Abstract
Peptides based on the complementarity-determining region 1 (CDR1) and CDR3 of an anti-DNA monoclonal antibody (mAb) carrying the 16/6 idiotype (Id) were shown to induce experimental systemic lupus erythematosus (SLE) in susceptible mouse strains. In the present study, T-cell lines specific to the pCDR1 and pCDR3 peptides were established in BALB/c and in SJL mice, respectively. The T-cell lines were characterized and analysed for their pathogenicity upon administration to syngeneic mouse strains. Both T-cell lines expressed the αβ T-cell receptor (TCR) and the CD4+ CD8– phenotype. Additionally, both cell lines secreted interleukin (IL)-4 and IL-10 upon stimulation with their specific peptide, thus belonged to the T helper 2 (Th2) subset. Upon immunization, the pCDR3-specific T-cell line induced experimental SLE in SJL mice. The animals produced high levels of autoimmune anti-DNA and antinuclear protein antibodies, as well as anti-16/6 Id antibodies (Abs). Furthermore, the mice developed clinical manifestations, including leukopenia, proteinuria and accumulation of immune complex deposits in their kidneys. The pCDR1-specific T-cell line failed to induce SLE when injected into BALB/c mice. It is thus suggested that pCDR3 is an immunodominant epitope in experimental SLE and that pCDR3-specific T cells initiate autoimmunity, leading to SLE, probably via epitope spreading.
Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease, which is characterized by the production of autoantibodies to nuclear proteins and nucleic acids, accompanied with clinical manifestations (e.g. leukopenia, thrombocytopenia and kidney damage.1 Induction of experimental SLE in mice has been previously reported in our laboratory, using the human or murine monoclonal anti-DNA antibodies that bear the common idiotype (Id) 16/6,2 or the murine anti-DNA 16/6 Id monoclonal antibody (mAb), 5G12.3 The immunized mice demonstrated high levels of autoantibodies, including anti-DNA and antinuclear protein antibodies, as well as anti-idiotypic antibodies belonging to the 16/6 Id network.
Much evidence supports the central role of T cells in the pathogenesis and development of SLE. BALB/c nude (BALB/c nu/nu) mice that lack T-helper function were resistant to induction of a lupus-like condition.4,5 Depletion of T cells was shown to prevent the development of SLE in SLE-prone mice.6–8 T-cell lines specific for the 16/6 Id were demonstrated to be capable of inducing experimental SLE in syngeneic mice. Furthermore, a 16/6 Id specific syngeneic (H-2b) T-cell line was capable of inducing experimental SLE9 in the resistant mouse strain, C57BL/6.2
Experimental SLE was found to share features with the (NZB × NZW)F1 spontaneous SLE model. Thus, the sequence of the variable regions coding for the heavy and light chains of anti-DNA mAbs isolated from mice afflicted with experimental SLE showed high homology with the variable regions of anti-DNA mAb isolated from (NZB × NZW)F1 mice.10
Based on this homology, we synthesized two peptides based on the sequence of the complementarity-determining regions (CDR) of the pathogenic murine monoclonal anti-DNA, 16/6 Id+ antibody (designated 5G12). The CDR-based peptides, namely pCDR1 and pCDR3, were shown to be immunodominant epitopes in BALB/c and SJL mouse strains, respectively, and induced a mild experimental SLE in the latter.11 In the present study, we established T-cell lines specific for the pCDR1- and pCDR3-based peptides of the high-responder mouse strains BALB/c (H-2d) and SJL (H-2), respectively. The T-cell lines were characterized for their peptide-specific proliferation, cytokine profile and pathogenicity upon inoculation into naive syngeneic mice.
Materials and methods
Mice
SJL/J inbred female mice were obtained from the Jackson Laboratory (Bar Harbor, ME). BALB/c inbred female mice were obtained from Olac (Oxon, UK). Mice were used at the age of 8–10 weeks.
Synthetic peptides
The CDR-based peptides of the murine anti-DNA anti-body, 5G12, were used. The CDR1-based peptide T GYYMQWVKQSPEKSLEWIG (pDCR1) and the CDR3-based peptide YYCAR FLWEPYAMDYWGQGS (pCDR3) (the CDRs are underlined) were prepared using an automated synthesizer (model 430A; Applied Biosystems, Weiterstadt, Germany) using the company’s protocols for t-butyloxycabonyl (t-BOC).12,13
mAbs
The human 16/6 Id anti-DNA mAb (immunoglobulin G1 [IgG1]/κ) was secreted by hybridoma cells grown in culture and the antibody was purified on a protein G–Sepharose column (Pharmacia, Uppsala, Sweden), according to the manufacturer’s instructions.
Immunizations
For the establishment of T-cell lines, BALB/c and SJL mice were injected intradermally (i.d.) in the hind footpads with 20 µg of pCDR1 or pCDR3, respectively, in complete Freund’s adjuvant (CFA) in a total volume of 100 µl. For the induction of experimental SLE, BALB/c mice were immunized with 2 µg of the human 16/6 Id mAb in CFA.
Establishment of antigen-specific T-cell lines
T-cell lines specific to peptide pCDR1 of BALB/c origin and to peptide pCDR3 of SJL origin, were established from popliteal lymph node (LN) cells of immunized mice (as detailed above), according to previously published methods.13 Cells were exposed to the stimulating peptide (15 µg/ml), presented by irradiated (3000 rads) syngeneic spleen cells, every 14 days.
Proliferative response of T-cell lines
Seven or 10 days after antigenic stimulation, cells of the T-cell line were tested for their specific proliferative responses. Cells (104/well) were cultured with 0·5 × 106 irradiated syngeneic spleen cells in the presence of different concentrations of the peptides. Cultures were established in flat-bottom microtitre plates in 200 µl of enriched medium containing 10% fetal calf serum (FCS). At the end of a 48-hr incubation period, 0·5 µCi of [3H]thymidine (5 Ci/mmole; Nuclear Research Center, Negev, Israel) was added. Sixteen hours later, cells were harvested and radioactivity counted by using a β-counter.14
Injection of T-cell lines
SJL mice were injected intravenously (i.v.) with 5 × 106 T cells from the pCDR3-specific line, and BALB/c mice were injected i.v. with 5 × 106 T cells from the pCDR1-specific line. Prior to inoculation, the T cells were exposed to their specific peptide for 3–4 days and then transferred to antigen-free medium containing 7·5% supernatant of concanavalin A (Con A)-stimulated splenocytes for 3–4 days. At this stage, mostly T cells are detectable in the culture. Prior to injection, cells were washed thoroughly and resuspended in phosphate-buffered saline (PBS). Mice were injected once (0·3 ml/animal). Mice were monitored and bled at 4-week intervals for a period of 6 months.
Fluorescence analysis of membrane T-cell markers
Direct staining was performed by incubating viable cells (1 × 106) of the T-cell lines at 4° in the dark with fluorescein isothiocyanate (FITC)-conjugated anti-αβ T-cell receptor (TCR), with FITC-conjugated different anti-TCR-Vβ chain antibodies, with FITC-conjugated anti-mouse CD4 and phycoerythrin (PE)-conjugated anti-mouse CD8 antibodies, or with the appropriate control isotype-matched antibodies (all antibodies were purchased from Pharmingen, San Diego, CA). Thereafter, the cells were washed twice with a cold PBS solution containing 0·05% sodium azide. Fluorescence of at least 5000 cells/sample was assessed by a FACSort cytometer and the data was analysed using the C ellquest software.
Detection of SLE-associated clinical and pathological manifestations
Proteinuria was measured by a semiquantitative method, using a Combistix kit (Ames Division, Bayer Diagnostics, Slough, UK). White blood cells (WBC) were counted after diluting the heparinized blood 10-fold in 1% acetic acid in distilled water (v/v). For immunohistology, frozen kidney cryostat sections (6-µm thick) were fixed and stained with FITC-conjugated goat antibodies to mouse IgG, γ-chain specific (Jackson Immunoresearch, West Groove, PA, USA).
Antibody detection assays
For the detection of specific antibodies in sera of immunized mice, enzyme-linked immunosorbent assay (ELISA) was carried out as described previously.3 Briefly, flat-bottom maxi-sorb plates (Nunc, Roskilde, Denmark) were coated with the appropriate antigens or peptides. For the detection of anti-DNA antibodies, plates were coated with 10 µg/ml of methylated bovine serum albumin (BSA) (Sigma, St Louis, MO). The plates were washed and coated with 10 µg/ml of denatured calf thymus DNA (Sigma). pCDR1 and pCDR3 were used for the detection of antipeptide antibodies at a concentration of 5 or 10 µg/ml. The human 16/6 Id+ antibody was used at (10 µg/ml) for detection of anti-16/6 Id antibodies. For antinuclear protein antibody detection, nuclear extract of HeLa cells10 (at 10 µg/ml) was used. After incubation with the antigen, plates were washed and blocked overnight with 10% milk, or 1% ovalbumin (Sigma), both in PBS, for the DNA assay. Diluted sera were then added for 2 hr, followed by incubation for 1 hr with 50 µl of 0·8 µg/ml of goat anti-mouse IgG (γ-chain Fc specific; Jackson ImmunoResearch). Analysis of specific isotypes of anti-DNA antibodies was carried out using peroxidase-conjugated anti-IgG1, anti-IgG2a and anti-IgG3 antibodies (Southern Biotechnology Associates, Birmingham, AL).
Stimulation of cells for cytokine production
Cells (1 × 106) of the pCDR1- or pCDR3-specific T-cell lines were taken 7 or 14 days after stimulation, washed and incubated for 24 hr at 37° in the presence of 5 × 106 irradiated (3000 rads) syngeneic splenocytes in fresh enriched RPMI medium supplemented with 10% FCS, in the presence or absence of either pCDR1 or pCDR3 peptides (50 µg/ml), or Con A (2·5 µg/ml). Supernatants were collected and stored at − 70° until used.
Detection of cytokines in supernatants of stimulated T cells
Interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10 and interferon-γ (IFN-γ) were quantified by using ELISA. Antibodies to the various cytokines (Pharmingen) were used according to the manufacturer’s instructions. Cytokines were detected using specific antibody pairs of capture antibody and biotinylated detection antibody, as described previously.15
Results
Establishment and characterization of pCDR-specific T-cell lines
Two T-cell lines were established and characterized. The first was specific to pCDR1 and was derived from LN cells of BALB/c mice. The second T-cell line was derived from LN cells of SJL mice and was specific to the pCDR3 peptide.
Figure 1 presents the results of typical proliferation assays of the T-cell lines. The BALB/c derived, pCDR1-specific T-cell line proliferated highly in response to pCDR1 and not to the control peptide (Fig. 1a). The SJL-derived, pCDR3-specific T-cell line proliferated highly in response to pCDR3 but not to pCDR1 (Fig. 1b).
Figure 1.
Proliferative responses of T-cell lines specific to complementarity-determining region (CDR)-based peptides (pCDR). Proliferative responses of the pCDR1-specific T-cell line of BALB/c origin (a), and of the pCDR3-specific T-cell line of SJL origin (b). (a) Proliferative response of the pCDR1-specific line was tested with different concentrations of pCDR1, with pCDR3 used as a control. (b) Proliferative response of the pCDR3-specific line was tested with different concentrations of pCDR3, with pCDR1 used as a control. Cells (104) of the T-cell lines were incubated for 48 hr in 96-well plates in the presence of irradiated syngeneic spleen cells (5 × 105) and various concentrations of the antigen. Thereafter, 0·5 µCi of [3H]thymidine was added to the cultures. Sixteen hours later, plates were harvested onto filter paper and radioactivity counted. Results are expressed as mean counts per minute (c.p.m.) of triplicate wells. Standard deviations did not exceed 10% of the mean c.p.m.
The T-cell lines were induced to secrete specific cytokines in response to stimulation with their specific peptides. Table 1 shows that in response to the pCDR1 peptide, the BALB/c-derived T-cell line secreted IL-4, IL-5 and IL-10, suggesting that this T-cell line is of the T helper 2 (Th2) cell subtype. As shown in Table 1, the SJL-derived pCDR3-specific T-cell line secreted large quantities of IL-4 and IL-10 in response to stimulation with pCDR3. Thus, based on its cytokine profile, the line is also of the Th2 subtype. It is noteworthy that the quantities of cytokines secreted by the pCDR3-specific T-cell line were much higher (by two logs for IL-10) than those secreted by the pCDR1-specific T-cell line.
Table 1.
Cytokine profile of the peptide complementarity-determining region (pCDR)-specific T-cell lines
| IL-2 (ng/ml) | IL-4 (ng/ml) | IL-5 (ng/ml) | IL-6 (ng/ml) | IL-10 (ng/ml) | IFN-γ (ng/ml) | |
|---|---|---|---|---|---|---|
| pCDR1-specific T-cell line (BALB/c) | ||||||
| Line | – | 0·1 | – | – | – | – |
| APCs | – | 0·25 | – | – | – | – |
| Line + APCs + pCDR1 | – | 3·45 | 3·2 | 1 | 1·5 | – |
| Line + Con A | – | 10 | 6·5 | 1·7 | 4 | – |
| pCDR3-specific T-cell line (SJL) | ||||||
| Line | – | – | ND | ND | 1·0 | – |
| APCs | – | – | ND | ND | – | – |
| Line + APCs + pCDR3 | – | 15 | ND | ND | 101·6 | – |
| Line + Con A | – | 27·2 | ND | ND | 204·8 | 0·32 |
Cells (106) of the T-cell lines were incubated with or without irradiated syngeneic splenocytes (5 × 106) and with or without 50 µg/ml of peptides or with concanavalin A (Con A; 2·5 µg/ml), for 24 hr in 24-well plates. Thereafter, plates were centrifuged and supernatants collected and stored at − 70° until used. Supernatants were tested for the different cytokines by using enzyme-linked immunosorbent assay (ELISA) as detailed in the Materials and methods.
APC, antigen-presenting cells; IFN-γ, interferon-γ; IL, interleukin.
Fluorescence analysis of membrane T-cell markers of the T-cell lines was carried out. As shown in Table 2, 99·8% of the cells of the BALB/c-derived pCDR1-specific T-cell line were αβ TCR positive, and 100% of the latter were CD4+ CD8–.Table 2 also presents results of the membrane marker analysis of the SJL-derived pCDR3-specific T-cell line. It was found that 99·2% of the cells possessed the αβ chains of TCR, and 95·04% of the cells were CD4+ CD8–.
Table 2.
T-cell membrane markers of peptide complementarity-determining region (pCDR)-specific T-cell lines
| pCDR1-specific T-cell line (% of positively stained cells) | pCDR3-specific T-cell line (% of positively stained cells) | |
|---|---|---|
| αβ TCR | 99·8 | 99·2 |
| CD4+/CD8– | 99·9 | 95·0 |
| TCR Vβ chain | ||
| Vβ2 | – | 35·8 |
| Vβ4 | 34 | 15·4 |
| Vβ8 | 30 | – |
| Vβ10 | – | – |
| Vβ14 | 26 | 7·6 |
| Vβ17 | – | 8·8 |
Cells of the two T-cell lines (106/sample) were taken 1 week after stimulation and were incubated, for 20 min on ice, with fluorescein isothiocyanate (FITC)-conjugated anti-mouse αβ T-cell receptor (TCR) antibody, with both FITC-conjugated anti-mouse CD4 and phycoerythrin (PE)-conjugated anti-mouse CD8 antibodies, or with different FITC-conjugated or PE conjugated anti-mouse TCR Vβ antibodies. After washing, the cells were analysed by using a fluorescence-activated cell sorter (FACS). Results are expressed as per cent of positively stained cells minus the control background fluorescence of unstained cells.
Analysis of the Vβ chain repertoire of the TCR of the cell lines is also summarized in Table 2. In the BALB/c-derived pCDR1-specific T-cell line, the three predominant Vβ chains used were Vβ4 (34%), Vβ8 (30%) and Vβ14 (26%). In the SJL-derived pCDR3-specific T-cell line the predominant Vβ chains were Vβ2 (35·8%), Vβ4 (15·4%), Vβ17 (8·8%) and Vβ14 (7·6%).
Pathogenicity of the T-cell lines
SJL/J mice (19 animals) and BALB/c mice (14 animals) were injected once i.v. with 5 × 106 cells/animal of pCDR3 or pCDR1, respectively. The T cells were injected 1 week after the in vitro activation with their stimulating peptides. Injected animals were followed monthly for the appearance of autoimmune-related antibodies and later for the appearance of clinical disease manifestations.
Figure 2 is representative of results obtained with sera taken from the injected SJL mice. As shown in Fig. 2(a), sera of SJL mice inoculated with the pCDR3-specific T-cell line did not contain any anti-pCDR3 activity. However, these sera had elevated titres of anti-DNA (Fig. 2b) and also of antinuclear protein antibodies (Fig. 2c), suggesting the induction of autoimmune processes following inoculation of the T-cell line. In addition, sera of these animals contained high titres of anti-16/6 Id antibodies (Fig. 2d).
Figure 2.
Antibody levels in sera of SJL mice inoculated with the pCDR3-specific T-cell line. Sera of individual line-inoculated SJL mice were taken 2 months after injection and from normal, age-matched, uninjected SJL mice. The sera were tested for anti-pCDR3 (a), anti-single-stranded DNA (b), antinuclear protein (c), and anti-16/6 idiotype (Id) (d) antibody titres by using enzyme-linked immunosorbent assay (ELISA). Results are expressed as mean absorbance (A) ± SE of each group.
Analysis of the isotype distribution of the anti-DNA antibodies showed that IgG1 was the dominant isotype appearing following inoculation with the pCDR3-reactive T-cell line (results not shown). No antibodies of either isotype IgG2a or IgG3 could be detected.
Sera of BALB/c animals injected with the pCDR1-specific T-cell line did not contain any antipeptide (pCDR1 in this case) antibodies (data not shown), as was observed with SJL mice inoculated with the pCDR3-specific T-cell line. However, in contrast to the situation with SJL mice, BALB/c animals did not develop any autoimmune anti-DNA antibodies (Fig. 3a) or antinuclear protein antibodies (data not shown) in response to injection with the pCDR1-specific T-cell line. The T-cell line-inoculated mice produced low titers of anti-16/6 Id antibodies (Fig. 3b) in concordance with the situation in line-inoculated SJL mice. Similar results were obtained in the analysis of all sera attained monthly from the inoculated animals.
Figure 3.
Antibody levels in sera of BALB/c mice inoculated with the pCDR1-specific T-cell line. Sera of individual line-inoculated BALB/c mice were taken 2 months after injection and from normal, age-matched, uninjected BALB/c mice. The sera were tested for anti-single-stranded DNA (a) and for anti-16/6 idiotype (Id) (b) antibody titres by using enzyme-linked immunosorbent assay (ELISA). Results are expressed as mean absorbance (A) ± SE of each group.
At 4 and 5 months following the pCDR3-specific T-cell line inoculation, SJL mice were analysed for the appearance of clinical manifestations typical of experimental SLE. As shown in Table 3, WBC counts in mice injected with the T-cell line were lower compared with those of normal age-matched SJL mice. It should be noted that normal WBC counts in SJL mice are higher than those measured in normal BALB/c mice, owing to a lymphoproliferative phenomenon typical to the SJL strain. The inoculated mice also had elevated levels of protein in their urine as compared to normal age-matched SJL mice.
Table 3.
Clinical manifestations in mice injected with peptide complementarity-determining region (pCDR)-specific T-cell lines
| Intensity of immune complex deposits† | |||||
|---|---|---|---|---|---|
| WBC/mm3 | Proteinuria* (g/l) | – | + | + + | |
| SJL mice injected with the pCDR3-specific T-cell line | 5970 ± 569 | 0·420 ± 0·098 | 6/19 | 8/19 | 5/19 |
| Normal SJL mice | 8340 ± 1488 | 0·091 ± 0·06 | 3/5 | 2/5 | – |
| BALB/c mice injected with the pCDR1-specific T-cell line | 4150 ± 333 | 0·300 ± 0·30 | 13/14 | 1/14 | – |
| Normal BALB/c mice | 4766 ± 378 | 0·200 ± 0·17 | 4/5 | 1/5 | – |
Mean values for urinary protein (± SD) were derived based on the numeric values specified in the diagnostic kit (see the Materials and methods).
Immune complex density: (–), no immune complexes; (+), low-intensity immune complex deposits; (+ +) high-intensity immune complex deposits.
In agreement with the elevated excretion of urinary protein in sick animals, evaluation of stained kidney sections showed that 13/19 of the inoculated animals had moderate to intense immune complex deposits, whereas six of 19 animals had no immune complexes in their kidneys (Table 3). Figure 4 represents typical immunohistology analysis of kidney sections from the line-inoculated animals (Fig. 4a) as compared to normal age-matched SJL mice (Fig. 4b). It should be noted that in repeated experiments a minority of normal SJL mice spontaneously developed immune complex deposits in their kidneys. However, the fraction of T-cell immunized animals bearing immune complex deposits and the intensity of the latter were highly different from those observed in the group of normal age-matched mice.
Figure 4.
Immunohistology of kidney sections from mice immunized with the pCDR3-specific T-cell line. SJL mice were inoculated with the line (a) and normal age-matched SJL mice (b) were used as a control. Five months later, mice were killed and their kidneys removed and analysed for the presence of immune complex deposits as described in the Materials and methods. (magnification × 400).
BALB/c mice analysed for the appearance of SLE-related clinical manifestations following the pCDR1-specific T-cell line injection did not have either leukopenia or proteinuria (apart from one animal that developed significant proteinuria (Table 3). Also, immunohistology evaluation of their kidney sections did not reveal accumulation of immune complex deposits in these organs at a level higher than the background found in normal age-matched BALB/c mice (Table 3). These data support analysis of the sera showing lack of autoimmune antibody induction following injection of BALB/c mice with the pCDR1-specific T-cell line.
As the pCDR1-specific T-cell line was unable to induce an SLE-like disease in BALB/c mice, it was of interest to examine whether it was capable of protecting these mice from experimental SLE induced by the human pathogenic 16/6 Id anti-DNA antibody. For this purpose, BALB/mice were injected with the pCDR1-specific T-cell line (5 × 106 cells/animal) once, and 2 weeks later the animals were challenged by immunization with the human 16/6 Id antibody, as detailed in Materials and methods.
Analysis of autoantibody induction in the sera of BALB/c animals (Fig. 5) showed that preinoculation with the pCDR1-specific T-cell line did not reduce, but rather increased, anti-DNA antibody levels, as compared to animals immunized with the human 16/6 Id antibody alone (Fig. 5a). Similar results were shown also when anti-16/6 Id antibody levels were evaluated in these animals (Fig. 5b). In addition, preinoculation with the pCDR1-reactive T-cell line did not modulate the values of WBC or protein measured in the urine of treated BALB/c mice, as compared to those measured in animals immunized with the pathogenic 16/6 Id antibody alone (Table 4). Also, immunohistology analysis of kidney sections taken from treated animals showed no reduction in the number or intensity of the immune complex deposits in line-inoculated animals, as compared to animals with experimental SLE induced by the 16/6 Id antibody (Table 4). Thus, under the experimental procedure described, inoculation with the pCDR1-reactive T-cell line, prior to immunization, could not protect BALB/c animals from the induction of experimental SLE by immunization with the 16/6 Id antibody.
Figure 5.
Antibody levels in sera of BALB/c mice injected with the pCDR1-specific T-cell line before immunization with the human 16/6 idiotype-positive (Id+) antibody. Sera were taken from: individual 16/6 Id+ immunized BALB/c mice, line-injected + 16/6 Id+ immunized BALB/c mice, line-inoculated BALB/c mice, and normal age-matched BALB/c mice. The sera were tested for anti-single-stranded DNA (a) and anti-16/6 Id (b) antibody levels by using enzyme-linked immunosorbent assay (ELISA). Results are expressed as mean absorbance (A) ± SE of each group.
Table 4.
Clinical manifestations in mice injected with the peptide complementarity-determining region (pCDR)-specific T-cell line before immunization with the 16/6 idiotype-positive (Id+) antibody
| WBC (/mm3) | Proteinuria (g/l) | Immune complex deposits in kidneys | |
|---|---|---|---|
| 16/6 Id+ immunized | 1633 ± 153 | 1·00 ± 0 | 3/3 |
| Pretreated with the pCDR-specific T-cell line and then 16/6 Id+ immunized | 1783 ± 292 | 0·65 ± 0·38 | 4/4 |
| pCDR1-specific T-cell line injected | 4666 ± 273 | 0·42 ± 0·28 | 1/6 |
| Normal | 4675 ± 95 | 0·18 ± 0·16 | 1/5 |
Discussion
We report here the establishment and characterization of T-cell lines, originating from high-responder mouse strains, to peptides based on the CDRs of a pathogenic mouse anti-DNA antibody (5G12) carrying the common idiotype 16/6. The peptides were previously found to trigger T-cell proliferation in various mouse strains and induce, upon immunization, the formation of autoantibodies and mild experimental SLE in mice.10 We further demonstrate here that these T-cell lines, each recognizing a strain-specific immunodominant T-cell epitope (namely the pCDR1 in BALB/c and the pCDR3 in SJL) differed in their ability to induce, upon inoculation, SLE. These findings propose that T cells specific to the pCDR3 epitope of the pathogenic antibody, play a major role in the antibody-induced model of experimental SLE.
A number of experimental models of autoimmune diseases have been shown to be mediated and/or regulated by CD4+ T cells. For example, in the spontaneous SLE model of (NZB × NZW)F1, the disease was shown to be CD4+ T-cell dependent.16 Furthermore, depletion of CD4+ T cells prior to disease induction with the 16/6 Id, inhibited experimental SLE.17 Adoptive transfer of T-cell lines and clones specific to myelin basic protein has been found to induce experimental autoimmune encephalomyelitis (EAE) in experimental animals.18,19 In another model, T-cell lines and clones specific to peptide sequences of the human acetylcholine receptor were able to induce experimental myasthenia gravis upon inoculation to naive mice.14
As shown in the results, pCDR3-specific and pCDR1-specific T-cell lines were successfully established in SJL and in BALB/c mice, respectively. The T-cell lines proliferated specifically to their immunizing peptides and not to control peptides. The T-cell lines established expressed exclusively the αβ TCR and were of the T helper subtype, as they expressed the CD4 (but not the CD8) murine antigen.
It has been previously shown that T-cell lines specific to the 16/6 Id pathogenic mAb used preferentially the Vβ8 TCR chain. Moreover, molecular analysis showed that these T-cell lines express a common motif in the junctional regions (CDR3) of the Vβ8 chain.20 As shown in the present work, peptide-specific T-cell lines were not restricted in their Vβ-TCR chain usage to one chain; however, they were limited in the Vβ-TCR families used. Thus, the predominant Vβ-TCR chains used by the pCDR1-specific T-cell line of BALB/c origin were Vβ4, Vβ8 and Vβ14, and the predominant Vβ-TCR chains used by the pCDR3-specific T-cell line of SJL origin were Vβ4 and Vβ14.
Both the pCDR1-specific T-cell line and the pCDR3-specific T-cell line secreted cytokines of the T helper 2 (Th2) type. It was previously shown in our laboratory that experimental SLE is initiated upon the induction of a T helper 1 (Th1) cytokine environment, followed by a shift towards a Th2 cytokine profile when clinical manifestations are detected.21 As inoculation of animals with the pCDR3-specific T-cell line induced autoimmune responses, it is suggested that the high levels of Th2 cytokines (especially IL-10) secreted by the cells contribute to the development of the pathogenic situation. In another autoimmune model, pathogenic Th2 T cells were demonstrated to induce acute diabetes mediated by local IL-10 production, upon inoculation to non-obese diabetic (NOD) severe combined immunodeficiency (SCID) mice.22 However, it is not clear whether the Th2-type cytokines were produced by the T-cell line in vivo, following its inoculation, because the cells were injected with no peptide stimulation, which normally induced cytokine secretion in vitro (Table 1).
Analysis of antibodies in the sera of mice inoculated with the pCDR3-specific line demonstrated that no anti-pCDR3 antibodies could be detected. Nevertheless, the immunization induced a number of other antibodies. First, inoculation with the pCDR3-specific T-cell line elicited anti-16/6 Id antibodies, probably through the phenomenon of epitope spreading.23 Second, the sera of line-inoculated mice contained antinuclear protein and anti-DNA autoantibodies, typical of experimental SLE. Analysis of the pathogenic anti-DNA antibodies showed that they were predominantly of the IgG1 isotype. This fact may be explained as a consequence of the production of high levels of Th2 cytokines by the pCDR3-specific T-cell line. These results are in accordance with data showing that SLE-related pathogenic anti-DNA antibodies induced in SJL mice following immunization with the 16/6 Id mAb are of both the IgG1 and the IgG2a isotypes (data not shown). Induction of autoimmune antibodies, following the pCDR3-specific T-cell line inoculation, led to the development of clinical manifestations associated with experimental SLE (Table 3 and Fig. 5).
Although the CDR1-based peptide was shown to be the immunodominant T-cell epitope in BALB/c mice and capable of inducing mild experimental SLE upon injection in CFA, inoculation of BALB/c mice with the pCDR1-specific T-cell line did not induce the development of a clinical disease. Analysis of the antibody repertoire in the sera of BALB/c mice inoculated with the pCDR1-specific T-cell line showed the production of small amounts of anti-16/6 Id. It is possible that the low anti-16/6 Id response did not reach a threshold to start an autoimmune cascade required for disease induction.
As the pCDR1-specific T-cell line was not capable of inducing experimental SLE, it was of interest to find out if it could protect from the induction of experimental SLE by the human pathogenic anti-DNA 16/6 Id antibody. Indeed, successful T-cell vaccination for EAE has been reported previously.24,25 The results (Fig. 5) indicate that, under the conditions of the present study, one preinoculation with the pCDR1-specific T-cell line was unable to inhibit disease induction by the 16/6 Id antibody. It is possible that because the 16/6 Id is a potent pathogenic antibody, either higher cell numbers or additional injections are required for protection.
It is noteworthy that SJL mice injected with a T-cell line specific to a peptide based on a sequence of the human acetylcholine receptor α-subunit, namely p159-212,13 did not develop clinical symptoms characteristic of SLE (data not shown). Furthermore, as demonstrated in the present report, BALB/c mice injected with the pCDR1-specific T-cell line did not develop experimental SLE. The latter supports the notion that it is the specificity to pCDR3 that renders the T-cell line pathogenic.
A series of results support the suggestion that although both the pCDR1 and the pCDR3 are disease-inducing epitopes of the 16/6 Id antibody, pCDR3 is more immunodominant. Striking similarities were determined between the V regions of both heavy and light chains of the murine anti-DNA 16/6 Id+ antibody and the V regions of the heavy and light chains of antibodies isolated from the (NZB × NZW)F1 lupus-prone mice.10 However, splenocytes of naive young (NZB × NZW)F1 lupus-prone mice were shown to proliferate following a trigger with pCDR3, but not with pCDR1. Furthermore, immunization of BALB/C or C3H.SW mice with the pCDR1 peptide induced LN cell proliferation also towards the pCDR3 peptide (N. Brosh, unpublished).
Several possible mechanisms by which the pCDR3-specific T-cell line induced experimental SLE may be considered. One possibility is that the T-cell line could provide help to B cells in vivo to produce the various autoantibodies. Another possibility is that the immunogenicity of the inoculated T cells by itself was sufficient to trigger an immune response directed against the TCR, namely, clonotypic, anti-idiotypic B-cell responses. The B cells arising secreted a cascade of autoantibodies (e.g. anti-16/6 Id, anti-DNA and antinuclear protein antibodies), which resulted in the initiation of experimental SLE. However, this possibility is rather unlikely because the T cells were injected into the animal only once in a small number (5 × 106 cells/animal) and in a ‘non-immunogenic’ (i.v. without CFA) manner.
Finally, autoimmune diseases are caused by aberrant T- and/or B-cell responses against self-antigens. Data from animal models of autoimmune diseases suggest that the immune responses in autoimmunity progress from an initial antigenic target and are extended towards new determinants (which include other epitopes on the same protein, or other proteins in the same tissue) as a result of epitope spreading.26,27 It is suggested that immunization of SJL mice with the pCDR3-specific T-cell line initiated experimental SLE via the epitope spreading process, thus altering immune responses from peptides towards new antigens, such as the whole 16/6 Id Ab (as evidenced by the production of anti-16/6 Id antibodies) and towards self-antigens, such as nuclear proteins and DNA (as evidenced by the production of antibodies to these antigens). Thus, the pCDR3-specific T cells may play a role in auto-immune processes leading to SLE in induced experimental models as well as in the (NZB × NZW)F1 lupus-prone mice.
Acknowledgments
This study was supported from TEVA Pharmaceutical Industries Ltd.
Glossary
Abbreviations
- Abs
antibodies
- APCs
antigen-presenting cells
- CDR
complementarity-determining region
- CFA
complete Freund’s adjuvant
- Con A
concanavalin A
- EAE
experimental autoimmune encephalomyelitis: FCS, fetal calf serum
- FITC
fluorescein isothiocyanate
- Id
idiotype
- IFN-γ
interferon-γ
- IL
interleukin
- i.v.
intravenous
- LN
lymph node
- mAb
monoclonal antibody
- PE
phycoerythrin
- TCR
T-cell receptor
- SLE
systemic lupus erythematosus
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