Autoreactive human T cell lines recognizing ribosomal protein L7

Johannes Donauer, Michael Wochner, Esther Witte, Hans-Hartmut Peter1, Michael Schlesier1 and Ulrich Krawinkel

Faculty of Biology, University of Konstanz, 74857 Konstanz, Germany
1 Department of Rheumatology and Clinical Immunology, University Hospital, Hugstetterstrasse 55, 79106 Freiburg, Germany

Correspondence to: J. Donauer, Department of Nephrology, University Hospital Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Sera of patients suffering from systemic lupus erythematosus (SLE) frequently contain oligoclonal IgG autoantibodies with high affinity for the ribosomal protein L7 (rpL7). The humoral autoimmune response to rpL7 apparently is driven by antigen and T cell dependent. In order to analyze the T cell response to rpL7 we cultured peripheral blood lymphocytes of healthy individuals and SLE patients in the presence of recombinant rpL7. After 10 days, the cytokine response to re-stimulation with rpL7 was examined using a spot-ELISA. Measuring IFN-{gamma} secretion, the T cells of two patients and four healthy donors showed a significant increase in the number of spots as compared to control cells. Secretion of IL-4 or IL-10 was not detected. From the antigen-stimulated primary cultures we established by limiting dilution cloning six rpL7-reactive, IFN-{gamma}-secreting T cell lines which show a CD3+CD4+CD8 phenotype. One line additionally was shown to be positive for HLA-DR and CD45R0, but negative for CD27 and CD31. The cell lines carry {alpha}ß TCR chains which differ from each other in sequence and specificity. rpL7 fragments rich in basic amino acids could be identified as epitopes recognized by the TCR of three cell lines. Recognition of rpL7 is HLA-DR6 restricted or respectively HLA-DP restricted in the two cell lines analyzed.

Keywords: autoreactivity, human T cells, ribosomal protein L7


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A major finding in systemic lupus erythematosus (SLE) is the loss of tolerance to nuclear and cytoplasmic self proteins, resulting in the production of autoantibodies (1), among them autoantibodies against ribosomal protein L7 (rpL7) (2,3). rpL7 associates with the large subunit of ribosomes and carries in the N-terminal region a basic region leucine-zipper-like binding domain which interacts in dimeric form with cognate mRNAs, thereby inhibiting their translation (47). It interacts specifically with ribosomal protein S7, and bona fide transcriptional regulators such as zinc-finger protein ZNF7 (8) and steroid receptors (9). Constitutive expression of rpL7 induces apoptosis in T lymphoma cells (10). Briefly, rpL7 is a multifunctional ribosomal protein which seems to play a role in translational control and transcriptional regulation.

SLE patients frequently produce oligoclonal anti-L7 IgG autoantibodies which target with one high-affinity immunodominant epitope overlapping with the RNA-binding domain of rpL7 (7,11,12). In the active phase of SLE, additional polyclonal autoantibodies are generated which recognize with low-affinity minor epitopes of rpL7 (12). This indicates that the autoantigen or a cross-reactive agent is available in quantities sufficient to induce B lymphocytes with low-affinity receptors for rpL7.

As the anti-L7 autoantibody response is driven by antigen, oligoclonal, and since all anti-L7 autoantibodies belong to the IgG subclass, it seems reasonable to assume an involvement of Th cells. In analogy to studies of the cellular immune response against other autoantigens in systemic autoimmune diseases (13,14), we established rpL7-reactive T cell lines from a SLE patient and healthy individuals in order to analyze their functional properties.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lymphocyte isolation and culture conditions
Peripheral blood mononuclear cells (PBMC) were obtained from 29 patients suffering from SLE according to the revised ACR diagnosis criteria (15). Blood cells of 30 healthy donors with no detectable serum antibodies against rpL7 served as controls. Mononuclear cells were isolated by Ficoll (Biochrom, Berlin, Germany) density gradient centrifugation and were cultured in RPMI 1640 containing bicarbonate (2 g/l), glutamine (2 mM) and HEPES buffer (25 mM) (Biochrom). The medium was further supplemented with 10% FCS, 2.5% autologous human serum or heat-inactivated human serum and antibiotics. After 6 days of incubation at 37°C, 5% CO2, 100% humidity, cells were stimulated with 10 µg/ml human rpL7 coupled to glutathione-S-transferase (GST–L7) (4), and cultured for additional 10 days in the presence of recombinant human IL-2 (Boehringer, Mannheim, Germany) at 20 U/ml.

Lymphocyte proliferation assay
Autologous peripheral blood lymphocytes (PBL) (4x105) were cultured in 96-well plates in order to obtain adherent antigen-presenting cells (APC). After 2 h, the wells were washed, cloned T cells were added to a density of 2x105 cells/well and the GST–L7 concentration was adjusted to 10 µg/ml. After another 8 h, 2x104 cells were transferred into new 96-well plates and cultured for additional 42 h. Their proliferative response was measured using a BrdU-labeling and detection kit (Boehringer). The amount of BrdU incorporated into newly synthesized DNA was measured with an ELISA reader using ABTS (Boehringer) as substrate.

Limiting dilution cloning
T cells responding to GST–L7 but not to GST alone were tested in a lymphocyte proliferation assay and then cloned by limiting dilution. Ninety-six-well U-shaped microtiter plates (Nunc, Roskilde, Denmark) were coated overnight with monoclonal goat anti-mouse IgG antibody (2,5 µg/ml; Dianova, Hamburg, Germany) in PBS. Plates were rinsed and further incubated for 2 h at 10 ng antibody/ml with mouse anti-human CD3 antibody (BMA030; Behring, Marburg, Germany) and mouse anti-human CD28 antibody (CLB, Amsterdam, Netherlands). After washing, cells were added at a mean density of 0.3 cells/well in RPMI 1640, 10% FCS, together with irradiated (30 Gy from a 60Co source) allogeneic PBL (1x105 /well). After 2 weeks, wells showing cell growth were analyzed for their proliferative response and cytokine release upon re-stimulation with GST–L7. Positive cultures were further expanded and kept in long-term culture by two weekly re-stimulations with irradiated allogeneic PBL, anti-CD3 mAb (10 ng/ml) and recombinant IL-2 (20 U/ml) in complete medium.

Determination of cytokine release
The release of IFN-{gamma}, IL-2, IL-4, IL-10 and tumor necrosis factor (TNF)-{alpha} was measured after antigen stimulation by a spot-ELISA (16) for the bulk cultures, and by a conventional ELISA for the cloned T cell lines.

For the spot-ELISA, 24-well cell culture plates (Greiner, Frickenhausen, Germany) were coated with a monoclonal anti-human cytokine antibody in coating buffer (Na2CO31.59 g/l, NaHCO3 2.93 g/l, NaN3 0.2g/l, pH 9.6). Plates were incubated overnight at 4°C. After washing, non-specific binding was blocked with PBS containing 10% FCS (Biochrom) for 1 h at 37°C and finally the plates were washed again with PBS. Cells to be analyzed were pre-cultured together with autologous APC and GST–L7. After 6–8 h, 2x105 cells were transferred to each well, and further incubated for 36 h for the analysis of IFN-{gamma} and TNF-{alpha} release, and respectively 18 h for the analysis of IL-2, IL-4 and IL-10 release. Plates were washed again with PBS/0.5% Tween 20 (PBS-T) and the respective secondary, biotinylated mouse anti-human cytokine antibody was added in PBS-T/1% BSA at a dilution recommended by the supplier. Plates were incubated for 1 h at 37°C and washed again. Finally, 1:2000 diluted streptavidin–alkaline phosphatase (Dianova, Hamburg, Germany) was added to the wells, and plates were incubated for another 2 h at 37°C. After washing, staining of the spots was performed using 5-bromo-4-chloro-3-indoyl phosphate (Sigma, Deisenhofen, Germany) as a substrate. After 1 h the number of spots was counted using a stereomicroscope.

For the conventional cytokine ELISA, cells were firstly incubated for 6–8 h at 2x105/well in 96-well plates, together with antigen and APC as described above, followed by a second incubation period at 2x104 cells/well. Then 100 µl of culture supernatant was harvested after 36 h for IFN-{gamma} and TNF-{alpha} detection, or respectively 18 h for IL-4 and IL-10 detection, or 12 h for IL-2 detection. Ninety-six-well plates (Greiner, Frickenhausen, Germany) were coated overnight with primary anti-cytokine antibodies in coating buffer at 4°C. After washing, the wells were blocked with PBS/1% BSA/0.1% NaN3 for 1 h at 37°C and washed again with PBS-T. Then 100 µl culture supernatant was added and plates were incubated at room temperature for 2 h. The secondary, biotinylated anti-cytokine antibody was added after another washing step and plates were again incubated for 1 h. Finally, streptavidin-conjugated horseradish peroxidase (Dianova, Hamburg, Germany) was added to the wells at a dilution of 1:2000. After washing 4 times, 100 µl p-nitrophenylphosphate solution (Sigma, Deisenhofen, Germany) was added and the absorbance was determined at 405 nm wavelength using a microplate reader.

For plate coating, the following monoclonal mouse anti-human cytokine antibodies were used in biotinylated and unbiotinylated form: anti-human IFN-{gamma} (M700A, M701; Biozol, Eiching, Germany), anti-human TNF-{alpha}, anti-human IL-4, anti-human IL-10 and anti-human IL-2 (all from Dianova, Hamburg, Germany).

MHC class II restriction analysis
T cell clones were stimulated with GST–L7 as described above in the presence of diluted inhibitory mAb against HLA-DR (L243; a gift from I. Melchers, University Freiburg, Germany) and HLA-DP (B7/21; Becton Dickinson, Heidelberg, Germany). As controls, isotype-matched antibodies with irrelevant specificities were used (Sigma, Deisenhofen, Germany). To characterize the restriction to a particular MHC class II subtype, proliferation experiments were performed using peripheral blood monocytes of HLA-typed donors as APC (17).

Purification of recombinant antigens
The expression and purification of rpL7 and fragments of rpL7 fused to GST of Schistosoma japonicum was performed as previously described (4). Protein concentration and purity of preparations were verified by Coomassie blue staining of SDS–polyacrylamide gels and Bradford assays. GST as a control protein was expressed and purified under the conditions used for GST fusion proteins. Bacterial strains expressing GST, GST–L7 and GST–L7 fragments (11) were kindly provided by A. von Mikecz and P. Hemmerich (University of Konstanz, Germany).

Phenotyping of T cell clones
T cells (1x106) of each clone were analyzed with a FACStar Plus using the PC Lysys II software (both Becton Dickinson, Heidelberg, Germany). Staining of cell surface antigens was performed with the following FITC- or phycoerythrin-labeled monoclonal mouse antibodies: anti-human CD3, anti-human CD4, anti-human CD8, anti-human TCR pan {alpha}ß, anti-human CD45R0, anti-human CD27, anti-human CD31 and anti-HLA-DR (all from Dianova).

Isolation of RNA, RT-PCR and anchor-ligated PCR for amplification of TCR chains
Total RNA was isolated from T cell clones by binding to silica gel-based membranes (RNeasy spin colums; Quiagen, Hilden, Germany). RT-PCR, anchor ligation and PCR were performed as described (18) using the 5'-RACE amplification protocol (Stratagene, Heidelberg, Germany). The following oligonucleotides were used as anchor oligonucleotide and PCR-primers: 5' anchor-oligonucleotide: 3'-GGAGACTTCCAAGGTCTTAGCTATCACTTAAGCAC-5', 3' C{alpha}-primer: 5'-CGGGATCCTCAGCTACACGGCAGGGT-3', 3' nested C{alpha}-primer: 5'-CGGGATCCGCAGACAGACTTGTCACTG-3', 3' Cß-primer: 5'-CGGGATCCGCTTCTGATGGCTCAAACAC-3', 3' nested Cß-primer: 5'-CGGGATCCACCTTGTTCAGGTCCTCTAC-3', anchor primer: 5'-CTGGTTCGGCCACCTCTGAAGGTTCCAGAATCGATAG-3'.

The amplified PCR products were electrophoresed on a 1.5% agarose gel. After staining with ethidium bromide, bands of 400–600 bp were isolated and the DNA was purified using the Jetsorb DNA extraction method (Genomed, Bad Oeynhausen, Germany).

Cloning and sequencing of amplified TCR gene products
Molecular cloning was performed according to standard protocols (19). Briefly, PCR products were digested with restriction enzymes EcoRI and BamHI, and re-purified on a 1.5% low-melting agarose gel. Ligation of restriction fragments into the appropriately cut pSK Bluescript cloning vector (Stratagene, La Jolla, CA) was performed in gel slices. Recombinant plasmids were amplified in Escherichia coli XL-2 Blue (Stratagene) and purified using the alkaline lysis method according to standard protocols (20). DNA sequencing was done employing the Sequenase 2.0 reagent kit (US Biochemicals, Cleveland, OH). Sequences of some amplified TCR chains were determined directly with the Sequenase PCR product sequencing kit (US Biochemicals, Cleveland, OH).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Establishment of rpL7-specific T cell lines
In order to establish rpL7-specific T cell lines, PBMC of healthy donors and SLE patients were cultured for 10 days in the presence of GST–L7 and IL-2. To reduce unspecific proliferation, cells were pre-cultured without antigen and IL-2 for a resting period of 7 days. Finally, their proliferative response and their cytokine production upon stimulation with GST–L7 were analyzed. Control stimulations were done with GST alone. Freshly prepared autologous mononuclear cells or, in the case of cells from donor KAS, an autologous Epstein–Barr virus-transformed B cell line, served as APC. Two of 29 cultures containing cells from SLE patients and five of 30 cultures with cells from healthy donors proliferated, showing 10–231 IFN-{gamma}-specific spots per 105 cells in the spot-ELISA upon stimulation by GST–L7. Between 3- and 12-fold less cytokine-producing cells were obtained upon GST stimulation in a given experiment (Fig. 1Go). The cells in the positive cultures were subjected to limiting dilution cloning. A total of six L7-reactive T cell lines were obtained, one from SLE patient HGR, and five from the healthy females KAS, UBK and EWE (Fig. 2Go).



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Fig. 1. Cytokine release and proliferation of primary T cell lines upon stimulation with rpL7. (A) Spot-ELISA to measure IFN-{gamma} production. The stimulation index is calculated from the number spots upon GST–L7 stimulation divided by the number of spots upon stimulation with GST alone (GST–L7 spots, 10–231/105 seeded cells; GST spots, 4–40/105 seeded cells). (B) Proliferation of primary T cell lines as measured by BrdU incorporation upon stimulation with GST–L7 or GST alone. The mean of three measurements and SD are documented here and in the following figures.

 


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Fig. 2. Cytokine-release and proliferation of cloned rpL7-reactive T cell lines. Response of cloned T cell lines to stimulation with GST–L7 and GST alone, measured as (A) IFN-{gamma} release and (B) BrdU incorporation.

 
Cytokine profile and surface markers of cloned T cell lines
After expansion of cloned T cells, their proliferative response and cytokine release upon antigen stimulation were determined. All six T cell lines showed in the FACScan analysis a pattern of surface markers characteristic of Th cells in that they were CD3+CD4+CD8 and TCR{alpha}ß+. Line KAS was further analyzed. It was positive for CD45R0 and HLA-DR, and negative for CD27 and CD31 (data not shown).

The rpL7-reactive T cell lines produced IFN-{gamma} in response to antigen stimulation, but no secretion of IL-4 and IL-10 could be detected. Clones KAS and UBK1 in addition to IFN-{gamma} also produced of IL-2 and TNF-{alpha} (data not shown).

TCR-specificity of rpL7-reactive T cell clones
To define the epitopes on rpL7 recognized by the T cell lines, proliferative responses and IFN-{gamma} production were measured upon stimulation with nine overlapping GST-fused fragments of rpL7 (P1–P9) (11) (Fig. 3Go). Line KAS recognized fragments P2, P3 and P4. The common sequence of these overlapping fragments, i.e. the sequence between positions 41 and 64, thus should carry the epitope recognized by the TCR of line KAS. Line UBK1 was stimulated by fragment P1 and weakly but significantly by fragment P2, but did not respond to the overlapping fragments P3 and P4. The epitope recognized by the TCR of UBK1 therefore should map to the sequence between positions 1 and 26. Line UBK2 was stimulated by fragment P7, and did not respond to the overlapping fragments P6 and P8. The rpL7 epitope that stimulates clone UBK2 consequently should lie between positions 118 and 167, or, in the case that the short overlapping regions of fragments P6–P8 are not involved, between positions 128–157. Lines UBK3, EWE and HGR did not respond significantly to the L7 fragments used in this study. The response pattern of L7-reactive lines to rpL7 fragments obtained by measuring BrdU incorporation was identical to the one obtained by measuring their cytokine release (data not shown).



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Fig. 3. Epitopes recognized by rpL7-reactive T cell lines. IFN-{gamma} release of rpL7-reactive T cell lines upon stimulation with GST-fused rpL7 fragments P1–P9, which represent the following rpL7 amino acid positions: 1–56 (P1), 12–64 (P2), 27–72 (P3), 41–80 (P4), 54–88 (P5), 78–127 (P6), 118–167 (P7), 158–207 (P8) and 198–248 (P9).

 
MHC class II restriction of rpL7-reactive T cell lines
In order to confirm that our T cell lines recognize rpL7 in a MHC class II-restricted fashion, stimulation by the antigen of lines KAS and UBK1 was determined in the presence of inhibitory monoclonal anti-HLA class II antibodies (Fig. 4Go). Stimulation of clone KAS was inhibited by the HLA-DP-specific antibody B7/21, whereas the isotype-matched control antibody failed to do so (Fig. 4aGo). Clone UBK1 showed reduced IFN-{gamma} production in the presence of the HLA-DR-specific antibody L243, but was not inhibited by the isotype-matched control antibody (Fig. 4bGo). Again, the corresponding experiments using BrdU incorporation showed identical results (data not shown).



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Fig. 4. MHC class II restriction of rpL7 recognition by T cell lines (A) KAS and (B) UBK1. rpL7-induced IFN-{gamma} production was measured in the presence of the mAb B7/21 against HLA-DP and L243 against HLA-DR. As controls, isotype-matched antibodies with irrelevant specificities were used.

 
To determine the MHC class II restriction of clones KAS and UBK1 in more detail, their IFN-{gamma} release upon rpL7 presentation by peripheral blood monocytes of a selected panel of HLA-typed donors was analyzed. In the case of line UBK1, the response to rpL7 was restricted to HLA-DR6. Line KAS responded to APC carrying HLA-DPw2 and -Dpw4 (data not shown). The exact HLA-DP subtype recognized by the TCR of line KAS could not be further resolved because homozygous donors were unavailable.

TCR usage of rpL7-reactive T cell lines
Finally, the nucleotide sequences of TCR {alpha} and ß chain gene segments expressed on rpL7-reactive Th1 cell lines were determined. The deduced amino acid sequences of V{alpha}J{alpha} and VßDßJß junctions are heterogeneous, although UBK1, 2 and 3 share the V{alpha}21 segment, and UBK1 and HGR share Vß22.1 (Table 1Go).


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Table 1. Junctional amino acid sequences of TCR of rpL7-reactive T cell lines
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We isolated six rpL7-reactive T cell clones, one from a SLE patient (clone HGR) and five from healthy donors (clones KAS, UBK1–3 and EWE). The lower cloning efficiency of rpL7-reactive T lymphocytes in PBMC from SLE patients as compared to the one in blood cells from healthy donors most likely is due to the treatment of the patients with cytostatic drugs before our study was initiated. T cell clones KAS and UBK1 could be further expanded, whereas the other T cell clones became increasingly labile and the proliferative response to stimulation with rpL7 declined. Replicative senescence has been suggested to be the reason for this behavior (21).

It is a general experience that autoreactive human T cells providing help for autoantibody-producing B cells (e.g. autoantibodies to nuclear and cytoplasmic autoantigens) are difficult to establish in vitro. Their precursor frequency is low even in patients with high autoantibody titers to the respective autoantigen (13,14). One reason for this observation may be sequestering of autoreactive Th cells in lymphoid organs. Another explanation, however, is the fact that nuclear and cytoplasmic autoantigens are usually taken up by autoreactive B cells not as single proteins but rather as large protein–nucleic acid complexes (e.g. nucleosomes, small nuclear ribonucleoprotein particles and ribosomes) (22). This increases the probability that the autoantigenic peptides presented by autoreactive B cells to their cognate Th cells do not derive from the autoantigen itself but from other proteins contained in the complex. It is therefore possible that our autoreactive T cell clones are not the physiological partners of anti-L7 autoantibody-producing cells in vivo.

The rpL7-reactive lines presented in this study show a CD3+CD4+TCR{alpha}ß+ phenotype. Line KAS in addition was shown to be positive for HLA-DR and CD45R0, but negative for CD27 and CD31. This pattern of surface markers on line KAS is believed to define mature Th2 cells (23,24) and we assume that the other cloned lines have the same phenotype because all cloned lines described here were isolated under the same conditions. However, upon stimulation with antigen all cell lines produced cytokines which at least in mice are characteristic of Th1 cells. The latter generally do not provide help for humoral immune responses. Moreover, in humans the functional meaning of Th1/Th2 phenotypes is less clear (25). CD45R0+HLA-DR+CD27CD31 cells as represented by line KAS may provide help in an anti-L7 autoantibody response. Most interestingly, donor KAS developed an anti-L7 autoantibody titer during the course of this study. Furthermore, the involvement of Th1-like cells in a humoral autoimmune response has been described: the expression of pathogenic anti-DNA IgG isotypes in murine SLE models, for instance, is dependent on Th1-derived cytokines, especially on IFN-{gamma} (26,27).

IL-4- and IL-10-producing rpL7-reactive T cells were either not stimulated under the culture conditions employed or existed in the original PBMC sample at a frequency that was too low to be detected. We also cannot exclude that cells producing very low amounts of IL-4 and IL-10 escaped detection. It has been suggested that in vitro culturing of T lymphocytes preferentially induces a Th1-like response (28). We do not believe that this is the case in our experimental system since IFN-{gamma} secretion was already detected in freshly isolated PBMC.

The specificity of the TCR on KAS, UBK1 and UBK2 has been determined. Lines KAS and UBK1 recognize epitopes that are rich in basic amino acids and have maximal sizes of 23 and respectively 26 amino acids. The length requirements of peptide presentation on MHC class II molecules is well fulfilled by these epitopes. Highly charged motives presumably assuming an {alpha}-helical conformation often seem to be recognized by autoreactive T cells in systemic autoimmune diseases (13,2931). The epitope recognized by the TCR of line UBK2 does not show particular sequence motifs. Three rpL7-reactive lines did not respond to the L7 fragments employed in this study. They may recognize peptides that cannot be processed from fragments P1–P9.

It has been claimed that an adjacent rather than an overlapping position of epitopes is mandatory for T cell help to autoreactive B cells (32). This study and others (33), however, demonstrate that TCR and BCR epitopes can fully overlap. The TCR epitopes recognized by lines UBK1 and KAS overlap with the immunodominant conformational epitope recognized on rpL7 by autoantibodies in SLE (12), and the TCR epitope of line KAS in addition overlaps with a minor autoantibody epitope (12) which happens to be the RNA binding domain of rpL7 (7) (Fig. 5Go). It is known that autoantibodies often are directed against the functional region of a given autoantigen (32), presumably because of selection of BCR recognizing solvent-exposed regions. In addition, and this should apply to BCR as well as to TCR, there may be an evolutionary selection for receptors specific for the functional regions of antigens because such regions are conserved.



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Fig. 5. Amino acid sequence of epitopes recognized by rpL7-reactive T cell lines. Epitopes of rpL7 recognized by lines UBK1, KAS and UBK2 are overlined. The immunodominant conformational autoantibody epitope (major ab-epitope) comprising two linear motives separated by 22 amino acids, a minor autoantibody epitope (minor ab-epitope) and the RNA-binding domain of rpL7 (RBD) are underlined. Amino acid positions of rpL7 are indicated. Basic amino acids are typed in bold letters.

 
MHC class II-restricted antigen recognition was verified for two rpL7-reactive lines. The antigen could be presented to line UBK1 on two subtypes of HLA-DR6 (DRB*1301 and DRB*1305), whereas line KAS responded to rpL7 in a HLA-DP restricted manner.

We also examined the TCR V-region structure of rpL7-reactive T cell lines. As these lines differ from each other in TCR specificity and MHC restriction, it is not unexpected that they express TCR which differ from each other in their junctional sequences, albeit their V gene usage is not random. Three of the L7-reactive lines, i.e. derived from donor UBK, use the V{alpha}21 gene but different Vß genes, whereas lines UBK1 and HGR share the Vß22.1 gene while differing from each other in their V{alpha} gene usage. During the thymic education process, selection for or against particular V gene segments occurs depending on the HLA type of the individual (3537). In the case of UBK this may explain the preferential usage of V{alpha}21.

Taken together, our findings on rpL7-autoreactive T cells show in accordance with studies on T cells specific for myelin basic protein (38,39) and U1 small ribonuclear protein (13,14) that (i) autoantigen-specific T cell lines derived from individual donors are heterogeneous in that they differ from each other in antigen fine specificity, TCR usage and HLA restriction, and that (ii) potentially autoagressive T cells are present within the normal immune repertoire. The precursor frequency of rpL7-autoreactive Th cells is low and their establishment and maintenance as cloned autoantigen-specific lines is delicate. Currently it is not clear whether our cloned Th cell lines represent the physiological cognate partners of anti-L7 autoantibody-producing cells in vivo.


    Acknowledgments
 
The authors wish to thank A. von Mikecz and P. Hemmerich for the gift of bacteria producing recombinant rpL7 fragments, UBK for frequent donations of blood, and K. Aviszus and S. Mauch for helpful discussion and advice. The technical support during T cell cloning by Ruth Dräger is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft through grant C7 to U. K. in Sonderforschungsbereich 156 and a postdoctoral fellowship no. Do 507/1-1 to J. D.


    Abbreviations
 
APCantigen-presenting cell
GSTglutathione-S-transferase
PBLperipheral blood lymphocytes
PBMCperipheral blood mononuclear cells
SLEsystemic lupus erythematosus
TNFtumor necrosis factor

    Notes
 
Transmitting editor: A. Radbruch

Received 8 April 1998, accepted 28 September 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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