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Multiple Sclerosis Research Laboratory, Department of Neurology, and Baylor-Methodist Multiple Sclerosis Center,
Department of 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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It is increasingly clear that T cell vaccination induces the Id anti-idiotypic network in regulating selected T cells used for vaccination. We demonstrated previously that T cell vaccination elicited CD8+ cytotoxic T cell responses that specifically recognized and lysed the immunizing T cell clones (8, 15). These anti-idiotypic T cell lines isolated from MS patients immunized with irradiated MBP-reactive T cell clones were found to react preferentially with complementarity-determining region 3 (CDR3) sequences of the immunizing T cells (16). In contrast to anti-idiotypic T cell responses, little is known about the occurrence and regulatory role of anti-idiotypic humoral responses in vaccinated patients. It has been speculated that the same vaccination protocol may be equally effective in eliciting anti-idiotypic Ab responses in MS patients. However, previous attempts to identify anti-idiotypic Abs in vaccinated patients were largely unsuccessful, even though the presence of such Abs was occasionally detected (8, 17). In these studies nonfractionated sera obtained from vaccinated patients were used to stain the immunizing T cells or cell lysates using flow cytometry or immunoblot analysis (8, 17). It was unclear whether the failure to detect the anti-idiotypic Abs in vaccinated patients was due to the lack of the humoral responses or whether it was associated with technical difficulties in detecting them using the whole T cells or T cell lysates as the Ags. Resolution of these issues is important, as anti-idiotypic humoral responses may potentially contribute to immune regulation induced by T cell vaccination. Identification and characterization of such anti-idiotypic Abs may help to understand the regulatory mechanism underlying T cell vaccination.
This study was undertaken to examine the occurrence and functional properties of anti-idiotypic Abs induced by T cell vaccination in patients with MS. Special considerations were given in the experimental design. First, in addition to TCR, the whole T cells or T cell lysates express numerous surface molecules that may interfere with the binding assay to detect anti-idiotypic Abs. Second, anti-idiotypic Abs of interest represent only a small fraction of serum Ig, which may account for the difficulties in detecting the specific Abs in sera. Based on these considerations, we synthesized a 20-mer TCR peptide incorporating the CDR3 sequence expressed by the immunizing MBP-reactive T cell clones and used it as a pure agent in the initial screening to identify anti-idiotypic Abs. Second, we employed a cell culture-based technique combining EBV transformation and limiting dilution to generate Ab-producing B cell lines. This technique was used successfully in previous studies to identify B cells producing autoantibodies, which are present at low frequencies in patient’s blood (18, 19). As a result, the precursor frequency of B cells producing specific Ab of interest can be estimated, and higher concentrations of relatively pure Abs can be obtained from individual B cell lines for detailed characterization. The study revealed for the first time that B cells producing anti-idiotypic Abs could be isolated from MS patients that received T cell vaccination. These Abs initially screened for their specific reactivity to the CDR3 peptide bound to and had an inhibitory effect on the immunizing MBP-reactive T cells expressing the CDR3 sequence. The findings have important implications in the understanding of the regulatory role of the B cell component of the Id anti-idiotypic network that can be modulated by T cell vaccination and in the development of a peptide-based vaccination approach.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The medium used for cell culture was AIM-V serum-free medium (Life Technologies, Grand Island, NY). Recombinant human IL-2 was purchased from Roche (Indianapolis, IN). The immunodominant peptide (residues 83–99) of MBP and two TCR peptides of 20 amino acids were synthesized by Chiron Mimotope (San Diego, CA). The purity of the peptides was >95%.
Estimation of the precursor frequency of MBP-reactive T cells
PBMC were plated at 200,000 cells/well (for a total of 96 wells)
in the presence of MBP (40 µg/ml). Seven days later, all cultures
were restimulated with MBP in the presence of irradiated autologous
PBMCs. After another week, each well was split into four aliquots
(
104 cells/aliquot) and cultured in duplicate
with 105 irradiated autologous PBMCs in the
presence and the absence of MBP. Cultures were kept for 3 days and
pulsed with [3H]thymidine (Nycomed Amersham,
Arlington Heights, IL) at 1 µCi/well during the final 16 h of
culture. Cells were then harvested using an automated cell harvester,
and [3H]thymidine incorporation was
measured.
A well/culture was defined as specific for MBP or the peptides of MBP when the counts per minute were >1000 and exceeded the reference counts per minute (in the absence of MBP) by at least 3-fold (8, 9, 11) The precursor frequency of MBP-reactive T cells was then estimated by dividing the number of specific wells by the total number of PBMCs (19.2 x 106 cells) seeded in the initial culture (8, 9, 11).
Myelin-reactive T cell clones
The positively identified T cell lines were cloned using limiting dilution assay in the presence of PHA-protein at 2 µg/ml. Cultures were fed with fresh medium every 3–4 days. Growth-positive wells were tested for specific reactivity to the MBP83–99 peptide in proliferation assays. The resulting MBP83–99-specific T cell clones were further characterized and used for T cell vaccination.
TCR V gene analysis and DNA sequencing
TCR V gene rearrangements of the immunizing MBP-reactive T
cell clones were analyzed using reverse transcribed PCR. TCR 
- and
ß-chain transcripts were amplified and directly sequenced as
previously described (20, 21). Briefly, total RNA was
extracted from 106 cells of each
MBP83–99-reactive T cell clone using the RNeasy
mini kit (Qiagen, Santa Clarita, CA). First-strand cDNA reverse
transcribed from total RNA was subjected to PCR amplification with a
set of primers specific for TCR V and Vß gene families whose
sequences were published previously (20, 21). 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). Template (1.5 µg) was sequenced with 2 pmol of the
corresponding V gene primer using the method of dideoxy chain
termination (20, 21).
Immunization of MS patients with irradiated autologous MBP-reactive T cell clones
Two patients with clinically definite MS confirmed by magnetic resonance imaging (22) were included in this study. They were diagnosed as having relapsing-remitting MS for >2 years. The patients had not taken any immunomodulatory drugs at least 3 mo before the study. Immunizations with irradiated autologous MBP83–99 reactive T cell clones was performed as previously described (8, 14). Briefly, MBP83–99-reactive T cell clones was activated and expanded in the presence of PHA 7 days before injection. T cells were then irradiated at 10,000 rad (a 60Co source) and thoroughly washed with sterile saline. A total of 4 x 107 cells of two autologous T cell clones was resuspended in 2 ml of sterile saline and was injected s.c. in the arms. Each patient received a total of four injections at a 2-mo interval to achieve adequate immune responses as defined by the proliferation of PBMC to the immunizing T cell clones. The protocol was approved by the institutional human subjects committee at Baylor College of Medicine. Consent forms were obtained from the patients after explaining the experimental procedures. The patients were evaluated for adverse events and disability score (Expanded Disability Scale Score) before and after each immunization. Gadolinium-enhanced magnetic resonance imaging scans were performed before and at different time points after immunization. The clinical and radiographic evaluation was part of a separate clinical study (J. Z. Zhang et al., manuscript in preparation).
Generation of Ab-producing B cell lines by EBV transformation
The method used was described previously (18, 19). Briefly, PBMC were plated out at 20,000 cells/well in microtiter plates (Costar, Cambridge, MA) in the presence of cell-free supernatant of a B95.8 line producing EBV (American Type Culture Collection, Manassas, VA) and 0.5 µg/ml cyclosporin A (Sandoz, Basel, Switzerland) to selectively inhibit T cell growth. Cells were cultured for 14 days with changes of medium every 3–4 days. On day 14, the growth-positive wells were visualized, and the culture supernatants were harvested for testing. The precursor frequency of B cells producing specific Abs was estimated by dividing the number of positive wells by the total number of PBMC plated. Positive B cell lines were transferred subsequently to 24-well plates (Costar) for expansion. As described previously, the B cell lines typically produced 2–10 µg/ml of relatively pure Abs.
Detection of anti-TCR Ab by ELISA
Culture supernatants were collected from individual B cell lines and tested in duplicate for the presence of anti-idiotypic Abs using ELISA. Briefly, microtiter plates were coated overnight at 4°C with the motif-positive TCR peptide or the control TCR peptide, respectively, at a concentration of 1 µg/well. Wells were then blocked at 37°C for 2 h with PBS containing 2% BSA (Sigma, St. Louis, MO) and washed four times with 0.02% Tween 20 in a 0.9% NaCl solution. Each sample and its control were added to the adjacent wells and incubated for 2 h. Plates were washed four times and incubated for 30 min with a goat anti-human IgG/IgM Ab conjugated with HRP at 1/1500 dilution (Sigma). Tetramethylbenzidine (0.0125%)/0.008% H2O2 in citrate buffer (pH 5.0) was used as a substrate, and color development was stopped using 2 N H2SO4. ODs were measured using an ELISA reader (Bio-Rad, Hercules, CA). Wells containing medium alone served as the background control. Supernatants were considered to contain specific anti-idiotypic Abs when mean OD in wells coated with the motif-positive peptide subtracted by background OD exceeded mean OD in wells coated with the control TCR peptide subtracted by background OD by at least three times (19).
Immunoblot analysis
Lysates were prepared from a representative MBP-reactive T cell clone (MS7-E2.6) expressing the common CDR3 sequence and a motif-negative T cell clone (MS7-D2.2), respectively, using a standard method described previously (23). Briefly, 5 x 106 T cells were lysed in 100 µl lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 7.6), 0.5% Triton X-100, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Cell debris were spun down at 13,000 x g for 20 min at 4 C. The resulting lysates were electrophoresed using 10% SDS-PAGE. After blotting, nitrocellulose membranes were cut into strips and then blocked with 5% low fat milk powder in TBS containing 0.1% Tween 20. The strips were then incubated with undiluted supernatants in mini-incubation trays for 1 h at room temperature. A goat anti-human IgG and IgM (H+L chains) coupled to HRP were used as secondary Ab (5% low fat milk powder in 100 ng/ml in TBS containing 0.1% Tween -20) and incubated with washed strips for 45 min, followed by ECL visualization of the proteins on membrane (Amersham, Arlington Heights, IL). Supernatant obtained from an EBV-transformed B cell line producing nonreactive Abs of the IgM type was used as a negative control. A rabbit polyclonal anti-human TCR-ß chain Ab (Santa Cruz Biotechnology, Santa Cruz, CA) was included as a positive control.
Flow cytometry
A representative MBP-reactive T cell clone (MS7-E2.6) expressing the common CDR3 sequence was incubated with the anti-idiotypic Abs derived from individual B cell lines at 4°C for 30 min. Supernatant obtained from an EBV-transformed B cell line producing nonreactive Abs of IgM type was used as a control. A motif-negative T cell clone (MS7-D2.2) was stained with the same anti-idiotypic Abs and the control Ab under similar experimental conditions. After washing with FACS buffer (PBS containing 5% FCS and 0.01% sodium azide) by centrifugation at 2300 rpm for 2 min at 4°C, cells were resuspended and stained with a goat anti-human IgG/IgM Ab conjugated with FITC. After two washes the cells were resuspended in 300 µl of FACS buffer and analyzed by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA). FITC-conjugated anti-IgG1 was used to detect background staining (Becton Dickinson).
The inhibition assay
Twenty thousand cells of the immunizing MBP-reactive T cell clones (motif-positive and motif-negative T cell clones) were cultured in 150 µl with irradiated autologous PBMC (100,000 cells/well) in the presence and the absence of the 83–99 peptide of MBP (20 µg/ml). Fifty microliters of undiluted supernatants were added to each well. Cell proliferation was measured after 72 h in [3H]thymidine incorporation assays. Supernatant obtained from an EBV-transformed B cell line producing nonreactive Abs was used as a control.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A panel of four MBP-reactive T cell clones was generated from two
patients with relapsing-remitting MS. These T cell clones expressed the
CD4 phenotype and recognized the 83–99 immunodominant peptide of MBP
in the context of DR4 or DR2 (DRB1*1501) molecules (Table I
). They were analyzed for TCR V gene
rearrangements by RT-PCR using V- and Vß-specific primers and
subsequently sequenced for the V-J and Vß-Dß-Jß junctional
regions. As shown in Table II, an
independent T cell clone (E2.6) derived from patient MS7 shared the
same TCR V17 and Vß13.1 genes with another T cell clone (C3.1)
obtained from a different patient (MS27). The two T cell clones had an
identical sequence (LGRAGLTY) within the Vß13.1-Dß-Jß junctional
region, while their V17 chains had two distinct V-J junctional
region sequences. As demonstrated previously, the identified LGRAGLTY
sequence represented a common CDR3 motif among Vß13.1 T cells that
recognize the 83–99 immunodominant region of MBP in different patients
with MS (24).
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Each patient received a total of four s.c. inoculations with two
irradiated autologous MBP-reactive T cell clones (2 x
107 cells/clone) at a 2-mo interval. The
proliferative responses of PBMC to the autologous immunizing T cell
clones were examined at two time points, corresponding to baseline and
2 mo after the last immunization. As shown in Fig. 1A, the proliferative
responses to both irradiated immunizing T cell clones were increased in
the patients after T cell vaccination and substantially exceeded the
baseline value. Furthermore, the responses to the TCR peptide
incorporating the common CDR3 sequence (motif-positive peptide), as
opposed to a control CDR3 peptide (motif-negative peptide) derived from
a nonimmunizing T cell clone, was apparent after vaccination. However,
the magnitude of the specific proliferation in response to the
motif-positive peptide was considerably lower than that induced by
irradiated immunizing T cells (Fig. 1A). The proliferative
response to the immunizing T cells correlated inversely with a decline
in the frequency of circulating MBP-reactive T cells in the immunized
patients (Fig. 1B), as demonstrated previously in other
studies (8, 15, 16).
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We then examined whether immunization with irradiated T cells would elicit specific anti-idiotypic Ab responses in the patients. As the whole T cells expressed an array of surface molecules that could interfere with the detection of serum anti-idiotypic Abs, the TCR peptide incorporating the common CDR3 sequence (motif-positive peptide) was used as the Ag for screening. A 20-mer TCR peptide derived from a nonimmunizing MBP-reactive T cell clone (motif-negative peptide) was included in all experiments as a control. The CDR3 sequence of the control peptide was not detected in the immunizing T cell clones.
As we experienced previously, no specific Ab reactivity to either the
TCR peptide or the original immunizing T cells could be detected using
ELISA or flow cytometry when tested with sera derived from the two
patients (data not shown). To further verify whether anti-idiotypic
Abs were present in the vaccinated patients, we generated a panel of
Ab-producing B cell lines from the postvaccination blood specimens
using a cell culture-based technique combining EBV transformation with
limiting dilution (see Materials and Methods). As
prevaccination PBMC were not available for the experiments, cells
obtained from two randomly selected healthy individuals were used as
control subjects and analyzed under the same experimental conditions.
Supernatants of the resulting B cell lines (92 cell lines from each
patient/individual) were tested for the presence of Abs to the
motif-positive TCR peptide and the control TCR peptide, respectively,
in ELISA. Abs were defined as anti-idiotypic when they exhibited
specific reactivity to the motif-positive TCR peptide but not the
control TCR peptide. As shown in Fig. 2, B cells producing specific anti-idiotypic Abs occurred at the
precursor frequency of 1.9 x 10-6 (7 of
92) and 0.82 x 10-6 (3 of 92) in patients
MS7 and MS27, respectively, compared with 0.27 x
10-6 (1 of 92) in both nonimmunized control
subjects. In contrast, no specific Ab reactivity to the control peptide
was detected in the same supernatants.
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Next, the resulting anti-idiotypic Abs were characterized in a
series of experiments for the reactivity pattern and potential
functional effect on the proliferation of the immunizing T cells in
response to the 83–99 peptide of MBP. We first addressed whether the
anti-idiotypic Abs reactive to the TCR peptide would bind to the
whole immunizing T cells expressing the common CDR3 sequence.
Supernatants collected from three representative B cell lines that
produced relatively high concentrations of anti-idiotypic Abs were
further characterized (Table III). These
independent Abs derived from the two immunized patients were of the IgM
isotype and were found to bind to original immunizing T cell clones but
not to control T cell clones that did not express the common CDR3
sequence. Such a representative experiment using flow cytometry is
shown in Fig. 3. Similarly, the selected
Abs also exhibited the specific reactivity to the TCR of the immunizing
T cell clones in immunoblot analysis (Fig. 4). Furthermore, the anti-idiotypic
Abs were examined for an inhibitory effect on the proliferation of the
original immunizing T cell clones. As shown in Fig. 5, all three Abs examined were found to
inhibit substantially the proliferation of the immunizing T cell clones
expressing the CDR3 motif (MS7-E2.6 and MS27-C3.1) but not the
motif-negative immunizing T cell clones (MS7-D2.2 and MS27-D4.4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The successful detection of anti-idiotypic Abs as described here
was attributable to the technical improvements made in the study, which
enabled us to estimate the precursor frequency of B cells producing
anti-idiotypic Abs in blood specimens and to obtain relatively high
concentrations of specific Abs from the identified B cell lines for
detailed characterization. There are several issues related to the
interpretation of the results. 1) The general efficiency of EBV
transformation of human B cells was reported to be <10%
(25). Therefore, the precursor frequency of B cells
producing Ab of interest is likely to be underestimated in the assays
involving EBV transformation. The actual frequency of B cells producing
anti-idiotypic Abs in the vaccinated patients was probably much
higher than that described in the present study. 2) The preferential
IgM expression of the obtained anti-idiotypic Abs is consistent
with the recent immunization in the patients. However, it should be
noted that EBV-transformed B cells usually produce Abs of predominantly
the IgM isotype (26). Therefore, the observed preference
of the resulting anti-idiotypic Abs for IgM is likely to result
from the isotype selection by EBV transformation and may not represent
the original isotype of the B cells. 3) EBV appears to
transform preferentially recently activated B cells (26)
that represent relevant subsets of B cells induced by T cell
vaccination, providing a technical advantage for the purpose of this
study. 4) Some of the B cell lines generated by EBV transformation as
described here are likely to contain multiple B cell populations that
may produce Abs to other Ags, which is indicated by the reactivity of
Ab MS7-C8 to an additional Ag present in the T cell lysates (Fig. 4
).
In the study described here only one TCR peptide incorporating the common CDR3 sequence motif was used to identify anti-idiotypic Abs induced by T cell vaccination. The selection of the 20-mer CDR3 peptide was based on the considerations that it contained the common Vß13.1 sequence motif among MBP83–99-reactive T cells and expressed by the immunizing T cell clones used for both patients. It is conceivable that the LGRAGLTY sequence contains the idiotypic epitope(s) recognized by the obtained anti-idiotypic Abs, as the TCR peptide incorporating the LGRAGLTY sequence shares similar franking residues with the control TCR peptide. However, in the absence of the data from overlapping peptides spanning the other regions of the TCR, the study does not address whether the LGRAGLTY sequence represents a dominant idiotypic determinant(s) responsible for eliciting anti-idiotypic Ab responses. It is likely that T cell vaccination may also induce anti-idiotypic Ab responses to other TCR regions, including CDR2 and the framework regions.
The study demonstrated that these anti-idiotypic Abs induced by T cell vaccination appear to have specific regulatory properties, as evident by the inhibition of the proliferation of MBP-reactive T cells used for vaccination. It is likely that the anti-idiotypic Abs bind specifically to the idiotypic determinants of target T cells, thus blocking the T cell recognition of the MBP peptide. The possibility is consistent with other reports describing similar blocking effects of anti-idiotypic Abs on T cell recognition and function (27). Therefore, the observed anti-idiotypic Ab responses are likely to contribute to the regulatory effects of T cell vaccination on MBP-reactive T cells, supporting the idea that multiple regulatory components are involved in T cell vaccination. The reactivity and the regulatory role of the anti-idiotypic Abs to the common Vß13.1 sequence among MBP83–99-reactive T cells have important implications. For example, a synthetic peptide(s) incorporating the common CDR3 sequence motif may be used to elicit both cellular and humoral anti-idiotypic responses to MBP-reactive T cells in a subset of MS patients. Indeed, the LGRAGLTY motif was identified in PBMCs of >31% randomly selected MS patients and was present in the majority of T cell lines recognizing the immunodominant 83–99 region of MBP isolated from different MS patients (24). It is hoped that the understanding of the anti-idiotypic regulatory mechanisms induced by T cell vaccination will help to develop a more practical peptide-based vaccination approach by selecting most relevant TCR sequences that can be used to target selected MBP-reactive T cells in a subset of patients with MS.
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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.
3 Abbreviations used in the paper: MS, multiple sclerosis; MBP, myelin basic protein; CDR3, complementarity-determining region 3; MBP83–99, the immunodominant 83–99 peptide of MBP.
Received for publication April 18, 2000. Accepted for publication September 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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  Z-G Li, R Mu, Z-P Dai, and X-M Gao T cell vaccination in systemic lupus erythematosus with autologous activated T cells Lupus, November 1, 2005; 14(11): 884 - 889. [Abstract] [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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