Download Length and Amino Acid Composition Complementarity

Selection of Ig µ Heavy Chains by
Complementarity-Determining Region 3
Length and Amino Acid Composition
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of August 3, 2017.
Denise A. Martin, Harald Bradl, Tara J. Collins, Edith Roth,
Hans-Martin Jäck and Gillian E. Wu
J Immunol 2003; 171:4663-4671; ;
doi: 10.4049/jimmunol.171.9.4663
http://www.jimmunol.org/content/171/9/4663
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References
The Journal of Immunology
Selection of Ig ␮ Heavy Chains by
Complementarity-Determining Region 3 Length and Amino
Acid Composition1
Denise A. Martin,* Harald Bradl,§ Tara J. Collins,* Edith Roth,§ Hans-Martin Jäck,2§ and
Gillian E. Wu*‡
B
lymphocyte development follows a complex series of
events and decisions in which genetics, environmental,
and stochastic processes all play a role in the outcome.
Integral to the survival of a developing B cell is the productive
rearrangement of the VH, D, and JH, and VL and JL gene segments
of Ig heavy chain (HC)3 and light chain (LC) loci, respectively (1,
2). The joining of VH, DH, and JH gene segments generates the
variable domain of a HC with its complementarity-determining
region 3 (CDR3) encompassing the sites of the joins. Formation of
a ␮HC demarcates a crucial checkpoint in B cell development. At
this checkpoint, called the pre-B cell receptor (pre-BCR) stage, the
␮HC and the surrogate LC (SLC) components, VpreB and ␭5,
assemble to form the pre-BCR. The ␮HC, if capable of pairing
with the SLC, appears on the surface of the B cell, where much
evidence implicates the successful formation of a pre-BCR in signaling events and the proliferative expansion associated with B
cell progression (3–5). The ability or inability of a particular ␮HC
to pair with SLC as well as the competence of that interaction
determines whether a ␮HC protein and ultimately a B cell are
*Department of Immunology, University of Toronto, and Ontario Cancer Institute,
Toronto, Canada; †Division of Molecular Immunology, Department of Internal Medicine III, Nikolaus Fiebiger Center, University of Erlangen-Nürnberg, Erlangen, Germany; and ‡Faculty of Pure and Applied Science, York University, Toronto, Canada
Received for publication April 10, 2003. Accepted for publication August 28, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by the Canadian Institutes for Health Research and
the Terry Fox Marathon of Hope (to G.W.) and by Project Grant SFB466 and Research Grant JA 968/2 from the Deutsche Forschungsgemeinschaft (to H.-M.J.). D.M.
is the recipient of a Canadian Institutes for Health Research Doctoral Research
Award. This work was performed in partial fulfillment of Ph.D. requirements of D.M.
2
Address correspondence and reprint requests to Dr. Hans-Martin Jäck, Division of
Molecular Immunology, Nikolaus Fiebiger Center, University of Erlangen-Nurnberg,
Gluckstrasse 6, D-91054 Erlangen, Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: HC, heavy chain of IgM; A-MuLV, Abelson
murine leukemia virus; BCR, B cell receptor; BM, bone marrow; pI, isoelectric point;
pre-BCR, pre-B cell receptor; SLC, surrogate light chain; VH81X-␮HC, ␮HC using
a VH81x-D-J rearrangement.
Copyright © 2003 by The American Association of Immunologists, Inc.
selected to proliferate and survive/persist into the mature pool (4,
6 – 8).
The importance of ␮HC deposition and the SLC in B cell progression has been demonstrated in various murine models with
targeted deletions. Mice with a deletion in the transmembrane exon
of the ␮HC are unable to produce cells that progress beyond the
pro-B cell stage of development (9). Mice that cannot make a
complete SLC due to the absence of ␭5 protein also have a block
in generating mature B cells, with most cells arrested at the pro-B
stage (10).
Evidence is accumulating that the ␮⌯C is assessed for its ability
to functionally associate with a LC before LC rearrangement, with
the SLC functioning as the assessor (8, 11, 12). The presence of the
SLC before LC rearrangement as well as its structural similarity to
a conventional Ig LC make it an ideal candidate for this function.
If a cell has passed the fitness test overseen by the pre-BCR, placement of a successful HC-LC pair in the plasma membrane provides
the B cell with a receptor necessary for survival and persistence in
the periphery (13). The features in the ␮⌯C protein important for
successful LC pairing, however, remain to be determined.
Some evidence suggests that the V-D-J joint that encodes CDR3
can impact HC folding and the ability to form HC-LC heterodimers (6). In particular, rearrangements using the most
D-proximal V gene segment, VH81X, have been found to generate
HC proteins largely incapable of pairing with SLC (8). Evidence
that impaired pairing has in vivo consequences comes from the
finding that VH81X is under-represented in productive rearrangements (7, 12, 14, 15). In addition, few mature cells have been
found with VH81X-containing BCRs, despite its presence in
⬎50% of the initial HC rearrangements in the bone marrow (11,
12, 14, 16, 17).
A diverse repertoire is paramount for immunocompetency.
Thus, it is crucial to understand the mechanisms restricting selection while ensuring an effective, functional repertoire. We have
used a system that allows us to track VH81X rearrangements
through stages of B cell development to identify the structure and
type of rearrangements that persist in the periphery. By breeding,
we have generated a mouse that carries one wild -type Ig HC allele
0022-1767/03/$02.00
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Although it is generally accepted that Ig heavy chains (HC) are selected at the pre-B cell receptor (pre-BCR) checkpoint, the
characteristics of a functional HC and the role of pre-BCR assembly in their selection have remained elusive. We determined the
characteristics of HCs that successfully passed the pre-BCR checkpoint by examining transcripts harboring VH81X and JH4 gene
segments from JHⴙ/ⴚ and ␭5ⴚ/ⴚmice. VH81X-JH4-HC transcripts isolated from cells before or in the absence of pre-BCR assembly
had no distinguishing complementarity-determining region 3 traits. In contrast, transcripts isolated subsequent to passage through
the pre-BCR checkpoint had distinctive complementarity-determining regions 3 of nine amino acids in length (49%) and a
histidine at position 1 (73%). Hence, our data define specific structural requirements for a functional HC, which is instrumental
in shaping the diverse B cell repertoire. The Journal of Immunology, 2003, 171: 4663– 4671.
4664
Materials and Methods
Mice
JH⫺/⫺ and ␭5⫺/⫺ mice have been previously described (10, 18). JH⫺/⫺,
␭5⫺/⫺, and C57BL/6 mice were maintained under specific pathogen-free
conditions in the animal colony at the Ontario Cancer Institute. JH⫹/⫺ mice
were generated by breeding C57BL/6 and JH⫺/⫺ mice. JH⫹/⫺ mice were
identified by Southern blot analysis as previously described (18). Briefly,
genomic DNA from mouse tails was digested with StuI, electrophoresed,
and transferred to Hybond nylon membranes (Amersham Pharmacia Biotech, Little Chalfont, U.K.). Membranes were hybridized with a 475-bp
EcoRI-StuI fragment of E␮, which reveals a band of 4.7 kb for the wildtype allele and of 3.0 kb for the mutant allele. Mice identified by Southern
analysis as having one mutant allele and one wild-type allele were used for
further analysis.
Flow cytometry and cell sorting
Single-cell suspensions were prepared from bone marrow (BM) and spleen
of the appropriate mice and were stained using standard procedures. BM
cells were stained with FITC-conjugated anti-B220 (mAb RA3-6B2; BD
PharMingen, San Diego, CA), PE-conjugated anti-CD43 (mAb S7; BD
PharMingen), and biotinylated anti-␮ IgH (mAb 33-60). Splenocytes were
stained with FITC-conjugated anti-IgD (mAb 11-26c.2a; BD PharMingen),
PE-conjugated anti-B220 (mAb RA3-6B2; BD PharMingen), and biotinylated anti-␮ IgH (mAb 33-60). Staining with biotinylated Abs was revealed with the secondary reagent streptavidin Quantum Red (SigmaAldrich, St. Louis, MO). All collected cells were B220⫹. To isolate B cell
precursors, BM populations were defined by the cell surface markers CD43
and ␮; collected cells were CD43⫹␮⫺ or CD43⫺␮⫺. To isolate newly
emerging B cells, spleen populations were defined by cell surface expression of IgM and IgD; collected cells were IgM⫹IgDlow/⫺. Cells were sorted
using FACStar Plus (BD Biosciences, Mountain View, CA) and MoFlo
(Cytomation, Fort Collins, CO) instrumentation. Abelson murine leukemia
virus (A-MuLV)-transformed pre-B cell lines, before (membrane staining)
or after (cytoplasmic staining) fixation and permeabilization with formaldehyde/Tween 20, were stained using standard staining protocols (6). Unconjugated Abs were detected with the appropriate fluorochrome-conjugated secondary Abs. Cells were washed, and fluorescence was analyzed
with a FACSCalibur (BD Biosciences). Flow diagrams were obtained by
analyzing the primary data with the CellQuest software program (BD Biosciences). Rat IgG mAbs directed against the pre-BCR (clone SL156) (19)
and the hamster anti-mouse ␭5 mAb, FS1 (20), have been previously described (21). FITC-conjugated as well as unconjugated affinity-purified
goat Abs against mouse ␮HC were purchased from Southern Biotechnology Associates (Birmingham, AL).
RNA isolation, amplification, and sequencing
Total RNA was extracted from sorted cells using Tri-Reagent (Molecular
Research Center, Cincinnati, OH). RNA was reverse transcribed using Superscript II (Life Technologies, Gaithersburg, MD), and the cDNA was
used for RT-PCR. The primary PCR globally amplified ⬎80% of VH gene
rearrangements; the secondary PCR specifically amplified transcripts harboring the VH81X gene segment. cDNA was amplified for the primary
PCR using VHALL sense 5⬘-AGGT(C/G)(A/C)A(A/G)CTGCAG(C/
G)AGTC(A/T)GG-3⬘ and JH4IN antisense 5⬘-GAGGAGACGGTGACT
GAGGTTCCTTG-3⬘, followed by a secondary PCR reaction using either
V81X145 sense 5⬘-TGGTCGCAGCCATTCATAGT-3⬘ or V81XEcoRI
sense 5⬘-GAATTCCCTTCCCATGACATGTCTTG-3⬘ and the JH4IN antisense primer. RT-PCR products were identified by resolution on a 1.0%
agarose gel and fragments of expected length were ligated into TA cloning
vectors (Invitrogen, Carlsbad, CA). Plasmid clones were sequenced with
the T7 sequencing kit (Amersham Pharmacia Biotech). IgH CDR3 regions
were identified as the amino acids between codon 94 and codon 102 (22).
Codon 94 is the second amino acid residue following the conserved YYC
motif coded by the VH gene segment (usually an arginine residue), and
codon 102 is the conserved tryptophan encoded by the JH gene segment.
Retroviral vector construction for expression of VH81X gene
rearrangements
The retroviral vector pELV81C␮ (Fig. 1A) was generated from pELVC (a
gift from F. Melchers, Basel, Switzerland) (4). pELVC contains the leader
(L) exon/intron sequence of the Sp6 ␮ gene (23) and a functional VDJH
sequence fused to the cDNA sequence encoding the membrane form of
␮HC. The rearranged VH81X segments were isolated from a number of
sources and inserted into pELV81. One set of VH81X sequences originated
from genomic VH81XDJ clones reported previously (24). TdT refers to
clones isolated from the BM of an adult transgenic TdT mouse (25), and
MTFL and MTBM refer to clones isolated from fetal liver and adult BM,
respectively, of a ␮MT mouse (9). Genomic VH81XDJ sequences were
converted into cDNA-type sequences (the JH abutting the C␮) by the following PCR cloning strategy. The forward primer was GAATTCCCTTC
CCATGACATGTCTTG (containing an EcoRI site that is present in all
VH81X sequences; Fig. 1A), and the backward primer is G A A G C T T T
GACTCTCTGAGGAGA CGGTGACTGAGGTTCCTTG
(containing HindIII, C␮, and JH4 sequences; Fig. 1A). The amplified products were TA cloned, and EcoRI/HindIII VH81X fragments were isolated
and inserted in the corresponding restriction sites of pELV81C␮. TdT4pELV81C␮, MTFL8-pELV81C␮, MTFL4-pELV81C␮, TdT1-pELV81C␮,
MTBM4-pELV81C␮, and MTBM27-pELV81C␮ were constructed in this
manner. Plasmid expression vectors encoding BC2-V81X-C␮ and 31AV81X-C␮ were gifts from J. Kearney (Birmingham, AL). F-V81X was cloned
from the 18-81 subclone F, an A-MuLV pre-B cell line, and inserted into a
conventional Ig expression vector (6). All VDJH regions were verified by complete sequencing.
Retroviral infection of the A-MuLV-transformed pre-B cell lines
38B9 and BINE4.8
The ectopic packaging line GP⫹E (26), grown to 80 –90% confluence in
25-cm2 flasks, was first transfected with 5 ␮g of the respective retroviral
plasmid vector with the calcium phosphate method using the Cal-PhosMaximizer kit (Clontech, Palo Alto, CA) for 2 h at 37°C. After infection,
the medium was removed, and the cells were washed with PBS and incubated in RPMI/10% heat-inactivated FCS at 37°C and 5% CO2. After 24 h
the medium was replaced with 5 ⫻ 106 pre-B cells (BINE 4.8 or 328B9)
in 5 ml of RPMI/10% heat-inactivated FCS supplemented with 40 ␮g of
polybrene (Sigma-Aldrich). The culture was incubated for 24 h and then
supplemented with 5 ␮g/ml puromycin. Single clones were generated from
puromycin-resistant bulk cultures by the limiting dilution method and were
analyzed for ␮HC production by flow cytometry.
Cell lines and culture conditions
The ectopic retroviral packaging line GP⫹E (26) and the A-MuLV-transformed mouse pre-B cell lines Bine4.8, TK, TK␮ (27), and 38B9 (2, 4)
were maintained at 37°C and 5% CO2 in RPMI medium supplemented with
50 U/ml penicillin, 50 ␮g/ml streptomycin, 5% FCS, 1 mM sodium pyruvate, and 2 mM L-glutamine. Bine4.8, TK, and 38B9 produce ␭5 and
VpreB proteins, but lack a ␮HC; TK␮ produces a completely assembled
pre-BCR and served as a positive control for ␮HC/SLC pairing.
Immunoprecipitation and gel electrophoresis
Metabolic labeling was performed as previously described (8). Briefly, 5 ⫻
106 of retrovirus-infected TK, TK␮, or 38B9 cells in 1 ml of methioninefree labeling medium were incubated overnight with 50 ␮Ci/ml of Tran35S
label (ICN Biomedicals, Eschwege, Germany). Cells were lysed for 30 min
on ice in 1 ml of lysis buffer. Cell lysates were incubated with either 5 ␮g
of affinity-purified goat anti-mouse ␮HC Abs (Jackson ImmunoResearch
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and one allele with a targeted deletion at the JH locus. This arrangement allows each B cell to make only one productive VDJH
rearrangement, ensuring that we are monitoring the gene structure
of only the used HC in various B cell compartments. By isolating
productive rearrangements using VH81X and JH4 gene segments
and determining their potential to associate with SLC and be expressed on the cell surface, we were able to determine which specific variable region structures, differing only at the CDR3, render
an HC successful.
These studies have revealed two characteristics common to successful HCs using VH81X and JH4 gene segments: a CDR3 length
of nine amino acids and the presence of histidine in position 1.
These traits have significant roles in determining which VH81X
␮HCs are selected into the peripheral pool. Other parameters, including isoelectric point (pI) and motifs, were not found to contribute substantially to HC selection. These data suggest a level of
selection before ␮HC deposition in the plasma membrane, followed by further selection subsequent to cell surface expression.
Our model implicates SLC in this paradigm, functioning to select
protein by shape and fit before surface expression and signaling
efficiency.
SELECTION OF Ig HC
The Journal of Immunology
4665
The theoretical models were then assembled identically and displayed together. The pI of the modeled VH81X-HC was calculated using Compute
pI/Mw, a tool, which allows computation of the theoretical pI and Mw
(http://www.expasy.ch/tools/pi_tool.html and (30, 31).
Statistics
Statistics were performed by the Statistics Department at University of
Toronto. Values were calculated using sampling distributions for counts
and proportions. Significance was calculated under the null hypothesis of
no difference in the proportion of occurrence of CDR3 lengths equal to 27
nt between the two populations or the null hypothesis of no difference in
the proportion of occurrence of amino acid histidine at position 1 between
the two populations.
Results
Laboratories, West Grove, PA) or FS1 mAb (anti-␭5) for 3 h on ice. Immunocomplexes were precipitated by protein G-Sepharose (Pierce, Rockford, IL) for 2 h on ice, separated on a 10% SDS-Laemmli polyacrylamide
gel, and detected by fluorography.
Modeling of IgH V regions
Virtually translated V regions of HCs were modeled using the SWISSMODEL Protein Modeling Server (http://www.expasy.ch/swissmod/
SWISS-MODEL.html). To analyze the structure of any VH region, the VH
regions of the VH81X-HCs, TdT4-pELV81C␮, V81XBC2-pELV81C␮,
and V81X31A-pELV81C␮, all of which could form a pre-BCR in the
A-MuLV line, 38B9 (see Results), were compared with all protein structures in the SWISS-PROT database. Protein 31AR1, a single HC Fv, had
the most sequence similarity (⬃72% amino acid identity overall, including
a structurally important cysteine) and was listed in each search. Protein
31AR1 was used as the template for the tested ␮⌯Cs (Table I). The data
received were converted and viewed using SWISS PDB viewer (28, 29).
VH81X HC rearrangements in BM and spleen
Mice heterozygous for a deletion of the JH gene segments are only
able to generate one productively rearranged allele per cell. In all
other respects examined, the B cell populations isolated from
spleen and BM were comparable to B cell compartments found in
wild-type animals (18). We examined the expressed ␮⌯C allele
first in cells from whole BM and spleen as described in Materials
and Methods (15). For each reaction, 2– 4% of the product was
cloned, sequenced, and evaluated. Using the guidelines of Kabat
and Wu (22), translated sequences were anchored at the conserved
YYC (tyrosine-tyrosine-cysteine) encoded by the VH gene segment. DH segments were identified by sequence homology according to published sequences (32–34), but only if sequence identity
could be demonstrated for 5 consecutive nt. CDR3 lengths were
measured from the amino acids commencing at the third residue
from the YYC motif and ending at (but not including) the conserved W (tryptophan) encoded by the JH gene segment.
The VH81X rearrangements, including DH usage, isolated from
unsorted BM were for the most part unremarkable. Their CDR3
lengths ranged from 6 –12 aa in length, with length values distributed without any significant bias (data not shown). The rearrangements in unsorted splenocytes were similar to those from whole
BM, except that CDR3 had a length bias toward 9 aa (data not
shown).
BM and spleen are heterogeneous populations containing B
cells at multiple stages of B cell development. To examine ␮⌯C in
the early stages of and subsequent to pre-BCR expression, it was
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FIGURE 1. Analysis of surface expression and assembly of VH81X␮HCs with SLC. A, The retroviral vector pELV81C␮ was generated from
pELVC by replacing its SalI/HindIII VHDJH fragment with a corresponding fragment of a VH81XDJH1 rearrangement cloned from the BC2 hybridoma (see Materials and Methods). Part of the VH81XDJH sequence can
be excised with EcoRI and HindIII and replaced with a corresponding
sequence of another VH81XDJH fragment. bla, gene encoding ␤-lactamase;
ori, origin of replication of pBR322; pac, gene encoding puromycin Nacetyltransferase. B, Flow cytometric analyses of ␮HC synthesis in stably
infected 38B9 pre-B cell clones. Cells were stained with FITC-conjugated
Abs against mouse ␮HC before (membrane) or after (cytoplasmic (cyto))
cell permeabilization. Fluorescence intensities (F) of cytoplasmic (cyto)
and cell surface (membrane) stainings are shown on the y-axis, and cell size
(forward scatter) is shown on the x-axis. C, Electrophoretic analysis of
␮HC and SLC assembly in stably infected 38B9 pre-B cell clones. Cells
were metabolically labeled with [35S]methionine, and labeled proteins
were precipitated from pre-B cell lysates with Abs against mouse ␮HC (␮)
or ␭5, followed by protein G-Sepharose. Radioactive bands were revealed
by fluorography. Molecular masses of protein standards are shown in kilodaltons on the left, and the positions of VpreB, ␭5, and ␮HC are shown on
the right of the blot.
The elements that dictate the configuration of biological systems
remain important issues in understanding the development of the
mature immune system. In any developing system, identifiable
characteristics of a population may reflect the outcome of stochastic or selective mechanisms. In this study we sought to identify
specific characteristics of ␮⌯C from splenic B cells and determine
whether these characteristics would yield insights into the structural requirements of Ig HCs. These HCs exhibit representative
traits that allowed the cell to passage through selection and persist
in the spleen. We addressed these issues using a mouse with a
single rearrangeable IgH allele that would allow us to compare
HCs that passed the pre-BCR checkpoint and persisted in the periphery to those present before selection in the BM. These experiments were followed up with analysis of functional Ig rearrangements isolated from peripheral B cells of ␭5⫺/⫺ mice, to examine
the properties of HCs that persist in the absence of SLC. We then
used an A-MuLV-transformed pre-B cell line that lacked its own
HC (but had SLC protein) to assess by flow cytometry and immunoprecipitation the ability of ␮HCs to associate with the SLC and
appear on the cell surface. The availability of modeling programs
allowed us then to determine the characteristics of a functional,
i.e., pairing, ␮HC that passes the pre-BCR checkpoint and persists
in the periphery.
4666
SELECTION OF Ig HC
Table I. VH81X-␮HCs and their potential to pair with SLC and to be transported to the cell surface.
VH81X
Sequence
Reference
No.
FR3
CDR3
TdT4
BC2
31A
MTBM4
MTFL8
MTFL4
TdT1
F
BFL23
24
6, 56
6, 58
24
24
24
24
8
6
YYCAR
YYCAR
YYCAR
YYCAR
YYCAR
YYCAR
YYCAR
YYCAR
YYCAR
HANWDAMDY
HGSSWYFDV
HYYGYAMDY
HPRMVTPYAMDY
HFTTATDY
HAMVTTYYAMDY
HGNYAMDY
HDYYVNF
EGNYAMDY
a
FR4
Intra
␮HC
Surface
␮HC
CDR3
Length
(aa)
pl
GRAVYa
WGOGT
WGAGT
WGOGT
WGOGT
WGOGT
WGOGT
WGOGT
WGOGT
WGOGT
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫺
⫺
⫺
⫺
⫺
9
8
9
12
8
12
8
7
8
4.20
5.08
6.08
6.64
5.08
5.06
5.08
5.08
3.67
⫺1.156
⫺0.433
⫺0.956
⫺0.658
⫺0.686
⫺0.033
⫺1.188
⫺0.829
⫺1.225
Grand average of hydropathicity.
CDR3 length in B cell developmental compartments
The sorted population of cells from the BM before pre-BCR expression (B220⫹, CD43⫹/⫺, surface ␮⫺) had ␮HC rearrangements
exhibiting random DH gene segment usage and variable junctional
sequences (n ⫽ 39; Fig. 3A). CDR3 lengths ranged from 5–15 aa
in length: 5% of clones had regions 5 aa long, 2.5% of clones had
regions 8 and 15 aa long, 23% were 9 and 10 aa long, 28% were
11 aa long, and ⬃8% were 12 and 13 aa long (Fig. 3A). Statistically, there was no significant bias toward any one particular
CDR3 length. These data are consistent with our observations in
total BM samples.
The splenic B220⫹IgMhighIgDlow/⫺ B cell population represents
cells that have survived selection in the BM and persisted in the
periphery. In 37 clones analyzed from the JH heterozygous mouse,
DH gene segment usage was random and comparable to VH81X
rearrangements in the BM compartments (Fig. 3B), since DH gene
segments from all families were represented in both populations,
and over-representation or the absence of specific DH gene segments was not evident. In addition, there were no significant differences in hydrophobicity values between the populations before
(average hydrophobicity, 0.82 ⫾ 0.8) and after (average hydrophobicity, 1.08 ⫾ 1.19) pre-BCR selection. The CDR3 length profiles were, however, distinct (Fig. 3B). There was a predominance
of CDR3 regions 9 aa in length. Approximately 49% of rearrangements in this immature B cell population encode a CDR3 of 9 aa.
Other CDR3 lengths isolated from this population were never
⬎14% of the total. The occurrence of CDR3 lengths of 9 aa in the
newly emerging splenic B cell compartment is significantly different from those in B220⫹, CD43⫹/⫺, surface ␮⫺ pro-B, and
early pre-B cells in the BM ( p ⬍ 0.02).
Histidine in the first position in CDR3
FIGURE 2. Flow cytometric sorting of B lymphoid populations from
spleen and bone marrow. Flow cytometric profiles of presort (A) and postsort (B) BM and spleen (Spl) populations are depicted. A, BM suspensions
(upper panels) were triple-stained with fluorochrome-conjugated Abs
against surface B220, surface CD43, and surface ␮HC. Cells were gated
for surface ␮HC-negative cells (a), and B220⫹/CD43⫹ as well as CD43⫺
cells were sorted from the surface ␮HC-negative population (see sorting
gate in b). Spleen cell suspensions (Spl; lower panels) were triple-stained
with Abs against surface B220, IgM, and IgD. Cells were gated for B220positive cells (c), and IgMhigh/IgDlow/⫺ cells were sorted (see sorting gate
in d). B, Staining profiles of representative populations isolated from BM
of JH⫹/⫺ mice (a and b) and from spleens of JH⫹/⫺ (c) and ␭5⫺/⫺ (d) mice.
Collected populations were used to isolate Ig VDJ rearrangements for subsequent sequencing of CDR3 regions. The staining profiles are representative of
four independent experiments. Sort purity was consistently ⬎98%.
The CDR3 of a VDJH gene rearrangement is the only region encoded by all three gene segments. This circumstance allows for
immense diversity due to combinatorial joining and end processing. Despite this extensive variability, examination of the amino
acid sequences of CDR3 allowed us to identify constraints on the
CDR3 structure imposed by the developmental selection process.
The 3⬘ end of the VH81X gene segment has nucleotides CA in
its germline configuration. In the absence of processing of the VH
gene during V to DJH joining, these nucleotides will encode histidine or glutamine at the first position of the CDR3. Processing,
theoretically, could introduce any amino acid. The earliest compartment analyzed, corresponding to the pro-B and pre-B cell compartments, appears to have no bias for any particular junctional
sequence or encoded residue (Fig. 4A). Position 1 of these CDR3s
encoded a range of amino acids varying in charge and polarity;
36% were histidine. The more mature population, however, exhibited an increasing propensity to encode histidine in CDR3 position
1 (Fig. 4B). Seventy-three percent of the HCs from cells of the
selected, newly emerging splenic B cell population, had histidine
at position 1. The predominance of histidine at this position is a
clear bias that was not evident in the pro- and pre-B cell population
( p ⬍ 0.0012). Glutamine, encoded by CAG and CAA, is not found
at equal frequency, as it would be if there were no preference for
CAC or CAT (both encode histidine). The presence of histidine
with a pI of 6.0 is interesting because it is unusual. CDR3 regions
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necessary to isolate populations at specific stages. For the BM
populations, we focused on the B220⫹CD43⫹surface ␮⫺ cells
(pro-B cells), and the large B220⫹CD43⫺ surface ␮⫺ cells, (transitional, early pre-B cells) as cells before and during pre-BCR
selection, respectively. For the spleen populations, we isolated the
B220⫹IgMhighIgDlow/⫺ cells (newly emerging, immature B cells)
as cells after pre-BCR selection. Fig. 2A shows typical profiles of
the presorted populations. Four independent experiments, with one
or two mice per group, generated the results presented. For each
group at least 2 ⫻ 106 cells were sampled. Sorted populations were
consistently ⬎98% pure (Fig. 2B).
The Journal of Immunology
4667
of VH regions rarely have charged amino acids and are generally
neutral or slightly hydrophilic (35, 36). Potentially, histidine is
selected for its association with SLC. This convergence of the
types of junctions that are preferred for the peripheral pool inevitably limits the VH81X-expressing clones that will survive and
may play a role in the reduced number of mature B cells that use
the VH81X gene segment. While the majority of sequences isolated from cells that have traversed the pre-BCR checkpoint code
for a histidine at position 1 of the CDR3, a small number of isolates do not conform to this strategy. However, hydrophobicity, pI,
and CDR3 analyses of His-less VH81X-␮HCs that have successfully traversed the pre-BCR checkpoint did not reveal any distinguishing characteristics of these HCs.
CDR3 characteristics in the absence of pre-BCR assembly
B220⫹IgMhighIgDlow/⫺ B cells were isolated from the spleens of
␭5⫺/⫺ mice. In the absence of SLC assembly, formation of the
pre-BCR is blocked, although immature and mature B cells gradually accumulate in the periphery of ␭5⫺/⫺ animals as the mice
age (10, 37). This population potentially represents cells that have
undergone premature LC assembly, but certainly represents cells
that have not undergone any pre-BCR-mediated selection events.
Analysis of 19 distinct clones isolated from ␭5⫺/⫺ mice reveals
that DH gene segment usage was random. The clones examined
exhibited CDR3 lengths ranging from 5–14 aa in length; 14% of
clones had regions 5 aa long, 24% of clones had regions 8 aa long,
28.5% of clones had regions 9 aa long, 5% of clones had regions
10 aa long, 14% of clones had regions 11 aa long, 9.5% of clones
had regions 13 aa long, and 5% of clones had regions 14 aa long
(Fig. 3C).
The propensity to encode the amino acid histidine in the first
position of the CDR3, observed in splenic B cells that possess an
intact SLC was not observed in splenic B cells from ␭5-deficient
animals. The shift of histidine usage, from 36 to 73%, observed
upon comparison of early BM and splenic B cell populations in the
JH heterozygote does not occur in the absence of ␭5. Immature B
cells from ␭5⫺/⫺ animals encode histidine at position 1 of the
CDR3 in ⬃32% of clones sequenced (Fig. 4C). This value is comparable to the level of histidine usage measured in JH⫹/⫺ B cells
before selection across the pre-B to immature transition.
Restrictions on pre-BCR assembly and cell surface expression
One requirement for a VH81X rearrangement to appear in the peripheral compartments includes the pairing-fitness of a VH81X-
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FIGURE 3. CDR3 length distribution in VH81X-bearing VDJ rearrangements from sorted B cell populations. Transcripts isolated from JH⫹/⫺ or ␭5⫺/⫺
mice were translated as depicted at the top of the figure. CDR3 boundaries were identified as outlined by Kabat and Wu (22). Charts at the bottom summarize
the distribution of Ig HC CDR3 lengths from the sequences described above. Distinct rearrangements were scored as individual clones. A, CDR3 length
distribution in VH81X-bearing VDJ rearrangements from sorted B220⫹CD43⫹/⫺surface ␮⫺ BM of JH⫹/⫺ mice. B, CDR3 length distribution in VH81Xbearing VDJ rearrangements from sorted B220⫹IgMhighIgDlow/⫺ spleen cells of JH⫹/⫺ mice. C, CDR3 length distribution in VH81X-bearing VDJ rearrangements from sorted B220⫹IgMhighIgDlow/⫺ spleen cells of ␭5⫺/⫺ mice. The pattern of CDR3 lengths in ␭5⫺/⫺ splenocytes contrasts with the distribution seen in the spleen compartment of JH⫹/⫺ mice, where most CDR3 lengths in the higher range are eliminated, and CDR3s 9 aa long clearly dominate
the profile.
4668
SELECTION OF Ig HC
Modeling of the CDR3 conformation
FIGURE 4. Amino acid usage in CDR3 regions of individual VH81X
rearrangements. Graphs show the percentage of clones that encode histidine (His) at position 1 of the CDR3 compared with the percentage of
clones that encode an alternate amino acid (X) at position 1. A, Histidine
usage in B220⫹CD43⫹/⫺␮⫺ BM cells from heterozygous JH mice. B, Histidine usage in B220⫹␮highIgDlow/⫺ spleen cells from heterozygous JH
mice. C, Histidine usage in B220⫹␮highIgDlow/⫺ spleen cells from ␭5deficient mice.
To examine whether the structures of VH81X-HCs might reveal a
clue as to why some were expressed on the surface of the A-MuLV
and some were not, we used the SWISS-MODEL Protein Modeling Server. As described in Materials and Methods, we searched
the protein databases for an Ig crystal structure with an amino acid
sequence similar to that of VH81X to use as a template for our
modeling. A single HC Fv, named in the database Protein 31AR1,
␮HC with SLC (8, 27). The repertoire changes that we observed as
a cell passes through SLC-dependent and independent compartments suggest a role for both a ␮HC and the SLC in choosing the
structure of the VH regions that are maintained in the peripheral
repertoire. To directly test the ability of various VH81X-␮HCs to
pair with SLC, we first cloned VH81X sequences (see Table I)
isolated from primary T cells, A-MuLV pre-B lines, and hybridomas into either retroviral or conventional plasmid expression vectors and tested the pairing capability of VH81X-␮HC with the SLC
after introducing the V␮81X-␮HC genes either by retroviral transduction (clones TdT-4, MTBM-4, MTFL-8, MTFL-4, and TdT-1
in Fig. 1 and Table I) or electroporation (clones BC2, 31A, F, and
BFL23 in Table I) in the ␮HC-negative A-MuLV pre-B lines 38B9
and Bine4.8 (see Materials and Methods). These VH81X-␮HC sequences differed from each other only in the CDR3 region, that is,
they used various DH gene segments with different joining sequences. ␮HC synthesis and association with SLC were evaluated
in stably infected or transfected pre-B cell subclones by flow cytometry and immunoprecipitation of metabolically labeled cell lysates. An example of a typical analysis is shown in Fig. 1, B and
C; Table I summarizes the findings of all analyzed sequences.
Nine V␮81X-␮HCs expression constructs were tested (see Table I). Although intracellular ␮HCs could be detected in all stable
clones (Fig. 1, B and C, and Table I), three had clear surface
expression of ␮HC, as revealed by Abs against ␮HC (clone TdT4
in Fig. 1B, and clones BC2 and 31A in Table I), one showed
intermediate levels (clone MTBM-4 in Fig. 1B) and five were negative (clones TdT-1, MTFL-4, and MTFL-8 in Fig. 1B and F and
BFL23 in Table I). Surface pre-BCR expression was verified with
an mAb (SL156) that recognizes an assembled pre-BCR (data not
shown). As expected, all four surface transport competent V␮81X-
FIGURE 5. Protein modeling of VH81X-␮HCs. Protein ribbon structures of VH81XDJ-HCs that are expressed on the cell surface with SLC (A)
or unable to pair with SLC (B). CDR3 loops are shown at the top left portion
of each structure. Solid lines superimposed upon the ribbon structures outline
the amino acids that are part of the hypervariable region. Protein modeling was
performed with the SWISS-MODEL Protein Modeling Server (SWISS-PROT
at http://www.expasy.ch/swissmod/SWISS-MODEL.html).
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␮HCs coprecipitated with the SLC components VpreB and ␭5 (results for TdT-4 and MTBM-4 are shown in Fig. 1C). Interestingly,
three of the four V␮81X-␮HCs (TdT-4, BC2, and 31A) that were
transported to the cell surface (Fig. 1B and Table I) and coprecipitated with the SLC components VpreB and ␭5 (Fig. 1C and Table
I) used a CDR3 region consisting of 9 aa (Table I; CDR3 defined
as above, according to Kabat’s nomenclature (22)). Consistent
with the trend observed in the splenic B cell compartment, all four
pairing VH81X-␮HCs contained a histidine residue at position 1 of
the CDR3 (Table I). One of the four pairing and surface-competent
VH81X-␮HCs (clone MTBM-4 in Fig. 1, A and B) used a CDR3
with 12 aa (Table I). In contrast to the TdT-4-␮HC, the MTBM4-␮HC showed a clearly reduced surface expression despite the
fact that intracellular levels of both ␮HCs were very similar (compare fluorescence intensities for cytoplasmic and membrane staining in Fig. 1B). Therefore, these two VH81-␮HC differ in their
propensity to be transported to the surface, which might be due to
CDR3-mediated differences in binding strength between a ␮HC
and the SLC. However, compared with transport-incompetent
VH81X-␮HCs, there was no bias in the pI or hydrophobicity values of those VH81X-␮HCs that were expressed on the cell surface
(summarized in Table I).
The Journal of Immunology
Discussion
This study reveals specific characteristics in the VH81X ␮HC that
have been allowed to passage through selection and be expressed
in splenic B cells. The majority of such HC have CDR3 lengths of
9 aa and a histidine in position 1. A computer-generated model
pictures the CDR3 backbone structure as planar.
The paradigm of B cell development in mice and humans is
characterized by checkpoints and selection events that ultimately
shape the mature repertoire. The structural complexity of the BCR
and its various functions necessitates a systematic and rigorous
testing of the receptor and its component parts. Many studies have
highlighted the importance of the pre-BCR checkpoint (9, 10, 18).
This stage, immediately following assembly of the HC gene segments, serves to select cells that have made productive rearrangements, expand clones with suitable receptors on the surface, and
regulate the structure of assembled HCs (6, 8, 10, 37). The SLC
components, VpreB and ␭5, are ideally positioned to play a role in
testing and selecting the ␮HCs that will proceed beyond this
checkpoint. Although the crystal structures of the SLC proteins
have not yet been elucidated, sequence and modeling have indicated that together they form a LC-like structure (3, 38, 39). An
amino terminal portion of the ␭5 protein appears to facilitate the
proper folding and assembly of the SLC, while forming an Ig
domain and a region that will be situated close to the CDR3 of the
␮HC upon displacement of BiP (40).
Other studies have revealed that the structure of a ␮HC, as dictated by its sequence, plays an important role in determining
whether a pre-BCR can form. A number of HCs produced by
V(D)J recombination are incapable of pairing with an assembled
SLC (4, 8, 27). Our studies here with the retrovirus expression
system revealed that the HC/SLC interaction seems to be largely
influenced by the structure of the HC’s CDR3. In conditions where
the VH sequence varies solely in the CDR3, only certain conformations allow for pairing with SLC. The finding of specific structural requirements for the CDR3 region of VH81X provides precise
evidence for Minegishi’s findings (40) that only when the SLC
achieves a particular shape can it interact with an HC. If the portion of the SLC that interacts in the vicinity of the CDR3 is disrupted by the conformation of the CDR3, it is reasonable to expect
that the two proteins would have a less efficient interaction. If the
CDR3 has a particular charge, it could potentially distort the SLC
and disrupt the proper fold of the constant domain, thereby inhibiting the displacement of BiP. Alternatively, a bulky CDR3 structure might impede the positioning of the SLC for BiP
supplantation.
We found that the persisting CDR3 structures of maturing B
cells are largely skewed to 9 aa. At this length, the hypervariable
region of the molecule takes on a structure distinct from the configuration assumed by CDR3 domains that were unable to pair
with SLC. Although other lengths can survive in vivo, the majority
are this length. HC that have other CDR3 lengths might bind the
SLC less efficiently, and as a result, fewer complexes might be
deposited on the surface. With a lower pre-BCR density, a B cell
clone would be signaled less efficiently, resulting in less expansion
or even deletion of that clone (37, 41).
A reduction in the average CDR3 length has been observed, as
human B cells progress through development (42). Some evidence
suggests that longer CDR3 regions are associated with autoreactivity (43– 45). Thus, testing of HCs by the SLC could function as
a screen to limit the production of autoreactive cells. In the JH⫹/⫺
mouse splenic compartment, there was a notable reduction in the
number of clones exhibiting longer CDR3s, a reduction not observed in the splenic compartment of ␭5⫺/⫺ animals. This observation provides support for the hypothesis that longer CDR3s are
associated with autoreactivity and consequently may be more
readily eliminated under normal circumstances (46). In this fashion, the SLC could function to minimize or regulate autoreactivity
by modulating the HC repertoire.
CDR3 length has been directly correlated to the potential for
interaction between Ig HC and LC (47). In these analyses there
were notable differences in the shape assumed by the coupled
CDR3 regions depending on the lengths involved. Both HC/LC
pairing and HC/SLC pairing may play a role in shaping the repertoire in the newly emerging B cell population. The correlations
and structural limitations of these studies can easily be extended to
the pairing of an HC and SLC. The fitness of the interaction would
be determined by the shape created by the CDR3.
Our experiments reveal that the repertoire shift observed in the
splenic B cell population subsequent to selection across the pre-B
cell transition does not occur in the absence of pre-BCR formation.
This finding directly implicates the pre-BCR and the SLC in particular in determining the final configuration of the B cell repertoire. This central role for the pre-BCR in repertoire selection extends the function of the receptor in the developmental paradigm.
The limitations imposed by the SLC may actually serve to maximize the efficiency of the developing immune system. The ultimate
goal of the system is to generate a B cell population where cells
produce HCs capable of pairing efficiently with LCs, forming receptors that broaden and expand Ag binding capabilities. Facilitating progression of only those cells capable of competent HC/LC
interactions later in development increases the probability of producing functional, viable cells. Testing through the SLC may also
serve to recruit groups or families of preferred LCs that augment
the diversity at the paired CDR3 domains. Studies have shown that
particular HC and LC combinations are capable of assuming multiple conformations depending on the components involved (48).
LCs that amplify this isomerism may have the potential to bind
multiple Ags and thereby increase the efficiency of an immune
response.
The biased representation of CDR3 lengths of 9 aa in the JH
heterozygotes and the absence of this skewing in ␭5-deficient animals may represent structural restrictions that have evolved over
time and are employed to augment the diversity of the HC in this
context. Using 9 aa in the CDR3 loop may provide the greatest
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was identified as having the most sequence similarity to the
VH81X variable domain (⬃72% identity). We then used protein
31AR1 as the template and modeled on it the 10 VH81X-HCs. The
data generated by the modeling program were converted, assembled, and viewed using the SWISS PDB viewer (28, 29). These
theoretical models were positioned to align the identical regions
(the ␤ strands of the framework regions), while outlining their
differences (the CDR3 regions). The results of this structure prediction analysis are presented in Fig. 5. The variable domains of
the four molecules that were expressed on the cell surface were
essentially superimposable in all regions, including the backbone
tracing of the hypervariable loop of CDR3 (shown with R groups
in blue). The CDR3 backbone is a simple loop in the single plane,
although the R groups of the residues are distinct. On the other
hand, the molecules that did not go on the surface have different
CDR3 structures (backbone is shown in red). CDR1, CDR2, the
framework region, and the C region, as expected by their near
identities, are more similar to each other, although not completely
superimposable; their positioning is probably influenced by the
CDR3 residues. Thus, the four molecules that are expressed on the
surface have a superimposable backbone structure despite differences in the sequences of their CDR3 regions.
4669
4670
selecting the cells that survive. In addition to the other factors that
restrict the presence of VH81X in the periphery, germline-encoded
features of the VH81X HC protein may disfavor a competent interaction with SLC. Only under certain conditions is the complex
suitably organized to warrant maintenance of the cell.
Functional assembly of a pre-BCR is an integral step in promoting the efficient transition of cells through the pro-B cell stage
to an immature stage of development. The facility with which a
particular HC associates with SLC has a direct impact on the survival of that cell (4). The studies that we have undertaken here
indicate that the length of the CDR3 region can have implications
for the suitability of fit and the subsequent fate of the cell. In these
studies the most striking evidence of CDR3 length selection was
observed across the pre-B transition; this was further confirmed by
the lack of length selection in the absence of ␭5 and the pre-BCR.
Further studies will reveal whether the absence of an SLC selection mechanism affects the characteristics of LCs that are able to
pair with HC at later stages in development. Comparison of the LC
repertoire in wild-type animals, in which pre-BCR-selective programs are intact, and in ␭5⫺/⫺ animals will reveal whether the
CDR3 restriction directly imposes identifiable features relevant to
compatibility and LC pairing.
Acknowledgments
We are grateful to Aaron Marshall, Anne Tran, Dirk Mielenz, and Ivan
Fong for valuable comments. We thank Queenie Lam and Stacy Hirano for
expert technical assistance. We thank Ana Cumano for critical reading of
the manuscript and thoughtful comments, and Ton Rolink, Fritz Melchers
and Jan Jongstra for providing mAbs against the pre-BCR and pre-BCR
components.
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diversity for the CDR3 without compromising the structural integrity of the receptor. Some studies have indicated that too many
residues in the loop may affect the stability and rigidity of the Ig
fold (49). The interaction between framework regions and CDRs
may also impact upon molecular structure in the vicinity of HC/LC
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at position 1 in the CDR3 of VH10 rearrangements, another HC variable region gene associated with restricted expression patterns (55).
Our conclusions differ slightly from those of Hayden et al. (56),
perhaps due to the approach taken by the authors. While their
studies looked at the characteristics of VH81X-HCs that used a
number of different JH gene segments (i.e., JH1 through JH4), we
have focused on VH81X rearrangements using JH4. In this manner,
we were able to attribute the selection or enrichment observed as
being due solely to the differences in the CDR3 regions used.
Therefore, structural contributions from amino acids encoded by
the JH region and their confounding effects can be negated. This
system allows us to directly test and assess the contribution of the
CDR3 to the HCs that are used/successful in the periphery. Based
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sequence. Clearly, the shape assumed by the HC plays a role in
SELECTION OF Ig HC
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