Immunotherapy directed against α-fetoprotein results in autoimmune liver disease during liver regeneration in mice

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Immunotherapy Directed Against ␣-Fetoprotein Results in Autoimmune Liver Disease During Liver Regeneration in Mice ¨ HLER,‡ MICHAEL GEISSLER,* LEONHARD MOHR,* ROBERT WETH,* GABRIELE KO CHRISTIAN F. GRIMM,* TIM U. KROHNE,* FRITZ VON WEIZSA¨CKER,* and HUBERT E. BLUM* *Department of Medicine II and ‡Institute of Pathology, University Hospital Freiburg, Freiburg, Germany

Background & Aims: Priming immune responses against ␣-fetoprotein (AFP) highly expressed in the majority of hepatocellular carcinomas results in significant antitumoral T-cell responses. Liver regeneration in humans and mice, however, is also associated with increased AFP expression. Therefore, we evaluated the risk of AFP-directed immunotherapeutic approaches to induce autoimmunity against the regenerating liver. Methods: Mice were immunized with DNA encoding mouse AFP. For induction of liver regeneration, partial hepatectomy was performed and mice were monitored by serial histopathologic examinations and measurements of serum ALT activities (U/L), and by determination of the kinetics of AFP-specific T-cell responses. Results: Livers of AFP immune mice without partial hepatectomy were characterized by minor lymphocytic infiltrations without transaminase elevations. By contrast, a significant hepatocyte damage was observed in regenerating liver that correlated well with the number of AFP-specific CD8ⴙ T cells, the activity of liver regeneration, and the level of AFP synthesis. Autoimmune liver damage was mediated by CD4ⴙ T cell– dependent CD8ⴙ cytotoxic T lymphocytes. Conclusions: These results show that priming of T-cell responses against shared tumor-specific self antigens may be accompanied by induction of autoimmunity dependent on the level of expression of the self antigen and have important implications for the development of antitumoral vaccines targeted against antigens that are not strictly tumor-specific.

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-Fetoprotein (AFP) is a tissue-specific tumor-associated self antigen, which is the predominant serum protein produced during early development of the liver. It is gradually replaced, later in development, by serum albumin, a protein closely related to AFP. Serum AFP levels fall 105–106-fold from 3–5 mg/mL in the fetal period to 5–10 ng/mL in adolescents and full adults. AFP is frequently re-expressed at high levels in patients with hepatocellular carcinoma (HCC) or embryonal malignancies1 depending on the degree of differentiation.2 It may, therefore, be a target for immunotherapy of HCC, which is one of the most common tumors in the

world.3 We and others have recently shown that breaking immunologic tolerance towards the HCC-specific self antigen AFP using the DNA-based immunization approach–induced cytotoxic T lymphocyte (CTL) and CD4⫹ T cell–mediated regression of AFP expressing HCCs growing subcutaneously in mice.4,5 In addition to AFP-expressing HCCs, AFP synthesis can be up-regulated also in normal mature hepatocytes under nonmalignant conditions. AFP may be expressed at high levels during the course of fulminant hepatic failure and other forms of hepatic injury, including viral hepatitis and cirrhosis, both conditions closely associated with the development of HCC.2,6 In addition, high level AFP synthesis can be observed after partial hepatectomy followed by rapid liver regeneration.6 Therefore, immunotherapeutic approaches against HCC inducing an efficient AFP-specific immune response may potentially be harmful, because immunity against normal hepatocytes expressing varying amounts of AFP may cause autoimmunity. Indeed, efficient tumor control in human patients may be associated with autoimmune phenomena.7–9 It is, therefore, important to determine the impact of immunity against tumor antigens that are also expressed by normal cells of peripheral organs. Such antigens may be ignored by T cells, may induce deletional tolerance by cross-presentation, or can be reacted against depending on the amount of circulating antigen, avidity of the T-cell receptor, or the time period present in secondary lymphoid organs.10,11 In this study, we evaluated the risk of AFP-directed immunotherapeutic approaches to induce autoimmunity in a nontransgenic pathophysiologically relevant murine Abbreviations used in this paper: AFP, ␣-fetoprotein; CTL, cytotoxic T lymphocyte; CTL-p, CTL precursor; ELISPOT, enzyme-linked immunospot; FACS, fluorescence-activated cell sorter; HCC, hepatocellular carcinoma; IFN, interferon; IL, interleukin; mAFP, murine alpha fetoprotein; MHC, major histocompatibility complex; MOI, multiplicity of infection. © 2001 by the American Gastroenterological Association 0016-5085/01/$35.00 doi:10.1053/gast.2001.28019

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model. Our results show that (1) DNA-based vaccination using expression plasmids encoding mouse AFP induces CD4⫹ T-cell dependent CTL responses against AFP, resulting in mild autoimmune hepatitis; (2) significant hepatocyte damage occurs in AFP-DNA immunized mice after partial hepatectomy compared with nonvaccinated mice; (3) liver damage correlates with the number of AFP-specific T cells, the activity of hepatocyte regeneration, and the level of AFP synthesis; (4) the autoimmunity is self-limiting after completion of liver regeneration; and (5) the autoimmune liver injury is primarily mediated by CD8⫹ CTL.

Materials and Methods Mice Male C57BL/6 and C3H mice were kept in the animal facility of the University Hospital Freiburg and used between the ages of 10 –25 weeks. Major histocompatibility complex (MHC) class I⌬/⌬ and MHC class II⌬/⌬ knockout mice of C57BL/6 background were kindly provided by Dr. Mossmann (Max-Planck-Institute for Immunobiology, Freiburg, Germany). Fluorescence-activated cell sorter (FACS) analysis of spleens confirmed the absence of H-2Kb/Db and I-A/I-E molecules in these mice, respectively (data not shown).

DNA Expression Vectors DNA expression plasmids expressing a cytoplasmic (pcD/3-mAFP) and secreted (pSec-mAFP) form of murine AFP (mAFP) and plasmids expressing murine interleukin (IL)-12 p70 (pApIL-12p70) or murine GM-CSF (pRJB-GM) have been described recently.4,12,13

Flow Cytometry MHC class I and II expression in Hepa1-6 cells and spleens of MHC class I and II knockout mice was examined by FACS analysis using an anti-mouse H-2Kb / H-2Db (clone 28-8-6) and anti-I-A/I-E (clone 06355A) specific antibody (all antibodies derived from PharMingen, San Diego, CA).

Generation of Recombinant Vaccinia Viruses To study CTL responses, a recombinant vaccinia virus expressing a cytoplasmic form of mAFP (rVV-mAFP) was generated as previously described in detail.4 rVV-pSc11 represents an empty vector negative control recombinant virus.

DNA-Based Immunization Facilitated DNA immunization was performed by injecting plasmid constructs into the tibialis anterior muscle of mice in a final volume of 50 ␮L 0.25% bupivacaine. One booster immunization was performed at day 14. Mice were killed for T-cell analysis, or partial hepatectomy was performed 5 days after the second booster immunization (e.g., 19 days after the first immunization).

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Partial Hepatectomy (50%) Mice were anesthetized with a single intraperitoneal injection (75 ␮L) of 100 mg/kg Ketanest (Parke-Davis/Pfizer, Freiburg, Germany) ⫹ 16 mg/kg Rompun (Prouet AG, Lyssach, Switzerland), which is a combination of an analgetic drug (ketamine) with a barbiturate (xylazine). After ligating and removing the lobus, sinister lateralis tissue remnants and coagulated blood were carefully removed with phosphate-buffered saline–soaked cotton swabs. To assay incorporation of BrdU, mice were injected intraperitoneally (IP) with BrdU (200 ␮g/g body wt), 4 and 2 hours before sacrificing. Hepatocellular injury was monitored by serially measuring serum alanine aminotransferase (ALT) activities (U/L) in each mouse using the Reflotron sytem (Roche, Basel, Switzerland). In addition, all animals from each experimental group were histopathologically examined for hepatocyte damage and hepatic lymphocyte infiltration.

Interferon ␥ Enzyme-Linked Immunospot Assays Multiscreen-HA 96-well filter plates were coated with 4 ␮g/mL rat anti-mouse interferon (IFN)-␥ antibody (PharMingen, San Diego, CA; clone R46A2) at 4°C overnight. CD8⫹ T cells (1 ⫻ 105/well) derived from DNA-immunized or unimmunized mice were separated from spleen cells using magnetic beads according to the manufacturer’s instructions (MACS; Myltenyi Biotech, Bergisch-Gladbach, Germany) and cultured in triplicates for 20 –25 hours with 1 ⫻ 104 irradiated stimulator cells (rVV-mAFP or rVV-pSC11 infected syngeneic spleen cells) per well in 200 ␮L medium. These stimulator cells derived from naive C57BL/6 mice were infected with vaccinia viruses (MOI 8) after inactivation of the virus using 300 mJ of UV-crosslinking energy to prevent replication and vaccinia virus induced cytopathic effects during the stimulation period. Spleen cells transduced with the inactivated rVVmAFP expressed AFP at high levels. After culture, the cells were washed out and 2 ␮g/mL biotinylated rat-anti-mouse IFN-␥ antibody (clone XMG1.2; PharMingen) was added, and the plates were incubated for 3 hours at room temperature. The plates were again washed, incubated with Streptavidin-alkaline phosphatase-polymer (Sigma, Taufkirchen, Germany) for 30 minutes at room temperature and then developed with alkaline phosphatase conjugate substrate solution. The spots in each well were counted under a microscope, and the values are expressed as numbers of spot-forming cells relative to the number of spleen cells added to each well at the start of the culture. As a control for specificity, spleen cells of DNAimmunized mice and the different irradiated stimulator cells were cultured alone.

Cytotoxicity Assays In vivo primed spleen cells derived from immunized mice were suspended, and after 8 days of in vitro stimulation in 24-well plates, the spleen cells were analyzed for cytotoxic activity. In vitro stimulation was performed by incubating 4 ⫻

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107 of in vivo primed spleen cells with 1 ⫻ 107 spleen cells of untreated syngeneic donor mice, which had been infected by UV-inactivated (300 mJ) rVV-mAFP at a multiplicity of infection (MOI) of 8 and then irradiated with 20 Gy (2000 rad) to prevent stimulator cells from proliferation. A 6-hour 51Cr release assay was performed. As target cells, rVV-mAFP– infected (MOI 8) H-2b–restricted MC57G fibroblasts were used at different effector:target (E:T) ratios. As a negative control, CTL activity was tested against target cells infected with rVV-pSC11. Results were expressed according to the formula: % specific lysis ⫽ (experimental release ⫺ spontaneous release) / (maximum release ⫺ spontaneous release). Experimental release represents the mean counts per minute released by target cells in the presence of effector cells. Total release represents the radioactivity released after total lysis of target cells with 5% Triton X-100. Spontaneous release represents the radioactivity present in medium derived from target cells only.

In Vivo Monoclonal Antibody Ablation CD4⫹ and CD8⫹ T-cell subpopulations were depleted by IP injection of purified hybridoma supernatant. A total of 1 mg per mouse per injection of anti-CD8 (clone YTS 169) or anti-CD4 (clone YTS 191.1)14,15 was injected on the days indicated at the different experiments. FACS analysis of peripheral blood mononuclear cell populations demonstrated that more than 95% of the CD4 and CD8 T cells were deleted after 4 injections.

Histology Livers of all mice were histologically examined. Freshly removed livers were snap-frozen in liquid nitrogen or fixed in 4% buffered formalin and subsequently embedded in paraplast. After section, they were stained with H&E. BrdU analysis was performed as previously described.16

Statistical Analysis All data were analyzed by the Wilcoxon signed rank test. A two-sided P value ⬍ 0.05 was considered statistically significant. Differences between immunized and control mice were calculated by the Mantel–Haenszel test.

Results DNA-Based Immunization Induces CD4ⴙ T-Cell Dependent CTL Responses Against AFP We have previously shown that DNA-based immunization against mouse AFP induces CTL responses, which are able to control AFP expressing HCCs growing subcutaneously in the syngeneic host.4 The maintainance of CTL activity by repetitive immunizations was an important parameter for efficacy of tumor growth control. In this context, it is important to note that immunization with a plasmid encoding a secreted form of

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mAFP induced a stronger antitumor response compared with a plasmid encoding a cytoplasmic form of mAFP. This phenomenon can be explained by a better activation of CD4⫹ T cells by secreted antigens in context of DNA-based immunization as demonstrated recently by our group.4,17 These specifically activated CD4⫹ and CD8⫹ T cells rejected the tumor-expressing high levels of AFP. In the present study, CTL activity was monitored by direct ex vivo analysis using the sensitive enzyme-linked immunospot (ELISPOT) technique in addition to the functional 51Cr-release assay, which requires extensive in vitro restimulation of AFP-specific CTLs.4 To evaluate AFP CTL precursor (CTL-p) frequencies, C57BL/6 mice were immunized 2 times at day 1 and 14 with plasmids encoding an intracellular (pcD/3-mAFP) and a secreted form (pSec-mAFP) of AFP, as previously shown.4 Five days after the second immunization, CD8⫹ T cells were analyzed for ex vivo AFP-specific CTL-p frequencies using the IFN-␥ ELISPOT technique after 20 –25 hours of in vitro stimulation using spleen cells infected with rVV-mAFP. AFP-specific DNA-based immunizations induced CD8⫹ CTL responses with splenic CTL-p frequencies of about 0.1% (Figure 1A). These IFN-␥– secreting CD8⫹ T cells were functionally active because they efficiently lysed AFP-expressing target cells in a CTL assay with lysis values up to 40% at an E:T ratio of 100:1 (Figure 1B and C). The CD8⫹ CTLs were AFP specific because restimulation with rVV-pSC11 or use of vaccinia-uninfected spleen cells for restimulation resulted in a background level of about 1 in 12,000 spot-forming cells in the ELISPOT assay (Figure 1A) and the lack of relevant CTL activity in the killing assay (data not shown). In addition, in mock DNA–immunized mice, no AFP-specific IFN-␥–producing spleen cells could be detected (Figure 1A). No significant differences in AFP-specific CTL activity were observed between pcD/3-mAFP or pSec-mAFP immunized mice (Figure 1A–C). Therefore, T-cell frequency data derived from pcD/3-mAFP and pSec-mAFP immunized mice were combined for the following experiments and are displayed as combined data in the following figures (AFPDNA). DNA-priming of AFP-specific CTL was dependent on CD4⫹ T-cell help because no IFN-␥–producing CD8⫹ T cells (Figure 2A) or CTL activity (Figure 2B and C) could be detected in AFP-DNA–immunized C57BL/6 MHC II knockout mice. As expected, no CD8⫹ T cell–immune reactivities were detected in MHC I knockout mice (data not shown) and there was no antiAFP antibody response in AFP-DNA immunized normal C57BL/6 mice.

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Figure 1. CTL activity in AFP-DNA immunized C57BL/6 mice. (A ) CD8⫹ T cells derived from AFP- or mock-DNA immunized mice (n ⫽ 4 in each group) were stimulated for 26 hours with rVV-mAFP or rVVpSC11, or left unstimulated. Subsequently, IFN-␥ ELISPOT assays were performed. The spots in each well were counted under a microscope, and the values are expressed as numbers of spot-forming cells relative to the number of spleen cells added to each well at the start of the culture. (B and C ) Single spleen cell suspensions derived from (B) pcD/3-mAFP– or (C ) pSec-mAFP–immunized mice (n ⫽ 4 in each group) were assayed after in vitro stimulation with rVV-mAFP–infected syngeneic spleen cells for 8 days. The effector cells were then tested against MC57G target cells infected with the indicated vaccinia viruses in a 51Cr-release assay at different E:T ratios. Values represent means of triplicate determinations.

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3B). For example, 5 days after the second immunization (day 19 after the first immunization), a time point when partial hepatectomy was performed in other experimental groups (Figure 5), livers of AFP-DNA immunized mice were characterized by microfocal or focal lymphocytic infiltrations, which were similar to day 6 lesions (see Figure 4B). These lesions can be classified as very mild autoimmune hepatitis because transaminase levels were in the normal range at any time (Figure 3B). Interestingly, AFP-specific CTL-p frequencies had increased to 0.1% at this time (Figure 3A), and the infiltrating lymphocytes belonged primarily to the CD8⫹ T-cell subset because similar microfocal lymphocytic infiltrations were observed in CD4⫹ T-cell depleted, but not in AFP-DNA immunized, MHC class I knockout mice (Figure 4F and E, respectively). This suggests that CD4⫹ T cells do not significantly contribute to the observed liver infiltrate. Again, transaminase levels were in the normal range in these mice and in CD8⫹ T cell– depleted mice (Figure 6B). CD4⫹ T-cell depletion did not interfere with priming of AFP-specific CD8⫹ CTL responses because in vivo depletion started 2 weeks after AFP-

AFP-Specific CD8ⴙ T Cells Induce a Mild Autoimmune Hepatitis The most important period for analysis of liver damage and CTL responses directed against AFP has been reported to be between 2 and 5 days after partial hepatectomy.18,19 During this time, maximal liver regeneration16,20 and AFP synthesis occurs. For determination of baseline AFP-specific CTL responses, we analyzed CTL-p frequencies at different time points after DNA immunization of normal mice without performing partial hepatectomy. At the time of immunization (day 1), no AFP-specific T cells were observed (Figure 3A). Serum transaminase levels were not elevated (Figure 3B) and no lymphocyte infiltrates of the liver were observed. Microfocal lymphocytic infiltrates of the liver started to appear 6 –7 days after the immunization, suggesting that some healthy AFP-expressing hepatocytes are recognized by activated AFP-specific T cells (Figure 3B and 4B). ALT levels, however, were in the normal range. Seven days later, lymphocytic infiltrates had disappeared. One booster immunization at day 14 was able to extend the presence of the lesions for an additional 7– 8 days (Figure

Figure 2. CTL activity in AFP-DNA immunized MHC II knockout mice. (A ) CD8⫹ T cells derived from AFP- or mock-DNA immunized MHC II knockout mice (n ⫽ 3 in each group) were stimulated for 20 hours with rVV-mAFP or rVV-pSC11, or left unstimulated. Subsequently, IFN-␥ ELISPOT assays were performed. (B and C ) Single spleen cell suspensions derived from (B) pcD/3-mAFP or (C ) pSec-mAFP immunized MHC II knockout mice (n ⫽ 3) were assayed after in vitro stimulation with rVV-mAFP–infected syngeneic spleen cells for 7 days. The effector cells were then tested against MC57G target cells infected with the different vaccinia viruses in a 51Cr-release assay at the E:T ratio indicated.

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Figure 3. Time course of AFPspecific CTL-p frequency and transaminases (ALT). (A ) Time course of AFP-specific CD8⫹ Tcell precursor frequency after immunization of C57BL/6 mice with AFP-DNA expression plasmids at day 1 and 14. Spleen cells derived from AFP- or mockDNA immunized mice (n ⫽ 4 in each group) were stimulated for 24 hours with rVV-mAFP. Subsequently, IFN-␥ ELISPOT assays were performed. (B) Time course of ALT levels and histologic examination of the liver of AFP-DNA–immunized mice for the presence of lymphocytic infiltrates (⫺, no infiltrates; (⫹), few scattered microfocal infiltrates; and ⫹, microfocal and focal infiltrates).

DNA priming (Figure 6A). These data thus provide direct evidence that low-level AFP expressed in the normal liver can be recognized by activated AFP-specific CD8⫹ T cells, resulting in mild autoimmune hepatitis. AFP Overexpression in the Liver Induces Autoimmunity Because AFP is expressed in relatively large amounts after hepatic injury,6,18 including viral hepati-

tis, cirrhosis, and partial hepatectomy followed by rapid regeneration, we studied the effect of an efficient AFPspecific immune response on nontumorous liver cells expressing high amounts of AFP and the potential to induce autoimmune liver disease. As a model of liver regeneration, we performed partial hepatectomy, which results in a well-characterized process of liver regeneration and transient AFP overexpression6,16,18,20 (Figure 5). In C57BL/6 mice, the peak of liver regeneration occurs

Figure 4. Histochemical analysis of livers. Bars in pictures A–F correspond to 0.2 mm, and in inserts of A, B, C, D, and F to 0.03 mm. (A ) Mock-DNA, C57BL/6 mice, 30 hours after partial hepatectomy. (Insert) BrdU-staining, bar corresponds to 0.063 mm. (B) AFP-DNA, C57B46 mice 6 days after the first immunization. (C ) AFP-DNA, C57BL/6 mice, 46 hours after partial hepatectomy. (D ) AFP-DNA, C57BL/6 mice, 120 hours after partial hepatectomy. (F ) AFP-DNA immunized CD4⫹ T cell– depleted mice, 5 days after the second immunization (day 19).

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Figure 5. Experimental design of the study. C57L/6 and C3H mice were DNA-immunized at days 1 and 14. Five days later, partial hepatectomy was performed and mice were monitored by serially measuring serum ALT, by serial histologic examinations of the liver, and by the determination of the time course of CTL responses directed against AFP. Identification of pathogenic immune effectors was done by using MHC class I knockout mice and in vivo T-cell depletion experiments.

between 32 and 42 hours after partial hepatectomy and DNA synthesis stops about 15–20 hours later. In fact, BrdU incorporation into regenerating hepatocytes was easily visible during this time period (Figure 4A). AFP expression after partial hepatectomy peaks between 24 and 48 hours and is undetectable after 96 hours.18,19 Therefore, we analyzed the AFP-specific CTL-p frequency 46 and 120 hours after partial hepatectomy, respectively, and serially measured ALT levels in AFPand mock-DNA immunized C57BL/6 mice. To examine the impact of AFP levels during liver regeneration on the severity of autoimmune liver disease, we performed identical experiments in C3H mice, which produce roughly

Figure 6. Time course of AFP-specific CTL-p frequency and transaminases (ALT) in CD4⫹ and CD8⫹ T cell– depleted mice. (A ) C57BL/6 mice were immunized with AFP- or mock-DNA expression plasmids at days 1 and 14. Spleen cells derived from AFP- or mock-DNA immunized mice (n ⫽ 2 in each group) were stimulated for 20 hours with rVV-mAFP. Subsequently, IFN-␥ ELISPOT assays were performed at the indicated time points. CD4⫹ and CD8⫹ T-cell subpopulations were depleted by IP injection of purified hybridoma supernatant. A total of 1 mg per mouse per injection of anti-CD8 or anti-CD4 was injected on days 14, 16, 18, 21, and 23 after the first immunization. (B) Time course of ALT levels in AFP-DNA immunized T cell– depleted mice (n ⫽ 2 in each group).

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10 times more AFP messenger RNA (mRNA) during liver regeneration than C57BL/6 mice.20 Partial hepatectomy was performed on day 5 after the second immunization (day 19 after the first immunization), which corresponds to 0 hours in Figure 7. As shown in Figure 7A, the ALT peak in mock-DNA immunized C57BL/6 mice occurs 6 hours after partial hepatectomy with a median value of 500 U/mL. Thereafter, ALT levels continuously decline to near normal levels after 120 hours. Elevated transaminases are caused by the hepatectomy procedure and no lymphocyte infiltrates could be observed at any time (Figure 4A). By contrast, in AFP-DNA immunized C57BL/6 mice, ALT levels continue to increase until 20 hours after partial hepatectomy and then slowly decline (Figure 7A). Elevated transaminases correlated well with microfocal, perivascular, and periportal lymphocytic aggregates and focal hepatocyte necrosis (Figure 4C). After 72 hours, no significant differences of ALT levels could be observed between mock- and AFP-DNA immunized C57BL/6 mice, and after 120 hours only microfocal lymphocyte infiltrates (Figure 4D ) were observed, which were similar to those before hepatectomy. In C3H mice, liver damage was more pronounced (ALT ⬎ 2000 U/mL) and characterized by a prolonged course of hepatitis with a duration of about 6 days (Figure 7A). These results, therefore, suggest that autoimmune liver damage is generally self-limiting under these experimental conditions.

Figure 7. ALT levels and AFP-specific CTL-p frequency after partial hepatectomy. Mock- (n ⫽ 13) or AFP-DNA (n ⫽ 17) immunized (days 1 and 14) C57BL/6 were partially hepatectomized at day 19 after the first immunization (corresponds to 0 hours in this figure; n ⫽ 7 and 10, respectively) or left without hepatectomy (n ⫽ 6 and 7, respectively). Similarly, C3H mice were immunized with mock- (n ⫽ 9) or AFP-DNA (n ⫽ 10), and partial hepatectomy was performed in 7 mockand 8 AFP-DNA immunized mice, respectively. (A ) ALT levels in C57BL/6 and C3H mice and (B) CTL-p frequencies in C57BL/6 mice were serially measured.

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Liver disease in AFP-DNA immunized C57BL/6 mice was accompanied by a significant increase of AFP-specific CD8⫹ CTL-p frequencies up to 1 in 125 spleen cells (Figure 7B) and correlated well with maximal liver regeneration, AFP synthesis, lymphocyte infiltrates, and ALT elevations. After 5 days, CTL-p frequency declined to numbers observed before partial hepatectomy. In mock-DNA immunized mice, no AFP-specific CTL could be detected after partial hepatectomy (Figure 7B). Autoimmunity Is Mediated by T Cells To prove that the observed hepatocyte damage is in fact mediated by CD8⫹ CTL, we performed the experiments described above in MHC class I knockout mice of C57BL/6 background. Liver damage in terms of transaminase levels was comparable between AFP-DNA and mock-DNA immunized MHC class I knockout mice after partial hepatectomy (Figure 8). Furthermore, ALT levels in MHC class I knockout mice after partial hepatectomy were comparable to those in mock-DNA immunized wild-type C57BL/6 mice independent of the immunization status of the MHC class I knockout mice (compare Figures 7A and 8). In addition, no AFP-specific IFN-␥–producing CD8⫹ CTLs were detectable in AFPDNA immunized MHC I knockout mice (data not shown). Finally, no anti-AFP antibody responses against denatured mouse AFP could be detected in AFP-DNA– immunized C57BL/6 or C3H mice. These results suggest that CD4⫹ T cell– dependent CD8⫹ T cells are required for mediating the hepatocyte damage. Taken together, these and previous data show that the self antigen AFP can be used as a target antigen for vaccination using DNA or dendritic cells.4,5 Significant

Figure 8. Identification of autoimmune mediators. Time course of ALT levels in naive or AFP-DNA (days 1 and 14) immunized MHC I knockout mice after partial hepatectomy performed at day 19 (corresponds to 0 hours).

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autoimmune hepatitis against this self antigen, however, is induced by DNA vaccination if AFP is expressed at high levels, e.g., during liver regeneration after partial hepatectomy.

Discussion Antitumor immunotherapy may be most promising if tumor antigen–specific T cells of high avidity have not been centrally or peripherally deleted. Whether antigens strictly expressed in peripheral tissues are immunologically ignored depends on antigen distribution and expression levels.10,21–24 Tumor antigens peripherally expressed at low levels may be ignored or recognized by CTLs if expressed at high levels and tracked to local secondary lymphoid organs.25 We and others have recently shown that tolerance to the peripheral tissue self antigen AFP can be broken by immunization with DNA encoding different forms of AFP.4,5 Furthermore, significant therapeutic antitumor responses could be elicited using a murine HCC tumor model.4 A potential problem of using AFP as a tumor antigen for immunotherapy may be the fact that AFP synthesis in the liver that is normally rapidly repressed at birth and expressed at very low levels in adult animals can be re-expressed not only during periods of uncontrolled cell growth, e.g., liver cancer, but also after hepatic injury, including chronic viral hepatitis and cirrhosis, both conditions closely associated with the development of HCC.2,6,26 Therefore, immunotherapeutic approaches against HCC inducing an efficient AFP-specific immune response may also target normal hepatocytes expressing varying amounts of AFP, thereby causing autoimmune liver disease. The present study shows that mild autoimmune hepatitis without transaminose elevations may develop in the healthy liver of AFP vaccinated animals as assessed by the presence of few microfocal and focal CD8⫹ lymphocytic infiltrates. This was an important finding because low AFP levels could be detected in the liver of these mice,4 suggesting that AFP expressing normal hepatocytes can be recognized by AFP-specific T cells activated in lymphoid organs distant from the liver. Contrary, in hepatitis B virus transgenic mice, none of the animals developed hepatitis or displayed suppressed viral gene expression or replication after either DNA or DC immunization, even in the presence of hepatitis B virus– specific antibodies or CTLs.15,27 These differences may be due to the mouse models used and the self-directed T-cell repertoires. At present, it is not clear whether the frequencies of functional AFP-specific lymphocytes are too low to induce elevation of transaminases or whether these infiltrating lymphocytes are tolerized within the

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liver. Even in adult Balb/c mice that express 15–20-fold higher AFP mRNA levels than other mouse strains,26 transaminases were not elevated,4 suggesting a high threshold of intrahepatic AFP levels for the efficient recognition and destruction of hepatocytes by AFP-specific T cells. The control of T cell–mediated autoimmunity has been investigated in transgenic mice, expressing model antigens under the control of tissue-specific promoters. The advantage of our model compared with the transgenic models is the fact that AFP is a naturally expressed antigen both in humans and in mice. The experiments described here indicate that AFP-specific autoimmune hepatitis primarily depends on the level of AFP production of hepatocytes. Partial hepatectomy in AFP immunized animals induced an immune response associated with significant liver damage as determined by histology and transaminase levels. This may be the result of abundant expression of AFP in the liver and more efficient uptake of AFP in local secondary lymphoid organs. The significant liver damage observed in our model using C57BL/6 mice is even more impressive because mice of the C57BL/6 strain produce roughly 10 times less AFP mRNA during liver regeneration than C3H mice.6 As a consequence, liver damage in the C3H mice was more severe and characterized by a prolonged course of autoimmune liver disease compared with C57BL/6 mice. Similar results have been recently published by Ludewig et al., who demonstrated that tumors expressing shared tumor antigens could be controlled by DC vaccination. However, antitumor treatment was accompanied by fatal autoimmune disease, i.e., autoimmune diabetes in transgenic mice expressing the model tumor antigen in pancreatic ␤ islet cells.11 In our study, it was an important finding that autoimmunity was self-limiting after completion of liver regeneration. Obviously, the T cell–mediated liver damage was not sufficient to induce a prolonged liver regeneration. An important issue is the type of immune cell mediating the hepatocyte damage. It has been suggested that low avidity CTLs against tumor self antigens may provide protection against subsequent tumor challenge but may not be sufficient to induce autoimmunity.28 Alternatively, it may be possible that CD4⫹ T cells mediate antitumor effects without causing autoimmunity.29 Our results support the view that some activated low avidity AFP-specific CD8⫹ T cells may infiltrate normal liver but do not cause significant hepatocyte damage. Furthermore, we could not detect anti-AFP antibody responses at any time in AFP vaccinated mice excluding antibodymediated autoimmune liver damage. As previously reported in the context of DNA-based immunization,14

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priming of AFP-specific CD8⫹ CTL responses was dependent on CD4⫹ T-cell help. To prove that the observed hepatocyte damage after hepatectomy is mediated by CTL, we repeated the experiments in MHC class I knockout mice of C57BL/6 background. Our results suggest that CD4⫹ T cell– dependent CD8⫹ T cells are required for mediating the hepatocyte damage after partial hepatectomy. AFP expressing hepatocytes may be directly killed by cytotoxic CD8⫹ T cells. Alternatively, proinflammatory cytokines such as tumor necrosis factor ␣ and IFN-␥ may be involved in mediating hepatic damage. These cytokines may be secreted by AFP-specific CD4⫹ and CD8⫹ T cells and subsequently result in cytokine-mediated hepatocyte damage, either directly or by attracting macrophages or natural killer cells.30 –32 Although normal hepatocytes are not particularly sensitive to direct tumor necrosis factor–mediated apoptosis, appropriate priming makes them highly susceptible. This could involve viral infections, cellular stress, or damage, e.g., hepatectomy, factors that could provide a danger signal and subsequently prevent deletion or anergy of self-reacting CD8⫹ T cells. Studies are ongoing to identify the pathogenetic mediators in detail. In conclusion, the use of nontumor-restricted antigens as targets for tumor immunotherapy may carry the risk for severe autoimmune disease. The risk for induction of autoimmunity depends largely on the relative size of the tumor versus the size and the importance of the target organ, on the relative precursor and effector T-cell frequencies, and on the expression level of the tumor antigen. Careful selection of HCC patients for AFP-directed immunotherapy may reduce the risk to induce autoimmune hepatitis.

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Received December 27, 2000. Accepted June 13, 2001. Address requests for reprints to: Michael Geissler, M.D., Department of Medicine II, University Hospital Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany. e-mail: [email protected]; fax: (49) 761-2703610. Supported by Bunderministerium fu ¨r Bildung und Forschung (BMBF) grant 01GE9611 from the German Center for Aeronautics and Space Travel (DLR), grants Ge824/5-1 and SFB364 from the Deutsche Forschungsgemeinschaft, and grant 10-1662-Re from the Deutsche Krebshilfe (to M.G.), and by grant SFB364 from the Deutsche Forschungsgemeinschaft (to L.M.).