VIROLOGY
191, 32 l-326
(1992)
Hepatitis B Virus-DNA Transfected Myeloma Cell-Specific Cytotoxic T Cells in Chronic Hepatitis B Patients’ YUMIKO KAMOGAWA,* KATSUMI YAMAUCHI,**’ HIROSHI OBATA,* OSAMU CHISAKA,t AND KENICHI MATSUBARAt *Division
of Medicine, Institute of Gastroenterology, Tokyo Women’s Medical College, 8- 1, Kawada-cho, Shinjuku-ku, Tokyo, Japan; and tlnstitute for Molecular and Cellular Biology, Osaka University, 3-2, Yamadaoka. Suita-shi, Osaka, Japan Received March 30, 1992; accepted fuly 28, 1992
To study the mechanisms of hepatitis B virus (HBV)-induced chronic hepatitis (B-CH), we took chronic hepatitis B patients’ peripheral blood lymphocytes (PBL) and examined their cytotoxic activities against human myeloma cells (ARH77) transfected by HBV-DNA. Two different transfected cells, one expressing HBV envelope antigens (S6) and the other expressing HBV core antigens (C4), were prepared and used as targets in the in vitro cytotoxic test. We found that PBL of B-CH patients had specific cytotoxic activity against these target cells (S6, 22.0 f 4.8%; C4, 21.6 t 4.8%), whereas no remarkable cytotoxic activity was observed in non-B chronic hepatitis patients as well as asymptomatic chronic HBV carriers. These specific cytotoxic activities were inhibited with anti-CD3 antibody, hence these killer cells belonged to T cells (cytotoxic T cells; CTL). The requirement of HLA class 1 antigens to exert these CTL activities was demonstrated by the absence of CTL activity with PBL obtained from HIA-nonidentical B-CH patients and by the inhibition of their activities with anti-HL4 class 1 antibody. Thus, our results indicate that, at least two different CTL, o 1992 one recognizing envelop antigen and the other recognizing core antigen, exist in chronic hepatitis B patients. Academic
Press,
Inc.
We prepared human myeloma cells expressing HBV-related antigen and used them to study the specific cytotoxic activity of a CH patients’ PBL in vitro cytotoxic test.
INTRODUCTION Hepatitis B virus (HBV) infection can lead to various outcomes in infected hosts (Hoofnagle, 1981), including induction of chronic hepatitis (CH), the acquisition of liver cirrhosis, and induced hepatocellular carcinoma, making the efforts important to understand the mechanism of HBV-induced CH. The existence of asymptomatic chronic HBV carriers suggests that HBV is not directly cytopathic. Rather, it is generally accepted that the host immune response, particularly the cell-mediated immune response, plays an important role in the generation of CH (Dudley et al., 1972). This possibility is supported by the fact that infiltration of CD8 (suppresser/cytotoxic) cells associates the hepatitis liver, particularly in the area of piecemeal necrosis (Montano et a/., 1983; Eggink et a/., 1982). In addition, it was demonstrated (Wands and Isselbacher, 1975; Mondelli et a/., 1982; Naumov et a/., 1984) that peripheral blood lymphocytes (PBL) of these patients or lymphocytes in the liver can act as HBV-related antigenspecific cytotoxic cells. However, the lack of a stable in vitro system makes it difficult to further study the mechanism of HBV-specific cytotoxic cells.
’ This work was supported Science and Culture. * To whom correspondence dressed.
PATIENTS Patients
Thirteen HBsAg-positive chronic hepatitis patients (B-CH) whose phenotypes of HLA class 1 were identical to ARH 77 (with a median age of 40.1 years; range, 17 to 50 years), were examined and compared with control patient groups. As controls, the following three patient groups were examined: (1) B-CH with different types of HLA to ARH 77 (median age was 42.7 years), (2) CH patients with the identical HLA phenotypes to ARH 77 having no HBV-related markers (NB-CH; median age was 46.7 years), and (3) asymptomatic chronic HBV carriers (ASC; median age was 40.9 years) with the identical HLA phenotype to ARH 77. Diagnosis of these patients was done based on their clinical features and histological examinations. ASC were diagnosed as having HBsAg in their sera without any evidence of liver injury for more than a year. HLA phenotypes of these patients and ARH 77 (phenotype; A24/33 B35 C4 DR8/12,) were determined by ordinary microcytotoxic test (Ray et a/., 1976).
by grants from Ministry of Education, and reprint requests
AND METHODS
should be ad-
321
0042-6822192
$5.00
CopyrIght 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
KAMOGAWA
322
ET AL.
cells (ARH-neo) were used as controls quent studies. Expression of HBV-related of transfectants
TK SW Enh
6 sv40 f PO’ ECOOP sv40 ori
Amp
FIG. 1. Construction of the plasmids pUc-HBc2, pUc-HBsl, and psT-neoB. (A) The pUc-HBc2 contains the complete Eco-gpt gene, human immunoglobulin heavy chain (IgH) enhancer gene, and the HBcAg coding region (BstE2-Dral fragment of PACYC HBadr4). (B) The pUc-HBsl contains the Eco-gpt gene, IgH enhancer gene, and the large envelop Ag coding regions (BarnHI-Bglll fragment of pBRHBadr4. (C) A control plasmid, psT-neoB. contains only the neomycin-resistant gene. (D) Locations of the transfected HBV genes.
Construction of human myeloma integrated HBV-DNA
cells having
ARH 77, a well-established human myeloma cell line (Burk eta/., 1978) was selected because its HLA class 1 phenotype (A24) was frequently found in Orientals (58.5%) and it was relatively resistant to NK cell activity (as shown in Fig. 5 legend). The vector used in this study was a derivation of pUc having the Eco-gpt gene and human immunoglobulin enhancer (Kudo et al., 1985). It was kindly provided by Dr. T. Watanabe, Kyusyu University. The expression vectors shown in Fig. 1 were linearized and ligated either with BarnHI-Bglll fragment (2.1 kb) containing large envelope antigen (HBsAg and pre-SAg) coding regions or with BstE2Dral fragment (1.98 kb) containing core antigen (HBcAg) gene. Both DNA fragments were originated from pBR HB adr4 (Fujiyama et a/., 1983). ARH 77 cells were transfected with 40 pg of one of these constructs along with Neomycin resistance gene (obtained from Dr. T. Taniguchi, Osaka University) using electroporation (Potter eta/., 1984). After the incubation of the culture for 48 hr, the cells were exposed to selection medium (RPM1 1640, GIBCO, USA, supplemented with 10% fetal calf serum and 1000 pgl ml of G418, Sigma, USA) for 2 to 3 weeks, and G418resistant colonies were selected. ARH-77 was also transfected with Neomycin resistance gene alone, and the resulting G418-resistant
in the subse-
markers on the surface
Expression of either HBsAg or HBcAg on the transfected cell was demonstrated by indirect immunofluorescence study. Cells (5 X 106) were incubated at 4’ for 30 min with either monoclonal anti-HBs or anti-HBc antibodies, obtained from Chemo-Sero-Therapeutic Co., Kumamoto, Japan. The cells were then washed and preincubated with FITC-labeled goat anti-mouse IgG at 4” for a further 30 min, fixed in 0.1% paraformaldehyde-PBS, and analyzed by FACStar. Finally, two different cell lines, HBsAg expressing transfected cell (S6) and core antigen expressing transfected cells (C4), were established. Southern
and Northern
blot analysis
A total cellular DNA was obtained from the transfected cells by a method reported previously (Maniatis et al., 1982). In brief, DNAs obtained from the cells, S6, C4, and ARH-neo, were digested with Sall, EcoRI, and Pstl, respectively, and subjected to agarose gelelectrophoreses, followed by and filter hybridization analysis. For Northern blot analysis, a total cellular RNA was extracted and purified by a centrifugation through guanidine isothianatecesium chloride density gradient (Chrgwin et a/., 1979). Poly(A)-tailed RNA was purified by oligo(dT) affinity chromatography and electrophoresed using 0.8% agarose-formaldehyde gels and then blotted onto nitrocellulose membrane. Hybridization analysis was performed using 32P-labeled cloned total HBV-DNA (Mulligan and Berg, 1980). In vitro cytotoxic
test
PBL were separated from heparinized venous blood using a Ficoll-Hypaque (Pharmacia Co., Upsala Sweden) gradient centrifugation. DNA transfected cells were suspended in 0.3 ml of RPMI 1640 containing 10% FCS and 100 &i of 5’Cr (Amersham Co.), incubated at 37” for 2 hr, and then washed extensively with PBS and resuspended with culture medium. Theywere used as targets in the in vitro cytotoxic test. ARH 77 cells transfected by the control plasmid containing the neomycin-resistant gene (ARH-Neo) were also used as control targets to calculate HBV-DNA transfected cellspecific cytotoxicity. An appropriate number of PBL was incubated with 1 X 1O4target cells in a round-bottomed 96-well culture plate at 37” for 4 hr. Subsequently, the level of 5’Cr in these cultures supernatants was measured and percent lysis was calculated by a
HBV-SPECIFIC
323
CTL IN B-CH PATIENTS
Expression of HBV-related of S6 and C4
on the surface
To identify expression of antigens on the surface of immunofluorescence study was performed. As shown in Fig. 3 both S6 and C4 were positively stained with either anti-HBs antibody or anti-HBc antibody, respectively, indicating that each of the transfecting DNA was expressing on the surface of these cells. No such signal was observed with the control cells, ARH-neo. These results indicate that both HBsAg and HBcAg were synthesized by these integrated HBV-DNAs and were expressed on S6 and C4, respectively.
+Z.lkb
FIG. 2. (A) Southern
blot analyses of transfected cells. Southern blot analyses of total cellular DNA (10 pg) prepared from each transfected cells was carried out using 32-P-labeled whole HBV-DNA probe. (B) RNA blot analyses of poly(A)-tailed RNA (10 pg) purified by oligo(dT) affinity chromatography was done by using the same probe.
subtraction from the percent lysis of either S6 or C4 to that of control plasmid transfectants. Inhibition of in vitro cytotoxicity monoclonal antibodies
antigens
with various
PBL were cultivated with various doses of monoclonal antibodies, either murine anti-CD3 or anti-HLA class 1 antibody (Cosmo Bio Co., Tokyo, Japan) and murine myeloma protein (Cape1 Co., USA) for 1 hr at 37RT” prior to mixing with the targets. Their cytotoxicity was then examined as described above.
Cytotoxic and C4
activities
of patients’
PBL against S6
We examined whether PBL obtained from two B-CH (B-CH 1, HLA-A24/33 B35/61 C3 DR4/6; and B-CH 2, HLA-Al 1 B15/55 Cl DR12/5) and from a NB-CH (NBCH 1, HLA-A24 B51/60 C3 DR4/6) might exhibit cytotoxicity against these transfectants. As shown in Fig. 4A, PBL from HLA-A locus identical B-CH (B-CH 1) killed both C4 and S6 but not ARH-Neo (in 50/l of effector/target ratio; 56.0 f 4.8% to S6 and 57.1 f 2.2% to C4, and 26.3 f 1.4% to ARH-neo, respectively). On the other hand, with PBL in other two patients, B-CH 2 and NB-CH 1, no significant difference existed in their killer cell activity against C4, S6 and ARH-Neo. (Figs. 4B and 4C). Thus, both S6 and C4 can be killed by PBL
RESULTS The state of the integrated the HBV gene
DNA and transcription
of
To see the state of integration of the transfecting plasmid, Southern blot analysis was done using high molecular DNA, which digested with Sell, Pstl, or EcoRI. As shown in Experiment A in Fig. 2, where a labeled full-length HBV DNA was used as a probe, a single band, 2.1 kb in size, was observed in S6, whereas two bands (4.1 and 1.8 kb) were observed in C4. No detectable HBV-DNA was observed in ARHneo. These results indicate that transfected DNAs integrated into cellular DNA in an expected configuration. The presence of transcripts of these integrated DNAs is demonstrated in Experiment B in Fig. 2 (lane 2, ARH-S6; lane 3, ARH-C4). In both C4 and S6, a fairly good amount of HBV-DNA transcripts were identified.
Fluorescence
Intensity
FIG. 3. Flow cytometry analysis of the HBV-DNA transfected cells. ARH-Neo and C4 were stained with either anti-HBc plus anti-mouse immunoglobulin(anti-Mlg)-FITC or with G-Mlg-FITC alone (a and b). ARH-Neo and S6 were stained with either anti-HBs plus anti-MlgFITC or anti-Mlg-FITC alone (c and d). A control FACS pattern (cells stained without first antibody) is shown by a solid line, and experiment groups (cells stained by monoclonal antibody) are shown by a dotted line.
324
KAMOGAWA
A
B
C
B-CH2
NB-CH
ET AL Exp-A Target
% inhibition Treatment
S6 HBc 50 -HBs
c4
Ne0 \
Exp-B Target
Treatment 0 (IlWml)
0
I
E/T ratio
50
75% I
1
50
25
12.5
50
25
12.5
50
25
12.5
of PBL from three CH patients against three transfected cells. Both B-CH 1 and NB-CH 1 share the same class 1 phenotype (A24) to these transfected cells, whereas B-CH 2 has the different HLA phenotype form the targets. Note significant cytotoxicities against HBV-DNA transfected cells, both S6 and C4, were only observed in B-CH 1’ PBL but not in others. (0) Cytotoxicity against C4, (0) cytototoxicity against S6, (x) cytotoxicity against ARH-Neo.
L
FIG. 4. Cytotoxicity
obtained from B-CH having the same HLA-class 1 phenotype to these targets but not the others. To further extend these observations, the cytotoxic activities of PBL obtained from 13 HLA identical B-CH patients (Gr. 1 in Fig. 5) were examined similarly and compared with those of PBL from five HLA-nonidentical B-CH patients (Gr. 2) seven HLA-identical NB-CH patients (Gr. 3) and nine HLA-identical ASC (Gr. 4). As
Target : C4
Target : S6 P
191, 32 l-326
(1992)
Hepatitis B Virus-DNA Transfected Myeloma Cell-Specific Cytotoxic T Cells in Chronic Hepatitis B Patients’ YUMIKO KAMOGAWA,* KATSUMI YAMAUCHI,**’ HIROSHI OBATA,* OSAMU CHISAKA,t AND KENICHI MATSUBARAt *Division
of Medicine, Institute of Gastroenterology, Tokyo Women’s Medical College, 8- 1, Kawada-cho, Shinjuku-ku, Tokyo, Japan; and tlnstitute for Molecular and Cellular Biology, Osaka University, 3-2, Yamadaoka. Suita-shi, Osaka, Japan Received March 30, 1992; accepted fuly 28, 1992
To study the mechanisms of hepatitis B virus (HBV)-induced chronic hepatitis (B-CH), we took chronic hepatitis B patients’ peripheral blood lymphocytes (PBL) and examined their cytotoxic activities against human myeloma cells (ARH77) transfected by HBV-DNA. Two different transfected cells, one expressing HBV envelope antigens (S6) and the other expressing HBV core antigens (C4), were prepared and used as targets in the in vitro cytotoxic test. We found that PBL of B-CH patients had specific cytotoxic activity against these target cells (S6, 22.0 f 4.8%; C4, 21.6 t 4.8%), whereas no remarkable cytotoxic activity was observed in non-B chronic hepatitis patients as well as asymptomatic chronic HBV carriers. These specific cytotoxic activities were inhibited with anti-CD3 antibody, hence these killer cells belonged to T cells (cytotoxic T cells; CTL). The requirement of HLA class 1 antigens to exert these CTL activities was demonstrated by the absence of CTL activity with PBL obtained from HIA-nonidentical B-CH patients and by the inhibition of their activities with anti-HL4 class 1 antibody. Thus, our results indicate that, at least two different CTL, o 1992 one recognizing envelop antigen and the other recognizing core antigen, exist in chronic hepatitis B patients. Academic
Press,
Inc.
We prepared human myeloma cells expressing HBV-related antigen and used them to study the specific cytotoxic activity of a CH patients’ PBL in vitro cytotoxic test.
INTRODUCTION Hepatitis B virus (HBV) infection can lead to various outcomes in infected hosts (Hoofnagle, 1981), including induction of chronic hepatitis (CH), the acquisition of liver cirrhosis, and induced hepatocellular carcinoma, making the efforts important to understand the mechanism of HBV-induced CH. The existence of asymptomatic chronic HBV carriers suggests that HBV is not directly cytopathic. Rather, it is generally accepted that the host immune response, particularly the cell-mediated immune response, plays an important role in the generation of CH (Dudley et al., 1972). This possibility is supported by the fact that infiltration of CD8 (suppresser/cytotoxic) cells associates the hepatitis liver, particularly in the area of piecemeal necrosis (Montano et a/., 1983; Eggink et a/., 1982). In addition, it was demonstrated (Wands and Isselbacher, 1975; Mondelli et a/., 1982; Naumov et a/., 1984) that peripheral blood lymphocytes (PBL) of these patients or lymphocytes in the liver can act as HBV-related antigenspecific cytotoxic cells. However, the lack of a stable in vitro system makes it difficult to further study the mechanism of HBV-specific cytotoxic cells.
’ This work was supported Science and Culture. * To whom correspondence dressed.
PATIENTS Patients
Thirteen HBsAg-positive chronic hepatitis patients (B-CH) whose phenotypes of HLA class 1 were identical to ARH 77 (with a median age of 40.1 years; range, 17 to 50 years), were examined and compared with control patient groups. As controls, the following three patient groups were examined: (1) B-CH with different types of HLA to ARH 77 (median age was 42.7 years), (2) CH patients with the identical HLA phenotypes to ARH 77 having no HBV-related markers (NB-CH; median age was 46.7 years), and (3) asymptomatic chronic HBV carriers (ASC; median age was 40.9 years) with the identical HLA phenotype to ARH 77. Diagnosis of these patients was done based on their clinical features and histological examinations. ASC were diagnosed as having HBsAg in their sera without any evidence of liver injury for more than a year. HLA phenotypes of these patients and ARH 77 (phenotype; A24/33 B35 C4 DR8/12,) were determined by ordinary microcytotoxic test (Ray et a/., 1976).
by grants from Ministry of Education, and reprint requests
AND METHODS
should be ad-
321
0042-6822192
$5.00
CopyrIght 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
KAMOGAWA
322
ET AL.
cells (ARH-neo) were used as controls quent studies. Expression of HBV-related of transfectants
TK SW Enh
6 sv40 f PO’ ECOOP sv40 ori
Amp
FIG. 1. Construction of the plasmids pUc-HBc2, pUc-HBsl, and psT-neoB. (A) The pUc-HBc2 contains the complete Eco-gpt gene, human immunoglobulin heavy chain (IgH) enhancer gene, and the HBcAg coding region (BstE2-Dral fragment of PACYC HBadr4). (B) The pUc-HBsl contains the Eco-gpt gene, IgH enhancer gene, and the large envelop Ag coding regions (BarnHI-Bglll fragment of pBRHBadr4. (C) A control plasmid, psT-neoB. contains only the neomycin-resistant gene. (D) Locations of the transfected HBV genes.
Construction of human myeloma integrated HBV-DNA
cells having
ARH 77, a well-established human myeloma cell line (Burk eta/., 1978) was selected because its HLA class 1 phenotype (A24) was frequently found in Orientals (58.5%) and it was relatively resistant to NK cell activity (as shown in Fig. 5 legend). The vector used in this study was a derivation of pUc having the Eco-gpt gene and human immunoglobulin enhancer (Kudo et al., 1985). It was kindly provided by Dr. T. Watanabe, Kyusyu University. The expression vectors shown in Fig. 1 were linearized and ligated either with BarnHI-Bglll fragment (2.1 kb) containing large envelope antigen (HBsAg and pre-SAg) coding regions or with BstE2Dral fragment (1.98 kb) containing core antigen (HBcAg) gene. Both DNA fragments were originated from pBR HB adr4 (Fujiyama et a/., 1983). ARH 77 cells were transfected with 40 pg of one of these constructs along with Neomycin resistance gene (obtained from Dr. T. Taniguchi, Osaka University) using electroporation (Potter eta/., 1984). After the incubation of the culture for 48 hr, the cells were exposed to selection medium (RPM1 1640, GIBCO, USA, supplemented with 10% fetal calf serum and 1000 pgl ml of G418, Sigma, USA) for 2 to 3 weeks, and G418resistant colonies were selected. ARH-77 was also transfected with Neomycin resistance gene alone, and the resulting G418-resistant
in the subse-
markers on the surface
Expression of either HBsAg or HBcAg on the transfected cell was demonstrated by indirect immunofluorescence study. Cells (5 X 106) were incubated at 4’ for 30 min with either monoclonal anti-HBs or anti-HBc antibodies, obtained from Chemo-Sero-Therapeutic Co., Kumamoto, Japan. The cells were then washed and preincubated with FITC-labeled goat anti-mouse IgG at 4” for a further 30 min, fixed in 0.1% paraformaldehyde-PBS, and analyzed by FACStar. Finally, two different cell lines, HBsAg expressing transfected cell (S6) and core antigen expressing transfected cells (C4), were established. Southern
and Northern
blot analysis
A total cellular DNA was obtained from the transfected cells by a method reported previously (Maniatis et al., 1982). In brief, DNAs obtained from the cells, S6, C4, and ARH-neo, were digested with Sall, EcoRI, and Pstl, respectively, and subjected to agarose gelelectrophoreses, followed by and filter hybridization analysis. For Northern blot analysis, a total cellular RNA was extracted and purified by a centrifugation through guanidine isothianatecesium chloride density gradient (Chrgwin et a/., 1979). Poly(A)-tailed RNA was purified by oligo(dT) affinity chromatography and electrophoresed using 0.8% agarose-formaldehyde gels and then blotted onto nitrocellulose membrane. Hybridization analysis was performed using 32P-labeled cloned total HBV-DNA (Mulligan and Berg, 1980). In vitro cytotoxic
test
PBL were separated from heparinized venous blood using a Ficoll-Hypaque (Pharmacia Co., Upsala Sweden) gradient centrifugation. DNA transfected cells were suspended in 0.3 ml of RPMI 1640 containing 10% FCS and 100 &i of 5’Cr (Amersham Co.), incubated at 37” for 2 hr, and then washed extensively with PBS and resuspended with culture medium. Theywere used as targets in the in vitro cytotoxic test. ARH 77 cells transfected by the control plasmid containing the neomycin-resistant gene (ARH-Neo) were also used as control targets to calculate HBV-DNA transfected cellspecific cytotoxicity. An appropriate number of PBL was incubated with 1 X 1O4target cells in a round-bottomed 96-well culture plate at 37” for 4 hr. Subsequently, the level of 5’Cr in these cultures supernatants was measured and percent lysis was calculated by a
HBV-SPECIFIC
323
CTL IN B-CH PATIENTS
Expression of HBV-related of S6 and C4
on the surface
To identify expression of antigens on the surface of immunofluorescence study was performed. As shown in Fig. 3 both S6 and C4 were positively stained with either anti-HBs antibody or anti-HBc antibody, respectively, indicating that each of the transfecting DNA was expressing on the surface of these cells. No such signal was observed with the control cells, ARH-neo. These results indicate that both HBsAg and HBcAg were synthesized by these integrated HBV-DNAs and were expressed on S6 and C4, respectively.
+Z.lkb
FIG. 2. (A) Southern
blot analyses of transfected cells. Southern blot analyses of total cellular DNA (10 pg) prepared from each transfected cells was carried out using 32-P-labeled whole HBV-DNA probe. (B) RNA blot analyses of poly(A)-tailed RNA (10 pg) purified by oligo(dT) affinity chromatography was done by using the same probe.
subtraction from the percent lysis of either S6 or C4 to that of control plasmid transfectants. Inhibition of in vitro cytotoxicity monoclonal antibodies
antigens
with various
PBL were cultivated with various doses of monoclonal antibodies, either murine anti-CD3 or anti-HLA class 1 antibody (Cosmo Bio Co., Tokyo, Japan) and murine myeloma protein (Cape1 Co., USA) for 1 hr at 37RT” prior to mixing with the targets. Their cytotoxicity was then examined as described above.
Cytotoxic and C4
activities
of patients’
PBL against S6
We examined whether PBL obtained from two B-CH (B-CH 1, HLA-A24/33 B35/61 C3 DR4/6; and B-CH 2, HLA-Al 1 B15/55 Cl DR12/5) and from a NB-CH (NBCH 1, HLA-A24 B51/60 C3 DR4/6) might exhibit cytotoxicity against these transfectants. As shown in Fig. 4A, PBL from HLA-A locus identical B-CH (B-CH 1) killed both C4 and S6 but not ARH-Neo (in 50/l of effector/target ratio; 56.0 f 4.8% to S6 and 57.1 f 2.2% to C4, and 26.3 f 1.4% to ARH-neo, respectively). On the other hand, with PBL in other two patients, B-CH 2 and NB-CH 1, no significant difference existed in their killer cell activity against C4, S6 and ARH-Neo. (Figs. 4B and 4C). Thus, both S6 and C4 can be killed by PBL
RESULTS The state of the integrated the HBV gene
DNA and transcription
of
To see the state of integration of the transfecting plasmid, Southern blot analysis was done using high molecular DNA, which digested with Sell, Pstl, or EcoRI. As shown in Experiment A in Fig. 2, where a labeled full-length HBV DNA was used as a probe, a single band, 2.1 kb in size, was observed in S6, whereas two bands (4.1 and 1.8 kb) were observed in C4. No detectable HBV-DNA was observed in ARHneo. These results indicate that transfected DNAs integrated into cellular DNA in an expected configuration. The presence of transcripts of these integrated DNAs is demonstrated in Experiment B in Fig. 2 (lane 2, ARH-S6; lane 3, ARH-C4). In both C4 and S6, a fairly good amount of HBV-DNA transcripts were identified.
Fluorescence
Intensity
FIG. 3. Flow cytometry analysis of the HBV-DNA transfected cells. ARH-Neo and C4 were stained with either anti-HBc plus anti-mouse immunoglobulin(anti-Mlg)-FITC or with G-Mlg-FITC alone (a and b). ARH-Neo and S6 were stained with either anti-HBs plus anti-MlgFITC or anti-Mlg-FITC alone (c and d). A control FACS pattern (cells stained without first antibody) is shown by a solid line, and experiment groups (cells stained by monoclonal antibody) are shown by a dotted line.
324
KAMOGAWA
A
B
C
B-CH2
NB-CH
ET AL Exp-A Target
% inhibition Treatment
S6 HBc 50 -HBs
c4
Ne0 \
Exp-B Target
Treatment 0 (IlWml)
0
I
E/T ratio
50
75% I
1
50
25
12.5
50
25
12.5
50
25
12.5
of PBL from three CH patients against three transfected cells. Both B-CH 1 and NB-CH 1 share the same class 1 phenotype (A24) to these transfected cells, whereas B-CH 2 has the different HLA phenotype form the targets. Note significant cytotoxicities against HBV-DNA transfected cells, both S6 and C4, were only observed in B-CH 1’ PBL but not in others. (0) Cytotoxicity against C4, (0) cytototoxicity against S6, (x) cytotoxicity against ARH-Neo.
L
FIG. 4. Cytotoxicity
obtained from B-CH having the same HLA-class 1 phenotype to these targets but not the others. To further extend these observations, the cytotoxic activities of PBL obtained from 13 HLA identical B-CH patients (Gr. 1 in Fig. 5) were examined similarly and compared with those of PBL from five HLA-nonidentical B-CH patients (Gr. 2) seven HLA-identical NB-CH patients (Gr. 3) and nine HLA-identical ASC (Gr. 4). As
Target : C4
Target : S6 P
