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The development of a host cytotoxic T-lymphocyte (CTL) response against adenovirus (Ad) proteins as well as immunogenic transgene products has been implicated in the transience of expression from Ad vectors in vivo (10, 11, 23). Several approaches have been explored to circumvent the CTL response, including immunosuppressive treatments (9, 12, 17) or modification of Ad vectors to reduce expression or simply delete CTL epitopes from the viral genome (3, 5, 6). In this study, a series of experiments were conducted to define further the relationship between Ad vector backbone and susceptibility to lysis by Ad-specific CTLs in vitro. Female C57BL/6 mice, 6 to 8 weeks of age (Taconic, Germantown, N.Y.), were treated with wild-type (wt) Ad and/or Ad vector to induce a CTL response. Spleen cells were restimulated in vitro with syngeneic SVB6KHA fibroblasts (gift from Linda Gooding, Emory University, Atlanta, Ga.) infected with E3-deleted wt Ad to expand virus-specific CTLs but not CTLs directed against the transgene product, when present (11). Cultures consisted of 5 × 10⁶ spleen cells incubated with 6 × 10⁴ mitomycin C-inactivated fibroblasts in the wells of a 24-well plate in 2 ml of RPMI 1640 medium supplemented with 100 U of penicillin per ml, 100 µg of streptomycin per ml, 2 mM glutamine, 5 × 10⁵ M 2-mercaptoethanol, and 10% heat-inactivated fetal calf serum (HyClone Laboratories, Inc., Logan, Utah). After 5 to 7 days of culture, effector cells were recovered and tested against target fibroblasts infected with a variety of Ad vectors in a standard chromium release assay (11). Effector cells generated in this manner displayed major histocompatibility complex (MHC)-restricted killing as effector cells from C57BL/6 mice were able to lyse infected C57BL/6 fibroblasts but not infected allogeneic BALB/c fibroblasts. All viral vectors were produced and purified, and their titers were determined, by the Virus Production Unit of Genzyme Corporation as described previously (1, 16). The Ad5-based vector constructs (Ad5/AAT and Ad5/Gal) were provided by Arthur Beaudet (Baylor College of Medicine, Houston, Tex.). Figure 1 depicts the structure of vectors used in our study.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Genomic structure of Ad vectors. The expression cassette replacing the E1 region is depicted on the left, and variations of the E3 and E4 regions are depicted on the right. The vectors contain a CMV promoter element (523 to 14) to drive -Gal or CFTR expression. Ad2/Gal-2, -4, -5, -7, and -8 all contain a wt E3 region. Ad2/Gal-4 also contains a wt E4 region, while Ad2/Gal-5 contains a complete E4 deletion. Ad2/Gal-2, -7, and -8 have deletions in E4 but retain ORF6, ORF4, and ORF6 and ORF6,7, respectively. Ad2/Gal/E32.9, Ad2/Gal/CMV-14.7K, and Ad2/CFTR-16 all contain wt E4 regions. Ad2/Gal/E32.9 has deletions of all E3 coding sequences (nucleotides 27971 to 30937 deleted) while Ad2/CFTR-16 retains E3 gp19K (nucleotides 29292 to 30840 deleted). The E3 region of Ad2/Gal/CMV-14.7K was deleted (nucleotides 27971 to 30937 deleted) for insertion of E3 14.7K under the control of a CMV promoter as depicted above. SV40, simian virus 40.

Results from several studies first indicated that cells infected with E1-deleted Ad vectors containing intact E3 and E4 regions (E3⁺ E4⁺) were relatively resistant to lysis by Ad-specific CTLs. Results from two such studies are shown in Fig. 2. It was found consistently that spleen cells from mice that had developed high levels of CTL activity against Ad, as evidenced by the lysis of target cells infected with E3-deleted wt virus, exhibited only weak lysis of target cells infected with various E3⁺ E4⁺ Ad vectors. This observation applied to Ad vectors with different serotypes (Ad2 and Ad5), encoding different transgenes (1-antitrypsin [AAT], -galactosidase [-Gal], and cystic fibrosis transmembrane conductance regulator [CFTR]), under the control of different promoters (phosphoglyceride kinase, cytomegalovirus [CMV], and E1a). A similar phenomenon was also observed in BALB/c mice (data not shown). Therefore, the observed resistance to in vitro lysis by Ad-specific CTLs was not limited to the C57BL/6 strain and appeared to be a property shared by E3⁺ E4⁺ Ad vectors in general.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Weak CTL lysis of target cells infected with Ad vectors containing intact E3 and E4 regions. Results from two separate experiments are shown. (A) A group of six C57BL/6 mice was treated intravenously with 2 × 1010 IU of a human AAT-encoding Ad vector (Ad5/PGK-AAT) on day 0 along with intranasal delivery of 109 IU of a -Gal-encoding vector (Ad5/CMV-Gal) on days 0 and 20 followed by an intravenous challenge with 109 IU of an Ad2 vector lacking a transgene on day 49. The animals were sacrificed on day 99, and spleen cells were pooled and stimulated in vitro with syngeneic fibroblasts infected with E3-deleted wt Ad5 (Ad52.9) to selectively expand virus-specific CTLs. The effector cells generated were tested against syngeneic fibroblasts that were either uninfected or infected with Ad52.9, Ad5/PGK-AAT, or Ad5/CMV-Gal. (B) A group of four C57BL/6 mice was instilled intranasally with 109 IU of wt Ad2, and spleens were collected 10 days later. Pooled splenocytes were stimulated in vitro with syngeneic fibroblasts infected with E3-deleted wt Ad2 (Ad22.9) and were then tested against target fibroblasts that were either uninfected or infected with Ad22.9, the -Gal-encoding Ad2/CMV-Gal-4 vector, or the CFTR-encoding Ad2/E1a-CFTR-11 vector. Percent lysis values shown represent the means of triplicate wells. E, effector; T, target.

Importantly, this anticytolytic effect was observed even in the presence of gamma interferon (IFN-), a cytokine which is induced by Ad vectors in vivo and is known to upregulate MHC class I expression leading to enhanced presentation of antigen to CTLs (22). As illustrated in Fig. 3, in the absence of IFN-, Ad-specific CTLs demonstrated only weak lysis of target cells infected with wt Ad. This phenomenon has previously been described and is attributed to downregulation of MHC class I expression by the E3 19,000-molecular weight glycoprotein (gp19K protein) (21). Treatment with IFN- was capable of counteracting this effect and of increasing lysis of fibroblasts infected with wt virus. In contrast, IFN- treatment of target cells infected with an E3⁺ E4⁺ vector failed to augment lysis by virus-specific CTLs. In fact, all studies presented here (Fig. 2 to 5) were conducted with target cells exposed to 100 U of recombinant mouse IFN- (Genzyme Corporation, Boston, Mass.) per ml for approximately 24 h prior to the assay. wt Ad and Ad2/Gal-4 vector both possess intact E3 and E4 regions, and the reason for the differential lysis of infected target cells following treatment with IFN- is unclear. One possibility is that the presence of the E1 region in wt Ad leads to comparatively high levels of expression of CTL target epitopes such as E1a and late viral protein determinants (10, 15), which, in combination with increased MHC class I expression by IFN-, may result in increased CTL recognition. By comparison, IFN--treated target cells infected with E1-deleted vectors may remain less susceptible to CTL lysis because of their lack of E1 target determinants and low levels of late viral protein expression.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Inability of IFN- to reverse protection from CTL lysis in target cells infected with Ad vectors containing intact E3 and E4 regions. A group of five C57BL/6 mice was instilled intranasally with 109 IU of wt Ad2, and spleens were collected 17 days later. Pooled splenocytes were stimulated in vitro with syngeneic fibroblasts infected with E3-deleted wt Ad2 (Ad22.9) and were then tested against target fibroblasts that were infected with wt Ad2 or the E3+ E4+ Ad2/Gal-4 vector with or without 24 h of exposure to IFN-. The background lysis of uninfected fibroblasts with or without exposure to IFN- was between 3.3 and 12.7% over the range of effector/target (E:T) ratios tested. Percent lysis values shown represent the means of triplicate wells.

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Elements from both the E3 and the E4 regions are required for protection against Ad-specific CTLs. A group of four C57BL/6 mice was instilled intranasally with 109 IU of wt Ad2, and spleens were collected 10 days later. Pooled splenocytes were stimulated in vitro with syngeneic fibroblasts infected with E3-deleted wt Ad2 (Ad22.9) and were then tested against target fibroblasts that were either uninfected or infected with -Gal-encoding vectors that either contained intact E3 and E4 regions (Ad2/Gal-4) or had deletions of E3 (Ad2/Gal-E32.9) or E4 (Ad2/Gal-5). Percent lysis values shown represent the means of triplicate wells. E, effector; T, target.

View larger version (23K): [in this window] [in a new window]   FIG. 5.   Identification of E3 and E4 elements involved in resistance to CTL lysis. (A) E4 elements. A group of six C57BL/6 mice was instilled intranasally with 109 IU of wt Ad2, and spleens were collected 10 days later. Splenocytes were stimulated in vitro with syngeneic fibroblasts infected with E3-deleted wt Ad2 (Ad22.9) and were then tested against target fibroblasts that were either uninfected or infected with matched E3+ vectors that differed only in the E4 region as outlined below panel A. (B) E3 elements. A group of five C57BL/6 mice was instilled intranasally with 109 IU of wt Ad2, and spleens were collected 24 days later. Splenocytes were stimulated in vitro with syngeneic fibroblasts infected with E3-deleted wt Ad2 (Ad22.9) and were then tested against target fibroblasts that were either uninfected or infected with E4+ vectors whose backbone differed in the E3 region as outlined below panel B. Percent lysis values shown represent the means of triplicate wells. E, effector; T, target.

Elements from both the E3 and the E4 regions appeared to be required for Ad vectors to convey resistance of infected cells to CTL lysis. As demonstrated in Fig. 4, deletion of one region or the other led to a significant increase in susceptibility of target cells to lysis by Ad-specific CTLs. The role of E3-encoded proteins in the protection of wt Ad from host immune responses is well documented, but the requirement for E4 was unexpected since no immunologically related activities had yet been ascribed to E4-encoded proteins. The exact function of E4, in this context, remains to be determined, but the construction of vectors expressing individual open reading frames (ORFs) from the E4 region allowed us to identify E4 elements involved in protection from CTL lysis. In Fig. 5A, virus-specific CTLs were tested against target fibroblasts infected with a series of Ad vectors possessing an intact E3 region in conjunction with a wt E4 region or only ORF6, ORF4, or ORF6,6/7 from the E4 region (1, 2). As observed previously, target cells infected with the E3⁺ E4⁺ vector showed resistance to cytolysis. Interestingly, E4ORF4 alone was sufficient to obtain potent protection against CTL lysis. Significant anticytolytic activity was also achieved with the vector expressing E4ORF6,6/7 although the effect was typically not as pronounced as that observed with ORF4. Finally, ORF6 from the E4 region failed to provide protection from lysis, suggesting that ORF6/7 from the ORF6,6/7-encoding Ad vector was likely to be responsible for the anticytolytic activity obtained. However, the possibility of a required interaction between ORF6 and ORF6/7 cannot be ruled out.

An element(s) from the E3 region was also required to achieve anti-CTL protection with E4-containing vectors. The involvement of known E3-encoded proteins was assessed by testing virus-specific CTLs against target cells infected with Ad vectors containing an intact E4 region along with either the entire E3 region; the E3a region, which encompasses the gp19K coding sequence; or only the E3 14.7K-encoding region under the control of a CMV promoter (18). The E3 gp19K protein is known to bind and retain nascent MHC class I molecules within the endoplasmic reticulum, resulting in decreased levels of surface MHC molecules available for antigen presentation, while the 14.7K protein has been reported to protect wt Ad-infected cells from tumor necrosis factor alpha- and FasL-mediated lysis, two potential pathways of CTL killing (4, 21). Expression of E3 gp19K alone offered partial protection but was not sufficient to attain the level of resistance achieved with a vector containing all of E3 (Fig. 5B). This finding suggests that an E3 protein(s) other than gp19K may also play a role in resistance of Ad vectors to CTL lysis. The E3 14.7K protein, in itself, did not confer any detectable protection from CTL lysis, suggesting that it is unlikely to play a major role (Fig. 5B). Another possibility is the involvement of the E3 10.4K-14.5K protein complex, which has also been implicated in protection from tumor necrosis factor alpha- and Fas-mediated killing (7, 19, 20).

The anticytolytic activity of E4 gene products described here is a novel finding, and the mechanism(s) involved remains to be defined. The 14K protein encoded by E4ORF4 is known to interact with protein phosphatase 2A (PP2A), resulting in downregulation of junB transcription (13). However, it appears unlikely that the anticytolytic activity of E4ORF4 is mediated through PP2A, since we were unable to reverse protection from CTL lysis in target cells exposed to okadaic acid, a specific inhibitor of PP2A and PP1 (data not shown). E4ORF6/7 codes for a 17K protein that activates the cellular transcription factor E2F (8), but it remains to be determined whether this activity contributes to the anticytolytic effect observed here.

Finally, the nature of the interaction between the E3 and E4 region proteins involved in in vitro protection from CTL lysis also remains unclear. E4-encoded proteins do not appear to act solely through upregulation of E3 protein expression, since immunoprecipitation of gp19K and 14.7K in cells infected with an Ad/E3⁺ E4⁺ vector or an Ad/E3⁺ E4 vector produced bands of similar intensity (data not shown). In addition, levels of surface MHC class I expression measured by fluorescence-activated cell sorting analysis were also found to be equivalent in cells infected with E3⁺ Ad vectors that contained an intact E4 region or had a complete deletion of E4 (data not shown). Interestingly, expression of E4ORF4 alone in rodent cells has been reported to induce p53-independent apoptosis (14), and it may be that the role of E3 proteins in our system is to counteract this particular pathway. Although further investigation will be necessary to elucidate the mechanisms involved, our findings have uncovered a previously undescribed role for E4 proteins in in vitro protection of Ad vectors from CTL lysis.