	WT	mutated	7.1	99.6	0.2	0.2
8705	pB-1a	4M	WT	non-mutated	93.8	99.7	0.3
8705	pB-1a	4M	WT	mutated	4.4	99.9
9870	pB-1a	4M	WT	non-mutated	86.4	99.9
9870	pB-1a	4M	WT	mutated	12.6	99.9
11165	pB-1a	5M	WT	non-mutated	98.1	99.9
11165	pB-1a	5M	WT	mutated	1.5	100
8707	pB-1a	5M	WT	non-mutated	91.6	97.2	0.1	2	0.5	0.1	0.1
8707	pB-1a	5M	WT	mutated	6.2	97.9	0.1	2
9861	pB-1a	6M	WT	non-mutated	82.4	99.6	0.4
9861	pB-1a	6M	WT	mutated	17.5	100

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 Id	subset	age	condition	non-mutated or mutated sequences (%)		IgM (%)	IgD (%)	IgG1(%)	IgG3 (%)	IgG2c (%)	IgG2b (%)	IgA(%)
9867	sB-1a	6M	SPF	non-mutated	50	95.8			2.7	0.6	0.8	0.1
9867	sB-1a	6M	SPF	mutated	50	65		0.03	16.8	4.3	4.8	9.1
8699	sB-1a	4M	SPF	non-mutated	56	99.5			0.3	0.1	0.1
8699	sB-1a	4M	SPF	mutated	44	89.9			3.4	2	1.3	3.4
9863	sB-1a	4M	SPF	non-mutated	74.1	93.9	0.2		3.6	1	1.3
9863	sB-1a	4M	SPF	mutated	25.9	44.2			41	9.7	3.7	1.4
13970	sB-1a	3.5M	SPF	non-mutated	74.1	92.7	0.5		3.7	1.7	1.3
13970	sB-1a	3.5M	SPF	mutated	25.9	92.1			2.5	0.5	4.8	0.1
13342	sB-1a	4M	SPF	non-mutated	88.9	97.6	0.5		0.8	0.6	0.2	0.3
13342	sB-1a	4M	SPF	mutated	11.1	85.2			0.3	0.2	7.3	7
13337	sB-1a	4M	GF	non-mutated	69.7	98.5	0.1		0.8	0.4	0.1
13337	sB-1a	4M	GF	mutated	30.3	79			15.3	5	0.7
13003	sB-1a	4M	GF	non-mutated	74.8	97.2	0.3	0.2	0.5	0.2	1.6
13003	sB-1a	4M	GF	mutated	25.2	89.8		1.1	2.3	1.2	5.6
13341	sB-1a	4M	GF	non-mutated	78.2	99	0.1		0.2	0.1	0.6
13341	sB-1a	4M	GF	mutated	21.8	72.2			9.6	5.5	12.6	0.1
13017	sB-1a	4M	GF	non-mutated	80.9	95.6	0.4		2	1	1
13017	sB-1a	4M	GF	mutated	19.1	79	0.2		7.9	3.9	8.9	0.1
13002	sB-1a	4M	GF	non-mutated	88.5	97.4	0.5		0.6	0.2	1.3
13002	sB-1a	4M	GF	mutated	11.5	63.8			14.8	8.4	13

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
sB-1a samples (4 months)		Peptide	V(D)J
germ-free #1	1	MRYGSSYWYFDV	V11-2 D1-1 J1
	2	ARGAY	V1-80 J2
	3	ARNPDGYYTYYYAMDY	V2-2 D2-9 J4
	4	ARDPFYYYGSSYWYFDV	V5-16 D1-1J1
	5	MRYSNYWYFDV	V11-2 D2-6 J1
	6	AITRAY	V1-55 J3
	7	ARRYYGSSYWYFDV	V1-55 D1-1 J1
	8	ARSDYYGSSSLSY	V1-26 D1-1 J2
	9	ASGGNYFDY	V1-75 J2
	10	ARSLYN	V1-9 J2
germ-free #2	1	ARNYGSSYDY	V1-53 D1-1 J2
	2	TRPSYYGSDY	V14-4 D1-1 J2
	3	TRESYDGYYVWYAMDY	V5-9-1 D2-9 J4
	4	ARGDY	V14-3 J2
	5	ASNWAY	V1-53 D4-1 J2
	6	MRYSNYWYFDV	V11-2 D2-6 J1
	7	AKGDYYGSSYYFDY	V1-9 D1-1 J2
	8	VRHGPRAFDY	V10-1 D3-2 J2
	9	ARLNGDY	V1-69 J2
	10	MRYGNYWYFDV	V11-2 D2-8 J1
specific pathogen free #1 (from Caltech)	1	ASYSNSDV	V3-6 D2-6 J1
	2	ARVSYSRAMDY	V14-3 D2-6 J4
	3	ARSGNYGAMDY	V1-7 D2-8 J4
	4	ASRLRSTFAY	V2-6-8 D1-1 J3
	5	ARVTTVHAMDY	V1-55 D1-1 J4
	6	ARNYGSSYWYFDV	V1-53 D1-1 J1
	7	ARTPNWEARDY	V1-55 D4-1 J4
	8	ARRYYGSSYWYFDV	V1-55 D1-1 J1
	9	ARPLLYRYYFDY	V1-75 D2-6 J2
	10	ARNYGSSYDWYFDV	V1-9 D1-1 J1
specific pathogen free #2 (from Caltech)	1	ARGGIYYDYDEVYYYAMDY	V1-55 D2-4 J4
	2	MRYSNYWYFDV	V11-2 D2-6 J1
	3	ARDYYGSSWYFDV	V1-26 D1-1 J1
	4	MRYGNYWYFDV	V11-2 D2-8 J1
	5	MRYGSSYWYFDV	V11-2 D1-1 J1
	6	ARYYDGYYGYYAMDY	V1-26 D2-4 J4
	7	ALITTWYFDV	V1-78 D1-2 J1
	8	ARHYYGSSWGY	V1-53 D1-1 J2
	9	ARSFSPYYFDY	V1-26 J2
	10	ARSHGYYPFDY	V1-54 D2-9 J2
specific pathogen free #1 (from Stanford)	1	ARSADYGGYFDV	V1-64 D2-4 J1
	2	ARGAY	V1-80 J2
	3	ARSYYDYPWFAY	V1-76 D2-4 J3
	4	ARRWLLNAMDY	V1-9 D2-9 J4
	5	ARPYYYGSSPWFAY	V1-69 D1-1 J3
	6	ARNDYPYWYFDV	V1-4 D2-4 J1
	7	ARSGDY	V1-64 J2
	8	ARVIGDY	V1-53 D2-14 J4
	9	ARANY	V1-55 J3
	10	AVNWDYAMDY	V1-84 D4-1 J4
specific pathogen free #2 (from Stanford)	1	ARGNY	V1-80 J2
	2	ARWVYYGSSSYWYFDV	V1-54 D1-1 J1
	3	ARSSNYAMDY	V1-78 D2-11 J4
	4	ARYYYGSNYAMDY	V7-3 D1-1 J4
	5	ARGAY	V1-80 J2
	6	ARRYYGSSYWYFDV	V1-55 D1-1 J1
	7	ARSPYYSNYEGYFDV	V1-72 D2-6 J1
	8	ARKNYGSSYWYFDV	V1-55 D1-1 J1
	9	ARLEIYYGNYGRVFDV	V1-80 D2-8 J2
	10	ARRDYYGSSYVLAY	V1-9 D1-1 J3

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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Figures

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.

FACS plots showing CD43⁺ CD5⁺ IgM⁺ B-1a cells in E19 fetal liver.

Recurrent V_H11-encoded PtC-binding V(D)J sequences.

CD11b expression on peritoneal B-1a (CD5⁺) and B-1b (CD5^-) is roughly 100-fold lower than the CD11b expression on myeloid cells.

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.

Comparison of V_H gene usage by splenic B-1a vs MZB B cells.

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

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.

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

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

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

The level of convergent recombination in the B-1a IgH repertoire declines with age.

Distinct V(D)J sequences encoding the same CDR3 peptide differ in V_H usage.

AID-mediated SHM accumulates on splenic B-1a IgV_H with age.

Splenic B-1a cells do not contain cells expressing GC phenotype.

Percentage of sequences containing > = 4 nucleotides changes for each B cell group.

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.

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

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.

Normal splenic B-1a compartment in GF mice.

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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.

FACS plots showing CD43+ CD5+ IgM+ B-1a cells in E19 fetal liver.

Recurrent VH11-encoded PtC-binding V(D)J sequences.

CD11b expression on peritoneal B-1a (CD5+) and B-1b (CD5-) is roughly 100-fold lower than the CD11b expression on myeloid cells.

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.

Comparison of VH gene usage by splenic B-1a vs MZB B cells.

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

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.

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

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

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

The level of convergent recombination in the B-1a IgH repertoire declines with age.

Distinct V(D)J sequences encoding the same CDR3 peptide differ in VH usage.

AID-mediated SHM accumulates on splenic B-1a IgVH with age.

Splenic B-1a cells do not contain cells expressing GC phenotype.

Percentage of sequences containing > = 4 nucleotides changes for each B cell group.

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.

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

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.

Normal splenic B-1a compartment in GF mice.

FACS plots showing CD43⁺ CD5⁺ IgM⁺ B-1a cells in E19 fetal liver.

Recurrent V_H11-encoded PtC-binding V(D)J sequences.

CD11b expression on peritoneal B-1a (CD5⁺) and B-1b (CD5^-) is roughly 100-fold lower than the CD11b expression on myeloid cells.

Comparison of V_H gene usage by splenic B-1a vs MZB B cells.

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

Distinct V(D)J sequences encoding the same CDR3 peptide differ in V_H usage.

AID-mediated SHM accumulates on splenic B-1a IgV_H with age.

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