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,
*
Multiple Sclerosis Research Laboratory, Department of Neurology, and Baylor-Methodist International Multiple Sclerosis Center, Baylor College of Medicine, Houston, TX 77030;
Neurology Research Laboratory, Veterans Affairs Medical Center, Houston, TX 77030; and
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although TCR V
and Vß gene usage of MBP-reactive T cells has been
shown to vary between patients with MS (8, 13, 14), the
V and Vß gene rearrangements of MBP-reactive T cells are highly
restricted in some MS patients. This finding is consistent with in vivo
clonal expansion of MBP-reactive T cells in some patients with MS
(8, 13, 14). It is well established that DNA sequence
within the complementarity determining region (CDR)-3 encodes
hypervariable V-J junctional region of -chain and
Vß-Dß-Jß junctional region of ß-chain, both of which are most
critical to Ag recognition (15, 16). It is hypothesized
that T cell clones with identical peptide specificity would share
conserved CDR3 amino acid sequence motifs that are characteristic of
TCR recognition of a given epitope(s). Although identical and related
CDR3 sequence motifs were reported previously in MBP-reactive T cell
clones, they were largely confined to and specific for given
individuals as a result of in vivo clonal expansion (14, 17). The CDR3 motif study has been unrevealing largely because
of the limited number of MBP-reactive T cell clones examined in the
previous studies and their functional and structural heterogeneity
(e.g., different epitope recognition and MHC restriction)
(18). However, recent studies indeed suggest that there
may be some structural similarities within TCR of T cells that
uniformly recognize a single peptide (e.g., MBP83–99) in the context
of the same MHC restriction element (17, 19). Oksenberg
and colleagues identified a number of repeated motifs in Vß5.2 T
cells isolated from the MS brain plaques of two HLA-identical patients,
one of which was identified previously in other human and rat
MBP-reactive T cell clones (2, 20).
The identification of common CDR3 motifs among MBP-reactive T cells is
critical to our understanding of T cell recognition of MBP and its
potential association with the disease processes in MS. It is also
important in our attempt to develop a useful TCR marker for identifying
and monitoring the activity of MBP-reactive T cells in MS. This study
was undertaken to address whether a common CDR3 motif(s) exists in
MBP-reactive T cells possessing similar characteristics. Twenty
independent T cell clones exclusively recognizing MBP83–99 were
analyzed for the CDR3 sequences using reverse-transcribed PCR and DNA
sequencing technique. The analysis revealed four shared CDR3 motifs in
both V-J and Vß-Dß-Jß regions. One of the motifs
(Vß13.1-LGRAGLTY) was identical among T cell clones derived from two
patients with MS, suggesting that the expression of this CDR3 motif was
not restricted to a given individual. A panel of oligonucleotides
complementary to various shared motifs were used as probes in a
combined PCR-hybridization detection system to trace identical sequence
in a large panel of T cell lines specific for MBP83–99 and unprimed
PBMC specimens. The study confirmed that, in contrast to other CDR3
motifs, the LGRAGLTY motif is preferentially expressed in
MBP83–99-reactive T cells in some patients with MS. The findings also
suggest that T cells expressing this common CDR3 motif are present but
at low precursor frequency in healthy individuals. The study provides
new evidence for further investigating common CDR3 motifs in
MBP-reactive T cells and their potential value in monitoring in vivo
activity and migration patterns of MBP83–99-reactive T cells in
different clinical stages of MS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rIL-2 was purchased from Boehringer Mannheim (Indianapolis, IN). Medium used for cell culture was RPMI 1640 supplemented with 10% heat-inactivated FCS and L-glutamine, sodium pyruvate, nonessential amino acids, and 10 mM HEPES buffer (HyClone, Logan, UT). A panel of overlapping peptides of human MBP (17 aa in length) and a panel of 17 peptides analogue to the 83–99 region of MBP were synthesized by the Merrified solid phase method and were purified by HPLC (courtesy of Dr. Stefen Boheme, Neurocrine Biosciences, San Diego, CA). The analogue peptides were synthesized with single alanine substitutions. The purity of all peptides used in this study was greater than 95%.
MS patients and healthy individuals
Forty-eight patients with clinically definite MS confirmed by magnetic resonance imaging were included in this study (21). The patients were characterized as having relapsing-remitting or chronic progressive MS for more than two years. The patients had not taken any immunomodulatory drugs 2 mo before the study. Consent forms were obtained from the patients after explaining the experimental procedures. The protocol was approved by the Institutional Human Subjects Committee at Baylor College of Medicine. The control blood specimens used in this study were obtained from healthy blood donors (Gulf Coast Blood Center, Houston, TX).
Generation of a MBP83–99-reactive T cell clone from PBMC in MS patients
To generate specific T cell lines (4, 6), PBMC were
plated out at 200,000 cells/well in U-bottom plates (Costar, Cambridge,
MA) in the presence of the 83–99 peptide (10 µg/ml). Seven days
later, all cultures were restimulated with irradiated autologous PBMC
pulsed with the peptide as a source of APC. After another week, each
culture was examined for specific proliferation to the 83–99 peptide
in proliferation assays. Briefly, each well was split into four
aliquots (
104 cells per aliquot) and cultured
in duplicate in the presence of 105 APC pulsed
with the 83–99 peptide or a control peptide, respectively. Cells were
cultured for 72 h and pulsed with
[3H]thymidine (Amersham, Arlington Heights, IL)
at 1 µCi per well during the last 16 h of the culture. Cells
were then harvested, and [3H]thymidine
incorporation was measured in a Betaplate counter (Wallac, Turku,
Finland). A T cell line was considered to be specific for the 83–99
peptide when the cpm were greater than 1500 (in the presence of
the peptide) and exceeded the reference cpm (in the absence of the
peptide) by at least threefold (4, 10). Some of these T
cell lines were used in the detection of the common CDR3 motif.
To establish stable MBP83–99-reactive T cell clones, the resulting T
cell lines were cloned by PHA (Sigma, St. Louis, MO) in the presence of
autologous PBMC as accessory cells (10). Briefly, T cells
were plated out at 0.3 cell per well under limiting dilution condition
and cultured with 105 irradiated autologous PBMC
and 2 µg/ml PHA. Cultures were fed with fresh medium containing 50
IU/ml rIL-2 every 3 to 4 days. After 10–12 days, growth-positive
wells became visible and were tested in proliferation assays for
specific responses to the 83–99 peptide.
Proliferation assays with peptides of MBP and analogue peptides
Twenty thousand cells of a given T cell clone were cultured with irradiated autologous PBMC (100,000 cells/well) in the presence of each alanine-substituted peptide (20 µg/ml) and the wild-type 83–99 peptide, respectively. Cultures were set up in duplicate for each peptide. In all cases, cell proliferation was measured after 72 h by [3H]thymidine incorporation assays as described above.
PCR amplifications and direct sequencing of PCR-amplified DNA products
Total RNA was extracted from 106 cells of
each MBP83–99-reactive T cell clone using RNeasy mini kit (Qiagen,
Santa Clarita, CA). TCR - and ß-chain genes were amplified and
directly sequenced as previously described (8, 17).
Briefly, extracted RNA was reverse transcribed into first-strand cDNA
using an oligo(dT) primer and Reverse Transcriptase (Life Technologies,
Gaithersburg, MD). cDNA was then subject to PCR amplification with a
set of primers specific for TCR V and Vß gene families, whose
sequences were published previously (8, 17). PCR was
performed with 1 µl of cDNA in the following amplification mixture: 5
µl of 10x PCR buffer II (100 mM Tris-HCl (pH 8.3) and 500 mM KCl), 3
µl of 25 mM magnesium chloride, 1 µl of 10 mM dNTP mix, 0.3 µl of
Taq polymerase (5 U/µl) (AmpliTaq Gold; Perkin-Elmer,
Norwalk, CT), 20 pmol of each 5'V or 5'Vß primer as the forward
primer, 20 pmol of 3'C or 3'Cß primer as the reverse primer. The
PCR amplification profile used was 1 min at 95°C for denaturation,
20 s at 56°C for annealing, and 40 s at 72°C for
extension in a total of 35 cycles. The amplified PCR products were
separated in a 1% agarose gel by electrophoresis and stained with
ethidium bromide. The visualized PCR products were cut and purified
subsequently using a QIAquick gel extraction kit (Qiagen) before
sequence analysis. The purified PCR products were directly sequenced
with the T7 sequencing kit (Pharmacia, Uppsala, Sweden). Ten nanograms
of template was sequenced with 2 pmol of the corresponding V gene
primer using the method of dideoxy chain termination
(22).
CDR3-specific oligonucleotide probes
Oligonucleotide probes corresponding to unique CDR3 sequences were synthesized (Genosis Biotechnologies, The Woodlands, TX) and subsequently labeled with digoxigenin-dUTP using terminal transferase according to the manufacturer’s instructions (Boehringer Mannheim). The sequences of the probes are underlined in Tables I and II. The specificity of these oligonucleotides was confirmed by cross-examination with original clones from which the sequences were derived and with unrelated clones. The labeled CDR3-specific oligonucleotide probes were used to trace similar/identical sequences in short-term T cell lines specific for MBP83–99 and unprimed PBMC specimens as described below.
Detection of CDR3 sequences in short-term T cell lines and PBMC specimens
cDNA products reverse-transcribed from total RNA of the T cell lines and PBMC specimens were analyzed by PCR with a corresponding 5'Vß forward primer and a 3'Cß reverse primer as previously described. The amplified PCR products were electrophoretically separated in a 1% agarose gel and transferred to a positively charged nylon membrane (Boehringer Mannheim) using vacuum blot (Bio-Rad, Hercules, CA) at 5 mm Hg for 90 min. DNA was fixed onto the membrane by 3 min-exposure to UV cross-linking and prehybridized at 68°C for at least 1 h. Poly(A) (0.1 mg/ml) was added to prehybridization solution (5x SSC, 1% blocking solution, 0.1% N-lauroylsarkosine, and 0.02% SDS) to reduce nonspecific binding of the probe to non-target DNA. Hybridization temperature and washing conditions were optimized according to different CDR3-specific probes to ensure a stringent hybridization condition. Hybridization was conducted in a buffer containing 5x SSC, 1% blocking solution, 0.1% N-lauroylsarkosine, 0.02% SDS, and 0.3 pmol/ml digoxigenin-labeled CDR3-specific probe for 6 h. The detection of DNA hybrid products was performed using the Digoxigenin Luminescent Detection Kit according to the manufacturer’s instruction (Boehringer Mannheim). The membrane was then exposed to x-ray film for 15–30 min at room temperature. To ensure the specificity, the original T cell clones from which CDR3-specific probes were derived were used as positive control, and MBP83–99 T cell clones of unrelated CDR3 sequences were used as negative control.
DNA cloning and sequencing of PBMC-derived PCR products
PCR products amplified by the 5'Vß13.1 primer and the 3'Jß primer from four PBMC specimens positive for the expression of the LGRAGLTY motif were cloned into the TA cloning vector pCR2.1 (Invitrogen, Carlsbad, CA). Plasmid DNA was prepared from transformed Escherichia coli using QIAprep Kit (Qiagen) and screened by PCR with a M13 primer as a forward primer and a reverse primer specific for the LGRAGLTY sequence. The positive plasmids that showed visible amplification by PCR were selected and sequenced for Vß-Dß-Jß sequences with a Vß13.1 primer.
Semiquantitative measurement of RNA containing the LGRAGLTY motif by RT-PCR and DNA hybridization
First strand cDNA from selected PBMC specimens was amplified by PCR with the 5'CDR3 primer specific for motif LGRAGLTY and a 3'Cß primer. TCR Cß was amplified with 5'Cß and 3'Cß primers in the same reaction as an internal control. To optimize PCR amplification condition, PCR was performed at different cycles in pilot experiments. A total of 30 cycles was used to ensure that Cß amplification did not reach a plateau level. The PCR products were then hybridized with a digoxigenin-labeled Cß-specific probe using the same protocol as described above. The chemiluminescent intensities of hybridized products were visualized on Kodak films and quantified using a Gel Doc 1000 scanning densitometer (Bio-Rad). The level of the LGRAGLTY gene expression was analyzed relative to that of Cß gene expression (reference gene expression) in each specimen. The results were presented as the ratio of the specific gene expression [(expression of the LGRAGLTY motif/expression of Cß) x 100%].
Genomic HLA-DR2b typing
HLA-DR2b (DRB1*1501) was identified by genomic typing of the second exon of DRB1*1501 gene that encodes the ß-chain of HLA-DR2b. The PCR method used for DR2b typing was described elsewhere with some modification (23). Briefly, cDNA products prepared from PBMC were amplified for the DRB1*1501 allele by specific oligonucleotide primers. The forward primer was 5'-TTCCTGTGGCAGCCTAAGAGG, and the reverse primer was 3'-ACCACCGAATTCTGCACTGTGAAGCTCTCCA, respectively. The primers amplified specifically the DRB1*1501 allele. The conditions used for PCR amplification were as follows: 1 min at 95°C for denaturation, 20 s at 65°C for annealing, and 40 s at 72°C for extension in a total of 30 cycles.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A panel of 20 CD4+ independent T cell clones
was generated from seven patients with MS. All T cell clones
exclusively recognized the 83–99 peptide of MBP in the context of
HLA-DR2, as determined by using mouse fibroblast cells (L cells)
transfected with DRB1*1501 as APCs. The T cell clones were analyzed for
TCR V gene rearrangements by RT-PCR using V- and Vß-specific
primers and were subsequently sequenced for the V-J and
Vß-Dß-Jß junctional regions. As shown in Table I
and Table II,
although the V and Vß rearrangements varied between individual
MBP83–99 T cell clones, many of these independent T cell clones
derived from a given individual expressed the same V- and
Vß-chains with identical V-J and Vß-Dß-Jß junctional
region sequences. This finding is consistent with in vivo clonal
expansion of MBP83–99-specific T cells in given patients with MS, as
reported previously (7, 8). Interestingly, four shared
motifs consisting of at least three identical amino acids were found
within V-J and Vß-Dß-Jß junctional regions of these T cell
clones derived from different patients with MS (Tables I and II). These
include the QDR motif shared by clones Vß9:MS7-E3.1 and
Vß7:MS37-D9.3, the FGN motif shared by V9:MS7-D2.2,
V17:MS27-C3.1, V17:MS27-D7.16, and V17:MS27-F3.4 and the
S-GGSN motif shared by two clones V17:MS7-E2.6 and V22:MS7-E3.1
derived from patient MS-7. Remarkably, as indicated in Table II, an
independent T cell clone (clone MS7-E2.6) derived from one patient
(MS7) expressed the same TCR V17 and Vß13.1 genes in 3/4 T cell
clones (MS27-C3.1, MS27-D7.16, and MS27-F3.4) obtained from another
patient (MS27). Vß13.1 of these T cell clones shared an identical
sequence (LGRAGLTY) within the Vß-Dß-Jß junctional region while
their V17 chains had two distinct V-J junctional region
sequences.
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). It
appeared that Val87 and the three C-terminal amino acids at
Arg97, Thr98, and
Pro99 were critical to peptide recognition of
clone MS7-E2.6 but were not
required by clone MS27-C3.1. Since both T cell clones shared the same
Vß-chain with identical Vß-Dß-Jß sequences, the finding
suggests that the structural difference in recognition of the 83–99
peptide is attributable to the distinct V-J junctional
sequences. Conversely, two independent clones, MS7-E2.6 and
MS7-E3.1, had distinct V-chains (V17 and V22) with a
similar V-J junctional sequence (SPGGSN vs
SRGGSN) while their Vß rearrangements were completely
unrelated. They also exhibited a distinct recognition pattern toward
alanine-substituted peptides (Fig. 1). All these T cell clones sharing
similar motifs were found to have distinct recognition patterns toward
alanine-substituted peptides (data not shown). Taken together, these
results suggest that identical Vß-Dß-Jß sequence and repeated
V-Jand Vß-Dß-Jß sequence motifs exist among
DR2-restricted MBP83–99 T cells derived from different patients with
MS.
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A set of 22 oligonucleotides, including 13 Vß-Dß-Jß
sequences and 9 V-J sequences, were synthesized according to the
identified CDR3 DNA sequences of independent MBP83–99 T cell clones.
The DNA sequences of these oligonucleotides are shown as underlined in
Tables I and II. The specificity of the CDR3-specific oligonucleotides
was examined in RT-PCR using the oligonucleotides as forward primers
and 3'C or 3'Cß as a reverse primer. The CDR3-specific
oligonucleotides were able to amplify the DNA sequences in the original
T cell clones from which the CDR3-specific oligonucleotides were
derived. Fig. 2 illustrates the
specificity of the TCR Vß CDR3-specific oligonucleotides in detecting
target DNA sequences in original MBP83–99-reactive T cell clones. No
cross amplification with unrelated MBP83–99 T cell clones was
observed, with the exception of the two clones MS7-E2.6 and
MS27-C3.1, which shared an identical Vß-Dß-Jß junctional sequence
(Fig. 2). Experiments with a set of nine V CDR3-specific
oligonucleotides revealed the same specificity (data not shown).
However, no cross-amplification was seen between clone MS-E2.6 and
clone MS27-C3.1 (the same V17 but distinct V-J sequences).
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/Vß
forward primer and a 3'C/Cß reverse primer. The pilot experiments
confirmed highly specific and sensitive detection of the expected
target DNA sequences in original PBMC specimens from which the
MBP83–99-reactive T cell clones were derived. As expected, there was
no cross-hybridization between unrelated CDR3-specific probes. Thus,
the experiments described above demonstrated that the combined PCR-DNA
hybridization detection system in which CDR3 region sequence served as
fingerprint was highly specific and sensitive in tracing
MBP83–99-reactive T cells present in PBMC by probing target DNA
sequences. The detection of a common CDR3 motif in unprimed PBMC specimens from different patients with MS and healthy individuals
Next, we examined whether target DNA sequences corresponding to
the identified CDR3 motifs could be detected in PBMC specimens randomly
selected from a group of patients with MS and healthy individuals. For
this purpose, the above-mentioned detection system that combined RT-PCR
and DNA hybridization using CDR3-specific oligonucleotide probes was
employed. As shown in Fig. 3, the results
indicate that only one oligonucleotide probe corresponding to the
Vß-Dß-Jß sequence (LGRAGLTY) shared by clone MS7-E2.6 and clone
MS27-C3.1 detected target DNA sequence(s) in the majority of PBMC
specimens (15/48, 31%) obtained from different patients with MS.
However, two CDR3-specific probes corresponding to their V17
sequences (MS7-E2.6 and MS27-C3.1) failed to detect target DNA
sequences in the same PBMC specimens. This finding suggests that
Vß13.1 T cells expressing LGRAGLTY motif probably pair with different
V-chains in PMBC specimens examined. No target sequences were
detected by the remaining CDR3-specific probes, including those that
exhibited the shared CDR3 motifs in the PBMC specimens, with the
exception of autologous PBMC specimens containing original
MBP83–99-reactive T cells from which the sequences of the probes were
derived (Fig. 3). Furthermore, the expression of the LGRAGLTY motif did
not closely correlate with the expression of DRB1*1501 in the PBMC
specimens examined (
2 = 1.71,
p > 0.05), even though 53% of the PBMC specimens that
exhibited the LGRAGLTY motif expressed the DRB1*1501 molecule. All
experiments described above were repeated with reproducible
results.
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, in contrast to other CDR3-specific
probes, the LGRAGLTY probe detected the target sequence in 5/20 control
PBMC specimens. Additional experiments to quantify the specific
expression of the LGRAGLTY motif using semiquantitative analysis
revealed lower expression of the motif in control PBMC specimens than
those from patients with MS (Fig. 5).
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Short-term MBP-reactive T cell lines are generated by limited
passages in culture, thus representing relatively preserved T cell
repertoire to MBP in a given individual. Therefore, it is of interest
to examine the percentage of short-term MBP83–99 T cell lines that
exhibited the LGRAGLTY in patients with MS. As showed in Fig. 6, the experiments revealed that the
LGRAGLTY motif was expressed in the majority of short-term
MBP83–99-reactive T cell lines (36/44, 82%) derived from five
patients with MS. In some patients with MS (MS26 and MS36), all T cell
lines expressed the LGRAGLTY motif. These results confirmed that the
LGRAGLTY motif is preferentially expressed in T cells recognizing
MBP83–99 in some MS patients.
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Next, we conducted a series of experiments to verify whether the
DNA products amplified from PBMC contained the LGRAGLTY sequence using
recombinant DNA cloning and sequencing techniques. The experiments were
performed with randomly selected PBMC specimens expressing the LGRAGLTY
motif using the experimental strategy illustrated in Fig. 7. PCR products were obtained by
amplification of cDNA using Vß13.1-Jß primers. The amplified PCR
products were then ligated into pCR2 vector and transformed into
E. coli. Plasmid DNA was prepared and screened for positive
insertions by PCR using the CDR3-specific primer. The plasmids that
contained Vß-Dß-Jß inserts were analyzed for DNA sequence. The
results confirmed that all PBMC specimens obtained from four MS
patients and one healthy individual contained the LGRAGLTY sequence in
the Vß-Dß-Jß junctional region.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The findings described in this study raised several important issues
pertinent to our understanding of the potential role and structural
characteristics of T cells recognizing the immunodominant 83–99
peptide of MBP in MS. First, it is remarkable that the identified
common CDR3 motif (LGRAGLTY) is specific for MBP83–99-reactive T cells
and is not restricted to a given individual with MS. In contrast, other
shared CDR3 motifs identified in this study were not detected in PBMC
specimens derived from different individuals. The findings indicate
that, although common CDR3 motifs are rarely detectable in T cell
clones, they do exist among T cells possessing uniform characteristics
in different individuals. Second, MBP83–99-reactive T cells commonly
expressing the LGRAGLTY motif carry distinct V-chains with unrelated
V-J sequences. This finding suggests that, although these
MBP-reactive T cells displayed the conserved Vß-Dß-Jß sequence,
they are structurally different from each other and most likely
originate from different clonal lineages. They also appear to exhibit
different recognition patterns toward the immunodominant region of MBP,
as demonstrated in their reactivity to alanine-substituted peptides of
MBP83–99. The results are consistent with considerable heterogeneity
in the recognition patterns of T cell clones specific for the
MBP-83–99 peptides, even though they display limited TCR V gene
rearrangements (17, 24). Furthermore, it is not surprising
that MBP83–99 T cells expressing the common CDR3 motif are also
present in some healthy individuals. Interestingly, our study
demonstrated that these T cells are present at much low precursor
frequency in healthy individuals, as evident by quantitatively low
expression of the LGRAGLTY motif in PBMC specimens derived from healthy
individuals as compared with that from patients with MS. This finding
is in agreement with the previous observation that some clonal
populations of MBP-reactive T cells undergo in vivo activation and
expansion in patients with MS, as opposed to healthy individuals
(7, 25, 26). Thus, the results suggest that the
MBP83–99-reactive T cells expressing the LGRAGLTY motif may undergo in
vivo activation and expansion in at least some patients with
MS.
These MBP83–99 T cells expressing the LGRAGLTY motif may represent a significant fraction of MBP83–99 T cells found in some patients with MS. This possibility is supported by the observation that T cells expressing this CDR3 motif seem to represent the majority of short-term MBP83–99 T cell lines in some patients with MS and are frequently found in unprimed PBMC specimens. The reason why this common CDR3 motif was not detected in MBP-reactive T cell clones in previous studies may be explained by several possibilities: 1) only a very limited set of long-term MBP83–99 T cell clones expressing Vß13.1 have been studied so far (18); and 2) the majority of MBP-reactive T cell lines do not survive repeated stimulation cycles and cloning procedure because of various inhibitory mechanisms (e.g., Ag-induced apoptosis) associated with cell culture and Ag stimulation (27, 28, 29). Thus, the current protocol used by most of the investigators dictates that the majority of MBP-reactive T cells would perish during the process in generating T cell clones and never reach the clonal stage that is necessary for TCR analysis. It is most likely that long-term MBP-reactive T cell clones available for TCR analysis represent a small fraction of MBP-reactive T cells. The TCR repertoire of long-term T cell clones that have survived repeated passages in culture is highly skewed. Therefore, it remains unclear whether the LGRAGLTY sequence described in this study represents the predominant motif in MS patients and whether there are more common CDR3 motifs among MBP-reactive T cells that are unidentified.
It is also tempting to consider potential clinical implications of the findings. The identified common CDR3 motif may be used as a specific marker in a quantitative PCR detection system to trace a subset of MBP83–99 T cells in the blood and cerebrospinal fluid in a large group of MS patients for the purpose of monitoring their activity in vivo. This method will be superior to conventional cell culture-based assays for the reasons discussed above. This is consistent with a recent study where the frequency of MBP-reactive T cells was found to be surprisingly high in patients with MS when direct ex vivo analysis was employed to quantify MBP-reactive T cells (29). Furthermore, synthetic peptides corresponding to the TCR V gene elements have been shown to induce antiidiotypic T cell responses to MBP-reactive T cells in patients with MS (30). Therefore, a TCR peptide containing a common CDR3 sequence may be of great potential in eliciting antiidiotypic T cells to suppress a specific subset of MBP-reactive T cells in a group of patients whose T cells bear the common CDR3 motif. Immunization with a common CDR3 peptide would be advantageous over CDR2 peptides or individual-dependent CDR3 peptides as a potential treatment procedure in patients with MS (11). At this time, however, these potential applications seem to be limited by the fact that the LGRAGLTY motif identified in this study is present only in a subset of Vß13.1 MBP83–99 T cells. Further investigations using the same approach may lead to discovery of additional common CDR3 motifs in MBP-reactive T cells, making these applications possible.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jingwu Zhang, Department of Neurology, Baylor College of Medicine, 6501 Fannin Street, NB302, Houston, TX 77030. E-mail address:
3 Abbreviations used in this paper: MS, multiple sclerosis; MBP, myelin basic protein; CDR, complementarity-determining region.
Received for publication April 27, 1999. Accepted for publication July 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex. Science 274:209. gene rearrangement in T cells recognizing an immunodominant peptide of myelin basic protein in DR2 patients with multiple sclerosis. Int. Immunol. 10:991.This article has been cited by other articles:
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  V. Venturi, H. Y. Chin, D. A. Price, D. C. Douek, and M. P. Davenport The Role of Production Frequency in the Sharing of Simian Immunodeficiency Virus-Specific CD8+ TCRs between Macaques J. Immunol., August 15, 2008; 181(4): 2597 - 2609. [Abstract] [Full Text] [PDF]
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H. Sakuma, K. Kohyama, Y. Jee, and Y. Matsumoto Tracking of V{beta}8.2-Positive Encephalitogenic T Cells by Complementarity-Determining Region 3 Spectratyping and Subsequent Southern Blot Hybridization in Lewis Rats after Neuroantigen Sensitization J. Immunol., October 1, 2004; 173(7): 4516 - 4522. [Abstract] [Full Text] [PDF]
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 E. L. Oleszak, J. R. Chang, H. Friedman, C. D. Katsetos, and C. D. Platsoucas Theiler's Virus Infection: a Model for Multiple Sclerosis Clin. Microbiol. Rev., January 1, 2004; 17(1): 174 - 207. [Abstract] [Full Text] [PDF]
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 Y. C. Q. Zang, J. Hong, V. M. Rivera, J. Killian, and J. Z. Zhang Human anti-idiotypic T cells induced by TCR peptides corresponding to a common CDR3 sequence motif in myelin basic protein-reactive T cells Int. Immunol., September 1, 2003; 15(9): 1073 - 1080. [Abstract] [Full Text] [PDF]
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N. E. Kilgore, J. D. Carter, U. Lorenz, and B. D. Evavold Cutting Edge: Dependence of TCR Antagonism on Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Activity J. Immunol., May 15, 2003; 170(10): 4891 - 4895. [Abstract] [Full Text] [PDF]
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Y. Matsumoto, W. K. Yoon, Y. Jee, K. Fujihara, T. Misu, S. Sato, I. Nakashima, and Y. Itoyama Complementarity-Determining Region 3 Spectratyping Analysis of the TCR Repertoire in Multiple Sclerosis J. Immunol., May 1, 2003; 170(9): 4846 - 4853. [Abstract] [Full Text] [PDF]
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 J. N. Kochenderfer, S. Kobayashi, E. D. Wieder, C. Su, and J. J. Molldrem Loss of T-lymphocyte clonal dominance in patients with myelodysplastic syndrome responsive to immunosuppression Blood, November 15, 2002; 100(10): 3639 - 3645. [Abstract] [Full Text] [PDF]
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A. M. Risitano, H. Kook, W. Zeng, G. Chen, N. S. Young, and J. P. Maciejewski Oligoclonal and polyclonal CD4 and CD8 lymphocytes in aplastic anemia and paroxysmal nocturnal hemoglobinuria measured by Vbeta CDR3 spectratyping and flow cytometry Blood, June 17, 2002; 100(1): 178 - 183. [Abstract] [Full Text] [PDF]
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J. Hong, Y. C. Q. Zang, M. V. Tejada-Simon, S. Li, V. M. Rivera, J. Killian, and J. Z. Zhang Reactivity and Regulatory Properties of Human Anti-Idiotypic Antibodies Induced by T Cell Vaccination J. Immunol., December 15, 2000; 165(12): 6858 - 6864. [Abstract] [Full Text] [PDF]
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 Y. C. Q. Zang, A. K. Samanta, J. B. Halder, J. Hong, M. V. Tejada-Simon, V. M. Rivera, and J. Z. Zhang Aberrant T cell migration toward RANTES and MIP-1{alpha} in patients with multiple sclerosis: Overexpression of chemokine receptor CCR5 Brain, September 1, 2000; 123(9): 1874 - 1882. [Abstract] [Full Text] [PDF]
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Y. C. Q. Zang, J. Hong, V. M. Rivera, J. Killian, and J. Z. Zhang Preferential Recognition of TCR Hypervariable Regions by Human Anti-Idiotypic T Cells Induced by T Cell Vaccination J. Immunol., April 15, 2000; 164(8): 4011 - 4017. [Abstract] [Full Text] [PDF]
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R. A. K. Singh, Y. C. Q. Zang, A. Shrivastava, J. Hong, G. T. Wang, S. Li, M. V. Tejada-Simon, M. Kozovska, V. M. Rivera, and J. Z. Zhang Th1 and Th2 Deviation of Myelin-Autoreactive T Cells by Altered Peptide Ligands Is Associated with Reciprocal Regulation of Lck, Fyn, and ZAP-70 J. Immunol., December 15, 1999; 163(12): 6393 - 6402. [Abstract] [Full Text] [PDF]
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