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P-377 Hepatitis C virus-specific cytotoxic T lymphocyte responses and viral status in chronic hepatitis C virus infection
Posters~International Hepatology Communications 3 SuppL (1995) $37-S169
P-376 IDENTIFICATION OF 'NATIVE' PEP'~IDE8 BOUND TO CLASS [ HLA MOLECULES ON HEPATOCYTE MEMBRANE J.Sakuma, O.Yokosuka, F. Imazeki,M.T~gawa, T. Eh~ta% N.Miyngim, M.Eikuchi, K. Imagawa, T.Takizawa, M.Aihara, T.Tomishige, Y. geike, F. Shimizu~,M. 0matam (1)lst Dept. of Internal Medicine, Chiba Univ. School of Med.(~Bioproduets Development Center, Takara Shuzo Co., Ltd.(8)Microbiological Research Institute, Otsuka Pharmaceutical Co.,Ltd.(4)2nd Dept. of Internal Medicine, Faculty of Med., Univ. of Tokyo, [AIM]Cxtotoxic T lymphocytes recognize the complexes of antigenic peptides and class I HLA molecules on the cell surface. To further understand the cellular immune mechanisms, the amino acid sequence of the peptides bound to HLA molecules from hepatocyte membrane were analyzed. [METHODS1 101° cells of Hep G2 (HLA typing, A2"A24) were cultured and HLA-A2 and A24 proteins were purified by affinity chromatography using the A2- and A24-specific monoclonal antibodies. The antigenic peptides were separated from the purified HLA proteins by acid elution method. Then, the amino acid sequence of the peptides were individually determined by HPLCelectrospray ionization-tandem mass spectrometry (LC/ MS/MS). [RESULTS] 20 peptides (6 from A2,14 from A24) of ca. M.N. I000 (equivalent to 9 A.A.) were identified. Of 6 peptides from HLA-A2, eOOH-terminus were supposed to be V for 2 and L/I for other 2. One of the 14 peptides from HLA-A24 was confirmed to have sequence NPLPQLPQL by using synthetic peptides. ]CONCLUSION]The novel ultra-sensitive method for analysis of 'native' peptides on the surface of hepatocyte might open a new perspective of immunopathogenesis of liver diseases.
P-378 IFN-a AND -1" PRODUCTION IN PATIENTS WITH CHRONIC HEPATITIS C. K.Unol, K. Kakimi2 Y. Suginoshita2, F. Moriyasu2, T. Kishida] 1Inst. Pasteur de Kyoto, 21st Dept. Int. Med., Kyoto Univ., Kyoto, Japan The response to interferon (IFN) therapy on chronic hepatitis C patients depends not only on the amount and subtype of virus but also on the immune status of patients. Thus, parameters which reflect the immune status of HCV patients have been required. Two types of IFN producing capacities, c~(induced by Sendal virus) and y (induced by PHA), were evaluated by the whole blood method in patients with HCV at various stages of the disease. IFN~ and ~, producing capacities of healthy subjects (n=101) were 8897 +4637 and 37+21 in males and 6390 + 3465 and 34 + 20 IU / ml in females, respectively. While IFN-c~ produicng capacity of various stages of HCV patients (n=117) were, CIH:8045 + 5492 (male), 6506 + 5621 (female), CAH: 4480 + 2772, LC:5100 + 3525, HCC:4191 + 2639 IU/ml, and IFN-y producing capacity were, CIH:48 _+38, CAH:44 + 36, LC:51 + 34, HCC:60 + 51 IU/ml, respectively. IFN-c~ producing capacity was observed to gradually decrease along with the progression of the disease while IFN-y producing capacity increased in all stages of the disease. Our research data on various diseases have demonstrated that defect of IFN-a production is related to the effect of virus infection while those of IFN-y reflect the enhancement of function of Thl cells. Periodical measurement of levels of IFN production, both c~and y, might be helpful in determining patients' immune status and would enable us to administer suitable therapy at the appropriate time.
S131
P-377 HEPATITIS C VIRUS-SPECIFIC CYTOTOXIC T LYMPHOCYTE RESPONSES AND VIRAL STATUS IN CHRONIC HEPATITIS C VIRUS INFECTION K.Hiroishi, M.Kojima, H.Kita, H.Okamoto, T.Moriyama, T.Kaneko, K.Ando, T.Aikawa, S.Ohnishi, N.Tanaka, K.Mitamura, M.Imawari Hepatol & I m m u n o l Div, Jichi Meal Sch, Minamikawachimachi; Aikawa Hosp, Mito; 3rd Dept of Intern Med, Univ of Tokyo, Tokyo; Inst of Clin Med, Univ of Tsukuba, Tsukuba; 2 n d Dept of Intern Med, Univ of Showa, Tokyo, Japan. Cytotoxic T l y m p h o c y t e s (CTL) play a role in a host d e f e n s e a g a i n s t viral infection. HLA B44-restricted CTL recognize a n epitope in HCV n u c l e o p r o t e i n r e s i d u e s 81 to 100. Relationships b e t w e e n CTL r e s p o n s e s a n d the status of HCV n u c l e o p r o t e i n residues 81 to 100 were s t u d i e d in 26 patients with g e n o t y p e l b / I I or 2a/III HCV infection mad HLA B44. T h r e e of t h e 26 patients h a d v a r i a n t HCV t h a t were r e c o g n i z e d less efficiently b y t h e CTL. Six of t h e r e m a i n i n g 23 p a t i e n t s (26%) d e m o n s t r a t e d t h e CTL response. Five of eight patients (63%) h a d low titers of s e r u m HCV RNA, while 14 of 15 patients (93%) w h o did n o t d e m o n s t r a t e the CTL r e s p o n s e h a d high titers of s e r u m HCV RNA (p=0.0086). Our findings s u g g e s t t h a t HCV-specific CTL m a y inhibit t h e o u t g r o w t h of HCV, or alternatively, t h a t h i g h - d o s e infection with HCV m a y s u p p r e s s t h e CTL r e s p o n s e . HCV v a r i a n t s that m i g h t escape t h e CTL r e c o g n i t i o n also existed.
P-379
EFFECT OF INTERFERON THERAPY ON THE RESPONSE OF PBMC FROM CHRONIC HEPATITIS C PATIENTS AGAINST HCV CORE-DERIVED PEPTIDES T.Arichi,M.Shirat,T Manabe,H.Okada,O.Fujii,K.Arima,SWatanebe, M.Nishioka Third Dept. of Medicine, Kagawa Medical School, Kagawa, Japan
Helper T lymphocyte (Th) is a critical component of the immune defense against viral infection including hepatitis C vtrus(HCV), because Th cells are necessary for the antibody response as well as the cytotoxic response. CD4 + T lymphocytes from HCV carriers have recently been shown to recognize antigens of HCV To investigate the epitope specifictty in the outcome of HCV infection and interferon (IFN) therapy, Tqymphoeyte responses to viral antigens were studied in infected individuals. Materials and MethodsHCV peptides were prepared by the simultaneous multiple peptide method of solid-phase peptide synthesis. We selected individuals with chronic hepatitis C. PBMC were separated on Ficoli and were introduced into wells of 96-well flat-bottomed culture plates containing each peptide After 78h of incubation, [3H]thymidme was added to all the wells. 18h later the cells were harvested and thymidine incorporated into DNA was determined by scintillation counting. Cytokme production of PBMC in response to peptides was measured by ELISA. Results and Conclusion Using 11 pepttdes from the core region, PBMC from 28 patients with chronic hepatitis C and 3 healthy individuals were assessed. Significant proliferative responses to peptides C2 (aa no.4560),C3(68-83),C5(111-126),C6(117-133).C7(129-144), and Cl1(172188) were observed before IFN therapy. The stimulation index (SI) were elevated in 6 patients after the therapy. 5 out of those 6 patients showed normal ALT level and HCV-RNA negative after the therapy. The level of IL2 production in response to the peptides was elevated in those patients after the therapy, conversely that of IL4 production were decreased. There was correlation between SI (or IL2 predominance) and HCV-RNA elimination. It is thought that a peptide ehctting HCV-speciflc Th may induce protective CTL or antibody against HCV.
P-376 IDENTIFICATION OF 'NATIVE' PEP'~IDE8 BOUND TO CLASS [ HLA MOLECULES ON HEPATOCYTE MEMBRANE J.Sakuma, O.Yokosuka, F. Imazeki,M.T~gawa, T. Eh~ta% N.Miyngim, M.Eikuchi, K. Imagawa, T.Takizawa, M.Aihara, T.Tomishige, Y. geike, F. Shimizu~,M. 0matam (1)lst Dept. of Internal Medicine, Chiba Univ. School of Med.(~Bioproduets Development Center, Takara Shuzo Co., Ltd.(8)Microbiological Research Institute, Otsuka Pharmaceutical Co.,Ltd.(4)2nd Dept. of Internal Medicine, Faculty of Med., Univ. of Tokyo, [AIM]Cxtotoxic T lymphocytes recognize the complexes of antigenic peptides and class I HLA molecules on the cell surface. To further understand the cellular immune mechanisms, the amino acid sequence of the peptides bound to HLA molecules from hepatocyte membrane were analyzed. [METHODS1 101° cells of Hep G2 (HLA typing, A2"A24) were cultured and HLA-A2 and A24 proteins were purified by affinity chromatography using the A2- and A24-specific monoclonal antibodies. The antigenic peptides were separated from the purified HLA proteins by acid elution method. Then, the amino acid sequence of the peptides were individually determined by HPLCelectrospray ionization-tandem mass spectrometry (LC/ MS/MS). [RESULTS] 20 peptides (6 from A2,14 from A24) of ca. M.N. I000 (equivalent to 9 A.A.) were identified. Of 6 peptides from HLA-A2, eOOH-terminus were supposed to be V for 2 and L/I for other 2. One of the 14 peptides from HLA-A24 was confirmed to have sequence NPLPQLPQL by using synthetic peptides. ]CONCLUSION]The novel ultra-sensitive method for analysis of 'native' peptides on the surface of hepatocyte might open a new perspective of immunopathogenesis of liver diseases.
P-378 IFN-a AND -1" PRODUCTION IN PATIENTS WITH CHRONIC HEPATITIS C. K.Unol, K. Kakimi2 Y. Suginoshita2, F. Moriyasu2, T. Kishida] 1Inst. Pasteur de Kyoto, 21st Dept. Int. Med., Kyoto Univ., Kyoto, Japan The response to interferon (IFN) therapy on chronic hepatitis C patients depends not only on the amount and subtype of virus but also on the immune status of patients. Thus, parameters which reflect the immune status of HCV patients have been required. Two types of IFN producing capacities, c~(induced by Sendal virus) and y (induced by PHA), were evaluated by the whole blood method in patients with HCV at various stages of the disease. IFN~ and ~, producing capacities of healthy subjects (n=101) were 8897 +4637 and 37+21 in males and 6390 + 3465 and 34 + 20 IU / ml in females, respectively. While IFN-c~ produicng capacity of various stages of HCV patients (n=117) were, CIH:8045 + 5492 (male), 6506 + 5621 (female), CAH: 4480 + 2772, LC:5100 + 3525, HCC:4191 + 2639 IU/ml, and IFN-y producing capacity were, CIH:48 _+38, CAH:44 + 36, LC:51 + 34, HCC:60 + 51 IU/ml, respectively. IFN-c~ producing capacity was observed to gradually decrease along with the progression of the disease while IFN-y producing capacity increased in all stages of the disease. Our research data on various diseases have demonstrated that defect of IFN-a production is related to the effect of virus infection while those of IFN-y reflect the enhancement of function of Thl cells. Periodical measurement of levels of IFN production, both c~and y, might be helpful in determining patients' immune status and would enable us to administer suitable therapy at the appropriate time.
S131
P-377 HEPATITIS C VIRUS-SPECIFIC CYTOTOXIC T LYMPHOCYTE RESPONSES AND VIRAL STATUS IN CHRONIC HEPATITIS C VIRUS INFECTION K.Hiroishi, M.Kojima, H.Kita, H.Okamoto, T.Moriyama, T.Kaneko, K.Ando, T.Aikawa, S.Ohnishi, N.Tanaka, K.Mitamura, M.Imawari Hepatol & I m m u n o l Div, Jichi Meal Sch, Minamikawachimachi; Aikawa Hosp, Mito; 3rd Dept of Intern Med, Univ of Tokyo, Tokyo; Inst of Clin Med, Univ of Tsukuba, Tsukuba; 2 n d Dept of Intern Med, Univ of Showa, Tokyo, Japan. Cytotoxic T l y m p h o c y t e s (CTL) play a role in a host d e f e n s e a g a i n s t viral infection. HLA B44-restricted CTL recognize a n epitope in HCV n u c l e o p r o t e i n r e s i d u e s 81 to 100. Relationships b e t w e e n CTL r e s p o n s e s a n d the status of HCV n u c l e o p r o t e i n residues 81 to 100 were s t u d i e d in 26 patients with g e n o t y p e l b / I I or 2a/III HCV infection mad HLA B44. T h r e e of t h e 26 patients h a d v a r i a n t HCV t h a t were r e c o g n i z e d less efficiently b y t h e CTL. Six of t h e r e m a i n i n g 23 p a t i e n t s (26%) d e m o n s t r a t e d t h e CTL response. Five of eight patients (63%) h a d low titers of s e r u m HCV RNA, while 14 of 15 patients (93%) w h o did n o t d e m o n s t r a t e the CTL r e s p o n s e h a d high titers of s e r u m HCV RNA (p=0.0086). Our findings s u g g e s t t h a t HCV-specific CTL m a y inhibit t h e o u t g r o w t h of HCV, or alternatively, t h a t h i g h - d o s e infection with HCV m a y s u p p r e s s t h e CTL r e s p o n s e . HCV v a r i a n t s that m i g h t escape t h e CTL r e c o g n i t i o n also existed.
P-379
EFFECT OF INTERFERON THERAPY ON THE RESPONSE OF PBMC FROM CHRONIC HEPATITIS C PATIENTS AGAINST HCV CORE-DERIVED PEPTIDES T.Arichi,M.Shirat,T Manabe,H.Okada,O.Fujii,K.Arima,SWatanebe, M.Nishioka Third Dept. of Medicine, Kagawa Medical School, Kagawa, Japan
Helper T lymphocyte (Th) is a critical component of the immune defense against viral infection including hepatitis C vtrus(HCV), because Th cells are necessary for the antibody response as well as the cytotoxic response. CD4 + T lymphocytes from HCV carriers have recently been shown to recognize antigens of HCV To investigate the epitope specifictty in the outcome of HCV infection and interferon (IFN) therapy, Tqymphoeyte responses to viral antigens were studied in infected individuals. Materials and MethodsHCV peptides were prepared by the simultaneous multiple peptide method of solid-phase peptide synthesis. We selected individuals with chronic hepatitis C. PBMC were separated on Ficoli and were introduced into wells of 96-well flat-bottomed culture plates containing each peptide After 78h of incubation, [3H]thymidme was added to all the wells. 18h later the cells were harvested and thymidine incorporated into DNA was determined by scintillation counting. Cytokme production of PBMC in response to peptides was measured by ELISA. Results and Conclusion Using 11 pepttdes from the core region, PBMC from 28 patients with chronic hepatitis C and 3 healthy individuals were assessed. Significant proliferative responses to peptides C2 (aa no.4560),C3(68-83),C5(111-126),C6(117-133).C7(129-144), and Cl1(172188) were observed before IFN therapy. The stimulation index (SI) were elevated in 6 patients after the therapy. 5 out of those 6 patients showed normal ALT level and HCV-RNA negative after the therapy. The level of IL2 production in response to the peptides was elevated in those patients after the therapy, conversely that of IL4 production were decreased. There was correlation between SI (or IL2 predominance) and HCV-RNA elimination. It is thought that a peptide ehctting HCV-speciflc Th may induce protective CTL or antibody against HCV.