Immunology 1990 70 197-202
HBsAg-induced interferon-gamma secretion in T cells from asymptomatic HBsAg carriers and HB-immune donors in vitro S. P. E. SYLVAN & U. B. HELLSTROM The Elias Bengtsson Research Unit, Department of Infectious Diseases, Karolinska Institute, Roslagstull Hospital, Stockholm, Sweden
Acceptedfor publication 14 February 1990
SUMMARY Peripheral T lymphocytes obtained from asymptomatic carriers of hepatitis B surface antigen (HBsAg) and donors immune to hepatitis B (HB) through natural infection or vaccination were induced by the envelope protein (HBsAg) of the hepatitis B virus (HBV) into secretion of interferongamma (IFN-y) in vitro. The kinetics of the IFN-y response varied between individuals, but was constantly found to be biphasic, with an early peak attained after 12 hr-A days and a late peak after 58 days of antigen stimulation. The early release of IFN-y activity was antigen-specifically induced, as it was in T cells from HB-immune and asymptomatic carriers of HBsAg but not HB-susceptible controls. The second peak of HBsAg-induced IFN-y secretion was induced in all three donor groups and the kinetics of IFN-y release were similar to that of the mitogen phytohemagglutinin(PHA)induced IFN-y production in similarly prepared T-cell cultures. Thus the late burst of IFN-y activity seems to result from a mitogenic property contained within the envelope material of HBV. The mitogenic response was three- to fivefold higher for 4/7 asymptomatic carriers of HBsAg compared to HB-immune donors and HB-susceptible controls, indicating that some patients with chronic asymptomatic carriership of HBsAg exhibit enhanced mitogenic responses to HBsAg.
(Dudley, Fox & Sherlock, 1972). Recently, however, circulatory immunocompetent T lymphocytes were demonstrated with reactivity against the outer coat of hepatitis B virus (HBV), as measured by lymphocyte transformation (Sylvan, Hellstr6m & Lundbergh, 1985) and interleukin-2 (IL-2) secretion (Sylvan & Hellstr6m, 1989), in asymptomatic carriers of HBsAg as well as donors with acquired immunity to HB. Production of IL-2 has been demonstrated to be associated with the generation of IFN-y (Northoffet al., 1980; Farrar, Johnson & Farrar, 1981). Therefore, it is conceivable that HBsAg-sensitized peripheral blood T lymphocytes with the capacity to produce IL-2, when stimulated with HBsAg in vitro should also be able to secrete IFN-y. Thus, a cohort of asymptomatic HBsAg carriers was analysed for IFN-y-producing capacity when specifically stimulated with HBsAg in in vitro conditions previously reported to be optimal for HBsAg-induced IL-2 secretion for these donors (Sylvan & Hellstrom, 1989). T lymphocytes from donors with acquired immunity to HB by infection or recent vaccination and HB-susceptible individuals were included as positive and negative controls.
INTRODUCTION Human peripheral blood lymphocytes produce interferon(IFN-y) in response to several non-viral and viral inducers (Wheelock, 1965; Nathan et al., 1981; Epstein, Cline & Merrigan, 1971; Epstein, Stevens & Merrigan, 1972; Valle et al., 1975; Ennis & Meager, 1981; Nakayama, 1983). The capacity of hepatitis B surface antigen (HBsAg) to induce IFN-y secretion has so far been studied only in T-cell clones obtained from hepatitis B (HB) vaccine recipients (Celis, Miller & Chang, 1984). It has been suggested that peripheral blood lymphocytes from patients with acute and chronic viral HB have a reduced capacity to produce IFN-a or IFN-y when stimulated with Newcastle disease (Tolentino et al., 1975), Influenza (Abb et al., 1985) and Sendai (Tolentino et al., 1975; Kinoshita et al., 1979; Kato et al., 1982; Ikeda, Lever & Thomas, 1986) viruses, Molt4 human leukaemia cells (Abb et al., 1985), polyinosinic-cytidilic acid (poly-IC; Levin & Hahn, 1982), mitogens (Levin & Hahn, 1982; Abb et al., 1985; Ikeda et al., 1986; Fuji et al., 1987) or interleukin-2 (IL-2; Fuji et al., 1987). However, no clear-cut correlation between IFN secretion and active viral replication or the severity of liver disease has emerged from these studies. It has been postulated that HBsAg carriers, particularly if asymptomatic, do not exhibit T-cell functions against HBsAg gamma
MATERIALS AND METHODS
Subjects Seven asymptomatic HBsAg carriers (five females; mean age 38 years, range 31-42 years) were studied. They had no detectable antibodies to HBsAg (anti-HBs) in serum and possessed HBsAg
Correspondence: Dr S. Sylvan, Dept. of Infectious Diseases, Karolinska Institute, Roslagstull Hospital, Box 5651, S-1 1489 Stockholm, Sweden.
197
198
S. P. E. Sylvan & U. B. Hellstrim
titres varying between 1/10,000 and 1/100,000 (subtype ad in all), IgG antibodies to hepatitis B core antigen (anti-HBc; 1/1000-1/10,000) and stable titres (1/10-1/1000) of antibodies to hepatitis B e antigen (anti-HBe). All had been found to be HBsAg positive through routine screening procedures of voluntary blood donors and none had a previous history of hepatitis or drug abuse. A percutaneous liver biopsy was available in one patient and showed normal liver histology. Repeated examination of their clinical status and liver-function tests over a 3-9 year period before being included in the study were consistently found to be normal. All were negative for HBV-DNA in serum, as measured by a method in which samples are hybridized in solution with an iodinated probe (Dr M. Kuhns, Abbott Laboratories, North Chicago, IL; Kuhns et al., 1988), and demonstrated no homosexual preferences. For comparison, seven HB-immune donors (all females; mean age 44 years, range 27-59 years), four with naturally acquired immunity to HB as judged by the absence of HBsAg and presence of anti-HBs (1/10-1/10,000) and anti-HBc (1/101/1000), three with artificially induced immunity after repeated vaccination with a plasma-derived HBV vaccine (Hep-B vax; Merck Sharp and Dohme) exhibiting anti-HBs titres between 1/100-1/10,000, and four HB-susceptible individuals (mean age 40 years, range 32-52 years) (lacking all HBV markers) were tested. No patient was receiving immunosuppressive or antiviral therapy at the time the blood samples were taken. HB serology was analysed by commercially available kits (AUSRIA II, AUSAB, CORAB and anti-HBe Diagnostics: Abbott Laboratories). Antigens Purified HBsAg, subtype ad (provided by Dr L. Kaplan, Biochemistry Research Department AB KABI, Stockholm), was used for T-cell stimulation. It was obtained by plasmaphoresis from chronic carriers of HBsAg who had anti-HBe antibodies and purified by affinity chromatography, as described elsewhere (Einarsson, Kaplan & Utter, 1978; Einarsson, Kaplan & Pertoft, 1981) and contained 25 pg antigen protein/ ml. Electron microscopy of the purified material showed a preparation of HBsAg spheres that was homogenous, although varying somewhat in size, with few filamentous HBsAg but no Dane particles. The antigen preparation was dialysed against Tris-buffered Hanks' salt solution, pH 7 4, filtered through a 0 45 pm Millipore filter and stored at - 200 before use. Phytohaemagglutinin (PHA) was purchased from Wellcome
Diagnostics (Dartford, Kent, U.K.).
and
HBsAg-induced interferon-gamma secretion in T cells from asymptomatic HBsAg carriers and HB-immune donors in vitro S. P. E. SYLVAN & U. B. HELLSTROM The Elias Bengtsson Research Unit, Department of Infectious Diseases, Karolinska Institute, Roslagstull Hospital, Stockholm, Sweden
Acceptedfor publication 14 February 1990
SUMMARY Peripheral T lymphocytes obtained from asymptomatic carriers of hepatitis B surface antigen (HBsAg) and donors immune to hepatitis B (HB) through natural infection or vaccination were induced by the envelope protein (HBsAg) of the hepatitis B virus (HBV) into secretion of interferongamma (IFN-y) in vitro. The kinetics of the IFN-y response varied between individuals, but was constantly found to be biphasic, with an early peak attained after 12 hr-A days and a late peak after 58 days of antigen stimulation. The early release of IFN-y activity was antigen-specifically induced, as it was in T cells from HB-immune and asymptomatic carriers of HBsAg but not HB-susceptible controls. The second peak of HBsAg-induced IFN-y secretion was induced in all three donor groups and the kinetics of IFN-y release were similar to that of the mitogen phytohemagglutinin(PHA)induced IFN-y production in similarly prepared T-cell cultures. Thus the late burst of IFN-y activity seems to result from a mitogenic property contained within the envelope material of HBV. The mitogenic response was three- to fivefold higher for 4/7 asymptomatic carriers of HBsAg compared to HB-immune donors and HB-susceptible controls, indicating that some patients with chronic asymptomatic carriership of HBsAg exhibit enhanced mitogenic responses to HBsAg.
(Dudley, Fox & Sherlock, 1972). Recently, however, circulatory immunocompetent T lymphocytes were demonstrated with reactivity against the outer coat of hepatitis B virus (HBV), as measured by lymphocyte transformation (Sylvan, Hellstr6m & Lundbergh, 1985) and interleukin-2 (IL-2) secretion (Sylvan & Hellstr6m, 1989), in asymptomatic carriers of HBsAg as well as donors with acquired immunity to HB. Production of IL-2 has been demonstrated to be associated with the generation of IFN-y (Northoffet al., 1980; Farrar, Johnson & Farrar, 1981). Therefore, it is conceivable that HBsAg-sensitized peripheral blood T lymphocytes with the capacity to produce IL-2, when stimulated with HBsAg in vitro should also be able to secrete IFN-y. Thus, a cohort of asymptomatic HBsAg carriers was analysed for IFN-y-producing capacity when specifically stimulated with HBsAg in in vitro conditions previously reported to be optimal for HBsAg-induced IL-2 secretion for these donors (Sylvan & Hellstrom, 1989). T lymphocytes from donors with acquired immunity to HB by infection or recent vaccination and HB-susceptible individuals were included as positive and negative controls.
INTRODUCTION Human peripheral blood lymphocytes produce interferon(IFN-y) in response to several non-viral and viral inducers (Wheelock, 1965; Nathan et al., 1981; Epstein, Cline & Merrigan, 1971; Epstein, Stevens & Merrigan, 1972; Valle et al., 1975; Ennis & Meager, 1981; Nakayama, 1983). The capacity of hepatitis B surface antigen (HBsAg) to induce IFN-y secretion has so far been studied only in T-cell clones obtained from hepatitis B (HB) vaccine recipients (Celis, Miller & Chang, 1984). It has been suggested that peripheral blood lymphocytes from patients with acute and chronic viral HB have a reduced capacity to produce IFN-a or IFN-y when stimulated with Newcastle disease (Tolentino et al., 1975), Influenza (Abb et al., 1985) and Sendai (Tolentino et al., 1975; Kinoshita et al., 1979; Kato et al., 1982; Ikeda, Lever & Thomas, 1986) viruses, Molt4 human leukaemia cells (Abb et al., 1985), polyinosinic-cytidilic acid (poly-IC; Levin & Hahn, 1982), mitogens (Levin & Hahn, 1982; Abb et al., 1985; Ikeda et al., 1986; Fuji et al., 1987) or interleukin-2 (IL-2; Fuji et al., 1987). However, no clear-cut correlation between IFN secretion and active viral replication or the severity of liver disease has emerged from these studies. It has been postulated that HBsAg carriers, particularly if asymptomatic, do not exhibit T-cell functions against HBsAg gamma
MATERIALS AND METHODS
Subjects Seven asymptomatic HBsAg carriers (five females; mean age 38 years, range 31-42 years) were studied. They had no detectable antibodies to HBsAg (anti-HBs) in serum and possessed HBsAg
Correspondence: Dr S. Sylvan, Dept. of Infectious Diseases, Karolinska Institute, Roslagstull Hospital, Box 5651, S-1 1489 Stockholm, Sweden.
197
198
S. P. E. Sylvan & U. B. Hellstrim
titres varying between 1/10,000 and 1/100,000 (subtype ad in all), IgG antibodies to hepatitis B core antigen (anti-HBc; 1/1000-1/10,000) and stable titres (1/10-1/1000) of antibodies to hepatitis B e antigen (anti-HBe). All had been found to be HBsAg positive through routine screening procedures of voluntary blood donors and none had a previous history of hepatitis or drug abuse. A percutaneous liver biopsy was available in one patient and showed normal liver histology. Repeated examination of their clinical status and liver-function tests over a 3-9 year period before being included in the study were consistently found to be normal. All were negative for HBV-DNA in serum, as measured by a method in which samples are hybridized in solution with an iodinated probe (Dr M. Kuhns, Abbott Laboratories, North Chicago, IL; Kuhns et al., 1988), and demonstrated no homosexual preferences. For comparison, seven HB-immune donors (all females; mean age 44 years, range 27-59 years), four with naturally acquired immunity to HB as judged by the absence of HBsAg and presence of anti-HBs (1/10-1/10,000) and anti-HBc (1/101/1000), three with artificially induced immunity after repeated vaccination with a plasma-derived HBV vaccine (Hep-B vax; Merck Sharp and Dohme) exhibiting anti-HBs titres between 1/100-1/10,000, and four HB-susceptible individuals (mean age 40 years, range 32-52 years) (lacking all HBV markers) were tested. No patient was receiving immunosuppressive or antiviral therapy at the time the blood samples were taken. HB serology was analysed by commercially available kits (AUSRIA II, AUSAB, CORAB and anti-HBe Diagnostics: Abbott Laboratories). Antigens Purified HBsAg, subtype ad (provided by Dr L. Kaplan, Biochemistry Research Department AB KABI, Stockholm), was used for T-cell stimulation. It was obtained by plasmaphoresis from chronic carriers of HBsAg who had anti-HBe antibodies and purified by affinity chromatography, as described elsewhere (Einarsson, Kaplan & Utter, 1978; Einarsson, Kaplan & Pertoft, 1981) and contained 25 pg antigen protein/ ml. Electron microscopy of the purified material showed a preparation of HBsAg spheres that was homogenous, although varying somewhat in size, with few filamentous HBsAg but no Dane particles. The antigen preparation was dialysed against Tris-buffered Hanks' salt solution, pH 7 4, filtered through a 0 45 pm Millipore filter and stored at - 200 before use. Phytohaemagglutinin (PHA) was purchased from Wellcome
Diagnostics (Dartford, Kent, U.K.).
and
