Adenoviral-mediated gene transfer in lymphocytes - PubMed (original) (raw)

Adenoviral-mediated gene transfer in lymphocytes

R P Leon et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Although adenovirus can infect a wide range of cell types, lymphocytes are not generally susceptible to adenovirus infection, in part because of the absence of the expression of the cellular receptor for the adenoviral fiber protein. The cellular receptor for adenovirus and coxsackievirus (CAR) recently was cloned and shown to mediate adenoviral entry by interaction with the viral fiber protein. We show that the ectopic expression of CAR in various lymphocyte cell lines, which are almost completely resistant to adenovirus infection, is sufficient to facilitate the efficient transduction of these cells by recombinant adenoviruses. Furthermore, this property of CAR does not require its cytoplasmic domain, consistent with the idea that CAR primarily serves as a high affinity binding site for the adenoviral fiber protein, and that viral entry is mediated by interaction of the viral penton base proteins with cellular integrins. As a demonstration of their functional utility, we used CAR-expressing lymphocytes transduced with an adenovirus expressing Fas ligand to efficiently kill Fas receptor-expressing tumor cells. The ability to efficiently manipulate gene expression in lymphocyte cells by using adenovirus vectors should facilitate the functional characterization of pathways affecting lymphocyte physiology.

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Figures

Figure 1

The expression of CAR in EL-4 cells confers susceptibility to adenovirus transduction. (A) Cells were transduced with the indicated MOI of either Ad-CON or with adenovirus expressing GFP from either a CMV, Rous sarcoma virus, or E1A promoter. After 24 hr, the live cells were analyzed by flow cytometry for the expression of GFP, as indicated by increased fluorescent intensity. The percentage of cells with fluorescence over background (denoted by C bar) is indicated.

Figure 2

Analysis of cell surface CAR expression, in vivo transduction of EL-4 CAR by adenovirus, and CAR-mediated adenovirus transduction of other lymphocyte cell lines. (A) The expression of CAR was determined on the indicated cells by incubation of the cells with RmcB monoclonal against hCAR (or no primary antibody as a control, shown as a dotted line overlay) followed by a fluorescein isothiocyanate-linked goat α-mouse IgG1 antibody. EL-4 mCAR 7 is a single cell clone from the pooled EL-4 cells expressing mCAR. (B) EL-4 CAR 13 or EL-4 LXSN cells were injected i.p. into C57BL/6 mice (5 × 106 in 0.5 ml of PBS per mouse). Seventy-two hours later, the mice were injected i.p. with 2 × 107 focus-forming units of AdCMV-GFP in 0.4 ml of PBS. The mice were sacrificed 12 hr later. EL-4 cells were removed from the peritoneum by instilling then immediately withdrawing 5 ml of PBS injected into the peritoneum cavity with a 22-gauge needle. These cells were stained with Cy-chrome linked anti-CD3 (PharMingen 01088A), and then analyzed by flow cytometry. Cells gated for both size (as determined for uninjected EL-4 cells) and CD3 expression were analyzed for GFP expression as shown in B. The percentage of fluorescent cells (F bar) is indicated. (C) FL5.12, DPK.C7, and the hCAR expressing derivatives of either of these cell lines were transduced with either Ad-CON or AdCMV-GFP at the indicated MOIs and analyzed by flow cytometry, as described in Fig. 1. The percentage of fluorescent cells (C bar) is indicated.

Figure 3

Adenoviral transduction is integrin dependent, but does not require the cytoplasmic domain of CAR. (A) Either EL-4 LXSN or EL-4 CAR 13 cells (105 cells per 0.1 ml in 96-well plates) were incubated for 1 hr at 37°C with 0 (Con), 0.1 (Lo), or 0.5 (Hi) mg/ml of GRGDS peptide (Sigma G4391) in 2% FBS in RPMI. AdCMV-GFP was added at an MOI of 5, and incubation continued for an additional hour at 37°C. Eight microliters of FBS then was added, and the cells were cultured at 37°C for 24 hr and analyzed on a flow cytometer. The percentage of fluorescent cells (C bar) is indicated, and the average fluorescent intensity of all of the cells is shown below the percentage. (B) Three different C-terminally truncated mutants of the hCAR receptor were generated by PCR amplification using internal primers, which resulted in a stop codon immediately following the indicated residue. The predicted transmembrane domain (TM) encompasses residues 236–258, as illustrated in the schemata (Upper Right). LXSN retroviruses encoding the CAR Δ mutants (or the LXSN and LXSN-hCAR as control viruses) were used to transduce EL-4 cells, and pooled NeoR cells were selected. The CAR Δ1 6 cell clone was derived from a single cell sorted from the highly fluorescent cells resulting from AdCMV-GFP transduction of the CAR Δ1 pool. The indicated cells were transduced with AdCMV-GFP at an MOI of either 200 (CAR Δ1 6 Hi) or 20 (all others) and analyzed by flow cytometry as described in Fig. 1. The percentage of fluorescent cells (C bar) is indicated.

Figure 4

EL-4-CAR cells transduced with an adenoviral FasL effectively kill FasR-expressing target cells. EL-4-CAR 13 cells were transduced (MOI of 100) with Ad-FasL, AdCMV-GFP, or no virus (control). The cells then were washed once and coincubated for 20 hr with 51Cr-labeled L1210-FasR cells at a 5:1 or 0.5:1 effector/target ratio. Each value represents the mean of triplicate measurements ± SD.

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Adenoviral-mediated gene transfer in lymphocytes - PubMed (original) (raw)