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Single-Cell Repertoire Analysis Demonstrates
that Clonal Expansion Is a Prominent
Feature of the B Cell Response in Multiple
Sclerosis Cerebrospinal Fluid
Gregory P. Owens, Alanna M. Ritchie, Mark P. Burgoon, R.
Anthony Williamson, John R. Corboy and Donald H. Gilden
J Immunol 2003; 171:2725-2733; ;
doi: 10.4049/jimmunol.171.5.2725
http://www.jimmunol.org/content/171/5/2725
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Copyright © 2003 by The American Association of
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References
The Journal of Immunology
Single-Cell Repertoire Analysis Demonstrates that Clonal
Expansion Is a Prominent Feature of the B Cell Response in
Multiple Sclerosis Cerebrospinal Fluid1
Gregory P. Owens,2* Alanna M. Ritchie,* Mark P. Burgoon,* R. Anthony Williamson,‡
John R. Corboy,* and Donald H. Gilden*†
T
he brain and cerebrospinal fluid (CSF)3 of multiple sclerosis (MS) patients contain increased amounts of intrathecally produced IgG and oligoclonal bands (OGBs) of
unknown specificity. B cells and plasma cells have been found in
active and late MS lesions (1, 2), and induction of immune effector
mechanisms by plaque Ig is evidenced by: 1) capping of surface
IgG on macrophages involved in myelin breakdown (3); 2)
codeposition of IgG and complement, particularly the activated
terminal lytic complex (4 – 6) at plaque borders; and 3) the presence in CSF of membrane attack complex-enriched membrane
vesicles, indicating a role for complement-mediated injury in MS
(7). Recent molecular studies to characterize the H chain V regions
(VH) of IgG expressed in MS plaques (8 –11) and CSF (12, 13)
revealed a limited repertoire with features of a targeted B cell
response. VH sequences from MS plaques and CSF were oligoclonal, extensively mutated, and derived in part from clonally expanded B cell populations, indicative of antigenic stimulation.
These properties are shared by the Ab response found in subacute
sclerosing panencephalitis brain (11), a chronic infectious disease
caused by measles virus and characterized by oligoclonal IgG directed against measles virus.
In this study, we used cell sorting and single-cell RT-PCR to
study clonal B cell expansion in CSF of four MS patients and two
control subjects with non-MS inflammatory disease. Analysis of V
region sequences expressed in randomly sorted, single B cells reduces potential bias when amplifying Ab repertoires from cDNA
or cDNA libraries (14, 15), which may overrepresent sequences
expressed by activated B cells or plasma cells. A second and more
distinct advantage of single-cell PCR is that the clonal B cell H and
L chain pairings found in vivo can be faithfully duplicated in vitro
and used to produce novel Abs potentially useful in identifying
disease relevant Ags.
Materials and Methods
Human CSFs
All MS and inflammatory control CSFs (2–30 ml) were collected in the
outpatient clinic of Department of Neurology using procedures approved
by the University of Colorado School of Medicine Institutional Review
Board. All MS patients had relapsing-remitting disease, magnetic resonance imaging scans that revealed multiple white matter lesions, and CSFs
containing two or more OGBs of IgG. The duration of MS varied from 1
to 22 years (Table I). None of the MS patients had received steroids or an
immunomodulatory drug for at least 1 mo before CSF sampling. The two
control CSFs were from patients with acute viral meningitis.
Fluorescence-activated cell sorting of CD19⫹ B cells in CSF
Departments of *Neurology and †Microbiology, University of Colorado Health Sciences Center, Denver, CO 80262; and ‡Department of Immunology, Scripps Research
Institute, La Jolla, CA 92037
Received for publication March 13, 2003. Accepted for publication June 26, 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 study was supported in part by Public Health Service Grant NS 32623.
2
Address correspondence and reprint requests to Dr. Gregory P. Owens, Department
of Neurology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Mail Stop Box B182, Denver, CO 80262. E-mail address: greg.owens@
uchsc.edu
3
Abbreviations used in this paper: CSF, cerebrospinal fluid; CDR, complementaritydetermining region; MS, multiple sclerosis; OGB, oligoclonal band; RT, reverse
transcription.
Copyright © 2003 by The American Association of Immunologists, Inc.
CSF obtained by lumbar puncture was immediately centrifuged for 10 min
at 1500 rpm. Pelleted cells were resuspended in 200 ␮l of residual CSF,
placed on ice, and sorted within 1–3 h. A three-color premixed combination (15 ␮l) of fluorescence-tagged murine Abs (Caltag Laboratories, Burlingame, CA) specific for the human white blood cell surface markers
CD45 (Tri-Color), CD19 (r-PE), and CD3 (FITC) was added to the CSF
cell suspension and incubated for 20 min at room temperature. Labeled
cells were diluted with 500 ␮l of sterile PBS, placed on ice, and sorted
using a MoFlo cytometer (Cytomations, Fort Collins, CO). Cells in the
approximate size range and granularity of lymphocytes were first selected
by light scattering, followed by a second selection for all CD45⫹ cells. B
cells (CD19⫹, CD3⫺) were further separated from T cells (CD19⫺, CD3⫹)
and other CD45⫹ cells (CD19⫺, CD3⫺), and individually expelled into
single wells of a 200-␮l, 96-well PCR plate (catalog no. T-3060; ISC
Bioexpress, Kaysville, UT) containing 20 ␮l of 1⫻ reverse-transcription
(RT) reaction buffer. From one to four plates of sorted cells were collected
0022-1767/03/$02.00
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Single-cell RT-PCR was used to sample CD19ⴙ B cell repertoires in cerebrospinal fluid (CSF) of patients with multiple sclerosis
(MS) or viral meningitis. Analysis of amplified Ab H and L chain products served to identify the rearranged germline segment and
J segment, and to determine the degree of homology for the H and L chain sequence of individual B cells. The B cell repertoire
of viral meningitis CSF was predominately polyclonal, whereas B cell clonal expansion was a prominent feature of the IgG
repertoire in three of four MS patients. Two dominant clonal populations in one MS CSF accounted for ⬃70% of the IgG H chain
V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed
multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS
patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately
recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS. The
Journal of Immunology, 2003, 171: 2725–2733.
2726
B CELL RESPONSES IN MS
Table I. Clinical features of MS and non-MS inflammatory disease control patients undergoing single B cell analysis of CSF
Subject
Age
Sex
Diagnosis
Years of MS at
Time of CSF
Exam
MS02-2
MS02-6
MS02-11
MS02-14
IC02-1q
IC02-3q
38
42
22
51
29
44
M
F
F
F
F
F
Relapsing-remitting MS
Relapsing-remitting MS
Relapsing-remitting MS
Relapsing-remitting MS
Acute viral meningitis
Acute viral meningitis
2
1
1–2
22
0
0
a
b
MRIa
CSF Cells
% IgG/Protein
OGBs
⫹
⫹
⫹
⫹
NDb
ND
1
9
18
10
560
778
8 (normal 3–13)
13
21
15
ND
ND
2–3
5
3
4 –5
ND
ND
Presence of multiple white matter lesions demonstrated by magnetic resonance imaging.
ND, not done.
for each CSF sample. When possible, the lysis and RT steps were conducted directly. Additional plates were stored at ⫺70°C until processed for
cDNA synthesis and PCR. To determine the probability of depositing more
than one cell per well, fluorescent beads were also sorted into 96-well
plates. Because none of the wells were found to contain more than one
bead, the deposition of more than one cell into a single well was unlikely.
The protocol used for cDNA synthesis and PCR amplification was a modification of that described by Wang and Stollar (15). cDNA synthesis and
PCR were performed using the I-Cycler (BioRad, Hercules, CA) thermocycler equipped with a 96-well block. Cells in each well were lysed in 4.5
␮l of a detergent mixture containing 2 ␮l of 5⫻ RT buffer (Invitrogen,
Carlsbad, CA), 0.5 ␮l of random hexamers (3 ␮g/␮l), 0.5 ␮l of an IgG
antisense C region primer CH5 (10 pmol/␮l) conserved among all four IgG
isotypes, and 1 ␮l of a 22.5% solution of Nonidet P-40. Plates were incubated for 3 min at 65°C, cooled to 25°C, incubated for 3 min in the thermocycler, and immediately placed on ice. A mixture of 3 ␮l of 0.1 M DTT,
0.5 ␮l RNAsin (50 U/␮l), 1 ␮l 10 mM dNTPs, and 0.5 ␮l of Superscript
II RT (200 U/␮l) was added to each well (final volume of 30 ␮l). Plates
were incubated in the thermocyler for 1 h at 37°C, heated at 70°C for 10
Table II. Sequences of oligonucleotide primers used for V region amplifications
VH leader primers
VH1L1
5⬘-CCATGGACTGGACCTGGAG
5⬘-ATGGACATACTTTGCTCCAC
VH2L1
5⬘-CCATGGAGTT(TG)GGGCTGAGCTGG
VH3L1
5⬘-ATGAAACACCTGTGGTTCTT
VH4L1
5⬘-ATGGGGTCAACCGCCATCCT
VH5L1
VH framework 1 primers
VH1FR1 5⬘-GGTGCAGCTGGT(GA)CAGTCTGGGGCTG
VH2FR1 5⬘-CAG(AG)TCACCTTGA(AG)GGAGTCTGGTCC
VH3FR1 5⬘-(GC)AGGT(GT)CAGCTGGTGGAGTCTGGGGG
VH4FR1 5⬘-GGTGCAGCTGCAG(GC)AGT(GC)GGGC(GC)CAGG
VH5FR1 5⬘-AGCTGGTGCAGTCTGGAGCAGAGG
IgG and IgM C region primers
5⬘-CCTCTCACCAACTTTCTTGTCC (cDNA priming)
CH5
CH␥1
5⬘-GTTGTCCACCTTGGTGTTGCTGG (primary PCR)
CH␥2
5⬘-CACCGGTTCGGGGAAGTAGACC (nested PCR)
5⬘-GAAGTAGTCCTTGACCAGGCAGCC (sequencing)
CH3
5⬘-GAAGCCAGCACCTGTGAGG (primary PCR)
CH␮1
5⬘-CTGCGTACTTGCCCCCTCTCA (nested PCR)
CH␮2
5⬘-GTATCCGACGGGGAATTCT (sequencing)
CHM2
␬ and ␭ C region primers
C␬1
5⬘-ACACTCTCCCCTGTTGAAGCTCTT (primary PCR)
C␬1D
5⬘-GCGCCGTCTAGAATTAACACTCTCCCCTGTTGAAGC
TCTTTGTGACGGGCGAACTCAGG (nested PCR)
C␬2
5⬘-GTAGGTGCTGTCCTTGCTGTCCTG (sequencing)
C␭1
C␭1D
5⬘-TGAACATTCTGTAGGGGCCAC (primary PCR)
5⬘-CGCCGTCTAGAATTATGAACATTCTGTAGGGGCCACTGTC
(nested PCR)
5⬘-CTTGTTGGCTTGAAGCTCCTC (sequencing)
C␭4
VL leader primers
V␬1L1
5⬘-CTCAGCTCCTGGGGCTCC
V␬2L1
5⬘-CTGCTCAGCTCCTGGGGC
V␬3L1
5⬘-GGAA(GA)CCCCAGC(AGT)CAGC
V␬4L1
5⬘-CTCTGTTGCTCTGGATCTCTG
V␬5L1
5⬘-GGGGTCCCAGGTTCACCTCCTC
V␭L1
5⬘-CCTCTCCTCCTCACCCTCCTC
V␭2L1
5⬘-CTCCTCACTCAGGGCACAG
V␭3L1
5⬘-ATGGCCTGGA(CT)CCCTCTCCT(GC)CT
V␭4aL1
5⬘-CTCCTC CTCTTCCCCCTCCCCCTC
V␭4bL1
5⬘-TGGCCTGGGTCTCCTTCTACCTAC
V␭5,6,9L1
5⬘-ATGGCCTGG(AG)CTCCTCTCCT(CT)CTC
V␭7,10L1
5⬘-ATGGCCTGG(AG)CT CCTCTCCT(CT)CTG
V␭8L1
5⬘-ATGGCCTGGATGATGCTTCTCCTC
VL framework 1 primers
V␬1/4 FR1
5⬘-G(AC)CATCC(AG)GATGACCCAGTCTC
V␬2FR1
5⬘-GATATTGTGATGACCCAG(AT)CTC
V␬3FR1
5⬘-GAAATTGTGTTGAC(AG)CAGTCTC
V␬5FR1
5⬘-GAAACGACACTCACGCAGTCTCC
V␭1FR1
V␭2FR1
V␭3FR1
V␭4aFR1
5⬘-CAGTCTGTGCTGAC(GT)CAGCC
5⬘-CAGTCTGCCCTGACTCAGCC
5⬘-CTTCCTATGAGCTGAC(AT)CAG
5⬘-CAGCCTGTGCTGACTCAATCG(CT)CC
V␭4bFR1
V␭5/9/
10FR1
V␭6FR1
V␭7/8FR1
5⬘-CTGCCTGTGCTGACTCAGCCCCC
5⬘-CAG(GC)CTG(GT)GCTGACTCAGCCA(GC)C
5⬘-AATTTTATGCTGACTCAGCCCCAC
5⬘-CAGACTGTGGTGAC(CT)CAGGAGCC
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Synthesis of cDNA and amplification of VH and VL sequences
min to inactivate RT, and cooled to 4°C. Plates containing cDNA were
stored at 4°C.
Table II lists the primers used for cDNA synthesis, specific amplification of VH and L chain V (VL) regions, and sequencing. The VH and V␬
leader and framework 1 PCR primers were family based and identical with
or modifications of well-established primer sets designed for single-cell
amplification of V region sequences from peripheral blood B cells (14, 16).
Conserved family-specific V␭ leader and framework 1 primers were obtained from alignments of family germline segments found in V BASE, an
online database (V BASE, http:/www.mrc-cpe.cam.ac.uk/) containing all
known human VH, V␬, V␭ germline segments. To amplify VH sequences,
conserved family-based leader sequence primers for VH families 1–5 were
used in a single primary PCR with the conserved C region primer CH␥1 for
IgG amplification. A separate primary PCR with the C region primer CH␮1
was used to amplify IgM sequences. Single-cell PCR amplifications were
performed in a 50-␮l vol containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl,
2 mM MgCl2, 0.01% gelatin, 100 ␮M dNTPs, 100 pmol of each primer, 2
U Taq polymerase, and 5 ␮l of cDNA reaction mix. Cycling conditions
included a single 5-min denaturation step at 94oC, followed by 34 cycles
of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, ending with a 7-min
incubation at 72oC. A nested PCR using 2 ␮l of the primary PCR product
The Journal of Immunology
2727
sequences were analyzed using PCGene or DNASIS Max software. Alignment of IgG sequences to the closest VH or VL germline segments was
performed using online services provided by Cambridge Center for Protein
Engineering with an alignment program (DNAPlot) that allows comparison
of H and L chain V regions to germline sequences in V BASE. This online
service was used to identify the closest V region germline segments and to
determine the extent of sequence homology for the Ab H and L chain
sequences analyzed. For consistency, homologies to germline segments of
donor DP (17) were used when appropriate. Otherwise, the gene locus was
used to identify the most homologous germline segment. A small number
of clones contained in-frame insertions or deletions in the CDR1 or CDR2
region of VH gene segments that were omitted when determining the extent
of somatic mutation for that sequence.
Results
Single CD19⫹ B cell sorting and amplification of arranged V
region sequences
Single CD19⫹ B cells were isolated from MS and inflammatory
control CSF using Abs to a combination of human white blood cell
markers (CD45, CD3, CD19), as shown in Fig. 1 for MS02-11
CSF. CD19⫹ cells accounted for 3.9% of the selected CD45⫹ cells
in this patient and varied from 1.5 to 3.9% among the four MS CSF
samples. In the lymphocyte population of control subjects with
Table III. Features of VH and VL sequences in MS CSF clonal populationsa
No.
VH CDR3 ⫺ IgG
Family
Germline
Homology
(%)
JH
Family
Germline
Homology
(%)
JL
WDDSLSGLV
VL1
DPL3
99
JL1
YAGSSSYV
VL2
DPL10
98.3
JL1
Nonproductive
QQYYSTPLT
MQGTHWPPPT
V␬1
V␬4
V␬2
ND
DPK24
DPK18
ND
98.3
100
ND
J␬5
J␬5
QQYGILPWT
QQYGILPWT
QQYVKLPWT
QQYGILPWT
QQYGILPWT
QQYGILPWT
QQYGILPWT
QQYGILPWT
V␬1
V␬1
V␬1
V␬1
V␬1
V␬1
V␬1
V␬1
DPK1
DPK1
DPK1
DPK1
DPK1
DPK1
DPK1
DPK1
96.1
96.1
97.1
96.1
96.1
96.1
96.1
96.1
J␬1
J␬1
J␬1
J␬1
J␬1
J␬1
J␬1
J␬1
VL CDR3 ⫺ IgG
MS02-2
1
5
6
14
22
26
38
39
42
48
51
3
8
29
40
44
12
46
50
18
25
Clone 1
VKASYFDY
VKASYFDY
VKASYFDY
VKASYFDY
VKASYFDY
VKASYFDY
VKASYFDY
VKASYFDY
VKASYFDY
VKASYFDY
VKASYFDY
Clone 2
PRNRYQDGLFDS
PRNRYQDGLFDS
PRNKYQDGLFSS
PRNRYQDGLFDS
PRNRYQDGLFDS
DGGSDYASQFYFDY
DGGSDYASQFTFDY
Clone 3
SPPDRGWDLLGDVFDI
SPPDRGWDLLGDVFDI
VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1
VH1
DP-10
DP-10
DP-10
DP-10
DP-10
DP-10
DP-10
DP-10
DP-10
DP-10
DP-10
92.9
92.5
92.5
92.9
92.9
92.9
92.9
92.9
92.9
92.9
92.9
JH4
JH4
JH4
JH4
JH4
JH4
JH4
JH4
JH4
JH4
JH4
VH4
VH4
VH4
VH4
VH4
VH1
VH1
DP-79
DP-79
DP-79
DP-79
DP-79
DP-14
DP-14
94.9
94.3
93.9
94.9
92.9
91.6
91.9
JH4
JH4
JH4
JH4
JH4
JH4
JH4
VH2
VH2
DP-28
DP-28
94.6
JH3
94.9
JH3
MS02-6
QQASF
V ␬1
L12
94.4
J␬4
Clone 1
DCSHGSCYSQYYYGMDV
DCSHGSCYSQYYYGMDV
DCSHGSCYSQYYYGMDV
DCSHGSCYSQYYYGMDV
Clone 2
FYGDHFDY
VH3
VH3
VH3
VH3
DP-54
DP-54
DP-54
DP-54
95.6
95.6
95.6
95.6
JH6
JH6
JH6
JH6
QQYDSFPM
QQYDSFPM
QQYDSFPM
QQYDSFPM
V␬1
V␬1
V␬1
V␬1
L12
L12
L12
L12
96.1
95.7
96.1
96.1
J␬1
J␬1
J␬1
J␬1
VH4
DP-79
94.3
JH4
V␬1
V␬1
DPK5
DPK5
95.4
95.4
J␬4
J␬4
4
27
Clone 3 ⫺ IgM
HRTISWFYY
HRTISWFYY
QQGNSFLLT
QQGNSFLLT
VH 4
VH4
DP-65
DP-65
93.6
JH5
93.6
JH5
MS02-11
QQYGSSSIF
QQYGSSSIF
V␬3
V␬3
DPK22
DPK22
96.2
96.2
J␬3
J␬3
35
95
Clone 1
EGPRIAAAGLD
EGPRIAAAGLD
VH3
VH3
DP-54
DP-54
98.3
98
FTGGYPWV
VL2
DPL12
97.9
J␬1
33
38
54
132
20
131
a
JH4
JH4
Each line identifies the PCR well number, CDR3 amino acid sequence, germline family, most homologous V and J germline segment, and degree of homology to the closest
germline segment for the H and L chain V region amplified from that single B cell.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
and a pool of family-based framework 1 primers in conjunction with a
second, more 5⬘-conserved IgG C region primer (CH␥2) or IgM C region
primer (CH␮2) was used to amplify VH region products. To amplify fulllength ␬ or ␭ L chain sequences, sets of family-based leader and framework
1 primers were used in conjunction with C region primers complementary
to the C-terminal portion of the L chain coding sequence.
To avoid PCR cross-contamination, we have a designated PCR area. All
primers and PCR components are aliquotted, and stored lyophilized at
⫺70oC until used. Fresh primers and PCR components are used for each
CSF repertoire analysis. Separate multichannel pipettes are used to prepare
and dispense PCR solutions, to dispense cDNA into PCR, and to analyze
PCR products. Analysis of PCR products is performed in an area physically
separated from the PCR area.
In analysis of patient MS02-2 CSF, specific VH sequences from some B
cell populations were reamplified using clone-specific primers complementary to known nucleotide sequences within the complementarity-determining region 3 (CDR3). For those studies, antisense CDR3 primers specific
for each MS02-2 clone 2 VH sequence (see Table III), identified by the
CDR3 amino acid sequences PRNRYQDGLFDS (5⬘-GTCCTGATACCT
GTTCCGGGG) and DGGSDYASQFYDY (5⬘-GAGACCGGTAGTCG
CTCCCACC), were used in conjunction with the appropriate VH framework 1 primer in nested PCR, as described above.
Amplification products were identified by agarose gel electrophoresis
and purified using the Qiaquick PCR Purification kit (Qiagen, Valencia,
CA). Purified PCR products were sequenced at the University of Colorado
Health Sciences Center Cancer Center sequencing core using the Ab H and
L chain C region antisense primers listed in Table II. Individual V region
2728
B CELL RESPONSES IN MS
FIGURE 1. FACS purification of CD19⫹ B
cells from MS02-11 CSF. Cells in CSF were
collected by centrifugation and incubated with a
mixture of labeled Abs to human CD45, CD19,
and CD3 (see Materials and Methods). Cells
were selected first by light scattering (FSC, forward scatter; SSC, side scatter) (A), then by
CD45 expression (B), and finally separated into
CD19⫹ (R3) and CD3⫹ (R6) populations (C).
Single CD19⫹ cells were expelled into separate
wells of a 96-well PCR plate.
A predominantly polyclonal CD19⫹ B cell repertoire in CSF of
two patients with acute viral meningitis
CD19⫹ B cell repertoires of CSF from two patients with acute
viral meningitis were analyzed as a non-MS inflammatory control.
In both patients, the H chain repertoires consisted of about equal
numbers of IgG- and IgM-expressing B cells. The amplified H
chain V region product from each positive PCR of the CSF as well
as L chain V region products amplified from VH-positive wells
were purified and sequenced. Approximately 80% of the VH-positive wells of the IC02-1 CSF and 54% of VH-positive wells from
IC02-3 CSF also revealed a L chain sequence. Only two VH and
two VL nonproductive rearrangements were detected among the
174 V region sequences analyzed from the two inflammatory
controls.
Each VH and VL sequence was aligned to a database containing
all known functional human H and L chain germline segments to
determine the family and most homologous germline segment, J
segment (MS patients only), and the extent of somatic mutation for
each sequence. Because of its unique nature, the CDR3 amino acid
sequence was used as a clonal marker to categorize V region sequences in the repertoire. No clonally related B cell populations
were detected among the 27 IgG and 22 IgM VH sequences analyzed from IC02-1 CSF, and only 2 VH sequences of the 50 analyzed in the IC02-3 repertoire were clonally related, indicating a
predominantly polyclonal response in CSF of both patients. Table
V gives the characteristics of V region sequences obtained from
the IC02-1 CSF analysis.
Table IV. V region sequences analyzed in MS and non-MS inflammatory disease control CSF B cells
CSF Donor
No. V region products amplified and
sequenced
No. VH sequences in clonal IgG
populationsa
a
IC02-1
IC02-3
MS02-2
MS02-6
MS02-11
MS02-14
IgG
IgM
27
22
26
24
25
4
13
22
18
21
30
15
V␬
V␭
IgG
20
19
0/27
17
10
2/26
15
6
20/25
36
13
5/13
41
20
2/18
56
8
15/30
IgM
0/22
0/24
0/4
2/22
0/21
0/15
Indicates the number of IgG VH sequences found in clonal populations relative to the total number of IgG VH sequences
analyzed. Clonal populations were defined as B cells from the same donor CSF expressing an identical VDJ recombination based
on the amino acid sequence of the CDR3 region. See Tables III and VI for the groupings and features of clonal IgG populations
in MS CSF.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
aseptic meningitis, CD19⫹ B cells represented 0.4% for donor
IC02-1 and 1.2% for donor IC02-3.
After cDNA synthesis, the expressed H and L chain V region
sequences of each cell were amplified with an established panel of
leader and framework 1 primers in conjunction with conserved Ig
C region primers (Table II). Table IV lists the number of V region
sequences amplified and sequenced for each MS and inflammatory
control CSF donor. Most H and L chain V region sequences in
each CSF donor were productive gene rearrangements and were in
frame into the respective C region domains. However, each analysis revealed a small number of nonproductive rearrangements
with either in-frame nonsense codons in the CDR3 coding region
or out-of-frame rearrangements. Amplification of both an IgG and
IgM V region sequence from the same PCR well was not observed
in any of the repertoires, and in the only case in which two different
L chain sequences were detected in the same cell, one was a nonproductive rearrangement. Thus, the deposition of more than one B
cell per well appeared to be a rare event.
The Journal of Immunology
2729
Table V. Features of VH and VL sequences amplified from IC02-1 donor CSFa
VH CDR3 ⫺ IgG
Family
Germline
Homology (%)
1
12
14
29
32
37
42
48
52
66
70
71
72
75
78
88
95
119
124
125
139
158
159
162
181
177
185
QGWYNWNLPSYY
DSRLTGTLGRVYYFDY
DHGGSSWYAGAFDI
WQGGSRVY
RRCSITACYNSGFNCFDY
VDGYNYNWEYYYDGLDV
GSEVRYVDLYYNGMDV
ESIIGRTFDC
DSYVDLREHYYNFGMYV
AVDIYNYGSNFDY
GQRYFDWSTSQDGFD
DETAADLTRLVAGSAYGMDV
GDVVGFSDMYFEF
ASEAYNLAPGDY
AKYGGNFEYFQH
DNWGSLDY
IPSIVAANPYAFDL
DRCSSNCFRERWFDP
Nonproductive
DDWVSSGWSNDY
DQSDYFDNFDYYPNAFDV
VGFGGSYYPGAFDV
QYGSGTWGLDAFDI
DSREFLGEWFFNY
DGEVGCGKVACRPDLHHYSSMDV
DRAQTRGQFLALNYYYYGMDV
GLLRGGWNDVDYYYGMDV
VH CDR3 ⫺ IGM
DRIEYSSSSGAFLYGMDV
RPKVGATATLFDY
DLGLGYCSSTSCYTGTIGMDV
GMDV
DSPQLRYKSKSISRGLLHAFDI
DSGGSGSYGDY
GYNSGSYYSVFDY
DPRDYGDYEGGN
AAWGEYSYGKATEY
Nonproductive
VKGSGSDTSWFRGFFDQ
HKVGGNDYGSLPYYFDY
AGRVAYNYLGQGAFDV
RYFDSSGYYYHYDMDV
DMGSTVVTPGIGGYYYGMDV
VISGNYWGGFDY
GPYYDNNSYYST
TLVALQPTSRSRYYCAMDV
RRGLGLRGDY
DRDYIAVAATGFDY
GHRWLYLYNWFDP
VH3
VH4
VH3
VH3
VH3
VH3
VH3
VH3
VH1
VH3
VH3
VH1
VH1
VH3
VH3
VH3
VH5
VH4
VH5
VH3
VH1
VH4
VH3
VH3
VH1
VH1
VH4
DP-51
DP-79
DP-86
DP-29
DP-54
DP-54
DP-47
DP-47
DP-10
DP-31
DP-54
DP-8
DP-75
DP-50
DP-86
DP-29
ND
DP-65
ND
DP-38
DP-8
DP-71
DP-54
DP-46
DP-14
DP-8
DP-63
91.4
97.3
95.9
92.9
95.2
89.4
93.9
92.2
91.9
93.3
97.6
88.1
88.4
85.6
93.8
91.5
ND
89.2
ND
91.1
90.9
90.8
97.3
94.6
83.2
93.6
96.2
VH 4
VH3
VH3
VH3
VH3
VH1
VH3
VH3
VH3
VH4
VH3
VH1
VH3
VH3
VH3
VH3
VH4
VH3
VH1
VH3
VH4
DP-79
DP-50
DP-47
DP-54
DP-38
DP-75
DP-35
DP-47
DP-46
DP-63
DP-47
DP-75
DP-49
DP-46
DP-31
DP-31
DP-63
DP-35
DP-15
DP-47
DP-79
97.6
100
100
96.9
100
98.3
96.2
94.3
95.5
ND
94.4
95.3
91.8
97.9
100
97.3
93.5
98
99
99
99.3
22
24
40
54
61
67
86
89
90
103
112
135
136
143
144
146
149
171
176
178
193
VL CDR3 ⫺ IgG
Family
Germline
Homology (%)
WDDSLNGMVF
QQSYSTPYYF
HDSSLSGF
QHYSDSPLSF
SSYAGSPSLVF
Nonproductive
WDDRLNGVVF
VL1
V␬1
VL1
V␬3
VL2
V␬1
VL1
DPL2
DPK9
DPL8
DPK22
2e
L1
DPK2
95.6
99.3
98.7
94.4
96.2
83.5
94.6
MQALQTTF
RDTNGDQYVF
QQSYGAPVTF
ADISGTYAIF
V␬2
VL3
V␬1
VL3
DPK15
DPL16
DPK9
3m
98.0
95.8
95.4
96.3
MQALQVPTTF
YDNSALSGYVF
Nonproductive
QQYSTHEFTF
QQTGDPFTF
WDSSLSVVVF
WDTSLSVGLF
FAGSYSPVAF
QQANSFPLTF
YRGMSTHTYVF
QQYHSLPLTF
V␬2
VL1
DPK15
1e
93.3
96.0
V␬1
V␬1
VL1
VL1
VL2
V␬1
VL2
V␬4
L12
DPK1
DPL5
DPL5
V1-2
DPK5
DPL10
DPK24
95.3
92.7
98.3
94.3
91.8
95.4
92.9
93.9
WDNSVSAGVF
QHSYNTPRTF
VL CDR3 ⫺ IgM
YAGSHTLVF
QQSY
WDSSTVVF
SDSSGNHRGVF
VL1
V␬1
DPL5
DPK9
94.2
97.2
VL2
V␬1
VL3
VL3
2e
DPK9
DP23
3p
98.7
98.2
100
96.3
QQSYSTPPTF
V␬1
DPK9
99.3
WDDSLSGRVF
VL1
DPL3
97.6
YTTSGTYVF
MQALQTPLTF
QQYYGTPLTF
MQALQTPYTF
YTSSSTLVF
VL2
V␬2
V␬4
V␬2
VL2
DPL11
DPK15
DPK24
DPK15
DPL11
95.9
97.6
99
94.3
99.0
QQYNSYSP
V␬1
L12
YTSSSTLVF
QQYNYYPWTF
VL2
V␬1
DPL11
L1
QQSYSTPAHF
V␬1
DPK9
100
97.3
98.6
100
a
Each line identifies the PCR well number, CDR3 amino acid sequence, germline family, most homologous germline segment, and degree of homology for the H and L chain
V region amplified from a single CD19⫹ B cell.
Clonal expansion of B cell populations in MS CSF
The relative number of IgG- and IgM-expressing B cells varied
among the four MS patients. Whereas the B cells from donors
MS02-2 and MS02-14 were predominantly IgG expressing, a more
balanced or IgM dominant response was observed in MS02-11 and
MS02-6 CSF, respectively (Table IV). Consistent with previous
characterization of OGBs in MS CSF, ␬ L chains represented the
predominant L chain of each MS donor. Of the combined 344 V
region sequences analyzed from the 4 MS donors, only 12 were
nonproductive rearrangements.
Clonally expanded B cell populations were detected in the IgG
repertoire of each of the MS samples, although to varying degrees
(Table IV). For example, the diversity of the IgG B cell repertoires
of both MS02-2 and MS02-14 CSF was limited, with clonal populations comprising 20 of 25 and 15 of 30 of the VH region sequences amplified from these respective donors. In contrast, the
repertoire of MS02-11 was predominately polyclonal, with only 2
of 18 IgG VH region sequences comprising a single clonal population. With the exception of a single clonal population detected in
MS02-6 CSF, clonally expanded IgM B cell populations were not
found in any other MS CSFs. Overall, and particularly for donors
MS02-11 and MS02-14, the amplification of VL regions from single B cells was relatively more efficient than the corresponding H
chain amplification. Thus, some amplified L chain sequences could
not be paired with the partnering H chain sequence (Table IV).
With the exception of donor MS02-11 in which two B cells expressing identical L chain sequence were detected, the sequencing
of these L chains revealed no additional clonal populations in the
other MS CSFs (data not shown).
Table VI compiles the entire VH and matching VL region repertoires amplified from B cells recovered from the CSF of donor
MS02-14. The VH sequences could be organized into seven
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
No.
2730
B CELL RESPONSES IN MS
Table VI. Features of VH and VL sequences amplified from MS02-14 donor CSFa
No.
58
171
201
375
83
177
358
108
170
252
25
241
126
90
209
360
142
147
149
156
163
216
220
235
295
320
325
329
343
344
367
109
144
164
217
226
238
239
249
278
292
307
309
345
379
388
Clone 1
VPRDGYNYFDF
VPRDGYNYFDF
VPRDGYNYFDF
VPRDGYNYFDF
Clone 2
SWDDSSGWPPENFYFDY
SWDDSSGWPPENFYFDY
SWDDSSGWPPENFYFDY
Clone 3
VSLSNIMVRGPPPVYGMDV
VSLSNIMVRGPPPVYGMDV
Clone 4
RGYYYDSANYYRVFDY
RGYYYDSANYYRVFDY
Clone 5
HKWDLASAALSWFGP
HWKDLASAALSWFGP
Clone 6
DGLGGSYSPYYSDY
Clone 7
MAGTYYYDSAGRGYIDH
IMVRGVISDHYYGMDV
GDDGDYFFQH
TLRGQGYYDSRLPYYHHVDV
GRRDDSAYVRGVIMTGEKYYNYGMDV
DLQGRAAWDVIAVPSDVSYYCALDV
FSAYTYGLPDSDLDY
SPHYGDYENYYFYGMDV
DTGYTSGCACDV
RPIAPPNTGYFDP
GEVSPYYDSSGFAYSRARMDV
QYCGGGSCYSVLDYYDF
TSGWGISA
DTKKEWELPWDAFDV
QLLGAEMATTPFDH
HSLIHPSAYRPRDDGFHM
CDR3 ⫺ IgM
AGPSGFLRSGPLDI
DGAVAGSRDH*YGMDV
GTTTTGAGDFSDGFEI
DPRGYSYGLFDY
VTRTPSTSIDY
LHGGNSPNWFDP
FFRYVSSPDAFDV
LPIPEEDVFEI
SGIYYDSSGYFDFDY
ELRQWLGHD
LNPSSIAVGGNWFDP
AVSVGTTFINY
GPPAYYYGSSGYFFEY
DQWLVQGYYYYGMDV
DFRPGYSSSWSFYYYGMDV
Family
Germline
Homology
(%)
JH
VH4
VH 4
VH 4
VH 4
DP-65
DP-65
DP-65
DP-65
93.9
93.9
93.9
93.9
JH4
JH4
JH4
JH4
VH4
VH 4
VH 4
DP-65
DP-65
DP-65
97.3
97.3
97.3
VH 3
DP-47
VH 3
Family
Germline
Homology
(%)
J␬
QQYNGFPYT
QQYNGFPYT
QQYNGFPYT
V␬1
V␬1
V␬1
L12
L12
L12
97.5
97.5
97.5
J␬2
J␬2
J␬2
JH4
JH4
JH4
QQYNSYPWT
QQYNSYPWT
QQYNSYPWT
V␬1
V␬1
V␬1
L12
L12
L12
97.9
97.9
97.9
J␬1
J␬1
J␬1
92.5
JH6
DP-47
91.8
JH6
MQALQTPCT
MQALQTPCT
MQALQTPCT
V␬2
V␬2
V␬2
DPK15
DPK15
DPK15
97.7
97.7
98.3
J␬2
J␬2
J␬2
VH 4
VH4
DP-78
DP-78
94.6
94.9
JH4
JH4
QQYGSPHLYT
V␬3
DPK22
95.7
J␬2
VH 4
VH4
DP-79
DP-79
91
91
JH5
JH5
Nonproductive
HQRGHWPPT
V␬4
V␬3
DPK24
L6
ND
96.4
J␬4
VH 1
DP-14
94.6
JH4
QQYNSYSGT
QQYNSYSGT
V␬1
V␬1
L12
L12
96.8
95.7
J␬1
J␬1
VH 3
DP-47
95.9
JH4
VH 3
VH 4
VH 4
VH 3
VH 3
VH4
VH 3
VH 3
VH 3
VH4
VH 5
VH4
VH4
VH5
VH4
3-66
DP-71
DP-79
DP-49
DP-46
DP-78
DP-54
DP-53
DP-35
DP-70
DP-73
DP-65
DP-65
DP-73
DP-65
ND
94.5
97.3
93.9
96.6
92.6
98.3
94.6
93.6
90.8
92.2
92.9
94.7
91.5
94.7
JH6
JH1
JH6
JH6
JH6
JH4
JH6
JH4
JH4
JH6
JH4
JH5
JH3
JH4
JH3
LQYYSSPRT
LQYYSSPRT
MQALQTSWT
QQSYILPRA
QQYSSYSLT
QQYANFPLT
V␬4
V␬4
V␬2
V␬1
V␬1
V␬1
DPK24
DPK24
DPK15
DPK9
L12
DPK1
98.6
98
ND
95.0
96.1
94.4
J␬1
J␬1
J␬2
J␬1
J␬4
J␬4
QHRSNWPPALT
MQTLQTPYT
QVHRNSLGT
V␬3
V␬2
V␬3
L6
DPK15
DPK22
99
98
96
J␬4
J␬2
J␬1
QQYDSSSGYT
V␬1
L12
95.5
J␬2
WDSSTVV
QQYHSYPVT
CDR3 VL ⫺ IgM
V L3
V␬1
DPL23
L12
93.1
96.8
JL2
J␬4
VH3
VH3
VH3
VH3
VH4
VH 5
VH3
VH3
VH3
VH3
VH3
VH3
VH3
VH3
VH3
DP-35
DP-35
DP-49
DP-46
DP-63
DP-73
DP-51
DP-31
DP-49
DP-51
DP-31
DP-47
DP-54
3-66
DP-50
90.5
97.3
95.6
98.6
99.7
98.0
97.6
96.6
100
96.6
94.2
96.6
97.3
98.3
99.7
JH3
JH6
JH3
JH4
JH4
JH5
JH3
JH3
JH4
JH4
JH5
JH4
JH4
JH6
JH6
YRSSSSYV
V L2
DPL11
98.0
JL1
LQHNSYPRT
YVGSYSFVV
Nonproductive
V␬1
V L2
V␬4
DPK3
DPL12
DPK24
96.5
98.0
ND
J␬1
JL2
YTSSSTYV
V L2
DPL11
99.0
JL1
QQYGSSPGIT
V␬3
DPK22
99.6
J␬3
CDR3 VL ⫺ IgG
a
Each line identifies the CDR3 amino acid sequence, germline family, most homologous V and J germline segment, and degree of homology of the H and L chain V region
amplified from that single CD19⫹ B cell.
discrete clonally expanded B cell populations. For example,
MS02-14 clone 1 (identified by the CDR3 sequence VPRDGYNYFDF) was amplified on four occasions and comprised the largest clonal population. Each B cell within this population used the
same VH and JH germline segments, and shared an identical set of
18 somatic mutations (data not shown). Three of these cells expressed an identical L chain sequence that shared a common set of
somatic mutations; no L chain product was amplified from the
fourth cell. Similar observations were made for B cells comprising
other clonal populations in MS02-14 CSF. However, limited se-
quence heterogeneity was detected in V region sequences of some
clonally related B cell progeny, indicating a degree of clonal variation among these expanded populations. Although the VH sequence of B cells in MS02-14 clones 6 and 7 was detected only
once, the L chain rearrangement expressed by each of these cells was
detected multiple times, indicating a clonally expanded population.
Table III lists the features of VH and VL sequences belonging to clonal
B cell populations rescued from the CSF of the three additional MS
donors studied. Three distinct clonal populations were detected in
MS02-2 and MS02-6 CSF, and one or possibly two distinct clonal
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
63
145
CDR3 ⫺ IgG
The Journal of Immunology
2731
FIGURE 2. Clone-specific PCR amplification of two distinct VH rearrangements in B cells comprising MS02-2 clone 2. The two VH sequences
(PRNRYQDGLFDS and DGGSDYASQFDY) found in MS02-2 clone 2 (Table III) were specifically amplified using primers complementary to nucleotide
sequences encoding their CDR3 regions and with the appropriate VH4 or VH1 framework 1 primer. PCR was performed on cells of MS02-2 clone 2 (wells
3, 8, 12, 40, 44, and 46) and MS02-2 clone 1 (wells 5, 6, 14, 22, 26, and 38). A and B, Show PCR amplifications using the PRNRYQDGLFDS CDR3 primer
and the DGGSDYASQFDY CDR3 primer, respectively. neg, Negative DNA controls.
Evidence of receptor editing in MS02-2 B cell repertoire
Several features of the MS02-2 IgG repertoire distinguished it
from the other clonal populations identified in Tables III and VI. In
B cells of MS02-2 clone 1, characterized by the H chain CDR3
sequence VKASFDY, no single dominant L chain sequence was
identified. Although it is possible that an existing dominant clone
1 L chain sequence failed to amplify, four different productive L
chain rearrangements and one nonproductive rearrangement were
detected in those B cells from which an L chain sequence was
amplified (Table III). It is unlikely that these sequences represented PCR contamination or artifact. These L chains were not
found in B cells from any other CSF repertoires analyzed in this
study, and MS02-2 clone 1 was the only clonal population in
which we detected multiple productive L chain rearrangements.
Interestingly, whereas the VH region of this population had accumulated 21–22 different somatic mutations, rearranged L chain
sequences were highly homologous to, and in one cell, identical
with their germline sequence. The difference in mutational frequencies between clone 1 H and L chain sequences (mean ratio
VH/VL ⫽ 0.94 ⫾ 0.01; n ⫽ 4) was significantly greater ( p ⬎
0.0002, nonpaired t test) than the differences found in the remaining MS02-2 B cell population (mean ratio VH/VL ⫽ 0.98 ⫾ 0.02,
n ⫽ 12), suggesting that some B cells of the clone 1 population had
recently undergone receptor editing to produce the multiple L
chain rearrangements detected.
A second clonal B cell population of MS02-2 CSF showed evidence of H chain receptor editing. All of the B cells expressing the
VH rearrangement with the CDR3 sequence PRNRYQDGLFDS
coexpressed the same L chain (QQYGILPWT) or a clonal variant
of this sequence. However, a subset of B cells that paired this L
chain with a different VH␥ rearrangement (DGGSDYASQFY
FDY) was also detected. Interestingly, an in-frame nonsense codon
was present in the first amino acid position of the CDR2 region of
each of this second group of H chain sequences (data not shown),
indicating that the VH␥ sequence was nonfunctional. To determine
whether B cells in the MS02-2 clone 2 population contained
mRNAs encoding both the PRNRYQDGLFDS and DGGSDY
ASQFYFDY IgG H chains, we repeated the nested H chain PCR
amplifications using clone-specific primers hybridizing to the distinct CDR3 nucleotide sequences of both these H chains. Fig. 2
shows that products corresponding to the PRNRYQDGLFDS H
chain were readily amplified from seven of seven B cells expressing the QQYGILPWT L chain, but not from B cells comprising
MS02-2 clone 1 (VKASYFDY). Products corresponding to the
nonfunctional DGGSDYASQFYFDY H chain were only detected
in the same clone 2 B cells from which this VH sequence was
originally identified. Thus, two B cells in the clone 2 population
were confirmed to contain distinct mRNA encoding both the functional PRNRYQDGLFDS VH rearrangement and the corrupted
DGGSDYASQFYFDY sequence. The lower frequency of a second nonfunctional VH rearrangement suggests that H chain receptor editing occurred in a precursor of the clone 2 B cell population.
This second H chain rearrangement most likely occurred during B
cell development, but could also have occurred peripherally in
response to generation of the nonsense mutation.
Discussion
In this study, we used fluorescence-activated cell sorting and single-cell RT-PCR to analyze the CD19⫹ B cell Ig repertoire in CSF
from four patients with relapsing-remitting MS, and two inflammatory controls with viral meningitis. Our goal was to evaluate the
extent of B cell clonal expansion within the CNS of MS patients.
Consistent with previous analyses of the IgG H chain repertoire
that identified overrepresented and clonally related IgG sequences
in MS plaques (8 –11) and CSF (12–13), we detected clonally expanded B cell populations in all of the MS CSFs. The largest
number of distinct IgG clonal populations was detected in CSF of
MS02-14, the patient with the longest disease duration of 22 years,
whereas the IgG repertoires of donors MS02-6 and MS02-2, individuals diagnosed with MS for 1 or 2 years, respectively, at the
time of sample collection contained markedly more limited clonal
populations. Whether these differences in complexity represent
disease heterogeneity or reflect disease duration is not known, and
necessitates the analysis of additional donors and tracking and repeated sampling of individual donors over time. The diverse B cell
response found in the inflammatory control CSFs probably reflects
recovery of CSF on the first day of illness before an Ab response
to the infectious agent develops. Further monitoring of the repertoire of those patients would most likely reveal the emergence of
clonal B cell populations, as previously described for CSF of viral
meningitis patients (13).
The synthesis of IgM in the CSF of inflammatory and infectious
diseases is thought to indicate active antigenic stimulation within
the CNS (18, 19). IgM OGBs have been detected in the CSF of
⬃50% of MS patients and appear to correlate with onset of relapses and a worsening disease course (20, 21). IgM-positive B
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
populations were detected in MS02-11 CSF. The repertoire in
MS02-2 CSF was the most restricted of the four MS CSFs studied.
Clones 1 and 2 accounted for ⬃70% of the H chain V regions
analyzed.
All the rearranged IgG sequences in B cells recovered from MS
donor CSF were somatically mutated. The degree of homology for
VH sequences from MS02-14 CSF ranged from 97.3 to 90.8%
when compared with their closest germline segment. Similar homologies were noted in VH repertoires rescued from the other MS
donors (data not shown). The average homology with nearest
germline sequence of VH␥ and VH␮ genes amplified from the MS
donors was ⬃94 and ⬃97%, respectively, irrespective of disease
duration.
2732
observed for B cells (29) and probably account for the bulk of Ab
production. Their similarity to B cell populations in MS CSF also
remains to be determined.
Overall, B cell clonal expansion is a prominent feature of the
MS humoral response and has been found in both MS plaques (8,
10) and CSF (12, 13). The clonal variation observed within some
populations and the somatic mutations found in rearranged V region segments indicate that a targeted response occurs in MS.
Identification of rearranged Ig sequences is the first step in accurately recreating recombinant Abs from these overrepresented H
and L chains for use in efforts to discover the relevant Ag(s) in MS.
Acknowledgments
We thank Marina Hoffman for editorial review, and Cathy Allen for preparing this manuscript. We also thank and acknowledge Karen Helm of the
University of Colorado Cancer Center for assisting in the protocol used for
sorting of CSF B cells.
References
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cells constituted a significant portion of the B cell repertoire in
three of the four MS patients studied in this work. Such IgMpositive cells were more abundant than IgG-positive B cells in
MS02-6 and MS02-11, and comprised ⬃33% of the B cell repertoire of MS02-14. Because we detected only one clonal IgM population in the four MS patients, it is still unknown whether the
presence of IgM-positive B cells is due to antigenic stimulation or
breakdown of the blood-brain barrier.
An interesting characteristic of the MS02-2 B cell repertoire was
evidence suggestive of L chain receptor editing. Receptor editing
occurs in primary lymphoid tissue during B cell development as an
attempt to rescue self-reactive B cells from destruction by altering
their receptor specificity (reviewed in Ref. 22). Through renewed
or continual expression of the recombinase genes, a secondary
rearrangement at the Ig loci can result in new Ab rearrangements
to form a functional, but nonautoreactive Ig receptor (23–26). Secondary rearrangements can also contribute to receptor diversification in germinal centers during an immune response. In clone 1 of
the MS02-2 repertoire, we did not detect a predominant L chain
sequence, but instead found four distinct and productive L chain
rearrangements expressed in different B cells with the same H
chain rearrangement (clone 1, Table III). Because an L chain product was detected in only 5 of 11 clone 1 B cells, it is possible that
our primers failed to amplify a dominant L chain sequence from
this population. However, at least some clone 1 B cells appear to
have undergone additional L chain rearrangements. Compared
with the extensively mutated VH sequence, the rearranged VL sequences did not deviate significantly from germline, suggesting
that rearrangement of these L chains did not occur during B cell
development, but rather as a more recent event. Ig receptor editing
might represent an attempt either to alter autoreactivity of this B
cell population or to improve affinity for Ag. Because there are no
organized germinal centers in brain, it is interesting that no dominant clonal L chain rearrangement was found in clone 1 and may
offer insights as to where B cell responses in the CNS of MS
patients mature. In model systems containing an intact blood-brain
barrier, specific B cell responses to Ag introduced intracerebrally
develop in peripheral germinal centers (27) and then migrate as
activated B cells back into the CNS, where they are retained and
differentiate at the site of Ag deposition (28). Our observations
suggest either that Ag recognition is largely independent of the L
chain sequence in clone 1, or that these secondary L chain
rearrangements may actually be occurring within the CNS. The
significance of L chain receptor editing in modifying the CNS
immune response in MS requires further study. Meanwhile, this
phenomenon does not appear to be widespread because MS02-2
clone 1 was the only MS B cell population in which evidence of L
chain editing was found.
The significance of a functional and nonfunctional VH rearrangement in MS02-2 clone 2 is not clear. This second rearrangement most likely occurred during B cell development to generate
a functional B cell receptor at the pre-B cell stage or to alter a
self-reactive B cell. The stop codon in the nonfunctional VH rearrangement may be present in one of the DP-14 germline alleles or
simply may have occurred during the germinal center reaction in
the absence of selective pressure.
The relationship between the clonal B cell populations in MS
CSF and OGB production remains to be determined. The number
of OGBs in MS02-6 and MS02-11 CSF exceeded that of clonal
populations detected in our CSF analysis. Some of these OGBs
may be synthesized by B or plasma cells located in plaques that
extravasate into the CSF. The degree of relatedness between B
cells populating plaques and those in CSF is still unknown. Plasma
cells are also present in CSF at levels that are ⬃25% of those
B CELL RESPONSES IN MS
The Journal of Immunology
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2733
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