Autoreactive T-Cell Responses in Primary Biliary Cirrhosis Are Proinflammatory Whereas Those of Controls Are Regulatory
2006; Elsevier BV; Volume: 131; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2006.05.056
ISSN1528-0012
AutoresShinji Shimoda, Fumihiko Ishikawa, Takashi Kamihira, Atsumasa Komori, Hiroaki Niiro, Eishi Baba, Kenichi Harada, Kumiko Isse, Yasuni Nakanuma, Hiromi Ishibashi, M. Eric Gershwin, Mine Harada,
Tópico(s)T-cell and B-cell Immunology
ResumoBackground & Aims: Autoreactive T cells that proliferate in response to autoantigens are found in both autoimmune disease and controls but have important qualitative differences in relative activation states, costimulation signal requirements, and pathogenetic significance. Understanding the mechanism for activation of autoreactive T cells will be critical in the treatment of autoimmune diseases. Methods: To understand the differences between autoreactive T cells in primary biliary cirrhosis (PBC) versus controls, we have developed autoreactive T-cell clones (TCCs) from patients with PBC and healthy controls and have used a peptide corresponding to the CD4 major autoepitope to define the relative proliferative and cytokine response. Results: Using an enzyme-linked immunosorbent spot assay, peripheral blood mononuclear cells (PBMCs) from PBC, but not from controls, produce interferon (IFN)-γ regardless of whether costimulation-competent or -incompetent antigen-presenting cells (APC) were used. In contrast, a significant number of IFN-γ–producing cells were found in PBMCs from controls but only if costimulation-competent PBMCs presented an autoantigenic peptide. In addition, costimulation-dependent autoreactive TCCs became anergic after a single round of stimulation in the presence of APC that did not provide a costimulatory signal, whereas some costimulation-independent autoreactive TCCs required repeated stimulation to become anergic and the others did not become anergic. Finally, anergic TCCs produced interleukin-10, but no IFN-γ, and exhibited regulatory functions in an antigen-dependent, cell contact–independent, and partially interleukin-10–mediated manner. Conclusions: These data relate specifically to the functional characteristics of autoreactive T cells in PBC but are also generically important for understanding the mechanisms for generating pathogenetic autoreactive T cells. Background & Aims: Autoreactive T cells that proliferate in response to autoantigens are found in both autoimmune disease and controls but have important qualitative differences in relative activation states, costimulation signal requirements, and pathogenetic significance. Understanding the mechanism for activation of autoreactive T cells will be critical in the treatment of autoimmune diseases. Methods: To understand the differences between autoreactive T cells in primary biliary cirrhosis (PBC) versus controls, we have developed autoreactive T-cell clones (TCCs) from patients with PBC and healthy controls and have used a peptide corresponding to the CD4 major autoepitope to define the relative proliferative and cytokine response. Results: Using an enzyme-linked immunosorbent spot assay, peripheral blood mononuclear cells (PBMCs) from PBC, but not from controls, produce interferon (IFN)-γ regardless of whether costimulation-competent or -incompetent antigen-presenting cells (APC) were used. In contrast, a significant number of IFN-γ–producing cells were found in PBMCs from controls but only if costimulation-competent PBMCs presented an autoantigenic peptide. In addition, costimulation-dependent autoreactive TCCs became anergic after a single round of stimulation in the presence of APC that did not provide a costimulatory signal, whereas some costimulation-independent autoreactive TCCs required repeated stimulation to become anergic and the others did not become anergic. Finally, anergic TCCs produced interleukin-10, but no IFN-γ, and exhibited regulatory functions in an antigen-dependent, cell contact–independent, and partially interleukin-10–mediated manner. Conclusions: These data relate specifically to the functional characteristics of autoreactive T cells in PBC but are also generically important for understanding the mechanisms for generating pathogenetic autoreactive T cells. T cells reactive to major autoantigens can be detected in healthy subjects, making it difficult to determine their pathogenetic role.1Scholz C. Patton K.T. Anderson D.E. Freeman G.J. Hafler D.A. Expansion of autoreactive T cells in multiple sclerosis is independent of exogenous B7 costimulation.J Immunol. 1998; 160: 1532-1538PubMed Google Scholar, 2Lovett-Racke A.E. Trotter J.L. Lauber J. Perrin P.J. June C.H. Racke M.K. Decreased dependence of myelin basic protein-reactive T cells on CD28-mediated costimulation in multiple sclerosis patients. A marker of activated/memory T cells.J Clin Invest. 1998; 101: 725-730Crossref PubMed Scopus (236) Google Scholar, 3Viglietta V. Kent S.C. Orban T. Hafler D.A. GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes.J Clin Invest. 2002; 109: 895-903Crossref PubMed Scopus (168) Google Scholar Recent data, however, indicate that autoreactive T cells from patients with type 1 diabetes mellitus or multiple sclerosis differ from those of healthy subjects in their state of activation; autoreactive T cells from patients are markedly less dependent on costimulation, suggesting that they are activated/memory cells.1Scholz C. Patton K.T. Anderson D.E. Freeman G.J. Hafler D.A. Expansion of autoreactive T cells in multiple sclerosis is independent of exogenous B7 costimulation.J Immunol. 1998; 160: 1532-1538PubMed Google Scholar, 2Lovett-Racke A.E. Trotter J.L. Lauber J. Perrin P.J. June C.H. Racke M.K. Decreased dependence of myelin basic protein-reactive T cells on CD28-mediated costimulation in multiple sclerosis patients. A marker of activated/memory T cells.J Clin Invest. 1998; 101: 725-730Crossref PubMed Scopus (236) Google Scholar, 3Viglietta V. Kent S.C. Orban T. Hafler D.A. GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes.J Clin Invest. 2002; 109: 895-903Crossref PubMed Scopus (168) Google Scholar Evidence has also been accumulated that regulatory T cells capable of actively suppressing autoreactive T-cell responses play a pivotal role in preventing autoimmune diseases.4Roncarolo M.G. Bacchetta R. Bordignon C. Narula S. Levings M.K. Type 1 T regulatory cells.Immunol Rev. 2001; 182: 68-79Crossref PubMed Scopus (733) Google Scholar, 5Sakaguchi S. Sakaguchi N. Shimizu J. Yamazaki S. Sakihama T. Itoh M. Kuniyasu Y. Nomura T. Toda M. Takahashi T. Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance.Immunol Rev. 2001; 182: 18-32Crossref PubMed Scopus (1388) Google Scholar, 6Shevach E.M. CD4+ CD25+ suppressor T cells more questions than answers.Nat Rev Immunol. 2002; 2: 389-400Crossref PubMed Scopus (1937) Google Scholar A variety of T-cell subsets that exert this suppressor activity have been implicated: (1) several types of CD4+ CD25+ T cells, (2) Th3 cells that secrete high levels of transforming growth factor (TGF)-β, and (3) Tr1 cells characterized by the production of interleukin (IL)-10 and TGF-β. Such cells are naturally present at a low frequency but can also be generated from mature T-cell populations under certain conditions of antigenic stimulation; the term "adaptive" regulatory T cells has been proposed for the latter set.7Bluestone J.A. Abbas A.K. Natural versus adaptive regulatory T cells.Nat Rev Immunol. 2003; 3: 253-257Crossref PubMed Scopus (1179) Google Scholar Previously we reported that the E2 component of pyruvate dehydrogenase complex (PDC-E2) 163–176 restricted by HLA-DR53 is the major autoepitope for CD4 T cells in primary biliary cirrhosis (PBC).8Shimoda S. Nakamura M. Ishibashi H. Hayashida K. Niho Y. HLA DRB4 0101-restricted immunodominant T cell autoepitope of pyruvate dehydrogenase complex in primary biliary cirrhosis evidence of molecular mimicry in human autoimmune diseases.J Exp Med. 1995; 181: 1835-1845Crossref PubMed Scopus (273) Google Scholar We also noted that, in patients with PBC, the precursor frequency of costimulation-independent CD4+ T cells that respond to this epitope is markedly elevated.9Kamihira T. Shimoda S. Harada K. Kawano A. Handa M. Baba E. Tsuneyama K. Nakamura M. Ishibashi H. Nakanuma Y. Gershwin M.E. Harada M. Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis.Gastroenterology. 2003; 125: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar We took advantage of our previously generated and stored T-cell clones (TCCs), as well as 3 additional clones produced herein, to rigorously address qualitative issues of these unique reagents. We report herein that costimulation-dependent TCCs stimulated without provision of the costimulatory signal do not proliferate but produce high levels of IL-10 but neither interferon (IFN)-γ nor IL-4. Indeed, qualitative characteristics that distinguish autoreactive T cells from patients with PBC compared with those from healthy controls are dependent on the costimulation signals provided by antigen-presenting cells (APCs). A total of 16 subjects were studied, including 8 consecutive but random patients with PBC (stage I/II disease) and 8 age- and sex-matched healthy controls. The diagnosis of PBC was based on international criteria, and all patients showed antimitochondrial antibodies and had previously undergone a liver biopsy.10Kaplan M.M. Primary biliary cirrhosis.N Engl J Med. 1996; 335: 1570-1580Crossref PubMed Scopus (504) Google Scholar, 11Scheuer P. Primary biliary cirrhosis.Proc R Soc Med. 1967; 60: 1257-1260PubMed Google Scholar From this group, we successfully generated a total of 9 TCCs from patients with PBC and 8 TCCs from controls. As described in the following text, of these 17 clones, 14 were produced and initially described in our earlier work.9Kamihira T. Shimoda S. Harada K. Kawano A. Handa M. Baba E. Tsuneyama K. Nakamura M. Ishibashi H. Nakanuma Y. Gershwin M.E. Harada M. Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis.Gastroenterology. 2003; 125: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar We have previously shown that the amino acid region 163–176 of PDC-E2, which is the major mitochondrial autoantigen of PBC, is the CD4-specific epitope. The PDC-E2 163–176 peptide (GDLLAEIETDKATI) was synthesized by F-moc chemistry using a peptide synthesizer (Model Synergy; Applied Biosystems Inc, Foster City, CA) and the peptide purified by reverse-phase high-pressure liquid chromatography; the purity was more than 90% as determined by high-pressure liquid chromatography. Purified protein derivative (PPD) was purchased from Nippon BCG Production Co (Tokyo, Japan). Peripheral blood mononuclear cells (PBMCs), prepared from heparinized venous blood samples by Ficoll gradient separation, were plated at a concentration of 1 × 106 cells/well into 24-well plates and cultured with irradiated autologous PBMCs (2 × 106 cells/well) or mitomycin C (MCC)-treated HLA-DR53–transfected mouse L cells (L-DR53 cells, a gift from Prof Y. Nishimura, Kumamoto University, Kumamoto, Japan) (3.5 × 105 cells/well) that were either pulsed or nonpulsed with PDC-E2 163–176 peptide. After 3 days of culture, viable T cells were harvested and ELISPOT assays were performed (BD ELISPOT Set, Human IFN-γ ELISPOT Set, Human IL-4 ELISPOT Set, Human IL-10 ELISPOT Set; BD Bioscience, San Diego, CA). Briefly, 96-well nitrocellulose plates were coated with an optimized capture monoclonal antibody (human anti–IFN-γ, anti–IL-4, or anti–IL-10 antibody) in phosphate-buffered saline and incubated overnight at 4°C. Unbound antibody was removed by washing with phosphate-buffered saline containing 0.05% Tween (PBS Tween). Viable T cells were added at 3 × 105 cells/well in 100 μL RPMI in triplicate. The plates were incubated at 37°C, 5% co2 for 24 hours, and then antigen stimulation was stopped. The plates were then washed; labeled with biotin-labeled anti–IFN-γ, anti–IL-4, or anti–IL-10; and developed by incubation with streptavidin/alkaline phosphatase, followed by incubation with final substrate solution (BD AEC Substrate Reagent Set). The reaction was stopped by rinsing the content with distilled water, and the number of spots was counted by using a KS ELISPOT Reader (Zeiss, Thornwood, NY). Known positive and negative samples were included throughout, and all 16 subjects were studied. In addition, antigen-specific spot-forming cells were quantitated as spot-forming cells in the presence of antigen minus spot-forming cells in the absence of antigen. The average number of spot-forming cells in the absence of antigen was less than 10 (data not shown). TCCs were developed according to our method previously described.8Shimoda S. Nakamura M. Ishibashi H. Hayashida K. Niho Y. HLA DRB4 0101-restricted immunodominant T cell autoepitope of pyruvate dehydrogenase complex in primary biliary cirrhosis evidence of molecular mimicry in human autoimmune diseases.J Exp Med. 1995; 181: 1835-1845Crossref PubMed Scopus (273) Google Scholar, 9Kamihira T. Shimoda S. Harada K. Kawano A. Handa M. Baba E. Tsuneyama K. Nakamura M. Ishibashi H. Nakanuma Y. Gershwin M.E. Harada M. Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis.Gastroenterology. 2003; 125: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Briefly, PBMCs were prepared in RPMI 1640 supplemented with 2 mmol/L l-glutamine, 50 ng/mL gentamycin, and 10% human AB positive sera (complete RPMI). Finally PDC-E2 163–176 peptide was added (10 μg/mL). After 7 days, the cells in each well were equally split and cultured with 5 × 104 freshly prepared and irradiated autologous PBMCs pulsed with PDC-E2 peptide 163–176 (10 μg/mL) or without any antigen for 72 hours. Thereafter, the cells were pulsed with 1 μCi of 3H-thymidine (3H-TdR)/well during the final 12 hours of culture and subsequently harvested with an automated cell harvester (Betaplate 1295-004; Pharmacia Fine Chemicals, Piscataway, NJ). 3H-TdR uptake was measured using a beta scintillation counter (Betaplate 1205; Pharmacia Fine Chemicals). T-cell lines were considered positive for proliferation when the Δ cpm (cpm values of T cells with irradiated autologous PBMCs pulsed with the PDC-E2 163–176 peptide minus cpm values of T cells with irradiated autologous PBMC alone) was more than 1000 cpm and the cpm values of T cells with the PDC-E2 163–176 peptide pulsed irradiated autologous PBMCs exceeded the cpm values of T cells with irradiated autologous PBMCs alone by at least 3-fold. T-cell lines showing positive proliferation in response to PDC-E2 163–176 were cloned at 1 cell/well in complete RPMI containing IL-2 (100 U/mL), in the presence of autologous irradiated (3000 rad) PBMCs as feeders and PDC-E2 163–176. A similar protocol was developed for TCCs responsive to PPD. In all cases, the established TCCs were expanded for use in several assays and the assays were performed in triplicate. TCCs were seeded at 5 × 104 cells/well in 96-well round-bottomed plates with irradiated (3000 rad) autologous PBMCs (1 × 105 cells/well) or MMC-treated L-DR53 cells (1 × 104 cells/well) as APCs in the presence or absence of the PDC-E2 163–176 peptide (final concentration of PDC-E2, 10 μg/mL) for 72 hours in the presence of 1 μCi of 3H-TdR/well during the final 12 hours. The cells were then harvested, and 3H-TdR incorporation was measured in a beta scintillation counter. TCCs were seeded at 1 × 106 cells/well in 24-well plates with irradiated (3000 rad) autologous PBMCs (1 × 106 cells/well) or MMC-treated L-DR53 cells (1 × 105 cells/well) as APCs in the presence or absence of the PDC-E2 163–176 peptide (final concentration of PDC-E2, 10 μg/mL) for 72 hours, and the supernatants were harvested. The supernatants were evaluated for either suppressor function or cytokine production. First, cytokine production was evaluated by a sandwich enzyme-linked immunosorbent assay, using a combination of unlabeled and biotin- or enzyme-coupled monoclonal antibody to IFN-γ, IL-2, IL-4, or IL-10 (all monoclonal antibodies from BD PharMingen). Second, suppressor function was assayed as noted in the following text on the evaluation of anergy/regulation. TCCs (5 × 106 cells/well) were cocultured with the PDC-E2 163–176 peptide (10 μg/mL) pulsed MMC-treated L-DR53 cells (5 × 105 cells/well) in a 6-well plate for 3 days. TCCs that were stimulated once with antigen (Ag)-pulsed L-DR53 cells were designated as L(1) state of TCCs; these cells were washed 3 times with phosphate-buffered saline and proliferative responses of 5 × 104 cells/well determined using PDC-E2 163–176 pulsed or nonpulsed irradiated autologous PBMCs (1 × 105/well) or MMC-treated L-DR53 cells (1 × 104/well) as APCs for 3 days in the presence of 1 μCi/well of 3H-TdR during the final 12 hours. In nested experiments, the L(1) state of TCCs were harvested and restimulated again with PDC-E2 peptide-pulsed MMC-treated L-DR53 cells for another 3 days; these cells were designated as the L(2) state of TCCs. The viable cells from the cultures were separated by Ficoll gradient and proliferative responses of the L(2) state of TCCs assessed using PDC-E2 163–176 peptide pulsed or nonpulsed irradiated autologous PBMCs (1 × 105/well) or MMC-treated L-DR53 cells (1 × 104/well) as APCs for 3 days in the presence of 1 μCi of 3H-TdR/well during the final 12 hours. The ability of anergic TCCs to regulate the proliferation of TCCs was assessed by coculturing 1 × 105 of the L(1) or L(2) state of TCCs/well with 1 × 105 of their corresponding TCCs in the presence of PDC-E2 peptide pulsed autologous irradiated PBMCs as APCs for 72 hours in the presence of 1 μCi of 3H-TdR/well during the final 12 hours. To determine whether the regulatory function of anergic TCCs required cellular interactions, the same experiments were performed with irradiated (3000 rad) or nonirradiated anergic TCCs. To assess whether soluble factors were responsible for the regulatory activity of anergic TCCs, the cell-free supernatants were harvested when anergy was induced, that is, after 3 days of culture of costimulation-dependent TCCs in the presence of Ag-pulsed MMC-treated L-DR53 cells. The proliferation of TCCs was assessed by coculturing 1 × 105 anergic TCCs/well or 50 μL of supernatants/well from the same anergic TCCs with 1 × 105 corresponding TCCs/well in the presence of PDC-E2 peptide pulsed autologous irradiated PBMCs (1 × 105 cells/well) as APCs for 72 hours in the presence of 1 μCi of 3H-TdR/well during the final 12 hours. In nested experiments, anti–IL-10 antibody (R&D Systems, Minneapolis, MN) was added with the supernatants at a final concentration of 10 μg/mL to investigate the function of IL-10 in the suppressor activity. Finally, to assess the antigen specificity of the regulatory function, in the presence or absence of PDC-E2 163–176 peptide, 1 × 105 anergic TCCs/well were cocultured with 1 × 105 PPD-responsive TCCs/well from the same subject with PPD pulsed autologous irradiated PBMCs as APCs. All assays were performed in triplicate, and the values between groups were compared using the Mann–Whitney U test. In all cases, the number of subjects studied in each assay was at least 4. Results of the ELISPOT assay for individual patients with PBC and controls are summarized in Figure 1. In particular, Figure 1 shows that the mean number (±SEM) of IFN-γ–secreting T cells is similar among patients with PBC whether they were pulsed with either autologous PBMCs or L-DR53 cells (P = .22). However, the number of IFN-γ–secreting T cells that recognize the same epitope in controls, when they were pulsed with autologous PBMCs, was significantly higher compared with pulsing with L-DR53 cells as APCs (P < .01). The number of IFN-γ–secreting T cells pulsed with autologous PBMCs was not significantly different between patients and controls (P = .35). In contrast, there were striking differences in the median number of IFN-γ–producing cells when the APCs were L-DR53 cells (P < .01). We also noted that the median number of IL-10– or IL-4–secreting T cells recognizing the autoepitope was similar whether they were pulsed with autologous PBMCs or L-DR53 cells in patients with PBC and controls (Figure 1). Hence, PBMCs from patients with PBC produced both IFN-γ and IL-10 regardless of whether costimulation-competent or -incompetent APCs were used to present PDC-E2 163–176. There were no clinical correlations with the data based in some measure on the difficulty and labor-intensive nature of developing these TCCs; a much larger patient population would be required. PBMCs from controls also contained IL-10–secreting cells regardless of the type of APC, but a significant number of IFN-γ–producing cells were only detected if costimulation-competent PBMCs presented PDC-E2 163–176. The number of IL-4–producing cells was very low in both patients with PBC and controls. TCCs were established from PBMCs from 4 healthy subjects as controls and from 7 patients with PBC. Eight TCCs were established from these 4 healthy controls and 9 from these 7 patients with PBC; all TCCs were restricted to HLA-DR53 as previously reported.8Shimoda S. Nakamura M. Ishibashi H. Hayashida K. Niho Y. HLA DRB4 0101-restricted immunodominant T cell autoepitope of pyruvate dehydrogenase complex in primary biliary cirrhosis evidence of molecular mimicry in human autoimmune diseases.J Exp Med. 1995; 181: 1835-1845Crossref PubMed Scopus (273) Google Scholar Of these 17 clones, 14 were produced and initially described in our earlier work.9Kamihira T. Shimoda S. Harada K. Kawano A. Handa M. Baba E. Tsuneyama K. Nakamura M. Ishibashi H. Nakanuma Y. Gershwin M.E. Harada M. Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis.Gastroenterology. 2003; 125: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Based on their ability to proliferate in the presence of antigen-pulsed L-DR53 cells, 5 of 8 of the TCCs from controls were classified as costimulation dependent (these were referred to as TCC dependent 1 from control 5, TCC dependent 2 from control 7, TCC dependent 3 from control 8, TCC dependent 4 from control 7, and TCC dependent 5 from control 8, as shown in Table 1) and 3 of 8 of the TCCs from controls were classified as costimulation independent (referred to as TCC independent 7 from control 3, TCC independent 8 from control 5, and TCC independent 9 from control 5, as shown in Table 2). Using the same criteria, 6 of 9 TCCs from patients with PBC were classified as costimulation independent (referred to as TCC independent 1 from PBC 4, TCC independent 2 from PBC 6, TCC independent 3 from PBC 1, TCC independent 4 from PBC 1, TCC independent 5 from PBC 8, and TCC independent 6 from PBC 2, as shown in Table 2, Table 3), while 3 were costimulation dependent (TCC dependent 6 from PBC 8, TCC dependent 7 from PBC 3, and TCC dependent 8 from PBC 7, as shown in Table 1). Hence, we developed from controls one costimulation-dependent TCC and 2 costimulation-independent TCCs from control 5, 2 costimulation-dependent TCCs from controls 7 and 8, and one costimulation-independent TCC from control 3.Table 1Profile of Costimulation-Dependent TCCsFirst cultureL(1)L(2)PBMCL-DR53PBMCL-DR53PBMCL-DR53Controls TCC dependent 1 (MN29)aThe nomenclature of the clones in parentheses corresponds to the original name designated in reference 9; newly established clones are so indicated. Proliferation (cpm)18,250510522420NTNT IFN-γ (pg/mL)48155027353NTNT IL-10 (pg/mL)3920373335032226NTNT IL-4 (pg/mL)22317NTNTNTNT TCC dependent 2 (IY46) Proliferation (cpm)21,2121008721631NTNT IFN-γ (pg/mL)38511828363NTNT IL-10 (pg/mL)7451495226152897NTNT IL-4 (pg/mL)18633NTNTNTNT TCC dependent 3 (TT16) Proliferation (cpm)15,736381361826NTNT IFN-γ (pg/mL)4213118103183NTNT IL-10 (pg/mL)5214384147194715NTNT IL-4 (pg/mL)14329NTNTNTNT TCC dependent 4 (IY65) Proliferation (cpm)9632381427309NTNT IFN-γ (pg/mL)824719172NTNT IL-10 (pg/mL)9371114683707NTNT IL-4 (pg/mL)35121NTNTNTNT TCC dependent 5 (TT9) Proliferation (cpm)16,522592825631NTNT IFN-γ (pg/mL)100611092113NTNT IL-10 (pg/mL)1102902742808NTNT IL-4 (pg/mL)9725NTNTNTNTPBC TCC dependent 6 (TY1) Proliferation (cpm)20,150892721838NTNT IFN-γ (pg/mL)38169288137NTNT IL-10 (pg/mL)4927400238724312NTNT IL-4 (pg/mL)27152NTNTNTNT TCC dependent 7 (TM1) Proliferation (cpm)8267631244190NTNT IFN-γ (pg/mL)942645231NTNT IL-10 (pg/mL)11739521078731NTNT IL-4 (pg/mL)8227NTNTNTNT TCC dependent 8 (HK15) Proliferation (cpm)10,963703396281NTNT IFN-γ (pg/mL)10737211183NTNT IL-10 (pg/mL)9581092703797NTNT IL-4 (pg/mL)19311NTNTNTNTNT, not tested.a The nomenclature of the clones in parentheses corresponds to the original name designated in reference 9Kamihira T. Shimoda S. Harada K. Kawano A. Handa M. Baba E. Tsuneyama K. Nakamura M. Ishibashi H. Nakanuma Y. Gershwin M.E. Harada M. Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis.Gastroenterology. 2003; 125: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar; newly established clones are so indicated. Open table in a new tab Table 2Profile of Costimulation Relative Independent TCCsFirst cultureL(1)L(2)PBMCL-DR53PBMCL-DR53PBMCL-DR53PBC TCC independent 1 (new clone)aThe nomenclature of the clones in parentheses corresponds to the original name designated in reference 9; newly established clones are so indicated. Proliferation (cpm)39,98029,7306411316448499 IFN-γ (pg/mL)556835811748138460 IL-10 (pg/mL)448654013409440124352941 IL-4 (pg/mL)978274NTNTNTNT TCC independent 2 (new clone) Proliferation (cpm)37,46222,17012,743284721508 IFN-γ (pg/mL)31822045326211869102 IL-10 (pg/mL)829447254621273520511795 IL-4 (pg/mL)421111NTNTNTNT TCC independent 3 (RS9) Proliferation (cpm)41,19238,79219,20611001205736 IFN-γ (pg/mL)69634851274810919283 IL-10 (pg/mL)715258904396390030122741 IL-4 (pg/mL)736281NTNTNTNT TCC independent 6 (HT7) Proliferation (cpm)20,74317,36217,4721174732462 IFN-γ (pg/mL)251128522073184211493 IL-10 (pg/mL)264122091793200711121090 IL-4 (pg/mL)328220NTNTNTNTControls TCC independent 7 (HH1) Proliferation (cpm)42,76140,10921,12919788521074 IFN-γ (pg/mL)58254485384111418396 IL-10 (pg/mL)638242104735386419462743 IL-4 (pg/mL)21489NTNTNTNT TCC independent 8 (MN3) Proliferation (cpm)19,35217,20316,392992660461 IFN-γ (pg/mL)193511741831119210388 IL-10 (pg/mL)19442001193215331024925 IL-4 (pg/mL)365231NTNTNTNT TCC independent 9 (MN5) Proliferation (cpm)18,25615,34216,2511321451583 IFN-γ (pg/mL)221519251775143692117 IL-10 (pg/mL)193620611437173510021189 IL-4 (pg/mL)28782NTNTNTNTNT, not tested.a The nomenclature of the clones in parentheses corresponds to the original name designated in reference 9Kamihira T. Shimoda S. Harada K. Kawano A. Handa M. Baba E. Tsuneyama K. Nakamura M. Ishibashi H. Nakanuma Y. Gershwin M.E. Harada M. Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis.Gastroenterology. 2003; 125: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar; newly established clones are so indicated. Open table in a new tab Table 3Profile of Costimulation Complete Independent TCCsFirst cultureL(1)L(2)PBMCL-DR53PBMCL-DR53PBMCL-DR53PBC TCC independent 4 (RS5)aThe nomenclature of the clones in parentheses corresponds to the original name designated in reference 9; newly established clones are so indicated. Proliferation (cpm)48,25142,31721,76215,28218,2119,218 IFN-γ (pg/mL)528648743118281431822842 IL-10 (pg/mL)622054974912401221853287 IL-4 (pg/mL)653423NTNTNTNT TCC independent 5 (new clone) Proliferation (cpm)38,92742,08535,08130,87920,00817,362 IFN-γ (pg/mL)396440023746194727352840 IL-10 (pg/mL)486550802984373630042147 IL-4 (pg/mL)432238NTNTNTNTNT, not tested.a The nomenclature of the clones in parentheses corresponds to the original name designated in reference 9Kamihira T. Shimoda S. Harada K. Kawano A. Handa M. Baba E. Tsuneyama K. Nakamura M. Ishibashi H. Nakanuma Y. Gershwin M.E. Harada M. Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis.Gastroenterology. 2003; 125: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar; newly established clones are so indicated. Open table in a new tab NT, not tested. NT, not tested. NT, not tested. In PBC, one costimulation-independent TCC was established from each of 3 patients with PBC (patients 2, 4, and 6) and 2 costimulation-independent TCCs from one patient with PBC (patient 1), one costimulation-dependent TCC from each of 2 patients with PBC (patients 3 and 7), and one costimulation-dependent and one costimulation-independent TCC from one patient with PBC (patient 8). Hence, in the data herein, our newly established TCC included TCC independent 1, 2, and 5; the other TCCs were based on previously established clones.9Kamihira T. Shimoda S. Harada K. Kawano A. Handa M. Baba E. Tsuneyama K. Nakamura M. Ishibashi H. Nakanuma Y. Gershwin M.E. Harada M. Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis.Gastroenterology. 2003; 125: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Our ability to establish TCCs was equal in all groups. IFN-γ, IL-4, and IL-10 production was measured by enzyme-linked immunosorbent assay using cell-free supernatants after stimulation with the PDC-E2 163–176 peptide in the presence of irradiated (3000 rad) autologous PBMCs or MMC-treated L-DR53 as APCs. All costimulation-dependent TCCs (TCC dependent 1–8) produced considerable IFN-γ and IL-10, but little IL-4, in the presence of Ag-pulsed PBMCs, whereas they secreted only IL-10 in the presence of Ag-pulsed L-DR53 cel
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