Brain, Vol. 122, No. 3, 523-535,
March 1999
© 1999 Oxford University Press
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Heterogeneity of T-cell receptor usage in experimental autoimmune neuritis in the Lewis rat
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1 Departments of Neurology and 2 Virology and Immunobiology, Julius-Maximilians Universität, Würzburg, Germany and 3 Immunology Department/IMM3 Scripps Clinic and Research Foundation, La Jolla, California, USA
Correspondence to:
Ralf Gold, MD, Department of Neurology, University of Würzburg, Josef-Schneider-Straße 11, D-97080 Würzburg, Germany E-mail r.gold{at}mail.uni-wuerzburg.de
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Abstract |
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In experimental autoimmune neuritis (EAN), T-cell receptor (TCR) variable (V)-region gene usage by neuritogenic T cells has been reported to be clonally restricted at the RNA level. This study was designed to verify TCR usage by neuritogenic T cells at the protein level. We generated two monoclonal antibodies (mAbs) 7H4 and 8G8 specific for a Vß4/V
11 associated idiotype expressed by the majority of neuritogenic cells of P2-specific T-cell lines. The remaining neuritogenic P2-specific T cells either exhibited a dominant usage of the TCR Vß13 chain recognized by the recently generated mAbs 17D5 and 18B1 or showed diverse Vß usage. Treatment of adoptive-transfer (AT)-EAN or of EAN actively induced with the neuritogenic P2 peptide by mAbs 7H4 and 8G8 led to a partial, but significant, reduction of clinical disease. Treatment with Vß13-specific mAb 17D5 had no clear effect on active EAN. Our data show that at least three different TCR are used by P2-specific pathogenic T cells in EAN, an animal model for human inflammatory neuropathies. Guillain–Barré syndrome; EAN therapy; antibodies; T-cell receptors
AT-EAN = adoptive transfer EAN; cDNA = complementary DNA; EAE = experimental autoimmune encephalomyelitis; EAN = experimental autoimmune neuritis; FACS = fluorescence-activated cell sorter; mAb = monoclonal antibody; MBP = myelin basic protein; PCR = polymerase chain reaction; rhP2 = recombinant human P2; RT–PCR = reverse transcription–PCR; TCR = T-cell receptor; V = variable
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* Present address: Department of Neurology, University of Regensburg, 93053, Regensburg, Germany
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Introduction |
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Experimental autoimmune neuritis (EAN) is an animal model for human Guillain–Barré syndrome, an acute demyelinating inflammatory disease of the peripheral nervous system mediated by autoantigen-specific T cells (summarized in Hughes, 1990
; Hartung et al., 1993). Major autoantigens in the peripheral nervous system include the myelin proteins P2 and P0, gangliosides and glycolipids (Hughes, 1990; Hartung et al., 1996). The neuritogenic epitope of the P2 protein is located within amino acids 53–78 (Olee et al., 1988; Rostami and Gregorian, 1991). In later studies, the minimum neuritogenic T-cell epitope of P2 was identified as residues 61–70 (Olee et al., 1990). EAN can be induced actively in Lewis rats by immunization with bovine P2 protein or recombinant human P2 protein, with a peptide (amino acids 53–78) spanning the neuritogenic epitope, or by adoptive transfer of neuritogenic T cells (AT-EAN) (Brostoff, et al., 1977; Kadlubowski and Hughes, 1979; Linington et al., 1984; Weishaupt et al., 1995).
The restricted usage of T-cell receptors (TCR) by autoreactive T-cell clones was observed in Lewis rat experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. In EAN, TCR usage is far less well defined. Initial studies suggested a preferential usage of TCR V (variable) ß8-family members in neuritogenic T cells (Clark et al., 1992; Zhang et al., 1994), but a wider pattern of TCR usage has recently been proposed by our group in neuritogenic T cells (Jung et al., 1993) and in inflamed sciatic nerve (Weilbach et al., 1997). TCR Vß8.2 chains could only be detected in nerve tissue when whole peripheral nerve myelin was used for immunization (Weilbach et al., 1997).
In view of these discrepancies, we sought to characterize TCR usage by neuritogenic T cells on the protein level by generating monoclonal antibodies (mAbs) raised against neuritogenic T-cell lines. These reagents, together with recently generated mAbs recognizing TCR Vß13 (M. Stienekemeier and R. Gold, unpublished results), were then used for cell sorting and therapeutic studies. Herein, we clearly show the heterogeneity of TCR usage in EAN.
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Material and methods |
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Reagents and antibodies
All tissue culture supplements were purchased from Gibco-BRL, except BSA (bovine serum albumin) fraction V (Roth, Karlsruhe, Germany) and concanavalin A (Sigma, Deisenhofen, Germany). Myelin basic protein (MBP) was prepared according to the protocol of Eylar et al. (1979). Recombinant human P2 protein (rhP2) was expressed and purified as described (Weishaupt et al., 1995
). Bovine P2 was isolated from peripheral nerve myelin according to the protocol of Kitamura et al. (1976). Human and bovine P2 peptides (amino acids 1–20, 14–25, 20–40, 35–60, 53–78, 70–90, 85–110, 100–120 and 115–131) were synthesized by Professor Dr D. Palm (University of Würzburg) using solid-phase stepwise elongation on an Applied Biosystems model 430. Table 1 shows the unconjugated rat-TCR-specific mAbs which were used in this study.
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FITC (fluorescein isothiocyanate)-labelled mAbs R78, G101, G177 and HIS42 were from Dr T. Herrmann. FITC-labelled mAb R73 and mAb OX-19 recognizing the rat CD5 antigen (R-PE anti-rat CD5, mouse IgG1) were purchased from Pharmingen (San Diego, Calif., USA).
Cells and culture
T-cell lines and T-cell hybridomas
P2- or MBP-specific CD4+ rat T-cell lines were established as described previously (Linington et al., 1984; Gold et al., 1995a). Briefly, female Lewis rats were immunized in the hind footpad with 200 µg bovine P2 or 100 µg mbp emulsified in CFA (complete Freund's adjuvant) (Difco, Detroit, Mich., USA). Ten days later, draining lymph nodes were removed and single-cell suspensions were prepared. Lymph node cells were then cultured at a density of 8 x 106 cells/ml in 60 mm plastic dishes (Nunc, Wiesbaden, Germany) in the presence of 20 µg/ml P2 or 10 µg/ml MBP in RPMI-1640 supplemented with 1% normal rat serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine. Antigen-specific T cells were selected by repeated propagation cycles in medium with 7.5% supernatant of concanavalin A-treated mouse spleen cells and 10% FCS (foetal calf serum), followed by antigen-specific restimulation using irradiated (40 Gy) syngeneic thymus cells. The P2- or MBP-specific CD4+ rat T-cell lines used for these experiments were characterized previously (Jung et al., 1991, 1992; Gold et al., 1995b; Zettl et al., 1995). T-cell hybridomas MBP-H1 and P2.48.1 were generated by fusion of activated T-cell blasts from T-cell lines MBP13 or P2.48 with a variant of the mouse thymoma BW5147 (BW1100.129.237) following standard methods (Torres-Nagel et al., 1993) and screened for expression of TCR by immunoflow cytometry.
For T-cell proliferation studies, 2 x 104/well resting T cells of unsorted or sorted T-cell lines were combined with 106 irradiated thymic APC (antigen presenting cells) and antigen in 100 µl medium in 96-well round bottom microtitre plates (Nunc). Triplicate cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 for 24 h and harvested following a 16 h pulse with 0.2 µCi/well 3H-dT (tritiated thymidine) (Amersham-Buchler; Braunschweig, Germany). The cells were collected on fibreglass filter paper by a Betaplate 96-well harvester (Pharmacia, Uppsala, Sweden), and radioactivity quantified with a 96-well Betaplate liquid scintillation counter (Pharmacia).
Generation of mAbs
mAbs directed to the rat TCR V region were generated as described previously (Torres-Nagel et al., 1993). Briefly, T-cell blasts from the neuritogenic P2-specific T-cell line G7 were used to immunize BALB/c mice (Charles River, Sulzfeld, Germany). After five weekly intraperitoneal injections of 1 x 107 cells, mice were rested for 3 weeks and challenged i.v. 3 days prior to PEG (polyethylenglycol)-mediated fusion of spleen cells with the Ag8 non-producer myeloma cell line. Cells were then seeded in 96-well flat-bottom microtitre plates in a medium containing HAT (hypoxanthine/aminopterin/thymidine medium) for selection and observed daily for clonal growth. Antibodies binding exclusively to neuritogenic T-cell lines, as screened by immunoflow cytometry, were further characterized and cloned. For isotype typing of mAbs, the mouse monoclonal antibody isotyping kit (Boehringer Mannheim, Germany) was used.
For therapy studies, B-cell hybridomas were propagated in RPMI 1640 supplemented with 2.5% FCS (
-globulin-free)/100 U/ml penicillin/100 µg/ml streptomycin/2 mM glutamine/0.12% glucose in `cell factories' (Nunc). mAbs were then purified from supernatants on protein A Sepharose following standard procedures.
Characterization of neuritogenic and naive T cells with mAbs
For immunoflow cytometry of TCR expression, 2 x 105 T cells were incubated with supernatants of the mAb diluted 1 : 1 in PBS/0.1% BSA in a final volume of 100 µl for 20 min on ice. After a washing cycle, antibody binding was detected with a fluorescein [DTAF (dichlorotriazinyl-amino-fluorescein)]-conjugated goat anti-mouse-IgG F(ab')2 fragment (Dianova, Heidelberg, Germany) at a dilution of 1 : 100 for 15 min on ice. Immunofluorescence was measured with a fluorescence-activated cell sorter (FACScan, Becton Dickinson, Sunnyvale, Calif., USA) using the CellQuest software (Becton Dickinson).
For immunoflow cytometry of naive T lymphocytes, a single cell suspension of popliteal and mesenterial lymph nodes from unimmunized Lewis rats was prepared. Cells were depleted from macrophages by adsorption on plastic dishes and further purified over nylon wool columns.
Neuritogenic T-cell lines were positively selected using the new mAb and the magnetic cell sorting `MACS' system (Miltenyi Biotech, Bergisch-Gladbach, Germany) basically following a previously described protocol (Miltenyi et al., 1990). Sorted T cells were then propagated in IL-2-containing medium. If necessary they were subjected to repeated sorting cycles as described.
To study the mitogenic effect of mAb 8G8 (IgG3), 96-well flat-bottom microtitre plates were precoated for 4 h with a goat anti-mouse-IgG (H + L) (Dianova) at a dilution of 1 : 1000 in borate buffer, pH 8.6, following incubation overnight at 4°C with either 100 µl of a sterile hybridoma supernatant (pH 8.0) or 100 µl of the respective isotype controls (Sigma; The Binding Site GmbH, Heidelberg, Germany) in borate buffer. After washing, G7-T-cell line cells were added. [3H]thymidine incorporation was determined as described above.
Immunoprecipitation
For TCR precipitation, 8 x 107 T-cell hybridoma cells were lysed in lysis buffer [PBS/1% Nonidet P40/1 mM PMSF (phenylmethylsulphonyl fluoride)] and immunoprecipitation was performed according to previous descriptions (Zavazava et al., 1990) and instructions given by the manufacturer (Dynal, Hamburg, Germany). After centrifugation, the lysate was incubated with 80 µg of the respective mAb bound to goat anti-mouse-IgG-conjugated dynabeads (Dynal) for 2 h at 4°C. The beads were washed three times with lysis buffer and boiled for 5 min in 30 µl reducing Laemmli sample buffer (Laemmli, 1970). The beads were removed and the supernatant was electrophoresed on 12.5% SDS–polyacrylamide gels. Precipitated TCR were analysed by semi-dry Western blotting using the biotinylated rat TCR-specific mAb R73 (Hünig et al., 1989) at a dilution of 1 : 100 and detected by a streptavidin-conjugated alkaline phosphatase (Dako, Hamburg, Germany). Colour reaction was visualized using NBT/BCIP (Boehringer Mannheim) as a chromogen.
Molecular biological studies
RNA extraction and complementary DNA (cDNA) synthesis
Extraction of total RNA was carried out with a Roti-Quick-Kit (Roth) following manufacturer's instructions. Approximately 20 µg of total RNA was reverse-transcribed into first-strand cDNA by using oligo-(dT)20 primers (Pharmacia) as described elsewhere in detail (Kruse et al., 1997).
Determination of TCR V and Vß sequences by reverse transcription–PCR (RT–PCR) and DNA sequencing
cDNA was used in dilutions of 1 : 10 for enzymatic amplification with specific TCR V or Vß element primers and a common C or Cß primer. Primer sequences are presented in Table 2.
Primers were synthesized on a gene assembler plus system (Pharmacia). The polymerase chain reaction (PCR) mixture consisted of 2.0 µl diluted cDNA, 5.0 µl x 10 PCR buffer, 1.25 U Taq polymerase (Applied Biosystems, Weiterstadt, Germany), 25 pmol of each primer and 10 nmol dNTP (Pharmacia) at a final volume of 50 µl H2O. Amplifications were performed with a model 9600 thermocycler (Perkin Elmer, Langen, Germany) with denaturation at 95°C for 40 s, annealing at 55°C for 120 s, and extension at 72°C for 60 s for five cycles. This was followed by denaturation at 95°C for 60 s, annealing at 55°C for 20 s, and extension at 72°C for 60 s for 20 or 30 cycles with a prolonged 10 min extension in the last cycle. In previous studies (Gold et al., 1995a), these primers detected all V and Vß families in Lewis rat lymph node cells. Five microlitres of amplification products were electrophoresed on a 2.0% agarose gel. Reaction products detected by ethidium bromide staining were purified using a QIA quick spin PCR purification kit (Qiagen, Hilden, Germany). The purified PCR products were either directly sequenced using the same primers as in the PCR reaction and ABI PRISMTM Dye Terminator Cycle Sequencing Kit (Perkin-Elmer) following manufacturer's instructions or were ligated into PCR-Script vectors and used to transform bacterial XL1-blue cells, as described by the manufacturer (Stratagene, Heidelberg, Germany). For each TCR ß-chain amplificate, two insert-positive plasmids were sequenced using T7 and T3 promoter sequencing primers. Sequencing probes were analysed using the ABI PRISM 373A sequencer (Applied Biosystems). For DNA sequence analyses, the DNASIS and PROSIS software (Hitachi, Yokohama, Japan) were used. All sequences were compared with previously published genes for unequivocal identification (Smith et al., 1991; Williams et al., 1991; Hinkkanen et al., 1993; Shirwan et al., 1993).
Determination of TCR Vß sequences by RNase protection assay
Two rat TCR Vß-chain-specific plasmid sets (`Vß riboprobes') were used as templates to synthesize antisense RNA with T7 RNA polymerase (MBI Fermentas, St Leon-Rot, Germany), as described (Smith et al., 1992). Riboprobe set 1 includes specific probes for Vß2, 6, 8.1, 8.3, 11, 12, 13, 16, 17 and 18, while set 2 entails probes for Vß 2, 3.3, 4, 8.2, 9, 10, 14, 15, 19 and 20. Briefly, probes were labelled to 2 x 106 c.p.m./µl using [-32P]CTP (10 mCi/ml, 400 Ci/mmol) and 2 µl were hybridized for 12 h at 56°C with 8 µg of RNA prepared from T cells or thymus. After incubation with RNase A (10.5 µg, Boehringer Mannheim) and T1 (70 U, Boehringer Mannheim) protected probe : target mRNA duplexes were phenol/chloroform-extracted, ethanol-precipitated and electrophoresed on a standard 3.5% polyacrylamide sequencing gel. Gels were autoradiographed on Kodak X-OMAT AR films with intensifying screens at –70°C for 3–48 h.
Animals and therapy studies
Female Lewis rats (4–8 weeks old) from Charles River (Sulzfeld, Germany) and Harlan (Borchen, Germany) were housed in plastic cages without grid floors and given commercial food pellets and water ad libitum. The blood of selected animals was serologically tested to verify they were free of relevant viral infections. All experiments were conducted according to approved Bavarian state regulations for animal experimentation.
For active EAN, rats were inoculated in the hind footpad with 100 µl of an emulsion of equal volumes of saline and CFA (1 mg/ml Mycobacterium tuberculosis H37Ra, Difco, Detroit, Mich., USA) containing 100 µg of P2 peptide (amino acids 53–78) or 300 µg of rhP2 protein. AT-EAN was induced by tail vein injection of 1 x 107 neuritogenic P2-specific, CD4-positive activated T cells from neuritogenic or sorted T-cell lines.
Animals were weighed and inspected for clinical signs of disease on a daily basis. Disease severity was assessed clinically employing a scale ranging from 0–10 (King et al., 1985; Hartung et al., 1988): 0 = normal; 1 = less active, reduced tone of tail; 2 = limp tail, impaired righting; 3 = absent righting; 4 = gait ataxia; 5 = mild paraparesis of the hind limbs; 6 = moderate paraparesis; 7 = severe paraparesis or paraplegia; 8 = tetraparesis; 9 = moribund; 10 = death.
For therapeutic studies, animals were injected in the tail vein with 500 µg/500 µl of purified mAb on days 5, 9 and 13 after induction of active EAN, or days 1, 3 and 5 after induction of AT-EAN. Control animals were injected i.v. with mouse IgG2a (kappa) (Sigma) or mouse IgG3 (The Binding Site GmbH) using an identical regimen.
Statistical evaluation
Statistical analysis was performed using the two-way ANOVA test for disease courses or Mann–Whitney U-test for body weights using the Prism computer program (GraphPad Software Incorporated, San Diego, Calif., USA). P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) were considered statistically significant.
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Results |
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Characterization of mAbs 7H4 and 8G8, and specific recognition of TCR on neuritogenic T-cell lines
B-cell hybridomas were generated from BALB/c mice immunized with rat T-cell blasts from the neuritogenic CD4+, P2-specific T-cell line G7. Clones 7H4 (IgG2a) and 8G8 (IgG3) were selected, as described above. Figure 1
shows that 40% of neuritogenic T-cell line G7 cells stained positive for mAb 7H4 in immunocytofluorometric analysis, while 44% were recognized by mAb 8G8. The specific binding of the rat TCR by the new mAbs 7H4 and 8G8 was clearly proved by immunoprecipitation of TCR using the new mAb 8G8 (Fig. 2; data for 7H4 are not shown).
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mAbs 7H4 and 8G8 stained <1% of naive T lymphocytes in immunoflow analysis (data not shown), indicating an idiotypic recognition by these mAb. The mitogenic effect of the new mAb 8G8 was tested on the G7 T-cell line by [3H]thymidine incorporation wherein an activating effect on the resting T cells was observed (SI
3, data not shown).
The specific usage of TCR chains, as recognized by the new mAbs, was further investigated by immunoflow cytometry using a panel of neuritogenic CD4+, P2-specific (Jung et al., 1991, 1992; Gold et al., 1995b; Zettl et al., 1995) or encephalitogenic, MBP-specific T-cell lines (Gold et al., 1995a). These lines were also typed with the existing rat TCR V-region-specific mAbs, and the results are summarized in Table 3. The staining pattern with the new mAbs differed from previously established mAbs. The mAbs 7H4 and 8G8 stained a dominant population in cell line P2.48 as well as the G7 cell line. Additionally, the mAb 8G8 fully recognized the T-cell line TKtsA6, which was not labelled by 7H4. The mAbs 17D5 and 18B1 recognizing TCR Vß13 stained a dominant part of the neuritogenic P2.6 line cells, but only a minor population in G7 T cells in most samples although more than 10% positive cells were seen during some restimulation cycles. The neuritogenic T-cell lines G5 and P2.76 were only rarely labelled by any of the available anti-TCR mAb. Encephalitogenic T-cell lines such as MBP13 or the non-neuritogenic P2-specific lines P2.7 (not recognizing the neuritogenic P2 epitope) and G6 were not labelled above controls. These results suggested that the new mAbs, in addition to the anti-Vß13 mAbs 17D5 and 18B1, specifically recognize TCR chains used by a large proportion of neuritogenic P2-specific T-cell lines.
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Analysis of TCR V-chain specificity of the new mAbs
To characterize further the TCR V chains recognized by the new mAbs, we sorted T-cell lines G7, P2.6 and P2.48 to more than 99% purity using the MACS system. Analysis of the TCR V
- and Vß-chain usage of sorted lines was then performed by RT–PCR. In T-cell lines G7 and P2.48 sorted by 7H4, we observed a Vß4 signal clearly enhanced compared with the unsorted lines (data not shown). For definite analysis, the RNase protection assay was used (see below). RT–PCR in T-cell line G5 or P2.76 yielded no clear results. Interestingly both Vß4 and Vß13 were associated with the V11 chain in sorted neuritogenic T-cell lines.
We used the RNase protection assay to confirm usage of TCR Vß chains recognized by the new mAb and extended characterization of lines G5 and P2.76. After hybridizing RNA prepared from unsorted/mAbs positive-sorted T-cell lines or thymus with rat TCR Vß-chain-specific probes (`Vß riboprobes'), we observed a Vß4 signal in T-cell lines G7, P2.48 and TKtsA6 (Fig. 3). After sorting with the mAb 7H4, only the Vß4 signal in the G7 line was detectable, while other contaminating bands disappeared. In T-cell line G5 signals for Vß2, 4, 17 and 19 were obtained. Furthermore, in T-cell line P2.6, a weak Vß8.1/8.3 signal but a dominant Vß13 signal was detectable (data not shown).
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Analysis of TCR Vß4–CDR3 region sequences amplified from neuritogenic P2-specific T-cell lines
mAbs 7H4 and 8G8 yielded similar staining patterns by immunoflow cytometry in most neuritogenic P2-specific T-cell lines, with the exception of the T-cell line TKtsA6, which showed a different staining pattern (Table 3
). To determine whether the mAb 7H4 recognized a CDR3-region epitope of the TCR Vß4 chain, in contrast to the mAb 8G8, we sequenced and compared the TCR Vß4 VDJ (variable–diversity–junctional) regions amplified by RT–PCR from neuritogenic P2-specific T-cell lines (Fig. 4). Sequence analyses showed that the TKtsA6, G7 and P2.48 line expressed identical TCR ß chain-VDJ-junctional regions, further underlining their specificity. For this reason, recognition of this region by mAb 7H4 is excluded. It is more likely that the idiotypic recognition of a conformational epitope is located on the Vß4 and the associated V11 chain.
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Characterization of epitope specificity of mAb 7H4- and 17D5-sorted cells from neuritogenic, P2-specific T-cell lines
The epitope of the P2 protein recognized by mAb 7H4- and 17D5-positive cell populations from P2-specific T-cell lines was further characterized by a panel of overlapping synthetic peptides from human P2 (Table 4
) and bovine P2 (not shown). The unsorted T-cell line G7 and the mAb 7H4-positive sorted line G7 were activated only by the human and bovine peptide (amino acids 53–78) enclosing the neuritogenic epitope and did not recognize any other peptides (Table 4). In addition, the 17D5-positive sorted line P2.6, the almost clonal, TCR Vß4-positive T-cell line P2.48 and the clone TKtsA6 responded only to these neuritogenic peptides. This peptide was also exclusively recognized by the neuritogenic line G5 whose TCR repertoire was heterogeneous (see above).
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Further cell transfer experiments corroborated the notion that all these mAb sorted T-cell lines were neuritogenic (see below).
In vivo studies
Positive selection of Vß4 or Vß13 in primary lymph node cultures from immunized rats
Primary lymph node cell populations from naive, rhP2- or P2 peptide (amino acids 53–78)-immunized Lewis rats were stained with the new anti-TCR mAb using a CD5-specific mAb (OX-19) as a gate parameter. In naive animals 4.6% of T lymphocytes were recognized by mAb 18B1. The 18B1-positive cell population increased to 7.3% after immunization with rhP2.
Similarly, the percentage of 7H4-positive T cells increased from less than 1% to 3.0% or 3.7%, respectively, in LN cells from rhP2 or P2 peptide immunized animals. After one antigen-specific selection cycle in vitro with the neuritogenic peptide, we found a stronger usage of TCRs recognized by mAb 18B1 (13.8%) or mAb 7H4 (4.0%), but there was no further selection with intact P2 protein. As controls, we included determination of Vß8.2-positive cells that did not expand either in vitro or in vivo (data not given).
Therapy studies
AT-EAN.
The prevention of AT-EAN induced by transfer of 7H4-positive sorted G7 cells with mAb 7H4 (anti-Vß4) or by transfer of P2.6 line cells using mAb 17D5 directed against TCR Vß13 implied almost full protection against clinical disease (Fig. 5A and B). Thus, both mAb-positive cell populations were neuritogenic and their TCR could be blocked by the respective mAb.
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Next, treatment of AT-EAN induced by unsorted G7 line cells with mAb 7H4 was investigated, and significant reduction of clinical signs was observed compared to controls (Fig. 5C
). Similar results were obtained with mAb 8G8 (not shown). In contrast, treatment with mAb 17D5 showed no clear effect when given alone or combined with mAb 7H4 (data not shown). This was perhaps due to the low incidence (3–12%) of 17D5-positive cells among line G7 (Table 3). Despite the significant reduction of clinical signs by mAb treatment, all animals still suffered from ataxia.
Active EAN.
Similarly, the treatment of EAN actively induced with the neuritogenic P2 peptide by mAb 8G8 (anti-Vß4) or 7H4 (not shown) led to a partial, but significant, reduction of clinical disease (Fig. 6A). mAb 17D5 (anti-Vß13) treatment had no clear effect on active EAN induced by P2 peptide (Fig. 6B). With anti-Vß13 therapy similar results were obtained when whole P2 protein was used for induction of EAN.
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Discussion |
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The topic of restricted TCR usage in disease is still significant to the potential use of TCR-directed, immunomodulatory strategies. In Lewis rat EAE, an animal model for multiple sclerosis, restricted TCR usage by autoreactive T cells was initially observed. T cells recognizing the major encephalitogenic epitope of MBP spanning amino acids 68–88 reportedly preferentially use the TCR Vß8.2 element, often in combination with the V
2 chain (Burns et al., 1989; Chluba et al., 1989; Gold et al., 1991; Sun et al., 1993) Subsequently, it was found that encephalitogenic, MBP-specific T cells do not exclusively use the TCR Vß8.2 (Sun et al., 1994), and that Vß8.2-positive cells were not essential for induction and progression of cell transfer EAE (Gold et al., 1995a). TCR usage by neuritogenic T cells is characterized to a far lesser degree than by encephalitogenic T cells. To further characterize the TCR repertoire of pathogenic T cells, specific for the peripheral nervous system-restricted protein P2, we generated the new mAbs 7H4 and 8G8. Both specifically recognized the TCR Vß4 chain as one major TCR element of neuritogenic T cells. Immunoprecipitation, flow cytometry, and activation studies clearly proved the specific binding of the TCR by the new mAbs 7H4 and 8G8. Characterization of the TCR V-chain specificity by molecular biological studies unequivocally revealed the recognition of the TCR Vß4 chain. In addition, TCR Vß13 was used by neuritogenic T-cell lines.
The neuritogenicity of TCR Vß4- or Vß13-chain-positive T cells was underlined by transfer experiments of mAb 7H4 (anti-Vß4)-positive sorted G7 line cells as well as mAbs 17D5 and 18B1 (anti-Vß13)-positive sorted P2.6 line cells, and by preventive treatment with the respective mAbs. TCR Vß4/Vß13-positive T cells were also enriched in lymph nodes from rats immunized with rhP2 or P2 peptide (amino acids 53–78), and a further positive selection of these TCR ß occurred after an antigen-driven selection cycle in culture.
Analysis of TCR Vß4–CDR3 region sequences amplified from P2-specific T-cell lines showed that all neuritogenic TCR Vß4-positive T-cell lines expressed almost identical VDJ-regions (amino acid motif QPGL) and it also revealed that the different staining pattern of Vß4-positive T-cell line TKtsA6 by mAbs 7H4 and 8G8 was not due to different VDJ-region recognition by these mAb. The TKtsA6 CDR3 sequence was identical to the CDR3 regions of other Vß4-positive P2 cells recognized by both mAbs. Moreover, staining of <1% of naive T lymph node cells favours an idiotypic binding of a conformational epitope located on the Vß4- and the associated V11 chain by mAbs 7H4 and 8G8. Interestingly, TCR V11 chain was not only associated with the Vß4 chain, as observed in previous studies from our group (F. X. Weilbach and G. Giegerich, personal communication), but was also coexpressed with Vß13. Similar to the predominant usage of the V2 chain by encephalitogenic T-cell lines in the Lewis rat (Gold, 1994; Gold et al., 1995a), the recognition of P2 by neuritogenic T-cell lines may be predominantly associated with usage of the V11 chain, regardless of the associated Vß chain.
In vitro, TCR Vß4- or Vß13 chain-positive cell populations only recognized the neuritogenic epitope (amino acids 53–78) of the P2 protein. This indicates that the recognition of the neuritogenic epitope is not exclusively determined by the Vß4-TCR. Although T-cell lines activated by the neuritogenic epitope showed a strong homology within their VDJ-regions of Vß4 TCR region (see above), the whole TCR repertoire of unsorted T cell lines activated by this peptide is heterogeneous. Data from immunoflow cytometry performed on a panel of P2-specific T-cell lines indicated that at least three different TCR chains were used by neuritogenic T-cell lines, since no dominant Vß4- or Vß13-positive subpopulation was detected in the pathogenic T-cell lines G5 and P2.76. RNase protection assays indicated a preferential usage of TCR Vß2/17/19 chains by T-cell line G5, in agreement with previous reports on diverse TCR usage by EAN T cells (Jung et al., 1993) and by T cells in inflamed sciatic nerve tissue (Weilbach et al., 1997).
Heterogeneity of TCR usage was also demonstrated in EAN actively induced by immunization with the neuritogenic peptide. Their preventive therapy with mAbs directed against TCR Vß4-region led to a significant, but partial, reduction of clinical disease. This, again, corroborates the notion that, although Vß4 might be the dominant recognition element, TCR usage in EAN is much more heterogeneous than previously assumed (Clark et al., 1992). Furthermore, the present results in mAb-treated peptide-induced EAN confirm our former suggestion that the failure of T-cell vaccination to induce resistance to EAN may rely on TCR-heterogeneity among neuritogenic T cells (Jung et al., 1993).
Thus, the diversity of TCR usage even for a single neuritogen in an inbred strain discourages hopes about the therapeutic potential of vaccination strategies or V-region-specific antibodies. Rather antigen-specific approaches using altered peptide ligands (Nicholson et al., 1995; Vergelli et al., 1997) or antigen-induced apoptosis of pathogenic T cells (Liblau et al., 1997; Weishaupt et al., 1997) may turn out to be more promising.
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and Cß primers
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Acknowledgments |
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We wish to thank Professor Dr K. V. Toyka and Professor Dr T. Hünig, Würzburg, for critical and stimulating comments and Professor Dr H.-P. Hartung, Graz, for help in initiating these studies. We also wish to thank Professor R. A. C. Hughes, London, for many discussions on disease mechanisms in EAN and GBS. We further thank Professor Dr D. Palm for synthesizing the P2 peptide, and Mrs A. Bunz and A. Hanold for skilful technical assistance. This study was supported by the Bundesministerium für Forschung und Technik (BMBF 01KS9603), the Gemeinnützige Hertie Stiftung (GHS 2/307/94) and the Dr med. Arthur-Arnstein-Stiftung.
The sequences reported in this paper have been deposited in the GenBank data base [accession nos. AF054144 (G5), AF054145 (G7), AF054146 (P2.6), AF054147 (P2.48), AF054148 (P2.76), AF054149 (TKtsA6)].
* Present address: Department of Neurology, University of Regensburg, 93053, Regensburg, Germany
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Received October 15, 1998. Accepted November 13, 1998.
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