Distinct roles of adenovirus vector-transduced dendritic cells, myoblasts, and endothelial cells in mediating an immune response against a transgene product

Abstract

Adenovirus-mediated gene delivery via the intramuscular route efficiently promotes an immune response against the transgene product. In this study, a recombinant adenovirus vector encoding beta-galactosidase (Ad beta Gal) was used to transduce dendritic cells (DC), which are antigen-presenting cells, as well as myoblasts and endothelial cells (EC), neither of which present antigens. C57BL/6 mice received a single intramuscular injection of Ad beta Gal-transduced DC, EC, or myoblasts and were then monitored for anti-beta-galactosidase (anti-beta-Gal) antibody production, induction of gamma interferon-secreting CD8(+) T cells, and protection against melanoma tumor cells expressing beta-Gal. While all transduced cell types were able to elicit an antibody response against the transgene product, the specific isotypes were distinct, with exclusive production of immunoglobulin G2a (IgG2a) antibodies following injection of transduced DC and EC versus equivalent IgG1 and IgG2a responses in mice inoculated with transduced myoblasts. Transduced DC induced a strong ex vivo CD8(+) T-cell response at a level of 50% of the specific response obtained with the Ad beta Gal control. In contrast, this response was 6- to 10-fold-lower in animals injected with transduced myoblasts and EC. Accordingly, only animals injected with transduced DC were protected against a beta-Gal tumor challenge. Thus, in order to induce a strong and protective immune response to an adenovirus-encoded transgene product, it is necessary to transduce cells of dendritic lineage. Importantly, it will be advantageous to block the transduction of DC for adenovirus-based gene therapy strategies.

FIG. 1.

In vitro transduction of DC, myoblasts, and EC with AdβGal. (A) Transduction efficiency of AdβGal on DC at MOI of 10, 100, 250, 500, and 1,000. Transduction was assayed by β-Gal expression 48 h after vector exposure. (Error bars, standard errors of the means; n = 2). (B) Myoblasts and EC, 48 h after infection with AdβGal at 10 and 50 MOI, respectively. Cells were stained with X-Gal (original magnification, ×50). (C) Immunophenotype of primary DC before (DC) and after (DC/Ad) adenovirus exposure. Flow cytometry analyses of CD11c, MHCII (I-A), and B7.2 (CD86) cell surface molecules were performed 24 h after exposure to adenovirus vector (MOI, 500). Isotype controls are presented as negative controls. Data are representative of results obtained from two independent experiments. (D) Immunophenotype of EC 48 h after AdβGal infection (MOI, 10). Isotype controls are presented as a negative control.

FIG. 1.

In vitro transduction of DC, myoblasts, and EC with AdβGal. (A) Transduction efficiency of AdβGal on DC at MOI of 10, 100, 250, 500, and 1,000. Transduction was assayed by β-Gal expression 48 h after vector exposure. (Error bars, standard errors of the means; n = 2). (B) Myoblasts and EC, 48 h after infection with AdβGal at 10 and 50 MOI, respectively. Cells were stained with X-Gal (original magnification, ×50). (C) Immunophenotype of primary DC before (DC) and after (DC/Ad) adenovirus exposure. Flow cytometry analyses of CD11c, MHCII (I-A), and B7.2 (CD86) cell surface molecules were performed 24 h after exposure to adenovirus vector (MOI, 500). Isotype controls are presented as negative controls. Data are representative of results obtained from two independent experiments. (D) Immunophenotype of EC 48 h after AdβGal infection (MOI, 10). Isotype controls are presented as a negative control.

FIG. 2.

β-Gal-specific antibody titers in mice immunized with AdβGal or with AdβGal-transduced muscle cells. Mice received one i.m. injection with either 10⁹ PFU of AdβGal (Ad) or 5 × 10⁴ AdβGal-expressing DC (DC/Ad), myoblasts (Myo/Ad), or EC (EC/Ad). Mice injected with untransduced DC, myoblasts, or EC (DC, Myo, or EC) were used as negative controls. Mice were bled once a week, and β-Gal-specific antibody titers were measured by ELISA. (A) Generation of β-Gal-specific IgG antibodies as a function of time. (B) Levels of IgG1- and IgG2a-specific antibodies at day 42. (C) Kinetics of IgG1 and IgG2a β-Gal-specific antibodies after immunization with AdβGal-transduced DC.

FIG. 3.

IFN-γ-secreting cells in mice immunized with AdβGal-transduced DC, EC, or myoblasts. The number of β-Gal peptide (I8V)-specific CD8⁺ T cells was determined directly ex vivo by an IFN-γ-specific ELISPOT assay. The number of IFN-γ SFC was determined after a 24-h incubation of PBMC pools with RMAS cells alone or pulsed with the I8V peptide. Data represent averages from duplicate or triplicate wells. (A) The frequency of IFN-γ SFC in mice injected with AdβGal-transduced muscle cells was compared, at 42 days postimmunization, with that in mice injected directly with AdβGal. (B) Numbers of IFN-γ SFC in mice injected with AdβGal directly (Ad), AdβGal-transduced DC (DC/Ad), and untransduced DC (DC) at days 11 and 40 postinoculation. Results are expressed as IFN-γ SFC per 10⁶ PBMC and are calculated as the differences between the numbers of spots observed in the presence and absence of β-Gal peptide I8V.

FIG. 4.

β-Gal-specific CD8⁺ T-cell effectors in AdβGal-immunized C57BL/6 mice. Splenocytes were harvested 13 days after inoculation, and CD8⁺ cells were depleted by MACS. Both CD8⁺ and CD8⁻ populations were collected. (A) CD8⁺ cell surface molecules were analyzed by flow cytometry before and after cell sorting. Staining with a nonspecific isotype control antibody is shown in each histogram. (B) The number of IFN-γ SFC was determined in the presence or absence of the β-Gal peptide I8V. Splenocytes from CD8⁺ and CD8⁻ fractions collected from immunized or control mice were plated at a graded density starting at 2 × 10⁵ cells/well, whereas unsorted splenocytes were plated at a graded density starting at 1 × 10⁶ cells/well. Data are means of duplicate samples.

FIG. 5.

Protective immunity against tumor challenge. C57BL/6 mice (six to seven per group) were immunized i.m. with either AdβGal (squares), AdβGal-transduced cells (triangles), or untransduced cells (circles). Forty-two days postimmunization, mice were challenged s.c. with 5 × 10⁴ B16-LacZ cells, and tumor formation was monitored every 2 days for 30 days. (A) Percent tumor-free mice. In order to facilitate the reading of the graphs, only points that determine the aspect of the curves were indicated, rather than each time point. (B) Tumor volume measured in cubic centimeters. At each time point represented, tumor sizes of all mice in the group were added up. Results are shown as mean tumor sizes ± standard errors of the means.

FIG. 6.

Histology of melanoma tumors in B16-LacZ-challenged mice. (A) B16-LacZ cells in culture after X-Gal staining (original magnification, ×50). (B) Tumor isolated from B16-LacZ-challenged mice previously vaccinated with AdβGal-transduced myoblasts (original magnification, ×360). The tissue section was stained with X-Gal and counterstained with neutral red. Black deposits correspond to melanin.

FIG. 7.

IFN-γ-secreting cells in BDF1 mice. F₁ (C57BL/6 × DBA2) mice were immunized as previously described with C57BL/6-transduced DC. The number of CD8⁺ T cells with a specificity for the I8V or T9L β-Gal peptide was directly determined ex vivo by an IFN-γ-specific ELISPOT assay 19 days after immunization. The number of IFN-γ SFC was counted after 24 h of stimulation. Results shown are mean numbers of IFN-γ-producing PBMC ± standard errors of the means from duplicate wells.