T cell receptor gene rearrangements of T lymphocytes infiltrating the liver in chronic active...

Eur. J. Immunol. 1990.20: 889-896 Liver-infiltrating human T lymphocytes 889

T cell receptor gene rearrangements of T lymphocytes infiltrating the liver in chronic active hepatitis B and primary biliary cirrhosis (PBC): oligoclonality of PBC-derived T cell clones*

Ulrich MoebiusO, Michael Manma, Georg Hewa, Gabriela KoberO, Karl-Hemnann Meyer zum Biischenfeldea, and Stefan C. MeuerO

Abteilung Angewandte Immunologie, Deutsches KrebsforschungszentrumO, Heidelberg and I. Med. Klinik, Johannes Gutenberg Universitat MainzA, Mainz

Immunological events are involved in the pathophysiology of chronic active hepatitis as indicated from the accumulation of T lymphocytes at the site of tissue damage. We generated Tcell clones from liver biopsies of 3 patients with chronic active hepatitis B and 2 patients with primary biliary cirrhosis. TheseTcell clones (n = 84) were analyzed by means of Tcell receptor (TcR) p gene rearrangements to determine whether the infiltrate consists of a polyclonal or oligoclonal Tcell population. The vast majority (62 of 64) of T cell clones from three different patients with chronic active hepatitis B showed no identical rearrangements of the TcR p chain genes. In marked contrast, in both patients with primary biliary cirrhosis,Tcell clones established were of limited diversity.Thus 5 out of 10 and 2 out of 10 Tcell clones from one patient and 3 out of 9 and 2 out of 9 Tcell clones from the second patient, respectively, showed identical TcR p gene rearrangements. These data suggest that a clonal dominance is characteristic for local Tcell responses in autoimmune liver disease such as primary biliary cirrhosis whereas in virus-induced chronic active hepatitis T cell activation occurs polyclonally.

1 Introduction

Chronic active hepatitis (CAH) has a number of causes including infection with viruses and autoimmune processes. Although the exact basis of liver cell injury in these situations is not known at present, it seems likely that immunological effector mechanisms are of central impor- tance. This view is supported by the presence of mononu- clear cells, predominantly T lymphocytes, at the typical sites of inflammation [l-51. Moreover, a correlation exists between the extent of T cell infiltration, disease severity and clinical prognosis. That in hepatitis B virus (HBV)- related liver disease viral replication can be extensive without clinical signs of liver cell damage argues strongly against cytopathic effects of HBV itself and in favor of immunologic attack [6-8].

Activated T lymphocytes that are found in liver tissue in both virus-induced and autoimmune forms of CAH are predominantly CD8+ [9-121. Employing cell cloning tech- niques it was recently demonstrated that humanT lymphocyte clones recovered from liver biopsies and expanded in vitro express cytotoxic activities [13, 141. Nevertheless, despite the apparent similarity of immunologic mechanisms being the cause of symptomatic chronic liver disease,

[I 82001 * Supported by Deutsche Forschungsgemeinschaft SFB 311lA3.

Correspondence: Ulrich Moebius, Abteilung Angewandte Immu- nologie, Institut fur Radiologie und Pathophysiologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg, FRG

Abbreviations: CAH-B: Chronic active hepatitis-B HBV: He- patitis B virus HBcAG: Hepatitis B virus core antigen HBsAg: Hepatitis B Virus surface antigen MBP Myelin basic protein PBC: Primary biliary cirrhosis

clinical differences exist between virally induced and autoimmune CAH. For example, the latter forms are associated with characteristic autoantibody formation [ 15-17].

One further step toward understanding of the pathophysiology of CAH with regard to the antigens being the targets of immunologic attack is the characterization of TcR expressed by lymphocytes in liver infiltrates that are involved in tissue damage. Moreover, the knowledge of those TcR specificities involved in mediating CAH could provide novel approaches to specific immune intervention. Such strategies, e.g. based on mAb, synthetic peptides or suppressor Tcells, respectively, have already been success- ful in animal models of autoimmune diseases [18-241.

Here we have analyzed a large panel of independently derived Tcell clones from liver biopsy cores from patients with HBV-induced CAH or primary biliary cirrhosis (PBC) with regard to their TcR gene rearrangements. The data reported below demonstrate that a striking difference exists between CAH-B and PBC regarding the clonal diversity of infiltratingT lymphocyte. In PBC, the local Tcell repertoire is rather limited whereas in CAH-B the infiltrate is essentially polyclonal.

2 Materials and methods

2.1 Patients

Patients with CAH-B, patient 3000 (20-year-old female), patient 7000 (63-year-old female) and patient 8000 (66-year-old female) had a history of circulating HBV- surface-specific Ab (anti-HBsAg) for at least one year. Anti-HBsAg-specific Ab were determined by commercially available in vitro assays (Abott, Chicago, IL). Histopatho- logical examination of liver biopsies revealed lympho- histiocytic infiltration of periportal areas. Erosion of

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990 0014-2980/90/0404-0889$02.50/0

890 U. Moebius, M. Manns, G. Hess et al. Eur. J. Immunol. 1990.20: 889-896

limiting plates and piecemeal necrosis of periportal hepa- tocytes was found. Patients with PBC, patient 12 000 (male, 70 years old) and 17000 (female, 58 years old), were negative for HBV-markers. They had circulating autoanti- bodies directed against mitochondria1 antigens [ 171. Histo- logically they corresponded to stage I1 and I11 as determined by the degree of lymphocyte infiltration in periportal areas, reduction of the number of bile ducts, degradation of cananiculi with disruption of basal membrane and changes in the overall structure of the hepatic tissues [3]. All patients gave informed consent statements.

2.2 Derivation of lymphocytes and clonal expansion

The derivation of T lymphocytes and their clonal expansion has been described previously [ 131. In brief, liver tissue was obtained by liver biopsy from which infiltrating T lymphocytes were separated. To this end, the biopsy specimen was washed prior to a 48-h incubation in RPMI (Gibco, Paisley, GB) supplemented with 10% human serum, 2 m~ L- glutamine (Gibco) and 5 U/ml penicillinkreptomycin (Gibco; standard culture medium). This incubation resulted in a spontaneous release of lymphocytes from the tissue which were separated by centrifugation. The remain- ing liver tissue was incubated in 0.5 ml of serum-free medium with 2.0 mg pronase E (Merck, Darmstadt, FRG) for 30 min to release infiltrating T lymphocytes. PBL were recovered by density centriguation with Ficoll-Hypaque (Pharmacia, Uppsala, Sweden).

After cell recovery, T lymphocytes were cloned by LD technique at one celVwel1 in 96-well V-bottom plates (Costar, Cambridge, MA) containing irradiated feeder cells (5 x 10" PBMC, 5 x 10" B lymphoblastoid cells) and 0.5 pg PHA (Wellcome, Burgwedel, FRG). IL 2 (generously provided by Dr. Luckenbach, Sandoz Research Institute, Vienna, Austria) was added at 3 ng/ml to each well on day 3. Growing T cells were expanded in ILZcontaining medium and restimulated approximately every 14 days at 10" T celldmicrowell in the presence of the feeder cells mentioned above.

2.3 Phenotype analysis

T cells were analyzed by indirect immunofluorescence by incubating T cells with anti-CD2, anti-CD3, anti-CD4, antLCD8 (anti-CD3: OKT3, hybridoma from American IIfrpe Culture Collection, Rockville, MD, anti-CD2, anti- CD4, anti-CD8 from our own laboratory), BMA031 (anti- TcR-a@, R. Kurrle, Behringwerke, Marburg FRG), anti- Ti-ylA (T. Hercend, [25]), 6-TcR1 (T Cell Sciences, Cam- bridge, MA), anti-CD54 (NKH1, T. Hercend, [26]) mAb, respectively, followed by an incubation with an FITC- labeled goat anti-mouse Ig (Coulter, Hialeah, FL). Fluo- rescence was determined in a Coulter Profile Analyzer by analyzing 10000 cells.

2.4 Determination of cytotoxicity

Cell-mediated cytotoxicity was analyzed in a standard Cr-release assay [27]. To determine the cytotoxic potential in a nonspecific manner, T lymphocytes were incubated at

different E/T ratios (60/1 to 2/1) with 103 51Cr-labeled B lymphoblastoid cells (Laz509) in the presence of 10 pg Con A. NK activity was determined by using K-562 as target cells at different En ratios. After 3 h of incubation at 37 "C released radioactivity was determined and % specific lysis calculated.

2.5 DNA isolation and Southern blotting

When not otherwise mentioned all chemicals were pur- chased from Sigma, Munich, FRG. Genomic DNA was isolated from lo7 cells by lysis in 0.5% SDS followed by phenol extraction as described [28]. Approximately 15 pg DNA was digested overnight with 60 U of Em RI and Hind 111 (Boehringer Mannheim, Mannheim, FRG). In some experiments DNA was digested with Bam HI (Boehringer Mannheim). DNA fragments were size separated overnight in 0.8% agarose gels and transferred to Gene Screen membranes (NEN, Dreieich, FRG) according to the manufacturer's instructions employing 0.025 M NaHC03, pH 6.5 as transfer buffer. Approximately 100 ng of aTcR Cg probe (C-p REX, kindly provided by H.-D. Royer; [29]) were labeled by nick translation employing 750 kBq [32P]adCTP (NEN) and a nick translation kit (Boehringer Mannheim). Filters were prehybridized overnight at 42 "C with 50% formamide, 1% SDS, 1 x Denhardt's solution (0.2 gA Ficoll, 0.2 g/l BSA, 0.2 g/l polyvinylpyrrolidone) and 1 X SSPPE solution. The TcR probe was diluted at 2 x 106 cpdml in fresh prehybridization buffer containing 5 pg/ml salmon sperm DNA and hybridized overnight at 42 "C [28]. Filters were washed with 2 x SSC at room temperature, 0.1 x SSC containing 1% SDS (65 "C), and 0.1 X SSC at room temperature and exposed to Kodak XAR films (Rochester, NY) for 3 days.

3 Results

3.1 Phenotypic and functional characterization of infiltrate-derived T cell clones

We have derived human T cell clones from the hepatic lymphocyte infiltrate (see Sect. 2.2) of several patients with different forms of chronic active liver disease [13]. T cell clones from three patients with CAH-B (30,18 and 17 Tcell clones, respectively) and two patients with PBC (10 and 10 T cell clones, respectively) were subjected to further investigations. The phenotypes and the cytotoxic activities of these liver-derived T cell clones are summarized in Table 1. As previously reported, the majority of T cell clones derived from liver infiltrates, regardless of the etiology of liver disease, are CD8+, activated CTZ [13, 141.

From one patient with CAH-B (patient 7000) we expanded 3 T cell clones expressing a TcR y/6 and 2 CD3- clones in addition to 12 TcR a@+ T cell clones (Table 1). ' h o y/6 T cell clones were found to be CD4-CD8-, the third one was CD4-CDW. These three TcR y/6+ clones and two CD3- clones expressed NK activity as determined by lysis of the NK target cell line K-562 (Table 1). It should be noted that lymphocyte cloning from biopsy specimens of the two other CAH-B patients did not yield TcR y/6+ Tcells or NK cells (Table 1). There, the majority of T cell clones generated

Eur. J. Immunol. 1990. 20: 889-896

Table 1. Phenotype and functional of liver infiltrate-derived cell clones

Liver-infiltrating human T lymphocytes 891

Diagnosis Patient Total no. of clones Phenotype@) Cytotoxicityb)

cD2 cD3 d p $6 CD4 CD8 LDCC NK RR

CAH B 3000 3oc) 30 30 30 0 9 18 26 1

CAH B 8000 18e) 18 18 18 0 9 7 12 1 PBC 12 000 10 10 10 10 0 3 7 10 0 PBC 17 000 10 10 10 10 0 4 6 6 0

CAH B 7000 17d) 17 15 10 3 4 5 12 6

a) Determined by indirect immunofluorescence. Samples with > 90% mab reactivity were scored as positive and samples with > 5%

b) Determined in cytotoxicity assays. LDCC: lectin-dependent cytotoxicity, NK: cytotoxicity towards the target cell ine K-562. Tcell

c) TcR a@+, CD4-CD8-; 1 clone; CD4CD8 mixed: 1 clone. d) CD3-/NK: 2 clones; TcR dP+. CD4-CD8-: 2 clones, R R a /p y/S mixed: 2 clones; CD4CD8 mixed: 1 clone. e) CD4CD8 mixed: 1 clone.

reactivity as negative.

clones showing > 30% specific lysis at an Em ratio of 20/1 were enumerated as cytolytic.

were CD4-CD8+ (n = 25). In addition to CD4TD8- T cells (n = 18), CD4-CD8- TcR a@+ Tcell clones (n = 3) could be expanded from the liver infiltrate in two cases (Table 1). These three CD4-CD8- TcR a/p T cell clones showed lectin-dependent cell lysis but no cytotoxic activity towards the NK target K-562. All CD4-CD8+ Tcell clones from CAH-B patients expressed cytolytic activity. A frac- tion of the Tcell clones with a CD4+CD8- phenotype was found to be also cytotoxic (patient 3000: 519 clones, patient 7000: 1/4 clone, patient 8000: 4/9 clones).

In the case of PBC patients, all Tcell clones obtained (n = 20) expressed a CD2+CD3+ phenotype (Table 1). The TcR of these Tcell clones were found to be exclusively a/p TcR. All CD8+ Tcell clones from both PBC patients (n = 13) were cytotoxic, and in addition the CD4+ clones from

patient 12 000 (n = 3 of 3 clones) showed cytotoxic activity in a lectin-dependent assay system (Table 1).

3.2 Analysis of TcR gene rearrangements of liver infiltrate-derived T cell clones

Sixty-four independently derived T cell clones from three different patients with CAH-B and 19 T cell clones from two PBC patients were subjected to TcR gene analysis by means of investigating restriction fragments of TcR p genes after digestion with Em RI and Hind HI, respectively, ([30-321; Fig. 1, Table 2). Hind I11 and Eco RI detect rearrangements of the Cgl and Cg2 gene segments, respectively. DNA isolated from a human B lymphoblastoid cell line (without any TcR gene rearrangements) demonstrated

Table 2. Summary of TcR p chain analysis of liver infiltrate-derived T cell clones

Patient Diagnosis No. of clones Clones with identical Phenotype Cytotoxicityb) investigated RR restriction

fragment@ R R CD4 cD8 LDCC NK

3000 CAH B 30 None 7000 CAHB 17 None 8000 CAH B 17 8406

8413 12 000 PBC 10 Group I 12402

12 407 12 414 12 415 12 416

Group II 12410 12423

10 Group I 17402 17 411 17 412

Group II 17408 17 409

17 00 PBC

+ + + +

+ + + + + + + + + + + + -

a) As determined by Southern blotting shown in Fig. 1 and Fig. 2. b) See legend to 'Rib. 1.

892 U. Moebius, M. Manns, G. Hess et al. Eur. J. Immunol. 1990.20: 889-896

Figure 1. Southern blot analysis of liver infiltrate-derived Tcell clones from patients with CAH B. (A) patient 3000, (B) patient 7000, (C) patient 8000. Restriction enzymes were Hind I11 and Eco RI, respectively, (D) DNA from threeTcell clones from patient 8000 after Bam HI digestion. The number on top of each lane represents the individual clone number, GL (germ line) represents DNA from B lymphoblasts. As DNA size marker, a Hind LII digest of h 'DNA was used.

germ-line bands of 10.5 and 4.0 kb after Eco RI digestion and 7.6,6.2 and 3.2 kb after Hind I11 digestion, respectively (lane GL in Figs. 1 and 2). In contrast, all TcR dfi+ Tcell clones showed additional bands and/or deletion of germ- line bands indicating TcR gene rearrangements. In two

patients with CAH B (3000 and 7000) none of the 47 Tcell clones investigated produced an identical rearrangement of TcR fi chain genes after incubation with either enzyme (Fig. 1A and B). As expected, TcR p genes of CD3- NK clones and TcR y/6+ T cell clones from patient 7000 were in

Eur. J. Immunol. 1990. 20: 889-896 Liver-infiltrating human T lymphocytes 893

the germ-line configuration. In the case of patient 8000, 3 In marked contrast toTcell clones derived from the hepatic out of 17 Tcell clones (8406, 8413, 8432) showed identical infiltrate of patients with CAH-B, multiple T cell clones restriction fragments after digestion of the DNA with Eco with identical TcR gene rearrangements were obtained RI and Hind I11 (Fig. 1C). However, subsequent TcR gene from the two patients with PBC. In one case (patient analysis of these three T cell clones by Bam HI digestion 12000) 5 out of 10 Tcell clones showed the same 7.5- and revealed that only 2 out of 17 T cell clones have identical 12-kb rearranged bands after digestion with Hind I11 and a TcR p gene rearrangements (Fig. 1D). germ-line configuration after digestion with Eco RI

23.3- Hind 111

(B) 9.5-

6.7-

Kb

4.4-

23.3 -

23.3-

9.5-

6.7-

9.5 - Eco RI

6.7-

4.4 -

Barn H I

4.4-

23.3-

23.3- 9.5-

9.5-

6.7-

6.7-

4.4-

4.4-

Hind Ill Eco R I

Figure 2. Southern blot analysis of liver infiltrate-derived Tcell clones from patients with PBC. (A) Patient 12 OOO, DNA digested with HInd I11 and Eco RI, respectively, (B) patient 12 OOO, DNA digested with Barn HI, and (C) patient 17 OOO, DNA digesed with Hind 111 and Eco RI, respectively. G L (germ-line) represents DNA from B lymphoblasts. As DNA size marker, a Hind I11 digest of h DNA was used.

894

(Fig. 2A). In addition, a second group of 2 T cell clones from the same patient with identical 12-kb (Hind In) and 6.7-kb (Eco RI) rearranged bands was observed.To confirm the identity of these TcR gene rearrangements, TcR restriction fragments generated by Bam HI were analyzed. As shown in Fig. 2B, all 7 Tcell clones showed an identically rearranged band of approximately 9.5 kb. The 5 clones in group I gave an additional germ-line band (23 kb) and the 2 clones of group 11 a band of slightly lower molecular weight (21 kb) strongly suggesting the identity of these 5 and 2 clones among the 10 Tcell clones obtained. Analysis of the T cell clones from the second patient with PBC gave similar results. From 9 Tcell clones investigated by Southern blot analysis, 3 presented identical rearranged 7.0-kb bands after digestion of their DNA with Hind 111 and 8.0-kb fragments after Eco RI digestion, respectively (Fig. 2C). In addition, a second group of two T cell clones showed identical TcR fi gene restriction fragments. Identitiy of these 3 and 2 T cell clones, respectively, could be confirmed employing a third restriction enzyme. Thus, after digestion with Bam HI, DNA of T cell clones 17302,17411 and 17412

U. Moebius, M. Manns, G. Hess et al. Eur. J. Immunol. 1990.20: 889-896

Table3. Analysis of TcR fi chain rearrangements of peripheral blood-derived Tcell clones from patients with PBC

contained an identical 14-kb rearranged band and DNA of Tcell clones 17408 and 17409 showed a germ-line configuration (not shown).The characteristics of Tcell clones with identical TcR p gene rearrangements are summarized in Table 2. TcR homology in PBC T cell clones was found in CD8+ as well as CD4+ T cell clones. Interestingly, in one instance identical TcR fi gene rearrangement were detected in one CD4+ and one CD8+ Tcell clone (clones 12410 and 12423 from patient 12 OOO).

3.3 Analysis of TcR gene rearrangements of T cell clones derived from peripheral blood

Eight T cell clones from the peripheral blood of PBC patient 12 000 and twenty-one T cell clones from peripheral blood of PBC patient 17000 that were generated at the same time and under the same conditions as the liver- derived T cell clones were analyzed for their TcR gene rearrangements. As shown inTable 3, none of the peripheral blood T cell clones from either patient showed an identical TcR p chain gene rearrangement. IIkroTcell clones from the peripheral blood of patient 12000 gave very similar rearranged bands after digestion of their DNA with Hind I11 (clones 12235 and 12241 in Table 3). However.

Clone +

Patient 12000 12 229 12 232 12 204

12 244 12 239 12 235 12241 Patient 17000 17221 17213 17210 17 236 17226 17212 17 227 17 230 17 225 17 203 17211 17217

17 214

17 234 17 215 17 237 17 204 17 207 17 231

12 m 5

17 2m

17 m 5

Em RI

9.0 8.5 8.5 8.2 7.0 6.7

GL GL

9.5, 7.6 9.2, 8.3 9.2, 8.1 9.2 8.1 8.0 7.6 7.6 Dc) D D D D D D GL GL GL GL GL GL

identity was excluded employing Bam HI. I n the case of patient 17 000, two T cell clones were found with identical restriction fragments after digestion of their DNA with Eco RI and Hind 111, respectively, (clones 17227, 17230 in Table 3). Further analysis of these two T cell clones

Size Of

(kb)8) Hind 111 Bam Hi

GLb) 9.5 7.3

11.3 7.3, 6.0

10.0 5.9 6.0

GL GL GL

GL 5.7

8.2, 6.8 8.0, 6.5 8.0, 6.5

20.0, 8.2 20.0. 9.0 14.0, 1.2 9.2, 7.3 7.4, 6.6 7.2, 6.7 7.2, 5.4

11.0 9.2 5.7 GL GL GL

employing Bam HI demonstrated a different TcR f3 chain gene rearrangement. It should be noted that those peripheral blood T cell clones of patient 17 OOO which showed restriction fragments of related size were investigated in neighboring lanes of a subsequent Southern blot. This clearly demonstrated the difference of rearranged TcR p genes in these T cell clones.

Incubation of liver biopsy specimens for 48 h in culture medium (prior to recovering tissue-infiltrating lymphocytes by means of enzymatic digestion of liver tissue) resulted in the spontaneous release of T lymphocytes which account for approximately 90% of T lymphocytes totally recovered from a biopsy [13]. T cell clones derived from these lymphocytes (designated as “pellet” in [13]) showed similar phenotypic and functional characteristics when compared to peripheral blood T cell clones suggesting that they represent peripheral blood Tcells [13]. Importantly, analysis of TcR p chain gene rearrangements of 20 “pellet”- derived T cell clones from patient 17000 (PBC) did not show any identical TcR gene rearrangement (not shown).

7.0

13.0 GL

27.0, 21.0, 16.0 27.0. 21.0

4 Discussion

Central to chronic liver diseases of different etiologies is the infiltration of the liver tissue withT lymphocytes which are believed to play a major role in tissue destruction [l-51 .The ‘specificity of infiltrating T lymphocytes is not known at present. Two recent reports suggest that T lymphocytes

a) Restriction fragments were analyzed by Southern blotting. b) GL: g e m - h e bands (10.5 kb and 3.9 kb for E~~ RI, 7.6 kb,

c) D = deletion of the 10.5-kb Eco FU germ-line band.

recovered from liver specimens from patients with CAHB where HBv is known as the disease-initiating agent, are specifically directed at soluble HBV Ag namely HBV core (nuclear; HBcAg) and HBV surface (envelope; HBsAg)

6.2 kb, 3.2 kb for Hind III).

Eur. J. Immunol. 1990.20: 889-896 Liver-infiltrating human T lymphocytes 895

was only found in biopsy specimens of patients with PBC but not in CAH-B. Generation of T cell clones from peripheral blood and “peUet”of the same patient with PBC did not yield clones with identical rearrangements. This indicates that the accumulation of distinct T lymphocytes was specific for the liver and did not occur in the peripheral blood.

proteins [33, 341. In the case of PBC, one form of autoimmune liver disease, no information is available concerning the target antigens of the Tcells. Knowledge of the mechanisms that lead to activation of lymphocytes at the local sites of tissue destruction would be useful to understand the pathophysiology of these diseases and, in addition, could eventually lead to better strategies of clinical immune intervention [1&24]. A first step towards this direction was to establish a protocol for recovery, expansion and subsequent functional characterization of the liver-infiltratingT lymphocytes from liver biopsy mater- ial [13]. A second step which is presented in this report is the analysis of TcR gene rearrangements in addition to further functional analyses.

It has been previously reported that T cell clones established from the infiltrate of CAH patients predominantly express a CD4-CD8+ phenotype [13-141. Further characterization of 84 Tcell clones from 5 individual patients (3 CAH-B, 2 PBC) confirmed these earlier findings. Almost all Tcell clones expressed anTcR a@ (79 out of 84). A small number of CD3- NK cells and TcR $15’ T cells were established from the infiltrate of only one patient with chronic active liver disease. They could represent a contam- ination with lymphocytes from peripheral blood since upon immunohistologic examination, y/S cells are extremely rare (own unpublished observations).

TcR gene rearrangements of individual T cell clones were analyzed investigating restriction length polymorphism of TcR p chain genes [31,32]. Rearrangements, either of Cgl or Cp2, were detected employing a TcR Cg probe which binds to both gene segments due to their high degree of sequence homology [29]. Analysis of TcR p chain gene rearrangements of Tcell clones from patients with CAH-B showed that the vast majority of these Tcell clones showed different TcR gene rearrangements. In contrast, T cell clones with identical rearrangements were generated from two patients with PBC. Although only a limited number of cases has been investigated so far, these data suggest that T lymphocytes infiltrating the liver in CAH-B are polyclonal whereas in PBC the infiltrate is composed of a T lymphocyte population with limited diversity. Identical TcR gene rearrangements employing three different restriction enzymes indicate a high degree of homology with regard to V-D-J rearrangements [31,32]. It seems likely that theseT cell clones with identical TcR p restriction fragments derive from the same T cell and therefore express identical TcR molecules. It can not be excluded, however, that those clones have rearranged homologous gene segments, and due to additional mechanisms leading to diversity, express a closely related TcR p chain. This may explain the finding of one particular rearrangement in a CD4+ and a CD8+ Tcell clone established from the same infiltrate. Whether Tcells with identical TcR p rearrangements express for instance identical V region genes (and J/D region genes) is presently under investigation. Moreover, the usage of TcR f3 gene segments in PBC-related T lymphocytes of different patients has to be determined; likewise it will be necessary to perform similar studies with regard to the TcR a genes.

Since biopsies are taken from one small area of the liver,T lymphocytes recovered from PBC patients may represent a local oligoclonal population. However, clonal dominance

T cell clones with specificity for HBV gene products have been reported to occur among liver-infiltrating T lymphocytes from CAH-B patients [33, 341. In these studies, however, and in contrast to the present study different procedures were employed for cloning and activating the tissue-derived T cells. Particularly, HBV-derived antigens were used to activate specific T cells. The T cell clones recovered by this technique were reported to recognize different HBV antigens. Ferrari et al. [33] demonstrated a CD4+/CD8- liver-derived Tcell clone specific for HBcAg. No attempts were made to separate blood-derived T cells from the real tissue-infiltrating cells. Given our results ([13] and this report) suggesting that the vast majority of T lymphocytes which are recovered from biopsy specimens represent peripheral blood T cells, it is not surprising to expand HBcAg-specific T cell lines since an anti-HBcAg response is characteristic for CAH-B. It should be noted that in our own investigations no HBcAg-specific response of liver-derived Tcells were observed (not shown). Employ- ing HBsAg to activate liver-derived Tcells, Barnaba et al. [34] reported on CD4+CD8- and CD4-CD8+ Tcell clones specific for HBsAg. Since PBL could not be activated by these antigens, response to HBsAg was suggested to be specific for liver-infiltrating T cells [34]. Interestingly, CD4+CD8- as well as CD4-CD8+ Tcell clones recognized the same pre-S2 region-derived 15 amino acids-containing peptide. It remains to be investigated whether these Tcell clones rearrange and use related TcR genes and whether T cells derived from the liver of CAH-B patients and expanded without any preselecting Ag show a related kind of specificity.

Several reports have shown that the diversity of T lymphocytes involved in experimental autoimmune animal models is limited. In experimental allergic encephalitis which can be induced by administration of myelin basic protein (MBP) and MBP-specificTcel1 clones, respectively [35-371, MBP-specific T cell clones showed a limited usage of TcR genes [38,39]. Similarly, in collagen-induced arthritis there is indirect evidence that a particular TcR V region gene determines disease susceptibility [40]. In human autoimmune diseases limited studies have also provided evidence for an oligoclonal T cell response. Thus, in patients with multiple sclerosis and myastenia gravis certain V, and Vg alleles were proposed to be associated with the disease [41]. Moreover, analysis of TcR p genes of uncloned T lymphocytes from joints of rheumatoid arthritis patients [42] and T cell clones established from the cerebrospinal fluid of patients with multiple sclerosis [43] suggest clonal dominance of infiltratingT lymphocytes. Our data indicate that an oligoclonal T cell response can exist in a solid organ, particularly the liver, and would agree with previous findings regarding the involvement of T cells of limited heterogeneity in other types of autoimmune diseases.

The knowledge that T cell specificities are limited in autoimmune diseases has been applied for specific modu-

896

lation of autoaggressive T cells in vivo. In this regard, peptides competing for autoantigens [ 18-20], suppressive T cells [21, 221, and TcR V region-specific mAb [23, 38, 391 and peptides [24] were shown to block autoreactiveTcells in vitro and in vivo. Our data on PBC-related Tcells raise the possibility to develop similar strategies for the treatment of autoimmune liver disease. In addition, the analysis of TcR involved in autoimmune diseases may help to concentrate particularly on frequent clones and to investigate their reactivity towards potential target antigens [15, 44, 451.

The mechanisms underlying the induction of an immune response which leads to the destruction of liver tissue may be entirely different in CAH-B and PBC. In CAH-B, a number of “immunologically equal” antigenic epitopes of foreign antigens, e.g. viral gene products in MHC associa- tion, may lead to a polyclonal activation of T cells possessing different specificities. In autoimmune disease such as PBC, however, T lymphocytes respond to an autologous antigen due to a breakdown of tolerance, i .e. by the escape of individual lymphocyte clones from those mechanisms leading to clonal deletion. This would explain why a (self directed?) immune response with dominance of a few clones is observed in the case of primary biliary cirrhosis.

U. Moebius, M. Manns, G. Hess et al. Eur. J. Immunol. 1990.20: 889-896

Received December 22, 1989.

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