Enhanced transduction and replication of RGD-fiber modified adenovirus in primary T cells

Abstract

Background: Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR). T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD).

Methodology/principal finding: A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35-45% of splenic T cells were transduced by Ad-RGD.

Conclusions: Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin.

Figure 1. β3 and β5 integrin expression on primary murine T cells.

(A) Flow cytometry profile of β3 and β5 integrin expression on naïve and activated primary murine T cells. Left panel shows histograms for β3 integrins on CD4⁺ (top) and CD8⁺ T cells (bottom). Right panel shows β5 integrins on CD4⁺ and CD8⁺ T cells. (B) Bar diagram representation of β3 and β5 integrin expression on T cells. Top panel shows integrin expression on CD4⁺ T cells while bottom panel represents CD8⁺ T cells. White bars indicate naïve T cells and black bars are activated T cells. Error bars indicate mean ± SD. (*p<0.05)

Figure 2. Primary murine CD8+ T cells can be transduced with RGD-modified adenovirus.

(A) Primary mouse T cells were treated for 48 h with replication-deficient fiber-modified adenoviruses encoding luciferase gene. Luciferase activity was measured from lysates of treated cells and is represented as relative luciferase unit (RLU) per milligram of cell protein. Cells transduced with RGD-modified adenovirus (AdRGD-Luc) showed maximum luciferase activity. (*p<0.05) (**p<0.001). (B) Flow cytometric dot-plots showing mean fluorescence intensity of viral hexon proteins expressed by transduced T cells. Gates were drawn by excluding the mock-transduced cells (Mock) represented in the left panel. T cells that were transduced with oncolytic replication-competent adenovirus with wild type fibers (Ad-WT) are represented in the middle panel while cells transduced with RGD-fiber modified oncolytic adenovirus (Ad-RGD) are shown in right panel. (C) Bar diagrammatic representation of viral hexon MFI in adenovirus induced murine T cells. Ad-RGD transduced T cells expressed 2.6-fold more viral hexon proteins in comparison to Ad-WT transduced cells (*p<0.01). Error bars indicate mean ± SD.

Figure 3. Evidence of adenovirus replication in Ad-RGD transduced T cells.

(A) Increased adenoviral E1A gene copy number in Ad-RGD treated cells (black bar) in comparison to Ad-WT treated cells (white bar) at different time-points after virus transduction calculated by qPCR. 24-fold higher viral E1A gene copy number was observed in Ad-RGD treated cells versus Ad-WT transduced cells at 24 h time-point (*p<0.001). (B) Bar diagram of flow-cytometric analysis showing percentage of T cells expressing viral hexon antigen at different time-points after virus transduction. Ad-RGD transduced T cells are represented by black bars and Ad-WT treated cells by white bars. (*p<0.0001; ns not significant). (C) Viral progeny was measured from cell lysate (cell-associated; upper panel) and culture supernantant (cell-free; lower panel) of adenovirus infected T cells. 16- to 21-fold increase in viral progeny was measured in T cell lysates and 64- to 77-fold higher viral progeny was observed in cell-free fractions of Ad-RGD treated cells (black bars) when compared to cells treated with Ad-WT (white bars) at different time-points after virus transduction (p<0.001; ND not detected). (D) Viability of virus-transduced T cells was assessed by Annexin/7AAD exclusion method. Frequencies of Annexin/7AAD double negative cells observed in flow-cytometry were plotted in a bar diagram. 1.8-fold and 2.8-fold higher death of Ad-RGD transduced T cells (black bars) was observed at 48 and 72 h after virus transduction respectively, when compared to Ad-WT transduced T cells (white bars). Mock (patterned bars) and Ad-WT showed similar viability through the entire time-course observation (*p<0.05; **p<0.001; ns not significant). Error bars represent mean ± SD.

Figure 4. Adenoviral E1A copy numbers in transduced human CD8⁺ T cells.

Increased adenoviral E1A gene copy number in Ad-RGD treated cells (black bar) in comparison to Ad-WT treated cells (white bar) at different time-points after virus transduction calculated by qPCR. Samples 1–3 are three independent experiments. (*p<0.001; ** p<0.05). Error bars represent mean ± SD.

Figure 5. Viral progeny release from transduced human CD8⁺ T cells.

Viral progeny was measured from culture supernantant of adenovirus infected human T cells. 36- to 60-fold increase in viral progeny was measured in supernatants of T cell lysates treated with Ad-RGD (black bars) when compared to cells treated with Ad-WT (white bars) at 24 h after virus transduction. Samples 1–3 are three independent experiments (*p<0.001; ** p<0.01). Error bars represent mean ± SD.

Figure 6. T cells transduced with oncolytic Ad-RGD undergo rapid cell cycle.

(A) Flow cytometric profile of propidium iodide (PI) dilution in virus-transduced T cells at different time-points after treatment. Gates drawn on different cell-cycle phases are M1 sub-G0, M2 G0/G1, M3 S, M4 G2/M and M5 quiescent. Left panel shows cells treated with adenovirus with wild-type fiber (Ad-WT). Right panel shows cells with RGD-modified oncolytic virus (Ad-RGD). (B). Bar diagram representation of the cell-cycle. Top panel represents cells treated with Ad-WT. Bottom panel represents cells treated with Ad-RGD. White bars are cells after 24 h, while patterned and black bars are cells after 48 and 72 h respectively after virus transduction. Error bars represent mean ± SD.

Figure 7. Efficient in vivo transduction of mouse T cells by Ad-RGD.

Three groups of B6 mice were injected via tail-vein with Ad-WT (open squares), Ad-RGD (open triangles) and PBS (open circles) respectively. Animals were sacrificed at 0, 3, 6, 9, 16, 24, 72 and 96 h after virus injection and spleens were harvested. Splenocytes were surface stained for CD4 and CD8 and then fixed and permeablized and stained with anti-hexon antibody for flow-cytometric analysis of adenovirus infected splenic T cells. Percentage of CD4⁺ T cells expressing adenoviral hexon antigen is represented in upper panel and CD8⁺ T cells are in lower panel. Error bars indicate standard deviation from three animals per group of study. (*p<0.01;**p>0.05; N = 3)