Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3737-3744
T-Cell Expansions With Conserved T-Cell Receptor 
Chain Motifs
in the Peripheral Blood of HLA-DRB1*0401 Positive Patients With
Necrotizing Vasculitis
By
Johan Grunewald,
Eva Halapi,
Jan Wahlström,
Ricardo Giscombe,
Soniya Nityanand,
Carani Sanjeevi, and
Ann-Kari Lefvert
From the Microbiology and Tumorbiology Center (MTC), Karolinska
Institute, Stockholm; and the Immunological Research Laboratory,
Department of Medicine and the Department of Molecular Medicine,
Karolinska Hospital, Stockholm, Sweden.
 |
ABSTRACT |
T lymphocytes are implicated in the pathogenesis of systemic
vasculitis such as Wegener's granulomatosis (WG) and polyarteritis nodosa (PAN). In the present study, we have characterized in detail the
T-cell receptor (TCR) of peripheral blood T cells from eight vasculitis
patients of known HLA class II genotypes. We used flow cytometry to outline the exact TCR V gene expression, complementarity determining region 3 (CDR3) fragment analysis to estimate the degree of
clonality and cDNA sequencing to define the exact TCR 
or 
chain
sequences. The TCR CDR3 region interacts with antigenic peptides
presented by HLA molecules, and it is normally immensely diverse. It
was therefore of particular interest to identify a common dominating
TCR BV8-F/L-G-G-A/Q-G-J2S3 chain sequence in the CD4+
T cells of four unrelated vasculitis patients. Furthermore, this BV8-associated CDR3 motif was linked to the HLA-DRB1*0401 allele, as
well as to active disease and/or an established
BV8+ CD4+ T-cell expansion. In contrast,
age- and HLA-matched patients with rheumatoid arthritis did not harbor
the described BV8 motif. These results strongly suggest that
BV8+ CD4+ T cells with the described CDR3
motif recognize a specific antigen presented by DR4 molecules,
indicating the existence of a common vasculitis-associated antigen.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
WEGENER'S GRANULOMATOSIS (WG) and
polyarteritis nodosa (PAN) are both systemic necrotizing vasculitis of
small and medium vessels of unknown etiologies.1 T
lymphocytes have been suggested to be important in the pathogenesis,
eg, they are found infiltrating vascular lesions. The majority of T
lymphocytes use the T-cell receptor (TCR) to specifically
recognize antigenic peptides in the context of major histocompatability
complex (MHC) class I or class II molecules.2 Vasculitis
patients often display a disturbed peripheral blood T-cell repertoire,
with a high frequency of T-cell expansions, ie, large T-cell
populations using a particular TCR V gene segment.3,4 In
contrast to healthy individuals, such T-cell expansions are frequently
found in the CD4+ subset, and phenotypic as well as
functional studies indicate their activated status.4,4a.
In human diseases of unknown etiology and thought to involve T
lymphocytes, detailed analysis of the TCR may indicate the nature of
postulated T-cell antigens. We thus decided to closer characterize the
TCR of T-cell populations from HLA typed patients with WG/PAN.
Complementarity determining region 3 (CDR3) fragment analysis and
sequencing were used for an exact determination of the and/or TCR chains. A strict correlation between the
presence of a common, dominating TCR chain CDR3-motif in several
unrelated systemic vasculitis patients and a certain HLA-DR allele is
described here. In addition, a remarkably high degree of clonality in
all cases of T-cell expansions in these patients was noted. The
described TCR motif was not found in age- and HLA-matched controls, and our findings therefore suggest the existence of a specific
vasculitis-associated antigen.
| |
MATERIALS AND METHODS |
Subjects.
Eight patients (five men, three women, age 45 to 70 years) with
necrotizing vasculitis treated at the Departments of Medicine at
Karolinska Hospital, Södertälje Sjukhus or Västerviks
Lasarett were included in the study. Five of these patients (patients 1 through 5) were chosen for this study because of their dramatic T-cell
expansions.3 Patients 6 through 8 were selected because they had signs of BV8+ CD4+ T-cell expansions
or because they were HLA-DR4 positive, and here only BV8+
CD4+ T cells were analyzed. For details on clinical
information, see Table 1.
Four patients with rheumatoid arthritis (RA) served as controls. They
were three men and one woman, age 62 to 78 years. The disease duration
was 5 to 20 years. They were all treated with nonsteroidal
antiinflammatory drugs and one with methotrexate and one with gold.
Three RA patients had active disease.
Cell separation.
Peripheral blood mononuclear cells (PBMC) were isolated from
heparinized venous blood by Ficoll-Paque gradient centrifugation (Pharmacia, Uppsala, Sweden). For analysis of clonality,
CD4+ and CD8+ T cells were separated by
magnetic beads (Dynabeads, Oslo, Norway) according to the
manufacturer's description. Briefly, 107 PBMC were
incubated end over end for 30 minutes at +4°C with the beads,
removed with a magnetic separator, and washed three times. This
procedure resulted in >95% enriched CD4+ or
CD8+ cell populations.
Immunofluorescence and monoclonal antibodies (MoAb).
For the identification of T-cell expansions (as defined in Grunewald
and Wigzell4), the following TCR V specific MoAb were included; BV2 from Immunotech S.A. (Marseille, France), AV12S1, BV3,
and BV8 from T Cell Sciences Inc (Cambridge, MA). MoAb specific for
CD3, CD4, and CD8 were purchased from Becton Dickinson (BD) (Mountain
View, CA). Fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragments of rabbit antimouse Ig were purchased
from Dakopatts A/S (Glostrup, Denmark). Normal mouse serum (NMS),
produced from BALB/c mice was used for negative control at a dilution
of 1:500 in phosphate-buffered saline (PBS) containing 0.2% bovine
serum albumin (BSA) and 0.01% sodium azide, in all cases staining < 0.5% of the cells. Lymphocyte subset analysis by flow cytometry was
performed as described.3
RNA preparation and polymerase chain reaction (PCR) amplifications.
Enriched CD4+ or CD8+ cells were lysed and RNA
was extracted using the RNA isolation kit (Stratagene, La Jolla, CA) or
RNAzol (Cinna/Biotek Laboratories Inc, Houston, TX) according to the manufacturer's recommendations. First strand cDNA was generated using
random hexanucleotides (Pharmacia) with reverse transcriptase (GIBCO
BRL Life Technologies Inc, Gaithersburg, MD).5
Amplifications of cDNA were performed in 20, 50, or 100 µL final
mixture volumes consisting of: 1x PCR buffer (10 mmol/L Tris-Cl pH 8.3, 50 mmol/L KCl, 0.1% gelatin), 0.2 mmol/L deoxynucleosidetriphosphate
(dNTP), 1.5 mmol/L MgCl2, 0.5 µmol/L
5
V specific primers and 0.5 µmol/L 3constant TCR or
primer, and 2.5 U of Ampli Taq-DNA polymerase (Perkin-Elmer, Roche
Molecular Systems Inc, Branchburg, NJ). TCR AV and -C specific primers
were from Genevee et al6 and Oksenberg et al,7
while TCR BV and -C specific primers were from Choi et al.8
The PCR profile for the TCR chain was as follows: denaturation at
94°C for 30 seconds, annealing at 61°C for 30 seconds, and
extension at 72°C for 30 seconds for 35 cycles with a final
extension of 5 minutes at 72°C.9 The PCR profile for the TCR chain was as follows: denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C
for 1 minute for 35 cycles with a final extension of 9 minutes at
72°C.5 An aliquot of each amplification reaction was
loaded in ethidium bromide-stained 2% agarose gel to confirm the
expected size of the amplified fragment.
CDR3 fragment analysis.
The size distribution of different TCR fragments was studied by using a
CDR3 length analysis.10 Briefly, TCR cDNA was amplified in
a PCR reaction with a 5BV or AV specific primer in conjuction with a 3constant or primer labelled with FITC. A
fraction of the PCR-amplified product was subsequently denatured (100% formamide, 3 minutes at 90°C) and loaded on a 6.75 % acrylamide gel (Readymix; Pharmacia) and run for 400 minutes at 42°C on an automated DNA sequencer, ALF (Pharmacia). CD4 or CD8 separated PBMC
from healthy individuals were run in parallel to show normal CDR3
fragment analysis patterns. The CDR3 length distribution was
analyzed with ALF Fragment Manager software version 1.1 (Pharmacia).
Cloning and sequencing.
PCR-amplified TCR V gene products were cloned and sequenced according
to standard procedures. Briefly, the PCR product was purified on a spin
column (Qiagen, Düsseldorf, Gemany). After phosphorylation with
T4 polynucleotide kinase (Pharmacia), the product was ligated to a
SmaI cleaved pUC18 vector that was subsequently used to
transform competent bacteria. Single colonies were used as templates
for solid phase sequencing11 using an Autoread Sequencing
Kit (Pharmacia) and analyzed with the ALF (Pharmacia). Alternatively,
PCR was performed with a biotinylated C or primer, using the
same conditions for PCR as described above, and the PCR products
were used for "direct" solid phase sequencing with a nonlabelled
AV or BV specific primer and Fluoro-dATP (Pharmacia) according to the
recommendation of the manufacturer. We defined the CDR3 region and
calculated CDR3 lengths using the formula by Rock et al,12
ie, the number of amino acids between the conserved GXG triplet in the
J region and the nearest preceding C in the V region, minus 4 amino
acids.
HLA typing.
HLA genotyping was performed by PCR-based techniques. Polymorphic
second exon of the DQA1 and DQB1 genes were amplified. The conditions
for amplification and the probes used were as described.13 The PCR-amplified products were manually dot blotted onto nylon membranes and 3 end-labelling of the synthetic oligonucleotide probes with 32P, hybridization, and stringency washes were
performed as described.13 HLA typing for DR was performed
by using allele specific primer pairs (PCR-SSP technique)14
and the subtyping for the DR4 was performed as described.15
Approval was obtained from the Institutional Review Board for these
studies. Informed consent was provided according to the Declaration of
Helsinki.
| |
RESULTS |
T-cell expansions.
Clinical information on the patients as well as data on the T-cell
populations analyzed and their respective percentage at the time for
sequence analysis is shown in Table 1.
Three of the patients showed signs of disease activity at the time of
the first analysis (patients 1, 4, and 5) and a fourth patient
(patient 3) had active disease at the second analysis. Three T-cell
expansions were also analyzed on a second occasion, where they in all
cases still constituted major T-cell expansions (Table 1).
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Table 1.
General Clinical Data, All of the Analyzed T-Cell
Populations and HLA-Haplotypes of the Patients Studied
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CDR3 fragment analysis.
The CDR3 fragment length analysis can be used to disclose the size
distribution of TCR products from T cells expressing a particular V
gene segment and thus estimate the degree of clonality within a T-cell
population. Polyclonality is usually seen in normal T-cell populations
with, in most cases, 6 to 12 peaks distributed in a gaussian pattern,
while dominant peaks showing T cells using a particular CDR3 length are
found in oligoclonal or clonal T-cell populations.
Fragment analyses of the two dramatic T-cell expansions of patient 1 (AV12 CD4 49% and BV3 CD8 33%) each showed a single dominant peak
(Fig 1), suggesting that these cells were
clonally derived. Similar results were found in the BV8 CD4 and BV8 CD8
T-cell expansions of patient 2 (data not shown). Patient 3 had a BV8
CD4 T-cell expansion that was analyzed on two occasions, 10 months
apart (Table 1). BV8 expression increased during this time from 15% to
29%, and in both cases, identical dominant peaks were detected (Fig 2A). A high stability over 4- and
2-month periods, respectively, was also seen in the clones of patients
4 and 5 (Figs 2B and C).
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| Fig 1.
CDR3 fragment analysis of (A) TCR AV 12S1 CD4 T-cell
expansion (49%) with AV 12S1 CD4 control and of (B) BV3 CD8 expansion
(33%) with BV3 CD8 control of patient 1.
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| Fig 2.
CDR3 fragment analysis of T-cell expansions at separate
time points, showing (A) BV8 CD4 T-cell expansions of patient three 10 months apart (15% and 29%) and (B) BV2 CD4 T-cell expansions of
patient four 4 months apart (50% and 60%), and (C) AV12S1 CD8 T-cell
expansion of patient five 2 months apart (41% and 35%). Relevant
controls of healthy subjects are shown at the bottom.
|
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TCR BV8 CD4 sequences.
Almost identical amino acid sequences, including the CDR3 regions as
well as the BJ gene segments, were found in BV8+
CD4+ T cells of patients 1, 2, 3, and 7 (Table 2). In patients 2, 3, and 7, these
clones clearly dominated the BV8+ CD4+
sequences, and moreover, the BV8+ T cells made up more or
less dramatic peripheral blood CD4+ T-cell expansions
either at the time of sequencing or before that (Table 1). In patient
1, who displayed two variants of the BV8-CDR3-motif, no data on BV8
expression before the sequence analysis was available. Additional
BV8-sequences of the same dominant CDR3 length were seen in all of
these patients, and in patient 3, the same dominating BV8 CD4 clone was
identified on two separate occasions, 10 months apart (Table 1).
Finally, the nucleotide sequences of the dominating BV8-CDR3-BJ2S3
clones all differed at several locations, ruling out any contamination
(Table 3).
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Table 3.
Nucleotide and Amino Acid Sequences of BV8
CD4-Associated CDR3 Region Motifs From Four Patients, All Being
HLA-DRB1*0401 Allele Positive
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Patients 5, 6, and 8 did not harbor the same sequence at all (Table 2).
These patients had a normal BV8 expression in CD4+ T cells
at the time of sequence analysis, although patients 5 and 6 had signs
of increased BV8 expression in the CD4+ T-cell subset
before that (Table 1). In patient 5, a different BV8-BJ2S7 clone,
making up 4 of 15 sequences, was detected.
All control individuals (1 through 4) had a normal TCR BV8 expression
in CD4+ peripheral blood cells (5.4%, 4.7 %, 2.8%, and
5.1%, respectively). Altogether 32 clones from these BV8+
CD4+ T cells of the four controls were sequenced. Only 3 of
32 sequences included the BJ2S3 gene segment, but in no
case did we identify the described sequence motif (data not shown).
TCR AV and BV sequences of T-cell expansions other than BV8.
Sequence analyses of the remaining T-cell expansions, where CDR3
fragment analyses had indicated a high degree of clonality, were in all
cases in concordance with the fragment analyses
(Table 4). The two AV12-associated chain sequences shared some CDR3 similarities, although they used
different AJ segments and were CD4+ and CD8+,
respectively. In addition, the two major clonal BV T-cell expansions in
the CD8 subset expressing BV3 (patient 1) or BV8 (patient 2) both used
CDR3 regions that were 10 amino acids long, and both had a positively
charged amino acid (arginin) at the beginning of the junctional region
(third and second position in the CDR3 region, respectively) and a
negatively charged amino acid (glutamic acid or aspartic acid) 5 amino
acids downstream (counting from the arginin). Thus, there were
additional similarities in CDR3 regions of T-cell expansions expressing
other TCR AV or BV gene segments from unrelated patients.
HLA-DR and -DQ and associations with TCR expression.
All patients were HLA typed for HLA-DR and -DQ (Table 1). Patients 1, 2, 3, 5, 7, and 8 were HLA-DR4 positive, and all except patient 5 had
the DRB1*0401 allele. Among the DRB1*0401 allele positive patients (1, 2, 3, 7, and 8), all except patient 8 had the above-described
BV8-CDR3-BJ2S3 sequence in their peripheral blood CD4+ T
cells (Table 5). Notably, patient 8, who
did not have the BV8-CDR3-BJ2S3 clone, was in clinical remission since
5.5 years and had no detectable BV8+ CD4+
expansion. In patient 5, who was HLA-DRB1*0403-0404 positive (Table 1),
we detected a repeated clone using a different TCR BV8-linked chain
(Table 2). Patient 6 had a BV8+ CD4+ T-cell
expansion, but was DRB1*0401 negative and did not have the BV8-motif
(Table 5).
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Table 5.
Summary of BV8-CDR3-BJ2S3 Sequences and Their
Associations With HLA-DRB1*0401 Alleles and BV8 CD4 T-Cell
Expansions
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All four control individuals had the DRB1*0401 allele, but lacked
completely the described TCR BV8 motif.
| |
DISCUSSION |
We have in this study closely characterized the TCR on T-cell
peripheral blood subpopulations of HLA-typed patients with necrotizing vasculitis, who are known to have a disturbed T-cell repertoire with a
high frequency of T-cell expansions.3 A large number of
such T-cell expansions, ie, big populations of T cells using a
particular TCR V gene segment, are frequently detected in the CD4+ T-cell subset, an extremely rare finding in healthy
individuals.4 Here we describe a common TCR
BV8-F/L-G-G-A/Q-G-J2S3 sequence in the CD4+ subsets of four
unrelated vasculitis patients, all being HLA-DRB1*0401 allele positive,
and moreover corresponding to active disease and/or
BV8+ CD4+ T-cell expansions. No such T cells
were found in age- and HLA-matched control subjects suffering from
another inflammatory disease, RA. These findings strongly suggest that
patients with systemic vasculitis have been exposed to and elicited a
cellular immune response against a common antigen.
Recent findings of Th1 skewed, activated CD4+ peripheral
blood T cells in WG patients,16 as well as previous reports
of activated CD4+ T cells in lesions,17,18
correlations between soluble interleukin-2 receptor (sIL-2R) and
disease activity,17,19,20 and the granuloma formation in
the lesions suggest a role for T cells in the pathogenesis of
granulomatous vasculitis syndromes. A central role for 
-interferon (IFN) producing T cells, infiltrating the vascular wall in giant cell
arteritis (GCA), has been discussed.21 The T lymphocytes may help in the production of the typically WG associated
antineutrophil cytoplasmic antibodies (ANCA).22,23
Analyses of the TCR may help in understanding the nature of a presumed
antigen responsible for eliciting T-cell responses, subsequently
creating T-cell expansions and may open the possibility of a highly
selective immunotherapy by targeting disease-mediating T
cells.24-26 T cells using a restricted TCR can expand after
interaction with certain well characterized antigens, as shown not only
in animal models,27 but also in humans. Thus, in the
recognition of certain Epstein-Barr virus (EBV) epitopes,28
mycobacterial heat shock proteins,29 influenza A
virus,30 or house dust mite allergens,31 human
T cells were shown to use a highly restricted TCR. Superantigens
are known to be able to selectively stimulate polyclonal T cells
expressing the appropriate TCR BV segment, regardless of the
composition of the rest of the TCR.32 The present finding
of a high degree of clonality in all of the investigated T-cell
expansions argues strongly against a superantigen as the cause of the
expansions in these patients.
What specific antigen, able to elicit strong T-cell responses, could be
associated with systemic vasculitides? An infectious cause for
vasculitis was implicated by findings in animal models where it has
been shown that certain herpes viruses, ie, -, -, or -herpes
viruses, can cause chronic vascular inflammatory
conditions.33-35 In humans, vasculitic complications can
emerge after chronic viral infections such as hepatitis C
infection.36 Interestingly, infections with the human
-herpes virus EBV has been shown to create dramatic T-cell
expansions in humans.37 Disease-associated antigens could be derived from an infectious agent itself, but the infection could
also lead to the release of sequestered self-antigens, which may
trigger an immune response.38 In line with this are reports of T cells of WG patients being autoreactive and specific for proteinase 3 (PR3), the most common target antigen for the
WG-associated autoantibodies (ANCA).39,40 Cross-reactivity
between a microbial antigen and PR3 as a possible pathogenetic
mechanism in WG was also suggested previously.22
We detected a BV8-BJ2S3 clone with a common amino acid CDR3 motif in
four of five unrelated DRB1*0401 allele positive patients, while in all
patients lacking the HLA-DRB1*0401 allele, this specific BV8-CDR3 motif
was absent. Interestingly, the only DRB1*0401 allele positive patient
without this specific clone had inactive disease since 5.5 years, and
moreover no signs of any BV8 CD4 T-cell expansion as analyzed 1 year
before the sequencing analysis. The four patients with the same T-cell
clone in their peripheral blood not only had the identical
HLA-DRB1*0401 allele in common, but also had signs of either a
BV8+ CD4+ T-cell expansion and/or
active disease. Importantly, a BV8+ CD4+ T-cell
expansion per se did not generate the specific BV8-motif, as seen in
the HLA-DRB1*0401 allele negative patient 6 (and also indicated in
patient 5). The randomly generated CDR3 region is under normal
circumstances immensely diverse and considered to interact with
antigenic peptides presented by HLA molecules.2,41-43 The
perfect positive correlation between a dominating TCR chain sequence and a certain HLA-DR molecule therefore strongly suggest that
CD4 positive T cells of these four unrelated patients have encountered
the same antigen, presented by the HLA-DR4 molecule and triggering BV8
CD4 positive T cells using the same CDR3 motif. The only differences in
amino acids in the BV8-associated CDR3 regions would in this respect
most likely not confer any significantly altered capabilities to
recognize a presumed antigen. In addition, the BV8-CDR3-BJ2S3 clones in
the four patients were coded for by different nucleotide sequences.
This finding rules out any contamination and more importantly, strongly
supports the suggestion that these cells have been selected for by a
specific (and shared) antigen.
A number of clones with identical CDR3 lengths, but using slightly
different amino acid sequences compared with the dominating clone, were
frequently detected in the T-cell expansions. This phenomenon can be
seen after antigen stimulation, where closely related T-cell clones are
selected in response to an antigen, as described in mice44
and also suggested in vivo in human peripheral blood T cells subsequent
to vaccination for hepatitis B.45 Moreover, although two of
the major T-cell expansions in the CD8 subset in these patients used
different BV genes, they had identical CDR3 lengths and positively and
negatively charged amino acids positioned similarly. Because charged
amino acids in the CDR3 region have been proven experimentally to be
important for antigen recognition,41 these results
altogether add to the suggestion that necrotizing vasculitis patients
have been exposed to a common antigen.
There is no association of the DRB1*0401 allele and vasculitis, but it
has been associated with a more systemic form of RA.46 The
DRB1*0401 allele may be associated with presentation of highly selected
sets of antigenic peptides, as has been described for the HLA-DR3
molecule.47 Such properties could influence the formation
of the TCR repertoire during selection, or could increase the risk for
presentation of certain autoantigens.
We previously described the occurrence of dramatic T-cell expansions in
patients with another necrotizing vasculitis, GCA.48 Interestingly, there was a preference for BV8 T-cell expansions also in
that group of patients, and in one case where we were able to
characterize the TCR chain BJ usage, the BV8 CD4 positive T-cell
expansion used the BJ2S3 gene segment to an unusually high degree.
Moreover, that particular patient was HLA-DR4 positive (the DR4 subtype
was not defined). It is therefore possible that the BV8-CDR3-J2S3
T-cell clone may exist in related systemic vasculitides, linked to
HLA-DR4 (DRB1*0401 allele) and disease activity/BV8 CD4 T-cell
expansions. RA patients, in contrast, did not harbor T cells with this
particular TCR motif, and one may accordingly speculate on the
existence of a common vasculitis-associated antigen.
Healthy individuals may accommodate (CD8 positive) T-cell
expansions,49-52 while in certain patient groups, T-cell
expansions seem more frequent and of a higher magnitude.4
In particular, patients suffering from systemic vasculitides frequently
harbor T-cell expansions, as described in patients with necrotizing
vasculitis,3 giant cell arteritis/temporal
arteritis,48 Takayasu's disease,53 Kawasakis
disease,54 and microscopic polyarteritis.55 The common occurrence of expansions in the CD4 positive T-cell subset clearly delineates the expansions of systemic vasculitis patients from
those found in healthy individuals. Although there was no clear
association between disease activity and the T-cell expansions, in one
patient (patient 3), the increase of BV8 CD4 cells in time correlated
with a relapse of the disease. Clearly, it would be necessary to study
a much larger group of patients to find any association of T-cell
expansions with disease activity. However, the BV8 motif
described here may become useful as a diagnostic marker, or a marker of
disease activity. Finally, studies on the specificity of the
BV8+ CD4+ T cells might lead to the
identification of a systemic vasculitis-associated antigen, which in
turn could be used for the development of new immunotherapies.
| |
ACKNOWLEDGMENT |
The authors wish to thank Louise Berg for her help with the control
subjects. Professor Hans Wigzell, Dr Olle Olerup and Dr Elisabeth
Svennungsson are acknowledged for their assistance in this work.
| |
FOOTNOTES |
Submitted March 17, 1998;
accepted July 10, 1998.
Supported by grants from AFA (Labour Market Insurance Company).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Johan Grunewald, MD, PhD,
Microbiology and Tumorbiology Center (MTC), Karolinska Institute, 171 77 Stockholm, Sweden; e-mail: Johan.Grunewald{at}mtc.ki.se.
| |
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Abstract
Introduction
