Cytotoxic T-lymphocyte target proteins and their major histocompatibility complex class I restriction in response to adenovirus vectors delivered to mouse liver

Abstract

The activation of cytotoxic T lymphocytes (CTLs) to cells infected with adenovirus vectors contributes to problems of inflammation and transient gene expression that attend their use in gene therapy. The goal of this study was to identify in a murine model of liver gene therapy the proteins that provide targets to CTLs and to characterize the major histocompatibility complex (MHC) class I restricting elements. Mice of different MHC haplotypes were infected with an E1-deleted adenovirus expressing human alkaline phosphatase (ALP) or beta-galactosidase as a reporter protein, and splenocytes were harvested for in vitro CTL assays to aid in the characterization of CTL epitopes. A library of vaccinia viruses was created to express individual viral open reading frames, as well as the ALP and lacZ transgenes. The MHC haplotype had a dramatic impact on the distribution of CTL targets: in C57BL/6 mice, the hexon protein presented by both H-2Kb and H2Db was dominant, and in C3H mice, H-2Dk-restricted presentation of ALP was dominant. Adoptive transfer of CTLs specific for various adenovirus proteins or transgene products into either Rag-I or C3H-scid mice infected previously with an E1-deleted adenovirus verified the in vivo relevance of the adenovirus-specific CTL targets identified in vitro. The results of these experiments illustrate the impact of lr gene control on the response to gene therapy with adenovirus vectors and suggest that the efficacy of therapy with adenovirus vectors may exhibit considerable heterogeneity when applied in human populations.

FIG. 1

Identification of target proteins for CTLs in C57BL/6 mice in response to adenovirus vectors. Spleens from three C57BL/6 mice injected with ALP-expressing adenovirus were restimulated in vitro for 5 days with ALP-expressing adenovirus. Effector cells were tested on MHC-compatible target cells (C57SV [H-2^b]), which had been infected with either ALP-expressing adenovirus or recombinant vaccinia virus and loaded with ⁵¹Cr. Each graph shows the percent specific lysis assayed at four different effector/target cell ratios. (A) Target cells were infected with ALP-expressing adenovirus (AdALP) or mock infected. (B) Target cells were infected with recombinant vaccinia virus expressing the ALP transgene or a control vaccinia virus expressing rabies glycoprotein (VRG). (C and D) Target cells were infected with ORF-specific vaccinia viruses expressing the early adenovirus proteins DNA-Pol, pTP, and DBP (C) or the late adenovirus proteins hexon, penton, and fiber (D). The VRG virus was used as a negative control for target cells expressing various recombinant vaccinia viruses in panels C and D. These data are from one of three experiments done. The standard deviations were less than 10%. Spontaneous release ranged from 10 to 15%.

FIG. 2

Comparison of CTL targets in C57BL/6 mice infected with wild-type or E1-deleted adenovirus. Splenocytes isolated on day 7 after infection of C57BL/6 mice with wild-type adenovirus (Ad5wt) (10⁹ particles per injection) (B and D) or E1-deleted adenovirus expressing ALP (AdALP) (10¹¹ particles per injection) were analyzed for specific lysis on target cells infected with recombinant vaccinia virus expressing hexon, penton, fiber, or a rabies glycoprotein (C and D) or on target cells infected with AdALP or Ad5wt (A and B) in a 6-h ⁵¹Cr release assay. Specific lysis was assayed at five different effector/target cell ratios. These data are from one of two experiments done. The standard deviations were less than 10%. Spontaneous release ranged from 10 to 15%.

FIG. 3

Identification of the MHC restricting element responsible for the recognition of hexon within adenovirus-infected C57BL/6 mice. Spleens from three adenovirus-infected C57BL/6 mice were harvested on day 7, and isolated splenocytes were used as effector cells after 5 days of in vitro restimulation. MHC congenic cells differentiating the K and D alleles within the H-2^b locus were used as target cells: C57SV cells (H-2K^bD^b) (A and B), KD2SV cells (H-2K^dD^d) (C and D), K5RSV (H-2K^bD^d) (E and F), and HTGSV (H-2K^dD^b) (G and H). For the determination of the MHC restricting element for E1-deleted adenovirus, the target cells were infected with ALP-expressing adenovirus (AdALP) or mock infected (negative control) (left panels). Target cells were also infected with recombinant vaccinia virus expressing hexon (VaccHexon) or recombinant vaccinia virus VRG expressing the rabies glycoprotein (negative control) (right panels). These data are from one of the two experiments done. The standard deviations were less than 10%. Spontaneous release values were 10% for C57SV and KD2SV cells and less than 18% for HTGSV and K5RSV cells.

FIG. 4

Adoptive transfer of primed splenocytes to congenic Rag-I mice infected i.v. with H5.010CMVlacZ. Splenocytes from C57BL/6 mice infected with recombinant vaccinia virus expressing the adenovirus hexon protein (D panels), fiber protein (E panels), or penton protein (F panels) or the lacZ transgene product (G panels) were isolated 7 days after infection, and cells (5 × 10⁷) were infused into the tail veins of Rag-I mice previously infected with H5.010CMVlacZ (10⁹ PFU). Liver tissues were analyzed for lacZ expression 12 days after the adoptive transfer by X-Gal histochemistry (upper panel of each pair of panels) or for the presence of CD8⁺ (FITC) and CD4⁺ (rhodamine) lymphocytes by double immunofluorescence (lower panel of each pair of panels). Control animals did not receive any splenocytes (A) or received splenocytes from naive C57BL/6 mice (B) or splenocytes from mice infected with recombinant vaccinia virus expressing the rabies virus glycoprotein (C).

FIG. 5

Identification of target proteins for CTLs in H-2^d mice. Splenocytes from three adenovirus-infected BALB/c mice were assayed on H-2^d-restricted target cells (KD2SV cells [H-2K^dD^d]). See the legend to Fig. 1 for the target cells used. These data are from one of three experiments done. The standard deviations were less than 13%. Spontaneous release ranged from 10 to 15%.

FIG. 6

Determination of the MHC restricting element for the presentation of hexon to CTLs in H-2^d mice infected with ALP-expressing adenovirus. Splenocytes from three adenovirus-infected BALB/c mice were assayed on various target cells differentiating the K and D determinants within the H-2^d haplotype. The following target cells were used in this study: KD2SV (H-2K^dD^d) (A and B), C57SV (H-2K^bD^b) (C and D), K5RSV (H-2K^bD^d) (E and F), and HTGSV (H-2K^dD^b) (G and H). The left panels show target cells infected with the ALP-expressing adenovirus (AdALP) or mock-infected cells. The right panels show results of target cells infected with recombinant vaccinia virus expressing the hexon transgene (VaccHexon) or with the recombinant vaccinia virus VRG used in all of the experiments as a negative control for recombinant vaccinia virus-infected cells. These data are from one of two experiments done; the standard deviations varied between 10 and 12%. In all of the target cells, spontaneous release was under 20%.

FIG. 7

Targets for CTLs in C3H mice infected with E1-deleted adenovirus expressing the ALP transgene. C3H mice (H-2^k) were injected with E1-deleted adenovirus expressing the ALP transgene. In each experiment, splenocytes from three spleens were pooled and used as effector cells. L929 cells (H-2^k) were used as target cells. See the legend to Fig. 1 for the target cells used. The data are from one of four experiments done; the standard deviations were less than 15%. Spontaneous release ranged from 8 to 12%.

FIG. 8

Identification of the MHC restricting element for ALP-derived epitopes in adenovirus-infected C3H mice. Splenocytes from three C3H mice infected with E1-deleted adenovirus expressing the ALP protein were isolated on day 7 after virus infection and restimulated in vitro for 5 days with ALP adenovirus. These effector cells were tested on target cells that differentiate H-2K^k and H-2D^k haplotypes. The MHC congenic target cell lines used were L929 cells (H-2K^kD^k) (A and B), KD2SV cells (H-2K^dD^d) (C and D), K2RSV cells (H-2K^kD^b) (E and F), and LC3H.OHSV (H-2K^dD^k) (G and H). Specific lysis was determined on target cells infected with adenovirus expressing the ALP protein (AdALP) (left panels) or recombinant vaccinia virus expressing the ALP protein (VaccALP) (right panels). Mock-infected cells were used as a negative control for adenovirus-infected target cells, whereas target cells infected with the recombinant vaccinia virus VRG expressing the rabies virus glycoprotein were used as a negative control for target cells infected with recombinant vaccinia virus. These data are from one of three experiments done; the standard deviations ranged from 8 to 12%, depending on the target cells. Spontaneous release was less than 10% for L929 and KD2SV cells, whereas it was 15 to 20% for K2RSV and LC3H.OHSV cells.

FIG. 9

Identification of proteins which activate CTLs in BALB/k mice (H-2^k) infected with E1-deleted adenovirus. Splenocytes were isolated from three BALB/k mice which had been previously infected with E1-deleted adenovirus, and CTL assays were performed with L929 cells as targets. See the legend to Fig. 1 for the target cells used. Spontaneous release of L929 cells was less than 10%. These data are from one of two experiments done; the standard deviations ranged from 10 to 14%.

FIG. 10

Adoptive transfer of primed splenocytes into C3H-scid mice infected i.v. with H5.010CBALP. Splenocytes (5 × 10⁷ cells) isolated from C3H mice infected with recombinant vaccinia virus expressing the ALP transgene (C panels) or the adenovirus hexon protein (D panels), penton protein (E panels), or fiber protein (F panels) were adoptively transferred into C3H-scid animals previously infected with H5.010CBALP (10⁹ PFU). Frozen liver tissues were evaluated for ALP expression 12 days later by ALP histochemistry (upper panel of each pair of panels) or for CD8⁺ (FITC-labeled) and CD4⁺ (rhodamine-labeled) infiltration by double immunofluorescence (lower panel of each pair of panels). Control animals either did not receive any splenocytes (A panels) or received naive splenocytes (B panels).