Ovine dendritic cells transduced with an adenoviral CTLA4eEGFP fusion protein construct induce hyporesponsiveness to allostimulation

Abstract

CTLA4 (CD152) is a transmembrane molecule expressed on activated T cells and functions as a negative regulator of T cell activation upon binding to the costimulatory molecules CD80/86. In this study, CTLA4eEGFP constructs were engineered by cloning the extracellular domains of ovine and human CTLA4 (CTLA4e) 'in frame' with the enhanced green fluorescent protein (EGFP). Recombinant adenoviral vectors were generated by incorporation of the CTLA4eEGFP sequence into the adenoviral genome using homologous recombination in Esherichia coli. The functional activity of the adenoviral vectors was shown by the secretion of the CTLA4eEGFP upon infection of ovine fibroblasts and the binding of the fusion protein to the target ovine and human dendritic cells expressing CD80/86 receptors by flow cytometry. The EGFP tag facilitated molecular size determinations and quantification of the secreted ovine CTLA4 fusion protein by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA), respectively, using anti-GFP mAbs. Ovine dendritic cells obtained from pseudoafferent lymphatic cannulation of sheep were characterized based on high major histocompatibility complex (MHC) class II expression and cross-reactivity with monoclonal antibodies to the human dendritic cell markers, CD83 and CMRF-56. In addition, ovine dendritic cells (DC) were transfected with the adenoviral CTLA4eEGFP and when used as stimulators in a mixed lymphocyte reaction showed a reduced capacity to induce allogeneic lymphocyte proliferation. This study verifies that the ovine CTLA4eEGFP fusion protein functions similarly to its human homologue and that DC modified with adenoviral CTLA4-EGFP may provide an effective therapeutic approach in targeting alloreactive T cells to prolong allograft acceptance in a preclinical ovine model of renal transplantation.

Figure 1

CTLA4_eEGFP expression by genetically modified CHO-cells and fibroblasts. Autoradiographs showing immunoprecipitation with an anti-GFP mAb of ³⁵S-methionine/cysteine-labelled proteins from CHO cells (a and b) transfected with povCTLA4_eEGFP, phuCTLA4_eEGFP or pEGFP-N1 plasmid DNA and ovine dermal fibroblasts (c) infected with recombinant adenoviral particles containing the ADVovCTLA4_eEGFP or ADV-EGFP gene constructs. Immunoprecipitates were subject to electrophoresis in 12·5% SDS-PAGE gels under non-reduced (a) and (c) or reduced (b) conditions. (d) Expression of green fluorescence by adenoviral-transduced fibroblasts under UV-microscopy (magnification × 20). Upper panel (d) shows transduction with ADV-EGFP and lower panel (d) with ADVovCTLA4_eEGFP. The autofluorescence of untransfected cells (not shown) was negligible.

Figure 2

Phenotypic analyses of ovine lymphatic dendritic cells and the binding activity ovCTLA4_eEGFP by flow cytometry (a) Ovine DC obtained by pseudo-afferent cannulation of the prefemoral lymph node were stained with control isotype antibodies (open histograms) and a panel of cell-surface antigen markers (filled histograms). Each histogram is representative of at least three different experiments. (b) Human or ovine DC were incubated with ovCTLA4_eEGFP derived from recombinant adenovirus modified fibroblasts. The bound ovCTLA4_eEGFP was detected with an anti-GFP mAb as shown in the shaded histogram and the negative-isotype matched mAb (X63) represented by the solid line. The dotted line represents blockade of ovCTLA4_eEGFP binding with CTLA4-Ig. (c)Human DC were stained with anti-CD80 and -CD86 mAbs to confirm the presence of the CTLA-4 receptors. Flow cytometric analysis of CD80 and CD86 staining is represented by the shaded histogram with X63 represented by the unshaded histogram.

Figure 3

Alloinhibitory properties of ovCTLA4_eEGFP. (a) Dose–response inhibition of a two-way ovine MLR by ovCTLA4_eEGFP. Responses are expressed as the percentage of inhibition relative to the untreated MLR. (b) Comparative alloinhibitory properties of ovCTLA4_eEGFP, huCTLA4_eEGFP and native EGFP. Fusion proteins were normalized to 5 µg/ml of EGFP and added to an ovine MLR. CsA was added at 100 ng/ml as an established inhibitory control. (c) Ovine DC were transfected with ADVovCTLA4_eEGFP or ADV-EGFP. Cell transduction efficiency is demonstrated as a dot-plot representation (FL-1 versus forward scatter). (d) DC modified with ADVovCTLA4_eEGFP were used as stimulators of ovine PBMC at ratios of 1:10 and 1:100. Unmodified DC and DC modified with ADV-EGFP were used as controls. Each treatment group consisted of triplicates and each graph is representative of three different experiments. *P < 0·05, unpaired Student's t-test.