T-cell clonality of peripheral blood from patients with chronic hepatitis C before and after treatment with interferon α

T-cell clonality of peripheral blood from patients with chronic hepatitis C before and after treatment with interferon α

Hepatology Research 17 (2000) 1 – 11 www.elsevier.com/locate/ihepcom T-cell clonality of peripheral blood from patients with chronic hepatitis C befo...

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Hepatology Research 17 (2000) 1 – 11 www.elsevier.com/locate/ihepcom

T-cell clonality of peripheral blood from patients with chronic hepatitis C before and after treatment with interferon a Hiromasa Ohira *, Hideharu Sekine, Masahito Kuroda, Jotaro Shinzawa, Tomohiro Suzuki, Jun Tojo, Masayuki Miyata, Reiji Kasukawa Department of Internal Medicine II, Fukushima Medical Uni6ersity School of Medicine, 1 -Hikarigaoka, Fukushima 960 -1295, Japan Received 30 March 1999; received in revised form 19 July 1999; accepted 26 July 1999

Abstract Immune-mediated liver cell damage has been likely to occur in patients with chronic hepatitis C virus (HCV) infection. To clarify the T-cell clonality in patients with chronic hepatitis C, we analyzed T-cell receptor (TCR) Vb chain messages expressed in peripheral blood mononuclear cells (PBMC) before and after treatment with interferon (IFN)-a. PBMC from 15 patients with chronic hepatitis C and from six healthy subjects were examined. The 15 patients were allocated to three groups based on their response to IFN-a treatment as follows: responders (group A, n= 5), transient responders (group B, n= 5), and non-responders (group C, n=5). Twenty-two TCR Vb repertoires Vb 1–Vb 20 expressed in PBMC were analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and single strand conformation polymorphism (SSCP). In responders, the mean total number of T-cell clones in 22 TCR Vb repertoires was significantly decreased from 97.4 9 40.1 to 62.69 32.1 (PB0.01) after treatment. In transient responders, although one patient showed a decrease, four patients showed an increase, with the mean total number of T-cell clones increasing from 66.4 928.0 to 79.8 928.8 after treatment. In non-responders, the mean total number of T-cell clones was almost unchanged between before and after treatment. No clear tendency towards the use of any specific Vb was observed in any group of patients. These results suggest that T-cell clones accumulated in the peripheral blood of patients with chronic

* Corresponding author. Tel.: +81-245-48-2111; fax: +81-245-47-2055. 1386-6346/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 9 9 ) 0 0 0 5 7 - 1

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hepatitis C and that the number of clones may change in response to IFN-a treatment. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hepatitis C virus; T-cell receptor; Single-strand conformation polymorphism; Polymerase chain reaction

1. Introduction The mechanism of hepatocellular injury in patients with chronic hepatitis C virus (HCV) infection has not yet been clearly elucidated. However, immune-mediated damage to liver cells, but no direct cytopathic effect of HCV, has been suggested in chronic HCV infection [1,2]. Recent studies have shown that HCV antigen-specific CD4 + T-cells and CD8 + T cells are present in peripheral blood mononuclear cells (PBMC) in patients with chronic hepatitis C and that HLA class I-restricted T cells mediate hepatocytotoxicity [3–5], indicating that antigen-specific T cells accumulate in both PBMC and liver tissue in these patients. The junctional regions of T-cell receptors (TCR) have been considered to play an important role in antigen recognition [6]. TCR consist of a and b chains generated by a recombination of non-contiguous gene segments from pools of variable (V), diverse (D, b only), and joining (J) segments [7,8]. This VDJ complex is then juxtaposed to a C region by RNA splicing. The b chain has three hypervariable regions within this complex that are responsible for binding to a specific antigen [8]. Moreover, all T cells originating from a clone bear a distinct TCR Vb chain with a specific VDJ complex. The reverse-transcription polymerase chain reaction and single-strand conformation polymorphism (RT-PCR/SSCP) system described by Yamamoto et al. [9] has shown that a single T-cell clone shows a clear single band of TCR Vb chain messages when PCR products of TCR Vb chain DNA are separated by electrophoresis. TCR Vb clonality has been investigated in several autoimmune diseases Table 1 Characteristics of 15 patients with chronic hepatitis C classified into three groups of responders, transient responders, and non-responders based on their response to IFN-a treatmenta Responders (n= 15)

Transient responders (n =5)

Non-responders (n =5)

Age (year) Gender (M/F)

389 17b 3/2

54 9 10 2/3

55 9 6b 2/3

HCV genotype II III IV HCV RNA (log10 copies/ ml)

1c 4 0 4.190.7d,e

2 1 2 5.1 90.2d

5c 0 0 5.2 90.3e

a

Values with same letters: b, c, PB0.05; d, e, PB0.01.

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Fig. 1. Representative results of SSCP analysis of TCR Vb messages Vb 1 to Vb 20 expressed in PBMC from a healthy subject (a) and from a patient with chronic hepatitis C (b). In the healthy subject (a), the messages of each Vb were expressed as a smear pattern with a few bands representing T-cell clones, while in the patient with chronic hepatitis C before treatment with IFN-a, many bands were seen in several Vb messages.

to determine the response of T cells to specific antigens [10,11]. However, the clonality of T cells in the peripheral blood or in the liver of patients with chronic hepatitis C has not been rigorously examined. Unbiased usage of TCR Vb region genes in patients with chronic hepatitis C has reported by Wang et al. [12]. The present study investigated the clonality of T cells in PBMC obtained from patients with chronic hepatitis C and compared the number of clones between responders, transient responders, and non-responders to interferon (IFN)-a treatment.

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Table 2 SSCP analysis for T-cell population with each Vb repertories in PBMCs before and after treatment with IFN-a of 15 chronic hepatitis C patients and 6 healthy controlsa Patient

No. of Distinct Bands in V3

Group A 1 2 3 4 5 Total Group B 6 7 8

2

3

4

5.1

5.2-3

6.1-3

7

8

9

10

11

12

13.1

13.2

14

15

16

17

18

19

20

Total

Before After Before After Before After Before After Before After

6 4 3 1 5 3 6 3 3 1

5 2 5 2 6 4 5 2 3 1

4 3 7 4 3 2 7 4 4 4

7 5 4 3 6 4 6 5 7 S

5 5 5 3 4 3 6 6 6 5

6 4 5 4 3 2 6 3 3 2

4 4 2 2 5 3 7 3 5 S

S 3 7 S 6 3 8 6 4 4

S 2 4 2 S 3 8 4 5 5

2 2 S S 5 2 6 4 4 2

4 3 7 4 6 5 10 7 2 2

3 3 3 S 6 5 3 4 S S

11 S S S 6 3 10 4 4 1

S 6 3 1 6 6 10 8 3 1

4 4 S S 6 3 7 6 3 3

S S S S 5 4 7 7 3 2

2 3 4 1 5 3 6 5 3 1

S S S S 6 2 12 9 4 1

S S 5 2 5 4 10 8 S S

S 2 6 3 5 5 9 4 S S

7 3 S S 6 3 13 8 S S

3 2 5 S 6 4 S S S S

73 60 75 32 111 76 162 110 66 35

Before After

23 15

24 11

25 17

30 17

26 22

23 15

23 12

25 16

17 16

17 10

29 21

15 12

31 8

22 22

20 16

15 13

20 13

22 12

20 14

20 14

26 14

14 6

487 313

Before After Before After Before After

4 4 6 S 4 4

S S 4 4 3 3

3 3 4 4 2 2

S S 3 3 3 3

3 2 3 3 2 3

S S S S 4 3

S S 3 3 S S

4 4 6 6 3 3

S S 6 6 2 2

5 5 3 4 3 S

4 2 2 4 3 3

3 3 6 10 3 3

2 2 4 4 4 4

S 3 3 3 3 3

S S 6 6 3 3

4 S 3 6 2 2

5 6 3 3 S S

4 6 3 3 3 3

4 4 3 6 3 4

S S 3 6 3 3

2 4 1 4 5 5

S S 3 3 4 4

47 48 78 91 62 60

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Table 2 (Continued) Patient

No. of Distinct Bands in V3 1

10 Total Group C 11 12 13 14 15 Total

3

4

5.1

5.2-3

6.1-3

7

8

9

10

11

12

13.1

13.2

14

15

16

17

18

19

20

Total

Before After Before After

3 5 5 10

2 5 3 6

2 4 4 S

3 3 4 4

3 5 7 9

S 3 7 8

3 3 5 S

3 3 3 4

S 4 4 4

S S 6 11

2 4 7 S

S S S 7

S 4 S 4

3 5 3 6

2 2 3 8

S 2 8 8

S S 6 S

S 4 9 8

1 4 5 8

4 6 9 6

3 6 10 11

3 6 S S

37 78 108 122

Before After

22 23

15 18

15 13

13 13

18 22

11 14

11 6

19 20

12 16

17 20

18 13

12 23

10 18

12 20

14 19

17 18

14 9

19 24

16 26

19 21

21 30

10 13

332 399

Before After Before After Before After Before After Before After

6 6 5 5 6 6 3 3 5 5

3 3 3 3 4 4 2 2 S S

4 4 4 4 3 3 5 3 4 4

4 4 4 4 4 4 3 3 6 6

4 4 4 4 5 5 3 2 4 4

3 3 3 3 2 2 4 4 3 3

6 6 4 4 5 4 S S 4 4

4 4 6 6 6 6 S S 8 8

4 4 4 4 4 4 3 3 3 3

2 2 4 4 2 2 4 4 4 4

5 5 6 6 6 6 5 5 3 3

2 2 6 6 2 2 3 3 2 2

3 3 3 3 4 4 S S S S

3 3 4 4 3 3 4 4 S S

5 5 5 5 6 5 6 6 5 5

4 4 4 4 5 5 3 3 4 3

3 3 2 2 4 4 S S 2 2

4 4 4 4 5 5 3 2 5 5

4 4 5 5 6 6 5 5 6 6

4 4 3 3 5 5 4 4 3 3

3 2 3 3 4 4 S S 3 2

4 4 4 4 2 2 3 3 4 4

84 83 90 90 93 91 63 59 78 76

Before After

25 25

12 12

20 18

21 21

20 19

15 15

19 18

24 24

18 18

16 16

25 25

15 15

10 10

14 14

27 27

20 19

11 11

21 20

26 26

19 19

13 12

17 17

408 399

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2

Six healthy controls Total a

1

2

3

4

5.1

5.2-3

6.1-3

7

8

9

10

11

12

13.1

13.2

14

15

16

17

18

19

20

1

1

1

2

S

1

2

S

S

1

2

2

4

2

2

1

1

2

3

1

2

1

S, smear (no distinct bands).

5

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Table 3 Average number of total SSCP bands in messages of TCR Vb repertoires from Vb 1 to Vb 20 expressed in PBMC obtained from patients before and after treatment with IFN-a Response to IFN-a

Responders (group A) Transient responders (group B) Non-responders (group C)

Number of patients

5 5 5

Average number of total SSCP bands Before

After

97.4 940.1* 66.4 928.0 81.6 911.9

62.6 932.1* 79.8 928.8 79.8 913.1

* PB0.01.

2. Materials and methods

2.1. Patients Fifteen patients with chronic hepatitis C and six healthy subjects participated in the study after giving written informed consent. The diagnostic inclusion criteria for chronic hepatitis C included: (1) histopathological evidence of chronic active hepatitis in a biopsied liver specimen; (2) seropositivity for both HCV-RNA (nested reverse-transcription polymerase chain reaction [RT-PCR]) and anti-HCV antibody (enzyme-linked immunosorbent assay of the second generation, Ortho Diagnostic Systems, Tokyo, Japan); and (3) the presence of abnormal ALT levels for longer

Fig. 2. The total number of distinct SSCP bands for messages of 22 TCR Vb repertoires Vb 1 to Vb 20 in responders (group A, n= 5), transient responders (group B, n = 5), and non-responders (group C, n = 5) before and after IFN-a treatment.

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Fig. 3. A, B, and C: Representative results of SSCP analysis for messages of TCR Vb repertoires expressed in PBMC from patients before and after IFN-a treatment. The number of SSCP bands for Vb 8 messages decreased from 4 to 2 in a responder (A), whereas the number of SSCP bands for Vb 16 messages increased from 4 to 6 in a transient responder (B), with two bands disappearing and four bands newly appearing. However, the number of bands for Vb 20 messages remained unchanged at 4 both before and after the treatment in a non-responder (C). a: before IFN-a treatment; and b: after IFN-a treatment.

than 6 months. Serum HCV-RNA was quantified by competitive RT-PCR, and genotype analysis of HCV was performed according to the method described by Okamoto et al. [13]. All patients tested were negative for serological markers of hepatitis B virus and had not received immunosuppressants or antiviral drugs within the 6 months prior to the study. Patients were treated initially with 10 million U/day of natural IFN-a (Otsuka Pharm. Co., Ltd., Tokyo, Japan) every day for 2 weeks and then 3 times weekly for the following 22 weeks. Serum HCV-RNA was checked by RT-PCR twice, once at the end treatment and again 6 months later. The patients were retrospectively allocated to three groups as shown in Table 1 based on the effect of IFN-a treatment as follows: responders, in whom HCV-RNA was negative at both points at the end of IFN-a treatment and 6 months later (group A, n = 5); transient responders, in whom HCV-RNA was negative at the end of IFN-a treatment but positive 6 months later (group B, n= 5); and non-responders, in whom HCV-RNA was positive at both points (group C, n= 5).

2.2. Isolation of RNA from PBMC and RT-PCR for TCR Vb expressed in PBMC Heparinized blood samples were collected before the start and immediately after the end of IFN-a treatment and PBMC were obtained by Ficoll-Hypaque density gradient centrifugation. Total RNA was isolated from the PBMC according to the method described by Chomczynski [14]. Briefly, the PBMC were suspended in Isogen-LS (Nippon Gene Inc., Tokyo, Japan) and extracted with chloroform, and the samples were centrifuged. RNA was precipitated from the aqueous phase using isopropanol and then washed and dissolved in water or stabilized with formamide.

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Complementary DNA (cDNA) was synthesized in a reaction containing the extracted RNA (approximately 2 – 4 mg), reverse transcriptase (Takara Biotechnology Co., Ltd., Tokyo, Japan), 10 mM dNTP (Gibco BRL, Life Technologies, Inc., Rockville, MD, USA) and a random primer (Gibco BRL). The cDNA obtained was amplified by PCR using Vb primer sets of 22 repetoires from 1 to 20 and Cb primer according to the method reported previously [15]. Briefly, the PCR reaction mixture consisted of 0.4 ml of cDNA, 0.4 ml of Cb primer (25 mM), 1.6 ml of dNTP (2.5 mM), and 1 U of Taq polymerase (Takara Biotechnology). The amplification proceeded for 35 cycles in a Perkin-Elmer Cetus thermocycler (Perkin Co., Inc., USA) under the following conditions: denaturation at 94°C for 1 min, annealing at 60°C for 2 min, and primer extension at 72°C for 3 min.

2.3. Single-strand conformation polymorphism analysis T-cell clonality was analyzed using the SSCP method described by Yamamoto et al. [9], with minor modification. Briefly, amplified cDNA was diluted 1:9 with 95% formamide containing 10 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol, and the mixture was heated at 95°C for 3 min. Denatured samples (2 ml) were resolved by electrophoresis in non-denaturing 5% polyacrylamide gel containing 10% glycerol at a constant power of 35 W for approximately 2.5 h. The resolved cDNA was transferred to a positively charged nylon membrane (Hybond-N+ , Amersham International Plc., Buckinghamshire, UK) at a constant current of 200 mA for approximately 1 h. The membrane was then hybridized with a 32P-labeled internal Cb1 and Cb2 oligonucleotide probe mixture (5%-GAG GAC CTG AAA/G AAG/C GTG TTC CCA CCC-3%) at 60°C overnight in a mixure of 5×Denhardt’s solution (Ficoll:BSA:polyvinyl pyrolidone, 1:1:1), 4× SSC (NaCl, sodium citrate mixture), 0.1% SDS, 10 mM EDTA, 40 mM Tris–HCl, and 100 mg of sonicated salmon sperm DNA. The membrane was washed at low stringency with 1× SSC containing 0.1% SDS, exposed to an autoradiogram, and then examined using a bioimage analyzing system (BAS 1000, Fuji Film, Tokyo, Japan). Each sharply stained bands were counted as a single band.

2.4. Statistical analysis Results were expressed as the mean9 S.D. Two values were compared using F-test, x 2-test or Mann – Whitney U-test. A two-tailed P value of B 0.05 was considered statistically significant.

3. Results The messages of TCR Vb repertoires Vb 1–Vb 20 expressed in PBMC obtained from patients with chronic hepatitis C before and after treatment with IFN-a were determined by SSCP analysis.

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Representative SSCP bands for TCR Vb messages Vb 1–Vb 20 for one healthy subject and for one patient (c 1) with chronic hepatitis C are shown in Fig. 1. PBMC obtained from healthy subjects showed a smear pattern for each Vb message with 1 – 2 bands. On the other hand, PBMC obtained from patients with chronic hepatitis C before treatment with IFN-a showed 2–13 bands for each TCR Vb in SSCP analysis, indicating the presence of T-cell clones. The number of distinct SSCP bands for messages of TCR Vb repertoires Vb 1–Vb 20 obtained from PBMC of responders (group A), transient responders (group B), non-responders (group C), and six healthy controls are summarized in Table 2. There was no particular bias regarding usage of TCR Vb repertoires in any group of patients, nor in the total of the three groups, either before or after treatment. As shown in Table 3 and Fig. 2, the mean total number of SSCP bands in the responders (group A) decreased significantly from 97.49 40.1 before treatment to 62.6 932.1 after treatment (P B 0.01), whereas that in the transient responders (group B) increased from 66.4 928.0 before treatment to 79.8 9 28.8 after treatment. In group B, there was no difference in the total number of SSCP bands between biochemical responders (patient c 6, 7, 9; serum ALT B 2 times the upper limit of normal at 6 month after the treatment) and the other patients (c8, 10). However, non-responders showed almost no change between before and after treatment. Representative results of SSCP analysis for messages of TCR Vb repertoires expressed in PBMC from patients are shown in Fig. 3. The number of SSCP bands for TCR Vb 8 messages expressed in PBMC from one of the responders (c 2) decreased from 4 to 2 after IFN-a treatment, whereas the number of SSCP bands for TCR Vb 16 messages increased from 4 to 6 in one of the transient responders (c 6), with two bands disappearing and four bands newly appearing after treatment. However, the number of bands for Vb 20 messages remained unchanged at 4 both before and after treatment in one of the non-responders (c 11).

4. Discussion In this study, PBMC obtained from patients with chronic hepatitis C demonstrated 2 – 13 T-cell clones for each TCR Vb in SSCP analysis. The observed absence of specific usage of any Vb repertoire in PBMC indicated that superantigenic stimulation did not occur in these patients. Wang et al. [12] also reported that TCR usage in patients with chronic hepatitis C was neither defective nor biased for any specific TCR Vb, as demonstrated by RT-PCR. Biased TCR Vb usage has been identified in other autoimmune diseases, such as rheumatoid arthritis [16,17], in which, however, the TCR Vb specificity was quite different among different patients. Thus, TCR Vb usage of T-cell clones may reflect a phase of disease activity or individual HLA differences. In clinical responders in this study, the mean total number of T-cell clones in PBMC decreased significantly from 97.4 to 62.6. Elimination of HCV-RNA in sera

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by IFN treatment, followed by the disappearance of stimuli with a specific-antigen, would possibly reduce the number of T-cell clones. However, in non-responders (group C), the positions of the observed SSCP bands for each TCR Vb message expressed in PBMC were quite similar between before and after treatment. Since DNA samples amplified from the same clone migrate to the same identical position in SSCP analysis [9,18], with the same amino acid sequence [19], these findings suggest that the number of common clones remained unchanged at 3.9 in the non-responders (group C) even after IFN-a treatment because of the absence of a decrease in the amount of serum HCV-RNA. On the other hand, in the transient responders (group B), the mean total number of T-cell clones expressed in PBMC increased from 66.4 to 79.8 after treatment. Interestingly, in these transient responders, a few of the SSCP bands present before treatment disappeared after treatment, while a larger number of new SSCP bands appeared at positions different from those of the original bands (Table 2). This would suggest that some T-cell clones disappeared during the transient response to IFN treatment and then new T-cell clones appeared as a result of a different type of antigenic stimuli originating from the IFN-resistant HCV that reappeared after treatment. The most important immunogenic T-cell epitope on HCV has not yet been identified. However, different variations in the hypervariable regions of quasispecies have been observed in individual patients with chronic hepatitis C, and these quasispecies change during IFN-a treatment [20,21]. Thus, the antigenicity of HCV may be changed during IFN-a treatment and IFN-resistant HCV may remain and multiply after the IFN-sensitive HCV is eliminated. On the other hand, further analysis of TCR may help to identify the population of the IFN-sensitive HCV. We were not able to examine the messages of TCR Vb repertoires in liverinfiltrating T cells. An accumulation of common clones in both the periphery and target organs has been identified in liver diseases [15,23,24] and cancers [22] tested by RT-PCR/SSCP analysis. The newly detected T-cell clones in the PBMC of the transient responder patients may reflect liver-infiltrating T-cell clones which were stimulated by newly developed HCV antigens. To clarify the pathogenic effect of these newly developed T-cell clones, more study will be needed, along with classification of these T cells as either CD 8 or CD 4. In conclusion, the clonal expansion of T cells in the peripheral blood of patients with chronic hepatitis C was verified by SSCP analysis of the messages of TCR Vb repetoires. The number of these T-cell clones decreased in responders, increased in transient responders, and remained unchanged in non-responders to IFN-a treatment. Further study is required to determine whether or not these T cells possess cytotoxic activity against HCV-infected hepatocytes and contribute to the progress of chronic hepatitis C. References [1] Cerney A, Chisary FV. Immunological aspects of HCV infection. Intervirology 1994;37:119 – 25. [2] Gonzalez-Peralta RP, Davis GL, Lau JY. Pathogenetic mechanisms of hepatocellular damage in chronic hepatitis C. J Hepatol 1994;21:255 – 9.

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