MHC class I-cestricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses

Immunity, Vol.

1. 4X3-442,

August, 1994, Copyright 0 1994 by Cell Press

MHC Class I-Restricted Cytotoxic T Lymphocytes to Viral Antigens Destroy Hepatocytes in Mice Infected with El-Deleted Recombinant Adenoviruses Yiping Yang,‘t Hildegund C. J. Ertl,t and James M. Wilson’t ‘Institute for Human Gene Therapy Department of Molecular and Cellular Engineering The University of Pennsylvania Medical Center tThe Wistar Institute Philadelphia, Pennsylvania 19104

Summary The use of El-deleted recombinant adenoviruses in gene therapy has consistently been associated with transient gene expression and inflammation due to immune-based destruction of the Infected cells. We have used murine models of adenovirus-medlated gene transfer to liver to investigate these immunologic mechanisms. Adoptive transfer experiments, as well as studies involving genetic knockout mice, confirmed the original hypothesis that cell-mediated immunity induced by El-deleted adenovlrus destroyed transgene-expressing hepatocytes and defined MHC class I-restricted CD@cytolytic lymphocytes as the primary immune effecters for hepatocyte destruction. Responses mediated by CD4+ cells per se were insufficient to mediate destruction of hepatocytes in vivo, despite the activation of virus-specific T helper cells of Thl subsets. A better understanding of the response of the host to in vivo gene therapy is important in evaluating its usefulness in humans. Introduction Successful human gene therapies require the development of efficient and safe in vivo gene transfer technologies. One promising approach is based on transfer of recombinant forms of human adenoviruses derived from serotypes 2 and 5. This family of nonenveloped viruses contains a 36 kb double-stranded DNA genome comprised of both early and late genes (Horwitz, 1990). Expression of the immediate-early genes El a and El b is the first step in virus replication that activates a cascade of events, which culminates in formation of new virions. The first generation of recombinant adenoviruses used in gene therapy has been rendered replication defective by deleting the essential genes within the El locu.s. These recombinants can be propagated in vitro in the packaging cell line 293, which has been stably transfected to provide El a and El b (Graham et al., 1977). Recombinant adenoviruses have many features that are useful in the development of gene therapies. High level gene transfer can be achieved in nondividing cells and in vivo administration is simplified because the virus is easily grown in large quantities, concentrated, and purified. Early applications of recombinant adenoviruses for gene ther-

apy focused on the treatment of cystic fibrosis by instillation of virus into the airway (Rosenfeld et al., 1992). Experiments in animal models were sufficiently promising to justify phase I clinical trials of adenovirus-mediated gene transfer to lungs of patients with cystic fibrosis (Wilson, 1993; Zabner et al., 1993). The observation that human adenovirus is hepatotropic when infused into the circulation suggested the use of this same technology for liverdirected gene therapy (Jaffe et al., 1992). This approach has been used to express a variety of therapeutic genes in animal models, including low density lipoprotein receptor, factor IX, ornithine transcarbamylase, and a-1-antitrypsin (Jaffeetal.,1992;Kozarskyetal.,1994;Smithetal., 1993; Stratford-Perricaudet et al., 1990). Localized delivery of recombinant adenovirus to other organ systems has yielded promising results in experimental models, enhancing the potential utility of this technology for treating a wide spectrum of disorders (Kozarsky and Wilson, 1993). Despite the promising results achieved with first generation recombinant adenoviruses, several significant problems remain. In virtually every in vivo application, expression of the adenoviral transgene is transient and associated with pathology (Kozarsky and Wilson, 1993). Based on previous studies, we proposed an hypothesis to explain the limitations in adenoviral technology (i.e., transient expression and pathology). We suggest that the primary problem is expression of El-like functions in the target cell that overcomes the block in transcription imposed by deletion of El a and El b. The adenovirus-transduced cells present the de novo synthesized viral protein fragments in association with major histocompatabiiity complex (MHC) determinants to the immune system, resulting in the activation of virus-specific cellular immune responses. This hypothesis has been confirmed in several models, including lung-directed gene transfer in mice, cotton rat, nonhuman primates, and human bronchial xenografts, as well as liver-directed gene transfer in mice (Engelhardt et al., 1993a, 1993b, 1994; Simon et al., 1993; Yang et al., 1994a, 1994b). In this study, we used the model of liverdirected gene transfer in the mouse to elucidate the immunological effector mechanisms responsible for the destruction of transgene-expressing cells. The results show that MHC class l-restricted CD6+ lymphocytes are the primary effectors mediating the destruction and, consequently, the turnover of transduced hepatocytes. In vitro analysis of splenocytes indicates that systemic delivery of recombinant adenovirus activates viral antigen-specific responses of both MHC class l-restricted cytolytic T lymphocytes (CTLs) and MHC class II-restricted T helper (Th) cells of Thl subsets. Further identification of the specificviral proteins and corresponding epitopes that provide immunodominant targets for recognition by CTLs might provide a basis for rational improvements in second generation recombinant adenoviruses.

Immunity 434

Figure

1. Adenovirus-Mediated

IacZ Expressi

on and CD6’

and/or

CD4’

Lymphocyte

Infiltration

in Mouse

Liver

euthanized, and the liver Suspensions of adenovirus H501OCBlacZ (2 x 1Oe pfu) were infused into the tail vein of mice that were subsequently tissues were evaluated for IacZ expression by X-gal histochemisby 3 (first column) and 26 (seco nd column) days later, and for presence of CDB’, CD4’. or both lymphocytes 7 days later (third I column) by double immunofluorescence. Open aind closed arrows indicate CD4+ and CD6’ cells, third row, RAG-P-mice respectively. First row, C57BU6 mice infused with H5.010CBlacZ; second row, RAG-P- mice in1fused with H501OCBlacZ; infused with H501OCBlacZand, subsequently, the total splenocytes (2 x IO’) from C57BU6 mice: fourth row, f&m- mice infused with H5.010CBlacZ; fifth row, MHC class II- mice infused with H5. OiOCBlacZ. Magnification for first and second col umn, 63 x ; third column, 332 x

C&otoxic

T Ceils in Adenovirus

3

Gene Therapy

7

14

28

Day Figure 2. Histopathology of Mouse Livers in Response to Recombinant Adenoviruses Liver tissues were harvested following infusion of H5.01 OCBlacZ (2 x lo9 pfu) and evaluated for evidence of pathological changes by light microscopic examination of hematoxylin and eosin sections. Samples were characterized with respect to periportal degeneration and focal necrosis, intralobular degeneration and focal necrosis, and portal inflammation. Extent of pathology was scored from 0 (no pathology) to 4 (severe pathology). This figure summarizes the extent of pathology observed in C57BU6 (closed boxes), RAG-P (open boxes), f&m- (stippled boxes), and MHC class II- (hashed boxes) mice as a function of time following infusion of virus (days 3, 7, 14, and 26).

Results The Rapid Loss of Transgene Expression upon Adenovirus-Mediated Gene Transfer Is Caused by MHC Class l-Restricted CD8+ Lymphocytes Genetically defined strains of mice deficient in important immunoregulatory genes were used to delineate the role of cellular immunity in adenovirus-mediated gene transfer to liver. The El-deleted virus H5010CBlacZ, which expresses IacZ from a cytomegalovirus-enhanced p-actin promoter, was infused into tail veins of the following inbred strains of mice that all carry the H-2b haplotype: fully immunocompetent C57BU6 mice; 62-microglobulin-deficient &rn-) mice that lack expression of MHC class I molecules

and functionally mature CD8 T cells (Zijlstra et al., 1990); MHC class II-deficient (MHC class II-) mice that are unable to express I-Ab determinants and thus fail to develop CD4+ T cell-mediated responses (Grusby et al., 1991); RAG-2deficient (RAG-T) mice that lack functionally active T and B lymphocytes due to a targeted disruption of a recombinase gene involving V(D)J rearrangement (Shinkai et al., 1992). Figure 1 summarizes a histochemical analysis of liver for expression of IacZ 3 (first column) and 28 (second column) days after gene transfer, in addition to immunocytochemical analysis for CD4+ and CD8+ cells at the time of inflammation (third column). The liver was the primary target of gene transfer in C57BU6 mice with over 80% of hepatocytes expressing high levels of IacZ 3 days after infusion of virus (Figure la). Over the subsequent lo-14 days, IacZ expression diminished to undetectable levels (Figure 1 b) concurrent with the infiltration of CD4+ and CD8+ cells (Figure lc) and the development of widespread hepatitis characterized by hepatocellular degeneration, focal necrosis, and periportal infiltration (Figure 2). The role of cellular immunity in these processes was demonstrated in experiments performed in RAG-P- mice in which infusion of H501OCBlacZ resulted in stable IacZ expression in the majority of hepatocytes (see Figure Id) for at least one month (see Figure le) with little or no pathology (Figure 2). Adoptive transfer of splenocytes from primed C57BU6 animals into RAGZ mice stably expressing adenoviral IacZ was associated with the subsequent loss of transgene expression and the appearance of a lymphocyte dominated hepatitis (see Figures 1 g-l i). Experiments were performed with &rn- and MHC class II- mice to analyze further the immunological basis of the apparent hepatocyte destruction upon transfer of the first generation recombinant adenoviruses into immune competent animals. LacZ expression was stable in Pnrn- animals (see Figures 1j and 1 k) despite substantial infiltration of CD4+ cells (see Figure II); hepatitis was present but reduced and delayed when compared with that in C57BU 6 mice (Figure 2). MHC class II- mice responded to infusion of recombinant adenovirus as did fully immunocompetent C57BU6 mice in that transgene expression was high initially (see Figure lm) and substantially reduced by day 28 (see Figure ln) with the concurrent development of significant hepatitis (Figure 2) mediated by the infiltration of CD8+ cells (see Figure lo). The destruction of hepatocytes in MHC class II- mice was slightly delayed in comparison to C57BU8 mice (see Figures 1b and 1 n). These experiments underscore the importance of MHC class I expression and CD8+ cells in the destruction of adenovirus-infected hepatocytes in vivo. More detailed adoptive transfer experiments were performed to dissect the relative contribution of MHC expression and function of CD8+ versus T lymphocytes in the murine model of liver-directed gene transfer. Immunoglobulin negative splenocytes from adenovirus-treated C57BU6 animals were separated into CD4 and CD8 fractions and infused into RAG-P- animals that were stably expressing adenovirusdirected IacZ in liver. Adoptively transferred CD6+ cells rapidly infiltrated the liver, leading

Immunity 436

Figure

3. Adoptive

Transfer

of CD4+ or CD8+ T Cells to Congenic

RAG.2

Mice Previously

Administered

with H5.010CBlacZ

CD8’ and CD4’ subsets were separated from primed splenocytes of C57BU6 mice by FACS with monoclonal antibody specific to either CD4 or CD8. Purified T cell subsets (2 x los) were then infused into the tail vein of RAG2recipient mice inoculated with H5.OlOCBlacZ (2 x lo0 pfu) 7 days before. Mice were subsequently euthanized, and the liver tissues were evaluated for IacZ expression by X-gal histochemistry 7 (first row) and 14 (second row) days later, and for presence of CD8+ or CD4+ lymphocytes 14 days later (third row) by double immunofluorescence. Open and closed arrows indicate CD4+ and CD8+ cells, respectively. First cotumn, mice transferred with serum-free DMEM; second column, RAG-2 mice transferred with CD8+ cells; third column, RAG-P- mice transferred with CD4’ cells. Magnification for first and second row, 87 x ; third row, 348 x

:f,otoxic

T Cells

in Adenovirus

Gene Therapy

A 13' 50 40

--e ----•----t

to the development of hepatitis and complete loss of IacZ expression (Figures 3b, 3e, and 3h) over a 14 day time interval in which no decrease in IacZ expression was observed in mock-treated RAG-2 animals (Figures 3a, 3d, and 39). Similar experiments with CD4+ cells led to a lymphocytic infiltrate comprised of CD4+ cells with only a slight diminution in IacZ expressing cells during the time interval. These experiments confirm the observations made in P2m- and MHC class II- animals and indicate that CD8+ cells are necessary and sufficient for the destruction of adenovirus-infected hepatocytes.

PlimedC57BU6 NaiveC57BU6 Pdmed fizrnPrimed MHC claq-II-

31

6:l

121

25:l

50:1

1003

31

6:l

12:i

25:1

50:1

loo:1

Adenovirus-Mediated Gene Transfer induces CD8+ MHC Class I-Restricted Virus-Specific Cytolytic T Cells To analyze further the functional activity of the cellmediated immune response to the adenovirus-infected hepatocytes, splenocytes from virus-infected mice were analyzed for the presence of virus-specific CTL using a standard 51chromium (“‘Cr) release assay upon in vitro restimulation. Substantial lysis of H5.01OCBlacZ-infected H-2compatible C57SV target cells that was proportional to the effector:target ratio was observed with splenocytes from adenovirus-treated C57BU6 and MHC class II- mice; no significant cytolysis was observed with splenocytes from naive C57BU6 or virus-treated Bern- mice (Figure 4A). This effect was clearly a virus-specific process, as evidenced by the absence of lysis to mock infected target cells (Figure 4A). Comparable lytic activity was observed to targets infected with a recombinant adenovirusexpressing alkaline phosphatase, indicating that CTL activity is mainly directed to viral proteins. Nevertheless, the experimental design did not allow exclusion of the presence of a minor population of CTLs directed to the product of the transgene (Figure 4A). As C57SV target cells lack expression of MHC class II determinants, the lysis mostly likely reflected CTL recognition of antigenic peptide associated with MHC class I determinants. The CTL activity from spleen is maintained at acomparable level within the duration of the experiment (Figure 4B).

iii/

10, 0

Effector:

Target

B 70 60 50

W

Day 7 Day14 Day28

I

40

8

30

p

20

G Q) K t Lx

10

O

s

l 3:1

6:l

12:1

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0

E

I

70

104,

,,,,,,,,,,I 3:l

Figure

4. Cytolytic

6:l

12:l

25:l

Effector:

Target

T Lymphocyte

Response

50:1

to Adenoviral

Adenovirus-Mediated Gene Transfer Activates CD4+ MHC Class II-Restricted Virus-Specific T Helper Cells of the Thl Subset Th cell responses in mouse recipients were evaluated by characterizing the release of cytokines from a population of splenocytes infected with the recombinant adenovirus. In this assay, the Th cell subsets, i.e., Thl and Th2, can be distinguished by release of interleukin-2 (IL-2) and interferon? (IFN$ or IL-4, respectively, upon in vitro restimulation with antigen.

1OO:l

Proteins

(A) Splenocytes harvested from C57BU6 (closed circles), bm- (closed squares) and MHC class II- (closed triangles) mice 7 days after administration of HS.OlOCBlacZ, as well as from naive C57BU6 mice (open circles), were restimulated in vitro for 5 days, and tested for specific lysis on mock-infected, H5.01OCBlacZinfected, and HS.OlOCBALPinfected C57SV cells in a 6 hr %r release assay.

(6) Splenocytes harvested from C57BU6 mice at day 7 (open circles), 14 (closed circles), and 28 (open squares) postinfusion were restimulated and tested for specific lysis on mock-infected and H5.01 OCBlacZinfected C57SV cells in a 6 hr Wr release assay. Percentage of specific lysis is expressed as a function of different effector to target ratios (3:1, 6:1, 12:1, 25:1, 50:1, and 1OO:l).

Immunity

430

x100

x10 Antigen

Xl Dose

X, UV-inacilvated

x0 1 for

In

Vttro Virus;

Yl

YO 1

Resilmulatlon Y,

lnfectlous

YO.01

--*-.

c57eL/6. antH1,

20

--*-.

C57W6, anti-IL2 62m** anti-IL4

18 1

-o-

f¶zm-.

anti-112

YO.001

(pfuicell)

2-

Virus 0. No Antibody

Figure 5. Induction of Lymphokine Secretion by Adenovirus Splenocytes from C57BU6 (open boxes), t%rn- (stippled boxes) and MHC class II- (hatched boxes) mice 7 days after administration of adenoviruses, as well as from naive C57BU6 mice (closed boxes), were restimulated in vitro for 24 hr with either UV-inactivated (X) or infectious (Y) H5.01OCBlacZ at different antigen doses (pfu/cell for UV-inactivated virus was determined before inactivation of virus). The culture supernatants were added to IL-2-or IL-4-dependent HT-2 cells, and the proliferation was measured 48-72 hr later by [3H]thymidine incorporation. Data reflect the mean of stimulation index, calculated by dividing [$H]thymidine counts (cpm) in presence of antigen by [3H]thymidine counts in absence of antigen, as a function of different antigen doses.

Supernatants from splenocytes stimulated in vitro with either live or UV-inactivated virus were incubated with the HT-2 cell line that is dependent on IL-2 or IL-4 for growth (Figure 5). Significant stimulation of HT-2 cells was accomplished with supernatants derived from splenocytes of adenovirus-infected C57BU6 and Bsrn- mice as opposed to

those from naive C57BU6 or virus-infected MHC class IImice. Antibody-mediated depletion of supernatants prior to incubation with the indicator cells suggested that IL-2 was primarily responsible for stimulating proliferation, indieating that virus-specific Thl cells dominate the Th cell response to the recombinant adenovirus (Figure 6). In both C57BU6 and Barn- mice, the Thl response was first observed 7 days after infusion of virus and progressively increased 2- to 3-fold during the subsequent three weeks (Table 1). Inactivated virus was as potent as live virus in stimulating the release of cytokines (see Figure 5) that are exclusively comprised of IL-2 (Figure 6; data not shown). Supernatants from in vitro stimulated splenocytes were also analyzed for the presence of functional IFNy. Splenocytes from C57BU6 and &.m- mice but not those from MHC class II- mice produced substantial quantities of IFNy in response to virus. Splenocytes from C57BU6 mice secreted constant levels of IFNy during the first month after infusion of virus, whereas the amount of IFNy produced from stimulated splenocytes of Pnrn- animals increased in proportion to the time interval between virus instillation and cell harvest (Table 1). The observed increase of IFNy secretion in 62m- might be related to the persistence of the recombinant viral gene expression. These data confirm the presence of a robust and stable Thl response in with time in Bsm- mice. C57BU6 mice that amplifies

1:5000

Antibody

1:5w

1:50

Dilution

Figure 6. Administration of Adenovirus Induces the Thl Subset of Th Cells Splenocytes from C57BU6 and P2m- mice 28 days after administration of adenoviruses were restimulated in vitro with W-inactivated H5.01OCBlacZ at 10 particles/cell for 24 hr. The culture supernatants were incubated without (“No Antibody”) or with serial dilutions (1:50, 1:500, and 1:5000) of ascites fluid containing monoclonal antibody either to IL-2 (S4.B6) or IL-4 (11811) prior to testing on HT-2 cells. The proliferation was measured 48-72 hr later by 13H)thymidine incorporation. Data represent the mean of stimulation index as a function of serial antibody dilutions

Enthusiasm for use of recombinant cations of human gene therapy, mendous efficiency of this type

adenoviruses in applibased in part on the treof gene transfer that can

be achieved in vivo, has been tempered by problems of transient expression and inflammatory responses. We have shown in several rodent and primate models that these problems can be explained by immunological responses to the adenovirus-transduced cells (Engelhardt et al., 1993a, 1993b, 1994; Simon et al., 1993; Yang et al., 1994a, 1994b). The current hypothesis is that first generation recombinant adenoviruses express viral proteins that stimulateantigen-specific cellular immune responses, resulting in the elimination of transgene-expressing cells. Mouse models of liver-directed gene therapy were used in this report to evaluate further this hypothesis and to define the primary effector of the destructive immune responses. Studies were designed to evaluate antigen-specific immune responses as well as those that are a nonspecific consequence of viral infection. Nonspecific immune effector cells, such as natural killer (NK) cells, macrophages, or both did not appear to contribute significantly to the transient nature of transgene expression, as destruction of target cells was not observed in strains of mice lacking functionally active B cells, T cells, or both but having intact NK and macrophage functions (e.g., nulnu, RAG-T, and SCID; Yang et al., 1994a, 1994b). Experiments in mice deficient in expression of either MHC class I or class II determinants, together with adoptive transfer of purified lymphocyte fractions, indicated that class l-restricted CD6+ cells are necessary and sufficient for the destruction

2fQotoxic

Table

T Cells

1. Induction

in Adenovirus

Gene

Therapy

of Adenovirus-Specific

Tn, Subset 13H-]Thymidine

Experiment C57BU6

II-

class II-

Experiment C57BU6 class

SD)

IFNy Titer (IU/ml)c

S.1.b

622 2004 574

f f f

105 169 258

6204 15274 519

f 1375 f 567 * 66

389 rt 58 669 zt 157 227 f 54

5688 5524 206

f f f

579 f 67 1295 f 164 225 k 40

14024 18553 276

f f f

9.9 7.6 0.9

80 80 0

146 695 21

14.6 8.3 0.9

40 160 0

1213 1279 68

24.2 14.3 1.2

40 320 0

Ill (Day 28)

bmMHC

f

II (Day 14)

B2mMHC

(cpm

I (Day 7)

BnrnMHC class

Incorporation

H501OCBlacZ

Medium Experiment C57BU6

in Mice

II-

* Splenocytes from mice 7, 14, and 21 days after administration of viruses were restimulated with UV-inactivated 24 hr. Supernatants were tested on HT-2 cells. b S.I., stimulation index; see legend to Figure 5. c Supernatants of H5.01OCBlacZ-restimulated splenocytes were tested on L929 cells for production of functional

of adenovirus-transduced hepatocytes in vivo. Activation of a virus-specific CTL response upon immunization with recombinant adenoviruses was confirmed by in vitro 51Crrelease assays. Together, these data confirm the original hypothesis that the inflammation and loss of transgene expression associated with first generation recombinant adenoviruses is caused by the development of class Irestricted CTLs to viral antigens, which rapidly destroy the genetically modified hepatocytes leading to repopulation of the liver with nontransgene-containing cells. The role of MHC class II-associated antigen presentation and consequent activation of CD4+ T cells in the biology of recombinant adenoviruses is less clear. Studies in the f&m- mouse indicate that class II expression and virus-specific CD4+ T cells are not sufficient for complete destruction of genetically modified hepatocytes in the absence of class I and functionally active CD8+ T cells. The delayed loss of transgene expression in MHC class IImice as compared with C57SU8 animals, and the incomplete but detectable diminution in IacZ expression from RAG-2- mice adoptively transferred with CD4+ T cells indicates that lymphocytes of Th subsets are capable of contributing to partial hepatocyte destruction under some experimental conditions. Splenocytes from immunized mice were analyzed in vitro using lymphokine release assays to evaluate the functional activity of CD4+ T cells. Spleen cells from immunized C57BL18 and Prim- mice secreted IL-2 and IFNy in response to stimulation with virus, consistent with the activation of virus-specific Th cells of Thl subsets. There was no detectable secretion of IL4, indicating that Th2 cells were not induced at detectable numbers. Thl cells could participate in the destruction of hepatocytes by promoting proliferation of activated CTL via secretion of IL-2; activating other effector cells such as NK cellsor macrophages through local production of IFNT; or supporting antibody-mediated lytic events, such as anti-

H5.OlOCBlacZ

at 10 pfulcell

for

IFNT.

body-dependent cell-mediated cytotoxicity (ADCC) or complement-mediated cytolysis (CMC), an unlikely explanation as the recombinant adenovirus (nonenveloped) fails to express viral antigens on the cell surface. Nevertheless, result8 in the Pem- animals illustrate the futility of the CD4+ T cell-mediated responses in the absence of CD8’ effector cells. Chronic expression of viral antigens in hepatocytes from these animals leads to massive infiltration of presumably antigen-specific CD4+ T cells that were shown at least in vitro to secrete over time, increasing the amount of IFNy without leading in vivo to the destruction of hepatocytes and loss of transgene expression. A chronic state of inflammation is established with persistence of this nonreplicating viral genome. Models of viral pathogenesis affecting other organs support the findings in this study that a highly efficacious host response to a primary virus infection is the generation of specific class l-restricted CTL to infected cells. Murine models of respiratory infections with Sendai virus and influenza virus used similar techniques to demonstrate the importance of class l-restricted CTL in the clearance of infected cells and resolution of the infection (Eichelberger et al., 1991; Hou et al., 1992). The well-characterized murine model of infection with lymphocytic choriomeningitis virus (LCMV) compares well with our model; during intracerebral infection of mice with LCMV, CD8+ T cells are assumed to be responsible for the initial pathogenic event either by direct lysis of infected cells or by the release of cytokines and a fatal delayed-type hypersensitivity (DTH) response (Doherty et al., 1990). Analysisof LCMV, Sendai, and influenza models of virus infection in mice deficient in CD4+ cells by germline inactivation, antibody depletion, or both confirms the finding in our study that an effective CD8+ CTL response can occur in the absence of CD4’ cells (Allan et al., 1990; Hou et al., 1992; Quinn et al., 1993; Rahemtulla et al., 1991). This contrasts with other

immunity 440

CD8+ CTL functions, such as responses to alloantigens, which are dependent on CD4+ cells (Rahemtulla et al., 1991; Grusby et al., 1991). In our model, MHC class l-deficient mice that are unable to develop a CD8’ T cell-mediated cytolytic response failed to clear the adenovirus-infected hepatocytes within the duration of the experiment. This is in contrast with other viral systems in which viral clearance by CD4+ T cells was observed using the same knockout mice (Eichelberger et al., 1991; Hou et al., 1992; Quinn et al., 1993). The viral clearance was explained by CD4+ T cell-mediated cytolysis of MHC class II-expressing virus-infected cells, by promoting B cell responses, or by activating other non-MHC class l-restricted cytolytic effecters such as yS T cells. Development of models for studying viral hepatitis has been difficult because most human viruses associated with hepatic pathology are uniquely tropic to human cells and are difficult to propagate in vitro. Virus-specific class l-restricted CTLs have been isolated from peripheral blood of patients with acute hepatitis B (Bertoletti et al., 1991). Evidence for the role of virus-specific CTLs in hepatocellular injury was provided in experiments in which cloned mouse CTLs to hepatitis B virus-specific antigens were adoptively transferred to mice expressing the hepatitis B virus envelope antigens under the control of mouse albumin promoter (Ando et al., 1993; Moriyama et al., 1990). In this model, antigen-specific CTLs are quickly mobilized to liver, leading to minor hepatocyte destruction; most of the destruction was caused by a DTH response elicited by IFNy secreted from the CTLs. The requirement of antigen-specific CTLs in this model is similar to the model of adenovirus-mediated gene transfer to mouse liver. However, the substantial role that IFNy-dependent DTH has in the CTL-induced destruction of hepatitis B virus expressing hepatocytes deserves further study in the model of adenovirus-mediated gene transfer to liver. The purpose of this study was to understand the cellular basis for the destruction of genetically modified cells that has been associated with the use of first generation viruses. Knowledge of the importance of virus-specific class l-restricted CTL will be useful in improving the potential of recombinant adenoviral technology for gene therapy. Experimental

Procedures

Animals C57BU6 mice were purchased from Jackson Laboratory (Bar Harbor, Maine). RAG-P-, P2rn-, and MHC class ii- mice were purchased from GenPharm internritionai (Mountain View, California). &m- and MHC class II- mice were bred onto a C57BU6 background (>5 generations)and carry the H-2b hapiotype. RAG-2- mice are in 129 background and also carry the H-2b hapiotype.

Delivery

of Recombinant

Adenoviruses

to Mouse

Liver

The viruses used in this study are based on human adenovirus type 5 sub 360 (H5d1360), in which sequences spanning Ela and El b from 1 to 9.2 map unit were deleted and replaced with a minigene cassette driven by a cytomegalovirus-enhanced B-actin promoter. H5di360 also contains a small deletion in E3b. H5.01OCBiacZ and H5.01OCBALP express Escherichia coii IacZ and human placental alkaline phosphatasegenes, respectively(Kozarskyet al., 1993). GroupsofG-to&weekold female mice were selected for this study. H501OCBiacZ (2 x 100 pfu) in 0.1 ml of phosphate-buffered saline (PBS [pH 7.41) was

administered to animals via the tail vein. When animals were necropsiecl 3, 7, 14, or 26 days later, liver tissues were prepared for paraffin and cryosections, while spleens were harvested for immunoiogicai assays. Uninfected naive mice were also sacrificed as controls. Ail animals that received recombinant virus survived to the time of necropsy.

Morphological Analyses X-Gal Histochamlstry Frozen sections (6 pm) were fixed in 0.5% giutaraidehyde and stained for B-gaiactosidase activity as described (Yang et al., 1993). Sections were counterstained in hematoxyiin.

Double Immunofluorescence and CD4’ Cells

for Detection

of CD@

Frozen sections (6 pm) were fixed in methanol as described (Yang et al., 1994b). After blocking with 10% goat serum in PBS (GS-PBS), sections were incubated with 10 pg/mi of rat anti-mouse CD4 (antiL3T4, Boehringer Mannheim) for 60 min, followed by the incubation with 5 pglml of goat anti-rat immunogiobuiin G (igG)-rhodamine for 30 min. Sections were then treated with 10 pg/mi of anti-L3T4 to block the unbound epitopes to CD4 on anti-rat IgG molecules prior to incubation of samples with 50 pg/mi of rat anti-mouse CDBa-fluorescein isothiocyanate (anti-Ly-2, Boehringer Mannheim) for 60 min. Sections were washed and mounted with the antifadent, Citifiuor (Citifluor, United Kingdom).

Pathology Paraffin sections (5 pm) were stained with hematoxyiin and eosin according to standard procedures. Random sections in a blinded fashion were evaluated for histopathoiogy using the criteria developed by Knodell et al. (1961) for describing viral hepatitis with minor modifications. Each section was evaluated according to three independent criteria, within each criterion the severity of pathology was quantified based on a scale of 0 (no pathology) to 4 (severe pathology). The three criteria included the following: periportal degeneration and focal necrosis; intralobuiar generation and focal necrosis; and periportai inflammation. Analyses were performed on three animals per time point encompassing two sections for each animal.

CTL Assay CTL assays were performed as described (Xiang et al., 1994). in brief, lymphocytes harvested from spleens were cultured for 5 days at 6 x lb ceils/well in 1.6 ml of DMEM supplemented with 5% fetal bovine serum (FBS) and 50 pM P-mercaptoethanoi in the presence of HS.OlOCBiacZ at multiplicity of infection (MOI) of 1 in 24-well Costar plates. Astandard 6 hr 51Cr release assaywas performed subsequently using different ratios of effector to target ceils (C57SV, H-29 in 200 pi DMEM supplemented with 10% FBS in V-bottomed swell plates. Prior to mixing with the effector ceils, target ceiis(1 x lmwereiabeied with 100 j&i of 5’Cr after a 24 hr infection with H5.01OCBiacZ or H5.01OCBALP at an MOI of 50 and used at 5 x lo3 cells/well. After incubation for 6 hr. aiiquots of 100 pl supernatant were removed for counting in a y-counter. Percentage of specific5’Cr release was calculated as the following: [(cpm of sample - cpm of spontaneous release) I (cpm of maximal release - cpm of spontaneous release)] x 100. Spontaneous release was determined by culturing target ceils in medium, and maximal release was established by culturing target ceils in a 1% solution of SDS. Ail sample values represent the average of quadruplicate wells; maximum (i.e., target ceils incubated with medium only) and spontaneous (i.e., target ceils incubated with 1% SDS) releases were averaged from at least 6 wells.

Cytoklne Release Assays IL-2 and IL-4 Release Assay Lymphokine release assays were performed by culturing 6 x 108 splenocytes in 1.6 ml of DMEM supplemented with 2% FBS and 50 KM Bmercaptoethanoi with or without antigen, i.e., H5.01OCBlacZ (UV-inactivated or infectious virus at various particles/ceil) in 24-well Costar plates. Ceil-free supernatants (100 pl) were transferred onto 2 x lo3 HT-2 ceils (IL-2- or iL+dependent ceils) on round-bottomed 96-well plates. Medium and 10% of rat concanavaiin A supernatant were used as negative and positive controls, respectively. Proliferation was measured 46-72 hr later by a 6 hr PH]thymidine (0.35 pCi/well)

C?&otoxic

T Cells in Adenovirus

Gene

Therapy

pulse. Data reflect the mean of quadruplicate samples. To determine whether proliferation of HT-2 cells was mediated by IL-2 or IL-4, serial dilutions (1:50, 1:500, and 1:5000) of ascites fluids containing monoclonal antibody to IL-2 (S4.66) or IL-4 (11 Bll) was added to the culture supernatants for 60 min at 4OC prior to addition of HT-2 cells (Xiang et al., 1994). IFNy Release Assay Presence of IFNy in the same splenocyte culture supernatant described above was measured using a functional assay. In brief, samples (100 pl) serially diluted in DMEM with 5% FBS were added to L929 cells grown on 96-well flat-bottomed plates at 1 x lO’cells/well. A series of standard IFNy samples and medium controls were also included. After a 24 hr incubation at 37OC. samples were removed and cells were washed once with DMEM containing 5% FBS prior to addition of 100 PI of encephalomyocarditis virus (EMC) at MOI of 0.1 in DMEM containing 5% FBS to all wells. Plates were read when medium controls (absence of IFN) showed >95% of cytopathic effect. Dilution of sample showing 50% cytopathic effect was denoted as the IFN titer. With the help of standard IFN samples, the IFN titer was converted to IUlml values. The identity of IFN was confirmed by preincubating of samples with a monoclonal antibody to IFNy (XMGI .2; Denkers et al.. 1993) which showed 100% inhibition.

tion of inflammatory 17, 55-59.

Adoptlve Transfer

Grusby, M. J., Johnson, R. S., Papaioannou, V. E., and Glimcher, L. H. (1991). Depletion of CD4+ T cells in major histocompatability complex class II-deficient mice. Science 253, 1417-1420.

Transfers of Total Splenocytes

Primed or naive splenocytes (2 x 10’ cells in 200 pl of serum-free DMEM) from C57BU6 mice were injected into the tail vein of RAG-T recipient mice. These recipients were injected intravenously with 2 x IO9 pfu of HB.OlOCBlacZ 7 days before.

Transfer

of CDS+ or CD4+ T Cell Subsets

Splenocytes were prepared from immunized C57BU6 mice, and immunoglobulin positive cells were removed by nylon wool adherence. lmmunoglobulin negative cells were incubated with fluorescein isothiocyanate- or phycoerithrin-labeled antibody to CD8 or CD4 cells (described above) according to the protocol of the manufacturer. CD4+ and CD8+ cells were subsequently separated by a fluorescence activated cell sorter (FACS). The purity of the individual populations was tested to be >98%. Purified T cell subsets (2 x 10 in 200 pl of serumfree DMEM) were then infused into the tail vein of RAG-T recipient mice inoculated with H5.01OCBlacZ 7 days before.

process

induced

by CD8+ T cells. Immunol.

Today

Eichelberger, M., Allan, W., Zijlstra, hf., Jaenisch, R., and Doherty, P. C. (1991). Clearance of influenza virus respiratory infection in mice lacking class I major histocompatability complex-restricted CD8+ T cells. J. Exp. Med. 774, 875-880. Engelhardt, J. F.. Simon, R. H., Yang, Y., Zepeda, M., WeberPendleton, S., Doranz, B., Grossman, M., and Wilson, J. M. (1993a). Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: biological efficacy study. Hum. Gene Ther. 4, 759-769. Engelhardt, J. F., Yang, Y., Stratford-Perricaudet, L. D., Allen, E.D., Kozarsky, K., Perricaudet, M., Yankaskas, J. R., and Wilson, J. M. (1993b). Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with El-deleted adenoviruses. Nature Genet. 4, 27-34. Engelhardt, J. E., Ye, X., Doranz, B., and Wilson, J. M. (1994). Ablation of E2a in recombinant adenoviruses improves transgene persistence anddecreasesinflammatoryresponse in mouseliver. Proc. Natl. Acad. Sci. USA 97, 61966200. Graham, F. L., Smiley, J., Russell, W. L., and Nairn, R. (1977). Characterization of a human cell line transformed by DNA from adenovirus 5. J. Gen. Virol. 36, 59-72.

Horwitz, M. S. (1990). Adenoviridae and their replication. In Virology, B. N. Fields, D. M. Knipe et al., eds. (New York: Raven Press, Limited), pp. 1679-l 721. Hou, S., Doherty, P. C., Zijlstra. M., Jaenisch, R., and Katz, J.M. (1992). Delayed clearance of Sendai virus in mice lacking class I MHCrestricted CDE’ T cells. J. Immunol. 149, 1319-1325. Jaffe, H. A., Danel, G., Longenecker, G., Metzger, M., Setoguchi, Y., Rosenfeld, M. A., Gant, T. W., Thorgeirsson. S. S., StratfordPerricaudet, L. D., Perricaudet, M., Pavirani, A., Lecocq, J. P., and Crystal, R. G. (1992). Adenovirus-mediated in vivo gene transfer and expression in normal rat liver. Nature Genet. 7, 372-378. Knodell, R. G., Ishak, K. G., Black, W. C., Chen, T. S., Craig, R., Kaplowitz, N., Kiernan, T. W., and Wollman, J. (1981). Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic hepatitis. Hepatology 7, 431-435.

Acknowledgments

Kozarsky, K. F., and Wilson, J. M. (1993). Gene vectors. Curr. Opin. Genet. Dev. 3, 499-503.

We wish to thank Q. Li and G. Krivulka for technical assistance, and Drs. L. Turka and Z. Xiang for helpful discussions. Support from the Vector, Cell Morphology, and Animal Models cores of the Institute for Human Gene Therapy was greatly appreciated. This work was funded by grants from the National Institutes of Health and the Cystic Fibrosis Foundation.

Kozarsky, K., Grossman, M., and Wilson, J. M. (1993). Adenovirusmediated correction of the genetic defect in hepatocytes from patients with familial hypercholesterolemia. Som. Cell Mol. Genet. 19, 449458.

Received

June

14, 1994; revised

July 7, 1994.

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