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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: autoreactivity, human T cells, ribosomal protein L7
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 GSTL7 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 GSTL7 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 GSTL7. 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-, IL-2, IL-4, IL-10 and tumor necrosis factor (TNF)-
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 GSTL7. After 68 h, 2x105 cells were transferred to each well, and further incubated for 36 h for the analysis of IFN- and TNF-
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 streptavidinalkaline 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 68 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- and TNF-
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- (M700A, M701; Biozol, Eiching, Germany), anti-human TNF-
, 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 GSTL7 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 SDSpolyacrylamide 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, GSTL7 and GSTL7 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 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-primer: 5'-CGGGATCCTCAGCTACACGGCAGGGT-3', 3' nested C
-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 400600 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The rpL7-reactive T cell lines produced IFN- 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-
also produced of IL-2 and TNF-
(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- production were measured upon stimulation with nine overlapping GST-fused fragments of rpL7 (P1P9) (11) (Fig. 3
). 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 P6P8 are not involved, between positions 128157. 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).
|
|
TCR usage of rpL7-reactive T cell lines
Finally, the nucleotide sequences of TCR and ß chain gene segments expressed on rpL7-reactive Th1 cell lines were determined. The deduced amino acid sequences of V
J
and VßDßJß junctions are heterogeneous, although UBK1, 2 and 3 share the V
21 segment, and UBK1 and HGR share Vß22.1 (Table 1
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 proteinnucleic 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+TCRß+ 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-
(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- 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 -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 P1P9.
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. 5). 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.
|
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 V21 gene but different Vß genes, whereas lines UBK1 and HGR share the Vß22.1 gene while differing from each other in their V
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
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 |
---|
![]() |
Abbreviations |
---|
APC | antigen-presenting cell |
GST | glutathione-S-transferase |
PBL | peripheral blood lymphocytes |
PBMC | peripheral blood mononuclear cells |
SLE | systemic lupus erythematosus |
TNF | tumor necrosis factor |
![]() |
Notes |
---|
Received 8 April 1998, accepted 28 September 1998.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|