| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
and Heavy Chain Gene Usage in Early Untreated Systemic Lupus Erythematosus Suggests Intensive B Cell Stimulation1
Department of Internal Medicine and The Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75235
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and J as well as
VH and JH gene usage in a patient with systemic
lupus erythematosus (SLE), productive and nonproductive VJ and V(D)J
rearrangements were amplified from individual peripheral
CD19+ B cells and were analyzed. No differences in the V
and J or the VH and JH gene usage in the
nonproductive gene repertoire of this SLE patient were found compared
with the distribution of genes found in normal adults, whereas marked
skewing of both V and VH was noted among the productive
rearrangements. The distribution of productive V rearrangements was
skewed, with significantly greater representation of the J distal
cluster C V genes and the V distal J7 element, consistent with
the possibility that there was receptor editing of the V locus in
this patient. Significant bias in VH gene usage was also
noted with VH3 family members dominating the peripheral B
cell repertoire of the SLE patient (83%) compared with that found in
normal subjects (55%; p < 0.001). Notably, a
clone of B cells employing the VH3-11 gene for the heavy
chain and the V1G segment for the light chain was detected. These
data are most consistent with the conclusion that extreme B cell
overactivity drives the initial stages of SLE leading to remarkable
changes in the peripheral V gene usage that may underlie on fail to
prevent the emergence of autoimmunity. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 rearrangements
(2), or, alternatively, whether abnormalities in somatic
hypermutation and/or subsequent selective influences can lead to the
generation of autoantibodies.
Recently, we found that the VJ recombination process, as judged
by analysis of nonproductive VJ rearrangements in an untreated
SLE patient, appeared to be comparable to that in normal subjects
(3). However, striking differences in the productive V
repertoire of this patient were noted, with enhanced usage of the J
distal V genes and a marked increase in the usage of J5, the most
V distal J gene. These data suggested that the replacement of
primary V rearrangements by subsequent rearrangements (receptor
editing) was more frequent in this SLE patient than had been observed
in normal subjects (4). Although these results were
obtained from a single patient, they were so strikingly different from
a previous VJ repertoire analysis in normal subjects
(4) that a more extensive examination of this patient was
conducted. Specifically an analysis of the V and
VH repertoire was conducted because of the
possibility that V and VH receptor editing in
SLE might also be abnormal.
The distribution of V/J and
VH/JH rearrangements in the
normal Ig gene repertoire has been delineated recently
(5, 6, 7). Therefore, to determine whether there was
increased receptor editing of V and VH genes
in SLE or other abnormalities in the V gene repertoire, the current
study analyzed and compared the usage of V/J and
VHDJH gene elements in this
same untreated SLE patient with the repertoire of normal donors. The
distribution of VH and V genes in the
nonproductive repertoire of this SLE patient was compared with that in
normal subjects, suggesting that there were no major molecular
abnormalities in VJ recombination. Striking abnormalities in the
distribution of V genes were noted in the productive repertoire,
however, consistent with accentuated receptor editing of V genes. In
addition, although no evidence of increased receptor editing of
VH genes was found, skewing of the expressed
VH repertoire, increased somatic hypermutation,
and clonal expansion was detected. These results are consistent with
the conclusion that there is marked overactivity of B cells in early
SLE that could contribute to the production of autoantibodies.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The method of cell purification, B cell staining and sorting, as well as the primer extension preamplification procedure have been reported in detail recently (6, 7). Briefly, B cells were obtained from a 54-yr-old Hispanic man with SLE, who was previously undiagnosed. Features of SLE included a typical butterfly rash, hyperkeratotic lesions of SCLE, increased fatigue, intermittent episodes of fever, and arthralgias of the proximal interphalangeal joints. The antinuclear Ab titer was 1:2560 (speckled pattern), and anti-Ro, -La, and -RNP Abs were present, whereas anti-dsDNA Abs were repeatedly absent. The patient did not have hypergammaglobulinemia. It should be noted that titers of these autoantibodies remained comparable for the next 1.5 yr. Clinically, disease manifestations began about 4 wk before the B cells were sorted, as evidenced by the onset of a typical rash. The patient had noted photosensitivity for many years. At this particular time point, there were no signs or symptoms of another connective disease. A reduction of complement factors C3 (62.6 mg/dl; normal, 65–203) and C4 (<10 mg/dl; normal, 16–54) was also found. The white blood cell count was 3.8 x 103/µl, with 20% lymphocytes. Because of the decreased blood cell count, only CD19+ B cells were isolated. The patient fulfilled the revised criteria for classification of SLE (8).
FACS sorting and PCR amplification
Altogether 276 individual CD19+ B cells
were sorted into wells of 96-well plates (Robbins Scientific,
Sunnyvale, CA) using a FACStar Plus flow cytometer with an automated
single cell deposit unit (Becton Dickinson, Mountain View, CA) as
described previously (4, 5, 6, 7). Twelve wells (four per plate)
that received no cells were used as negative controls. Rearranged
VJ and VHDJH genes
were then amplified as described recently (5, 6, 7). The PCR
amplification included a primer extension preamplification
(7) and subsequent nested PCR steps (5, 7).
After column purification of PCR products (GenElute Agarose Spin
Column, Supelco, Bellefonte, PA), all PCR products were directly
sequenced using the ABI Prism Dye Termination Cycle Sequencing Kit
(Perkin-Elmer, Palo Alto, CA) and analyzed with an automated sequencer
(ABI Prism 377, Perkin-Elmer). Sequences were analyzed using the V BASE
Sequence Directory (9) to identify the respective germline
gene. For the identification of the underlying germline segments, the
software programs GeneWorks (release 2.45; IntelliGenetics, Mountain
View, CA) and Sequencher (Gene Codes, Ann Arbor, MI) were employed.
The usage of VH and JH
genes as well as V and J rearrangements segments from two healthy
normal male donors (26 and 45 yr old) that had been published
previously (5, 6) served as a comparison. Both the
nonproductive and productive repertoires of these two normal,
age-disparate donors exhibited an overall similar usage of V and J gene
elements.
Determination of Taq polymerase fidelity and the frequency of potential sequence errors
The maximal PCR error rate for this analysis has been documented to be 1.2 x 10-3 mutations/bp, and the minimal error to be 1 x 10-4 (5, 10).
Statistical analysis
Sequences were analyzed with the 
2 test
to compare the differences in the distribution of particular gene
segments as well as mutational frequencies between the VJ and
VHDJH rearrangements of the
SLE patient and the normal subjects. The goodness of fit
2 test (11) was used to compare
the actual distribution of V and J as well as the
VH and JH family gene usage
in the SLE patient to the frequency that might be expected based upon
the number of genes in the genome (6). P < 0.05 was
considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene usage in SLE
A total of 104 productive and 58 nonproductive VJ
rearrangements were analyzed. The distribution of individual V
families is shown in Table I
. Of
importance, no major differences in V gene usage between the
nonproductive repertoire of the SLE patient and that of the normal
subjects was found. Some minor differences between the nonproductive
repertoire of normal subjects and that of the SLE patient were noted.
For example, V6 genes were found significantly more often in the
nonproductive repertoire of the SLE patient (9%) than expected by
chance alone, whereas this V gene family was not over-represented in
the nonproductive repertoire of the normal subjects (3.6%).
|
4 family members occurred
significantly more often in the productive repertoire of the normal
subjects than in that of the SLE patient (5.8 vs 0%; p
= 0.012), whereas members of the V6 (p <
0.001) and V8 (p < 0.001) families were
more frequently employed in the productive repertoire of the SLE
patient. These differences suggest that factors dependent on expression
of a productively rearranged V gene may have influenced the
distribution of V genes in the SLE patient.
J gene usage in SLE
The distribution of rearranged J elements in the SLE patient is
summarized in Table II. Of note, the
usage of J2/3 and J7 was significantly different between the
nonproductive repertoire of the SLE patient and that of the normal
subjects. Whereas J2/3 genes were employed more often in the patient
(58.6%) than in normal subjects (34.5%; p = 0.01),
J7 was found more frequently in normal subjects (60.0%) than in
nonproductive rearrangements of the SLE patient (36.2%;
p = 0.011). In general, the usage of J1 was
significantly less than expected by chance alone regardless of whether
nonproductive or productive rearrangements were analyzed.
|
7 was found in the
SLE patient than in normal subjects (67.3 vs 54.1%; p
= 0.03). Moreover, the frequency of J7 was significantly greater in
the productive than in the nonproductive repertoire of the SLE patient
(67.3 vs 36.2%; p < 0.05).
Distribution of individual V genes in B cells from SLE
As shown in Table III, there were no
differences in the distribution of individual V genes in the
nonproductive repertoire of the SLE patient and the normal donors. When
the distribution of particular V gene segments in the productive
repertoire of the SLE patient was compared with that in the normal
subjects, four V genes, 2A2 (p = 0.001), 3H
(p = 0.045), 1B (p =
0.001), and 4B (p = 0.012), were found
significantly less often in the SLE patient. Moreover, a significant
over-representation of 3L (p = 0.025), 1G
(p = 0.04), 6A (p =
0.001), and 8A (p = 0.001) was found in the
productive repertoire of the SLE patient. In general, the
over-represented V genes in the productive repertoire of the SLE
patient tended to be J distal. To analyze this in greater detail,
the use of V genes in the three major gene clusters was assessed
(Table IV). The usage of the V gene
clusters, A, B, and C, was similar in the nonproductive repertoires of
the patient and the normal subjects. In the productive repertoires,
however, the usage of V genes of the most J proximal cluster, A,
was significantly less frequent in the SLE repertoire (33.7%) than in
the normal repertoire (48.8%; p < 0.05). Moreover,
genes belonging to the most J distal cluster, C, were found
significantly more frequently in the SLE patient (30.8%) than in
normal subjects (14%; p < 0.001). Thus, the
productive rearrangements of the SLE patient employed the J distal
V gene cluster C as well as the V distal J7 gene element
significantly more frequently than those of the normal subjects.
|
|
The mutational frequencies of nonproductive and productive V
rearrangements from the SLE patient were 3.12 and 3.38%, respectively
(Table V). Thus, there were no major
differences between the mutational frequencies of the productive and
nonproductive repertoires. Moreover, the nonproductive V
rearrangements using J7 exhibited a mutational frequency of 3.05%
compared with a mutational frequency of 3.04% in the productive
repertoire. Thus, the distribution of mutations in rearrangements using
the most 3'-proximal J element was comparable in productive and
nonproductive repertoires. This compares with a mutational frequency of
3.43% for nonproductive V rearrangements using J1–3 and 3.53%
for productive rearrangements using J1–3. Analysis of mutational
frequencies in V genes in this patient indicated that productive
V rearrangements were mutated significantly less than nonproductive
V rearrangements (2.80 vs 3.60%; p < 0.01).
Moreover, productively rearranged V genes using the distal J5
element were less mutated (1.99%) than rearrangements using J1–4
(3.08%; p < 0.001) (3).
|
Further analysis sought to compare N nucleotide addition at the
joins of V and J elements as an estimate of TdT activity
operative on the rearrangements (Table VI). Remarkably, in the nonproductive
repertoire, there were no J7 rearrangements that failed to contain N
additions, implying that TdT activity was active during the
rearrangement of these gene segments. In the productive repertoire,
20% of rearrangements employing J7 exhibited no evidence of TdT
activity. The opposite tendency was noted when rearrangements employing
J1–3 were analyzed, with a higher frequency exhibiting no TdT
activity in the nonproductive compared with the productive repertoire.
Since TdT activity decreases during B cell ontogeny (12),
these results suggested that the productive repertoire may be enriched
in J7-containing rearrangements that were generated later in
ontogeny, as might be anticipated if central receptor editing accounted
for their introduction.
|
A total of 41 productive and six nonproductive
VHDJH rearrangements were
analyzed. The comparison of the productive VH
repertoires between the SLE patient and the normal subjects revealed
differences, in that there was a striking over-representation of
VH3 family members (82.9 vs 54.9%;
p < 0.001) and under-representation of the
VH4 family (7.3 vs 22.0%; p <
0.03) in the peripheral B cell repertoire of the SLE patient (Table VII). Of note, members of the
VH5, -6, and -7 families were not found in the
productive repertoire of the SLE patient, whereas only one
VH1 family member (2.4%) and three members of
the VH2 family (7.3%) were found.
|
) in the nonproductive repertoire was similar to that in normal
subjects. Of note, the ratio of nonproductive/productive rearrangements
(0.15%) was identical in the SLE patient and the normal subjects. JH gene usage
As shown in Table VIII, the
distribution of JH genes did not differ between
the SLE patient and the normal subjects. In general,
JH4 and JH6 dominated the
repertoire of productive rearrangements in the SLE patient (53.7 and
22.0%, respectively) and the normal subjects (54.0 and 25.1%,
respectively).
|
There was a significantly increased usage of VH3-11 in the productive repertoire of the SLE patient (p < 0.001), whereas the frequency of occurrence of all other genes did not differ compared with that in normal subjects. This indicated a preferential expansion of B cells using the VH3-11 element in this patient (see below). Of note, the VH3-11 gene segment has previously been documented to be negatively selected in the normal peripheral B cell repertoire (7).
Detailed analysis of individual gene usage in the nonproductive
repertoire of the SLE patient revealed that
VH3-08 (p < 0.002),
VH3-11 (p < 0.001), and
VH3-64 (p < 0.002) were
detected significantly more often than in normal subjects (Table IX). However, the small number of
nonproductive rearrangements from the SLE patient does not permit a
firm conclusion, although the significantly increased detection of
these three VH genes suggests the possibility of
preferential gene usage not seen in normal subjects.
Identification of a clonal expansion of B cells in early, untreated SLE
Sequence analysis of the 8 VH3-11
rearrangements obtained from the SLE patient revealed a high degree of
sequence homology in six cases (92.2–99.2%; Fig. 1B) consistent with the
possibility that B cells expressing this VH3 gene
may have derived from of a single B cell precursor. These
rearrangements used VH3-11,
JH6 and the D elements LR5 and inverse D12/9. The
CDR3 length was 45 bp (15aa) and with the exception of putative
mutations was identical in each member of the clone. In addition, a
productive V light chain rearrangement employing V1G/J7 (Fig. 1, A and C) was found in five of these same B
cells. The CDR3 length of this V rearrangement was 33 bp (11 aa) and
was also identical in each clone member, with the exception of putative
mutations. In one of the six potential members of this clone, the light
chain could not be amplified (D2-2g3E5). The two remaining
VH rearrangements using
VH3-11 differed from the clone in that two of
them used JH4 genes (D2-1g3D11 and D2-1g3E8) with
CDR3 lengths of 14 and 8 aa, respectively.
|
The nonproductive VH rearrangements of the
SLE patient exhibited a mutational frequency of 6.5% (87
mutations/1330 bp) compared with 4.4% for the productive
VH rearrangements (446 mutations/10,172 bp;
p < 0.001, by 2 test). Of
note, there were only two productively rearranged
VH genes (one VH3 and one
VH4 family member, respectively) that did not
contain nucleotide substitutions, indicating that
VH genes of the vast majority of the
CD19+ B cells analyzed had undergone somatic
hypermutation. The clonally related VH3-11 genes
(see Fig. 1B) acquired 82 mutations/1446 bp for a
mutational frequency of 5.67%. The mutational frequency of the
clonally related VH3-11 genes was significantly
higher compared with that of the remaining genes (3.7%;
p < 0.001).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and J as well as the
VH and JH gene usage in a
patient with SLE and documented that there are few molecular
differences apparent in the peripheral B cell repertoire of this
patient compared with that of normal controls as indicated by the
distribution of V and heavy chain genes in the nonproductive
repertoires. Analysis of genomic DNA made it possible to obtain a
representative number of nonproductive gene rearrangements and thereby
permitted the comparison with productive rearrangements.
V analysis
Marked differences were noted between the productive VJ gene
repertoire of the SLE patient and that of the normal subjects and
between the productive and nonproductive repertoires of the SLE
patient. These differences could have been attributed to a variety of
influences that are dependent on expression of a V gene product,
including selection and receptor editing.
Detailed analysis of the productive V repertoire revealed
significant deviations in the distribution of both V and J
elements in the SLE patient compared with normal subjects, in that the
frequencies of both the J distal V elements and the V distal
J segment were increased. In detail, there was an
under-representation of the J proximal genes, 2A2, 3H, 1B, and 4B in
the SLE patient, whereas the J distal genes 3L, 1G, 6A, and 8A were
over-represented compared with those in normal subjects. This
contributed to a significantly different usage of the J proximal
gene cluster A and the J distal V gene cluster C between the SLE
patient and the controls. The number of V genes that exhibited
biased representation as well as their locations were most consistent
with the conclusion that receptor editing of the V locus might have
been frequently used in shaping the productive repertoire of the SLE
patient. The over-representation of J7 in the productive repertoire
of the SLE patient was consistent with augmented receptor editing.
Analysis of the V repertoire of the same patient also revealed
evidence of extensive receptor editing of the V locus
(3). In both circumstances, receptor editing was
significantly increased in degree in the SLE patient compared with that
in the normal controls.
Despite the evidence that receptor editing of V and V genes was
increased in this SLE patient compared with that in normal subjects,
the mechanisms appeared to be different. The data suggested that V
receptor editing was most marked in the periphery of this SLE patient
based on the higher mutational frequency of productive rearrangements
employing J1–4 compared with those using J5 (3).
This finding implies that receptor editing of V in SLE occurs in the
periphery after somatic hypermutation has been initiated. The current
analysis supports this conclusion, since productively rearranged V
genes exhibited a lower frequency of mutations than nonproductive V
rearrangements. This is markedly different from the situation in normal
subjects, in which the mutational frequency of productive V
rearrangements is significantly greater than that for the nonproductive
repertoire (4). This finding implies that receptor editing
of V rearrangements after mutation is sufficiently robust to result
in an overall decrease in the mutational frequency of productive V
rearrangements in this SLE patient.
The distribution of V genes also implied that receptor editing of
V had occurred in this patient. As opposed to the V genes,
however, analysis of the mutational pattern suggested that the dominant
influence was central, and not peripheral, receptor editing of V
genes. Thus, there was an increase in the usage of 5' V genes and
the 3' J7 segment, but there was no decrease in the mutational
frequency of productive V rearrangements using these elements or of
the entire productive V repertoire. These results imply that the
bulk of V receptor editing in this SLE patient occurred before the
mutational machinery had been activated and therefore most likely
occurred in the bone marrow during B cell ontogeny. This contention was
supported by an analysis of apparent TdT activity exerted on
rearrangements employing J1–3 vs J7. As TdT activity diminishes
during B cell ontogeny (12), it would be anticipated that
productive rearrangements that were introduced later in B cell
development as a result of receptor editing would contain fewer
additions because of waning TdT activity. The increased number of J7
containing productive rearrangements with no N segment additions is
consistent with this conclusion. The combination of J7 containing
rearrangements with fewer N nucleotides but a comparable mutational
frequency is most consistent with the conclusion that their
over-representation in the productive repertoire resulted from central
receptor editing.
Whether additional peripheral editing of V genes also occurred
cannot be determined from this analysis, although it should be noted
that if such a process occurred, it was of insufficient magnitude to be
detected by this approach. Similarly, we cannot determine whether
central receptor editing of V rearrangements occurred because
peripheral editing was so dominant.
It is noteworthy that recombined V4B and J2/3 genes were found
exclusively in the nonproductive repertoires of both the normal
subjects and the SLE patient, suggesting that they were eliminated from
the productive gene repertoire of each comparably. This implies that
some elements of negative selection or receptor editing operated
normally in the SLE patient. Similarly, A30/J2 was exclusively found
in the nonproductive repertoire of this SLE patient (3).
Productively rearranged A30/J2 genes have been shown to bind dsDNA
in their germline configuration (2, 13). Although the
binding specificity of 4B/J2/3 gene rearrangements has not been
delineated, it was detected only in the nonproductive and not the
productive repertoires, suggesting that it might bind an autoantigen.
Its elimination from the productive repertoire might, therefore, result
from negative selection and/or receptor editing. Whatever the mechanism
of elimination, this process appears to be intact in this SLE patient
and comparable to that in normal subjects.
Data from V transgenic mice have shown that central receptor editing
can operate to replace light chains of B cells expressing
autoantibodies (14), although there are no previous
examples of central receptor editing of V chains. There is, however,
no conceptual reason that central receptor editing of V chains could
not occur if these V gene products encoded autoantigen recognition.
This suggests that emergence of V-containing autoantibodies during B
cell ontogeny may have been the stimulus for central V receptor
editing in this SLE patient. In this context, V genes have been
shown to be critical parts of a number of human autoantibodies,
including Abs to dsDNA (15, 16, 17, 18), Abs to La/SS-B and
Ro/SS-A (19, 20), rheumatoid factor (21, 22, 23, 24),
and Abs to laminin (25), phospholipids (26),
collagen, and histone 2A (27). It should be noted,
however, that there are insufficient data on the light chain usage of
autoantibodies to conclude that receptor editing differentially effects
the use of V or V.
Heavy chain gene usage
In contrast to analyses of VL gene usage in this same patient, the current examination of the VH gene usage revealed no evidence of increased VH receptor editing, but detected other differences in the VH gene repertoire that could contribute to autoantibody formation.
VH gene usage
Receptor editing of the VH gene locus has been observed in a site-directed manner in a transgenic mouse model (28, 29). Defects in receptor editing have been suggested to play a role in retaining autoreactive B cell receptors (BCRs) in autoimmune diseases (29, 30, 31, 32, 33). Despite this, no evidence of abnormalities in receptor editing of VH genes was detected in the current analysis by assessment of the distribution of VH genes, but the possibility that this process is impaired in this SLE patient cannot be completely excluded. Only defects in receptor editing of sufficient magnitude to alter the distribution of VH genes in the productive repertoire were detected. It remains possible that a defect in site-directed receptor editing of VH genes could contribute to autoimmunity in this SLE patient. Of note, however, evidence of enhanced receptor editing of VL chains was easily detected in this patient, making it unlikely that there was an overall defect in the receptor-editing process in this SLE patient.
Comparison of the VH gene usage in the productive repertoire provided evidence that the gene segment VH3-11 was found significantly more often in this SLE patient than in normal subjects. This over-representation was accounted for by the above noted preference to rearrange this germline gene in this patient as well as by the expansion of a B cell clone expressing this VH gene segment. Moreover, other B cells expressing VH3-11 rearranged to other JH segments were also over-represented, suggesting that the negative selection of rearrangements employing VH3-11 observed in normal subjects (7) was disturbed in this patient. Negative selection of VH3-11 in normal subjects has previously been suggested in other studies (34, 35) regardless of the genetic background of the donor. The over-representation of VH3-11 in this SLE patient, therefore, is unusual and mandates an analysis of other SLE patients to determine whether this is a consistent feature of this disease. The possibility that this patient manifested a generalized enhancement in positive selection of VH3-expressing B cells was suggested by the analysis of the entire VH3 family, as well as of the VH3-23 (DP-47) gene. VH3-23 (DP-47) is the most frequently used VH3 family member (6, 7, 34, 35), accounting for 12–14% of the normal repertoire (6). In this SLE patient, VH3-23 was even more frequently used, being expressed by 22% of the B cells. Of importance, VH3-23 has previously been noted to encode anti-DNA Abs frequently, especially the 16/6 Id (36). Whether an abnormal mechanism, such as B cell superantigen stimulation (37, 38), is causing expansion of VH3-expressing B cells in this SLE patient will require carefully analysis of other patients.
One of the remarkable findings of this study is the identification of
six B cells that expressed BCRs using VH3-11 and
in five cases V1G rearrangements with a high degree of sequence
homology. Of note, unique patterns of mutations and, with the exception
of putative mutations, identical CDR3s were identified, suggesting that
these resulted from clonal expansion and Ag-mediated selection. The
usage of VH3-11 and V1G by this clone requires
emphasis, since both genes have been reported to be negatively selected
in normal subjects (5, 7). Although proof of
autoreactivity of these resulting BCRs is lacking, these data are
consistent with the conclusion that clonal expansion of B cells can
occur in the initial stages of SLE, suggesting an overwhelming
antigenic stimulus. Studies in mice have extensively documented that
clonal expression of autoreactive B cells occurs in early lupus
(39, 40). In addition, this clone used two D elements, one
of which was employed in an inverted orientation. Although the use of
inverted D segments has been suggested to increase the frequency of
arginines in CDR3s and thereby contribute to the development of
anti-dsDNA Abs (41, 42), inverted D elements have also
been detected in normal subjects (6, 7). The current data
do not allow a firm conclusion about whether an enhanced rate of
inverted D elements is a hallmark of clonally expanded B cells in SLE
or whether these clones encode anti-DNA Abs. However, the expansion
of a B cell clone in the initial stages of SLE in this patient is
consistent with findings noted in autoimmune-prone mice (40, 43).
Analysis of mutations provided further insights into the generation of
diversity in this SLE patient. The marked degree of somatic
hypermutation of the VH rearrangements of this
untreated SLE patient is obvious. The mutational frequencies of the
CD19+ B cells from the SLE patient (6.5% for
nonproductive and 4.4% for productive rearrangements) were
significantly greater than those found in normal subjects
(CD19+ peripheral B cells from a female Caucasian
donor exhibited mutational frequencies of 3.8% for nonproductive and
3.3% for productive rearrangements) (44, 45). Thus, both the
nonproductive (p < 0.001) as well as the
productive repertoire (p < 0.001) were
significantly more mutated in the SLE patient than in normal controls
(45). Since previous analyses provided evidence that age
influences the number of mutations in memory B cells, we compared the
mutations in the 54-yr-old SLE patient with that previously reported in
a 45-yr-old male donor (6, 45). The mutational frequency
in the SLE patient significantly exceeded that in the nonproductive
(p < 0.001) and the productive repertoire
(p < 0.005) of this normal donor
(nonproductive: 245 mutations/6528 bp; mutational frequency, 3.8%;
productive: 1601 mutations/47872 bp; mutational frequency, 3.3%).
Since mutational activity, in general, is induced in response to
T-dependent Ags, and the frequency of mutations in the nonproductive
repertoire reflects the activity of the mutational machinery without
subsequent selection (44), the B cells of this patient
appear to have been stimulated in a T cell-dependent manner more
intensively or more persistently than in normal subjects. Whether this
reflects the intensity or persistence of stimulation or a defect in
apoptosis of B cells expressing mutated BCRs, as has been suggested
(46, 47), remains to be determined. Preliminary data
analyzing the mutational frequency of nonproductive Vk
rearrangements revealed a marked increase compared with that in normal
subjects (3.6 x 10-2 vs 4.8 x
10-3; p < 0.001). As the
mutational frequency of the nonproductive rearrangements is an
indication of the immediate impact of the mutator without the
subsequent influence of selection or B cell survival, these results are
most consistent with the conclusion that the mutational machinery was
overactive in this patient.
The difference in the frequency of mutations in the productive and nonproductive repertoires reflects the influence of selection, with elimination of mutation-generated defective BCR normally more evident than positive selection of those with increased avidity (6, 44, 45). This process seems to be generally intact in this SLE patient, even though the overall resulting frequency of mutations in the productive repertoire is much greater than normal.
In summary, skewing of the VH repertoire toward utilization of VH3 genes, clonal expansion of B cells, and a generalized increase in somatic hypermutation may all contribute to the emergence of autoimmunity in this SLE patient. These data are most consistent with the conclusion that extreme B cell overactivity is found in the initial stages of SLE, leading to remarkable changes in peripheral V gene usage and, despite extensive light chain receptor editing, predisposes to the emergence of autoimmunity.
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Peter E. Lipsky, The Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9054. E-mail address:
3 Abbreviations used in this paper: SLE, lupus erythematosus; BCR, B cell Ag receptor.
Received for publication February 3, 1999. Accepted for publication May 3, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain receptor editing in systemic lupus erythematosus. J. Clin. Invest. 102:688.[Medline]
gene repertoire of IgM+ B cells. J. Clin. Invest. 99:1614.[Medline]
and J gene usage in normal individuals. J. Immunol. 162:2137.II gene family and maps to the vicinity of L chain CDR1. J. Immunol. 149:3588.[Abstract]
I and VII light chain genes in the expressed B cell repertoire. Ann. NY Acad. Sci. 764:301.[Abstract]
3 gene in monoclonal rheumatoid factors derived from the synovila cells of rheumatoid arthritis patients. Arthritis Rheum. 37:860.[Medline]
IIIb. J. Exp. Med. 179:1445.VIII light chains. J. Immunol. 153:1658.[Abstract]
-light chain deficient mice. Autoimmunity. 20:113.[Medline]
-deficient animals: a potential mechanism of B cell tolerance. J. Exp. Med. 180:1805.This article has been cited by other articles:
|
|
 |
|
  J. L. Lamoureux, L. C. Watson, M. Cherrier, P. Skog, D. Nemazee, and A. J. Feeney Reduced receptor editing in lupus-prone MRL/lpr mice J. Exp. Med., November 26, 2007; 204(12): 2853 - 2864. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L Cassady-Cain and A. K Kaushik Increased negative selection impairs neonatal B cell repertoire but does not directly lead to generation of disease-associated IgM auto-antibodies Int. Immunol., May 1, 2006; 18(5): 661 - 669. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. L. Hippen, B. R. Schram, L. E. Tze, K. A. Pape, M. K. Jenkins, and T. W. Behrens In Vivo Assessment of the Relative Contributions of Deletion, Anergy, and Editing to B Cell Self-Tolerance J. Immunol., July 15, 2005; 175(2): 909 - 916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yurasov, H. Wardemann, J. Hammersen, M. Tsuiji, E. Meffre, V. Pascual, and M. C. Nussenzweig Defective B cell tolerance checkpoints in systemic lupus erythematosus J. Exp. Med., March 7, 2005; 201(5): 703 - 711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Rahman Autoantibodies, lupus and the science of sabotage Rheumatology, November 1, 2004; 43(11): 1326 - 1336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Seagal, E. Edry, H. Naftali, and D. Melamed Generation and selection of an IgG-driven autoimmune repertoire during B-lymphopoiesis in Ig{micro}-deficient/lpr mice Int. Immunol., July 1, 2004; 16(7): 905 - 913. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Rahman, I Giles, J Haley, and D Isenberg Systematic analysis of sequences of anti-DNA antibodies--relevance to theories of origin and pathogenicity Lupus, December 1, 2002; 11(12): 807 - 823. [Abstract] [PDF]
|
|
|
|
|
|
M. R. Edwards, W. Brouwer, C. H. Y. Choi, J. Ruhno, R. L. Ward, and A. M. Collins Analysis of IgE Antibodies from a Patient with Atopic Dermatitis: Biased V Gene Usage and Evidence for Polyreactive IgE Heavy Chain Complementarity-Determining Region 3 J. Immunol., June 15, 2002; 168(12): 6305 - 6313. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Sekiguchi, S. M. Jainandunsing, M. L. Fields, M. A. Maldonado, M. P. Madaio, J. Erikson, M. Weigert, and R. A. Eisenberg Chronic Graft-Versus-Host in Ig Knockin Transgenic Mice Abrogates B Cell Tolerance in Anti-Double-Stranded DNA B Cells J. Immunol., April 15, 2002; 168(8): 4142 - 4153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Meffre, M. Chiorazzi, and M. C. Nussenzweig Circulating Human B Cells That Express Surrogate Light Chains Display a Unique Antibody Repertoire J. Immunol., |
