Download Human Ig heavy chain CDR3 regions in adult

International Immunology, Vol. 9, No. 10, pp. 1503–1515
© 1997 Oxford University Press
Human Ig heavy chain CDR3 regions in
adult bone marrow pre-B cells display an
adult phenotype of diversity: evidence for
structural selection of DH amino acid
sequences
Frank M. Raaphorst1,4, C. S. Raman2, Joseph Tami1,5, Michael Fischbach1,3 and
Iñaki Sanz1,6
Departments of 1Medicine and 2Biochemistry, The University of Texas Health Science Center at San
Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, USA
3Audie L. Murphy Division, South Texas Veterans Health Care System, San Antonio, TX 78284, USA
4Present
address: Department of Microbiology, The University of Texas Health Science Center at San
Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, USA
5Present address: ISIS Pharmaceuticals, Carlsbad Research Center, 2292 Faraday Avenue, Carlsbad,
CA 92008, USA
6Present address: University of Rochester Medical Center, Rheumatology/Immunology Unit, 601 Elmwood
Ave, Box 695, Rochester, NY 14642, USA
Keywords: antigen receptor, B lymphocyte, repertoire, VDJ rearrangement
Abstract
Ig repertoires generated at various developmental stages differ markedly in diversity. It is well
documented that Ig H chain genes in human fetal liver are limited with regard to N-regional
diversity and use of diversity elements. It is unclear whether these characteristics persist in pre-B
cell H chain genes of adult bone marrow. Using Ig H chain CDR3 fingerprinting and sequence
analysis, we analyzed the diversity of Ig H chain third complementarity determining regions
(HCDR3) in adult bone marrow pre-B and mature B lymphocytes. Pre-B cell HCDR3 sequences
exhibited adult characteristics with respect to HCDR3 size, distribution of N regions and usage of
diversity elements. This suggested that pre-B cells in adults are distinct from fetal B cell
precursors with regard to Ig H chain diversification mechanisms. At the DNA sequence level,
HCDR3 diversity in mature B cells was similar to that in pre-B cells. Pre-B HCDR3s, however,
frequently contained a consecutive stretch of hydrophobic amino acids, which were rare in mature
B cells. We propose that highly hydrophobic pre-B HCDR3s may be negatively selected on the
basis of structural limitations imposed by the antigen binding site. At the same time, usage of
hydrophilic HCDR3 sequences (thought to support HCDR3 loop formation) may be promoted by
positive selection.
Introduction
The variable (V) region of Ig H chains is generated by
recombination of variable (VH), diversity (DH) and joining (JH)
gene segments (1). Ig H chains bind antigen through three
complementarity determining regions (HCDR1–3) (2,3).
HCDR1 and HCDR2 are encoded by VH segments and are
not affected by recombination (1,3–8). By contrast, the HCDR3
is primarily generated by rearrangement and modification of
DH genes (1). The borders of rearranging gene elements can
be processed extensively during gene assembly: nucleotides
can be randomly removed from VH, DH and JH segments by
exonuclease, and two types of nucleotides can be inserted
between recombination partners. N segments are randomly
Correspondence to: I. Sanz
Transmitting editor: L. Steinman
Received 26 February 1997, accepted 19 June 1997
1504 HCDR3 diversity in human adult bone marrow
added by terminal deoxynucleotidyl transferase (TdT) and
are not encoded in the germline (9–11). P nucleotides reflect
a palindromic repeat of the germline sequence at the border
of VH, DH or JH elements (12). Additional HCDR3 diversity
can be produced by DH inversion, DH–DH fusion and gene
conversion (13,14).
The combined variation in sequence and size of the HCDR3
generates an enormous diversity in Ig antigen binding sites,
especially in humans (7,14–17). The importance of HCDR3
diversity is illustrated by the fact that this region forms the
center of the antigen binding site and provides essential
residues for antigen binding (2,3,18–22). Given the central
location of the HCDR3 within the antigen-binding pocket,
major changes in HCDR3 length, sequence and/or bulk are
thought to change antibody specificity directly, and may
impose conformational changes in the Ig molecule.
An interesting aspect of HCDR3 structures is that their
diversity is dependent on the developmental stage of the
individual. Human fetal HCDR3 regions are generally smaller
than those found in the adult, which can be partly explained
by distribution of N regions. Up to 25% of human fetal Ig H
chain genes lack N-regional diversity and the N regions that
are generated in the remainder of fetal VH–DH–JH junctions
are generally smaller than those in adult Ig H chain genes
(14,23–27). In addition, fetal HCDR3 sequences frequently
express the JH-proximal DH element, DQ52, which is virtually
undetectable in neonatal and adult Ig H chain rearrangements
(14,23–36). Finally, adult HCDR3 regions generally favor the
expression of the JH4 and JH6 segments over the JH3 segment,
whereas in fetal sequences this ratio is reversed.
Despite the relative absence of N-regional diversity, some
human fetal liver (FL) HCDR3s are of adult size. The ability
to add N segments is thought to increase gradually during
human fetal development and peripheral Ig H chain assemblies in neonates are as diverse as those in adults (14,28,29).
It is unclear whether the transition from a ‘fetal-type HCDR3’
to an ‘adult-type HCDR3’ reflects intrinsically different diversification mechanisms operating at different timepoints in ontogeny. A significant gap in our understanding of this issue in
humans is HCDR3 diversity in adult bone marrow (BM) B cell
progenitors. In this study we address the question whether
fetal-type HCDR3s persist in B cell precursors of human adult
BM, and we present a detailed analysis of HCDR3 assemblies
in pre-B and mature B lymphocytes. Both these populations
expressed HCDR3 sequences of an adult nature, suggesting
that pre-B cells in adults are distinct from fetal B cell precursors
with regard to Ig H chain diversification mechanisms. In
addition, we provide evidence suggesting that the adult BM
Ig H chain repertoire may be shaped by selection mechanisms
imposed by structural requirements of the antigen binding site.
Methods
Isolation of B cell populations
Fetal liver (FL), adult peripheral blood mononuclear cells
(PBMC) and adult BM were collected as described previously
(14,26). Pre-B and mature B lymphocytes were isolated from
adult BM (obtained with IRB approval from consenting adults)
as described in detail elsewhere (37). Cell isolation was
Fig. 1. Isolation of adult BM B cell populations using FACS. CD10–/
CD201 cells represent immature B cells from the BM and mature B
cells from the periphery; CD101/CD201 cells represent pre-pre-B/
pre-B lymphocytes (38).
independent of Ig H chain production and based on the
expression of the membrane markers CD10 and CD20. Briefly,
53107 buffy-coat adult BM cells were stained with phycoerythrin-labeled anti-CD20 antibody (clone L27; Becton
Dickinson, San Jose, CA) and FITC-labeled anti-CD10 antibody (clone W8E7; Becton Dickinson) and subjected to FACS
sorting using a FACStar Plus (Becton Dickinson) (37). The
cells were illuminated with 200 mV, 288 nm laser light from
an argon laser. FITC fluorescence was collected through a
530/30 nm band pass filter, phycoerythrin fluorescence
through a 575/26 nm band pass filter (Oreil Optical, Stamford,
CT). The FACStar was calibrated with glutaraldehyde-fixed
chicken erythrocytes. Data was analyzed with DP2 and 3
software from the NIH DCRT on a PDP 11/73 computer
(Digital Equipment, Maynard, MA), and collected in log format
(contours represent cell counts). Data collection was done
on viable cells determined by forward light scatter pulse
height and width, and right angle light scatter pulse height.
Figure 1 illustrates the populations which were isolated:
CD10–/CD201 cells represent immature B cells from the BM
and mature B cells from the periphery; CD101/CD201 cells
represent pre-pre-B/pre-B lymphocytes (38).
PCR amplification of Ig H chain rearrangements
Methods used for RNA isolation and cDNA synthesis have
been described elsewhere (37). H chain rearrangements
were amplified in a primary PCR using 2 µl cDNA and 20
pmol of a 59 primer recognizing nucleotides 1–24 of the
framework region (FR) 1 of VH1 and VH3 subgroups (VH59:
59-CAGGTGCAGCTGCTCGAGTCTGGG-39) and 20 pmol of
a 39 primer specific for nucleotides 334–358 of Cµ (Cµ1; 59TGGGACGAAGACGCTCACTTTGGGA-39). The 59 primer was
originally developed for the construction of phage expression
libraries of Ig H chain genes employing VH1 and VH3 genes,
but may cross-hybridize with other VH groups (39). Phage
expression libraries generated with this primer contained VH1,
VH3, VH4 and VH6 elements, including frequently expressed
genes like V3–23 (40–44). Since up to 80% of the human Ig
H chain genes employs either a VH1, VH3 or VH5 segment
(35,36,45), the primary PCR should amplify a large number
of rearrangements employing different VH genes. PCR was
HCDR3 diversity in human adult bone marrow 1505
performed in a final volume of 100 µl, in the presence of
50 mM KCl, 10 mM Tris–HCl (pH 9.0), 0.1% Triton X-100,
1.5 mM MgCl2, 0.2 mM of each dNTP and 2.5 U of Taq DNA
polymerase (Promega, Madison, WI). Cycles consisted of
1 min denaturation at 94°C, 45 s annealing at 62°C and 1 min
extension at 74°C (final cycle 10 min). Since the amount of
Ig H chain transcripts could vary between isolates, we took
samples from the primary PCR reactions to assess when the
PCR was in the linear phase of amplification. For FL, adult
BM, APBL, a product became detectable by ethidium bromide
staining in an agarose gel after ~30 cycles; pre-B and
mature B cells generally needed four cycles more. PCR were
saturated six cycles later and the cycles between these two
timepoints were taken as the linear phase of amplification.
This correlated well with a similar PCR on the constant region
of Ig H chain genes, performed with a 59 Cµ primer (59AAGTACGCAGCCAACTCA-39) and the 39 Cµ1 primer. Then,
1 µl of the primary PCR, diluted 50 times in water and sampled
in the log phase, was used in a nested PCR in order to
generate PCR fragments that consisted of the HCDR3 region
and part of the constant region. Nested PCR was performed
with 20 pmol of a 59 primer which recognizes the 39 end of
.95% of all known VH gene elements [panVH: 59ACGGCCGTGTATTACTGT-39 (14)] and 20 pmol of a 39 Cµ
primer situated at 254 bp from the beginning of Cµ (Cµ2: 59TATTTCCAGGAGAAAGTGAT-39); the reaction conditions were
similar to those of the primary PCR, except that annealing of
the primers was performed at 45°C.
HCDR3 fingerprinting and cloning of rearrangements
HCDR3 fingerprint profiles were generated essentially as
described previously (37). Briefly, nested PCR products were
obtained in the log phase of amplification (cycles 21–25).
Then 10 µl of the 23 cycle reaction was separated on a 5 or
6% denaturing polyacrylamide gel containing 7 M urea in
0.53TBE. The DNA was visualized by silver staining using the
Promega DNA silver staining system according to Promega
protocols (Promega).
The nested PCR products were also used for sequence
analysis by random cloning and isolation of size-selected
HCDR3 segments (ù25 amino acids). These procedures were
performed as described (37). Recombinant clones were
identified by colony hybridization with a DNA probe specific
for the human JH elements. JH1 colonies were sequenced
using double-stranded templates with Sequenase version 2
(USB, Cleveland, OH) and an internal Cµ primer (Cµ3: 59AATTCTCACAGGAGACGAGG-39) specific for the constant
region of the rearrangements.
Analysis of HCDR3 regions
The HCDR3 length was assessed by counting the number of
amino acids between the end of FR3 (amino acid position
94) and the first residue of FR4, indicated by a Trp residue
that is conserved in all JH segments (residue 102). DH and
JH elements in the HCDR3 regions were identified using
DNASTAR software (DNASTAR, Madison, WI) by searching
for homology with all known human Ig H chain VH, DH and JH
germline sequences, as made available through the V BASE
directory (website: http://www.mrc-cpe.cam.ac.uk/imt-doc/
vbase-home-page.html) (45). Also included in the analysis
were the currently known sequences of DIR elements, allelic
forms of JH elements, and the recently identified DM3 and
DM5 DH elements (46–52). Identity of DH segments was
assessed in straight and inverted orientation using the DNASTAR COMPARE and SEQCOMP programs. For HCDR3
regions ,12 amino acids, a minimum of six consecutive
bases identity in the alignment was scored as positive.
For HCDR3 regions .12 amino acids, nine consecutive
bases minimally were required. In the case of DIR genes, 1
bp deletion or insertion and one mismatch were allowed in
the sequence. Sequences that did not match to any known
VH, DH, DIR or JH element were counted as N or P regions.
N regions .12 nucleotides were analyzed in more detail by
comparing them to the most recent update of GenBank using
the BLAST sequence similarity searching tool, provided by
the National Center for Biotechnology Information. The blastn
program was used to identify DNA sequence similarity and
the blastx program was used to screen the nucleotide query
sequence, translated in all reading frames, against Ig H chain
protein sequences (53). Reading frame 1 (RF1) of the DH
elements was defined as the DH amino acid sequence starting
with the first codon of the DH coding region. RF2 and RF3
were the amino acid sequences obtained after a 1 and 2
nucleotides frameshift respectively. Hydropathy profiles for
the HCDR3 regions were created using the CAMELEON
program (Oxford Molecular, Oxford, UK) according to the
Kyte and Doolittle hydrophobicity scale (54). The profiles
were generated with a 7 amino acids scanning window. Chi
square analysis of HCDR3 amino acids sequences was
performed with the Sigma Stat program, version 1.0.
Results
HCDR3 fingerprinting on adult BM pre-B and mature B
lymphocyte subpopulations
As a first step in the analysis of adult BM HCDR3 diversity,
we used HCDR3 fingerprinting (37 and references therein) to
visualize the general size distribution of HCDR3 regions in
one full adult BM, two adult PBMC (PBMC1–2), and adult BM
pre-B (CD101/CD201) and mature B (CD10–/CD201) cells.
We also included three 12-week-old FL samples (FL1–3),
where the HCDR3 regions are expected to be smaller. The
result of this analysis is presented in Fig. 2. The majority of
HCDR3 in pre-B cells fell in the range of from 7 to 30 amino
acids [with faint bands visible of 31 and 32 amino acids (data
not shown)]. The corresponding mature B cells (obtained
from the same adult BM) exhibited HCDR3 in a slightly
narrower range of 7–25 amino acids, while HCDR3s in adult
PBMC and total adult BM ranged from 7 to 22 amino acids.
The longest human HCDR3s reported in the literature can
reach lengths of up to 26 amino acids (14,16,17). In the
current study, HCDR3 regions .26 amino acids were detected
in the adult BM pre-B cell population. In order to determine
whether the number of PCR cycles affected size distribution
of pre-B and mature B cell HCDR3 regions, we repeated
the analysis in a second sample. Fingerprint profiles were
generated in the logarithmic phase of amplification and
at saturation of the PCR. Both PCR conditions produced
fingerprint patterns that were not different from those shown
1506 HCDR3 diversity in human adult bone marrow
Fig. 2. Ig HCDR3 fingerprinting on human fetal and adult B lymphocytes. ABM 5 full adult BM; APBMC 5 adult peripheral blood mononuclear
cells. CD10– 5 CD10–/CD201 mature B cell subpopulation; CD101 5 CD101/CD201 pre-B cell subpopulation, both obtained by FACS sorting
from the same adult BM. Profiles were generated on a denaturing 5% polyacrylamide gel. MW 5 mol. wt marker, prepared by fingerprinting
five clones with HCDR3 of known size. Lengths of HCDR3 regions are given in nucleotides (nt) and amino acids (aa).
in Fig. 2 (data not shown). In contrast to the adult samples,
FL-derived HCDR3 regions were smaller and generally ranged
in size from 4 to 16 amino acids. These results extended earlier
findings (37) and were representative of four experiments
performed on the same samples with varying amounts of
cDNA (data not shown). The size distributions correlated well
with previous estimations of fetal and adult HCDR3 sizes
based on random DNA sequencing (see Discussion).
Sequence analysis of HCDR3 diversity of adult BM pre-B and
mature B rearrangements
HCDR3 diversity in adult BM B cell precursors was further
investigated by random sequencing. We obtained 63 independent rearrangements from CD101/CD201 pre-B cells (PBT
clones). From the corresponding CD10–/CD201 mature B
cells, 42 clones were generated (MBT clones). The majority
of the rearrangements (.95%) were productive.
General characteristics of the randomly cloned pre-B and
mature B sequences are summarized in Table 1. The mean
length of the HCDR3 region in pre-B and mature B clones
was 15 and 14 amino acids respectively. Pre-B HCDR3
sequences ranged from 6 to 31 amino acids and mature B
cell HCDR3 sequences ranged from 7 to 24 amino acids.
These sizes were consistent with the fingerprinting results
(Fig. 2). All of the rearrangements exhibited N-regional diversity. Seventy percent of the pre-B clones and 79% of the
mature B clones contained N regions at both the VH–DH
junction and DH–JH junction. The remaining clones lacked N
nucleotides at one of these junctions (Table 1). The average
length of N regions in pre-B and mature B clones was 5
nucleotides at the VH–DH junction and 4 nucleotides at the
DH–JH junction. The size distribution of N regions in clones with
an identifiable DH element is illustrated in Fig. 3. Approximately
12% of the clones contained long N regions ranging in size
from 13 to 26 nucleotides. Since TdT has a preference for G
and C nucleotides (11,55), and these long N regions were
not always GC-rich, they could contain an unidentified DH
element with N regions of undetermined length. In addition,
some DIR-like sequences were identified in junctional regions
that fell outside the limits we set. In these instances the N
regions could reflect TdT activity or rearrangement of a DIR
element, which would make the clone a DH–DIR or DIR–DH
fusion (exemplified by PBT-52 in Fig. 4). Finally, 29% of the
pre-B cell sequences and 44% of the mature B cell sequences
contained possible P nucleotides which were in most
instances from the VH junctions. The mean length of the P
regions was 2 nucleotides, but they could be up to 7 nucleotides in size (as illustrated by clone PBT-180 in Fig. 4).
Except for three clones, all rearrangements contained an
identifiable DH element. We rarely observed mutations in the
DNA sequence of these DH genes. Up to 90% of the
sequences contained a single DH gene (Table 1). Approximately 3% of the pre-B clones and 12% of the mature B
clones exhibited good DIR homology, although in the case of
the sequences from mature B cells this may be an overestimation since many of these sequences were GC-rich HCDR3.
These values rose to 16 and 29% if sequences with less than
optimal DIR homology were counted as well (data not shown).
DH–DH and DH–DIR fusions were detected in 11 and 10% of the
pre-B and mature B clones respectively (Table 1). Inclusion of
DH–DH or DH–DIR rearrangements where one of the fusion
partners exhibited less than optimal homology raised these
values to ~30% for the pre-B clones and 29% for the mature
B clones. Examples of DH–DH and DH–DIR rearrangements
are given in Fig. 4 (good homology: MBT-186; less than
optimal homology: PBT-52). Only two inversions of a conventional DH gene (DLR4) were found.
The usage frequencies of DH elements in the pre-B and
mature B sequences are presented in Table 2. Both pre-B
and mature B clones employed DH-elements in a non-random
fashion. The DA family and DH elements belonging to the minor
D5 locus were never detected. By contrast, 40% of the preB clones and 60% of the mature B clones used DXP genes.
Likewise, DLR genes were employed in 25 and 14% of the
rearrangements in PBT and MBT clones respectively. The
DQ52 gene, frequently expressed in fetal rearrangements,
was absent from all the adult BM HCDR3 sequences. The DH
elements were used in all three RF (see below).
HCDR3 diversity in human adult bone marrow 1507
Table 1. General characteristics of Ig H chain CDR3 regions in adult BM
Length HCDR3
N regions
Size N regions
DH elements
JH elements
mean
range
at VH–DH and DH–JH
at VH–DH only
at DH–JH only
no N regions
N mean at VH–DH
N mean at DH–JH
% single DH
% DH–DH fusion
% DIR
% J H1
% JH2
% J H3
% J H4
% J H5
% J H6
Pre-B (PBT)
(n 5 63)
Mature B (MBT)
(n 5 42)
Pre-B (PBUF)
(n 5 11)
15
6–31
70
15
15
0
5
4
89
11
3
0
4
14
32
17
33
14
7–24
79
18
3
0
5
4
90
10
12
0
5
14
57
12
12
27
25–31
90
10
0
0
13
8
70
30
10
0
0
0
0
0
100
Length of HCDR3s is given in amino acids; size of N regions is given in nucleotides. PBT (pre-B cell total repertoire): HCDR3 sequences
derived from randomly cloned human adult BM CD101CD201 pre-B cells. MBT (mature B cell total repertoire): HCDR3 sequences derived
from randomly cloned human adult BM CD10–CD201 mature B cells. PBUF (pre-B cell upper fraction): HCDR3 sequences obtained from
human adult BM pre-B cells selected for HCDR3 ù25 amino acids. The distribution of DH elements is given for clones that contained an
identifiable DH segment. The percentages of clones with a single DH or DH–DH fusion includes clones with DIR-like regions; ‘% DIR’ reflects
the clones with DIR-like sequences.
Table 2. Usage of conventional DH families in adult BM
HCDR3 sequences
DH family
PBT
MBT
PBUF
DA
DHFL16
DK
DLR
DM
DN
DXP
DQ52
–
2
12
25
7
14
40
–
–
9
0
14
11
6
60
–
–
–
–
58
–
–
42
–
Usage frequencies are given as percentages in rearrangements
with an identifiable DH element. For legend: see Table 1.
JH usage profiles in PBT and MBT sequences were skewed
to the more 39 JH segments (Table 1). None of the rearrangements contained JH1, while JH2 was detected once in the
pre-B clones and 3 times in the mature B clones. JH3, JH4
and JH5 were found in 14, 32 and 17% of the pre-B sequences
respectively, and in 14, 57 and 12% of the mature B
sequences. Usage of JH6 was 33% in pre-B clones and 12%
in mature B clones. The sequences of the recombined JH
elements reflected the germline sequence of JH alleles.
Analysis of HCDR3 diversity in pre-B Ig H chain rearrangements with HCDR3 ù25 amino acids
Although CDR3 fingerprinting demonstrated HCDR3 .25
amino acids in pre-B cells (data not shown), only three of the
randomly cloned pre-B cell sequences (PBT-180, PBT-52 and
PBT-51) contained a HCDR3 of this size. Since HCDR3 .25
amino acids have not been studied in detail, we isolated
these HCDR3 regions directly from the polyacrylamide gels
(37). Eleven unique rearrangements were obtained (PBUF
sequences). Two of these, PBUF-BC2 and PBUF-DE1, had
been identified previously by random sequencing of pre-B
cell rearrangements (PBT-180 and PBT-52 respectively). The
sequences are characterized in Tables 1–3. The HCDR3 sizes
ranged from 25 to 31 amino acids; the mean length of N
regions was 13 nucleotides at the VH–DH junction and 8
nucleotides at the DH–JH junction. The greater length of N
regions in PBUF clones, as compared to pre-B and mature
B sequences, is likely related to the fact that they were
obtained by selection for a larger HCDR3. Several PBUF
clones expressed long N regions which could represent an
unknown DH sequence. These sequences frequently shared
limited homology with DIR genes in the N regions (e.g. PBUFDE-47 in Fig. 4). P nucleotides were detected 4 times at the
VH–DH junction and twice at a DH–DH and a DH–JH junction.
The mean length of these regions was 3 nucleotides, with
seven consecutive P nucleotides detected at the VH–DH
junction of PBUF-DE1 (Fig. 4). The PBUF sequences exclusively used the longer DH elements, belonging to the DLR and
DXP families. All of these had rearranged by deletion. As
noted earlier for the pre-B and mature B clones, DH elements
generally exhibited a germline DNA sequence and none of
the PBUF sequences used the DQ52 gene. Two sequences,
PBUF-BC35 and BC2, provided clear evidence for DH–DH
fusion (Fig. 4). The remaining sequences contained a single
DH element. Finally, all of the PBUF clones used JH6.
Amino acid usage profiles of HCDR3 regions
The predicted amino acid sequence of HCDR3 segments
encoded by the N regions and DH elements is shown in
Fig. 5. In agreement with previously published studies of
1508 HCDR3 diversity in human adult bone marrow
Fig. 3. Distribution and size of N regions in adult BM HCDR3 sequences. (A) N-regional diversity at the VH–DH junction. (B) N-regional diversity
at the DH–JH junction. Open bars represent PBT clones (CD101CD201 pre-B cells) and black bars represent MBT clones (CD10–CD201
mature B cells). nt 5 length in nucleotides, # 5 number.
mouse and human HCDR3s (56,57), Gly, Tyr and Ser residues
were detected at high frequency; together these amino acids
accounted for ~40% of the amino acids. Twenty-six percent
of the pre-B HCDR3 regions and all of the pre-B HCDR3
sequences .26 amino acids (PBUF clones) contained 39
Tyr-rich regions (of four to five consecutive Tyr residues)
originating from the JH6 gene element. In contrast, only
three MBT HCDR3 sequences (7%) displayed a 39 Tyrrich sequence. In addition, pre-B cell HCDR3 sequences
frequently encoded continuous stretches of hydrophobic
amino acids. This is best illustrated by the occurrence of Val
and Ala. When only continuous stretches of at least four Val/
HCDR3 diversity in human adult bone marrow 1509
Fig. 4. Examples of HCDR3 sequences cloned from human adult BM pre-B and mature B cells. Shown is the N DH–N sequence, flanked at
the 59 side by the VH element and at the 39 side by the JH element. Sequences in bold type exhibit homology to a known DH or DIR element,
the sequence of which is given under the HCDR3 sequence. All other sequences are N regions or P nucleotides; P nucleotides are boxed.
Lower case letters indicate a difference of the HCDR3 sequence with the germline sequence; sometimes gaps were introduced for optimal
alignment (indicated by a dash). A square bracket indicates a germline end. For details and comments on individual sequences: see text.
Accession numbers are given in Fig. 5.
Ala residues were considered, 13 PBT clones (21%) and four
PBUF sequences (37%) contained a stretch of four to nine
highly hydrophobic amino acids. In contrast, only two MBT
clones (5%) encoded consecutive hydrophobic amino acids
(4 and 5 amino acids respectively). The difference between
PBT and MBT clones was statistically significant (χ2, P 5
0.046) and suggested a shift from hydrophobic to more
hydrophilic amino acids in the mature B cell HCDR3s.
The impact of the amino acids composition on the HCDR3
can be analyzed by hydropathicity plots. These reflect the
mean hydrophobicity value of overlapping amino acid
sequences within windows of defined length, spanning the
HCDR3. Since the PBUF HCDR3 regions are of sufficient size
to allow such an analysis, the effect of hydrophobic amino
acids is illustrated in Fig. 6 for two PBUF clones. PBUF-DE7
carries a Val-rich sequence in the HCDR3 and displays a
positive value in a hydropathicity plot. This is indicative of a
highly hydrophobic HCDR3 sequence. By contrast, PBUFDE47 does not express a Val-rich amino acid sequence and
displays primarily negative values (corresponding with a
hydrophilic amino acid sequence).
Continuous hydrophobic amino acid sequences in pre-B
HCDR3 segments originated from DH elements used in the
rearrangements. Human DH elements exhibit three character-
1510 HCDR3 diversity in human adult bone marrow
Fig. 5A. Amino acid sequences of HCDR3 regions derived from adult BM pre-B and mature B cells. HCDR3 regions in CD101/CD201 pre-B
cells (PBT and PBUF clones). VH 5 sequence derived from the variable region segment; N(D)N 5 sequence derived from N-regional diversity
and diversity elements; JH 5 sequence derived from the JH element. AA 5 length of the HCDR3 in amino acids. Amino acids in bold type
represent continuous highly hydrophobic sequences. The sequences of the rearrangements described in this paper are available from EMBO
GenBank/DDBJ under accession numbers listed as ACC#.
HCDR3 diversity in human adult bone marrow 1511
Fig. 5B. HCDR3 regions in CD10–/CD201 mature B cells (MBT clones). See legend to Fig. 5(A).
istics, distributed over the three RFs: one RF encodes a
hydrophilic amino acids sequence (usually rich in Gly and
Tyr), another RF encodes a hydrophobic sequence (rich in
Val, Ala, Met and Leu) and a third RF usually contains a
stop codon (48,49,58). The shift to more hydrophilic HCDR3
sequences in the mature B clones was reflected in a shift in
DH RFs. This was, however, obscured by the fact that the
amino acids characteristic of DH RF sequences exhibit a
family-specific distribution (48,49,58). For instance, DLR
genes carry a hydrophobic sequence in RF3, whereas DK
genes encode a hydrophobic sequence in RF1 and DA genes
in RF2. We therefore tabulated usage of the DH amino acids
sequences not according to RF, but according to biochemical
characteristics of the amino acid sequence (Table 3). The DH
sequence that usually encodes a stop codon was used at
the lowest frequency in both pre-B cells and mature B cells
(7.1 and 5.8% of the sequences respectively). Distribution of
the other two RFs showed a significant shift from hydrophobic
DH sequences in pre-B cells to hydrophilic DH sequences in
mature B cells. The frequency of the hydrophobic sequence
dropped from 35% in the pre-B clones to 14.4% in the mature
B clones (χ2, P 5 0.034). In most instances, the mature B
cell clones that did use the hydrophobic sequence had lost
most or all of the hydrophobic amino acids, presumably
by exonuclease. Finally, the frequency of the hydrophilic
sequence (encoding Gly and Tyr) increased from 57.9% in
the pre-B sequences to 79.8% in the mature B sequences
(χ2, P 5 0.029). The RF distribution in PBUF clones was
similar to that in the PBT clones.
Discussion
Adult BM rearrangements in pre-B and mature B cells are of
an adult phenotype
We have shown that B cell progenitors in adult BM generate
an Ig H chain repertoire with adult characteristics that is
distinct from the repertoire of B cell progenitors in fetal tissue.
Fetal and adult repertoires can be distinguished by size of
the HCDR3 regions and usage of particular DH and JH
elements. Approximately 42% of HCDR3s in FL are 9 amino
acids or smaller (14,23–27,31), whereas an estimated 80%
of adult HCDR3 sequences are .9 amino acids (32–36). The
distribution of HCDR3 sizes in adult BM pre-B and mature B
lymphocytes, as assessed in the current study by HCDR3
fingerprinting, fell in the range of HCDR3 lengths in adult
PBMC. The fingerprinting results further demonstrated that
pre-B and mature B cell HCDR3s were significantly larger
1512 HCDR3 diversity in human adult bone marrow
Fig. 6. Examples of hydropathicity plots of pre-B lymphocyte HCDR3 regions. Plots were generated using a 7 amino acid window according
to the Kyle and Doolittle algorithm. Peaks displaying a positive value of hydropathicity are strongly hydrophobic; peaks with negative values
are hydrophilic. Consecutive hydrophobic amino acid residues are underlined in the amino acid sequence.
Table 3. Distribution of biochemical characteristics of DH amino acid sequences in HCDR3 regions of adult BM B lymphocytes
DH gene
PBT
MBT
PBUF
Hydrophobic
Hydrophilic
Stop
Hydrophobic
Hydrophilic
Stop
Hydrophobic
Hydrophilic
Stop
DHFL16
DK1
DK4
DLR1
DLR2
DLR3
DLR4
DM1
DM2
DM3
DM4
DN1
DN4
DXP’1
DXP1
DXP4
D21-9
D21-10
–
–
1.8
1.8
7
–
3.5
–
–
–
–
10.3
–
5.3
–
1.8
3.5
–
1.8
3.5
7
3.5
5.3
1.8
1.8
1.8
–
1.8
3.5
3.5
–
1.8
3.5
3.5
10.3
3.5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5.3
1.8
–
–
–
–
–
5.7
–
–
2.9
–
–
–
–
–
–
2.9
–
2.9
–
–
8.6
–
–
–
–
–
5.7
2.9
–
–
8.6
5.7
–
11.3
11.3
–
20
5.7
–
–
–
–
–
–
–
–
–
–
–
–
–
2.9
–
2.9
–
–
–
–
–
–
16.7
–
16.7
–
–
–
–
–
–
–
–
–
–
–
–
–
–
8.3
–
8.3
8.3
–
–
–
–
–
–
8.3
–
–
16.7
8.3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
8.3
–
–
Σ
35
57.9
7.1
14.4
79.8
5.8
33.4
58.2
8.3
Representation of DH elements is given in percentages. For legend: see Table 1. DH amino acid sequences are tabulated according to
biochemical characteristics of the amino acid sequence encoded in each of the three possible reading frames (hydrophobic or hydrophilic
amino acid sequences, or a sequence containing a stop codon). For details: see text. Σ 5 the total of DH elements employed as the
hydrophobic, hydrophilic or stop codon-containing sequence.
HCDR3 diversity in human adult bone marrow 1513
than FL HCDR3s. These findings were supported by sequence
analysis of adult BM pre-B and mature B cell HCDR3 regions,
which showed that .90% of the pre-B and mature B cell
HCDR3s were .9 amino acids. Previous studies have shown
that HCDR3 size differences between fetal and adult repertoires are reflected in the distribution of N nucleotides: up to
25% of fetal HCDR3 sequences lack N regions at either the
VH–DH or DH–JH junction (14,23–27,31), whereas .98% of
adult PBMC HCDR3 regions have N regions at both these
junctions (14,32–36). Like adult Ig H chain sequences, adult
BM pre-B and mature B cell HCDR3 sequences contained N
regions at both junctions.
The adult nature of HCDR3s in adult BM pre-B and mature
B cells was further supported by the pattern of DH and JH
gene utilization. A defining property of fetal B lymphocytes is
frequent usage of DQ52, which can be as high as 55% in
human FL (14,23–27,31). By contrast, ,2% of the adult
rearrangements employs DQ52 (14,32–36). None of the preB or mature B lymphocyte rearrangements in our study
used DQ52. A majority of the studies indicate that JH4 is
overexpressed in fetal and adult repertoires. JH3 tends to be
utilized more than JH6 in fetal rearrangements, whereas the
opposite is true in cord blood and adult PBMC Ig H chain
genes. The adult BM pre-B and mature B cell JH usage
profiles reflected the adult pattern, wherein JH4 was used at
higher frequency than JH3. Similar DH and JH usage patterns
were recently described in a study of adult BM pre-B cells
isolated on the basis of Vpre-B expression (59). These observations demonstrate that DH and JH usage patterns in adult
PBMC are established by the pre-B cell stage, as previously
suggested by analysis of Ig H chain rearrangements in preB acute lymphoblastic leukemias (60).
Use of DH elements and rearrangement frequency
The most frequently used DH elements in fetal or adult
Ig repertoires belong to the DXP family (24–60% of the
rearrangements) and the DLR family (11–60% of the
rearrangements), whereas DA and DM genes are generally
expressed at low levels. (14,23–27,31–36,59,60 and this
study). The recurrent usage of DLR and DXP genes in
fetal and adult pre-B cells suggests that rearrangement
frequencies may contribute to increased representation of
these elements in the Ig H chain repertoire. The DH recombination signal sequences (RSS) may be important in this regard.
The heptamer and nonamer sequences that constitute RSS
contain several conserved residues that greatly affect recombination frequencies (61,62). Indeed, all DLR and DXP genes
carry conserved 39 RSS, whereas DA and DM genes have
substitutions in these conserved residues (48–50). These
mutations are frequently located in the heptamer, which is
considered most important for rearrangement (61,62). Other
factors that might influence recombination frequency, such
as the chromosomal location of particular DH elements (the
proximity to the JH locus), do not seem to play a determining
role in rearrangement of DH elements in adult BM. DXP4, the
most telomeric DXP element, is expressed with equal or
higher frequency than DXP1, DXP’1 or D21–10. Similarly,
DLR2 was more frequently expressed in ABM pre-B cells
than the more JH-proximal DLR3 gene, while DLR4 (the most
telomeric DLR gene) was at least as frequently expressed as
more JH-proximal DLR genes. Finally, since DQ52 was not
detected in adult BM pre-B cells (despite perfect RSS and a
JH-proximal position), other mechanisms must influence DH
usage frequencies in the adult.
Adult BM HCDR3 amino acid sequences and structure of the
antigen binding site
The differences between pre-B cell and mature B cell HCDR3
regions in the current study suggest that pre-B cell Ig H
chains may be subject to selection during differentiation into
mature B cells. Selection of pre-B Ig H chain genes purely
on the basis of the length of the HCDR3 region, as suggested
by the fingerprinting experiments and HCDR3 sequences,
is unlikely because HCDR3 regions .25 amino acids are
detectable in mature B cells (data not shown and Raaphorst,
unpublished observations). The composition of the HCDR3
sequence may be a more likely target for selection. PreB cell HCDR3s frequently contained extended 39 Tyr-rich
sequences, originating from JH6. In addition, a significant
proportion of pre-B cell HCDR3s encoded continuous
stretches of four or more highly hydrophobic amino acids.
These sequences were particularly rich in Val and Ala, and
were encoded by DLR and DXP genes used in RF3. The
relative absence of continuous hydrophobic amino acids in
mature B cell HCDR3 regions suggests that hydrophobicity
of HCDR3 sequences may have been negatively selected.
Although no canonical structures have been established
for HCDR3 (4–6,8,15), some observations hold for all known
HCDR3 structures. Most peripheral HCDR3 regions contain
at least one Gly and Tyr residue, thought to be essential for
HCDR3 loop formation (56,57). This feature is also reflected
in the current set of adult BM sequences, especially in the
mature B cell clones. The diminished hydrophobicity of mature
B cell HCDR3 might result from positive selection of more
hydrophilic amino acids sequences. In addition to this mechanism, we propose that hydrophobic pre-B cell HCDR3s are
negatively selected. In order to understand this in more detail,
we have performed a search of the high resolution X-ray and
NMR structures in the protein data bank (63) for Val-rich
amino acids stretches and their conformation (58). Val-rich
stretches were frequently detected in α helices of membrane
proteins. In addition, in almost all cases of water-soluble
proteins, Val-rich regions were buried in the molecule and
exhibited a very high propensity for forming β sheets. This
is in disagreement with the presumed expression of such
sequences in a solvent-exposed HCDR3 loop.
Several possibilities for negative selection of hydrophobic
HCDR3 structures can be proposed. Consecutive hydrophobic amino acids in pre-B cell HCDR3s may disrupt the
structure of the antigen binding site. Highly hydrophobic preB HCDR3s may be internalized in the H chain molecule or
may become part of flanking FR structures. Either possibility
could drastically alter the conformation of the HCDR3 and
might render this region unsuitable for interaction with antigen
or L chains. Alternatively, cell surface expression of Ig H
chains with hydrophobic HCDR3s may be prevented as a
result of intracellular aggregation. In this regard it is interesting
to note that molecular chaperones, which preferentially bind
to solvent-exposed hydrophobic groups, have the ability to
retain misfolded proteins in the endoplasmic reticulum (64).
1514 HCDR3 diversity in human adult bone marrow
Continuous hydrophobic amino acids were detected to a
lesser extent in HCDR3 from adult BM Vpre-B1 pre-B cells (59)
and in some published human adult PBMC HCDR3 sequences
(58). These sequences, however, were not particularly Valrich and may have been stabilized by the linkage to Vpre-B or
a L chain molecule.
Concluding remarks
We have demonstrated that the extent of N-regional diversity
and usage frequencies of DH and JH genes, as described for
adult peripheral blood B cells, is established by the pre-B
cell stage in the BM. In addition, the RF of the DH element in
pre-B HCDR3 sequences may be selected by structural
demands of the antigen binding site. Strongly hydrophobic
DH sequences can be incompatible with HCDR3 structure
and may be negatively selected. Hydrophilic DH sequences
could be positively selected because of their ability to form
solvent-exposed HCDR3 loops.
Acknowledgements
We gratefully acknowledge Scott Breitlow (Promega) for stimulating
discussions regarding silver staining and Robert Schelonka
(UTHSCSA, Pediatrics) for statistical analysis of the data. We thank
Gregg Silverman and Dennis Burton for helpful suggestions. F. M. R.
is thankful to Judy Teale for her enthusiasm and support.
Abbreviations
BM
HCDR
DIR
FL
FR
MBT
PBMC
PBUF
PBT
RF
RSS
TdT
bone marrow
complementarity determining region of Ig H chain
DH element with irregular RSS
fetal liver
framework region
mature B cell total repertoire
peripheral blood mononuclear cell
pre-B cell upper fraction
pre-B cell total repertoire
reading frame
recombination signal sequence
terminal deoxynucleotidyl transferase
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