Autoreactive CD4+ LKM-specific and anticlonotypic T-cell responses in LKM-1 antibody-positive autoimmune hepatitis

Autoreactive CD4/ LKM-Specific and Anticlonotypic T-Cell Responses in LKM-1 Antibody–Positive Autoimmune Hepatitis HANNS F. LO¨HR, JO¨RG F. SCHLAAK, ANSGAR W. LOHSE, WULF O. BO¨CHER, MONIKA ARENZ, GUIDO GERKEN, ¨ SCHENFELDE AND KARL-HERMANN MEYER ZUM BU

Peripheral blood mononuclear cells (PBMC) of patients with autoimmune hepatitis (AIH) and controls were studied for their proliferative response to six overlapping synthetic peptides covering the 33–amino acid immunodominant region of cytochrome P450IID6, the main target antigen of LKM-1 antibody–positive type II AIH. PBMC from 8 of 8 type II AIH patients (100%), 6 of 12 LKM-1 antibody–negative type I AIH patients (50%), but only 4 of 31 patients with chronic hepatitis C (12.9%) reacted with a 23–amino acid LKM peptide and mainly with a shorter 18–amino acid LKM peptide. Follow-up showed that LKM-specific T-cell responses decreased after immunosuppression had started. Fine specificity, HLA restriction, and cytokine release of LKM-specific T cells were analyzed with 16 CD4/ peptide-specific T-cell lines and 21 CD4/ T-cell clones isolated and expanded from blood and liver tissue of six AIH patients. Activated LKM-specific T cells released interferon gamma (IFN-g) but no or little interleukin-4. In three AIH patients, PBMC showed specific recognition of autologous LKMspecific T cells, suggesting the presence of a regulatory T-cell network. These T cells also showed the CD4/ phenotype and secreted large amounts of IFN-g. Furthermore, it was assessed that the regulatory T-cell response is clonotypic. To conclude, we describe a major T-cell epitope in AIH that was recognized by Th1 helper cells isolated from blood and liver tissue. This autoreactive T-cell response correlated widely with disease activity and LKM-1 antibody status and seemed to be regulated by anticlonotypic T cells. (HEPATOLOGY 1996;24:14161421.) Autoantibodies against intracellular structures such as nuclear and microsomal antigens, cytoskeleton, and cytosol serve as diagnostic markers in autoimmune hepatitis (AIH).1,2 Cytochrome P450IID6 is the major antigen of antibodies directed against microsomal antigens derived from liver and kidney (LKM-1) that characterize a subgroup of AIH (type II).3,4 However, LKM-1 antibodies are also found in a small proportion of patients with chronic hepatitis C

Abbreviations: AIH, autoimmune hepatitis; HCV, hepatitis C virus; IL-4, interleukin4; PBMC, peripheral blood mononuclear cell; CHC, chronic hepatitis C; CHB, chronic hepatitis B; PBC, primary biliary cirrhosis; ELISA, enzyme-linked immunosorbent assay; Ig, immunoglobulin; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; OD, optical density; PL, peptide-specific T-cell lines; LL, liver-infiltrating T-cell lines; PHA, phytohemagglutinin; mAb, monoclonal antibody; TCR, T-cell receptor; TNF-a, tumor necrosis factor a; IFN-g, interferon gamma; SI, stimulation index. From the Department of Internal Medicine, Johannes Gutenberg-University Mainz, Mainz, Germany. Received May 9, 1996; accepted July 31, 1996. Supported by the Deutsche Forschungsgemeinschaft (Lo 621/1-1 and SFB 311/A14). Address reprint requests to: Hanns F. Lo¨hr, M. D., I. Department of Internal Medicine, Johannes Gutenberg-University Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2406-0019$3.00/0

virus (HCV), and the sequence homology between HCV and LKM suggests cross-reactivity or HCV-related autoimmunity.4-7 Immunohistological studies in AIH patients showed a predominance of CD4/ T cells within dense lymphocytic infiltrations.8-10 In vitro, T-cell lines and clones derived from the blood and liver tissue of AIH patients showed a preferential activation of CD4/CD80 cells compared with the accumulation of CD40CD8/ T cells in the liver of patients with chronic active hepatitis B or C.11,12 The clonal analysis showed that in AIH patients the T cells predominantly showed the Th1-like cytokine release, but the frequency of interleukin-4 (IL-4)–producing Th2 helper T cells was also increased.12 It has been demonstrated recently that liver-infiltrating T-cell clones from patients with type II AIH recognized a recombinant LKM-1 antigen carrying the immunodominant 33–amino acid region of cytochrome P450IID6, the main target of LKM-1 antibodies.4,13 Thus, the LKM-specific T-cell response in AIH may contribute to the immunopathogenesis, although further functional characterization is needed to show pathogenetic significance. This study focused on (1) the fine specificity of the cellular immune response to main T-cell epitopes within the immunodominant LKM-1 antigen, (2) analysis of the cytokine release of T cells in response to LKM peptides, and (3) demonstration of anticlonotypic T cells that may control autoreactivity. PATIENTS AND METHODS Patients. Peripheral blood mononuclear cells (PBMCs) from 20 patients with AIH, characterized by liver histology, serum autoantibodies, hypergammaglobulinemia, and response to immunosuppressive therapy, were studied. Eight of the 20 (40%) had LKM-1 antibody– positive type II AIH, and 12 of the 20 (60%) had LKM-1 antibody– negative type I AIH showing other serum autoantibodies. In addition, 31 patients with chronic hepatitis C (CHC), 4 of 31 patients (12.9%) with LKM-1 serum antibodies, 9 patients with chronic hepatitis B (CHB), 7 patients with primary biliary cirrhosis (PBC), and 12 healthy blood donors served as controls. All diagnoses were based on clinical, serological, and histological data. The patients gave informed consent to the experiments in conformation with the Declaration of Helsinki ethical guidelines (Table 1). LKM-1 Antibody Assay. To search for antibodies against the microsomal protein LKM-1, an enzyme-linked immunosorbent assay (ELISA) was used based on the inhibition of the binding of purified LKM immunoglobulin (Ig) G isolated from a reference serum to microsomal antigens.14 The g-globulin fraction of LKM reference serum was precipitated three times by a one-third volume of saturated (NH4)2SO4 and dialyzed against 0.01 mol/L phosphate-buffered saline (PBS). Then the IgG fraction was labeled with avidine, chromatographed on a Sephadex G100 column, and adjusted to 1 mg protein/ mL. Microtiter plates were then coated with 0.1 mg of unlabeled LKM IgG before 50 mL of a crude human microsomal protein preparation was added as antigen solution, followed by an incubation step for 60 minutes at room temperature. Patient serum, 50 mL diluted in 450 mL PBS/10 mmol/L ethylenediaminetetraacetic acid (EDTA), was added and incubated for 2 hours at room temperature. Then, 50 mL of peroxidase-labeled LKM IgG was added together with 10 mL of biotinylated goat antihuman IgG (DAKO, Copenhagen, Denmark).

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TABLE 1. Clinical Data From AIH Patients and Controls Patients

Type II AIH Type I AIH PBC CHB CHC Healthy controls

n

Male (n)

Age (yr)

ALT (U/L)

g-Globulins (%)

Total Protein (g/L)

Treatment* (n)

LKM-1 Antibody Positive (n)

8 12 7 9 31 12

1 3 1 6 20 8

32.2 50.1 60.0 41.0 42.7 28.7

146.6 292.3 59.6 39.0 89.0 15.1

29.7 25.1 19.7 20.5 18.3 15.0

83.6 73.5 78.9 73.5 74.2 68.0

1 3 4 8 17 0

8 0 0 0 4 0

NOTE. Clinical data are from LKM-1 antibody–positive type II and LKM-1 antibody–negative type I patients with AIH, PBC, CHB, or CHC. * AIH patients were treated by a combination of steroids and azathioprine; PBC patients were treated with ursodesoxycholic acid. Some patients with chronic viral hepatitis B or C had received IFN-a previously.

After intensive washings, 50 mg of freshly prepared substrate 2,2*acino-bis-(3-ethylbenthiazoline-6-sulphonic acid) ammonium salt was added and the reaction was stopped after 30 minutes by 300 mL of 2 mmol sodium azide in 0.01 PBS. Finally, optical densities (OD) were measured by a Titertek Multiscan ELISA reader. T-Cell Lines and Clones. PBMCs were separated from heparinized blood by Ficoll density centrifugation. The cells at the interface were recovered and then washed twice with RPMI 1640. Liver-infiltrating T cells were derived from diagnostic liver biopsies as described.15 Briefly, 2-mm specimens were brought to 96 flat-bottom wells and incubated with 200 mL of medium for 48 hours before outgrowing T cells were harvested and washed twice. For the generation of peptidespecific T-cell lines from the peripheral blood (PL), 1 1 105 PBMCs were stimulated with 50 mg/mL peptide and 200 mL of medium in 96 round-bottom plates. Alternatively, liver-infiltrating T-cell lines (LL) were generated by the stimulation with LKM peptides and 4,000-rad irradiated autologous PBMCs serving for antigen presentation. On day 5, recombinant IL-2 (Euro-Cetus, Amsterdam) was added at a final concentration of 20 U/mL. By weekly stimulation with oxidized and irradiated allogeneic feeder cells in the presence of IL-2, the peptide-specific PL/LL were expanded.16 T-cell clones were obtained from PL or LL that had shown a proliferative response to LKM peptides before. Briefly, T cells were brought to 60-well microtiter plates in a final concentration of 0.3 cells per well together with 1 1 104 irradiated allogeneic feeder cells and medium supplemented with 10% human serum, 20 U/mL IL-2, and 50% supernatant of phytohemagglutinin (PHA) stimulated T cells. After 10 days of incubation the outgrowing T-cell clones were harvested, brought to a 96 round-bottom well plate, and further expanded by weekly splitting and restimulation with oxidized allogeneic feeder cells. Phenotypical Analysis of the LKM-Specific T Cells. Cell lines were analyzed phenotypically by indirect flow cytometry using monoclonal antibodies (mAbs) against the CD4- (OKT4) and CD8- (OKT8) molecules as well as the a/b and g/d chains of the specific T-cell receptor (TCR). After incubation with the primary antibodies the positive T cells were stained with a fluorescein-conjugated goat anti-mouse immunoglobulin antibody (Dianova, Hamburg, Germany). The flow cytometry analysis was performed in a FACScan (Becton & Dickinson, Heidelburg, Germany). Forward and side scatters were set to exclude nonvital cells and debris.11 To save cells quantitatively for further functional analysis, some LKM-specific T-cell clones were phenotyped using a single-cell ELISA as described.17

Peptides and Microsomal Antigens. Six overlapping peptides (LKM-P1 to LKM-P6) with lengths between 8 and 24 amino acids were synthesized covering the 33–amino acid (aa) immunodominant aa 254-285 region of the LKM-1 antigen.4,18 The purification of the peptide synthesis was proven by high-performance liquid chromography (HPLC) analysis (Table 2; Professor Mu¨ller-Esterl, Mainz). In previous experiments, optimal peptide concentrations for T-cell stimulation had been evaluated (10 mg/well). In addition, we used crude preparations of human microsomal liver antigens as natural LKM antigen (nLKM) to stimulate PBMC.14 mAbs and HLA-Restriction Analysis. HLA restriction was analyzed by blocking the proliferative T-cell response with antibodies to HLA class II, DR, DP, or DQ molecules.15 Hybridoma supernatants with anti-CD4 (OKT4), anti-CD8 (OKT8), anti-HLA II (DA6.231), and anti-HLA-DR (L243) mAbs were kindly provided by B. Fleischer, Hamburg, Germany. Anti-HLA-DQ, anti-TCRa/b chains, anti-TCRg/ d chains were obtained from Dianova, Hamburg, Germany. Proliferation Assay. The proliferative T-cell response was studied by culturing 1 1 105 PBMCs in triplicate in 96 round-bottom microtiter plates in the presence or absence of a synthetic LKM peptide (50 mg/mL) or crude liver microsomal antigens (5 and 15 mg/mL). Medium and PHA-induced PBMCs served as controls. After 6 days of incubation, PBMC cultures were pulsed with 25 mCi 3H-thymidine (Amersham, Braunschweig, Germany), and after another 20 hours the cells were harvested and the rate of incorporated radioactivity was determined by liquid scintillation counting. The proliferative responses of peptide-specific T-cell lines and clones were investigated similarly: 5 1 104 autologous or allogeneic irradiated PBMCs were incubated in triplicate together with 2 1 104 T cells in the presence or absence of 50 mg/mL peptide. mAbs directed against HLA class II, -DR, -DP, or -DQ molecules were added at a final dilution of 1:100 to block the specific proliferative response. After 48 hours of incubation, cell cultures were pulsed, and 20 hours later the 3H-thymidine incorporation was measured by liquid scintillation counting. Stimulation indices higher than four times the medium control were regarded as positive. Statistical analysis was carried out using Student’s t test for impaired data. Cytokine-Release Assays. To analyze the cytokine release of specifically activated liver-infiltrating and circulating T cells, 5 1 104 T cells were stimulated in triplicates in 200 mL of RPMI medium supplemented with 1% glutamine and 5% human heat-inactivated AB serum in 96 round-bottom plates together with autologous antigen-

TABLE 2. Synthetic Overlapping LKM Peptides Peptide

Position*

LKM-P1 LKM-P2 LKM-P3 LKM-P4 LKM-P5 LKM-P6 HCV-E1 HCV-NS5 HCV-core GOR 47-1

262-285 266-285 267-277 272-281 254-277 263-270

Sequence†,‡

Length†

23 18 10 10 24 8

aa aa aa aa aa aa

DPAQPPRDLTEAFLAEMEKAKGN PPRDLTEAFLAEMEKAKGN PRDLTEAFLA TEAFLAEMEK LLTEHRMTWDPAQPPRDLTEAFLA DPAQPPRD HRMAWD DPPQPEYDL KNKRNT GRRGQKAKSNP

* Amino acid position within the cytochrome P450IID6 according to Yamamoto et al.18 † Sequences and length of the synthetic LKM peptides used for analysis of the cellular immune response. ‡ Sequence homologies between HCV, GOR 47-1, and LKM according to Mishiro et al.26

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FIG. 1. Relative frequencies of patients with type I or II AIH or CHC who showed a proliferative PBMC response to LKM peptides P1-6 or the crude microsomal protein fraction (nLKM). Mean SI { SD.

presenting cells and peptide at a concentration of 10 mg/well. In some experiments, mAbs against monomorphic HLA class II and HLA-DR molecules were added. After 24 and 48 hours of incubation, 75 mL of cell culture supernatants were taken and frozen at 0207C until cytokine assays were performed. The amounts of secreted tumor necrosis factor a (TNF-a), interferon gamma (IFN-g), and IL-4 were determined by ELISA, as described recently.11,12 RESULTS Epitope Mapping. All eight LKM-1 antibody–positive type II AIH patients (100%) and six of 12 (50%) type I AIH patients showed a proliferative response to at least one LKM peptide. In detail, of eight type II AIH patients, all eight PBMCs responded to the 23–aa peptide LKM-P1 and five responded to the N-terminal–deleted 18-aa peptide LKM-P2 and the nLKM antigen. In addition, peptides LKM-P5 (24 aa) and LKM-P6 (8 aa) were recognized by two of eight PBMCs each. All six LKM-responsive type I AIH patients showed a T-cell response to LKM-P1, and four also responded to the nLKM antigen. Peptides LKM-P2 was recognized by one, LKM-P5 and -P6 by two type I AIH patients (Fig. 1). The PBMCs from seven PBC patients, nine patients with chronic hepatitis B virus, and 12 healthy blood donors did not show an LKMspecific autoreactive T-cell response. However, the T cells from four of 31 patients with CHC (12.9%), including two of four LKM-1 antibody–positive patients, showed a proliferative response to at least one LKM peptide. Two LKM-1 antibody–positive CHC patients reacted with LKM-P1, one of them also with LKM-P2 and nLKM antigen, and two further LKM-1 antibody–negative CHC patients recognized peptides LKM-P2 and -P4. The clinical data from all LKM peptide– reactive patients were listed in Table 3. The mean stimulation index (SI) of PBMCs in response to the most immunogeneic peptide LKM-P1 was higher in type II and type I AIH patients than in the LKM-reactive CHC patients (SI { S.D., 10.8 { 10.9 and 19.5 { 16.0 vs. 6.1 { 0.9; P õ .05). With regard to peptide LKM-P2, the SI was not significantly different between AIH and responsive CHC patients (6.0 { 1.0 and 4.5 { 1.5 vs. 5.2 { 2.7; P ú .05). To investigate whether the autoreactive T-cell response correlated with the clinical course LKM-P1–specific T-cell response were followed up in two LKM-1 antibody–positive AIH patients before and during immunosuppressive therapy. In both patients, the LKM-P1–specific T-cell response declined within 3 to 6 weeks paralleled by the rapid decrease of serum ALT levels (Fig. 2). HLA-Restriction Analysis. For further phenotypical and functional analysis of the antigen-specific autoreactive cellular immune response, LKM-P1– and LKM-P2–specific T-cell lines were established from the peripheral blood and the liver

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tissue of six AIH patients whose PBMCs reacted with peptides LKM-P1 and/or LKM-P2. Phenotyping by flow cytometry showed that all 16 expanded T-cell lines consisted predominantly of CD4/CD80 cells (ú90%). Furthermore, 13 LKM-P1– and eight LKM-P2–specific CD4/ T-cell clones could be isolated from LKM-specific T-cell lines by limitingdilution technique. The HLA restriction of the LKM-specific T-cell response was studied by the addition of antibodies to class II molecules. The T-cell activation of eight tested Tcell lines and 12 T-cell clones could be blocked by antibodies against monomorphic determinants of HLA class II and HLADR but not HLA-DQ molecules (Fig. 3). Cytokine Release. For functional characterization of LKMspecific T cells, the cytokine release in response to LKM peptides was analyzed. After antigenic stimulation, five tested T-cell lines and five T-cell clones secreted large amounts of IFN-g (450-13,000 pg/mL) and TNF-a (100-1,700 pg/mL) but no or little IL-4 (0-750 pg/mL) in their cell supernatants. The strong release of IFN-g was inhibited by the addition of antiHLA class II antibodies (Fig. 4). Moreover, the IFN-g/IL-4 ratio was not affected by mitogenic activation of the LKMspecific T cells (IFN-g, 1,000-46,000 pg/mL; TNF-a, 300-3,200 pg/mL; IL-4, 0-1,700 pg/mL). Thus, the LKM-specific T cells can be classified functionally as Th1-/Th0-like cells. Regulatory T-Cell Response. To analyze whether the expansion of autoreactive T cells may be regulated by peripheral T cells, PBMCs of three AIH patients were studied for their proliferative response to autologous irradiated LKMP1–specific T cells; PBMCs from the three patients reacted with irradiated LKM-P1–specific T-cell lines, suggesting the existence of regulatory cells. In two AIH patients, T-cell lines could be established from PBMCs by repeated stimulation with washed and irradiated autologous LKM-P1–specific T cells. Flow cytometry showed that these T-cell lines consisted predominantly of CD4/CD80 T cells (91.9% and 68.9%) that expressed the a/b T-cell receptor ú98% (Fig. 5). When activated, these T cells produced large amounts of IFN-g (mitogenic activation, 6,000/24,000 pg/mL; stimulation with LKMspecific T cells, 11,500/27,000 pg/mL). To exclude antigen-independent mixed lymphocyte reaction or LKM-unrestricted T-cell response of the ‘‘regulatory’’ T cells, PBMCs derived from the third type II AIH patient were studied for their proliferative response to LKM-specific and irrelevant T-cell clones. Three LKM-specific and another autoreactive T-cell clone, isolated by repeated stimulation with autologous PBMCs, were specifically recognized by PBMCs, whereas none of 13 mitogen-induced irrelevant Tcell clones and four other autoreactive T-cell clones were able to induce a proliferative response (Fig. 6). DISCUSSION

The immunopathogenesis of autoimmune liver diseases is not clearly understood. Serum autoantibodies against a liver and kidney microsomal antigen (LKM-1) characterize the subgroup of type II AIH that occurs predominantly in young females with hypergammaglobulinemia.1,2 However, a small proportion of patients with CHC, often elderly and male patients with anti-HCV antibodies, also had LKM-1 antibodies.5-7 Recently, a 33-aa long region of cytochrome P450IID6 (aa 254-285) was identified as the main epitope for these autoantibodies.3,4,18 However, it is controversial whether the LKM-1 antibodies are directed against the same epitopes in type II AIH and LKM antibody–positive CHC.4,18 In addition, it was discussed whether epitopes of the cytochrome P450 complex have been expressed on the membrane of the hepatocytes.19,20 Thus, LKM-1 antibodies may reflect HCV-related autoimmunity in these patients, although the pathogenetic relevance is not clear.21 Immunohistological data of lymphocytic infiltrations, which consist predominantly of CD3/CD4/ T cells, and the increased expression of HLA class I and II

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TABLE 3. Clinical Data From Patients With AIH or CHC With T-Cell Response to LKM Peptides Patient

Sex

Age (yr)

ALT (U/L)

g-Globulins (%)

Serum Protein (g/L)

Histology

Therapy

MHC

1 2 3 4

F F F F

12 30 74 44

225 283 82 43

48 27 29 20

98 85 84 75

CAH CAH CPH CAH

No No No ster/aza†

A25,33 B8,18 C7 DR3,4 A1 B8 C7 DR3,5 A1,3 B51,62 C3,4 DR4,8 A3,26 B51,56 C1 DR3,6

5 6 7 8 9 10

F M F F F F

26 18 36 45 18 68

303 63 7 56 167 224

29 36 16 22 33 18

76 86 66 86 99 83

CAH CAH CPH CAH CAH CAH

No No No No No No

A3 B7 C7 DR2,3 DQ1 A3,32 B7,14 C7 DR3,7 A23,30 B13,44 C4,6 DR4,7 A1,2 B8 C4 DR3,4 A1,2 B8,51 C3,7 DR3,11 A1,2 B8,18 C6,7 DR3

11 12 13

M F M

19 48 53

72 44 24

15 21 19

73 81 75

CAH CAH CPH

ster/aza† ster/aza† No

A1,2 B7,8 C5 DR2,3 A2,3 B7,14 C4 DR3 A1,2 B8,51 C7 DR3,7

14

F

48

293

22

75

CAH

No

A26 B38,44 C4 DR4,7

15

M

34

50

19

74

CPH

No

A2 B41,60 C3 DR8,13

16

M

56

16

28

90

CAH

No

A1,2 B51 C5 DR3,4

17 18

M F

25 34

132 8

14 16

49 71

CAH CPH

No No

A3 B7,60 C3 DR1,4 A2,24 B44,45 C4 DR1,4 DQ1,3

Autoantibodies*

LKM-1 LKM-1 LKM-1 LKM-1 ANA 1:80 SMA 1:160 LKM-1 LKM-1 LKM-1 LKM-1 ANA 1:160 ANA 1:160 AMA 1:80 ANA 1:320 ANA 1:160 ANA 1:80 SMA 1:160 ANA 1:160 SMA 1:320 HCV-RNA LKM-1 HCV-RNA LKM-1 HCV-RNA HCV-RNA

NOTE. Characteristics from patients with LKM-specific T-cell response: patients 1-8, type II AIH; 9-14, type I AIH; 15-18, CHC. * Autoantibodies to LKM-1, nuclear antigens (ANA), cytoskeletal antigens (SMA), and mitochondrial antigens (AMA). Viremic patients with HCV RNA in serum. † Treatment with steroids and azathioprine.

molecules indicated that AIH appears to be mediated largely by cellular immune responses.8,22 In vitro studies found that most of the liver-infiltrating T cells in AIH are CD4/CD80 and secrete IFN-g and TNF-a after mitogenic activation, although the relative proportion of Th2-helper cells was increased.11,12 Recently, the asialoglycoprotein receptor and the pyruvate dehydrogenase complex have been characterized as liver-derived cellular antigens in AIH and PBC.15,23,24 In addition, liver-infiltrating T-cell clones from LKM-1 antibody– positive AIH patients reacted with a recombinant LKM antigen preparation.13 In this study we showed that T cells from all patients with type II AIH and half of the patients with type I AIH recognized at least the peptide LKM-P1 (aa 263285). Furthermore, a significant number of these responsive patients also reacted with the N-terminal shortened peptide LKM-P2 (aa 268-285) and/or a crude preparation of human liver microsomes that may contain natural LKM antigens.

FIG. 2. ALT levels (—, U/mL) and LKM-P1–specific T-cell response ( , mean SI) in the clinical course of a type II AIH patient (patient 3) after immunosuppressive therapy has started.

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However, because the antigen concentration was variable in the crude microsomal preparation, some patients preferentially reacted only with the synthetic peptides. In the control group, two of four LKM-1 antibody–positive and two of 27 LKM-1 antibody–negative CHC patients had T cells that responded to LKM-P1 and/or LKM-P2. Although the T-cell response to this main epitope of the LKM antigen correlated widely with AIH and positive LKM-1 antibody status, this may reflect that some patients with CHC, especially those with LKM-1 antibodies, shared the T-cell epitopes with AIH patients. There are some sequence homologies between the aa 254-285 LKM-1 region and HCV antigens (õ70%) or the 11-aa GOR peptide (45%). Therefore, molecular mimicry could be the reason for the T-cell response in some anti– LKM-1 antibody–seronegative patients4,25-27 (Table 2). Simultaneous with clinical improvement, the proliferative Tcell response to the LKM peptides decreased in two previously untreated LKM antibody–positive AIH patients after

FIG. 3. Representative data of liver- and blood-derived LKM-P2–specific T-cell lines (LL, PL 330) and clone (PC 330) derived from a patient with type II AIH (patient 5). T cells showed a significant response to LKM-P2 and LKMP1 that was blocked by anti-class II and anti-DR but not anti-DQ antibodies. j, medium control; h, PHA; , LKM-P1; , LKM-P2; , P2 / anti-11; ≠, P2 / anti-DR; À, P2 / anti-DQ. cpm, 3H-thymidine uptake in counts per minute.

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FIG. 4. Cytokine release of LKM-P1– and LKM-P2–specific T cells after specific stimulation. Liver-derived (LL, LC 330; patient 5) and blood-derived (PL386, patient 1) T-cell lines and clones. j, IL-4 (pg/mL); h, TNF-a (pg/mL); ≠, IFN-g (pg/mL); , IFN-g / anti–class II antibody.

FIG. 5. Flow cytometry analysis of two T-cell lines from AIH patients established by repeated stimulation with autologous LKM-P1–specific T-cell lines. Antibodies against CD4, CD8, and the a/b chains of the T-cell receptor (TCRab).

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FIG. 6. Anticlonotypic recognition of autologous T-cell clones in a patient with type II AIH (patient 3). The proliferative response of PBMCs to irradiated individual T-cell clones (stimulator-to-responder ratio, 1:10) is shown. PHA 1 and 2 are two representatives of 13 mitogen-driven nonspecific T-cell clones. Auto 22 and 29 were two autoreactive T-cell clones that recognized autologous antigen-presenting cells and Epstein-Barr virus–transformed B-cell lines but not LKM peptides. Anti-LKM 34, 42, and 48 were three individual autologous LKM-P1–specific T-cell clones.

immunosuppressive therapy had started. This may reflect that the LKM-specific T-cell response correlates with inflammatory disease activity. The isolation of peptide-specific T-cell lines and clones from the liver tissue and the blood may give additional information about the specificity and functional capacities of the LKMspecific T-cell response. The LKM-P1– or LKM-P2–specific T-cell lines and clones described herein acted in a class IIand DR-restricted manner. Thus the response against LKM peptides could be regarded as highly specific.28,29 To characterize the functional significance, we determined the cytokine release of T-cell lines and clones after peptide-specific and mitogenic activation. The specific stimulation of all tested Tcell lines and clones induced a significant increase of IFNg and TNF-a but no or little IL-4. Furthermore, mitogenic activation of the LKM-specific T cells did not change the IFNg/IL-4 ratio. Therefore, it can be suggested that the LKMspecific T cells belong to the Th1- or TH0-like–helper cell subsets that have also been demonstrated in other organspecific autoimmune diseases.30 Because Th1-helper cells may induce class I–restricted cellular cytotoxicity and immunoglobulin synthesis by B cells, the demonstration of HLADR–restricted autoreactive T cells, specific to a 23-aa main LKM epitope, that correlate with disease activity and LKM1 antibody status may have pathogenetic significance.31-33 It appears that the autoreactive immune response is controlled on multiple levels.34 Deletion of autoreactive T cells in the thymus is the most central selection step of an immune repertoire physiologically tolerant toward self-antigens.35 It has been recognized recently that autoreactive cells are part of the normal immune repertoire in healthy individuals, suggesting the presence of peripheral control mechanisms.34,36 Studies in experimental animals have demonstrated the presence of regulatory antiidiotypic and anticlonotypic cellular network in the control of autoreactive T cells as well as T cells responsive to the activation state, defined as antiergotypic.37-39 Vaccination with attenuated autoreactive T cells offers the prospect of specific immunotherapy in autoimmune disease.40 Our findings show recognition of LKM-specific autoreactive T cells in AIH patients by another population of Th1 cells. Therefore, it can be suggested that the autoimmune reactions in these patients underlie peripheral control mechanisms. Mixed lymphocyte reactions and antigen-independent T-cell activation were excluded by the demonstration

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that the proliferative response was restricted to the presence of LKM-specific T-cell clones. Further studies are needed to define whether the anticlonotypic T-cell response is directed to T-cell receptor molecules and what are the functional characteristics of these cells in the clinical course of AIH. Acknowledgment. The authors thank Sabine HerzogHauff and Sandra Weyer for their excellent technical assistance.

18. 19. 20. 21.

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hpta

WBS: Hepatology