Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7257-7261, August 1995 Immunology Upregulation of class I major complex by y is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo YIPING YANG*t, ZHIQUAN XIANGt, HILDEGUND C. J. ERTLt, AND JAMES M. WILSON*t# *Institute for Human Gene Therapy and Departments of Medicine and Molecular and Cellular Engineering, the University of Pennsylvania, and tthe Wistar Institute, Philadelphia, PA 19104-4268 Communicated by Robert Austrian, University ofPennsylvania, Philadelphia, PA, March 30, 1995 (received for review January 30, 1995)

ABSTRACT Recombinant adenoviruses are attractive ve- mediated destruction through upregulation of major histocom- hicles for liver-directed gene therapy because of the high patibility complex (MHC) class I expression. efficiency with which they transfer genes to hepatocytes in vivo. First generation recombinant adenoviruses deleted of El sequences also express recombinant and early and late viral MATERIALS AND METHODS genes, which lead to development of destructive cellular Animals. C57BL/6 mice (H-2b) were obtained from The immune responses. Previous studies indicated that class I Jackson Laboratory. MHC class II-deficient mice were pur- major histocompatibility complex (MHC)-restricted cytotoxic chased from GenPharm International (Mountain View, CA). T (CTLs) play a major role in eliminating IFN-y-deficient mice were obtained from Genentech. CD4- virus-infected cells. The present studies utilize mouse models and perforin-deficient mice were kindly provided by Barbara to evaluate the role of T-helper cells in the primary response Knowles (The Jackson Laboratory) and William Clark (Uni- to adenovirus-mediated gene transfer to the liver. In vivo versity of California, Los Angeles), respectively. MHC class ablation of CD4+ cells or interferon 'y (IFN-y) was sufficient II-deficient, IFN-y-deficient, CD4-, and perforin-deficient to prevent the elimination of adenovirus-transduced hepato- mice were bred onto the C57BL/6 background. CD4+ T cells cytes, despite the induction of a measurable CTL response. and IFN-,ywere depleted by inoculating mice intraperitoneally Mobilization of an effective THI response as measured by in with 0.5-ml aliquots of a 1:10 dilution of mouse ascites fluid vitro proliferation assays was associated with substantial containing the GK1.5 (anti-CD4; American Type Culture upregulation of MHC class I expression, an effect that was Collection) and XMG1.2 (anti-IFN--y; American Type Culture prevented in IFN-y-deficient animals. These results suggest Collection) monoclonal (mAb). For depletion of that elimination of virus-infected hepatocytes in a primary CD4+ cells, the mAb was given 3 days before, on the day of exposure to recombinant adenovirus requires both induction infection, 3 days after, and then at 3-day intervals until of -specific CTLs as well as sensitization of the target completion of the experiment; for depletion of IFN-y, the cell by TH1-mediated activation of MHC class I expression. mAb was administered 3 days before, 3 days after, and then at 6-day intervals until completion of the experiment. Human type C adenoviruses have been rendered replication Recombinant Adenoviruses. The recombinant adenoviruses defective for gene therapy by deleting the first early gene locus H5.OOCMVlacZ [lacZ gene expressed from the cytomegalovirus that encodes Ela and Elb. An extensive literature has (CMV) promoter in the sub360 backbone] and H5.01OCBALP emerged demonstrating the utility of El-deleted viruses for (alkaline phosphatase gene expressed from the CMV-enhanced accomplishing efficient gene transfer in a variety of cells in vivo (3-actin promoter in the sub360 backbone) were used in this study. (1). In most models, recombinant gene expression has been Virus (2 x 109 plaque-forming units in 100 ,ul of phosphate- transient, lasting less than 3-4 weeks, and associated with buffered saline) was administered into female mice (6-8 weeks development of inflammation characterized by lymphocytic old) via the tail vein. When mice were necropsied 3, 10, or 24 days infiltrates. Our work in murine models indicates that El- later, liver tissues were prepared for cryosections, while spleens deleted adenoviruses express viral proteins and potentially were harvested for immunological assays. immunogenic recombinant proteins that elicit destructive class Morphological Analyses. Histochemistry. Sections of fresh I-restricted cytotoxic T lymphocytes (CTLs) (refs. 2 and 3; data frozen tissue (6 ,um) were fixed in 0.5% glutaraldehyde and not shown). Immune-mediated clearance of the corrected cell analyzed for expression of 13-galactosidase or alkaline phos- could explain the limitations of transient expression and phatase histochemistry as described (3). pathology that characterized first generation adenoviral tech- Immunofluorescence. Frozen sections (6 ,um) were fixed in nology. Humoral immune responses to proteins of the input methanol. CD4+/CD8+ double immunofluorescence was per- recombinant adenovirus underlie an unrelated but equally formed as described (4). For MHC class I staining, sections important problem of neutralizing that prevent were incubated with 1:10 diluted mouse hybridoma superna- gene transfer upon a second administration of virus (3). tant to H-2KbDb (20-8-4S) for 60 min, followed by incubation This study uses a mouse model of adenovirus-mediated gene with goat anti-mouse IgG-conjugated fluorescein isothiocya- transfer to liver to define the precise cellular immune re- nate (FITC) (5 ,tg/ml) for 30 min. Sections were washed and sponses responsible for destruction of the genetically modified mounted with the antifadent Citifluor (Canterbury Chemical hepatocytes. Data presented here show that CD4+ cells work Lab., Canterbury, U.K.). in concert with virus-activated CTLs to eliminate the target CTL Assays. CTL assays were performed with splenocytes cells. This occurs, in part, through activation of T-helper cells pooled from three mice as described (2). Briefly, splenocytes of the THI subset and of interferon -y (IFN-,y), which were restimulated in vitro for 5 days with H5.OlOCMVlacZ and sensitizes the genetically modified hepatocyte to CTL- Abbreviations: CTL, cytotoxic T ; IFN-,y, interferon y; The publication costs of this article were defrayed in part by page charge MHC, major histocompatibility complex; mAb, monoclonal antibody; payment. This article must therefore be hereby marked "advertisement" in IL, interleukin. accordance with 18 U.S.C. §1734 solely to indicate this fact. iTo whom reprint requests should be addressed. 7257 Downloaded by guest on September 28, 2021 7258 Immunology: Yang et aL Proc. Natl. Acad. Sci. USA 92 (1995) assayed on MHC-compatible, 51Cr-labeled, H5.OlOCMVlacZ class I-compatible targets infected with the recombinant virus infected, C57SV (H-2b) cells using different effector/target or another recombinant virus expressing a different transgene. cell ratios. Percentage specific 51Cr release was calculated as Animals deficient in MHC class I and CD8+ cells by virtue of described (2). a germ-line interruption of f32-microglobulin failed to elimi- Cytokine Release Assays. Interleukin (IL)-2 and IL-4 release nate transduced hepatocytes. In addition, adoptive transfer of assay. Splenocytes were restimulated in vitro with UV- activated and purified CD8+ cells into an adenovirus-trans- inactivated H5.OlOCMVlacZ for 24 hr. Cell-free supernatants duced RAG-2-deficient mouse was sufficient to eliminate the were assayed for the presence of IL-2 or IL-4 on HT-2 cells (an transgene-expressing hepatocytes. IL-2- or IL-4-dependent cell line) as described (2); the relative The function and relative importance of CTLs in adenovi- contribution of each cytokine to stimulation of HT-2 was rus-mediated gene transfer to mouse liver were further eval- assessed by specific neutralization of either IL-2 or IL-4 with uated in the current study by using mice genetically deficient mAbs. in perforin (11), a molecule that mediates one mechanism of IFN-,y release assay. The presence of IFN-,y in the same CTL killing (12). Perforin-deficient mice were injected with splenocyte culture supernatant was measured as described (2). the lacZ adenovirus and liver tissue was analyzed for stability of transgene expression and infiltration of lymphocytes (Fig. RESULTS AND DISCUSSION 1). Congenic immune-competent animals developed a mixed CD4+ and CD8+ lymphocytic infiltrate in liver (Fig. lc), while First generation recombinant adenoviruses were developed for expression of the transgene diminished to undetectable levels gene therapy based on the premise that deletion of El by day 24 (Fig. lb); this is in contrast to the perforin-deficient sequences should be sufficient to inactivate other viral genes animals who did not eliminate significant numbers of trans- and render the virus replication defective. Previous in vitro duced hepatocytes (Fig. le) despite mobilizing a full CD4+ and studies suggested that this is not the case when infections are CD8+ lymphocyte response (Fig. lf). This same animal model performed under high multiplicity of infection or in hepato- in cyte-derived cells where El transcription-like factors, such as has been used to demonstrate the importance of perforin NF-IL-6, are abundant (5). Studies in a variety of preclinical CTL-mediated destruction of cells infected with lymphocytic models including mouse lung and liver (6, 7), cotton rat lung choriomeningitis virus and vaccinia (11, 13). (8), human bronchial xenografts (9), baboon lung (10), and CD4+ Cells Are Necessary for a Fully Competent CTL rhesus monkey liver (data not shown) have detected expression Response. Murine models of infection by a variety of patho- of both early and late viral genes in animals infected with genic viruses have suggested that, under some conditions, El-deleted recombinant adenoviruses. Experiments in mice CD4+ cells may contribute to the host's cellular immune indicate that cellular immune responses to newly synthesized response to virus-infected cells. This can occur through anti- viral proteins contribute to destruction of hepatocytes and gen-specific activation of T-helper cells, which enhance the development of inflammation (2). This study uses adenovirus- activity of other effector cells such as macrophages and CD8+ mediated gene transfer to mouse liver as a model to expand our CTLs (14-17). CD4+ cells can emerge as primary CTL effec- understanding of the precise mechanisms responsible for tors, especially when CD8+ CTLs are depleted (18-20). The transient gene expression and the associated pathology. role of CD4+ cells in the immune response to infection with Clearance of Virus-Infected Hepatocytes Is Primarily Me- recombinant adenovirus has been restricted to our previous diated by Antigen-Specific CTLs. Several previous observa- murine studies. Animals deficient in CD4+ cells because of a tions suggested a primary role for virus-specific CTLs in genetic defect in class II expression were able to clear virus- elimination of hepatocytes infected with El-deleted recombi- infected cells, suggesting that a fully competent CTL effect was nant adenoviruses (2, 6). Infusion of first generation recom- possible in the absence of CD4+ cells in this genetic back- binant adenoviruses into peripheral blood leads to high trans- ground (ref. 4; see also Fig. 2 g and h). Adoptive transfer of gene expression in hepatocytes that diminishes to undetectable activated CD4+ cells into adenovirus-transduced RAG-2- levels by day 21, concurrent with the development of mono- deficient mice failed to completely diminish transgene expres- nuclear infiltrates. Loss of transgene expression is associated sion, arguing against CD4+ cells as the only effectors (2). with the appearance of CTLs in spleen capable of lysing MHC Neither experiment, however, directly evaluated the specific

FIG. 1. Adenovirus-mediated gene transfer to liver of mice heterozygous or homozygous for disruption of the perforin gene. Suspensions of lacZ adenovirus (2 x 109 plaque-forming units) were administered into the tail vein of mice, which were subsequently euthanized, and liver tissues were evaluated for lacZ expression by ,B-galactosidase histochemistry 3 (a and d) and 24 (b and e) days later and for CD4+ and CD8+ cells by immunofluorescence 10 days later (c and f). (a-c) Mouse heterozygous for perforin mutation. (d-f) Mouse homozygous for perforin mutation. (c andf) Solid arrows, green fluorescence indicating CD8+ cells; open arrows, red fluorescence indicating CD4+ cells. (a, b, d, and e, x46; c and f, x185.) Downloaded by guest on September 28, 2021 Immunology: Yang et aL Proc. Natl. Acad. Sci. USA 92 (1995) 7259

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i FIG. 2. Role of IFN-'y in adenovirus-mediated gene transfer to mouse liver. H5.OlOCMVlacZ-infected liver tissues were evaluated for lacZ expression by 5-bromo-4-chloro-3-indolyl iS-D-galactoside histochemistry 3 (a, c, e, g, and i) and 24 (b, d, j h, andj) days later. C57BL/6 mice (a and b), IFN-y-deficient mice (c and d), C57BL/6 mice depleted of IFN-'y (e andf), class II-deficient mice (g and h), class II-deficient mice depleted of IFN--y (i and j). (x60.) role of CD4+ cells in the context of a primary administration several effectors capable of target cell elimination. One mech- of virus. anism is activation of macrophages by IFN-,y and mobilization More direct murine models were used in the current study of a destructive delayed-type hypersensitivity reaction (22, 23). to evaluate the specific role of CD4+ cells in this application Alternatively, TH1 cells can contribute to development of a of liver-directed gene therapy. Animals specifically ablated of competent CTL response by enhancing induction and ampli- CD4+ cells through germ-line interruption of the gene or upon fication of activated CTLs via IL-2 and possibly IFN--y (24, 25) antibody-mediated depletion of CD4+ cells were infused with as well as sensitizing the target cell to cytolysis through recombinant alkaline phosphatase adenovirus and analyzed IFN-y-mediated upregulation of MHC class I expression (26, for stability of transgene expression and infiltration of lym- 27). phocytes (Fig. 3). Transgene expression was stable for at least Attempts to define the role of CD4+ cells in our model of 28 days in both CD4 knockout animals (Fig. 3e) and C57BL/6 liver-directed gene therapy focused on the THI-specific cyto- animals depleted ofCD4+ cellswith antibody (Fig. 3h), despite kine IFN--y, which is capable of augmenting both delayed-type the infiltration of CD8+ cells into the liver (Fig. 3 f and i). hypersensitivity and CTL responses. Virus was administered to Primary Response to Recombinant Adenovirus Requires animals deficient in IFN--y through either germ-line interrup- Activation of TH1 and Secretion of IFN-'y. CD4+ cells could tion of the gene (28) or antibody-mediated depletion of the contribute to the destruction of virus-infected cells through cytokine. Analysis of splenocytes from C57BL/6 animals one of several mechanisms. Exogenous viral proteins are harvested 10 days after virus administration revealed selective presented by MHC class II to T-helper cells that differentiate activation of TH1 cells as evidenced by the secretion of both into either TH1 subsets, which secrete IL-2 and IFN-'Y, or TH2 IFN--y and IL-2 (Table 1). Similar studies of splenocytes from subsets, which secrete a variety of cytokines including IL-4, IFN-y-deficient animals revealed virus-activated secretion of IL-6, and IL-10 (21). TH1 have a primary role in regulation of IL-4 with no production of IFN-y, consistent with a shift from

FIG. 3. Role of CD4+ cells in adenovirus-mediated gene transfer to mouse liver. Alkaline phosphatase virus (2 x 109 plaque-forming units) was infused into the tail vein of mice, which were subsequently euthanized, and liver tissues were evaluated for transgene expression by alkaline phosphatase histochemistry 3 (a, d, and g) and 24 (b, e, and h) days later and for CD4+ and CD8+ cells by immunofluorescence 10 (c, f, and i) days later. (a-c) C57BL/6 mice; (d-f) CD4- mice; (g-i) C57BL/6 mice depleted of CD4+ cells with antibody. (c, f, and i) Solid arrows, green fluorescence CD8+ cells; open arrows, red fluorescence CD4+ cells. (a, b, d, e, g, and h, X70; c, f, and i, X185.) Downloaded by guest on September 28, 2021 7260 Immunology: Yang et al. Proc. Natl. Acad. Sci. USA 92 (1995)

TH1 to TH2 (Table 1). Analysis of liver tissue revealed a high i80 . Mock *- C57BL/6 level and stable expression of lacZ in both the IFN--y knockout 70. -0- IFNy(-/-) animals (Fig. 2 c and d) as well as C57BL/6 animals depleted 60. °-O- C57BL/6 + anti-IFN-y of IFN-,ywith antibody (Fig. 2 e andf). These data indicate that 50. IFN--y is essential for complete elimination of the virus- 40. infected cells. 30. IFN-y Sensitizes Target Cells to CTL-Mediated Destruction 920. by Activation of Class I MHC. Additional studies were per- CO I10. formed to define the precise role IFN-,y plays in the cellular' p.11 immune response to adenovirus-infected mouse hepatocytes. 0 !E 3:1 6:1 12:1 25:1 Our studies indicate that activation of macrophages and de- 9 layed-type hypersensitivity responses contribute little to the CL j80 destruction of virus-transduced hepatocytes. Immunocyto- co chemical analysis of liver tissue revealed few infiltrating mac- 70 rophages in either immune-competent or IFN-y-depleted an- 60 / 9 imals infected with recombinant adenoviruses (data not I50 shown). Furthermore, animals with intact macrophage and/or 40 / T-helper functions but deficient CTL activity [i.e., /32- 3-0 microglobulin (2) and perforin knockout mice (Fig. 1)] failed f 920- to eliminate transgene-expressing cells. Another potential role of TH1 cells and IFN--y is in gener- I10 ation of an effective CTL response. Previous studies have 3:1 6:1 12:1 25:1 suggested that IFN-,y contributes to the actual activation and Effector/target amplification of CTL precursors (24, 25, 29). Lymphocytes harvested from spleen were evaluated for class I-restricted FIG. 4. CTL responses in IFN-y-deficient mice. Splenocytes har- vested from C57BL/6 mice (solid circles), IFN-y-deficient mice (open cytolysis against virus-infected target cells by the chromium squares), and C57BL/6 mice depleted of IFN-,y (open circles) 10 days release assay. Splenocytes from C57BL/6 animals restimu- after administration of lacZ virus were restimulated in vitro for 5 days lated in vitro with viral antigens yielded substantial cytolytic and tested for specific lysis on mock-infected or lacZ virus-infected activity that was specific to virus-infected, class I-compatible C57SV cells in a 6-hr 51Cr release assay. Percentage specific lysis is targets (Fig. 4). Studies performed with splenocytes from expressed as a function of different effector/target cell ratios (3:1, 6:1, animals deficient in IFN-,y (i.e., IFN-y knockout mice and 12:1, and 25:1). C57BL/6 animals depleted of IFN-,y with antibody) revealed depletion completely prevented the upregulation of class I CTL activity equal to or greater than that from C57BL/6 mice RNA (data not shown) or protein (Fig. Sc) that occurred after (Fig. 4). We conclude that the role of IFN-,y in in vivo administration of adenovirus. activation and amplification of virus-specific CTLs is not Results obtained in the class II-deficient animals were significant. puzzling in that transgene expression was transient, reflecting The remaining mechanism by which IFN-,y can promote a fully competent cellular immune response, despite the ab- CTL activity is through sensitization of the target cell by sence of both class II expression and functionally active CD4+ upregulating expression of class I MHC and enhancing the cells. Additional experiments were performed to better un- presentation of antigen (26). Livers from C57BL/6 and IFN- derstand this observation. The ability of this animal to elim- y-depleted animals infected with lacZ virus were analyzed for inate the target cells was independent of IFN-,y based on in vivo class I MHC expression by RNA blot analysis (data not shown) antibody-depletion studies (Fig. 2 i and j). Direct analysis of and immunofluorescence (Fig. 5) using a probe and antibody liver tissue from class I1-deficient mice revealed low levels of specific for H2b heavy chain, respectively. Class I heavy-chain MHC class I expression at baseline that was substantially up- RNA in C57BL/6 animals progressively increased after infec- tion with recombinant adenovirus to a peak 10 days after gene transfer with a subsequent diminution to baseline (data not shown). Immunocytochemical studies on liver tissue demon- strated a concurrent increase in class I protein from virtually undetectable levels in naive animals (Fig. Sa) to very high quantities of protein that appeared to localize to the basolat- eral surface of hepatocytes (Fig. Sb). This pattern is identical to that obtained in animals systemically infused with IFN--y (26). Depletion of IFN-,y by genetic interruption or antibody Table 1. Cytokine secretion by in vitro secondary bulk splenocyte cultures Cytokine B6 + produced C57BL/6(B6) IFN-y- anti-IFN-y IFN-y* 80 0 0 IL-2t 40.5 49.5 45.2 IL-4t 0 12.8 8.5 Splenocytes from mice 10 days after administration of adenoviruses were restimulated with H5.010CMVlacZ for 24 hr. Supernatants were FIG. 5. MHC class I expression in recombinant adenovirus- tested for cytokine secretion. infected mouse liver. Liver tissues harvested from naive C57BL/6 mice *Supernatants were tested onL929 cells forproduction of functional (a), lacZ virus-infected C57BL/6 mice at day 10 (b), lacZ virus- IFN-,y (units/ml) (see ref. 2). infected C57BL/6 mice depleted of IFN-,y at day 10 (c), and lacZ tSupernatants were tested on HT-2 cells, and proliferation was virus-infected class II-deficient mice depleted of IFN-,y at day 10 (d) measured 72 hr later by [3H]thymidine incorporation; results are were stained for MHC class I expression by immunofluorescence with expressed as stimulation index (see ref. 2). mAb to H-2KbDb. (x70.) Downloaded by guest on September 28, 2021 Immunology: Yang et aL Proc. Natl. Acad. Sci. USA 92 (1995) 7261 regulated after adenovirus-mediated gene transfer in the 5. Spergel, J. M., Hsu, W., Akira, S., Thimmappaya, B., Kishimoto, presence (data not shown) or absence (Fig. 5d) of IFN-,y. This T. & Chen-Kiang, S. (1992) J. Virol. 66, 1021-1030. suggests that the class II-deficient animal is capable of both 6. Yang, Y., Nunes, F. A., Berencsi, K, Furth, E. E., Gonczol, E. & activating virus-specific CTLs and upregulating MHC class I in Wilson, J. M. (1994) Proc. Natl. Acad. Sci. USA 91, 4407-4411. 7. Yang, Y., Nunes, F. A., Berencsi, K., Gonczol, E., Engelhardt, a manner that is independent of IFN-y and CD4+ cells. These J. E. & Wilson, J. M. (1994) Nat. Genet. 7, 362-369. experiments support the hypothesis that upregulation of class 8. Engelhardt, J. F., Litzky, L. & Wilson, J. M. (1994) Hum. Gene I is directly involved in sensitization of the hepatocyte to CTLs Ther. 5, 1217-1229. rather than an unrelated correlate of IFN--y secretion. 9. Engelhardt, J. F., Yang, Y., Stratford-Perricaudet, L. D., Allen, Our model proposes that TH1 cells are the source of IFN-,y E. D., Kozarsky, K., Perricaudet, M., Yankaskas, J. R. & Wilson, required for target cell elimination. Another possibility that J. M. (1993) Nat. Genet. 4, 27-34. cannot be formally excluded is secretion of IFN--y from CD8+ 10. Engelhardt, J. F., Simon, R. H., Yang, Y., Zepeda, M., Weber- CTLs. Analyses of cloned CTL cell lines generated in other Pendleton, S., Doranz, B., Brossman, M. & Wilson, J. M. (1993) models have demonstrated the capacity of these cells to secrete Hum. Gene Ther. 4, 759-769. IFN--y, although its importance in setting a primary exposure 11. Walsh, C. M., Matloubian, M., Liu, C., Ueda, R., Kuahara, C. G., Christensen, J. L., Huang, M. T. F., Young, J. D., Ahmed, R. & to virus has not been demonstrated in vivo (30, 31). This Clark, W. R. (1994) Proc. Natl. Acad. Sci. USA 91, 10854-10858. contrasts with our observation made on class II-deficient mice 12. Henkart, P. A. (1994) Immunity 1, 343-346. in that IFN-,y is not detectable in splenocyte culture upon 13. Kagi, D., Ledermann, B., Burki, K., Seiler, P., Odermatt, B., adenovirus infection (2) but is consistent with the study of Olsen, K. J., Podack, E. R., Zikernagel, R. M. & Hengartner, H. Sendai virus infection in this same mouse model, which (1994) Nature (London) 369, 31-37. detected IFN--y production (32). A primary role of CTL- 14. Matloubian, M., Concepcion, R. J. & Ahmed, R. (1994) J. Virol. derived IFN-,y in our system would leave unexplained, how- 68, 8056-8063. ever, the dependence on CD4+ cells for a fully functional CTL 15. Jennings, S. R., Bonneau, R. H., Smith, P. M., Wolcott, R. M. & response. Chervenak, R. (1991) Cell. Immunol. 133, 234-252. Model of Cellular Immunity in Adenovirus-Mediated Gene 16. Leist, T. P., Kohler, M. & Zinkernagel, R. M. (1989) Scand. J. Immunol. 30, 679-686. Transfer to the Liver. These studies have identified essential 17. Battegay, M., Moskophidis, D., Rahemtulla, A., Hengartner, H., components ofthe cellular immune response that contribute to Mak, T. W. & Zinkernagel, R. M. (1994) J. Virol. 68,4700-4704. elimination of adenovirus-transduced hepatocytes. Important 18. Muller, D., Koller, B. H., Whitton, J. L., LaPan, K E., Brigman, effectors in this process are CD8+ CTLs to new proteins K. K & Frelinger, J. A. (1992) Science 255, 1576-1578. synthesized in the viral transduced cells; perforin plays an 19. Eichelberger, M., Allan, W., Zijlstra, M., Jaenisch, R. & Doherty, essential role in target cell destruction. that direct the P. C. (1991) J. Exp. Med. 174, 875-880. activation of these CTLs have not been identified; however, 20. Hou, S., Doherty, P. C., Zijlstra, M., Jaenisch, R. & Katz, J. M. chromium release assay to infected targets suggests that some (1992) J. Immunol. 149, 1319-1325. epitopes reside on viral proteins (4). CTL responses to the 21. Paul, W. E. & Seder, R. A. (1994) Cell 76, 241-245. transgene protein product may contribute to target cell de- 22. Ando, K., Moriyama, T., Guidotti, L. G., Wirth, S., Schreiber, R. D., Schlicht, H. J., Huang, S. & Chisari, F. V. (1993) J. Exp. struction in some experimental models. Our model also sug- Med. 178, 1541-1554. gests an essential role for antigen-specific activation of TH1 23. Issekutz, T. B., Stoltz, J. M. & Meide, P. V. D. (1988)J. Immunol. cells that are mobilized to the liver where they sensitize 140, 2989-2997. hepatocytes to CTL-mediated cytolysis by secreting IFN--y and 24. Maraskovsky, E., Chen, W.-F. & Shortman, K. (1989) J. Immu- upregulating MHC class I presentation of antigens. nol. 143, 1210-1214. 25. Wille, A., Gessner, A., Lother, H. & Lehmann-Grube, F. (1989) We wish to thank Q. Su for technical assistance, Dr. Barbara Eur. J. Immunol. 19, 1283-1288. Knowles for CD4- mice, and Dr. William Clark for perforin knockout 26. Skoskiewicz, M. J., Colvin, R. B., Schneeberger, E. E. & Russell, mice. This work was supported by grants from the Cystic Fibrosis P. S. (1985) J. Fxp. Med. 162, 1645-1664. Foundation and the National Institute of Diabetes and Digestive and 27. Fellous, M., Nir, U., Wallach, D., Merlin, G., Rubinstein, M. & Kidney Diseases of the National Institutes of Health. Revel, M. (1982) Proc. Natl. Acad. Sci. USA 79, 3082-3086. 28. Dalton, D. K., Pitts-Meek, S., Keshav, S., Figari, I. S., Bradley, A. 1. Kozarsky, K. F. & Wilson, J. M. (1993) Curr. Opin. Genet. Dev. & Stewart, T. A. (1993) Science 259, 1739-1742. 3, 499-503. 29. Simon, M. M., Hochgeschwender, U., Brugger, U. & Landolfo, S. 2. Yang, Y., Ertl, H. C. J. & Wilson, J. M. (1994) Immunity 1, (1986) J. Immunol. 136, 2755-2762. 433-442. 30. Morris, A. G., Lin, Y.-L. & Askonas, B. A. (1982) Nature (Lon- 3. Yang, Y., Li, Q., Ertl, H. C. J. & Wilson, J. M. (1994) J. Virol. 69, don) 295, 150-152. 2004-2015. 31. Yamasaki, T. H., Handa, J., Yamashita, Y., Watanabe, Y., 4. Yang, Y., Devor, D. C., Engelhardt, J. F., Ernst, S. A., Strong, Namba, Y. & Hanaoka, M. (1984) Cancer Res. 44, 1776-1783. T. V., Collins, F. S., Cohn, J. A., Frizzell, R. A. & Wilson, J. M. 32. Hou, S., Mo, X. 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