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The Journal of Immunology, 2001, 166: 5456-5463.
Copyright © 2001 by The American Association of Immunologists

Distinct Autoreactive T Cell Responses to Native and Fragmented DNA Topoisomerase I: Influence of APC Type and IL-21

Timothy B. Oriss, Paul Q. Hu and Timothy M. Wright2

Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, University of Pittsburgh Arthritis Institute, Pittsburgh, PA 15261


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Systemic sclerosis (SSc) is an autoimmune connective tissue disease of unknown etiology in which T cell responses to various autoantigens, including DNA topoisomerase I (Topo I), have been implicated. We investigated whether dendritic cells, generally considered to be the most potent APCs for the initiation of immune responses, would present either of two forms of Topo I to T cells more efficiently than PBMC APCs. Using cells from healthy controls and SSc patients, several important observations were made. First, neither APC type was able to initiate T cell proliferative responses to full-length native Topo I unless exogenous IL-2 was added. This is in contrast to vigorous T cell proliferation in response to Topo I polypeptide fragments presented by either APC type. Second, T cell responses to the full-length form of Topo I presented by dendritic cells were considerably lower than responses to Ag presented by PBMC APCs. Finally, no secondary T cell responses were observed unless the same Ag/APC combination as that used in the primary stimulation was maintained. These data indicate that different peptides are generated based upon the form of the Topo I and the APC that processes it. Taken together, these results suggest that a very specific combination of antigenic form and APC may be involved in breaking tolerance to Topo I in the early stages of development of SSc.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Scleroderma, or systemic sclerosis (SSc),3 is an autoimmune disease characterized by the presence of Abs against a variety of autoantigens including, but not limited to, DNA topoisomerase I (Topo I; also known as Scl-70), centromere, RNA polymerase I and III, and U3 RNA/fibrillarin (1). SSc is characterized by overproduction of collagen by affected fibroblasts, resulting in fibrosis of the skin and internal organs including the heart, lungs, kidneys, and gastrointestinal tract (2). Ten-year mortality rates can approach 50% in the most severe systemic forms of the disease (3).

The role of T cells in the pathogenesis of SSc is supported by: 1) the presence of CD4+ T cells in early skin lesions and increased T cells in bronchoaveolar lavage fluid of patients with pulmonary fibrosis; 2) disease-associated autoantibodies of multiple isotypes that correlate with clinical findings and disease course; and 3) responses to T cell-targeted therapies (e.g., cyclosporin A and anti-thymocyte globulin), although thus far this is limited to case reports and small series (4, 5, 6, 7, 8). In several autoimmune diseases in which autoantigens have been identified, including multiple sclerosis and SSc, autoreactive T cells can be detected and expanded in vitro (9, 10). Interestingly, these same autoreactive T cells can also be found in the peripheral circulation of healthy individuals (9, 11). The finding of autoreactive cells in healthy individuals suggests that additional immunologic events must occur to break T cell tolerance during the initiation of disease. These events may involve cross-reactivity of bacterial or viral Ags with autoantigens (12, 13, 14, 15), environmental influences such as chemical toxins (3, 16), altered forms of autoantigen as may occur during apoptosis (17, 18, 19), and changes or defects in Ag processing (20, 21, 22, 23, 24, 25, 26).

Our laboratory has studied the autoreactive T and B cell responses to the SSc-associated Ag DNA Topo I. We previously reported that Topo I-specific T cells can be identified in the peripheral blood of SSc patients and healthy subjects whose cells bear one or more MHC class II HLA-DR responder alleles, notably HLA DR*11, DR*15, or DR*7 (9). These prior studies were performed using a Topo I Ag preparation consisting of a set of five overlapping maltose binding protein (MBP) fusion proteins, termed F3 through F7, spanning the entire length of the 766 amino acid Topo I molecule. A series of CD4+ T cell clones derived using this Ag were specific for a portion of the Topo I molecule represented by fragments F5 and F6 (spanning aa 209–386 and aa 363–563, respectively), and all but one of 15 clones expressed the same TCR {beta}-chain variable gene fragment (v{beta}20.1) (27). The biologic relevance of the Ag preparation used in these studies was confirmed when it was determined that Abs in the serum of Topo I Ab-positive SSc patients also reacted with this portion of the molecule and that the T cell clones could provide help to autoreactive B cells, leading to anti-Topo I Ab production (28).

As part of our efforts to understand the initial phases of SSc, we examined dendritic cell (DC) presentation of Topo I to T cells, because DCs are believed to prime immune responses in vivo (29). As a component of these studies, we undertook the production of a native full-length form of Topo I using a baculovirus vector in insect cells. The initial goal of these studies was to compare the presentation of Topo I to T cells by DCs and APCs present in PBMC. However, unexpected differences were found regarding the form of the Topo I Ag and the APC type involved in presentation. These findings are presented herein, and their potential significance is discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human subjects

Peripheral blood was obtained from healthy adults and SSc patients by venipuncture according to all applicable Institutional Review Board guidelines. SSc subjects fulfilled the American College of Rheumatology preliminary criteria for the classification of SSc (30) and were judged to be either positive or negative for serum anti-Topo I Ab (31). Data presented herein represent results obtained with cells from healthy individuals. In certain cases, cells from SSc patients were used to confirm results that had been obtained using healthy donors’ cells, as noted in the text.

Ag preparation

An overlapping set of five MBP fusion proteins representing the entire length of the human Topo I molecule was prepared by subcloning Topo I cDNA fragments into the pMAL-C2 expression vector (New England Biolabs, Beverly, MA). These were expressed in Escherichia coli strain DH5{alpha} (Life Technologies, Rockville, MD) and were purified as previously described (30). This set of fusion proteins was used together in equimolar concentrations and was termed MBP-Topo I mix. In several experiments, one of the MBP fusion proteins, MBP-F5 (aa 209–386), was used alone. MBP was similarly generated by transforming E. coli with pMAL-C2 vector lacking Topo I cDNA and was purified for use as a control Ag.

Purified native full-length Topo I was prepared using the MaxBac baculovirus expression system (Invitrogen, Carlsbad, CA) as described by P. Q. Hu T. A. Medsger, Jr., and T. M. Wright, (manuscript in preparation). Briefly, cDNA corresponding to the entire open reading frame of Topo I (30) was cloned into the multiple cloning site of the shuttle vector pBlueBac4.5 (Invitrogen), which was then cotransfected along with Bac-N-Blue DNA (Invitrogen) into insect Sf9 cells to produce recombinant virus. Full-length recombinant baculovirus-derived Topo I (rTopo I) was produced by infection of Sf9 cells with the recombinant virus, followed by isolation of cell nuclei, precipitation of cellular genomic DNA, and purification of the rTopo I from the resulting supernatant using three columns in succession: a heparin Sepharose CL-6B column (Amersham Pharmacia Biotech, Uppsala, Sweden), followed by phenyl Sepharose 6 and Mono S HR 5/5 columns (Amersham Pharmacia Biotech). The concentration of rTopo I was measured by a colorimetric protein assay (Bio-Rad, Richmond, CA), and purity was estimated to be >95% by SDS-PAGE and Coomassie blue staining. Purified rTopo I was reconstituted in 50% glycerol with 1 mM DTT, filter-sterilized, and stored at -80°C.

The MBP-F5 fusion construct was also produced using the baculovirus system as described above. Tetanus toxoid, used as a control Ag in some experiments, was purchased from Massachusetts State Biological Laboratories (Jamaica Plain, MA).

Synthetic peptides representing the entire length of the Topo I molecule were purchased from Chiron Mimotopes (Victoria, Australia). The peptides were synthesized as 257 15-mers, each overlapping the next by three amino acids. The peptides were resuspended in 0.1 M HEPES (pH 7.4), 40% acetonitrile, and were used either alone (5 µM) or in 25 pools of 10 peptides/pool.

DC generation and Ag loading

PBMC were obtained from heparinized peripheral blood by isolation on Ficoll-Paque gradients (Amersham Pharmacia Biotech). DCs were obtained by first incubating PBMC (5–10 x 107 cells/flask) in 75 cm2 plastic cell-culture flasks (Falcon; BD Biosciences, Franklin Lakes, NJ) for 2 h at 37°C in AIM V culture medium (Life Technologies) with penicillin/streptomycin (100 U/ml, 100 µg/ml; Life Technologies), followed by removal of nonadherent cells and culture of adherent cells for 7 days in AIM V supplemented with IL-4 (30 ng/ml; Life Technologies) and GM-CSF (10 ng/ml; BD PharMingen, San Diego, CA). Additional AIM V and cytokines were added on days 4 and 6 of culture. DC were harvested by removing nonadherent cells and collecting adherent cells by incubation with PBS/15 mM EDTA for 15 min at 4°C, followed by scraping with a cell scraper. The phenotype of DC preparations was monitored by staining and flow cytometry for CD11c, CD14, CD80, CD86, and HLA-DR. DCs expressed all of the markers except CD14, which was expressed by fresh monocytes/macrophages or cells cultured in GM-CSF alone. The cells, 90–95% of which had a DC phenotype by flow cytometry, were not further purified and demonstrated no proliferative response to Ag without the addition of T cells.

DCs were exposed to Ag directly in the 96-well plates that were to be used for the assays. For some experiments, variable numbers of cells (30–30,000 cells/well) were used, but for most experiments 104 DCs/well were used. Cells were plated in 100 µl of AIM V medium with 1000 U/ml TNF-{alpha} (0.9 U/ng; BD PharMingen) and the indicated Ag and were incubated overnight at 37°C. The following day the medium was gently removed from the wells, leaving behind the plastic-adherent DCs.

Primary T cell stimulation

DCs were exposed to Ags as described above. Plastic-nonadherent PBMCs were used as a source of T cells and were added to wells (105 cells/well) containing Ag-exposed DC in a total volume of 200 µl of RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated (60°C, 1 h) human AB serum (Cellgro, Herndon, VA) and penicillin/streptomycin (RPMI/huAB). The T cell-enriched plastic-nonadherent cell population typically was 72.4 ± 11.4% T cells, 6.6 ± 3.9% monocytes, 2.8 ± 0.4% B-cells, and 23.2 ± 3.5% NK cells (n = 10) as analyzed by flow cytometry. Using a wide range of Ag concentrations (0.1–10 µg/ml), no detectable T cell proliferation was observed in the plastic nonadherent fraction unless additional APCs were added (data not shown).

Primary stimulation of T cells with PBMC APC was conducted by directly plating whole, unseparated PBMC (2 x 105 cells/well) with Ag in RPMI/huAB. Twice as many undepleted PBMC were used as for DC stimulation because roughly one half of the PBMC were typically plastic nonadherent. In some experiments, rIL-2 was also added to the cultures (20 U/ml, 106 U/µg; Life Technologies) as indicated. Primary stimulation cultures with either APC type were incubated for 7 days at 37°C with the addition of [methyl-3H]thymidine ([3H]thymidine) (1 µCi/well; NEN, Boston, MA) during the final 18 h of incubation. Incorporation of [3H]thymidine was determined by harvesting cells onto filters followed by liquid scintillation counting. Allogeneic immune responses were measured in the absence of exogenous Ag using an MLC with T cells and DCs from two individuals with mismatched MHC class II alleles. Cells that were to undergo secondary stimulation were treated in a manner identical to that described above, except that the [3H]thymidine addition was eliminated.

Secondary T cell stimulation

T cells were stimulated with Ag and APC for 7 days, were then washed three times with PBS, and were used in secondary stimulation assays. DCs were exposed to Ag as described for primary T cell stimulation. PBMC APCs in these assays consisted of unseparated PBMCs (105 cells/well) that were {gamma} irradiated at 2000 rad and were mixed with Ag at the time of adding T cells. Irradiated, unseparated PBMC did not proliferate in response to any Ag without the addition of T cells. Primary-stimulated T cells (3 x 104 cells/well) were added in RPMI/huAB, and culture volumes were maintained at 200 µl. Secondary stimulation assays were incubated for 48 h, and the [3H]thymidine addition, harvesting, and scintillation counting was performed as described above.

T cell line generation

Short-term T cell lines were generated by repeated stimulation with Ag. Plastic-nonadherent PBMC were incubated with Ag-loaded DCs under conditions identical with those described for primary T cell stimulation. At intervals of 7–10 days, the T cells were restimulated with Ag, APC, and IL-2 (10 U/ml) as described for secondary T cell stimulation above. After three to four rounds of stimulation, the cells were routinely found to be >95% CD4+ T cells by flow cytometry (data not shown). These cells were tested in proliferation assays (3 x 104 cells/well) with DCs (1 x 104 cells/well) as the APC for their ability to be stimulated by synthetic peptides. In these assays, the peptides, DCs, and T cells were added simultaneously because Ag uptake and processing by the APCs was not required. These assays were incubated for 48 h, and the [3H]thymidine addition, harvesting, and scintillation counting was performed as described above.

Factor Xa digestion

The MBP fusion partner was removed from the MBP-Topo I fusion protein MBP-F5 by enzymatic cleavage with Factor Xa (New England Biolabs). Cleavage was performed according to the manufacturer’s instructions. Briefly, 1 mg of MBP-F5 was suspended in a total volume of 1 ml reaction buffer (20 mM Tris-HCl, 100 mM NaCl, and 2 mM CaCl (pH 8.0)) to which 20 µg of Factor Xa was added. The reaction was allowed to proceed for 1 h at 23°C. Reaction products, as well as uncut control, were visualized by electrophoresis on 10% SDS-polyacrylamide gels followed by staining with Coomassie blue.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DC phenotype and function

The mature myeloid DCs used in this study were generated from peripheral blood monocytes by the commonly used method of culture with GM-CSF and IL-4 (29). DCs generated by this method had the expected gross morphology and cell surface phenotype, including expression of MHC class II, costimulatory molecules CD80 and CD86, CD11c, and the absence of CD14 expression (data not shown). DCs initiated vigorous allogeneic immune responses (Fig. 1GoA) and efficiently presented various Ags, including recombinant Topo I fusion proteins and tetanus toxoid to T cells (Fig. 1Go, B–D). Also, as is typical of DCs, these cells presented Ags to T cells much more efficiently than PBMC APCs (Fig. 1Go, C and D). Thus, the cells used in our experiments have phenotypic and functional properties typical of DCs described by others (29). This is important because our findings using a full-length rTopo I clearly differ from these typical observations (see the following section and Fig. 2DGo).



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  		FIGURE 1. In vitro-generated DCs are functional in T cell assays measuring allogeneic and Ag-specific responses and are more potent stimulators of T cell responses than PBMC APCs. DCs were generated from peripheral blood monocytes as described in Materials and Methods. A, Varying numbers of either autologous (filled symbols) or allogeneic (open symbols) DCs (300–30,000 cells/well) were plated into 96-well plates overnight with TNF-{alpha} (1000 U/ml). T cells (plastic-nonadherent PBMC; 105 cells/well) were added the following day. B–D, DCs (104 cells/well) were plated as described above with the addition of increasing concentrations of tetanus toxoid (B–C) or the MBP-Topo I mix (D). T cells (105 cells/well) were added after overnight incubation with Ag. B, DCs from three different donors were plated and incubated with increasing concentrations of tetanus toxoid (0.01–10 µg/ml). C, DCs ({square}) were compared with PBMCs (2 x 105 cells/well; {circ}) for their ability to stimulate T cell proliferation in response to increasing concentrations of tetanus toxoid (0.03–20 µg/ml). D, DC ({square}) and PBMC ({circ}) were compared for their ability to stimulate T cell proliferation to increasing concentrations of the MBP-Topo I mix (0.003–10 µg/ml). Each of the above assays was incubated for 7 days with the addition of [3H]thymidine during the final 18 h of incubation.

 


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  		FIGURE 2. The rTopo I does not initiate T cell responses in the absence of exogenous IL-2 and induces a relatively weak T cell response when presented by DCs. A, DCs were plated as in Fig. 1Go with no Ag ({circ}), rTopo I (30 µg/ml; {square}), or the MBP-Topo I mix (10 µg/ml; {triangleup}) with T cells (1 x 105 cells/well) added the following day. B, DCs (1 x 104 cells/well) were plated with increasing concentrations of rTopo I (1–30 µg/ml), with ({square}) or without ({circ}) the addition of exogenous IL-2 (20 U/ml). C, Unseparated PBMCs (2 x 105 cells/well) that had not been irradiated were similarly plated with rTopo I with ({square}) or without ({circ}) IL-2. D, Increasing concentrations of rTopo I (1–30 µg/ml) were used to stimulate T cells (1 x 105 cells/well) using DC alone ({circ}), DC plus IL-2 (20 U/ml; {square}), or PBMC plus IL-2 (•). In this experiment, T cells were prepared from the same individual after a single blood draw and were assayed simultaneously. All assays were incubated for 7 days with the addition of [3H]thymidine during the final 18 h of incubation. Data are representative of experiments from seven healthy subjects and two SSc patients.

 
T cell response to rTopo I requires exogenous IL-2 and is preferentially initiated by PBMC APCs

Previous work in our laboratory used Topo I Ag in the form of a mix of five MBP fusion proteins, which together constitute the entire length of the Topo I molecule (MBP-Topo I mix) in an overlapping fashion (31). The MBP-Topo I mix Ag preparation stimulated T cell proliferation from both SSc patients as well as healthy subjects with certain MHC class II alleles and reacted with Ab in the sera of SSc patients but not healthy controls (9, 28, 31). As part of our efforts to better understand the events that initiate disease pathogenesis in SSc, we wanted to examine T cell responses to the native form of Topo I. Therefore, we produced full-length human TopoI protein in insect cells (rTopo I). We found that rTopo I was recognized by anti-Topo I Ab-positive SSc patient sera in ELISA, had the expected Mr of 100 kDa on SDS-PAGE, and was enzymatically active in a plasmid relaxation assay (P.Q. Hu, T. A. Medsger, Jr., and T. M. Wright, manuscript in preparation). Therefore, the rTopo I represented native, functional DNA Topo I, which was recognized by disease-specific sera.

We next examined the T cell proliferative response to native rTopo I and compared it to the response to the MBP-Topo I mix. In contrast to proliferative responses to Topo I fragments, T cell responses were not observed to rTopo I presented by DCs (Fig. 2GoA). This experiment was repeated with cells from seven healthy subjects and two SSc patients, and the results were similar (data not shown). T cells from all individuals tested failed to respond to rTopo I at Ag concentrations as high as 100 µg/ml and at DC concentrations as high as 3 x 104 cells/well (data not shown). We hypothesized that the autoreactive T cells specific for peptides generated from rTopo I may have been deleted or rendered anergic. To address the possibility that these T cells were anergized in vivo, we examined T cell proliferation in response to rTopo I with the addition of IL-2, which is reported to reverse T cell anergy (32, 33, 34, 35). As shown in Fig. 2GoB, cultures of T cells stimulated with Ag presented by DCs and supplemented with IL-2 (20 U/ml) responded to rTopo I in a concentration-dependent manner.

Similar observations were made regarding the requirement for IL-2 when PBMCs were used as the source of APCs (Fig. 2GoC). However, in contrast to the T cell responses to Topo I fragments (Fig. 1GoD), PBMC APCs stimulated greater T cell response to rTopo I in the presence of IL-2 compared with DCs (Fig. 2GoD). This may reflect the ability of PBMC APCs to process and present rTopo I peptide fragments to T cells much more efficiently than DCs. Similar observations, including the requirement for IL-2 and the preference for PBMC APCs, have been made using cells from a limited number of SSc patients examined to date (data not shown).

For both APC types, magnetic bead depletion studies determined that the proliferative response to rTopo I plus IL-2 was mediated by CD4+ T cells (data not shown), as had been previously observed for the MBP-Topo I mix Ag (5). Addition of IL-2 to the MBP-Topo I mix resulted in an increased rate of [3H]thymidine incorporation but did not alter the shape of the Ag dose-response curve (data not shown). Even in the presence of exogenous IL-2, T cell responses from any given individual were generally much lower in response to rTopo I plus IL-2 than to the MBP-Topo I mix Ag regardless of the APC used (data not shown). Together, these data raise important questions about the form of the Topo I Ag and the APC that are responsible for the initiation of the autoimmune response in SSc.

The protein expression system is not important in the differential T cell response to different forms of DNA Topo I

An important consideration regarding the two Topo I Ag preparations was whether the different methods by which the proteins were expressed affected their relative immunogenicity. Full-length Topo I could not be produced as an MBP fusion protein in bacteria due to bacterial toxicity and enzymatic degradation (31), prompting the use of a baculovirus expression system that should promote natural glycosylation and folding of the protein (36). When rTopo I failed to elicit T cell proliferation, several possible explanations for the greater efficiency of MBP-Topo I mix presentation to T cells relative to rTopo I were considered. First, because the MBP-Topo I mix Ag was produced in a bacterial expression system and the rTopo I in a baculovirus expression system, there could have been differences in protein folding and glycosylation between the two systems. Second, the bacterially expressed Topo I fragments may contain LPS, which could affect APC function and possibly enhance T cell responses to this form of Ag. Third, the MBP fusion partner itself could have directed proteins into a processing pathway that allowed for more efficient Ag presentation to T cells and/or the presentation of different epitopes. Finally, MBP might have a direct effect on the APC and enhance Ag processing and/or presentation.

To address these issues, we performed a series of experiments using one of the Topo I fragments contained in the MBP-Topo I mix Ag preparation, MBP-F5, which was used because we have observed strong T cell proliferative responses to this Ag, and most of the Topo I-specific T cell clones generated in our laboratory previously have reacted with this portion of the Topo I molecule (27). The construct that was used to generate bacterial MBP-F5 was cloned into a baculovirus vector and was expressed in the Sf9 insect cell line. No difference in T cell response was observed regardless of the expression system used to generate MBP-F5 (Fig. 3GoA). We also found that the addition of purified LPS did not stimulate or enhance T cell responses to any of our Ag preparations (data not shown). Furthermore, enzymatic cleavage of MBP from F5 also did not reduce T cell response to this fragment (Fig. 3GoC). Finally, exogenous MBP produced in the bacterial expression system and used at the same molar concentration as that in the MBP-Topo I mix preparation failed to augment T cell responses to rTopo I or the control Ag tetanus toxoid (Fig. 3GoB). Taken together, these results indicate that the observed differences in T cell responses to rTopo I and the MBP-Topo I mix do not arise from differences in methods by which the proteins were expressed, and that MBP itself does not act as an "adjuvant" or a chaperone of proteins into a particular APC Ag-processing pathway.



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  		FIGURE 3. The method of Ag preparation does not account for the observed differences in T cell responsiveness. A, One fragment (MBP-F5) of the five that constitute the MBP-Topo I mix was produced using the baculovirus expression system, which had been used to produce rTopo I. The DCs were incubated with Ag (0.3–10 µg/ml) followed by addition of T cells. B, DCs were incubated with the MBP-Topo I mix (0.5 µM), MBP (0.5 µM), rTopo I (50 µg/ml) ± MBP (0.5 µM), or tetanus toxoid (10 µg/ml) ± MBP (0.5 µM) before the addition of T cells. C, MBP was cleaved from F5 using Factor Xa enzymatic digestion. The Coomassie Blue-stained polyacrylamide gel shows that the MBP-F5 was completely cleaved. The MBP plus F5 ({triangleup}) mix was compared with uncleaved MBP-F5 ({square}) and MBP ({circ}) for the ability to induce T cell proliferation using DC APCs.

 
IL-2, which is required for rTopo I response, appears to act directly on T cells

The absolute requirement for exogenous IL-2 to elicit T cell responses to rTopo I may be due to effects on the T cells or on the APCs. To examine this further, DCs were incubated overnight with Ag in the presence or absence of exogenous IL-2. Unbound IL-2 was washed away before the addition of T cells. Untreated and IL-2-treated DCs both failed to induce T cell responses to rTopo I (Fig. 4GoA). As shown in Fig. 4GoB, we observed variable levels of background (no Ag) incorporation of [3H]thymidine in T cell cultures supplemented with IL-2. In this figure, only the samples in which IL-2 was added at time zero resulted in T cell responses with a stimulation index (cpm of Ag-stimulated cultures/cpm of cultures with no Ag added) >2. However, as shown in Fig. 4GoB, exogenous IL-2 added as late as 48 h following T cell/DC coincubation resulted in significant T cell proliferation (Fig. 4GoB). These results indicate that the exogenous IL-2 did not affect Ag uptake, processing, or MHC class II loading because these events are largely complete in DC after overnight incubation with TNF-{alpha} before the addition of T cells. The results suggest that IL-2 acted directly on T cells to promote proliferation following Ag-specific TCR engagement.



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  		FIGURE 4. IL-2 acts on the T cells, not on the APCs. A, DCs were incubated with increasing concentrations of rTopo I (1–30 µg/ml) alone ({circ}) or with exogenous IL-2 (20 U/ml; {square}). After overnight incubation, the DCs were washed to remove IL-2, then T cells (105 cells/well) were added. B, DCs were incubated with rTopo, as described. Exogenous IL-2 (20 U/ml) was either not added ({circ}) or added at 0, 12, 24, or 48 h ({square}, {triangleup}, •, and {blacksquare}, respectively) after the addition of T cells.

 
T cells stimulated by the MBP-Topo I mix and rTopo I are specific for Topo I peptide amino acid sequences

In order make certain that the observed T cell responses were specific for the Topo I molecule, we generated short-term T cell lines by three to four rounds of stimulation with either rTopo I or the MBP-Topo I mix. Overlapping synthetic peptides representing the entire length of the rTopo I molecule were then presented to the T cell lines to determine whether they could induce proliferation. The first step in the epitope mapping process involved screening pools of 10 peptides each. Fig. 5GoA demonstrates that for a single rTopo I-responsive line, a single peptide pool stimulated T cell proliferation. In the second step of mapping, the individual peptides within the pool that was stimulatory in Fig. 5GoA were each tested for their ability to induce T cell line proliferation. Three overlapping peptides (Fig. 5GoC) were found to stimulate the T cell line (Fig. 5GoB). Each of these peptides is within the F6 fragment of the MBP-Topo I mix (9). Similar results were obtained with lines generated using the MBP-Topo I mix (data not shown). Additionally, we have previously shown that T cell lines stimulated by the MBP-Topo I mix are very likely to be peptide-specific because they can provide help to SSc patient B cells for the production of anti-Topo I Ab (28).



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  		FIGURE 5. T cells originally stimulated with rTopo I plus IL-2 respond to peptides derived from the Topo I molecule. A short-term T cell line was generated by stimulation of plastic-nonadherent PBMC with rTopo I-loaded DC plus IL-2 as described for primary stimulations. The line was restimulated every 7–10 days with Ag and IL-2 (10 U/ml) and was used for experiments after four rounds of stimulation. A, The line was tested for specificity for the rTopo I molecule in the absence of IL-2 using pools of synthetic peptides (10 peptides/pool; 5 µM each) by proliferation assay. T cells (3 x 104 cells/well) were added to peptide-loaded DC (1 x 104 cells/well) and controls with no DCs, no Ag, or rTopo I (30 µg/ml) were included. B, Peptides (5 µM) in the lone stimulatory peptide pool (pool 13) were individually tested for ability to stimulate the T cell line. Proliferation assays were incubated for 48 h, and [3H]thymidine was added during the final 18 h of incubation. C, The amino acid sequence and amino acid numbers of three peptides that were able to stimulate the T cell line in B.

 
Unique antigenic responses are generated from the MBP-Topo I mix and rTopo I

T cell responses are generally amplified in secondary stimulations relative to primary stimulations, due to higher numbers of Ag-specific T cells that were expanded during the initial stimulation. We next examined whether there were differences between rTopo I and the MBP-Topo I mix in secondary T cell stimulations and whether the responding T cells were specific for the form of Topo I Ag. Secondary stimulations were performed using each of the Topo I Ag preparations and resulted in higher levels of T cell proliferation than primary stimulations (data not shown). In the case of rTopo I, the absolute requirement for exogenous IL-2 in primary stimulations was abrogated in secondary stimulations, suggesting that a nonresponsive state to this Ag was reversed or that the small number of IL-2-producing rTopo I-specific T cells were expanded during the primary stimulation (Fig. 6GoB). Interestingly, cells that received a primary stimulation with one Ag preparation were unable to respond to the other Ag preparation in the secondary assay (Fig. 6Go). The results in Fig. 6Go were obtained using DCs as the APCs, and similar results were obtained when PBMC APCs were used (data not shown). In addition, several short-term T cell lines were generated using DC or PBMC APCs, and these lines only responded to the Ag with which they were initiated (data not shown). These results suggest that unique sets of antigenic peptides are derived from the two forms of Topo I Ag.



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  		FIGURE 6. Secondary T cell responses to Topo I Ag preparations are specific for the Ag used in the primary stimulation. T cells were cultured with DCs that had been incubated with either the MBP-Topo I mix (10 µg/ml; {circ}) or rTopo I (30 µg/ml) plus IL-2 (20 U/ml; {square}). After 7 days, the T cells were harvested and used (3 x 104 cells/well) in a secondary proliferation assay containing DCs (104 cells/well) incubated with increasing concentrations of (A) MPB-Topo I mix (0.03–10 µg/ml) or (B) rTopo I (1–30 µg/ml). Secondary proliferation assays were incubated for 48 h with the addition of [3H]thymidine during the final 18 h of incubation.

 
DC and PBMC APCs generate unique epitopes from the same form of Topo I Ag

As described above, the T cell responses to the two Topo I Ag preparations were distinct and not cross-reactive. We next examined the role of the APC in determining the response to the two different forms of Topo I autoantigen. For typical Ags, such as tetanus toxoid (Fig. 7Go), primary stimulation with DCs results in a population of T cells that is able to respond the Ag presented by either DC or PBMC APCs in a secondary stimulation. However, we found that for both the MBP-Topo I mix and rTopo I, secondary proliferative responses were dependent upon using the same APC as that used in the primary stimulation (Fig. 8Go). This was true whether DCs (Fig. 8Go, A and B) or PBMCs (Fig. 8Go, C and D) were used as the APC in the primary stimulation. This was unexpected in the case of DCs, because these cells are widely believed to initiate immune responses in vivo. These results indicate that different sets of antigenic peptides are generated from each of the two forms of the autoantigen Topo I by DC and PBMC APCs, resulting in unique responder T cell populations.



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  		FIGURE 7. Secondary T cell response to tetanus toxoid is not specific for the APC used in the primary stimulation. T cells were cultured with DCs that had been incubated overnight with tetanus toxoid (20 µg/ml). After 7 days, the T cells were harvested and used (3 x 104 cells/well) in a secondary proliferation assay containing DCs (104 cells/well) or irradiated (2000 rad) PBMC (105 cells/well) that were not Ag-pulsed ({square}) or were pulsed with 0.08 µg/ml ({blacksquare}) or 6.7 µg/ml () tetanus toxoid. The secondary proliferation assays were incubated for 48 h, and [3H]thymidine was added during the final 18 h of incubation.

 


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  		FIGURE 8. Secondary T cell responses to Topo I Ag preparations are specific for the APC used in the primary stimulation. Primary T cell stimulations were performed using (A) the MBP-Topo I mix (10 µg/ml) presented by DCs (104 cells/well), (B) IL-2 plus rTopo I (30 µg/ml) presented by DCs, (C) the MBP-Topo I mix presented by irradiated (2000 rad) PBMC (105 cells/well), or (D) IL-2 plus rTopo I presented by PBMC. After 7 days, the T cells were harvested and assayed (3 x 104 cells/well) for response to each Ag presented by DCs (1 x 104 cells/well) or irradiated PBMC (105 cells/well) as indicated. These secondary stimulation proliferation assays were incubated for 48 h, and [3H]thymidine was added during the final 18 h of incubation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To date, there is limited information regarding the processing and presentation of disease-related autoantigens and the mechanisms regulating autoreactive T cell responses. In this regard, there are several significant findings in the present work. First, two forms of the autoantigen Topo I were very different in their ability to induce T cell proliferation. We found that a fragmented form (the MBP-Topo I mix) efficiently elicited a response, but a full-length form requires the addition of exogenous IL-2 (Fig. 2Go, A–C). Second, even in the presence of IL-2, T cell responses to rTopo I were much lower when presented by DC vs PBMC APCs (Fig. 2GoD). Finally, the two forms of Ag (native full-length vs fragmented) appear to be processed differently, with each resulting in a unique set of antigenic peptides being presented to T cells in the context of MHC class II. T cells primed with one Ag preparation were completely unable to subsequently respond to the second preparation (Fig. 6Go). DCs and PBMC APCs (monocytes and B cells) also appear to process the Topo I Ags differently, because T cells stimulated by one APC type could not subsequently respond to the same Ag presented by a different APC (Fig. 8Go).

These findings raise questions about which form of Ag is important in vivo to initiate and perpetuate autoreactive T and B cell responses in SSc (31). Our results of increased proliferative responses to Topo I fragments and the requirement for IL-2 to induce response to full-length native Topo I suggest that cryptic epitopes generated by processing fragmented Topo I may be involved in autoimmune responses in vivo. This is supported by our previous observation that T cells from SSc patients stimulated in vitro with the MBP-Topo I mix had rapid proliferation kinetics (peak proliferation at 3–5 days after stimulation) compared with healthy controls (9).

Self proteins do not normally elicit immune responses, a state that is known as immunologic tolerance. For example, in the case of T cell-mediated diseases such as multiple sclerosis or SSc, breaking of immunologic tolerance appears to involve the activation of autoreactive T cells that are present in the peripheral circulation of otherwise healthy adults (9, 10). In animal models of autoimmune disease, such as experimental allergic encephalomyelitis and collagen-induced arthritis, tolerance is broken by activating T cells present in the repertoire by immunization with appropriate Ags under inflammatory conditions (37, 38, 39, 40, 41). In the case of human autoimmune diseases, many factors have been proposed to mediate the breakage of T cell tolerance including cross-reactive microbial proteins or superantigens, environmental irritants, nonspecific tissue injury with exposure of normally cryptic Ags, and modification of protein Ags, which renders them immunogenic (3, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26). Several proteins associated with autoimmune disorders are, for example, cleaved during apoptosis (17, 19, 20, 23). However, simply being cleaved may not be adequate to induce immunogenicity of an autoantigen. The pattern of cleavage may be very important. For instance, the altered cleavage of autoantigens has been proposed in SSc as the result of ischemia/reperfusion and apoptosis and, thus, could be involved in initiation and progression of this disease (18, 19). Indeed, the fragmented form of Topo I used in our studies was far more immunogenic in vitro than the full-length protein. Preliminary experiments in which rTopo I was chemically cleaved by cyanogen bromide (42) and presented by DCs failed to demonstrate a T cell proliferative response (data not shown), but, again, cleavage at specific sites may be required for immunogenicity (18, 19). Therefore, the fragmented Topo I mix Ag may mimic the cleavage necessary for T cell responses to occur.

DCs are now generally accepted to be the most potent APC yet described (29) and are believed to play a major role in the initiation of immune responses, including autoimmune responses (43, 44). The full-length Topo I Ag used in these studies was generated using a baculovirus expression system and has properties of the native form of the protein (36). Therefore, it was unexpected that DCs should fail to stimulate T cell proliferation when this full-length form of Topo I was used as Ag (Fig. 2GoA), which was in contrast to the strong proliferative response to the Topo I fragments. These observations are not entirely without precedent because autoreactive T cells have been described that are capable of responding to synthetic peptides derived from autoantigens but not to the whole native form of the Ags from which they are derived (45). In defining the reason for the different proliferative responses to rTopo I and the MBP-Topo I mix Ags, an important initial consideration was to eliminate any potential contribution of the MBP fusion partner to the immunogenicity of the MBP-Topo I mix fragments. It was possible that MBP mediated the uptake of the MBP-Topo I mix Ag into APC, and that this effect was absent in the case of the rTopo I Ag. However, we found that MBP did not alter immune responses to the Ags used in these studies regardless of whether it was present in soluble form or covalently linked to the Ag (Fig. 3Go, B and C). Our findings are consistent with the concept that there are multiple pathways by which protein Ags are taken up by DCs, resulting in potentially different immune responses (29, 46, 47, 48, 49).

T cell responses to rTopo I were detected only when exogenous IL-2 was added to cultures, regardless of whether DCs (Fig. 2GoB) or PBMCs (Fig. 2GoC) were used as APC; however, the addition of IL-2 was no longer required in secondary stimulations (Figs. 6Go and 8Go). There are at least two possible mechanisms by which T cell responses could have been elicited to rTopo I by IL-2. First, T cells specific for rTopo I may be tolerized in vivo. Most autoreactive T cells are thought to undergo clonal deletion in the thymus. This process is not completely efficient as evidenced by the presence of autoantigen-specific T cells in the peripheral blood of healthy individuals (9, 10). However, the fact that the incidence of autoimmune diseases is quite low in general strongly suggests that some mechanism(s) of peripheral tolerance prevents these autoreactive cells from having deleterious effects. Such "tolerized" T cells may be activated from their anergized state by treatment with IL-2, a concept supported by animal models (32, 33, 34, 35).

The second possible mechanism explaining the role IL-2 involves the expansion of a small number of nontolerized Ag-specific T cells, which could not be detected in the absence of the cytokine. Against this hypothesis was the magnitude of the Ag-specific T cell proliferative response in cultures containing rTopo I and IL-2, which was similar to the response to tetanus toxoid. It seems unlikely that a small number of responder T cells that could not produce adequate amounts of IL-2 to provide an accessory signal for proliferation would expand in the presence of IL-2 over a 2-day period to yield [3H]thymidine incorporation comparable to a recall Ag. It may be possible to clarify these issues in future studies by examining the responses of naive vs memory T cells to the full-length Ag. Tolerized T cells would be expected to have a memory phenotype, and expansion of Topo I-specific cells from this population would indicate that they had previously encountered the Ag but were now tolerized.

In summary, we have shown that T cell responses to Topo I vary depending upon the nature of the Ag and the APC type that presents it. The mechanism by which Topo I-specific T cells do not normally become activated in vivo but do so in the setting of SSc is not known. However, our present data suggest that a specific form of Ag and APC type may be preferentially involved in the autoimmune pathogenesis of SSc.


   Acknowledgments
 
We thank Drs. Olivera Finn and Elizabeth Hiltbold for assistance with DC culture, Dr. Joseph Ahearn and Jeanine Navritil for assistance with flow cytometry, Dr. Thomas A. Medsger, Jr., and Carol Blair, R.N., for assistance with obtaining blood samples, and Drs. Dana Ascherman and Carol Feghali for thoughtful reading of the manuscript and helpful discussions.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants PO1-CA73743 and RO1-AR46764. T.B.O. was supported by an National Research Service Award Institutional Training Grant. Back

2 Address correspondence and reprint requests to Dr. Timothy M. Wright, S711 Biomedical Science Tower, 3500 Terrace Street, University of Pittsburgh, Pittsburgh, PA 15261. Back

3 Abbreviations used in this paper: SSc, systemic sclerosis; Topo I, DNA topoisomerase I; DC, dendritic cell; MBP, maltose binding protein; [3H]thymidine, [methyl-3H]thymidine; RPMI/huAB, RPMI 1640 plus 10% heat-inactivated human AB serum and penicillin/streptomycin; rTopo I, full-length recombinant baculovirus-derived Topo I. Back

Received for publication October 30, 2000. Accepted for publication February 22, 2001.


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