Contrasting effects of myeloid dendritic cells transduced with an adenoviral vector encoding interleukin-10 on organ allograft and tumour rejection

Abstract

Mouse bone marrow-derived myeloid dendritic cells (DC) propagated in granulocyte-macrophage colony-stimulating factor and transforming growth factor-beta1 (TGF-beta1) (so-called 'TGF-beta DC') are phenotypically immature, and prolong allograft survival. Interleukin-10 (IL-10) has been shown to inhibit the maturation of DC by down-regulating surface major histocompatibility complex (MHC) class II, co-stimulatory and adhesion molecule expression. Genetic engineering of TGF-beta DC to overexpress IL-10 might enhance their tolerogenic potential. In this study, adenoviral (Ad) vectors encoding the mouse IL-10 gene were transduced into B10 (H2b) TGF-beta DC. Transduction with Ad-IL-10 at a multiplicity of infection (MOI) of 50-100 resulted in a modest reduction in the incidence of DC expressing surface MHC class II, CD40, CD80 and CD86. Paradoxically, Ad-IL-10 transduction enhanced the allostimulatory activity of DC in mixed leucocyte reactions and cytotoxic T lymphocyte assays, and increased their natural killer cell stimulatory activity. Systemic injection of normal C3H recipients with Ad-IL-10-transduced B10-DC 7 days before organ transplantation, exacerbated heart graft rejection and augmented circulating anti-donor alloantibody titres. Contrasting effects were observed in relation to tumour growth. All mice preimmunized with Ad-IL-10-transduced, tumour antigen (B16F10)-pulsed DC developed palpable tumours, associated with significant inhibition of splenic anti-tumour cytotoxic T lymphocyte generation. Animals pretreated with control Ad-LacZ-transduced, B16F10-pulsed DC however, remained tumour free. These findings are consistent with the multifunctional immunomodulatory properties of mammalian IL-10.

Figure 1

Expression of IL-10 in TGF-β DC transduced with Ad-IL-10. B10 bone marrow-derived DC were propagated in the presence of GM-CSF and TGF-β₁ for 5 days, and then transduced with (a) Ad-LacZ or (b) Ad-IL-10 at 50 MOI as described in the Materials and Methods. An avidin–biotin complex–alkaline phosphatase immunocytochemical staining procedure was used to identify intracellular IL-10, as seen in (b); counterstained with fast red. Magnification: × 70 (a); × 140 (b).

Figure 2

Flow cytometric analysis of TGF-β DC propagated from B10 (H2^b) bone marrow for 5 days and then transduced with Ad-LacZ or Ad-IL-10 (50 MOI), as described in the Materials and Methods. Data indicate the incidence of cells expressing molecules of interest on the cell surface (a), and the mean fluorescence intensity of these positive cells (b). The data are means ± 1SD of three separate experiments.

Figure 3

Ad-IL-10 transduction enhances the allostimulatory function of TGF-β DC. Unmodified, bone marrow-derived TGF-β DC (B10, H2^b) or DC transduced with either Ad-LacZ or Ad-IL-10 (50 MOI), were tested as stimulators of naïve, allogeneic (C3H, H2^k) T cells in 3-day MLR. Data are mean c.p.m. ± 1SD of triplicate cultures, and are representative of three separate experiments.

Figure 4

TGF-β DC transduced with Ad-IL-10 (50 MOI) show markedly increased capacity to induce the generation of (a) CTL and (b) NK cell activity. Bone marrow-derived TGF-β DC (B10; H2^b) were transduced with either Ad-LacZ or Ad-IL-10. (a) The DC were used as stimulators of CTL induction in 6-day MLR, with naive C3H (H2^k) T cells as responders. Non-transduced DC were used as controls. Cytotoxicity against EL4 targets (H2^b) was determined at various effector : target cell ratios in a 4-hr ⁵¹Cr-release assay. (b) The DC were used as inducers of NK cell activity in 6-day MLR, with naive C3H (H2^k) spleen cells as responders. Non-transduced DC were used as controls. Cytotoxicity against YAC-1 targets was determined at various effector : target cell ratios, in a 4-hr ⁵¹Cr-release assay. Data are means ± 1SD of triplicate cultures, and are representative of three separate experiments.

Figure 5

Influence of Ad-IL-10 transduction on the capacity of TGF-β DC to prolong allogeneic vascularized organ graft survival. Bone marrow-derived TGF-β DC (B10; H2^b) were transduced with either Ad-LacZ or Ad-IL-10 then injected (2 × 10⁶) intravenously, 7 days before C3H (H2^k) recipients received B10 cardiac allografts. Non-transduced DC were used as controls. Graft survival in the Ad-IL-10 DC group was reduced significantly compared with either the non-transduced DC or Ad-LacZ-transduced DC groups (P < 0·01). Numbers of mice per group are shown in parentheses.

Figure 6

Serum complement-dependent lymphocytotoxic antibody titrrs against B10 (H2^b) targets in normal C3H (H2^k) mice (no transplant), or C3H recipients of B10 heart allografts, pretreated (day 7) with unmodified B10 TGF-b DC, Ad-LacZ-transduced DC (Ad-LacZ), or Ad-IL-10-transduced DC (Ad-IL-10). The sera were obtained 4 days after cardiac transplantation. The results (means ± 1SD) are representative of two separate experiments, with three animals per group at each time point.

Figure 7

Influence of Ad-IL-10 transduction on the anti-tumour activity of tumour antigen-pulsed DC. B6 mice were either untreated (no DC; n = 8) or injected i.p. with 10⁶ TGF-β DC, that had been pulsed with Bl6F10 tumour antigen, and transduced with Ad-LacZ (n = 8), or Ad-IL-10 (n = 8), 14 days before challenge with 10⁴ B16F10 tumour cells by subcutaneous injection. Non-transduced DC pulsed with tumour antigen gave similar results to those shown for Ad-LacZ DC (data not shown). Data are percentages of tumour-free animals in each group.

Figure 8

Influence of Ad-IL-10 transduction of tumour antigen-pulsed TGF-β DC on the tumour-specific cytotoxic activity induced by these cells in spleens of immunized mice. Effector cells were harvested from mice 14 days after immunization with tumour antigen-pulsed DC transduced with either Ad-LacZ or Ad-IL-10. They were cultured with tumour target cells for 5 days. Cytotoxicity against tumour antigens was determined in a 4-hr ⁵¹Cr-release assay. Data are means ± 1SD of triplicate cultures, and are representative of three separate experiments.