Brain, Vol. 123, No. 3, 508-518,
March 2000
© 2000 Oxford University Press
Invited review |
Repertoire dynamics of autoreactive T cells in multiple sclerosis patients and healthy subjects
Epitope spreading versus clonal persistence
1 Department of Neuroimmunology, Max Planck Institute for Neurobiology, Martinsried and 2 Department of Neurology and Institute for Clinical Neuroimmunology, Ludwig-Maximilians University, Munich, Germany
Correspondence to:
Dr Reinhard Hohlfeld, Institute for Clinical Neuroimmunology, Klinikum Grosshadern, Marchioninistrasse 15, D-81366 Munich, Germany E-mail: hohlfeld{at}neuro.mpg.de
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Abstract |
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Autoantigen-specific T-lymphocytes are present in patients with autoimmune disease and in normal subjects. Little is currently known about the temporal variation (dynamics) of the immune repertoire of these autoreactive T cells. We analysed the long-term variation of the immune repertoire of T cells specific for myelin basic protein (MBP) in five untreated patients with multiple sclerosis and four normal control subjects over a mean observation period of 6 years. MBP-specific CD4+ T-cell lines were selected with purified human MBP, and their epitope specificity was mapped with overlapping synthetic peptides. Three distinct patterns of repertoire development were observed. (i) Two patients and three control subjects maintained a broad epitope response with fluctuations over time. (ii) Two patients initially showed a focused response that broadened over the course of 6 years; this finding could be described as intramolecular epitope spreading. (iii) In one patient and one control subject, a strikingly focused response, which was directed to a cluster of nested epitopes in the MBP region 83–102, persisted over time. T-cell receptor Vß sequence analysis allowed us to trace individual clones of MBP-specific T cells for up to 7 years in the peripheral circulation in four of the five patients and three of the four controls, suggesting that the long-term persistence of MBP-specific T-cell clones is a common feature of the T-cell repertoire not unique to multiple sclerosis. The persisting MBP-specific T-cell clones were not detectable in the blood of one of the patients by complementarity-determining region (CDR)-3 spectratyping, indicating that their frequency does not exceed 1 in 5000 T cells. The temporal characteristics of the MBP-specific T-cell repertoire described here are relevant to therapeutic strategies targeting autoantigen-specific T cells in multiple sclerosis and other autoimmune diseases.
multiple sclerosis; autoimmunity; T lymphocytes; repertoire; immunotherapy
BJ = joining region of the ß chain of the T-cell receptor (also called Jß); BV = variable region of the ß chain of the T-cell receptor (also called Vß); CD = cluster of differentiation; CDR3 = complementarity-determining region 3 (of the T- or B-cell receptor for antigen); D = diversity region of the TCR; J = joining region of the TCR; MBP = myelin basic protein; N-D-N = diversity region of the TCR flanked by N-nucleotides; PBMC = peripheral blood mononuclear cells; PCR = polymerase chain reaction; PLP = proteolipid protein; RT–PCR = reverse transcription–polymerase chain reaction; TCL = T-cell line; TCR = T-cell receptor for antigen; V = variable region of the TCR
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Introduction |
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Myelin basic protein (MBP) is an extensively studied human autoantigen. It is not only considered a candidate autoantigen of multiple sclerosis, but also serves as a paradigm for investigating the general features of antigen recognition by human T cells. Previous studies have demonstrated that, in most patients and healthy subjects, the T-cell response is directed to a range of different epitopes along the entire length of the 170 amino acids of the MBP molecule (Martin et al., 1992
; Hohlfeld et al., 1995; Hafler et al., 1996; Stinissen et al., 1997). In some individuals, however, the T-cell response is strikingly focused on only one or a few immunodominant clusters of nested epitopes, as has been observed in the Lewis rat (Meinl et al., 1993; Salvetti et al., 1993; Wucherpfennig et al., 1994; Vandevyver et al., 1995).
Little is known about the time course of the human T-cell response to MBP or other autoantigens. Studies in animals immunized with MBP or proteolipid protein (PLP) (Tuohy et al., 1998) or the acetylcholine receptor (Vincent et al., 1998) showed that the T-cell response is initially directed to a few immunodominant determinants of the immunizing protein or peptide. Later it spreads to other determinants of the same antigen (intramolecular epitope spreading), or to other antigens expressed in the target tissue (intermolecular epitope spreading) (Sercarz et al., 1993). Recent reports have indicated that epitope spreading may also occur in the immune response to PLP in patients with multiple sclerosis (Tuohy et al., 1997, 1999). However, PLP is not the only candidate autoantigen of multiple sclerosis (Lassmann and Wekerle, 1998) and virtually nothing is known about the temporal dynamics of the T-cell response to other myelin autoantigens in multiple sclerosis patients. In the future, longitudinal studies of the T-cell repertoire will be increasingly difficult to perform in untreated patients, because multiple sclerosis is now often treated with immunomodulatory agents such as interferon-ß (for review, see Hohlfeld, 1997).
In 1993 we published a detailed analysis of the MBP-specific T-cell repertoire in multiple sclerosis patients and normal subjects (Meinl et al., 1993). To gain insight into the temporal changes of the T-cell repertoire, we re-examined the MBP-specific T-cell response in an untreated subgroup of the same patients and in control subjects whom we had initially studied 6–7 years earlier (Meinl et al., 1993).
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Material and methods |
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Peripheral blood mononuclear cells (PBMC)
PBMC were isolated by density gradient centrifugation (Nycomed, Oslo, Norway) from five patients with laboratory-supported definite, relapsing–remitting multiple sclerosis and from four healthy donors (for details, see Table 1
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Purification of human MBP and synthesis of MBP peptides
MBP was purified from adult human brain according to established protocols (Eylar et al., 1979
). Briefly, the purification steps included homogenization, delipidation, acid extraction, neutralization, ammonium sulphate precipitation and acetone precipitation (Eylar et al., 1979). Purity of the MBP preparations was assessed by gel electrophoresis. MBP peptides were synthesized with an automated peptide synthesizer (431A; Applied Biosystems, Foster City, Calif., USA) according to the fluorenyl methoxycarbonyl method. The following overlapping peptides, covering the entire human MBP molecule, were synthesized (confirmed by amino acid analysis): MBP amino acids 1–20, 7–26, 16–38, 50–68, 61–82, 71–89, 83–102, 94–117, 108–131, 124–141, 131–145, 139–153, 148–162 and 153–170.
Isolation of MBP-specific T-cell lines (TCLs)
MBP-specific CD4+ TCLs were isolated using the standard split-well method exactly as described previously (Meinl et al., 1993). Peptide specificity was determined and documented in duplicate proliferation assays using 2 x 105 irradiated (5000 rad) autologous feeder cells and 2 x 104 T cells per round-bottomed well. Human MBP was used at a final concentration of 30 µg/ml and synthetic peptides at 10 µg/ml (Meinl et al., 1993). After 72 h, 0.22 µCi [3H]thymidine (2 Ci/mmol specific activity; Amersham, Braunschweig, Germany) was added for 18 h. Incorporation of [3H]thymidine was measured with a direct ß-counter (Matrix TM 96 Direct Beta Counter; Packard, Frankfurt, Germany). It should be noted that the absolute counts measured by the direct counting system are only 
20% of the counts obtained by conventional liquid scintillation.
All TCLs included in the repertoire analysis were first stimulated with whole MBP. Those TCLs recognizing more than one MBP peptide are listed separately for each recognized peptide epitope. After a few rounds of restimulation with MBP and after determination of the immunodominant peptide epitope, TCLs were restimulated with their respective peptide. In particular, they were restimulated at least three times with their individual peptide before reverse transcription–polymerase chain reaction (RT–PCR) analysis of their usage of T-cell antigen receptor (TCR) Vß. TCLs were also isolated from some of the patients by primary stimulation with synthetic peptides. Primary stimulation with synthetic peptides mostly recruited the same T-cell clones (sequences) as stimulation with full-length MBP. However, to avoid selection bias, the peptide-selected TCLs were not included in the repertoire analysis, but their TCR sequences were added to the sequence bank.
Analysis of TCR Vß sequences
Total RNA was prepared from 5 x 105 to 1 x 106 cells with Trizol-LS reagent (Gibco BRL, Gaithersburg, Md., USA). Oligo (dt)-primed cDNA was prepared from 2–5 µg total RNA using Superscript II reverse transcriptase (Gibco BRL), as recommended by the manufacturer. PCR was performed with one Cß-specific primer and 26 individual Vß family-specific primers, as described elsewhere (Monteiro et al., 1996). TCR sequences were obtained from four multiple sclerosis patients and three healthy donors. PCR products of TCLs with persisting peptide specificity and Vß usage were purified with Microcon® 30 microconcentrators (Amicon, Beverly, Mass., USA). Sequencing was performed with an ABI377 automated sequencer using the ABI dRhodamine terminator cycle sequencing kit according to the recommendations of the manufacturer (Applied Biosystems, Weiterstadt, Germany). CDR3 sequences identified at more than one point in time (on average 6 years apart) represented persisting T-cell clones.
CDR3 spectratyping
CDR3 spectratype analysis was done essentially as described (Puisieux et al., 1994; Liu et al., 1995; Pannetier et al., 1995; Monteiro et al., 1996), with the exception that Amplitaq Gold® (Applied Biosystems) with an appropriate preincubation time was used for all PCR procedures. Briefly, CD4+, CD8+, CD25+, CD26+ or CD38+ subpopulations of PBMC or CD4+ T lymphocytes from patient HK prestimulated for various time periods with MBP or MBP-peptide P83 were isolated with magnetic beads (Dynabead®, Dynal, Hamburg, Germany). TCR-specific PCR analysis of cDNA from these lymphocyte populations was performed using one TCR Cß-specific and 26 different Vß-specific oligonucleotides. From each Vß-specific PCR product, fluorescence-labelled runoff transcripts were generated using one TCR Cß- and 13 different Jß-specific oligonucleotides. Spectratype analysis of these runoff transcripts was performed on an ABI 377® automated sequencer using Genescan® software (Applied Biosystems). Expanded candidate Vß–Jß subpopulations were subamplified with the respective Vß–Jß primer pair from the initial Vß-Cß amplification product and purified with Microcon 30 columns. Sequence analysis was performed using BigDye® Sequence Mix and an ABI 377® automated sequencer according to the manufacturer's recommendations (Applied Biosystems).
In order to estimate the sensitivity of the CDR3 spectratyping method, we performed the following spiking experiment. Total RNA was prepared from a mouse T-cell hybridoma which lacks its endogenous TCR, but was transfected with a BV9.1/BJ1.5 human TCR (kindly provided by Dr Klaus Dornmair and Dr Heinz Wiendl, Department of Neuroimmunology, Max Planck Institute for Neurobiology, Martinsried, Germany), and from magnetic bead-separated polyclonal CD4+ T cells of a healthy donor. RNA concentrations were determined photometrically, and a log3 dilution series was prepared by diluting RNA from the transfected T-cell clone into aliquots of the RNA from polyclonal CD4+ T cells. RNA mixes containing from 100% down to 0.0001% of the RNA from the transfected T-cell clone were individually reverse-transcribed. Aliquots of the resulting cDNA were amplified using BV9 and Cß PCR primers. Runoff products were produced with fluorescence-labelled Cß and BJ1.5 primers and electrophoresed on an ABI377 automated sequencer as described above.
On the (more sensitive) BJ level of the CDR3 spectratyping (immunoscope) procedure, RNA from the transfected T-cell clone could still be clearly identified at a dilution of ~1 : 3000 (1 : 2187), whereas a dilution of ~1 : 10 000 (1 : 6561) gave a normal Gaussian spectratype pattern (data not shown). These estimates are consistent with previous findings by Cochet and colleagues, who detected one T-cell clone cell in 5000 T cells (Cochet et al., 1992). As the authors indicated, these levels of sensitivity may vary with BV/BJ usage and the size class of the individual clone's PCR product (Cochet et al., 1992).
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Results |
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Dynamics of epitope recognition
We probed the MBP-specific T-cell repertoire at different time points in five patients with multiple sclerosis and four normal control subjects (Table 1
), using the split-well technique to isolate CD4+ MBP-specific TCLs (Meinl et al., 1993; Pette et al., 1990a, b). In patient HK, MBP-specific TCLs were isolated at more than six time points over the course of 7 years. In four additional patients and three controls, MBP-specific TCLs were isolated at two time points separated by 6 years. In control subject IH, MBP-specific TCLs were isolated at two time points 3 years apart. None of the patients was treated with long-term immunomodulatory or immunosuppressive agents during the observation period.
All TCLs used for repertoire analysis were initially stimulated and selected with full-length human MBP. Epitope specificity of the MBP-reactive TCLs was established with a panel of overlapping ~20-mer synthetic peptides. We observed three basic patterns of repertoire dynamics (Figs 1A, and 2A and B). (i) Patients BM and MH and control subjects FP, HW and RV maintained a broad epitope response with evidence of shifts and variation over time (e.g. patient MH). (ii) Two patients (AS and SS) initially had a relatively focused response that broadened over the course of 6 years. (iii) Patient HK and control subject IH initially focused on region 83–102 and essentially maintained this focused response throughout the observation period.
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Long-term persistence of individual T-cell clones
To see whether the persistence of epitope recognition reflects the persistence of individual T-cell clones, we sequenced the TCR Vß chains of several TCLs that recognized the same epitope and had been isolated at different points in time. The most detailed analysis was performed in patient HK, who had a strikingly stable dominant epitope cluster in the MBP region 83–102 (Fig. 1A
). As reported previously (Meinl et al., 1993), this epitope cluster is recognized by several distinct T-cell clones in patient HK. Figure 1B shows that identical TCR Vß sequences were obtained from MBP-specific TCLs isolated at different points in time separated by up to 7 years (e.g. CDR3 sequence LLGDG in Fig. 1B). Further data on the persisting TCLs of patient HK are provided in Table 2. At least one persisting TCR Vß sequence was identified in three other patients and in three of the four normal controls (Table 3).
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Repertoire analysis by TCR-CDR3 spectratyping
To establish whether the persisting MBP-specific T-cell clones were detectably expanded in the peripheral blood, we performed CDR3 spectratyping analyses. This technique allows the detection of expanded T-cell clones in TCR CDR3 length spectragrams as conspicuous peaks differing from the normal Gaussian distribution (Pannetier et al., 1995
). According to our own experiments, consistent with previously published results (Cochet et al., 1992), this technique allows the clonal detection of ~1 in 5000 T cells. We freshly isolated CD4+, CD25+, CD26+, and CD38+ subpopulations of PBMC from patient HK for CDR3 spectratype analysis. In addition, we analysed cryopreserved CD4+ (1992 and 1997) and CD25+ (1997) subpopulations of PBMC from the same patient. Furthermore, we performed CDR3 spectratyping on CD4+ T lymphocytes from patient HK, which had been stimulated in vitro with MBP or MBP peptide 83 for 0, 3, 7 or 37 days according to our standard protocol (Pannetier et al., 1995). We especially looked for potential clonal expansions of TCR Vß–Jß combinations that we had previously identified in the 10 different MBP-specific T-cell clones in earlier experiments (i.e. the persisting T-cell clones BV2-BJ2.7, BV5.1-BJ2.2, BV5.1-BJ2.5, BV9-BJ1.2 and BV9-BJ2.1, and the sporadic T-cell clones BV2-BJ1.1, BV5.1-BJ1.1, BV5.1-BJ1.3, BV7-BJ1.5 and BV17-BJ1.1) (Fig. 1B). The TCR Vß rearrangements that were detectable as conspicuous peaks in the CDR3 spectratypes of different cell preparations were either not readable by direct sequencing (indicating that they were still polyclonal), or represented T-cell clones different from the previously identified MBP-specific sequences (data not shown). Only one CDR3 spectratyping sequence (from a T-cell clone expanded by in vitro stimulation with MBP) was identical to one of the previously identified MBP-specific T-cell clones: BV9-CASS-QDLWNIA-NYG-BJ1.2 (cf. Fig. 1B).
Figure 3 shows that this MBP-specific T-cell clone gradually expanded in vitro and became more conspicuous as a distinct peak in the CDR3 spectragrams after in vitro stimulation with MBP. These results indicate that the frequency of the persisting MBP-specific T-cell clones in the blood was <1 in 5000. However, the T cells were readily expanded to detectable levels by in vitro stimulation with MBP.
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Discussion |
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Our study of the long-term dynamics of the MBP-specific T-cell repertoire (performed in the same laboratory with identical techniques over the course of 6–7 years) reveals three basic patterns of epitope recognition: (i) persistence of a broad response with shifts and fluctuations over time; (ii) broadening of an initially focused response to a wider spectrum of MBP epitopes, and (iii) persistence of a focused anti-MBP T-cell response. In the majority of patients and controls, some clones of MBP-specific T cells persist for many years in the peripheral circulation. CDR3 spectratyping analysis indicates that the frequency of these persisting T-cell clones is <1 in 5000 T cells in the blood.
As regards epitope dynamics, we note that two of the three observed patterns, i.e. the persistence of a focused response and the persistence of a broad response, occurred both in patients and in healthy subjects. The third pattern, the broadening of an initially focused response, was seen only in the patients AS and SS. Although this might represent a form of disease-related epitope spreading (Sercarz et al., 1993), the data must obviously be interpreted with caution in view of the small number of cases and controls. Evidence of epitope spreading in multiple sclerosis has been reported previously in a longitudinal study of the T-cell response to synthetic peptides of another candidate myelin autoantigen, PLP (Tuohy et al., 1997, 1999). In contrast to our approach, however, that study relied on the stimulation of PBMC with synthetic peptides rather than the selection of TCLs with the full-length protein.
Clearly, if epitope spreading occurs, it is not observed in all patients. Patient HK, for example, maintained an extremely focused epitope response that remained essentially stable for >7 years. A few additional clones of persisting MBP-specific T cells have been described by other investigators in a few other patients with multiple sclerosis (Salvetti et al., 1993; Wucherpfennig et al., 1994; Uccelli et al., 1998). However, it appears that the pattern of an extremely focused and stable epitope response is the exception rather than rule in multiple sclerosis (Meinl et al., 1993; Lovett-Racke et al., 1997).
In patient HK, a small cluster of nested epitopes in the MBP region 83–102 are recognized by several distinct T-cell clones. Sequence analysis of the TCLs obtained over the course of 7 years provides insight into the clonal dynamics of the T-cell response to this small immunodominant region (Fig. 1B). It is remarkable that several T-cell clones with specificity for this region persisted for many years in HK's peripheral circulation. For example, the clone with the TCR Vß CDR3 sequence LLGDG persisted for the entire observation period of 7 years (Fig. 1B).
Although the clinical course of patient HK is relatively mild, his disease is clearly active. Between 1990 and 1999 he had two clinical exacerbations with sensory symptoms. Furthermore, frequent MRI scanning showed clear evidence for ongoing disease activity. Monthly cranial MRI with gadolinium enhancement, obtained between 1992 and 1993, revealed six active and eight inactive scans (Fig. 1B). Thus, in this patient the remarkably stable anti-MBP repertoire with clonal persistence is associated with active disease. One possible interpretation is that the persisting MBP-specific T-cell clones are irrelevant in this patient, perhaps because the pathogenic T-cell response is directed to other autoantigen(s). Alternatively, the persisting MBP-specific T cells may have an unknown regulatory or protective function.
Our previous studies in a primate (rhesus monkey) model provided direct evidence that the MBP-specific CD4+ T-cell lines that can be isolated from normal (non-immunized) donors do differ in their encephalitogenic potential (Meinl et al., 1997). It is therefore conceivable that at least some of the autoantigen-specific T cells that exist in the immune repertoire have some beneficial effect. It is interesting in this connection that the transfer of MBP-specific T cells can protect CNS neurons after experimental crush injury of the optic nerve (Moalem et al., 1999). A possible explanation for such a protective effect is that MBP-specific T cells produce neurotrophic factors such as brain-derived neurotrophic factor (Besser and Wank, 1999; Kerschensteiner et al., 1999). A protective role of at least some of the autoreactive T cells would help to explain why these cells are maintained as a regular and apparently stable component of the normal immune system (Cohen, 1992).
An unexpected and, to our knowledge, novel observation made during the course of our study is that, in principle, a focused anti-MBP epitope response can occur in normal subjects (subject IH in Fig. 2B), and that MBP-specific T-cell clones can persist for many years both in healthy controls and in patients. It thus appears that clonal persistence is a more general feature of the human T-cell repertoire and is not necessarily related to the pathogenic (or protective) potential of (auto)antigen-specific T cells. That human T cells can have a lifespan of many decades has been inferred from observations on memory responses after vaccination and the persistence of chromosomal alterations after irradiation for ankylosing spondylitis (Sprent, 1994; Ahmed and Gray, 1996; Zinkernagel et al., 1996; Dutton et al., 1998). Whether the long-term survival of antigen-specific T cells requires continued stimulation with antigen is still open to debate (Viret et al., 1999). Our observations of clonal persistence in healthy subjects could indicate that MBP-specific T cells are stimulated by MBP fragments processed and presented outside the CNS, perhaps in peripheral nerves or in immune organs, where MBP may be also expressed (Voskuhl, 1998). Alternatively, the MBP-specific T cells that persist in normal subjects might be cross-stimulated by viral or bacterial `mimicry determinants'. It seems relevant in this regard that T-cell recognition of MBP is surprisingly degenerate (Wucherpfennig and Strominger, 1995; Hemmer et al., 1997; Gran et al., 1999). For example, it has been demonstrated in experiments with experimental autoimmune encephalitis that a viral antigen, depending on its nature, dose and number of exposures, may select antigen-specific T cells that survive in vivo and can trigger autoimmune disease after adoptive transfer (Ufret-Vincenty et al., 1998). Whatever the mechanism favouring the survival of these cells, our results indicate that the long-term persistence of MBP-specific T cells is a relatively common feature of the human T-cell repertoire both in patients with multiple sclerosis and in normal subjects. Further studies are required to establish whether at least some of the persisting MBP-specific T cells play a role in multiple sclerosis and whether they are harmful or protective.
To estimate the frequency of the persisting T cells, we employed the CDR3 spectratyping technique (Pannetier et al., 1995). This technique, also referred to as the immunoscope technique, is based on the fact that in a normal, undisturbed T-cell repertoire, the PCR products of TCRs sharing a particular Vß (or Vß and Jß) chain show a Gaussian distribution of their CDR3 length (Pannetier et al., 1995). Any significant clonal expansion is revealed by a conspicuous peak that deviates from the Gaussian distribution. The sensitivity of this method is ~1 in 5000 T cells for the detection of single clones (our own results; Cochet et al., 1992). We focused on patient HK, who has the most strikingly persisting anti-MBP T-cell response. Although we detected several peaks in different PCR products of various T-cell preparations (including T cells selected for activation markers like CD25), either the peaks did not correspond to the previously identified persisting MBP-specific TCR sequences or they could not be read by direct sequencing, indicating that they were polyclonal. Only after in vitro stimulation with MBP did a CDR3 length peak appear that corresponded to the previously identified persisting TCR amino acid sequence BV9-CASS-QDLWNIA-NYG-BJ1.2 (Figs 1B and 3). These results indicate that the frequency of the MBP-specific T cells persisting in patient HK is below the sensitivity of the CDR3 spectratyping technique, i.e. <1 in 5000 T cells. It should be noted, however, that the lack of a detectable extent of clonal expansion does not, of course, rule out a possible pathogenic role of the persisting T cells.
Patients like HK, who maintain a focused and stable T-cell response to an immunodominant region of antigen, may seem obvious candidates for selective immunotherapy targeting autoreactive T cells. Such therapies include `vaccination' with T cells, TCR peptides or TCR DNA, or application of the autoantigen in a tolerogenic form (for reviews, see Hohlfeld, 1997; Stinissen et al., 1997; Steinman, 1999). However, our results demonstrate that, even in seemingly ideal candidate patients, the T-cell response may still be clonally diverse, although it is directed only to a narrow region of the target antigen, as is strikingly illustrated in patient HK (Fig. 1B). Furthermore, as discussed above, some of the persisting T cells may have a suppressive or protective rather than a pathogenic role. Obviously, it would not be desirable to curb such a protective response.
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Acknowledgments |
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We thank Ms M. Sölch and I. Eiglmeier for excellent technical assistance, Drs H. Wiendl and K. Dornmair for kindly providing a mouse hybridoma transfected with human TCR for control experiments and Drs K. Dornmair, A. Flügel, E. Meinl and Mrs J. Benson for helpful comments on the manuscript. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 217, C13; Goe 514/4–1), Hertie Stiftung (GHS 339/95) and European Community (BMH4-CT96–0893: Immunoregulatory aspects of T cell autoimmunity in multiple sclerosis). This work is part of the doctoral thesis of Harald Hofstetter. The Institute for Clinical Neuroimmunology is supported by the Hermann and Lilly Schilling Foundation.
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Received July 13, 1999. Revised September 2, 1999. Accepted September 15, 1999.
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