Transgenic expression of the coxsackie/adenovirus receptor enables adenoviral-mediated gene delivery in naive T cells

Abstract

The inability to easily and efficiently introduce genes into primary T cells has hampered the investigation of the pathways controlling T cell fate. To enable adenoviral-mediated gene transfer into normal naive T cells, transgenic (Tg) mice expressing the coxsackie/adenovirus receptor (CAR) in their T cell compartment were constructed. Whereas naive T cells are resistant to adenoviral infection, Tg expression of CAR on T cells greatly facilitates adenoviral-mediated gene expression ex vivo, in vivo, and in differentiated T helper cells. Thus we have developed a technology for efficient gene delivery to naive T cells. By using adenoviral vectors encoding specific inhibitors, we show that G1 cyclin-dependent kinase, NF-kappaB, and caspase activities are required for the proliferation of primary T cells. In addition, by expressing Bcl-x(L) protein at a level that closely approximates mitogen-induced levels, we demonstrate that Bcl-x(L) expression is sufficient to account for mitogen-mediated survival of primary T cells. Thus, adenoviral-mediated gene delivery to CAR Tg T cells should be useful for the analysis of many genes controlling T cell fate.

Figure 1

Expression of CARΔ1 and wt CAR on Tg lymphocytes. (A) CAR expression does not perturb T cell development. Lymphocytes were isolated from the spleen or thymus of either non-Tg, wt CAR Tg, or CARΔ1 Tg mice, stained with fluorescent-conjugated antibodies to CAR, CD4, CD8, and/or B220, and analyzed by flow cytometry (CD4 and CD8 expression in the spleen and thymus is shown). The percentages shown for cells in each quadrant represent the average for four sets of age- and sex-matched mice. Thymic and splenic cellularity did not differ significantly between mice of the different genotypes. Average cell number ± SD (×10⁷) for the thymus: non-Tg, 7.7 ± 3.6; CARΔ1, 8.6 ± 4.7; and wt CAR, 7.5 ± 3.4. For the spleen: non-Tg, 9.9 ± 2.3; CARΔ1, 12.0 ± 5.6; and wt CAR, 14.1 ± 1.6. (B) The expression of CAR on lymphocyte subsets. The expression of CAR on peripheral blood T cells (B220-negative lymphocytes), B cells (B220 positive), CD4⁺CD8⁺ thymocytes, or CD4⁻CD8⁻ thymocytes was determined by flow cytometry as in A. The mean value of fluorescence for the entire cell population is indicated inside each histogram. (C) The CAR Tg mRNA is specifically expressed in the thymus and peripheral lymphocytes. Total RNA was isolated from the indicated tissues from either non-TG (−) or CARΔ1 Tg mice and analyzed for the expression of the human CARΔ1 transgene by Northern blotting. Lymphocyte RNA was isolated from the spleen and lymph nodes combined. The positions of RNA size markers are indicated. The expression of 28S and 18S rRNA (methylene blue stain) is shown as a loading control.

Figure 2

CAR Δ1 Tg mature T cells and thymocytes are efficiently transduced by recombinant adenovirus. (A) Transduction of mature lymphocytes. Lymphocytes from CAR Tg or non-Tg mice were harvested from the spleen and lymph nodes (pooled), transduced with either AdCMV-GFP or AdUbC-GFP at a MOI of 10, cultured overnight, and stained with fluorescent-tagged antibodies to CD4 and CD8. GFP expression (displayed on the x axis; the y axis represents cell number) was monitored by flow cytometry in lymphocytes gated for the expression of either CD4, CD8, or neither (B cells). In A and B, percentages of GFP-positive cells are indicated inside each histogram. Gating for GFP-positive cells was determined based on the fluorescent intensity of mock-transduced cells. (B) Transduction of CAR Tg thymocytes. Thymocytes from CARΔ1 Tg and non-Tg mice were transduced with AdUbC-GFP at a MOI of 5 as described in A. GFP expression was determined in thymocytes gated for the expression of both CD4 and CD8 or neither (CD4⁻CD8⁻ thymocytes). (C) Dose-dependent gene expression. Lymphocytes from CARΔ1 Tg mice were transduced with AdUbC-GFP at the indicated MOIs as described in A. The mean value of GFP intensity for transduced T cells is plotted relative to the MOI. (D) T cell activation potentiates CAR-mediated adenoviral delivery. Peripheral lymphocytes (DO11.10 TCR and CARΔ1 double Tg) were harvested and either immediately transduced with AdUbC-GFP at a MOI of 1 (Unactiv.), or cultured in RP10 together with 5 μg/ml OVA (ISQAVHAAHAEINEAGR) or 4 μg/ml Con A for 2 days before transduction with AdUbC-GFP at a MOI of one. Twenty-four hours after transduction, the cells were harvested, stained with anti-B220, and analyzed for GFP expression in lymphocytes by flow cytometry. The upper and lower quadrants represent B cells and T cells, respectively. The percentages in each quadrant are indicated. (E) Manipulation of gene expression in T cells in vivo. Lymphocytes from CARΔ1 Tg mice were harvested and transduced with AdUbC-GFP at a MOI of 10 ex vivo as described in A. Transduced cells (or mock-transduced cells) were washed twice with PBS and immediately transferred into recipient FvB mice by subocular injection; 1, 3 and 5 days after transfer, splenocytes from recipient mice were harvested and stained with antibodies to CAR and B220. The expression of CAR and GFP was determined in T cells (B220 negative) by flow cytometry.

Figure 3

Generation of CAR Tg Th1 and Th2 cell clones. (A) CAR expression on Th1 and Th2 cell clones. T cell clones CAR.Th1.12 and CAR.Th2.5 were stained with antibodies to CAR and CD3 and analyzed by flow cytometry. The previously established wt Th1 clone pGL10 is shown for comparison. Cytokine ELISA OD after stimulation with anti-CD3 for 24 h are displayed in the Inset. (B) Transduction of CAR.Th1.12. The Th1 clone was transduced with AdCMV-GFP at a MOI of 10, and flow cytometric analysis of GFP expression was performed 16 h later. (C) IL-2 production. CAR.Th1.12 cells were transduced with increasing concentrations of AdCMV-GFP, cells were stimulated with either anti-CD3 plus anti-CD28 mAbs or 50 ng/ml PMA/0.5 μM ionomycin, and IL-2 production was measured at 24 h.

Figure 4

Adenoviral-mediated manipulation of T cell proliferation and survival pathways. (A) The expression of p27KIP, CrmA, and IκB can block T cell proliferation. Lymphocytes from CAR Tg mice (BALB/c) were harvested and either mock transduced or transduced with adenovectors expressing either p27, CrmA, IκB, Mdm2, or GFP at the indicated MOI. One day after transduction, T cells were cultured with PMA and Con A in RP10 with [³H]thymidine for 2 days, and the amount of incorporated [³H]thymidine (±SE) was determined by scintillation counting. The first two bars represent mock-transduced cells that did not receive PMA or Con A. (B) Adenoviral-mediated expression of Bcl-x_L in T cells. Lymphocytes from CARΔ1 Tg mice (BALB/c) were harvested and transduced with AdUbC-GFP or AdCMV-Bcl-x_L at a MOI of 10, and then cultured in RP10 for 2 days. (Left) Cells were mock transduced and cultured without (−) or with 4 μg/ml Con A. Cells were stained with anti-B220, fixed, permeabilized, and then stained with an antibody to Bcl-x_L. The cells were analyzed by flow cytometry for the expression of Bcl-x_L in T cells (B220 negative). (C) Bcl-x_L expression increases T cell survival in the absence of mitogenic stimulation. Apoptosis was determined in the cells from the same experiments described in B. The cells were harvested, stained with allophycocyanin-linked anti-B220, stained with propidium iodide, and DNA content (x axis) was determined in T cells (B220 negative) by flow cytometry. The percentage of T cells with a sub-G₁ DNA content (apoptotic) representing the average of two experiments (±SE) is indicated.